Comparative evaluation of As, Se and V removal technologies for the treatment of oil refinery wastewater.

In this study, a broad range of readily deployable metal removal technologies were tested on a US refinery's wastewater to determine vanadium, arsenic and selenium removal performance. The bench-scale treatability studies were designed and performed so that test conditions could be as uniform as possible given the different mechanisms of action and engineering applications of each technology. The experimental data show that both ferric precipitation and reactive filtration were able to remove As, Se and V more efficiently from the wastewater than other tested technologies. Additionally, granular ferric hydroxide (GFH) adsorption was also effective in both V and As removal. Although the thiol-SAMMS adsorbent was developed for mercury removal, it also demonstrated appreciable selenium removal. None of the tested membrane filtration technologies showed any significant metals removal. This was attributed to the dissolved form of the metals as well as the wastewater's fouling characteristics.

The Separative Bioreactor: A Continuous Separation Process for the Simultaneous Production and Direct Capture of Organic Acids.

The replacement of petrochemicals with biobased chemicals requires efficient bioprocesses, biocatalysis, and product recovery. Biocatalysis (e.g., enzyme conversion and fermentation) offers an attractive alternative to chemical processing because biocatalysis utilize renewable feedstocks under benign reaction conditions. One class of chemical products that could be produced in large volumes by biocatalysis is organic acids. However, biocatalytic reactions to produce organic acids typically result in only dilute concentrations of the product because of product inhibition and acidification that drives the reaction pH outside of the optimal range for the biocatalyst. Buffering or neutralization results in formation of the acid salt rather than the acid, which requires further processing to recover the free acid product. To address these barriers to biocatalytic organic acid production, we developed the "separative bioreactor" based on resin wafer electrodeionization, which is an electro-deionization platform that uses resin wafers fabricated from ion exchange resins. The separative bioreactor simultaneously separates the organic acid from the biocatalyst as it is produced, thus it avoids product inhibition enhancing reaction rates. In addition, the separative bioreactor recovers the product in its acid form to avoid neutralization. The instantaneous separation of acid upon formation in the separative bioreactor is one of the first truly one-step systems for producing organic acids. The separative bioreactor was demonstrated with two systems. In the first demonstration, the enzyme glucose fructose oxidoreductase (GFOR) was immobilized in the reactor and later regenerated in situ. GFOR produced gluconic acid (in its acid form) continuously for 7 days with production rates up to 1000 mg/L/hr at >99% product recovery and GFOR reactivity >30mg gluconic acid/mg GFOR/hour. In the second demonstration, the E. coli strain CSM1 produced lactic acid for up to 24 hours with a productivity of >200 mg/L/hr and almost 100% product recovery.

9-Nitrocamptothecin inhibits HIV-1 replication in human peripheral blood lymphocytes: a potential alternative for HIV-infection/AIDS therapy.

The ability of the anti-cancer drug, 9-Nitrocamptothecin (9NC), to inhibit replication of HIV-1 in clinically relevant primary lymphocytic cells was studied. Primary peripheral blood lymphocytes (PBLs) from a non-infected donor were freshly infected with HIV-1 and treated with 9NC by using three different treatment schedules. Cells were monitored for cytotoxicity by the XTT metabolic cell proliferation assay and a sensitive flow cytometric assay that was capable of measuring cell cycle changes and apoptosis. 9NC inhibited replication of HIV-1 in PBLs by greater than 95% in a dose-dependent manner as measured by the level of extracellular HIV-1 p24 release. Similar results were observed, whether 9NC was applied in a single, double, or triple dose regimen. Minimal cytotoxicity was observed for both non-infected and infected PBLs, as determined by the XTT assay. Moreover, 9NC induced apoptosis within 24 hours of drug treatment in freshly infected, but not non-infected, PBLs. The data showed that 9NC reduced replication of HIV-1 in primary human lymphocytes; thus, it indicates the potential clinical utility of this drug as an alternative or adjunct therapy for HIV-infection/AIDS.

Predictors of smoking cessation during pregnancy.

