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New Jersey Academy of Science |
2000 Senior Academy Abstracts |
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**DISCLAIMER: These abstracts were uploaded from Word to HTML format. Some of the formatting and symbols were lost. Although most were restored, we cannot guarantee that there are no errors. Also, some of the chemistry abstracts included structures that are not shown here. For "official" versions of the abstracts, please contact the authors or consult The Bulletin of the New Jersey Academy of Science, in which the abstracts are published. Thank you!** POSTER PRESENTATIONS
The endometrial layer of the uterus undergoes cyclic changes in
response to female sex homones, such as estrogen and progesterone. Further, estrogens and
progesterones have been used as hormone replacement therapy (HRT) in postmenopausal women.
The aim of this research was to examine the responsiveness of normal and carcinogenic
uterine endometrial cells to 17b-estradiol (estrogen) and medroxyprogesterone acetate
(progesterone) in terms of cellular proliferation and production level of collagenase and
growth factors. Normal, unaffected endometrial cells (cell line AG02101) and endometrial
carcinogenic cells (cell line CRL95-2) were obtained from Coriell Cell Repositories and
American Tissue Culture Collection, respectfully. The normal and cancer cells were treated
separately with optimal doses (minimal toxicity) of estrogen, progesterone or
estrogen/progesterone combination in serum-free media. Results showed differential effects
of hormones on normal and cancer cells. In normal uterine endometrial cells, estrogen and
estrogen/progesterone combinations inhibited cell proliferation and inhibited secretion of
interstitial collagenase and growth factors [transforming growth factor (TGF) beta, and
TGF but not epidermal growth factor (EGF)]. Opposite effects were seen in cancer cells,
with stimulation of cell proliferation and secretion of interstitial collagenase and
growth factors on treatment with estrogen/progesterone combinations. Estrogen was
inhibitory to cancer cells whereas progesterone had minimal effects on both normal and
cancer cells. Hence, while estrogen and progesterone combinations may be beneficial to
normal uterine endometrial cells, they may facilitate proliferation and metastasis of
cancer cells. NONTOXIC EFFECTS OF BENZENE METABOLITES ON HUMAN DERMAL
FIBROBLASTS Benzene, a cytotoxic chemical, is known to be metabolized in the liver through the cytochrome P450 2E1 to phenolic compounds. Once oxidized, the metabolites play a substantial role in benzene toxicity by inducing either DNA alkylation or oxidative stress which leads to many systemic disorders. Skin contains an aromatic hydrocarbon responsive (Ah) battery and is a major route of entry of benzene into the body. We had observed by gas chromatography coupled with mass spectrometry that the benzene metabolites formed in skin cells are not toxic phenol compounds but alkyl benzenes such as toluene, benzaldehyde, benzamine and benzoic acid. In this study we examined the effects of these skin specific benzene metabolites on cell toxicity, DNA apoptosis and expression of extracellular matrix proteins. We observed that these metabolites were not toxic to skin cells for the parameters studied. Hence skin can act as a barrier to detoxify benzene and reduce its detrimental effects in the body.
A poly(anhydride-ester) was synthesized by incorporating a
bioactive molecule, salicylic acid into the polymer backbone. The polymer was synthesized
in several steps. First, we combined benzylated salicylic acid with sebacoyl chloride to
give benzylated diacid. Second, catalytic hydrogenation was used to deprotect the
benzylated diacid by removing the benzyl protecting groups. Third, the deprotected diacid
was activated by acetic anhydride. Later, all compounds were analyzed by 1H NMR
and IR spectroscopy, as well as by GCMS confirming the diacid as our final compound. Melt
condensation polymerization was performed to yield the polymer. The polymer undergoes
hydrolytic degradation to release salicylic acid, which for its medicinal properties has
potential for a variety of medical treatments (e.g., inflammatory bowel disease,
periodontal prosthetics). We are also interested in controlling the rates of polymer
hydrolysis by changing the linker chain length. Progress towards synthesis of several
novel poly(anhydride-esters) incorporating these new linkers will also be presented. USING GEOGRAPHIC INFORMATION SYSTEMS TO ANALYZE THE MOVEMENT OF
HORSESHOE CRABS (Limulus polyphemus) IN DELAWARE BAY Delaware Bay is the population epicenter of horseshoe crabs (Limulus polyphemus). Currently it is known that there is an important connection between the spawning horseshoe crabs and their eggs and the migratory shorebirds. The best evidence indicates that there is a decrease in horseshoe crabs and their eggs, which can seriously impact the horseshoe crab population, migratory shorebirds, fishermen, and the biomedical industry. To further our understanding of the horseshoe crab, over 10 years of tagging data from Benji Swan of Limuli Laboratories, Inc. was geographically analyzed to determine distances and direction traveled by horseshoe crabs, to verify that the proportion of males returning to the same beach within days of release is greater than females returning to the same beach, and to ascertain if horseshoe crabs display fidelity to an area within days, months, and years since release. Horseshoe crabs were released from New Jersey, Delaware, Maryland, and New York and recovered in New Jersey, Delaware, Maryland, Virginia, and other offshore locations, traveling a maximum distance of 260.3 km. The proportion of males returning to the same release site within days is greater than the proportion of females (z = 1.90, a = 0.05). Within days, months, and years between release and recovery, horseshoe crabs do display fidelity to the area of release (days: C 2 = 345.1, df = 1, a = 0.01; months: C 2 = 48.7, df = 4, a = 0.01; years: C 2 = 22.7, df = 2, a = 0.01). The fidelity to an area appears strongest within days of release, diminishing, though still significant, as time goes on with more crabs traveling across the bay to either New Jersey or Delaware.
The decline in fisheries over the last several decades led the U.S. Congress to pass the Sustainable Fisheries Act of 1996. The Act stresses the need to identify and map essential fish habitat as part of restoring fishery resources. Recently acquired sidescan sonar imagery of the seafloor in the New York Bight Apex provides continuous coverage of the benthos over a 1,976 km2 area and has the potential to meet guidelines of producing essential fish habitat maps over large spatial regions. The Rutgers University Center for Remote Sensing and Spatial Analysis, in cooperation with the USGS Woods Hole Field Center and the NOAA Northeast Fisheries Science Center, has instigated the New York Bight Apex Seafloor Habitat Mapping Project to develop and test strategies for remotely mapping the benthic habitats of commercial fish species and investigate the influence of sea floor geology and seafloor disturbance on the distribution, abundance and diversity of fishery resources. Traditional classification algorithms and geographic information system techniques, which have been applied extensively to terrestrial and nearshore regions, are being utilized to characterize seafloor type. The relationship of sonar signal strength is not highly correlated with benthic sediment type, which complicates the interpretation of the imagery. However, general categories of seafloor type can be created based on classifying sonar signal, bathymetry and secondarily derived spatial heterogeneity. A seafloor type map is produced and assessed for accuracy that will be used with in situ observations of biota and the seafloor (photography and video) to map habitats in future research.
Ictacalcin is a novel calcium binding protein found exclusively in chemosensory tissue of catfish (olfactory epithelium, barbel and gills). Isolation of the promoter region of the ictacalcin gene may give insight as to the regulation of its expression. In other words, why is this gene only turned on in chemosensory tissues of catfish and not in other tissues (brain, liver, muscle, for example)? In order to isolate the promoter region of the ictacalcin gene, a genomic DNA library needs to be screened using the ictacalcin cDNA clone already in hand. Genomic DNA was prepared from both catfish liver and muscle using a commercially available kit from Stratagene. The integrity of the DNA was assessed using agarose gel electrophoresis. The DNA was digested using increasing concentrations of the restriction enzyme EcoRI in order to determine the concentration of enzyme needed to obtain DNA fragments in the 9 to 21 kilobase size range. This is the size range needed to insert the DNA into the lambda DASH vector that will be used in the library preparation.
Ictacalcin is a novel calcium binding protein found exclusively in chemosensory tissues of the catfish, Ictalurus punctatus. In order to isolate the promoter region of the ictacalcin gene, a catfish genomic library will be screened using ictacalcin cDNA as a probe. In order to visualize the genomic clone containing the ictacalcin promoter sequences, biotin labeled ictacalcin cDNA will be hybridized to the library clones and the positive clones detected by a colorimetric reaction. This abstract describes the preparation of biotinylated ictacalcin cDNA and its detection. Ictacalcin cDNA is isolated from the plasmid pST18-ictacalcin by restriction digestion using BamHI and EcoRI. The 327 base pair ictacalcin cDNA is isolated by running the digest on a 1.5% agarose gel, transfer of the cDNA to a DEAE membrane, and elution of the cDNA into a high salt buffer followed by ammonium acetate/ethanol precipitation. The ictacalcin cDNA is biotinylated by annealing random primers to the denatured cDNA and extending using Klenow fragment in the presence of biotin 14-dCTP. Unincorporated nucleotides are separated from biotinylated ictacalcin cDNA probe by Sephadex G-50 column chromatography. The biotinylated ictacalcin cDNA is detected by incubation with streptavidin-alkaline phosphatase conjugate, which binds to the biotin, followed by incubation with the substrates for alkaline phosphatase, NBT and BCIP. These form a purple precipitate that irreversibly binds to the nitrocellulose membrane, indicating the presence of biotinylated ictacalcin cDNA.
