Several CD4 domains can play a role in human immunodeficiency virus infection in cells.

The human immunodefiency virus (HIV) uses the human CD4 glycoprotein as a receptor for infection of susceptible cells. Cells expressing a series of mutated forms of the CD4 gene have shown a variability in their ability to support replication of three HIV type 1 (HIV-1) and three HIV-2 strains. Moreover, when different stages of virus production were examined by a variety of assays, a consistent delay was observed in all cell lines containing CD4 mutants compared with those with intact full-length CD4. Cells expressing the CD4.415 mutant (modified at the serine 415 corresponding to a phosphorylation site of the cytoplasmic domain) showed only a minimal effect on virus replication. Cells expressing CD4.403 and CD4.401 mutants (lacking the whole cytoplasmic domain) manifested a moderate delay in production of virus progeny. The most substantial effect on HIV replication was observed in cells expressing a chimeric hybrid containing sequences corresponding to the first 177 residues of the N-terminal CD4 fused to CD8 sequences encoding the hinge, transmembrane, and cytoplasmic domains of the human CD8. Furthermore, in a cell-to-cell contact assay, fusion was absent when the CD4 proximal membrane domain was replaced by the CD8 counterpart. In addition, a strong correlation between the down-modulation of the surface CD4 and HIV expression was observed. These observations suggest that in addition to the known binding region, other domains of CD4 could play an important role in regulating HIV entry of cells.     

Immunological responsiveness against tumors induced by avian sarcoma virus: reduced expression of pp60src kinase activity in regressing tumors.

Tumors which are induced in chickens by avian sarcoma virus frequently grow progressively for several weeks and then regress. We showed that tumor cells which are derived from the progressively growing phase of tumor growth produce large quantities of progeny-transforming virus, are reactive with antiviral antibody, and are susceptible to lysis in cell-mediated cytotoxicity assays by splenic lymphocytes of sensitized hosts. In contrast, tumor cells derived from regressing sarcomas are poor producers of progeny virus and are relatively unreactive with both antiviral antibody and sensitized lymphocytes. We further found that pp60src kinase activity was reduced by about 75% in regressing compared with progressively growing tumor cells. The half-lives of directly precipitable pp60src in tumor cells derived from progressively growing and regressing neoplasms were 6 and 1.5 h, respectively. Studies on each of three other cellular enzymes did not reveal any regression-associated decreases in enzyme activity. These data support the notion that expression of adequate levels of long-lived pp60src kinase activity is essential to progressive tumor growth.     

Environmental formation of methylmercury is controlled by synergy of inorganic mercury bioavailability and microbial mercury-methylation capacity

Methylmercury (MeHg) production is controlled by the bioavailability of inorganic divalent mercury (Hg(II)_i) and Hg-methylation capacity of the microbial community (conferred by the hgcAB gene cluster). However, the relative importance of these factors and their interaction in the environment remain poorly understood. Here, metagenomic sequencing and a full-factorial MeHg formation experiment were conducted across a wetland sulfate gradient with different microbial communities and pore water chemistries. From this experiment, the relative importance of each factor on MeHg formation was isolated. Hg(II)_i bioavailability correlated with the dissolved organic matter composition, while the microbial Hg-methylation capacity correlated with the abundance of hgcA genes. MeHg formation responded synergistically to both factors. Notably, hgcA sequences were from diverse taxonomic groups, none of which contained genes for dissimilatory sulfate reduction. This work expands our understanding of the geochemical and microbial constraints on MeHg formation in situ and provides an experimental framework for further mechanistic studies.     

Prévalence de l'infection cervicale à Chlamydia trachomatis dans une population féminine consultant pour contraception.

OBJECTIVES: To determine the prevalence and risk indicators of cervical infection due to Chlamydia trachomatis among female patients consulting for contraception and to evaluate an enzyme immunoassay for the detection of C. trachomatis in this setting. DESIGN: Prevalence study. Endocervical specimens were analysed by means of culture and enzyme immunoassay. C. trachomatis infection was diagnosed through culture. SETTING: A hospital family planning clinic in Trois-Rivières, Que. SUBJECTS: All 533 female patients who consulted for contraception between November 1986 and March 1988. Results of culture were available for 495 patients. MAIN OUTCOME MEASURE: Demographic, epidemiologic and clinical information was collected by means of a standard questionnaire and a gynecologic examination. MAIN RESULTS: The prevalence rate of chlamydial infection was 9% (45/495). Enzyme immunoassay detected 37 (82%) of the infections. The mean age of the patients was 19.8 years, and 98% of the infections were diagnosed in those aged 25 years or less. The variables significantly associated with C. trachomatis infection were having more than one sexual partner in the preceding year (odds ratio [OR] 2.9; 95% confidence limits [CL] 1.7 and 5.0) and having more than one partner in the preceding 3 months (OR 2.3; 95% CL 1.2 and 4.3). These two indicators would have detected 58% and 22% of the infections respectively. CONCLUSIONS: Screening for C. trachomatis infection by means of enzyme immunoassay should be proposed to all female patients aged 25 years or less consulting for contraception in our clinic. Such screening may prove to be an effective preventive measure in other similar clinical settings.     

