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TNF-alpha is crucial in defense against intracellular microbes. Host immune cells use type 3 complement receptors (CR3) to regulate excess TNF-alpha production during physiological clearance of apoptotic cells. BAD1, a virulence factor of Blastomyces dermatitidis, is displayed on yeast and released during infection. BAD1 binds yeast to macrophages (Mphi) via CR3 and CD14 and suppresses TNF-alpha, which is required for resistance. We investigated whether blastomyces adhesin 1 (BAD1) exploits host receptors for immune deviation and pathogen survival. Soluble BAD1 rapidly entered Mphi, accumulated intracellularly by 10 min after introduction to cells, and trafficked to early and late endosomes. Inhibition of receptor recycling by monodansyl cadaverine blocked association of BAD1 with Mphi and reversed TNF-alpha suppression in vitro. Inhibition of BAD1 uptake with cytochalasin D and FcR-redirected delivery of soluble BAD1 as Ag-Ab complexes or of wild-type yeast opsonized with IgG similarly reversed TNF-alpha suppression. Hence, receptor-mediated entry of BAD1 is requisite in TNF-alpha suppression, and the route of entry is critical. Binding of soluble BAD1 to Mphi of CR3(-/-) and CD14(-/-) mice was reduced to 50 and 33%, respectively, of that in wild-type mice. Mphi of CR3(-/-) and CD14(-/-) mice resisted soluble BAD1 TNF-alpha suppression in vitro, but, in contrast to CR3(-/-) cells, CD14(-/-) cells were still subject to suppression mediated by surface BAD1 on wild-type yeast. CR3(-/-) mice resisted both infection and TNF-alpha suppression in vivo, in contrast to wild-type and CD14(-/-) mice. BAD1 of B. dermatitidis thus co-opts normal host cell physiology by exploiting CR3 to subdue TNF-alpha production and foster pathogen survival.

Eight tetrabrominated to hexabrominated diphenylethers were present at ppb levels in liver from cetaceans found stranded on the beaches of the Mediterranean Sea, Italy. The highest concentration was found in striped dolphin (sum polybrominated diphenyl ethers [PBDE] 8133 ng/g l.w.) and the lowest concentration in bottlenose dolphin (sum PBDE 66 ng/g lipid weight [l.w.]). The predominant congener in all samples was 2,2',4,4'-tetraBDE (PBDE # 47) followed by, in decreasing order, the pentaPBDE # 99 and 100 and the hexaPBDE # 154 and 153. In 12 of the 14 analyzed samples, 3 different methoxylated PBDEs (MeO-PBDE # 1, 2, and 3) were detected at semiquantitatively calculated concentration ranges of 2 to 14 ng/g l.w.; 5 to 167 ng/g l.w.; and 7 to 628 ng/g l.w., respectively. In addition, several unidentified bromine compounds were seen when screening the samples in negative-chemical ionization (NCI) mode monitoring m/z 79 and 81, which illustrates the importance of running both electron-impact ionization and NCI when analyzing environmental samples. Electron-impact ionization is more specific for monitoring the molecular ion compared with NCI, which might overestimate the concentration of certain PBDE congeners.

Human melanoma mortality is associated with the growth of metastasis in selected organs including the lungs, liver, and brain. In this study, we examined the consequences of overexpression of pigment epithelium-derived factor (PEDF), a neurotrophic factor and potent angiogenesis inhibitor, on both melanoma primary tumor growth and metastasis development. PEDF overexpression by melanoma cells greatly inhibited subcutaneous tumor formation and completely prevented lung and liver metastasis in immunocompromised mice after tail vein injection of metastatic human melanoma cell lines. Whereas the effects of PEDF on primary tumor xenografts appear mostly associated with inhibition of the angiogenic tumor response, abrogation of melanoma metastasis appears to depend on direct PEDF effects on both migration and survival of melanoma cells. PEDF-mediated inhibition of melanoma metastases could thus have a major impact on existing therapies for melanoma.