Biochemisty I EFFECTS OF ANTI-TGF-b ANTIBODIES ON
TGF-b SECRETION, EXTRACELLULAR (ECM) PROTEINS AND DNA APOPTOSIS
IN DERMAL FIBROBLASTS Transforming growth factor –beta (TGF-b ) has been reported to generate reactive oxygen species (ROS) in cells. ROS is a major mediator of photoaged skin, which is characterized by alterations in ECM proteins. The aim of this study was to examine the effects of anti-TGF-b antibodies [TGF-b and latent associated peptide (LAP)] on the expressions TGF-b and extracellular matrix proteins, as well as on DNA apoptosis, in normal dermal fibroblasts. In three independent studies, dermal fibroblasts were untreated or treated with anti- TGF-b antibodies for 24 hours, in incubation medium without serum or with serum. The incubation media was analyzed for TGF-b , LAP and ECM proteins by an enzyme linked immunosorbant assays (ELISA) using specific antibodies. The cells were harvested, counted and examined for apoptosis. In the absence of serum there were dramatic increases in expressions of TGF-b , L AP and elastin (500 to 1000 fold), while there were decreases in both collagenase and fibronectin (FN). In the absence of serum, the TGF-b antibodies decreased apoptosis in fibroblasts. In contrast, in the presence of serum there were slight decreases in collagenase, elastin and FN and there were no differences in the expression of TGF-b and LAP. In the presence and absence of serum, there was an increase in cell proliferation with TGF-b antibodies. In conclusion, TGF-b antibodies can induce TGF-b secretion, perhaps by autocrine stimulation, and can remodel the extracellular matrix favorably in dermal fibroblasts.
Nitric oxide, a cellular signaling molecule, can cause oxidative damage by generating reactive nitrogen species or protect cells by neutralizing reactive oxygen species generated in cells. The aim of this study was to determine the effects of NO on (a) the promoter activities of interstitial collagenase, ICAM-1 and a transcription factor, NF-K B and (b) the cellular expressions of growth factors and ECM proteins in human dermal fibroblasts. A secondary aim was to determine the effects of NO on hydrogen peroxide (H2O2) mediated alterations in dermal fibroblasts. Dermal fibroblasts were transfected with interstitial collagenase, ICAM-1 or NF-K B – chloramphenicol acetyl transferase (CAT) plasmids. Forty eight hours following transfection, cells were untreated or exposed to hydrogen peroxide (H2O2), a combination of nitric oxide donors [Sodium nitroprusside(SNP) and a S-nitroso derivative of glutathione (GSNO)], or with a combination of H2O2 and SNP/GSNO for 24 hours. Three independent experiments were performed and analyzed. CAT activities in cells were measured to determine promoter activities. Growth factors and ECM proteins, secreted into the cell incubation media, were determined by enzyme linked immunosorbant assays (ELISA), using specific antibodies. Nitric oxide significantly inhibited the promoter activities of ICAM-1 and NF-K B, and was inhibitory to the collagenase promoter to a lesser extent. Further, NO inhibited the secretion of growth factors and extracellular matrix proteins, known to be upregulated on photoaging of skin. The H2O2 did not induce significant changes and could be the result of the low dosage used in experiments. In conclusion, nitric oxide has photoprotective properties in dermal fibroblasts.
Rapid decay of mRNAs is an important mechanism of regulating gene expression. Many mRNAs encoding proteins involved in cell growth, cell division, and the inflammatory response are particularly unstable. These mRNAs share common sequences, termed A+U-rich elements (AREs), in their 3'-untranslated regions which target the mRNAs for rapid decay in the cytoplasm. AREs are specifically recognized by the RNA-binding protein AUF1, and the binding affinity of an ARE for AUF1 correlates with the potential of the ARE to destabilize mRNA. Previously, we observed that the affinity of AUF1 binding to AREs in vitro was influenced by the presence of magnesium ions. The purpose of this study was to test the hypothesis that Mg2+-induced changes in RNA structure are involved in regulating AUF1 binding affinity. Using fluorescence anisotropy to monitor changes in molecular motion in solution, we found that Mg2+ restricted the motion of an ARE in a dose-dependent manner. The sensitivity of this response was influenced by both temperature and RNA sequence. Stoichiometric analyses indicated that the decrease in RNA mobility resulted from a 1:1 association of RNA and Mg2+ and was consistent with the adoption of a Mg2+-induced intramolecular RNA structure. Using this model, the affinity of Mg2+ for different RNAs was evaluated and compared to AUF1-binding affinity. In each case, we observed that strong binding affinity of Mg2+ for an ARE correlated with inhibition of AUF1-binding by Mg2+. This indicated that the recognition of an ARE by AUF1 is not solely dependent on RNA sequence, but may be influenced by higher-order RNA structures near the binding site. Potential regulatory implications of these findings are discussed.
Twenty-six essential oils were tested for antimicrobial activity against two Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, two Gram-positive bacteria Staphylococcus aureus and Bacillus subtitis and the fungus Candida albicans. Two antibiotics, chloramphenicol and streptomycin, and one antifungal agent, nystatin, were used as zone diameter interpretive standards. Our results demonstrate that the two Gram-negative bacteria were susceptible to Cinnamon oil. The two Gram-positive bacteria were susceptible to the essential oils of: Thyme, Wintergreen, Geranium, Peppermint, Cinnamon, Grapefruit, Allspice and Clary Sage. B. substitis was also susceptible to Rosewood oil. Candida albicans was susceptible to the essential oils of Thyme, Wintergreen, Geranium, Sage, Spearmint, Peppermint, Bay, Cinnamon, Grapefruit, Clove, Allspice, Clary Sage, Balsam Fir and Geranium Egyptian. Screening of essential oils for antimicrobial activity may provide an additional source of organic chemicals active in inhibiting the growth of bacteria and fungi as well as providing insight as to the biochemical mechanism which produce the inhibition.
The aim of this study was to examine the effects of hydrogen peroxide, a reactive oxygen species, and ascorbic acid, an antioxidant or oxidant on cancer cells (renal and uterine cancer cells). Cells were exposed to different doses of hydrogen peroxide or ascorbic acid or combinations of hydrogen peroxide and ascorbic acid for 24 hours. The cells were examined for cell proliferation and DNA apoptosis. In renal adenocarcinoma cells, lower doses of hydrogen peroxide and ascorbic acid inhibited cell growth. Higher doses of ascorbic acid were stimulatory to renaladenocarcinoma cells whereas higher doses of hydrogen peroxide were less toxic to the renal cells than lower doses. In uterine endometrial cells, hydrogen peroxide alone caused cell toxicity and ascorbic acid could prevent toxicity caused by hydrogen peroxide. Further, in uterine cancer cells, ascorbic acid reversed the effects of higher doses of ascobic acid in relation to secretion of growth factors and collagenase expression to levels observed with ascorbic acid alone. Hence, renal adenocarcinoma cells and uterine endometrial cells differ in their responsiveness to hydrogen peroxide and ascorbic acid. Further studies are ongoing to determine the beneficial versus harmful effects.
Incellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein that mediates cellular adhesion and plays a key role in the regulation of cutaneous inflammation, immunologic responses and wound repair. The function of ICAM-1 in fibroblast culture is significant to understanding the cell-cell mediated immune mechanisms. There is evidence that reactive oxygen species can upregulate ICAM-1 expression in keratinocytes. There is further evidence for antioxidant effects of nitric oxide donors and antibodies to certain growth factors (i.e. TGF-b ). The purpose of this study was to investigate the effects of oxidants (hydrogen peroxide and benzene metabolites), antioxidants (nitric oxide donors), and combinations of oxidants and antioxidants on ICAM-1 expression in fibroblasts. The cell cultures were transfected with ICAM-1/Chloramphenicol acetyl transferase (CAT) plasmid. Eleven different reagents were added to individual cell samples in serum free media. The reagents were hydrogen peroxide, hydroquinone, phenol, t-butyl hydroquinone, ceramide, S-nitroso derivative of glutathione (GSNO), sodium nitropruside (SNP), hydrogen peroxide/GSNO, hydrogen peroxide/SNP, hydrogen peroxide/anti-TGF-b /anti-LAP, and anti-TGF-b /anti-LAP. The ICAM-1 expression in response to oxidants, antioxidants, and combinations of antioxidants and oxidants was assessed by measuring ICAM-1 protein levels in the media and CAT activity in cells by Enzyme Linked Immunoabsorbant Assay (ELISA) using specific ICAM-1 antibodies and CAT antibodies. Fibroblasts demonstrated an inhibition of ICAM-1 expression to the variety of reagents. The ICAM-1 promoter inhibition was not as marked with ceramide, GSNO, and anti-TGF-b /anti-LAP treated cultures. In addition, GSNO and hydrogen peroxide/GSNO treatments stimulated ICAM-1 protein secretion into the media. Overall, our data demonstrated an inhibitory effect of the ICAM-1 promoter activity when treated with oxidants, antioxidants, and combinations of antioxidants and oxidants.
