Effects of feminization of male F344 rats on induction of tumors and on nucleic acid alkylation by nitrosobis-(2-oxopropyl)amine

Groups of male and female F344 rats were treated twice weekly by gavage with 2.5 mg of nitrosobis-(2-oxopropyl)amine (BOP) for 35 weeks. Additional groups given the same treatment were male rats castrated at birth, male rats bearing an implant of a pellet containing estradiol and castrated male rats bearing an estradiol pellet. Most rats died with tumors related to the treatment; intact male rats survived the least well of the five groups. Most rats in all groups had alveolar/bronchiolar neoplasms of the lung. Many of the male rats also had follicular cell neoplasms of the thyroid and transitional cell neoplasms of the urinary bladder and kidney pelvis; there were no liver tumors in intact male rats. Almost all female rats and castrated male rats had liver neoplasms, including hepatocellular, cholangiocellular and hemangiosarcomatous neoplasms, but few neoplasms of the thyroid, kidney or bladder. The male rats feminized with estradiol, intact or castrated, had liver neoplasms, mainly cholangiocellular, and also neoplasms of the thyroid. Two rats of each of the five groups were treated at 20 weeks of age with [14C]BOP. As measured by respiration of 14CO2, metabolism of BOP was faster in the two groups of male rats with the estradiol implant than in the other groups. DNA and RNA of the liver were isolated 6 h after treatment. The extent of methylation of liver DNA as 7-methylguanine and O6-methylguanine was higher in the females and in the feminized males than in the intact male rats, but when normalized to the dose of nitrosamine per unit body weight there was little difference among the five groups.     

Chemical and physical characterization of rabbit sperm acrosome stabilizing factor

Rabbit Acrosome Stabilizing Factor (ASF) is an epididymal product that reversibly inhibits the process of sperm capacitation. The native molecular weights of the monomer and dimer ASF were determined from sedimentation and diffusion data at 129,000 and 259,000 Mr. The monomer is composed of 92,000 and 38,000 Mr subunits according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with size heterogeneity demonstrated for the latter. The stoichiometry of the subunits appears to be one-to-one by gel scanning. Amino acid and carbohydrate compositions are characteristic of a globular glycoprotein, which is high in cysteine content and is 8.3% carbohydrate by weight. The sugar composition suggests the presence of both high mannose and complex N-linked oligosaccharides with the unusual feature of appreciable amounts of glucose. The isoelectric character of ASF spans a range from 5.3 to 7.0.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

Foraging-efficiency-predation-risk trade-off in the grey squirrel

Many animals must often respond to environmental patterns that simulatneously influence both foraging efficiency and predation risk. We noted that grey squirrels (Sciurus carolinensis) sometimes immediately consume food items in areas of relatively great exposure to predators, and at other times carry food items to the safety of a tree prior to consumption. We outlined a hypothesis that the squirrels were somehow trading-off energy intake rate against predation risk. A simple model shows that maximal energetic efficiency is associated with immediate consumption, whereas (under the field conditions studied) carrying items to the safety of trees provides for minimal exposure to predation. Our analysis of the model predicts that the tendency to carry a food item should decrease with distance of food from cover (travel time) and increase with item size (handling time). To test our predictions, we presented free-roaming grey squirrels with patches containing a fixed number of identical food items. We estimated the proportion of items carried to trees before consumption for 12 different combinations of distance to the nearest tree and item size. The results support our hypothesis and indicate that a simple behavioural criterion based solely on foraging rate or time exposed to predators is insufficient to explain the variation in the data.     

Nonrandom catabolism of proteins in the formation of antigenic peptide fragments

To examine the role of protein catabolism in the formation of antigenic peptide fragments, human fibrinopeptide-immune guinea pig T cells were stimulated with the large native molecule, human fibrinogen. Two different systems were tested. In the first, we determined responses by human fibrinopeptide B (hFPB)-immune T cells, to which strain (St.) 2 guinea pigs are responders and St. 13 are nonresponders, and by human fibrinopeptide A (hFPA)-immune T cells to which St. 13 are responders and St. 2 are nonresponders. Of interest in this comparison is that both hFPA and hFPB are amino terminal peptides on the A and B chain of fibrinogen, respectively, and are readily cleaved by thrombin during fibrin formation and by other trypsin-like enzymes, leaving a carboxyl terminal Arg. Thus, if fibrinogen catabolism occurred, both antigenic peptides should be equally represented for availability in T cell responses. It was found that hFPB-immune St. 2 T cells responded to fibrinogen, but no response was observed with hPFA-immune St. 13 T cells cultured with fibrinogen. To rule out that there was a general catabolic defect in St. 13 antigen-presenting cells, fibrinogen was presented by (2 X 13)F1 macrophages to fibrinopeptide-immune parental T cells. Again it was found that F1 macrophages could present fibrinogen to hFPB-immune T cells but failed to present hFPA. In another comparison, responses with fibrinogen were also determined with des-ARg-hFPB, which lacks the carboxyl terminal Arg of hFPB, to which St. 13 are responders and St. 2 are nonresponders. The advantage of this comparison is that both antigenic determinants are contained within the same small peptide. St. 13 des-Arg-hFPB-immune T cells failed to respond in vitro by culture with human fibrinogen, suggesting that these antigenic determinants are not produced from larger peptides or proteins containing those determinants. To rule out the possibility that this was only an in vitro phenomenon, guinea pigs were immunized with the larger protein, the B chain of fibrinogen, and the immune T cells were examined for responses to fibrinopeptides derived from the B chain. Immune St. 2 T cells responded to hFPB but not to des-Arg-hFPB, whereas St. 13 T cells remained unresponsive with both peptides. These results indicate that proteolysis of larger proteins to form small antigenic peptides is not a random event and that not all potential antigenic determinants contained in a protein are produced during antigen processing.     