AIMS: The purpose of this study was to determine predictors of smoking cessation from a sample of pregnant Medicaid recipients. Of special interest was whether patient stage of change, based on the transtheoretical model, was predictive of smoking behavior change during pregnancy. PARTICIPANTS/SETTING: The sample was drawn from a cohort of pregnant smokers who were participants in a prospective, randomized clinical trial conducted in four public health maternity clinics in Birmingham, Alabama, USA. DESIGN/MEASUREMENTS: The 435 participants entered prenatal care on or before their 24th week of gestation and had saliva collected for cotinine assays at baseline and follow-up. In this secondary analysis, descriptive statistics defined the sample, cross-tabulation procedures identified a preliminary set of predictor variables, and discriminant function analyses predicted group membership--quitter or smoker. FINDINGS/CONCLUSIONS: Discriminant function analyses revealed that patient baseline cotinine value, duration of smoking habit, self-efficacy, exposure to environmental tobacco smoke, and exposure to patient education methods were predictive of non-smoking status assessed during the third trimester of pregnancy.

Domain structure analysis of elongation factor-3 from Saccharomyces cerevisiae by limited proteolysis and differential scanning calorimetry.

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultracentrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 degrees C, and a minor transition at 58 degrees C. Ultracentrifugation shows that EF-3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, generating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N-terminal fragment slowly undergoes further digestion generating two major bands, one at approximately 75 kDa and the other at approximately 55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58 degrees C transition, and a loose structure of the C-terminus, giving rise to the 39 degrees C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N-terminal ATP-binding cassette (Gly463-Lys622), and the aminoacyl-tRNA-synthase homologous domain (Glu820-Gly865). Both the basal and ribosome-stimulated ATPase activities were inactivated by trypsin, but the ribosome-stimulated activity was inactivated faster.

Fat distribution, physical activity, and cardiovascular risk factors.

The purpose of this study was to report the relationship between fat distribution, physical activity (PA), and cardiovascular disease (CVD) risk factors. Percent fat, computed tomography intra-abdominal adipose tissue (IAF), anthropometrics, Baecke activity questionnaire, and CVD risk (blood pressure, cholesterol, HDL, HDL2, HDL3, IDL, LDL, VLDL, and triglycerides) were evaluated in 137 men 30-71 yr old. IAF was consistently more highly related to CVD risk than other fat distribution variables including percent fat and waist:hip ratio (r = 0.3-0.45). IAF was significantly related to CVD risk after adjusting for other fat distribution variables. With the exception of the sum of biceps, triceps, thigh, and calf skinfolds (peripheral skinfolds), which was negatively related to CVD risk, no other fat distribution variable had consistent significant partial correlations with CVD risk. PA was related to IAF after adjusting for peripheral skinfolds, but PA was not related to peripheral skinfolds after adjusting for IAF, indicating more active men have relatively low IAF. IAF was related to CVD risk after adjusting for PA, but PA was not related to CVD risk after adjusting for IAF. These results indicate that IAF is directly related to CVD risk while the lower CVD risk found with more active men is more directly related to the low IAF found in more active men.

Initial characterization of autoprocessing and active-center mutants of CMV proteinase.

Human cytomegalovirus (CMV) encodes a unique serine proteinase that is required in the maturation of the viral capsid. The CMV proteinase can undergo autocatalytic activation and is subject to proteolytic self-inactivation. Mutant enzyme forms were prepared to eliminate the initial autoprocessing site and thus form an active single-chain protein for structure-function studies. Two mutants of CMV proteinase were cloned and expressed in Escherichia coli. The A143V mutant was a conservative substitution at the first internal cleavage site. The S132A mutant modified one of the triad of residues responsible for catalytic activity. Through the use of computer-controlled high-cell-density fermentations the mutant proteins were expressed in E. coli at approximately 170 mg/L as both soluble (approximately 40% of total) and inclusion-body forms (approximately 60% of total). The soluble enzyme was purified by standard methods; inclusion-body protein was isolated by standard methods after refolding and solubilization in guanidine or urea. Sedimentation equilibrium and sedimentation velocity analyses reveal that the enzyme undergoes concentration-dependent aggregation. It exhibits a monomer <==> dimer equilibrium (Kd = 1 microM) at low concentrations and remains dimeric at high concentrations (28 mg/ml). Differential scanning calorimetry data for protein thermal unfolding fit best to a non-two-state model with two components (Tm = 52.3 and 55.3 degrees C) which subsequently aggregate upon unfolding. Analysis of the short-UV circular dichroism spectra of protein forms resulting from expression as soluble molecules (not refolded) reveals that the two mutants have very similar secondary structures which comprise a mixed structural motif of 20% alpha-helix, 26% beta-sheet, and 53% random coil. Though soluble and active (A143V mutant only), CD analysis revealed that protein refolded from inclusion bodies did not exhibit spectra identical to that of protein expressed only in soluble form.

Immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1. The National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group.

Phase I studies of volunteers not infected with human immunodeficiency virus type 1 (HIV-1) have shown that immunization with envelope subunit vaccine products elicits antibodies that neutralize laboratory-adapted (prototype) HIV-1 strains in vitro. Prototype strains are adapted to grow in continuous (neoplastic) cell lines and are more susceptible to neutralization than are primary isolates cultured in human peripheral blood mononuclear cells. In this study, 50 sera from nine phase I vaccine trials and 16 from HIV-1-infected persons were evaluated for neutralizing antibody activity against 3 laboratory-adapted and 5 primary HIV-1 isolates. Of 50 sera, 49 neutralized at least 1 of the prototype strains; however, none displayed neutralizing activity against primary isolates of HIV-1. Serum from most HIV-1-infected persons neutralized both laboratory-adapted and primary HIV-1 isolates. These data demonstrate a qualitative, or large quantitative, difference in the neutralizing antibody response induced by envelope subunit vaccination and natural HIV-1 infection.

The nanometer-scale structure of amyloid-beta visualized by atomic force microscopy.

Amyloid-beta (A beta) is the major protein component of neuritic plaques found in Alzheimer's disease. Evidence suggests that the physical aggregation state of A beta directly influences neurotoxicity and specific cellular biochemical events. Atomic force microscopy (AFM) is used to investigate the three-dimensional structure of aggregated A beta and characterize aggregate/fibril size, structure, and distribution. Aggregates are characterized by fibril length and packing densities. The packing densities correspond to the differential thickness of fiber aggregates along a zeta axis (fiber height above the x-y imaging surface). Densely packed aggregates ( > or = 100 nm thick) were observed. At the edges of these densely packed regions and in dispersed regions, three types of A beta fibrils were observed. These were classified by fibril thickness into three size ranges: 2-3 nm thick, 4-6 nm thick, and 8-12 nm thick. Some of the two thicker classes of fibrils exhibited pronounced axial periodicity. Substructural features observed included fibril branching or annealing and a height periodicity which varied with fibril thickness. When identical samples were visualized with AFM and electron microscopy (EM) the thicker fibrils (4-6 nm and 8-12 nm thick) had similar morphology. In comparison, the densely packed regions of approximately > or = 100 nm thickness observed by AFM were difficult to resolve by EM. The small, 2- to 3-nm-thick, fibrils were not observed by EM even though they were routinely imaged by AFM. These studies demonstrate that AFM imaging of A beta fibrils can, for the first time, resolve nanometer-scale, zeta-axis, surface-height (thickness) fibril features. Concurrent x-y surface scans of fibrils reveal the surface submicrometer structure and organization of aggregated A beta. Thus, when AFM imaging of A beta is combined with, and correlated to, careful studies of cellular A beta toxicity it may be possible to relate certain A beta structural features to cellular neurotoxicity.

Clusterin (apoJ) alters the aggregation of amyloid beta-peptide (A beta 1-42) and forms slowly sedimenting A beta complexes that cause oxidative stress.

Clusterin (apoJ), a multifunctional apolipoprotein made by cells in the brain and many other locations, is associated with aggregated amyloid beta-peptide (A beta) in senile and diffuse plaques of Alzheimer's disease (AD). We observed that purified human serum clusterin partially blocked the aggregation of synthetic A beta 1-42, as shown by centrifugal assays (14,000g x 10 min) and by atomic force (scanning probe) microscopy. Slowly sedimenting A beta complexes were formed in the presence of clusterin, which included aggregates > 200 kDa that resist dissociation by low concentrations of SDS. Clusterin enhanced the oxidative stress caused by A beta, as assayed by oxidative stress in PC12 cells with MTT, which is widely used to estimate neurotoxicity. These indications of enhanced neurotoxicity by the MTT assay were observed in the highly aggregated rapidly sedimenting fraction, but also in more slowly sedimenting "soluble" forms. This novel activity of slowly sedimenting A beta may enhance the neurotoxicity of A beta deposits in AD brains, because soluble complexes have a potential for diffusing to damage distal neurons.