Mexico City, with a population of 18 million, has been sending its wastewater for more than 100 years to the Tula Valley where it is used to irrigate 90,000 ha. Due to the large wastewater volume (60 m3/s) sent through unlined channels, combined with the use of very high irrigation rates, artificial recharge of the local aquifer has been occurring. This recharge is estimated in more than 25 m3/s. As a consequence, the water table has raised and several springs have appeared in the last decades with flows between 100 to 600 L/s. These springs and several wells are the water sources in the region. An evaluation of the Tula Valley aquifer quality was performed to analyze the use of such water as source of drinking water for Mexico City. The work is divided into 5 individual projects: (a) drinking water quality in the Tula Valley; (b) water availability in the Tula Valley; (c) wastewater treatment due to its use for irrigation, (d) use of membrane processes to treat groundwater; and (d) biota developed in the new surface water reservoirs. Results show that it is feasible to use this reclaimed water as drinking source.

Worldwide, unsanitary conditions are responsible for more than three million deaths annually. One of the reasons is the low level of sanitation in developing countries. Particularly, sludge from these regions has a high parasite concentration and low heavy metal content even though the available information is limited. Different issues needed to achieve a sustainable sludge management in developing nations are analysed. Based on this analysis some conclusions arise: sludge management plays an important role in sanitation programs by helping reduce health problems and associated risks; investments in sanitation should consider sludge management within the overall projects; the main restriction for reusing sludge is the high microbial concentration, which requires a science-based decision on the treatment process, while heavy metals are generally low; adequate sludge management needs the commitment of those sectors involved in the development and enforcement of the regulations as well as those that are directly related to its generation, treatment, reuse or disposal; current regulations have followed different approaches, based mainly on local conditions, but they favour sludge reuse to fight problems like soil degradation, reduced crop production, and the increased use of inorganic fertilizers. This paper summarises an overview of these issues.

Ammonia is a disinfectant which can diffuse through the membrane of highly resistant structures like helminth ova. Thus, it can be considered an alternative disinfectant of wastewater sludge with high pathogenic content. In this study, the kinetic parameters of the Hom model were used to describe the inactivation with ammonia of faecal coliforms, Salmonella spp. and viable helminth ova. These were obtained in processes considering the addition of ammonia alone as well as for ammonia combined with an increase in temperature. The sludge was sampled from a municipal wastewater treatment plant using an APT (Advanced Primary Treatment) or CEP (Chemical Enhanced Primary) process. With 20% w/w of ammonia, 7 logs of faecal coliforms, 6 logs of Salmonella spp., and 83% of viable helminth ova were reduced in 2 hours contact time. To eliminate 100% of the helminth ova from samples having 88-132 ova/g TS it was needed to combine 20% of ammonia with 50 degrees C. The analysis of parameters k, n and m indicate higher resistance to inactivation of helminth ova compared to bacteria and a better performance of the ammonia process than lime stabilization to inactivate microorganisms. In addition, ammonia increased the agricultural value of the biosolids produced.

Worldwide, raw or treated wastewater is used for irrigation. However, this practice implies that the microbial content must be controlled. Unfortunately, detection techniques for microorganisms are costly, time consuming, and require highly trained personnel. For these reasons, this study used particle size distribution to measure the microbial quality of wastewater through correlations between the number or volume of particles and the concentration of fecal coliforms, Salmonella spp. and helminth ova. Such correlations were obtained for both raw and chemically treated wastewater. The best fit was the one for helminth ova, which applies for both the influent and effluent and also for all the coagulants involved. This technique allows the on-line quantification of helminth ova at a cost of US$3 and it takes only 5 minutes, instead of the US$70 and 5 days for the standard technique. With respect to the coagulants applied, their behavior is different only for particles smaller than 8 microm, and thus this value is considered as the critical size for this particular treatment. The best coagulant was the aluminium polychloride. In addition, this work establishes the distribution of COD, TSS, nitrogen, and phosphorous for particles smaller and larger than 20 microm.