Ultraviolet radiation can damage and age skin by remodeling the extracellular matrix. The aim of this study was to determine the effects of ultraviolet radiation and NO on dermal fibroblasts, with regard to the modulation of interstitial collagenase, a collagen degrading enzyme, and elastin, an exrtracellular matrix protein. Photoaged skin is characterized by deposition of elastotic material and loss of collagen. The nitric oxide donors used were a combination of Sodium nitroprusside (SNP) and a S-nitroso derivative of glutathione (GSNO). Ultraviolet radiation inhibited collagenase secretion and upregulated elastin expression in dermal fibroblasts. Nitric oxide did not alter the ultraviolet irradiation induced changes in dermal fibroblasts. These results are different from those observed for keratinocytes. Hence ultraviolet radiation and nitric oxide differentially regulate expression of extracellular matrix genes in keratinocytes and fibroblasts. Biochemisty II THE EFFECT OF THE FEMALE SEX HORMONES PROLACTIN AND ESTROGEN ON THE
SECRETION OF MMP1 AND TGF IN BOTH NORMAL AND TURMOIGENIC MAMMARY EPITHELIAL CELLS Prolactin works synergistically with estrogen and progesterone to control neoplastic growth of mammary cells. Excessive levels of prolactin have been shown to cause cancer. The extracellular matrix, a reservoir of growth factors, hormones and matrix degrading enzymes, is suspected of having an influence over the prolactin-estrogen pathway but the details are unknown. The aim of our research was to determine the effects of prolactin, estrogen or a combination of each on the level of the extracellular matrix proteins, matrix metalloproteinase-1 (MMP1), and transforming growth factor beta (TGFb ), expression. MMP1 is a collagen-degrading enzyme that may play a role in tumorigenic cell metastasis and TGFb is a pleiotrophic growth factor with predominately antiproliferative qualities. The cells were transfected with a plasmid that contained either a normal or mutated (AP-1 site) MMP1 promoter behind chloramphenicol acetyl transferase (CAT) gene. After 48 hours the cells were exposed to 10 nM prolactin, 0.5 mM estrogen or a combination of each. The secreted levels of MMP1 and TGFb were tested using indirect ELISAs with specific antibodies and the cells were tested for CAT expression. Prolactin increased the MMP- promoter activity. Both MMP1 and TGFb secretions were decreased to nearly a quarter of control levels in prolactin treated cells. Estrogen treatment did not greatly alter the levels of MMP1 or TGFb . The combination of estrogen and prolactin also decreased levels to nearly one quarter of control levels. Prolactin was also shown to inhibit DNA apoptosis to nearly a third of control levels. Studies are ongoing to delineate the role of prolactin and estrogen on the proliferation and secretion of MMP1 and TGFb in normal cells.
Dioxins are a group of suspected carcinogens that are present in the environment. The aim of this study was to examine oxidative effects of dioxins, tetrachlorodibenzo dioxin (TCDD) and dibenzo dioxin (DD), on dermal fibroblasts and the ability of anti-TGFb to ameliorate these effects. Fibroblasts were exposed to either TCDD or DD with or without the anti-TGFb antibodies. After 24 hours media and/or cells were analyzed for lipid peroxidation, DNA apoptosis, membrane damage and extracellular matrix proteins. Cells toxified with either TCDD or DD showed increased levels of lipid peroxidation but in the presence of anti-TGF antibodies lipid peroxidation was prevented. TCDD or DD and anti TGFb antibodies did not significantly alter DNA apoptosis in cells. TCDD and DD enhanced elastin expression but in combination with anti TGFb antibodies there was further stimulation. Thus, it appears that both TCDD and DD can cause oxidative damage to cells and that the anti TGFb antibodies can counteract the oxidative effects.
Compared to adult rats, juvenile Long-Evans rats showed faster onset and greater severity of alcohol withdrawal symptoms after chronic alcohol consumption. The present study tested the hypothesis that juvenile rats are less efficient in alcohol metabolism than adults and therefore juveniles develop faster and more severe signs of alcohol dependency. Alcohol metabolism, blood alcohol levels and acute alcohol intoxication were studied in juvenile (<35 days old) and adult (>65 days old) Long-Evans rats. Activities of the NAD+-dependent alcohol dehydrogenase (AlcD) and aldehyde dehydrogenase (AldD) were measured by UV spectroscopy using soluble liver extracts. Both AldD and AlcD activities (Vmax) were significantly higher in the juvenile rats. The results are consistent with higher levels of enzyme in tissue from the juveniles. Consistent with liver enzyme activities, the rate of decrease in blood ethanol concentration was faster in juveniles as was the decline in intoxication score following acute alcohol exposure (2.5 g/kg, ip). These results eliminate the hypothesis that alcohol metabolism is deficient in juvenile rats, but raise new questions about the possible relationship between alcohol dependency and the tendency to enter full alcohol withdrawal during periods of intoxication.
Much research in sexology is of the verbal-descriptive kind despite the existence of decades of measurements of various kinds. A reasonably comprehensive mathematal/dynamic model does not exist. In this paper, a nonlinear differential equation model and its associated catastrophe is shown to model the simplest version of the sexual response of humans; simple in the sense that it is for males (whose response patterns are simpler than that of females), that there are no dysfunctions, and that no multiple orgasms are being modeled. Other than these the mathematical model is derived via well-known and non-controversial aspects of sexual orgasm as can be found in the literature. Two different and independent derivations of the equation are given; the equilibrium-oscillation model and the slow-fast dual excitation model. The associated catastrophe and response surface is related to commonly known facts, including various sexual dysfunctions. Although the model is simple (which is only simple in relation to how complex a total model that captures all aspects of sexuality including various possible dysfunctions could be) it does take into account physical, psychological and cognitive aspects of sex and orgasm. The model, has the extra benefit in that it makes possible a unification of the various measurements involving sexual excitement and orgasm in terms of the coefficients of the equations modeling the process.
Organophosphate insecticides seem less toxic to male than to female Oriental fruit moths in field trials. The molecular mechanisms of this apparent sex-linked difference are under investigation. Data indicate that male moths have higher levels of acetylcholinesterase and general esterase activities than females.
Tamoxifen, an antiestrogen, is used for breast cancer. The objective of this study was to determine the effects of Tamoxifen on interstitial collagenase expression in normal uterine endometrial cells. Expression of interstitial collagenase was examined at the transcriptional and protein levels. The cells were transfected with a plasmid containing either a normal or mutated (lacking AP-1 site) collagenase promoter linked to chloramphenicol acetyl transferase (CAT) gene. Experiments were perfomed in the absence or presence of serum in the incubation media. Following transfection (48 hours), the cells were either treated with 0.03 mM Tamoxifen or left untreated for 24 hours. Cells were analyzed for collagenase promoter activity and the media for secreted collagenase protein. The collagenase promoter activity, but not the mutated promoter, was remarkedly inhibited by Tamoxifen in normal cells lacking serum in the growth media. This indicates not only the inhibition of collagenase expression by Tamoxifen in normal cells, but also the involvement of the AP-1 response element in the promoter in the inhibitory response. In the presence of serum, the collagenase promoter activity was inhibited but the mutated promoter was stimulated indicating involvement of transcription factors, other than AP-1. These results indicate that Tamoxifen may not be harmful to normal uterine endometrial cells.