Intra-abdominal adipose tissue values associated with risk of possessing elevated blood lipids and blood pressure.

Central fat distribution and more recently intra-abdominal adipose tissue (IAF) have been associated with elevated cardiovascular risk factors (CRF). Despite increased interest in use of LAF for screening for CRF, interpretation of risk found in a specific IAF is difficult since regressions for estimating CRF from IAF have not been published. The purpose of this paper is to report IAF values that are likely to be associated with elevated CRF. One hundred forty-six healthy male subjects 30-71 years were evaluated for IAF and subcutaneous fat (computed tomography scan at 4th lumbar vertebra), height, body weight, % fat, various anthropometric measures, blood cholesterol (CHOL), HDL cholesterol (HDL), systolic blood pressure (SBP), and diastolic blood pressure (DBP). Receiver-Operating-Characteristic curves (ROC) were used to develop IAF cutpoints associated with elevation of at least one established CRF criteria (CHOL=200, HDL=<35, SBP=140, DBP=90). A sensitivity/(1- specificity) curve established the value of using IAF cutpoints for detecting elevated CRF. Likelihood ratios were used to identify optimal cutpoints. Two cutpoints were identified, 131 cm2 with a relatively high Lpos ratio and 71 cm2 with a relatively low Lneg. False positives associated with 131 cm2 were 14% for one or more elevated CRF. False negatives associated with 71 cm2 were 9% for one or more elevated CRF, 4% for two or more CRF, and 0% for three or more elevated CRF. This study clearly indicates that IAF above 131 cm2 is related to elevated CRF and IAF below 71 cm2 is associated with reduced cardiovascular risk.

Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths.

One of the clinical manifestations of Alzheimer's disease is the deposition of the 39-43 residue amyloid-beta (A beta) peptide in aggregated fibrils in senile plaques. Characterization of the aggregation behavior of A beta is one of the critical issues in understanding the role of A beta in the disease process. Using solution hydrodynamics, A beta was observed to form three types of species in phosphate-buffered saline: insoluble aggregates with sedimentation coefficients of approximately 50,000 S and molecular masses of approximately 10(9) Da, "soluble aggregates" with sedimentation coefficients of approximately 30 S and masses of approximately 10(6) Da, and monomer. When starting from monomer, the aggregation kinetics of A beta 1-40 (A beta 40) and A beta 1-42 (A beta 42), alone and in combination, reveal large differences in the tendency of these peptides to aggregate as a function of pH and other solution conditions. At pH 4.1 and 7.0-7.4, aggregation is significantly slower than at pH 5 and 6. Under all conditions, aggregation of the longer A beta 42 was more rapid than A beta 40. Oxidation of Met-35 to the sulfoxide in A beta 40 enhances the aggregation rate over that of the nonoxidized peptide. Aggregation was found to be dependent upon temperature and to be strongly dependent on peptide concentration and ionic strength, indicating that aggregation is driven by a hydrophobic effect. When A beta 40 and A beta 42 are mixed together, A beta 40 retards the aggregation of A beta 42 in a concentration-dependent manner. Shorter fragments have a decreasing ability to interfere with A beta 42 aggregation. Conversely, the rate of aggregation of A beta 40 can be significantly enhanced by seeding slow aggregating solutions with preformed aggregates of A beta 42. Taken together, the inhibition of A beta 42 aggregation by A beta 40, the seeding of A beta 40 aggregation by A beta 42 aggregates, and the chemical oxidation of A beta 40 suggest that the relative abundance and rates of production of different-length A beta and its exposure to radical damage may be factors in the accumulation of A beta in plaques in vivo.

Cleavage at the amino and carboxyl termini of Alzheimer's amyloid-beta by cathepsin D.