To identify the frequency of and factors associated with early detection of HIV infection in Los Angeles County, data were evaluated from interviews of a population-based sample of adult persons with AIDS. Early detection was defined as greater than 5 years between the first reported positive HIV test and an AIDS diagnosis. The associations between early detection and sociodemographic and behavioral factors were assessed for the period January 1997 through June 2002. Over the study period, only 20% (253/1268) of persons interviewed met the criterion for early detection. Early HIV detection was less likely for women (adjusted odds ratio [AOR] = 0.6, 95% confidence interval [CI]: 0.4, 0.9), blacks (AOR = 0.5, 95% CI: 0.4, 0.8), foreign-born Latinos (AOR = 0.2, 95% CI: 0.1, 0.3), U.S.-born Latinos (AOR = 0.3, 95% CI: 0.2, 0.6, and heterosexuals (AOR = 0.5, 95% CI: 0.3, 0.7). Trends of increasing early detection with older age groups (p < 0.001) and higher educational levels (p < 0.001) were also observed. Our findings indicate an overall low level of early HIV detection and suggest that major sociodemographic and risk group disparities exist in the likelihood of early detection among HIV-infected persons in Los Angeles. These differences have important implications for reducing the level of community HIV transmission and for improving individual health outcomes among people with HIV. Aggressive efforts are needed to expand HIV testing and early detection for women, minorities, heterosexuals, younger age groups, and persons of lower education. Links to treatment and behavioral intervention programs should accompany such expanded testing efforts.

BAD1, an adhesin and immune modulator of Blastomyces dermatitidis, is an essential virulence factor that is released extracellularly before association with the yeast surface. Here, deletion of the C-terminal EGF-like domain profoundly affected BAD1 function, leading to non-association with yeast, extracellular accumulation and impaired yeast adherence to macrophages. In equilibrium binding assays, DeltaC-term BAD1, lacking an EGF-like domain, bound poorly to BAD1 null yeast, yielding a low affinity (Kd, 3 x 10(-7) M versus 5 x 10(-8) M) and Bmax (1.9 x 10(5) versus 7.9 x 10(5)) compared with BAD1. Similar protein binding profiles were observed using chitin particles, reinforcing the notion that chitin fibrils are a receptor for BAD1, and that the EGF-like domain is critical for BAD1 interactions with chitin on yeast. DeltaC-term strains bound poorly to macrophages, compared with parental or BAD1-reconstituted null strains. However, DeltaC-term strains and the purified protein itself sharply suppressed tumour necrosis factor (TNF)-alpha release by phagocytes in vitro and in lung in vivo, and the strains retained pathogenicity in a murine model of blastomycosis. Our results illustrate the previously undefined role of the EGF-like domain for BAD1 localization to yeast surfaces during cell wall biogenesis. They also demonstrate that the requirements for host cell binding and immune modulation by BAD1 can be dissociated from one another, and that the former is unexpectedly dispensable in the requisite role of BAD1 in pathogenesis.

We investigated how BAD1, an adhesin and virulence factor of Blastomyces dermatitidis, suppresses phagocyte proinflammatory responses. Wild-type yeast cocultured with murine neutrophils or macrophages prompted release of a soluble factor into conditioned supernatant that abolished TNF-alpha production in response to the fungus; isogenic, attenuated BAD1 knockout yeast did not have this effect. Phagocytes released 4- to 5-fold more TGF-beta in vitro in response to wild-type yeast vs BAD1 knockout yeast. Treatment of inhibitory, conditioned supernatant with anti-TGF-beta mAb neutralized detectable TGF-beta and restored phagocyte TNF-alpha production. Similarly, addition of anti-TGF-beta mAb into cultures of phagocytes and wild-type yeast reversed BAD1 inhibition of TNF-alpha production. Conversely, TGF-beta treatment of phagocytes cultured with knockout yeast suppressed TNF-alpha production. Hence, TGF-beta mediates BAD1 suppression of TNF-alpha by wild-type B. dermatitidis cultured in vitro with phagocytes. In contrast to these findings, neutralization of elevated TGF-beta levels during experimental pulmonary blastomycosis did not restore BAD1-suppressed TNF-alpha levels in the lung or ameliorate disease. Soluble BAD1 was found to accumulate in the alveoli of infected mice at levels that suppressed TNF-alpha production by phagocytes. However, in contrast to yeast cell surface BAD1, which induced TGF-beta, soluble BAD1 failed to do so and TNF-alpha suppression mediated by soluble BAD1 was unaffected by neutralization of TGF-beta. Thus, BAD1 of B. dermatitidis induces suppression of TNF-alpha and progressive infection by both TGF-beta-dependent and -independent mechanisms.