The purpose of this study was to examine the effects of tamoxifen on expression of interstitial collagenase and growth factors (TGFb, TGF-a , EGF, MMP-1) in breast cancer cells. Experimental cells were transfected with collagenase promoter-chloramphenicol acteyl transferase plasmid, as well as collagenase promoter mutated at the AP-1 site, in the presence and absence of serum. The results indicated that both in presence and absence of serum collagenase gene transcription was downregulated by Tamoxifen. Further, the mutated collagenase promoter showed similar down regulation, indicating the lack of involvement of the AP-1 site in the downregulation of the collagenase promoter activity by Tamoxifen. Tamoxifen also inhibited collagenase and growth factor levels in serum free incubation media. However, Tamoxifen caused a dramatic increase in the secretion of collagenase and growth factors, in the presence of serum in the media. These results indicate differential effects of Tamoxifen in the absence and presence of serum and of the collagenase gene expression, at transcriptional versus post-transcriptional levels in the presence of serum.
Chemistry SYNTHESIS OF A NOVEL AROMATIC POLY(ANHYDRIDE-ESTER) OF
5-AMINOSALICYLIC ACID FOR ENHANCED DRUG DELIVERY 5-Aminosalicylic acid (5ASA) is known to be an effective anti-inflammatory agent in treating diseases like Crohns disease and related inflammatory bowel diseases. Current drug delivery systems for 5ASA can cause allergic side effects, as is the case with Sulfasalazine. We are striving to synthesize a novel poly(anhydride-ester) incorporating 5-aminosalicylic acid based on existing methods to create a poly(anhydride-ester) from salicylic acid. Degradative studies of salicylic acid-containing polymer predict slowed digestion in the acidic environment of the stomach and direct delivery in the basic environment of the colon. We anticipate similar degradative behavior of 5ASA-containing polymer. The step-wise synthesis is as follows. The amino group of 5ASA is protected with a carbobenzoxy moiety using sodium bicarbonate as the base. Next, the carboxylate group is protected with a benzyl moiety, also using sodium bicarbonate as the base. These protections allow two units of 5ASA to be linked with sebacoyl chloride in the presence of sodium carbonate. In subsequent steps, the protecting groups are removed, the compound activated, and then polymerized. Chemical structures are confirmed by techniques including proton NMR and FTIR spectroscopy.
Concentrated nitric acid diluted to 10-15% strength with acetic acid has been found to be a very effective nitrating agent under microwave irradiation. In connection with the synthesis of a dinitro marine natural product (3), the nitration of p-hydroxycinnamic acid (1) was studied under different conditions. Reaction of dilute nitric acid with 1 in an unmodified domestic microwave oven (which provides multimodal microwave irradiation) leads to the mononitro cinnamic acid derivative (2) and the dinitrostyrene derivative (3). On the contrary, when o-hydroxycinnamic acid (4) and nitric acid/acetic acid mixture were subjected to multimodal microwave irradiation, a single product (5), which is a dinitrohydroxycinnamic acid, was formed. Comparative nitration studies were also conducted using computer controlled monomodal microwave instruments (Prolabo Synthewave S402 and S1000, and Milestone Ethos Plus). Details will be provided on controlled nitration in a few minutes of several electron rich aromatic compounds.
The preparation of 5-(oxy–propane–tert–butyldiphenylsiloxy)isophthalic acid, a propagating unit, is accomplished in four synthetic steps. The construction of polyurethane wedges of generation one is successful via a modified Curtius rearrangement using diphenylphosphoryl azide (DPPA) followed by deprotecting the siloxy group at the focal point with tetra n-butylammonium fluoride (TBAF). Materials are characterized by NMR and IR methods.
Magaly Huarotte (student). Ashoke Bhattarcharjee, Maghar Manhas, Ajay K. Bose. George Barash Bioorganic Research Laboratory, Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, NJ 07030 Aromatic nitro compounds are readily deoxygenated by triethyl phosphite. Many examples of this general reaction has been known for sometime, but the reaction involved refluxing under nitrogen or argon for long periods of time. A faster approach has been developed using microwave irradiation and utilizing open glassware, resulting in faster reaction time with good yields. A series of the nitro imines were reacted with triethyl phosphite in clorobenzene under microwave irradiation, the reaction was completed in minutes compared to the usual 9 to 10 hours.
The efficacy of the B3LYP and B3PW91 methods in conjunction with Pople type bases for the calculation of 73Ge quadrupole coupling constants in gaseous state molecules is discussed. Assessment of the several models is made by linear regression analysis of the calculated electric field gradients versus the experimental coupling constants. For the B3PW91/6-311G(2d) model, the residual standard deviation is 0.84 MHz, which is 1.0% of the average experimental coupling constant. In the case of germylacetylene [1], our calculations indicate that the experimental coupling constant has been incorrectly assigned with respect to algebraic sign. [1] E. C. Thomas and Victor W. Laurie, J. Chem. Phys. 44, 2602 (1966).
Micropatterned protein surfaces can be used to direct cell growth, which has potential application in peripheral nerve repair after injury. Previous studies illustrate that nerve cells can be guided by depositing specific proteins on inorganic substrates such as glass or gold. The inorganic substrates are patterned to create alternating regions of proteins that enhance cell adhesion with regions that do not enhance cell growth. The patterns are created using microlithography, a technique also utilized in the microelectronics industry. Although it is known that microlithographically patterned inorganic substrates direct cell growth, the chemistry at each processing step and the interactions responsible for cell guidance are unknown. In this project, we examine the chemical content of the surfaces at several crucial steps in the microplithographic procedure. We are using x-ray photoelectron spectroscopy (XPS), a surface analysis tool that enables us to quantify the chemical composition of the surfaces. Since certain elements are signatures of chemicals used in the process, we can correlate the XPS data with chemical events occurring on the surface. The understanding of chemical interactions on the surfaces is crucial to optimizing chemically modified surfaces that direct and enhance cell growth. Marine Science I
Dredging in New Jersey coastal bays has been principally conducted at tidal inlets, the Intracoastal Waterway, and channels leading to these areas. In the Manasquan, Barnegat Bay, Little Egg Harbor region, for example, the amount of sediment dredged annually from the tidal inlets has ranged from ~10,000 m3 to more than 1,000,000 m3, with most of the sediment removed from Barnegat Inlet and dumped by barge in the nearshore ocean. Dredging maintains the Intracoastal Waterway to a depth of 1.5 to 3.7 m below mean low water and a width of 30 m over most of its length. The U.S. Army Corps of Engineers is responsible for dredging at the tidal inlets and the Intracoastal Waterway, whereas the New Jersey Department of Environmental Protection oversees dredging elsewhere in the system. Much sediment removed from the estuarine floor has been dumped at dredged spoil islands which now provide valuable habitat for many organisms. Although the volume of sediment removed from Barnegat Inlet and Manasquan Inlet has been large in past years, these physically stressed habitats support less extensive biotic communities than backbay regions. As a result, the overall biotic impacts of dredging at the tidal inlets may be far less severe than in open bay areas. The potential impacts of dredging in the coastal bays are discussed.
The Barnegat Bay-Little Egg Harbor estuary, a shallow lagoon-type system along the central New Jersey coastline, is the focus of an ongoing comprehensive assessment and management program. Designated as a National Estuary Program site in July 1995 because of its exceptional ecological, commercial, and recreational value, this critically important coastal waterbody is now threatened by pollution, development, and overuse. Results of a four-year study have identified the following priority impacts on the system: (1) nutrient (nitrogen) loading, which is problematical in the northern estuary; (2) elevated numbers of fecal coliform bacteria, which periodically cause the closure of bathing beaches and shellfish harvesting areas; (3) habitat loss and alteration in the estuary and its watershed, which may adversely affect the structure and function of major components of the system; and (4) altered watershed contributions of freshwater and water quality constituents, which play a significant role in the dynamics and overall health of the estuary. These impacts will be the subject of a number of management initiatives. Details of this ongoing investigation are discussed, together with the management strategies being formulated to address environmental concerns in the estuary and watershed.
Common beach management practices affect the ecological values of coastal dunes. Mechanical beach cleaning eliminates incipient dunes maintained by pioneer plant species. Artificially restricting foredunes to small, stabilized forms reduces the microhabitat variability that helps support biodiversity. Beach nourishment replaces sand but not necessarily the habitats needed to restore a natural landscape. Ecological research is needed to complement sedimentological, geomorphological, and attitudinal studies in an initiative to support beach nourishment projects and inform coastal restoration planning. The objective is to restore a dynamic and ecologically viable landscape to the New Jersey shore that is compatible with human use. This study examines plant communities resulting from different management techniques, including beach nourishment, implemented in developed areas at Ocean City, NJ. Eight dune types were sampled using quadrats along shore perpendicular transects. Species richness, percent cover, and their relevance to beach management techniques were evaluated for microhabitats defined by geomorphological features. A dune and associated backbeach that is compressed in size because of reduced beach width were found to have similar species richness to a wide beach and dune. Ecological costs including reduced percent cover of vegetation and height of Ammophila breviligulata did occur within the compressed environmental gradient of the narrower dune. Dune stability and relative age of the backdune were also found to affect richness in other dune types.