Amyloid beta (A beta) is a 39-43-residue protein that originates from proteolysis of the beta-protein precursor (beta PP) and accumulates in senile plaques in brains of Alzheimer's disease (AD) patients. Mutant beta PP, which incorporates an AD-causing double mutation at positions 687-688, has been shown to enhance A beta production in transfected cells. In this work we investigate the susceptibility of the mutant beta PP sequence to proteolytic cleavage by proteinases from human brain. Internally quenched fluorogenic substrates were used that encompass the NH2-terminal sequence of A beta from wild-type beta PP, the double mutant, and the two single substitutions. Proteinase activity in brain extract cleaved the mutant substrate 100-fold faster than the wild-type substrate and the partial mutants 25-fold faster. The major cleavage site in all substrates was at the amyloidogenic Asp1 site. The brain activity appeared to be cathepsin D (CD), as indicated by similarities to purified CD in 1) the rate and site of substrates cleavage, 2) the pH optima, and 3) the sensitivity to pepstatin A. The increased activity against the mutant substrate was not shared by cathepsins B and C, pepsin, HIV proteinase, and Candida albicans Asp-proteinase. Furthermore, CD cleaved a substrate that incorporates the COOH terminus of A beta at positions equivalent to Thr43 and Ala42, at ratios of 68% and 32%, respectively. CD degraded A beta 1-40 into six fragments but A beta 1-42 was completely resistant to digestion, probably because of its aggregation characteristics. These results indicate that CD is capable of producing the cleavages resulting in A beta production and that it may prove to be a suitable therapeutic target.

Complement C1q does not bind monomeric beta-amyloid.

The tendency of both labeled and unlabeled beta-amyloid to bind in solution to C1q, the recognition species in the complement cascade, was examined using both hydrodynamic and spectroscopic methods. Potential binding interactions were evaluated using a purified synthetic beta-amyloid 1-40 sequence, alone, and selectively labeled at the amino terminus with spectroscopic probes. The probes permitted both absorbance and fluorescence analyses of beta-amyloid binding interactions. Under conditions used for the analyses beta-amyloid exists exclusively as a monomer in solution, and C1q retains an intact quaternary structure and is capable of binding to IgM. When mixed together the monomeric beta-amyloid does not bind to, or interact with, the complement C1q at concentrations below approximately 100 microM. The data suggest that if beta-amyloid toxicity is associated with complement activation in Alzheimer's disease then monomeric beta-amyloid is likely not responsible for activation through the classical complement pathway.

Similarities between beta amyloid peptides 1-40 and 40-1: effects on aggregation, toxicity in vitro, and injection in young and aged rats.

Peptides corresponding to the first 40 amino acids of beta amyloid peptide (beta 1-40) and the reverse sequence (beta 40-1) were synthesized, purified, and compared for their ability to aggregate and cause toxicity in vitro to human neuroblastoma cells (SH-SY5Y), as well as for effects following injection into young or aged rats. Aggregation of both peptides produced similar sedimentation velocity profiles and resulted in significant toxicity in vitro with no observable differences between beta 1-40 and beta 40-1. In addition, when injected into the cortex of young rats, beta 1-40 was more toxic than beta 40-1 although both resulted in significant lesions. However, in aged rats the two peptides resulted in lesions of similar size. Alz 50 staining and abnormal neurites were associated with both beta 1-40 and beta 40-1 lesions; however, no evidence of plaques or tangles was found in either age group. While both peptides were toxic in vitro, only beta 1-40 elicited Alz 50 staining of SH-SY5Y cells. Electron microscopic examination of beta 1-40 and beta 40-1 aggregates showed that beta 1-40 formed fibrillar structures whereas beta 40-1 resulted in amorphous particles. Thus, although both peptides were toxic to cultured cells and aged rats, the toxicities may have resulted from different mechanisms.

Evidence for decidual modulation of contractile function in the pregnant rat uterus.