Many normal human cells produce thrombospondin-1 (TSP-1), a potent antiangiogenic protein that promotes vascular quiescence. In various organ systems, including the brain, breast and bladder and in fibroblasts, TSP-1 secretion is reduced during tumorigenesis, thereby allowing induction of the vigorous neovascularization required for tumor growth and metastasis. Full-length and short TSP-1-derived peptides inhibit angiogenesis by inducing endothelial cell apoptosis and thus disrupting the vasculature of the growing tumor. CD36 expressed on the surface of endothelial cells functions as the primary antiangiogenic receptor for TSP-1. A D-isoleucyl enantiomer of a TSP-1 heptapeptide specifically inhibits the proliferation and migration of capillary endothelial cells. DI-TSP, an approximately 1 kDa capped version of this peptide, is also antiangiogenic in vitro, with a specific activity approaching that of the 450 kDa parental molecule. Here, we show that DI-TSP delivered systemically dose-dependently inhibits the growth of murine melanoma metastases in syngeneic animals and that its more soluble isomer, DI-TSPa, similarly blocks the progression of primary human bladder tumors in an orthotopic model in immune-deficient mice. Like intact TSP-1, these peptide mimetics had no effect on cancer cells growing in vitro but markedly suppressed the growth of endothelial cells by inducing receptor-dependent apoptosis. Antibodies raised against CD36 blocked the ability of peptides to induce apoptosis in endothelial cells but had no effect on tumor necrosis factor-alpha-induced apoptosis. In vivo, the peptide mimetics were associated with a significantly reduced microvessel density and increased apoptotic indices in both the endothelial and tumor cell compartments. Such short peptides targeted to a specific antiangiogenic receptor, potent and easy to synthesize, show great promise as lead compounds in clinical antiangiogenic strategies., (Copyright 2002 Wiley-Liss, Inc.)

The WI-1 adhesin is indispensable for pathogenicity of Blastomyces dermatitidis and is thought to promote pulmonary infection by fixing yeast to lung tissue and cells. Recent findings suggest that WI-1 confers pathogenicity by mechanisms in addition to adherence. Here, we investigated whether WI-1 modulates host immunity by altering production of pro-inflammatory cytokines. Production of TNF-alpha in lung alveolar fluids of mice infected with B. dermatitidis was severalfold higher for WI-1 knockout yeast compared with wild-type yeast, and in vitro coculture of unseparated lung cells with these isogenic yeast disclosed similar differences. Upon coculture with purified macrophages and neutrophils, wild-type yeast blocked TNF-alpha production, yet WI-1 knockout yeast stimulated production. Coating knockout yeast with purified WI-1 converted them from stimulating TNF-alpha production to inhibiting production. Addition of purified WI-1 into stimulated phagocyte cultures led to concentration-dependent inhibition of TNF-alpha production. Neutralization of TNF-alpha in vivo exacerbated experimental pulmonary infection, particularly for the nonpathogenic WI-1 knockout yeast. Inducing increased TNF-alpha levels in the lung by adenovirus-vectored gene therapy controlled infection with wild-type yeast. Thus, the WI-1 adhesin on yeast modulates host immunity through blocking TNF-alpha production by phagocytes, which fosters progression of pulmonary infection.

In recent years, several hypotheses have emerged to explain the toxicologic activity of particulate matter. Organic compounds, ultrafine particles, biologic components, and transition metals are some of the constituents that reportedly exert some type of adverse effect on human health. A considerable fraction of the urban particulate matter consists of carbon compounds, which originate mostly from anthropogenic sources. The toxicity of organic fractions from particulate matter have been mainly evaluated by considering their mutagenic activity. This research expands on the toxicologic profile of organic compounds adsorbed to particulate matter, specifically in Puerto Rico, by using the cytotoxic neutral red bioassay (NRB). The NRB uses normal human epidermal keratinocytes or other types of cells to measure the effect on cell viability when exposed to organic compounds associated to the particles in the air. We validated the NRB for particulate matter by using a standard reference material (SRM 1649). We used the NRB to determine toxicologic differences of extracts between an urban industrialized site with anthropogenic activity versus a coastal region with less human activity. The cytotoxicity associated with organic compounds in particulate matter collected at the urban industrialized site was detected in both the particulate matter (3/4) 10 microm in aerodynamic diameter (PM(10)) and particulate matter (3/4) 100 microm in aerodynamic diameter (PM(100)). Greater toxic effects were observed in PM(10) extracts than in PM(100) extracts, but PM(10) toxic effects were not significantly different from those in PM(100). The extracts from the industrialized site were more cytotoxic than the extracts from coastal reference site, although in the summer, extracts from both sites were significantly cytotoxic to normal human epidermal keratinocytes. In addition, the nonpolar extracts of both PM(10) and PM(100) exerted the greatest cytotoxicity, followed by the polar, and, finally, the moderately polar extract. This study demonstrates that extracts from the Guaynabo industrialized site were more toxic than similar extracts obtained from a reference coastal site in Fajardo, Puerto Rico.