Shoreline erosion and recovery seldom occur as a linear losses and gains of sediment. This is especially true along the Fire Island, New York shoreline, where nearshore circulation cells of several hundred meters are responsible for mobilizing great quantities of sand from the beach and occasionally eroding portions of the foredune as well. In December 1992 a large circulation cell produced a major indentation of the shoreline and caused beach and dune erosion along a distance of 400m at Talisman, Fire Island. Parts of the foredune were completely eroded and other parts were severely scarped. The recovery sequence in the foredune consisted of large inland transfers of sediment in the area of complete dune loss, accumulation on the dune face and dune crest in adjacent areas, and build out of the dune toe in the marginal locations. Compartmentalization of the foredune was established on the basis of degree of erosion. Subsequent recovery in volume and morphology were tracked in each of the compartments and for entire foredune system.
General theory of process-response modeling indicates that the distribution of sediments on a dune-beach profile vary according to the energetics acting upon that profile. Sediments were collected from fifteen dune-beach profiles surveyed along the shoreline of a washover lobate formation on the bayside of Island Beach State Park, New Jersey. The surface samples were measured and statistically analyzed for mean grain size, standard deviation, and skewness. Fifty-eight data points were grouped into six categories based on profile characteristics and plotted to determine clustering of sediment/form assemblages. Typically, dune and surf zone sediments had a mean phi of 1.54 with a 0.41-0.45 standard deviation. Beach sediments fined to 1.63 phi with a 0.39 standard deviation. The small (0.1 meter) berm surfaces had the finest sediments (1.86 phi) with the best sorting (0.30 standard deviation). Near offshore and far offshore sediments had equal sediment sizes (1.39 and 1.40 phi, respectively) with a slight decrease in sorting in the far offshore (0.52 and 0.58 standard deviation, respectively). Additionally, a qualitative interpretation of bi-variate plots of mean grain size, standard deviation, profile type and exposure was conducted on the sediment/form response to the direction of incident waves. Resulting analyses showed a general characteristic of similar source material throughout the lobe with evidence of a sinusoidal distribution of sediment characteristics from north to south. There is a trend of increased scattering in the bi-variate plot seaward, with dunes showing the tightest clusters and the offshore showing the largest range of variation.
The Mahantango Formation of the Hamilton Group (mid-Devonian Age, about 380 million years ago) extends from south-central Pennsylvania into New York where it is differentiated into the Skaneatales and Ludlowville Formations representing regressive and transgressive-regressive cycles, respectively. These formations are recognized as facies in Monroe Township, Pennsylvania where a locale was selected and faunal changes observed through an approximately eight meter vertical stratigraphic section. Examination of faunal assemblages revealed a marked change from diverse shallow water communities, to deeper water fauna where species numbers decrease dramatically and neritic forms appear. This distinct communal change suggests the Skaneatales and Ludlowville facies contact occurs on–site. The dominant biomarker for shallow bottom-dwelling communities is the brachiopod Spirifera. Less abundant forms, particularly crinoids, gastropods, and trilobites also occur in the lower portion of the stratigraphic section. Deeper water biofacies constituting the upper portion of the stratigraphic section are dominated by a trilobite-cephalopod-gastropod assemblage primarily defined by Phacops sp./Greenops sp.-Stracocera sp.- Cyclonema sp. Ammonites, brachiopods, and crinoids are estimated to compose less than 10 percent of the species represented in the upper faunal assemblage. It is our interpretation that the transgression limited available light to the ocean floor so that neritic and burrowing forms replaced diverse shallow water faunal communities.
Marine Science II
Hemocytes act as an immune system for hard-shell clams Mercenaria mercenaria by phagocytosis of foreign particles. Optimal production of these blood cells, hematopoiesis, can be altered by many environmental conditions, such as salinity, temperature, and heavy metals. The parameter considered in this paper is temperature; clams maintained at lower temperatures were found to have the highest concentration of hemocytes. The process of phagocytosis occurs in all three hemocyte cell types: large granulocytes, small granulocytes, and agranulocytes but the three cytotypes phagocytose at different rates. Polystyrene microspheres (1.0 m m) were used in a 7:1 ratio to the hemocytes in order to observe phagocytosis and make differential counts of cells. Small granulocytes phagocytosed the most microspheres in comparison to the other cytotypes. Although agranulocytes were the cytotype least present, these cells had high numbers of microspheres phagocytosed. During the process of counting hemocytes, inexplicable spherical-like objects were noticed and documented with the temperature change. Contrary to hemocyte numbers, the quantity of spheres had a direct correlation with the increase in temperature. Further research into the identification of the unknown spheres revealed that they may possibly be an unknown parasite. Some of these parasites were found to be phagocytosed by various hemocytes but were not digested over a time course of one hour. The parasites also seemed to multiply and develop despite the presence of hemocytes. The cytology of the hemocytes did not appear altered by the presence of the parasite.
The restoration of areas dominated by the invasive plant Phragmites australis (common reed) to native salt marsh grasses is ongoing throughout much of the northeastern United States. However, we know little about the response of fishes to this restoration. As a preliminary step in evaluating fish response to vegetation type and associated microtopography, common mummichog larvae/small juvenile (~ 5-25 mm TL) and larger juvenile/adult (~ 26 mm+ TL) abundance and distribution were investigated among three marsh types. Biweekly pit trap samples (n=18) were collected June, July, and August of 1999 from Spartina alterniflora, Phragmites and bare substrate. Dramatically higher larval and small juvenile abundances were found in S. alterniflora (overall cpue = 5.92) relative to the Phragmites (overall cpue = .08) and bare substrate stations (overall cpue = 1.58). Biweekly juvenile trap samples showed larger juveniles and adult mummichog are similarly abundant in all three habitats. Marsh type appears to play an important role for larvae and small juveniles, although less important for larger juveniles and adults.
Twilight Lake in Bayhead, New Jersey, receives large volumes of surface drainage water from the Point Pleasant municipal area through an overflow drain entering the lake below water level. The lake is thus potentially at risk for diminished water quality from organic loading, as well as from pollutants such as oil, pesticides, etc. This study was carried out to determine whether input of surface water from the overflow drain to the lake has measurable effects on water quality. Data used to assess the quality of water included turbidity, depth, temperature, pH, nitrate content, dissolved oxygen, salinity, algal index, submerged aquatic vegetation, and wildlife observations. A second sampling site, the canal adjacent to the Bayhead Yacht Club, was sampled and used for comparison. The second sampling site was chosen because the water near the Yacht Club was not in the immediate vicinity of the drainage outlet, but was close enough to the original sample site to possess similar water quality characteristics. The second site is also the area where lake-water flows into the larger Barnegat Bay system. Beginning in March of 1999, samples were taken on an approximately weekly basis for a period of nine months. During this time, both sampling sites frequently displayed reduced dissolved oxygen levels, with near-surface oxygen concentrations often falling below saturation, particularly during the summer months. However, excessive nutrient inputs were not detectable and high levels of phytoplankton growth were not evident. Thus the cause of the observed hypoxic conditions could not be unequivocally determined. However, our preliminary results suggest that there is a potentially significant problem with water quality at this site that warrants further study.
The Musconetcong River and the South Branch of the Raritan River support viable populations of brown trout, Salmo trutta, rainbow trout, Onchorhyncus mykiss, and brook trout, Salvelinus fontinalis. To assess habitat suitability for these cold- water species computerized temperature loggers were initially installed at ten locations on the Musconetcong and ten locations on the South Branch, which coincide with studies conducted on the rivers by the Musconetcong Watershed Association, the South Branch Watershed Association, and the Ken Lockwood Chapter of Trout Unlimited. The loggers record temperature information every hour and are visited approximately every two months by Trout Unlimited volunteers, equipped with a laptop computer to download the data. The information is stored in graphical form in Logbook software, and data from individual samples is stored in Lotus spreadsheets. This information will be used by the New Jersey Division of Fish and Wildlife for management decisions and New Jersey Trout Unlimited for habitat rehabilitation projects and by respective watershed associations to add supporting information for future studies. Molecular and Cellular Biology I
Ice worms (Mesenchytraeus solifugus) inhabit coastal glaciers in North America. They are among a small group of metazoans that complete their life cycle in glacial ice, a micro-climate that does not deviate significantly from 0o C. Survival under these conditions requires adaptations at multiple levels ranging from metabolism to morphology. We are interested in understanding the relationship between several morphological features of the ice worm with their behavior(s) on ice. Specifically, ice worms penetrate several feet of hard glacial ice daily and return to the surface of the glacier at dusk, presumably to feed. Their mechanism of navigation in ice is unknown, but it is likely to involve some form of photo-, thermo- and/or chemotaxis. To address this question, we constructed an apparatus that establishes a photo and/or temperature gradient. Surprisingly, ice worms moved up the temperature gradient (toward heat), but did not respond to light of various wavelengths. Ice worm heads have been sectioned in different orientations in an effort to identify morphological structures that respond to environmental cues, and which may also aid the worm in its navigation through ice. Serial thick and thin sections have been analyzed by light and transmission electron microscopy, respectively.