This study tested the hypothesis that the decidua of pregnant uterus modulates contractile behavior of the underlying myometrium. Isometric contractile function was measured in transverse rings of pregnant rat uterus with or without the decidua. Observations were made of spontaneous contractions after in vitro isolation, and also of contractile responses to oxytocin and prostaglandin F2 alpha (PGF2 alpha) after the tissues had ceased spontaneous mechanical activity. The amplitude and frequency of spontaneous uterine contractions progressively declined after in vitro isolation; during this period, amplitude of spontaneous contractions was greater in the presence of the decidua, whereas contraction frequency was similar in the decidua-intact and decidua-removed tissues. Reinsertion of donor decidua reproduced contractile characteristics of intact tissue. PGF2 alpha and oxytocin stimulated myometrial contractions, but contraction frequency was greater in the absence than in the presence of the decidua. Depending upon the stimulus, the decidua seems able to express both excitatory and inhibitory factors which can selectively modulate either strength or frequency of uterine contractions.

Alkylamides as inducers of human leukemia cell differentiation: a quantitative structure-activity relationship study using comparative molecular field analysis.

Computer assisted quantitative structure-activity studies using comparative molecular field analysis (CoMFA) were performed on a series of alkylamides that induce cell differentiation. The series included alkylformamides, alkylacetamides, alkylureas, and substituted hexyl analogues of acetamide. The biological activity studied for correlation with structure was the ability of each compound to induce differentiation of the human promyelocytic leukemia cell line, HL-60, to granulocyte-like cells. In the CoMFA study, both steric and electrostatic fields were used along with molecular weight to determine a correlation between biological activity of the compounds and their structural features. The CoMFA results indicated a linear structure-activity correlation with a high predictive value. There was almost an even contribution towards activity from steric interactions, electrostatic potential, and molecular weight. These findings confirm a previous report by Langdon and Hickman (S. P. Langdon and J. A. Hickman, Cancer Res., 47: 140-144, 1987) that the ability to induce cell differentiation is highly dependent on molecular weight. Additionally, CoMFA contour maps provided information about regions of the molecule that are favorable to increased steric bulk and electrostatic charge. CoMFA was used to predict the activities of six hexamethylene acetamide analogues: ethyl 6-acetamidohexanoate; 6-acetamidohexanol; 1,5-bis(acetamido)hexane; 6-acetamidohexanonitrile; 6-acetamidohexanoic acid; and caprolactam. Although the model incorrectly predicted high activity for 6-acetamidohexanoic acid, the predicted activities for the remaining compounds were 0.3 to 1.5 times that of the corresponding experimental activities, which is comparable to the results obtained from other published CoMFA studies.

Coronavirus species specificity: murine coronavirus binds to a mouse-specific epitope on its carcinoembryonic antigen-related receptor glycoprotein.

Like most coronaviruses, the coronavirus mouse hepatitis virus (MHV) exhibits strong species specificity, causing natural infection only in mice. MHV-A59 virions use as a receptor a 110- to 120-kDa glycoprotein (MHVR) in the carcinoembryonic antigen (CEA) family of glycoproteins (G. S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G. S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991; and R. K. Williams, G. S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). The role of virus-receptor interactions in determining the species specificity of MHV-A59 was examined by comparing the binding of virus and antireceptor antibodies to cell lines and intestinal brush border membranes (BBM) from many species. Polyclonal antireceptor antiserum (anti-MHVR) raised by immunization of SJL/J mice with BALB/c BBM recognized MHVR specifically in immunoblots of BALB/c BBM but not in BBM from adult SJL/J mice that are resistant to infection with MHV-A59, indicating a major difference in epitopes between MHVR and its SJL/J homolog which does not bind MHV (7). Anti-MHVR bound to plasma membranes of MHV-susceptible murine cell lines but not to membranes of human, cat, dog, monkey, or hamster cell lines. Cell lines from these species were resistant to MHV-A59 infection, and only the murine cell lines tested were susceptible. Pretreatment of murine fibroblasts with anti-MHVR prevented binding of radiolabeled virions to murine cells and prevented virus infection. Solid-phase virus-binding assays and virus overlay protein blot assays showed that MHV-A59 virions bound to MHVR on intestinal BBM from MHV-susceptible mouse strains but not to proteins on intestinal BBM from humans, cats, dogs, pigs, cows, rabbits, rats, cotton rats, or chickens. In immunoblots of BBM from these species, both polyclonal and monoclonal antireceptor antibodies that block MHV-A59 infection of murine cells recognized only the murine CEA-related glycoprotein and not homologous CEA-related glycoproteins of other species. These results suggest that MHV-A59 binds to a mouse-specific epitope of MHVR, and they support the hypothesis that the species specificity of MHV-A59 infection may be due to the specificity of the virus-receptor interaction.