Gene 33 (g33) is a non-tissue-specific gene regulated in rat liver and hepatoma cells by insulin and other agents. It is thought to participate in the transition from quiescence to proliferation in mitogen-treated cells. The mechanism(s) by which insulin exerts its action on g33 are not totally understood; it is unclear whether a functional insulin receptor is required for this action. In this study, we evaluate the mechanism for insulin induction of g33 mRNA in Chinese hamster ovary (CHO) cells transfected with the neomycin-resistant plasmid (CHONeoB), human insulin receptor (CHONewIRa), and a kinase-defective insulin receptor mutated at the ATP-binding site (CHOK1018A). Transfected cells had higher levels of insulin binding than that of CHONeoB cells; insulin-induced phosphorylation of the insulin receptor and its intracellular substrates were impaired in CHOK1018A cells. Maximal insulin induction of mRNA(g33) occurred 3 h after hormonal exposure in all cell lines. The degree of insulin stimulation of g33 mRNA levels was four- to sixfold higher in CHONewIRa than in CHONeoB or CHOK1018A cells, which had minimal levels of insulin-stimulated g33 mRNA levels. Half-maximal stimulation of g33 mRNA levels was observed at 0.06 +/- 0.01 nM in CHONewIRa cells, consistent with insulin interaction with its own receptor. Wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI3K), had some effects on insulin stimulation of g33 mRNA in CHO NewIRa cells. PD98059, an inhibitor of mitogen-activated kinase kinase (MAPKK), and rapamycin, a p70 S6 kinase inhibitor, had minimal effect on insulin stimulation of g33 mRNA in all cells tested. By contrast, hydroxy-2-naphthalenylmethyl)phosphonic acid triacetoxymethyl ester (HNMPA(AM)(3), a selective inhibitor of the insulin receptor tyrosine kinase, caused complete inhibition of insulin stimulation of g33 mRNA levels. These data indicate that the insulin receptor with intact kinase activity is required for insulin stimulation of g33 mRNA levels. They also suggest that AKT, a PI 3-kinase downstream effector molecule, could mediate insulin stimulation of g33 mRNA. The mechanism(s) of insulin regulation of g33 expression downstream of receptor do not seem to rely entirely on the classic insulin receptor transduction pathway, as a minor effect was observed upon inhibition of MAPKK, suggesting that multiple pathways may be involved., (Copyright 2000 Wiley-Liss, Inc.)

Keratinocyte growth factor (KGF), also called fibroblast growth factor-7, is widely known as a paracrine growth and differentiation factor that is produced by mesenchymal cells and has been thought to act specifically on epithelial cells. Here it is shown to affect a new cell type, the microvascular endothelial cell. At subnanomolar concentrations KGF induced in vivo neovascularization in the rat cornea. In vitro it was not effective against endothelial cells cultured from large vessels, but did act directly on those cultured from small vessels, inducing chemotaxis with an ED50 of 0.02-0.05 ng/ml, stimulating proliferation and activating mitogen activated protein kinase (MAPK). KGF also helped to maintain the barrier function of monolayers of capillary but not aortic endothelial cells, protecting against hydrogen peroxide and vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induced increases in permeability with an ED50 of 0.2-0.5 ng/ml. These newfound abilities of KGF to induce angiogenesis and to stabilize endothelial barriers suggest that it functions in microvascular tissue as it does in epithelial tissues to protect them against mild insults and to speed their repair after major damage.