A monoclonal antibody that neutralizes poliovirus by cross-linking virions.

The neutralization of type 1 poliovirus by monoclonal antibody 35-1f4 was studied. The virions were rapidly linked by antibody into oligomers and larger aggregates, followed by slow redistribution of antibody between the immune complexes. The antibody content and infectivity of immune complexes were determined. Remaining single virions were fully infectious and free of antibody. The oligomers and larger aggregates did not significantly contribute to the residual infectivity, which therefore correlated with the number of remaining single virions. Papain digestion of neutralized poliovirus released fully infectious, antibody-free virions from the immune complexes. Anti-immunoglobulin antibodies reneutralized these virions. Polymerization was shown to occur even at virus concentrations of less than 10(3) PFU per ml.     

Seasonality,abundance, and biomass of bacteria in a southwestern reservoir

The seasonality, abundance, and biomass of planktonic bacteria was investigated in a south temperate zone reservoir. Epilimnetic samples were collected periodically throughout 1983 from 5 locations within Lake Arlington, TX. Total bacteria were determined from epifluorescence microscopy and averaged 1.1 × 10¹³ cells m^–3 of water. Planktobacteria accounted for 85% of total cell counts and 73% of total bacterial biomass. Cell volumes were substantially larger in winter than in summer and were negatively correlated with temperature. Cell volumes ranged from 0.076 to 0.330 µm³ and averaged 0.160 µm³. The average biovolume corresponded to a sphere 0.670 µm in diameter. Bacterial biomass was high, averaging 172 mg C m^–3 of water and reached seasonal maximum during winter months. Correlation analysis (simple linear and multiple linear) revealed that approximately 50% of the variation in bacterial biomass could be accounted for by variation in temperature and dissolved organic carbon.     

Characterization of monoclonal antibodies to bromodeoxyuridine

The characteristics of three mouse monoclonal antibodies to halogenated uridine derivatives are presented. Two, IU-1 and IU-2, are produced by hybridomas derived in our laboratory, and the third is the B-44 hybridoma described by Gratzner (7) and obtained commercially from Becton-Dickinson Monoclonal Center. Hybridomas IU-1 and IU-2 were derived from the fusion of spleen cells from a Biozzi High Responder mouse immunized with iododeoxyuridine (IdUrd) conjugated to bovine serum albumin and SP2/0 mouse myeloma cells. This paper presents methods and results for enzyme-linked immunosorbent assays (ELISA) against whole cells labeled with bromodeoxyuridine (BrdUrd), ELISA against BrdUrd-labeled DNA, and a competition ELISA for free BrdUrd. All three antibodies show similar binding affinities and specificities. The IU antibodies react with BrdUrd and IdUrd when the nucleosides are either free in solution or incorporated into single-stranded DNA (ss-DNA). The antibodies do not recognize either halogenated base in double-stranded DNA (ds-DNA), nor do they react with uracil or bromocytidine. Weak binding to thymidine, 5-fluorodeoxyuridine, and unsubstituted ss-DNA occurs.     

Evidence from cDNA clones that the rat leukocyte-common antigen (T200) spans the lipid bilayer and contains a cytoplasmic domain of 80,000 Mr

The leukocyte-common antigen (L-CA or T200) includes a family of lymphoid and myeloid cell surface glycoproteins with apparent molecular weights from 180,000 to 240,000. We report a partial protein sequence for thymocyte L-CA containing 1073 amino acids predicted from cDNA clones isolated using an oligonucleotide probe. Only one segment (residues 347-368) is likely to cross the membrane, and peptide data suggest that sequences N-terminal to this are outside the cell, with residues 369-1073 inside. The cytoplasmic domain includes possible phosphorylation sites and an internal homology between residues 385-671 and 676-986. Analysis of B lymphocyte cDNA clones suggests that B cell and thymocyte mRNAs are identical in 3' sequences, but size differences in Northern blots suggest 5' sequences may differ.