1H-15N heteronuclear NMR studies of Escherichia coli thioredoxin in samples isotopically labeled by residue type

Ten samples of Escherichia coli thioredoxin were individually isotopically enriched by residue type via growth of an appropriate auxotrophic strain on media supplemented with one 2H, 15N-enriched amino acid. 1H observe-heteronuclear decoupling experiments were conducted on these samples making use of the 95-Hz 1H-15N amide J1 coupling. Subtraction of near-resonance from off-resonance 15N decoupled spectra generated difference patterns corresponding only to protons directly bonded to 15N nuclei. For the ten different enriched residue types observed to date, every labeled position (60) has been observed as a resolved resonance. The spectral dispersion in both the 1H and the 15N dimensions was roughly 1500 Hz (at 500-MHz field strength) with rather little apparent dependence on residue type. With the exception of the glycine-enriched sample, the range of the J1 coupling constants was not much greater than the precision of the measurements (1.5-2.0 Hz). However, for the glycine residues the J1 amide coupling values varied over a range of 10 Hz.     

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.     

Transport of chlorine in the proximal tubule. Its effects on water-electrolyte absorption

Several studies in rat kidney have established that an appreciable fraction of proximal absorption is passive in nature and occurs across the highly conductive paracellular pathway. Passive absorption is generally ascribed to the transepithelial Cl- distribution, luminal Cl- activity (alpha lCl) being higher than plasma Cl- activity (alpha pCl). The inequality alpha lCl greater than alpha pCl generates a transepithelial diffusion potential, lumen positive, which taken together with the chemical potential differences of Cl- and Na+ across the epithelium gives rise to transepithelial electrochemical potential differences for Cl- and Na+ favoring their absorption. The alpha lCl greater than alpha pCl distribution is traditionally ascribed to preferential bicarbonate absorption. We argue that HCO3- absorption alone cannot generate a non equilibrium transepithelial Cl- distribution. Other mechanisms are necessary. Our measurements in amphibian proximal tubule demonstrate that the intracellular Cl- activity, alpha cCl, is higher than the theoretical value predicted for equilibrium. This distribution is the result of two basolateral coupled transport processes (Cl-/HCO3- exchange and Cl-/Na+ cotransport). It contributes to the exit of Cl- from cell to lumen (by passive diffusion and K+/Cl- cotransport), yielding alpha lCl values higher than the theoretical value for equilibrium with regard to plasma. Thus, a small transcellular flux of Cl- (without solvent) proceeds from interstitium to lumen. It compensates the dissipative tendency of a much higher paracellular Cl- absorptive flux (in association with water) on the transepithelial Cl- gradient. The result is a steady-state luminal Cl- distribution above equilibrium, along the major part of the proximal tubule.