Angiostatin is a circulating inhibitor of angiogenesis generated by proteolytic cleavage of plasminogen. In this study we have used recombinant human and murine angiostatins (kringles 1-4) as well as native human angiostatin (prepared by elastase digestion of plasminogen [kringles 1-3] or by plasmin autocatalysis in the presence of a free sulfhydryl donor [kringles 1-4]). We report that angiostatin reduces endothelial cell number in a 4-day proliferation assay without affecting cell cycle progression into S-phase (as determined by bromodeoxyuridine labeling). This suggested that the reduction in cell number in the proliferation assay might in part be due to cytotoxicity. This was confirmed by the observation that ethidium homodimer incorporation (a measure of plasma membrane integrity) into endothelial cells was increased by angiostatin in a manner similar to that seen with tumor necrosis factor- (TNF-) and transforming growth factor-beta1 (TGF-beta1), both of which induce apoptosis in endothelial cells. In contrast to TNF- and TGF-beta1, angiostatin did not induce cytotoxicity in human MRC-5 fibroblast, rat smooth muscle, canine MDCK epithelial, or murine B16-F10 melanoma cell lines. Angiostatin-induced apoptosis was confirmed by endothelial cell nuclear acridine orange incorporation as well as by annexin V and TUNEL staining. These in vitro findings point to endothelial cell apoptosis as a mechanism for the antiangiogenic effect of angiostatin in vivo.

The aim of this study was to develop and validate a method for assessing the toxicological risk of endangered populations of pinnipeds based on a nondestructive biological tool, the skin biopsy specimen. Skin biopsies can be obtained from pinnipeds by anaesthetising the animals and taking a small amount of skin in the anterior flipper area, or by shooting a biopsy dart with a crossbow. Skin biopsy material is suitable for a wide range of chemical and biomarker analysis. Organochlorines and polycyclic aromatic hydrocarbons can be analysed in subcutaneous fat and MFO activity (BPMO), Cyt.P450 isoforms, and DNA damage can be detected in epidermis.

The fingerprint of 14 polycyclic aromatic hydrocarbons (PAHs) was investigated in biopsy, fur, blood, liver and faeces of live and dead specimens of two Argentinian population of southern sea lion (Otaria flavescens). One colony lives in Mar del Plata harbour which is particularly polluted with petroleum, the second (control) colony lives at Punta Bermeja (Patagonia). The highest concentrations of the five carcinogenic PAHs were found in the Mar del Plata sea lions.

Study Objective: To evaluate the usefulness of two new parameters for separating pleural transudates and exudates: pleural fluid cholinesterase level and pleural fluid to serum cholinesterase ratio, and to compare the results with the other well-established criteria., Design: Prospective evaluation of the patients referred for diagnostic thoracentesis., Setting: Pulmonary sections of a community hospital and a university hospital., Patients: One hundred ninety-three consecutive patients. Forty were excluded for different reasons., Measurements: The following criteria for separating the pleural effusions in transudates and exudates were analyzed: Light's criteria, the pleural fluid cholesterol level, the pleural fluid to serum cholesterol ratio, the pleural fluid cholinesterase level, and the pleural fluid to serum cholinesterase ratio., Results: One hundred fifty-three patients had conditions diagnosed. Thirty-five were classified as having transudates and 118 as exudates. The percentage of effusions misclassified by each parameter was as follows: Light's criteria, 7.8%; pleural fluid cholesterol, 7.8%; pleural fluid to serum cholesterol ratio, 6.5%; pleural fluid cholinesterase, 8.5%; and pleural fluid to serum cholinesterase ratio, 1.3%., Conclusions: The pleural fluid to serum cholinesterase ratio is the most accurate criterion for separating pleural transudates and exudates. If further studies confirm our results, the cholinesterase ratio could be used as the first step in the diagnosis of pleural effusions.

Genomic and cDNA clones coding for elongation factor-1 beta (eEF-1 beta) from Arabidopsis thaliana (At) were isolated and characterized. eEF-1 beta was found to be encoded by a single-copy At gene. Chimeric genes fusing the promoter and the 5' untranslated region of the At eEF-1 beta gene to the gus reporter gene were constructed and used to study the expression of this gene in transgenic tobacco plants. Interestingly, it was found that the first intron of this gene is required for high levels of expression. Experiments using chimeric promoters showed that an enhancer-like element is present in the first intron of At eEF-1 beta. Gel-shift assays were used to demonstrate that this intron is specifically bound by putative transcription factors present in nuclear protein extracts.