Photosynthetic electron-transfer reactions in the green sulfur bacterium Chlorobium vibrioforme: evidence for the functional involvement of iron-sulfur redox centers on the acceptor side of the reaction center.

The green sulfur bacterium Chlorobium vibrioforme was cultured in the presence of ethylene to selectively inhibit the synthesis of the chlorosome antenna BChl d. Use of these cells as starting material simplified the isolation of a photoactive antenna-depleted membrane fraction without the use of high concentrations of detergents. The preparation had a BChl alpha/P840 of 50, and the spectral properties were similar to those of preparations isolated from cells grown with a normal complement of chlorosomes. The membrane preparation was active in NADP+ photoreduction. This indicated that the fraction contained reaction centers with complete electron-transfer sequences which were then characterized further by flash kinetic spectrophotometry and EPR. We confirmed that cytochrome c553 is the endogenous donor to P840+, and at room temperature we observed a recombination reaction between the reduced terminal acceptor and P840+ with a t1/2 = 7 ms. Oxidative degradation of iron-sulfur centers using low concentrations of chaotropic salts introduced a faster recombination reaction of t1/2 = 50 microseconds which was lost at higher concentrations of chaotrope, indicating the participation of another iron-sulfur redox center earlier than the terminal acceptor. Cluster insertion using ferric chloride and sodium sulfide in the presence of 2-mercaptoethanol restored both the 50-microseconds and 7-ms recombination reactions, allowing definitive assignments of these centers as iron-sulfur centers. Following the suggestion of Nitschke et al. [(1990) Biochemistry 29, 3834-3842], we associate these two kinetic phases to back-reactions between P840+ and iron-sulfur centers FX and FAFB, respectively. The iron-sulfur cluster degradation and reconstitution protocols also led to inhibition and restoration of NADP+ photoreduction by the membrane preparation, providing unequivocal evidence for the function of the centers FX and FAFB in the physiological electron-transfer sequence on the acceptor side of the Chlorobium reaction center. At 77 K we observed a recombination reaction of t1/2 = 20 ms that we suggest occurs between Fx- and P840+. Degradation of the iron-sulfur clusters resulted in replacement of the 20-ms phase with a faster reaction of t1/2 = 80 microseconds that was most likely a recombination between the early acceptor A1- and P840+ or decay of 3P840. Analysis of the iron-sulfur centers in the preparation by EPR at cryogenic temperature supports the optical measurements. EPR signals originating from the terminal acceptor(s) were not observed following treatment of the membrane preparation by chaotropes, and a modified signal was restored following cluster reinsertion.(ABSTRACT TRUNCATED AT 400 WORDS)

Substance abuse during pregnancy in a rural population.

Drug abuse during pregnancy in rural populations has received less attention than that in urban populations. Urban studies have reported alarming rates, but it is unknown whether the situation is the same in rural areas. To investigate this, urine samples were collected anonymously from 181 pregnant women who presented to the University of Missouri clinics for care and who resided in communities of less than 25,000. Each urine specimen was tested for cocaine, marijuana, amphetamines, barbiturates, opiates, phencyclidine, benzodiazepines, ethanol, and nicotine. Of the 181 specimens, 83 (46%) contained nicotine, 17 (9.4%) contained marijuana, and one each (0.6%) tested positive for cocaine, barbiturates, ethanol, and benzodiazepines. No other tested substances were detected. Excluding nicotine and ethanol, 20 (11%) of the urine samples tested positive for the screened substances. Review of the prenatal records revealed that 46% of the women reported using tobacco, 15% reported using alcohol, and 8.3% reported illicit drug use during pregnancy. This study indicates that there is a substantial drug abuse problem in rural populations, and that the profile of abuse differs from that of urban populations. Tobacco, ethanol, and marijuana were the most prevalent substances abused during pregnancy, but cocaine was a minor problem. This information may help in directing resources to reduce drug abuse during pregnancy.