Parasites, aggression and dominance in male upland bullies

The effects of the digenean trematode parasite Telogaster opisthorchis on aggression and competitive ability in male upland bullies ( Gobiomorphus breviceps ) were examined using mirror-image stimulation (MIS) techniques and dyadic contests for nest sites between pairs of males. Parasite load had no significant effect on male aggression or success in dyadic contests, nor was aggression a predictor of the likely winner of these contests. The results are discussed with relation to the role of parasites in male-male competition and to possible problems in using MIS techniques for studies on dominance and aggression in fish.     

An early enlargement of the putrescine pool is required for growth in L1210 mouse leukemia cells under hypoosmotic stress

Hypoosmotic stress is a potent inducer of ornithine decarboxylase (ODC) activity in a variety of mammalian cells, but the physiological relevance of this response has not been determined. To test whether an increased putrescine content confers a growth advantage at lower osmolarities, we compared the ability of L1210 mouse leukemia cells and of ODC-overproducing variants obtained from this cell line (D-R cells) to proliferate after a hypotonic shock (325----130 mosmol/kg). The growth rate of D-R cells at 130 mosmol/kg was greater than or equal to 5-fold higher than in L1210 cells; and unlike the ODC-overproducing strain, L1210 cells underwent up to a 90% loss of viability over time as seen after restoration of normosmotic growth conditions and by trypan blue exclusion tests. The addition of putrescine or L-ornithine stimulated the proliferation of both cell sublines up to 5-fold in a concentration-dependent manner, with a maximal effect observed at about 10 and 100 microM, respectively. Putrescine restored virtually normal growth rates in both sublines at osmolarities as low as 190 mosmol/kg. No other alpha,omega-diamine was active in that respect whereas spermidine was markedly inhibitory. Furthermore, D-R cells incubated at 130 mosmol/kg showed a marked growth inhibition by 1-aminooxy-3-aminopropane (potent ODC inhibitor to which they are resistant in isotonic media) as a result of putrescine but not spermidine depletion. Whereas ODC was strongly and rapidly induced by hypotonic shock there was a precipitous decline in S-adenosylmethionine decarboxylase activity. Putrescine synthesis and accumulation were nevertheless reduced in D-R cells incubated at 130 mosmol/kg because of a decreased availability of L-ornithine. When either putrescine or L-ornithine was added to hypotonic media, D-R cells accumulated putrescine massively for extended periods together with a reduction in spermidine and spermine contents. Putrescine transport patterns were altered by hypotonic shock, net excretion of the diamine being reduced by about 80%, with a concurrent enlargement of the intracellular pool. Finally, parental L1210 cells incubated with an irreversible inhibitor of S-adenosylmethionine decarboxylase for 24 h until hypotonic shock and supplemented with putrescine in the presence of the drug thereafter exhibited a greatly exaggerated growth stimulation by the diamine. These results demonstrate an essential role for an early increase in putrescine content in the growth adaptation of a mammalian cell line to a lower osmolarity.     

Determinants of host-specificity in parasites of freshwater fishes.

Factors responsible for interspecific variability in host-specificity were investigated within 15 genera (including 176 species) of metazoan parasites found in Canadian freshwater fish. For each species in a genus, the parasite's number of known hosts was determined from published host-parasite records. The effects of the total number and mean size of potential hosts (i.e. all fish species belonging to the family or families that include a parasite's known hosts) on number of hosts of congeneric species were evaluated using multiple regressions. Since parasite species that have been recorded often tend to have greater numbers of known hosts than do seldom-recorded parasites, it was necessary to control for the confounding effect of study intensity. In all parasite genera, whether from highly specific taxa such as monogeneans or from less host-specific ones, there was a positive relationship between the number of potential hosts and the number of known hosts. However, no consistent relationships were observed between the mean size of potential hosts and number of known hosts. These results suggest that the availability of suitable host species may have been a key factor limiting the colonization of new hosts by fish parasites.     

Mechanism of the irreversible inactivation of mouse ornithine decarboxylase by alpha-difluoromethylornithine. Characterization of sequences at the inhibitor and coenzyme binding sites.

Mouse ornithine decarboxylase (ODC) was expressed in Escherichia coli and the purified recombinant enzyme used for determination of the binding site for pyridoxal 5'-phosphate and of the residues modified in the inactivation of the enzyme by the enzyme-activated irreversible inhibitor, alpha-difluoromethylornithine (DFMO). The pyridoxal 5'-phosphate binding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoenzyme form with NaB[3H]4 followed by digestion of the carboxymethylated protein with endoproteinase Lys-C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptide sequencing. This lysine is contained in the sequence PFYAVKC, which is found in all known ODCs from eukaryotes. The preceding amino acids do not conform to the consensus sequence of SXHK, which contains the pyridoxal 5'-phosphate binding lysine in a number of other decarboxylases including ODCs from E. coli. Using a similar procedure to analyze ODC labeled by reaction with [5-14C]DFMO, it was found that lysine 69 and cysteine 360 formed covalent adducts with the inhibitor. Cysteine 360, which was the major adduct accounting for about 90% of the total labeling, is contained within the sequence -WGPTCDGL(I)D-, which is present in all known eukaryote ODCs. These results provide strong evidence that these two peptides form essential parts of the catalytic site of ODC. Analysis by fast atom bombardment-mass spectrometry of tryptic peptides containing the DFMO-cysteine adduct indicated that the adduct formed in the enzyme was probably the cyclic imine S-(2-(1-pyrroline)methyl)cysteine. This is readily oxidized to S-((2-pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction. This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between the pyridoxal 5'-phosphate, the enzyme, and the inhibitor.