Levels of PCDDs, PCDFs and non-ortho PCBs are reported for fish oils available in Spain which are consumed by the population as a dietary supplement. Trace enrichment methodology based on the use of modified silica and activated carbon chromatography was combined with on-column extraction (FMS 100) to separate PCDDs, PCDFs and PCBs from lipid material. The extracts were further cleaned up on disposable Florisil columns and analysed by HRGC/HRMS (PCDD/F and non-ortho PCB fractions). Mean levels found in the samples were 10.50 ppt for total PCDDs and 9.95 ppt for total PCDFs. Calculated PCDD/F I-TEQ values were found to be 2.11 ppt on a lipid weight basis. Total mean levels found for co-planar PCBs (#77, 126 and 169) were 18.26 ppt and the calculated I-TEQ value was 0.31 ppt on a lipid weight basis.

The incorporation of purine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in nucleotide salvage biosynthetic pathways were investigated by radioactive tracer analysis and nuclear magnetic resonance spectroscopy. The organism took up the nucleobases adenine, guanine and hypoxanthine, and the nucleosides adenosine, guanosine and deoxyadenosine. Any incorporation of deoxyguanosine by the cells was below the detection limits of the methods employed. The activities of adenine-, guanine- and hypoxanthine-phosphoribosyl transferases were established. The bacterium showed high levels of adenosine and guanosine nucleosidase activities and lesser activity for deoxyadenosine; no hydrolysis of deoxyguanosine was detected. Phosphorylase activities were not observed with any of the nucleosides. Phosphotransferase activities with similar rates were demonstrated for adenosine, guanosine and deoxyadenosine; and a weaker activity was detected for deoxyguanosine. No nucleoside kinase activities were observed with any of the nucleosides. The presence of adenylate kinase was established, but no guanylate kinase activity was observed. The study provided evidence for the presence in H. pylori of salvage pathways for the biosynthesis of purine nucleotides.

Sexually experienced male rats were injected IP with 0, 0.3, 1.5, 3.0, or 6.0 mg/kg cadmium chloride. The highest dose was fatal within 48 h of injection. A dose-related deficit in erectile function was observed in ex copula tests 48 h after injection. Copulatory dysfunction was evident in mating tests 72 h after injection. Hepatic mixed function oxidase activity after 0.3 mg/kg injections was not different from controls, but was diminished by 50% in rats treated with 3.0 mg/kg. The higher cadmium doses (1.5 and 3.0 mg/kg) were associated with equivalent and marked reductions in circulating testosterone levels, and lesser decrements in circulating corticosterone levels. The rats treated with 1.5 or 3.0 mg/kg cadmium chloride lost weight (32 +/- 12 and 39 +/- 9 g, respectively). Relative decapsulated adrenal gland weights were markedly increased in rats treated with 3.0 mg/kg. These data indicate that exposure to cadmium is associated with alterations in sexual, hepatic, and adrenal function, with erectile dysfunction occurring at the lowest dose. The effects on copulatory behavior are especially striking because it requires some weeks for copulatory behavior to wane following castration.

The incorporation of pyrimidine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in their synthetic pathways were investigated by radioactive tracer analysis and 31P nuclear magnetic resonance spectroscopy. The bacterium was found to take up aspartate and bicarbonate and to incorporate carbon atoms from these precursors into its genomic DNA. Orotate, an intermediate of de novo pyrimidine biosynthesis, and uracil and uridine, precursors for pyrimidine pathways, were also incorporated by the micro-organism. Radiolabelled substrates were used to assess the activities of aspartate transcarbamoylase, orotate phosphoribosyltransferase, orotidylate decarboxylase, CTP synthetase, uracil phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase in bacterial lysates. The study provided evidence for the presence in H. pylori of an operational de novo pathway, and a less active salvage pathway for the biosynthesis of pyrimidine nucleotides.

The subcellular localization of rap proteins in resting human neutrophils was investigated by immunoblot analysis with specific anti-rap2 and anti-rap1 antibodies of the membrane proteins obtained from distinct subcellular fractions. Rap2 protein was mainly located in gelatinase-containing granules, whereas rap1 protein was detected both in gelatinase-containing granules and in fractions enriched in plasma membrane. Neither rap1 nor rap2 proteins were found in the cytosol or in azurophilic granules. Rap2B, not rap2A, appeared to be the major rap2 protein in human neutrophils. The identification and subcellular localization of rap1 and rap2 proteins at the membranes of gelatinase-rich granules suggest that these proteins could play a role in the regulation of the rapid and selective mobilization of gelatinase-containing granules in human neutrophils.