The nervous system of glossiphoniid leeches comprises a relatively simple network of longitudinal and segmentally iterated nerves. Despite its simplicity, leeches display sophisticated forms of learning and memory including habituation, sensitization, etc. The aim of this study is to identify cellular processes associated with nervous system development in the glossiphoniid leech, Theromyzon rude. Axon development in leech can be monitored by epifluorescense using a mouse monoclonal alpha-acetylated tubulin antibody. By comparing leech embryos in which selective cell lineages were ablated, it has been possible to determine the requirement of subsets of cells in the developing nervous system. These results indicate that cells derived from both the mesoderm and ectoderm are required for establishing the normal distribution of axonal process, and that a subset of neuroectodermal cells that express the leech engrailed-class gene are required for forming the posterior segmental nerve
In a proposed animal model of obsessive-compulsive disorder (OCD), Yadin, Friedman and Bridger (1991) used serotonin agonists to disrupt spontaneous alternation behavior (SAB) in rats. In the present study, agonists selective for different serotonin (5-HT) receptor subtypes were tested for effects on SAB in Sprague-Dawley and Long-Evans strains of rats. Rats were scored for alternation or repetition in their choice of arms of a T-maze equally baited with chocolate milk. The 5-HT1A agonist 8-OH-dipropylaminotetralin (8-OH-DPAT; 2 mg/kg, ip) significantly increased repetitive choices (disrupted SAB) in both rat strains. In contrast, injections with the 5-HT2/1C agonist dimethoxyiodophenylaminoethane (R(-)-DOI; 1 mg/kg, ip) or the 5-HT3 agonist N-methyl quipazine (NMQ; 3 mg/kg, ip) had no significant effect on SAB. Increases in the time spent at the decision point and in vicarious trial and error (VTE) prolonged the time required for each rat to choose an arm of the T-maze when injected with 8-OH-DPAT, or another 5-HT1A agonist buspirone (2 mg/kg, ip). The disruption of SAB was reversible as behavioral scores returned to preinjection levels within 48 hours after injections. The results extend the use of SAB in an animal model of OCD and point to a specific role of serotonin 5-HT1A receptors in the induction of repetitive behavioral patterns.
Hypoxia induced erythropoiesis allows mice to achieve a greater red cell mass than animals induced with recombinant human erythropoietin (rEPO). Furthermore, splenectomized mice fail to demonstrate a significant peripheral erythroid response to rEPO compared to splenectomized hypoxic mice, with a normal erythroid response. Availability of hematopoietic tissue space may, therefore, determine overall erythroid carrying capacity. During hypoxic exposure, increased specific bone marrow volume, with no change in bone density, occurs at the interface of the bone with the bone marrow. This may explain the increased erythroid carrying capacity of hypoxic mice. Hypoxia induced, Interleukin l (lL-1) mediated, T cell dependent bone modulation has been suggested as a mechanism for increasing marrow erythroid carrying capacity during hypoxic exposure. Occlusion of marrow space by estrone induced osteosclerosis, initiates a shift in hematopoiesis from marrow to the spleen. Splenectomized osteosclerotic mice demonstrate a significant decrease in hematopoietic carrying capacity. This supports a direct relationship between marrow space and hematopoietic carrying capacity. Circulating levels of lL-1, a bone modulating inflammatory cytokine, increase during hypoxic exposure in mice and likely mediate the bone modulations needed to increase erythropoietic carrying capacity. Inhibition of EPO synthesis by lL-1 has been reported. During hypoxic exposure, inhibition of EPO synthesis may serve to spare stem cells in individuals with limited marrow hematopoietic carrying capacity, e.g., splenectomized mice. Inhibition of EPO synthesis in hypoxic mice would permit the slower, rate limiting bone modulations needed to increase marrow carrying capacity.
Alzheimer's disease (AD) is a neurodegenerative disorder that preferentially affects the cerebral cortex and hippocampus of the brain and is accompanied by loss of memory, neurons and synaptic contacts. Mechanisms by which synaptic contacts are lost are poorly understood. Astrocytes, the most common type of glial cell in the cerebral cortex, have been implicated in both synapse maintenance and synaptic plasticity. Here, we used immunohistochemistry and digital image analysis to examine the effects of AD pathogenesis on the behavior of astrocytes. In AD brains, astrocytes were found to simultaneously and progressively accumulate amyloid, a7 nicotinic cholinergic receptor, and choline acetyltransferase, three substances well-known to be neuron-specific and associated with synapses. This finding suggests that, in AD brains, astrocytes take on an enhanced phagocytic role aimed at removing defective synapses from the dendritic branches of pyramidal neurons. In late-stage AD brains, excessive accumulation of these materials within astrocytes apparently becomes toxic and these cells undergo lysis, leading to the formation of glial-derived amyloid plaques.
Alzheimer's Disease (AD) is characterized by progressive memory loss, deterioration of cognitive functions and preferential loss of neurons, especially in the cerebral cortex and hippocampus. This condition is hallmarked by the prevalence of focal, extracellular deposits of amyloid called amyloid plaques (APs). How APs form in AD brains is unknown. In the present study, we have used immunohistochemistry and digital image analysis to examine the origin and distribution of amyloid during AD pathogenesis. We report here that, in the entorhinal cortex and hippocampus of AD brains, amyloid primarily accumulates intracellularly within pyramidal cells and is localized to large cytoplasmic granules that occupy a substantial fraction of the total cytoplasmic volume. Co-localization of intraneuronal amyloid with cathepsin D suggests that most amyloid is associated with lysosomal elements. Amyloid-burdened PCS were found to engage in cell lysis, resulting in the release and radial dispersion of their cytoplasmic contents, including amyloid-positive material, into the surrounding extracellular space. Based on these observations we suggest that intracellular accumulation of amyloid followed by neuron lysis may be an important mechanism of neuronal loss and AP formation in AD brains.
William J. Anderson (student), Daniel H. Shain, Dennis J. Joslyn, Hsin-yi Lee, Michael R. D'Andrea and Robert G. Nagele. Biology Department, Rutgers University, Camden, NJ and Molecular Biology Department, UMDNJ-SOM, Stratford, NJ Accumulation of amyloid occurs intracellularly (i.e., within pyramidal neurons), rather than extracellularly, leading to AP formation and the pathogenesis of Alzheimer's disease (AD). Thus, identification of factors or conditions that mediate amyloid accumulation is of paramount importance. Previous in vitro studies have shown that amyloid binds with high affinity to the a7 nicotinic acetylcholine receptor (a7nAChR). In the present study, we used an in vitro system to investigate the possible role of this binding in facilitating the entry and accumulation of amyloid into neurons in AD brains. In a time-course study, intracellular entry and accumulation of exogenous amyloid was studied in the human neuroblastoma cells (SK-N-MC cell line) transfected with human a7nAChR cDNA and overexpressing a7nAChR. Amyloid accumulated in these cells within 2 hours of treatment and this accumulation was completely blocked with of 1 mM a-bungarotoxin (a specific a7nAChR competitive antagonist) and phenylarsine oxide (a specific inhibitor of endocytosis). Results suggest that binding of amyloid to the a7nAChR followed by endocytosis of this complex is the primary mode of entry of amyloid into neurons during AD. Molecular and Cellular Biology II
We are interested in examining the relationship between cancer and a gene called telomerase. In all known cancers, the telomerase gene is re-activated from its quiescent state in somatic cells. Our approach is to compare telomerase DNA sequences from diverse animal phyla in an effort to identify functionally important coding and regulatory regions. We propose to clone the telomerase gene in an annelid, Theromyzon rude, and compare its DNA sequence to the human telomerase gene. Our strategy is to synthesize degenerate oligonucleotides to conserved regions of the telomerase gene, and use the polymerase chain reaction (PCR) to amplify intervening telomerase DNA sequences. This DNA fragment will be used as a probe to clone the regulatory element of the leech telomerase gene.
