Kinetic analysis of an E.coli phenylalanine-tRNA synthetase mutant.

A mutation in the pheS gene, encoding phenylalanyl-tRNA synthetase, in E. coli NP37 confers temperature-sensitivity on the organism. A five-fold increase in tRNA(phe) levels complements the mutation. Analysis of the kinetic properties of the mutant enzyme indicates that the KM is 20-fold higher than the wild-type and the dissociation constant of the tRNA(phe)-synthetase complex for the mutant is at least 10-fold higher. These results indicate that the mutation in E. coli NP37 directly affects the tRNA(phe) binding site on the cognate synthetase.     

The fidelity of base selection by the polymerase subunit of DNA polymerase III holoenzyme.

In common with other DNA polymerases, DNA polymerase III holoenzyme of E. coli selects the biologically correct base pair with remarkable accuracy. DNA polymerase III is particularly useful for mechanistic studies because the polymerase and editing activities reside on separate subunits. To investigate the biochemical mechanism for base insertion fidelity, we have used a gel electrophoresis assay to measure kinetic parameters for the incorporation of correct and incorrect nucleotides by the polymerase (alpha) subunit of DNA polymerase III. As judged by this assay, base selection contributes a factor of roughly 10(4)-10(5) to the overall fidelity of genome duplication. The accuracy of base selection is determined mainly by the differential KM of the enzyme for correct vs. incorrect deoxynucleoside triphosphate. The misinsertion of G opposite template A is relatively efficient, comparable to that found for G opposite T. Based on a variety of other work, the G:A pair may require a special correction mechanism, possibly because of a syn-anti pairing approximating Watson-Crick geometry. We suggest that precise recognition of the equivalent geometry of the Watson-Crick base pairs may be the most critical feature for base selection.     

Autologous antibodies to human bladder cancer

Summary The autologous serologic reactivity of 13 patients with bladder cancer was evaluated using cell lines derived from each individual's own tumor as targets. Protein A and immune adherence assays were employed to determine antibody binding to the tumor targets at varying passage numbers. Autologous reactivity was found in 6 of the 13 cell lines tested. However, the titer was usually low regardless of the passage number. Seven autologous serum/cell line combinations were tested using both low and high passage cells as targets. In six of these combinations, the degree of antibody binding was similar with both low and high passage target cells. The incidence of autologous reactivity in the 12 patients with urothelial tumors was 50%.This investigation was supported by PHS Grant number CA36933, awarded by the National Cancer Institute, DHHS.     

Lack of a relationship between immune function and chemically induced hepatocarcinogenesis in B6C3F1 mice

Summary The relationship between immune function and chemically induced hepatocarcinogenesis was studied employing an in vivo murine model. Neonatal B6C3F₁ mice were given a single carcinogenic dose of diethylnitrosamine (DEN) and the time-response kinetics for the early (foci of alteration) and late (adenomas/carcinomas) phases of hepatocellular carcinogenesis were compared to changes in hematopoiesis and immune functions associated with immune surveillance and natural resistance. Increases in hematopoiesis occurred just prior to or concurrent with the appearance of hepatocellular carcinomas, while increased macrophage and natural killer cell cytotoxicity and suppression of cell-mediated immunity occurred following tumor appearance and progressed with increasing tumor burden. Neither immunological nor hematopoietic changes were associated with early phases of hepatocarcinogenesis, as monitored by the appearance of altered hepatocellular foci. Although changes in hematopoiesis may represent an early indicator for hepatocarcinogenesis in the mouse tumor model, the data suggest that altered immune surveillance and natural resistance are not factors in the development of chemically induced hepatocellular tumors, and the changes in immune function are probably secondary to tumor development.     

Antibody directed targeting of methotrexate-containing small unilamellar vesicles

Summary The potential of antibody-linked SUVs containing MTX in anticancer therapy was investigated. The SUVs, mean diameter 50±20 nm, were prepared by probe sonication of MTX-containing MLVs and were covalently linked either to a RAMG or NRG. After incubation with M21 melanoma cells for 2 h, RAMG-linked SUVs showed 2 and 4 times more binding than NRG-linked MTX-containing SUVs or MTX-containing SUVs unlinked to any Ig. Furthermore, on incubating M21 melanoma cells with RAMG-linked ³H MTX-containing SUVs for 2, 4, and 8 h at 4° C or 37° C, a higher radioactivity was associated with cells at 37° C than at 4° C. Membrane immunofluorescence revealed aggregation of and cap formation by RAMG-linked SUVs after 2 h (37° C) and endocytosis at 4 and 8 h at 37° C. Electron microscopic and autoradiographic studies confirmed aggregation of ³H MTX-containing SUVs around and on the surface of M21 cells. Electron microscopy also revealed these SUVs inside invaginations of and under the plasma membrane of melanoma cells. A colony inhibition assay showed that RAMG-linked, MTX-containing SUVs were 60 times, 8 times, and 4.5 times more growth inhibitory than free MTX, NRG-linked MTX-containing SUV, and MTX-containing SUVs unlinked to any Ig, but not toxic to a human kidney cancer line (that did not react with RAMG). Abbreviations used: DPPC, DL- -dipalmitoyl phosphatidylcholine; DTT, dithiothreitol; MTX, methotrexate; (MTX)SUV or MLV, MTX-containing SUV or MLV; MLV, multilamellar vesicle; NRG, normal rabbit immunoglobulin G; RAMG, rabbit antimelanoma IgG; SA, stearylamine; SPDP, N-succinimidy1-3-(2-pyridyldithio)propionate; SUV, small unilamellar vesicle; CHOL, cholesterol; LUV, large unilamellar vesicle; Ig, immunoglobulin; PDP-SA, N-[3-(2-pyridyldithio)-propinyl]stearylamine     

Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drosophila

The fasciclin I, II, and III glycoproteins are expressed on different subsets of axon bundles (fascicles) in insect embryos and are thus candidates for surface recognition molecules involved in growth cone guidance. Here we present the sequence of grasshopper fasciclin I and the identification and sequence of the Drosophila fasciclin I homolog. In both species, fasciclin I appears to be an extrinsic membrane protein with a signal sequence but no transmembrane region; the protein comprises four homologous domains of approximately 150 amino acids each. Antibodies against Drosophila fasciclin I reveal that it is expressed on the surface of a subset of commissural axon pathways in the embryonic central nervous system and on all sensory axon pathways in the peripheral nervous system. This pattern of expression is similar to that in grasshopper.     

Effects of Cellulolytic Ruminal Bacteria and of Cell Extracts on Germination of Euonymus americanus L. Seeds

In past attempts, the experimental germination of the seeds of Euonymus americanus L. in vitro has had little success. However, treatment of seeds with ruminal fluid containing viable microflora has been successful in stimulating germination. In the presence of the cellulolytic ruminal bacterium, Clostridium cellobioparum ATCC 15832, seeds of E. americanus were stimulated to germinate. Subsequent studies were designed to determine whether the bacterium synthesized a cellulolytic enzyme responsible for initiating germination. The cell-free endocellulase from C. cellobioparum induced germination of the seeds. To support the hypothesis that the endocellulase from C. cellobioparum was responsible for triggering germination, a 1,4-beta-d-glucan glucanohydrolase (EC 3.2.1.4) from Penicillum funiculosum was used to treat the seeds. In addition, no germination was obtained from seeds treated with a commercial exocellulase enzyme. Also, Ruminococcus flavefaciens FD-1 was found to initiate germination of E. americanus seeds. Thus, cellulase activity is indicated in the degradation of the testa of the seed, allowing imbibition and germination.     

Diversity of the mouse T cell receptor Cγ1 gene: structural analysis in C57BL/Ka

We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor.     

Effects of temperature and moisture during incubation on carcass composition of hatchling snapping turtles (Chelydra serpentina)

Summary Flexible-shelled eggs of common snapping turtles (Chelydra serpentina) were incubated on each of two substrates (vermiculite, sand) at each of three temperatures (26.0°C, 28.5°C, 31.0°C) and three moisture regimes (wet, intermediate, dry). Embryos developing in cool, wet environments mobilized the largest amounts of protein from their yolk and attained the largest size before hatching, whereas turtles developing in warm, dry environments mobilized the smallest quantities of protein and were the smallest in body size at hatching. Embryos on wet substrates mobilized more lipid from their yolk than did embryos on dry media, but ambient temperature had no demonstrable influence on patterns of lipid mobilization. The total reserve of neutral lipid available in residual yolk plus carcass to sustain neonates in the interval prior to the beginning of feeding was largest in hatchlings from dry environments and smallest in animals from wet environments, but was unaffected by temperature during incubation. Hydration of tissues in hatchlings was higher when incubation was in cool, moist conditions than when incubation was in warm, dry settings, thereby indicating that some of the effects of moisture and temperature on mobilization of nutrients by embryos may be mediated by differences in intracellular water. Patterns of response to temperature and moisture recorded for turtles emerging from eggs on sand were similar to those recorded for hatchlings on vermiculite, so no important conclusion would have been affected by incubating eggs on one medium instead of the other.     

Differential amplification of antifreeze protein genes in the pleuronectinae

Summary The organization of antifreeze protein (AFP) genes in the yellowtail flounder was investigated by Southern blotting and the characterization of clones from a genomic library. This flounder, like the closely related winter flounder, has a set of 10–12 linked but irregularly spaced AFP genes. However, it lacks the tandemly amplified set of 20 such genes that are present in the winter flounder. DNA sequence analysis of a tandemly repeated gene from winter flounder showed that it can code for one of the two most abundant AFP components in the serum. Consistent with this higher AFP gene dosage, the peak serum AFP level in midwinter was 9 mg/ml in the winter flounder and only 4 mg/ml in the yellowtail flounder. A recent amplification of the AFP gene in the winter flounder lineage might be responsible for the higher serum AFP levels in this fish. This increase in gene dosage might have helped the winter flounder colonize the ice-laden, shallow-water niche that it currently occupies along the east coast of North America. Genomic Southern blotting of two other righteye flounders, the smooth flounder and the American plaice, illustrates another example of a differential amplification of AFP genes that correlates with a species' exposure to ice.