Effects of chemically induced hepatic injury on biotransformation enzymes in fish were studied. Sunfish hybrids (Lepomis macrochirus x L. cyanellus) were dosed per os with allyl formate (ALF) and carbon tetrachloride (CCl4), and the induction of liver EROD (7-ethoxyresorufin O-deethylase) activity was subsequently challenged by injections of beta-naphthoflavone (BNF) and benzo[a]pyrene (B[a]P). Hepatotoxicity of chemical treatments was assessed using blood enzymes (ASAT, ALAT, and LDH) along with other biochemical variables. Both hepatotoxicants partially abolished the induction of EROD (maximally by 76-89%), and the decrease in induction was dose related. The cytosolic activity of glutathione S-transferase (GST) in the liver decreased in parallel with the decrease in EROD induction. Fish receiving high doses of ALF exhibited significantly less microsomal and blood plasma proteins and, occasionally, were jaundiced. These symptoms, however, were less sensitive indicators of hepatotoxicity than alterations in liver EROD and GST. Both ALF and CCl4 increased the activities of hepatic enzymes in the blood plasma, indicating cytotoxicity. In addition B[a]P, unlike BNF, also increased plasma activities of LDH and ALAT at a dose inducing liver EROD, implying simultaneous hepatotoxicity at high sublethal levels of this xenobiotic. These data suggest that hepatotoxic chemicals absorbed by fish may act antagonistically by decreasing the degree of induction of the cytochrome P450 system relative to the inherent capacity of inducing xenobiotic chemicals present in the environment. Therefore, when assessing the toxicological status of water using fish health biomarkers, it is advisable to measure a concert of metabolic and biochemical variables instead of any single biomarker.

Ras oncogenes encode 21-kDa (p21s) GTP binding proteins that are capable of transforming immortalized cells in culture. The ras-related rap1A/Krev-1/smgp21A protein, that exhibits a similar structural organization and contains the same effector domain as ras proteins, antagonizes ras-transformation. In order to investigate whether the closely related (61% identical) rap2 protein had similar capacities, the corresponding cDNA was inserted into constitutive as well as inducible mammalian expression vectors. Neither the wild-type, nor an "activated" mutant carrying a glycine-to-valine substitution at position 12, had any transforming activity. Several independent lines of evidence demonstrate that the rap2 protein exhibits neither growth-promoting nor growth-inhibitory effects, and that its over-expression does not interfere with ras-induced transformation. Thus, in spite of their great similarities, the rap1A/Krev-1/smgp21A and rap2 proteins have distinct physiological properties.

The study examined the performance of 71 university students on the Fullerton Language Test for Adolescents subtest on Idioms. The subjects were assigned to one of three age categories, and the performances of the groups were compared. The results demonstrated that 51% of the 18-21-year-old group fell within the "Competence Range" for this subtest, while 84% of the 22-29-year-old group and 91% of the 30+ year-old group fell within this range. As chronological age increased, performance improved.

The efflux and exchange of beta-alanine were studied in synaptic plasma membrane vesicles from rat brain. The mechanism of beta-alanine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride ions while influx is dependent on the presence of these two ions on the outside [Zafra, F., Aragón, M. C., Valdivieso, F. and Giménez, C. (1984) Neurochem Res. 9, 695-707]. These data show that the release of beta-alanine occurs via the carrier system and that it is cotransported with sodium and chloride ions. beta-Alanine efflux from the membrane vesicles is stimulated by external beta-alanine. This exchange does not require external sodium and chloride but it is dependent on the external concentration of beta-alanine. Half-maximal stimulation is obtained at a beta-alanine concentration similar to the Km for beta-alanine influx. Results of the direct measurements of the coupling of sodium and chloride to the transport of beta-alanine by using a kinetic approach allow us to propose a stoichiometry for the translocation cycle catalyzed by the beta-alanine transporter of three sodium ions and one chloride ion per beta-alanine zwitterion. To account for all the observed effects of external ions, beta-alanine concentrations and membrane potential on beta-alanine influx and efflux, a kinetic model of the Na+/Cl-/beta-alanine cotransport system is discussed.