Histones H3 and H4 have been shown to play critical roles in the formation of heterochromatin-like repressive structures at yeast telomeres and the silent mating loci. It has been previously shown through genetic and biochemical analysis that the non-essential N-terminal tails of H3 and H4, which structurally extend out from the nucleosome, make specific contacts with silencing factors Sir3 and Sir4, creating a chromatin structure which inhibits transcriptional activity. While much work has been done to dissect the role of the histone N-termini, little has been done to address the role of the essential core domains. To address the potential role for the histone H3 core domain in telomeric heterochromatin, the gene encoding H3 (HHT2) was randomly mutagenized by PCR mutagenesis, and the mutated copies were introduced into yeast lacking the endogenous copies of HHT1 and HHT2. A total of 25 mutant strains were identified, exhibiting a broad range of effects on yeast telomeric and silent mating locus repression. While some of the mutations appear to specifically affect heterochromatin formation, other mutations have been found to exhibit partial alleviation of basal repression at the GAL1 promoter, as well as temperature-sensitive growth. A notable number of the sequenced mutations map to looped regions of H3 that are believed to serve as DNA docking sites on the nucleosome, suggesting that specific contacts between the DNA and the nucleosome core particle can influence chromatin structure.
Primitive cells had to divide with very little biology. We have proposed physicochemical mechanisms based upon electrostatics which explain and unify the basic motions during mitosis and cytokinesis: (1) centrioles to poles, (2) chromosomes to metaphase "plate", (3) alignment of chromosomes, (4) anaphase A motion of chromosomes, (5) rounding up of cells, (6) cell elongation, and (7) furrowing. The present work is concerned with a self-consistent model for anaphase B motion, which (like the seven processes mentioned above) introduces no new assumptions and relies solely on electrostatic forces. In particular, we propose that microtubules transmit the forces for anaphase B motion through electrostatic interactions at their disassembling ends. This mechanism is based on excess charge at the negatively charged ends of the electric dipolar microtubule subunits, and is completely consistent with the dynamics that we have proposed for the anaphase A motion of chromosomes. Thus, a minimal assumptions model based solely on known electrostatic interactions is able to explain all of the basic motions of mitosis and cytokinesis, including timing, sequencing, and force generation for each type of motion.
To regulate the large number of genes in the eukaryotic cell, the eukaryotic genome is believed to be organized into discrete domains with independent regulation. It has been suggested that these domains are separated by boundaries to prevent regulatory activities within one domain from affecting adjacent domains. When located between an enhancer and a gene, boundary elements are known to block transcriptional activation. Boundary elements also may function by preventing the spread of different forms of chromatin structure. We have developed an in vivo boundary assay utilizing the yeast Saccharomyces cerevisae. Heterochromatin in yeast forms at the telomeres and propagates centromerically for about 3-4 kilobases. Genes found in this area are transcriptionally silenced. If boundary elements function to prevent the spread of heterochromatin into transcriptionally active domains, then the insertion of the element between the telomere and the normally silenced genes should allow the genes to be expressed. We have introduced two potential boundary elements into yeast: scs from the Drosophila hsp70 locus, and a regulatory element located upstream of the mouse H19 gene. A plasmid was constructed with one of these boundary elements and the reporter gene URA3, which was integrated adjacent to the telomere of chromosome VII in yeast. We have designed a polymerase chain reaction to identify integrated candidates. Expression of the reporter gene will be evaluated by a growth assay to determine the effect of the boundary element on the transcriptionally silenced region.
Early embryonic development in metazoa is characterized by cascades of gene expression that initially define the dorsoventral and anteroposterior axes. Central to this process is the deposition of maternally derived proteins and mRNAs that initiate these molecular events. Among the few maternal mRNAs that have been characterized, bicoid, oscar and nanos are associated with anteroposterior fate in Drosophila, and VGA defines the vegetal pole in Xenopus. We are interested in identifying maternal mRNAs in the leech, Theromyzon rude, which is among the most well-characterized members of the phylum Annelida. T. rude eggs are large, robust and develop rapidly outside of the mother which, collectively, provides a suitable experimental system for examining the function of maternally-derived products. Due to the relatively small genome size of leech (~2 x 108), our approach has been to survey abundant maternal mRNAs present in fertilized eggs. We have constructed several maternal cDNA libraries from T. rude eggs by employing PCR-based techniques. Preliminary sequencing of clones has not revealed any cDNAs that are in the DNA sequence databank (GenBank).
Studies have shown that approximately 1 in 16 human newborns has a birth defect. More than 80% of these birth defects are caused by teratogens (e.g., drugs, chemicals and microbes) that have crossed the placenta and altered normal development. The vertebrate embryo, however, is not equally susceptible to a particular teratogen at all developmental stages. For example, during the first month of development, the human embryo is highly susceptible to many drugs when (1) neural tube formation (the early development of the brain) takes place and (2) many women are unaware of their pregnancy. However, other drugs have detrimental effects only much later in the postnatal life of individuals exposed to them during the early stages of the intrauterine period of development. Since it is not feasible to study effects of drugs on human embryos, researchers have resorted to using other vertebrates such as the chick, mouse, or rat in their work. In the present study, we studied the toxicity and teratogenicity of two commonly used drugs for treatment of hypertension, Prinivil (an angiotensin-converting enzyme inhibitor) and Norvasc (a calcium channel inhibitor) on rat embryos. In general, Prinivil was a more potent teratogen than Norvasc at all the concentrations tested. In particular, Prinivil, at a concentration as low as 160 mg/ml, significantly increased the incidence of neural tube closure defects. Heart development was affected only in more severely affected embryos. Growth retardation was rarely observed among those embryos treated with either Prinivil or Norvasc at teratologic doses. A combination of non-teratologic doses of the two drugs often increased the incidence of embryos with defective neural tube closure and somite formation. The significance of our findings will be discussed.
Fibrocystic breast disease occurs in at least one in four women and increases the likelihood of developing breast malignancy by 200-400% compared to those without the condition. The pathology is characterized by solid and fluid-filled cysts; the fluid contains high levels of gross cystic disease fluid proteins (GCDFPs) 15, 24, 44, and 70 (named for their masses in kD). Presence of solid masses predisposes development of breast carcinoma, although these masses are not known to be precancerous. GCDFP expression is also associated with breast carcinoma and some correlations to metastasis have been suggested. To address the role of GCDFPs in fibrocystic disease, hormonal control of GCDFP expression was examined in the fibrocystic breast cell line MCF-10A. GCDFPs 15 and 24 are hormonally regulated in breast cancer cells, but were not found to be produced by cystic epithelial cells. In contrast, MCF-10A cells produced GCDFP-70 (also known as serum albumin), and production was stimulated by prolactin. Both prolactin and albumin have been implicated in mitogenic activation of breast cancer cells in vitro, and nulliparous women, who have higher prolactin levels, are at greater risk for breast cancer than those who have given birth. Implications for breast cancer pathogenesis when fibrocystic tissue is present will be discussed.
Science Education I
The Department of Health and the Environmental Commission of the City of Clifton, N.J. recently completed a two-year watershed characterization study of a portion of the Third River sub-watershed in the Lower Passaic River Watershed. The study was funded by a Watershed Management Public Education and Outreach Grant from the New Jersey Department of Environmental Protection (NJDEP) in 1997. The focal point of the study was instream benthic macroinvertebrate sampling using the USEPA Rapid Bioassessment Protocol. A composite New Jersey Impairment Score (NJIS) was obtained for eight physico-chemical/biomonitoring stations. The total NJIS for all stations ranged from 6 to 15, indicating a moderately impaired bioassessment. Concurrent water chemistry results were in good agreement with the bioassessment results; there were few contraventions of NJDEP Surface Water Quality Standards. Other watershed characterization activities included a land-use study using G.I.S. mapping in cooperation with the Passaic County Department of Health and an electrofishing survey conducted by the NJDEP, Division of Fish and Wildlife. In general, the results of the Third River Watershed Characterization Study revealed that the Third River is a recovering urban stream within a developed watershed that is capable of supporting a moderate diversity of aquatic and terrestrial biota. Various educational and public outreach activities were also successfully administered as part of the project.
Aberrant specimens of three fish species, obtained from New Jersey Fish and Game (DEP), were examined and X-rayed. The three species include muskellunge (Esox masquinongy), striped bass (Morone saxatilis), and channel catfish (Ictalurus punctatus). The musky is a member of the pike family. It is a popular game fish (reaches a weight of 34 kg, and a total length (T.L.) of 150 cm), and is raised and stocked by the NJ Department of Fish and Game. Aberrant specimens of this species included, 1.a fish 22.3 g, T.L. 190 mm, with kyphosis between vertebrae (V) 15 and 18, and scoliosis from V seven to 21. Its weight was 40% of that of normal fish. 2.this fish weighed 81.2 g, T.L. 255 mm, with kyphosis from V 34-36. 3.weight 18.9 g, T.L. 165 mm, opercula incomplete. 4.weight 14.8 g, T.L. 155 mm, operculum on left side incomplete. 5.weight 56.3 g, T.L. 246 mm, dentary bone on left side mishaped, branchistegal rays spread, mouth closes incompletely. A channel catfish, weight 12.2 g, T.L. 107 mm, had scoliosis from V 6 to 8. This resulted in a distended stomach. A striped bass, weight 31.9 g, 135 mm, was doubly kyphotic, from V 6 to 12 and V 24 to 28. Some aberrant fishes appear to develop normally (e.g. mouth and opercular aberrations) while others do not (e.g. those with severe spinal aberrations). The frequency of fish abnormalities appears to be less than 1%.
Eight benthic surveys were made in 1998, and three in 1999. Triplicate samples were taken at each of four sites in the Morris Canal, and from one site each in the embayment and in the Hudson River. A Ponar Grab sampler was used for collection. Samples (one pint) were fixed in a formaldehyde X Rose Bengal solution. Samples were sieved through a #40 mesh. Species collected were primarily Phylum Annelida, Class Polychaeta. Few specimens from the Phylum Mollusca and Nemathelminthes were collected. The largest number of polychaetes collected in 1998 was in May (1012), and in September, 1999 (939). The dominant polychaete in the 1998 collection was Streblospio benedicti (74% of the total), followed by Capitella capitata (17%), Haploscoloplos sp. (6%), and Eteone heteropoda (1%). Twelve other species made the remainder. The westernmost station (i.e. at the end of the canal) yielded only four specimens. At this station the substrate is muddy and oily. Dissolved oxygen concentrations are low. The station to its immediate east has a similar substrate, but yielded 1158 specimens. In 1999, 1184 polychaetes were collected. The same species dominated (S. benedicti, 76%, C. capitata, 12%, Haploscoloplos sp., 10%, E. heteropoda 1%). In 1999 no specimens were collected from the westernmost station.
A benthic survey was conducted in the Port Liberte canal system, Jersey City, New Jersey, in 1999. Four sample collections in duplicate were made at five stations. Three stations were located within the canal system, one station at the northeast entrance to the canal system, and one station in Claremont Terminal channel, opposite the southern circulation canal (Dinghy Canal). Ponar grab samples (one pint) were fixed with formaldehyde X Rose Bengal solution, and the organisms removed by sieving (#40 mesh) in the laboratory. The four sampling events yielded 1871 annelids representing 13 species. A few arthropods and molluscs were also collected. The dominant polychaetes included Streblospio benedicti (65% of the total), Capitella capitata (17%), Haploscoloplos sp. (14%), Eteone heteropoda (2%), and Cirratulus cirratus (1%). Fewest polychaetes were collected in May (282), most in November (651). The station at the northeast entrance contained 47% of the worms, the Claremont Terminal channel station 13%, and the canal stations 10%, 19%, and 11%. Opening Dinghy Canal to Claremont Terminal channel provides better water circulation and concomitant better water quality, which is conducive to a healthier biotic environment, although the waters are not aesthetically pleasing.
A fisheries survey of the Morris Canal was made during March, May, August, and November, 1999. The Morris Canal is located off Jersey City, in Upper New York Bay. Six stations were sampled using a 16 foot otter trawl. The easternmost station is located in the Hudson River. A second station is in the embayment. The remaining stations are located in the canal. Fish numbers and numbers of species collected were; March, 30 fishes, nine species, May, 66 fishes, 11 species, August, 66 fishes, 12 species, November, 48 fishes, 11 species. The dominant fishes were striped bass (Morone saxatilis), representing 34 percent of the total, followed by winter flounder (Pseudopleuronectes americanus), 15 percent, northern king (Menticirrhus saxatilis), nine percent, and bay anchovy (Anchoa mitchili), seven percent. The river station (i.e. outside the canal) yielded 59 percent of the fishes caught. This was followed by the easternmost station in the canal, with 23 percent of the total. The westernmost stations each yielded one percent of the total fish catch. These two stations have the poorest water quality. The most westerly station also yields few, if any, benthic organisms. The lack of food availability, lower dissolved oxygen, and poor circulation is probably the cause of fewer fishes at this site.
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A fisheries survey was conducted at Port Liberte, Jersey City, New Jersey. The survey consisted of duplicate sample collections from five sites in and around a canal system, using a 16-foot otter trawl. Three stations were within the canal system, one station at the canal entrance, and one in the Claremont Terminal channel. A total of 725 fishes were collected representing 28 species. The most abundant species were striped bass (24% of the total), silversides (20%), northern kingfish (11%), weakfish (11%), blueback herring (10%), winter flounder (3%), striped sea robin (3%), bay anchovy (2%). The remaining 16% of the catch was made up of 20 species. The greatest number of fishes were collected from the station at the canal entrance (32%), followed by a station furthest within the canal system (27%), then the Terminal channel station (25%). The two remaining canal stations had the fewest fishes (12% and 4%). The fewest fishes were collected during the March survey (8). The most during the August survey (284). Several unusual fishes were caught, including a three foot shortnose sturgeon, and several young-of-the-year cobia. Most of the fishes collected were young-of-the-year or zero year class fishes.
Wreck Pond is located in southern Monmouth County, NJ. It originates as a freshwater pond. The pond water flows over a dam, downstream, and mixes gradually with saltwater prior to entering a flume leading to the Atlantic Ocean. Fishes were collected in a shallow (two foot deep) section of freshwater, and included Fundulus diaphanus, F. heteroclitus, Lucania parva, Lepomis macrochirus, Cyprinodon variegatus, and Menidia beryllina. The appearance of distended abdomens is some fishes and subsequent analysis showed heavy infestation of the nematode Eustrongylides sp. These were encysted in the body cavities. When present, one to five worms up to length of 100 mm occurred in fishes 30 to 40 mm total length. In the cavity the worms may develop into parthenogenetic females. Embryonated eggs were associated with some nematodes in the cavities. The nematode life cycle in the pond may be crustacea-fish-bird in the non-parthenogenetic cycle. If the infected bird defecates in the pond, crustacea can eat the eggs, then fishes eat the crustacea. More than 50% of F. diaphanus were infected with nematodes, while 10% of F. heteroclitus and Lucania parva were infected. The other fish species were not infected. This suggests resource partitioning, or selective feeding, even though different species are in close proximity.
Water quality was examined on six occasions from five stations at Port Liberte, Jersey City, New Jersey, in the Hudson River, between January and November, 1999. Each station was sampled at surface, middle, and bottom depths. Parameters examined, with a Hydrolab, included temperature, salinity, pH, and dissolved oxygen (D.O.). Water samples were examined for fecal coliforms and fecal streptococcus in the laboratory. Water temperatures ranged from 3.8 C (January) to 26.4 C (August). Waters were usually colder at the bottom. Salinities ranged from 18.4 ppt (September) to 26.4 ppt (August). Salinity was usually higher at the bottom except for September, when two stations had salinities seven ppt lower at the bottom than at the surface. Values for pH ranged from 7.5 (July) to 6.9 (January). With one exception, pH was lower at the bottom than at the surface. D.O. ranged from 4.3 mg/l (September) to 12.2 (January). D.O. stratification occurred in July and August, with lower readings at the bottom. D.O. stratification was not evident during the other sampling periods. Highest fecal coliforms (1230 colonies/100 mls) and fecal streptococcus (870 colonies/100 mls) occurred at a control station, that is, at an offsite (out of canal) location.
Water quality was examined on twelve occasions from six stations in the Morris Canal, Jersey City, NJ, in the Hudson River, between January and October, 1999. Each station was sampled at surface, middle, and bottom depths. Parameters examined with a Hydrolab included temperature, salinity, pH, and dissolved oxygen (D.O.). Water samples were returned to the laboratory to determine fecal coliforms and fecal streptococcus. Water temperatures were coldest in March (3.1C), and warmest in August (26.3C). Generally, surface waters were colder until May, then bottom waters were colder through September. Salinity ranged from 8.1 ppt (March) to 25.4 ppt (July). Higher salinities always occur at the bottom, with the greatest differences in March (e.g. 9.6 surface vs 16.3 bottom). Values for pH were generally higher at the surface from January throughout June, then slightly lower from July through October. A clear pattern for pH is not evident. D.O. was highest in June (12.8 mg/l) and lowest in August (1.9 mg/l). In January bottom D.O. was higher than surface values. At other sampling times bottom D.O. was lower. A difference of 7.1 mg/l occurred at one station between surface and bottom at a 5.8 meter depth. Fecal coliform and fecal streptococcus were higher in the canal than in the two outer (control) stations.
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