Structural and functional differentiation of two clinally distributed glucosephosphate isomerase allelic isozymes from the teleost Fundulus heteroclitus

The teleost Fundulus heteroclitus (L.) possesses two loci, Gpi-A and Gpi-B, for the glycolytic enzyme, glucose-phosphate isomerase (GPI; D- glucose-6-phosphate ketol-isomerase; E.C. 5.3.1.9). The Gpi-B locus is polymorphic in Fundulus, with two common alleles, Gpi-Bb and Gpi-Bc, distributed in a clinal manner in populations along the east coast of North America. Since this clinal distribution is strongly correlated with a temperature gradient, we asked whether the GPI-B2 allozymes were functionally adapted to the thermal environment in which a given phenotype predominated. The two major GPI-B2 allozymes were purified to homogeneity and were characterized as to molecular weight, isoelectric pH, thermal denaturation, and kinetic parameters. Both GPI-Bb2 and GPI- Bc2 allozymes have molecular masses of 110 kD, and they have isoelectric pHs of 6.4 and 6.6, respectively. The GPI-Bb2 allozyme was more stable to thermal denaturation than was the GPI-Bc2 enzyme. Kinetic properties of the allelic isozymes were investigated both as a function of pH and as a function of temperature. At 25 degrees C, over the pH range considered, there were no significant differences between allozymes, either in Km for fructose-6-phosphate or in Ki for 6- phosphogluconate, but apparent Vmax values differed between pH 7.5 and pH 8.5. All steady-state kinetic parameters showed strong temperature dependence, but the allozymes differed only in the Ki for 6- phosphogluconate at temperatures greater than 30 degrees C. On the basis of the observed structural and functional differences alluded to above, the hypothesis that the major allelic isozymes of the Gpi-B locus were functionally equivalent was rejected. However, it is not yet known whether these structural and functional differences have any significance at higher levels of biological organization.      

Coaggregation of urogenital bacteria in vitro and in vivo

The working hypotheses of the present study were that (1) bacterial coaggregates exist in the urogenital tract of healthy and infected women, and (2) coaggregation reactions can occur in vitro between members of the urogenital flora. Examination of urogenital specimens from 25 healthy women showed that lactobacilli were the dominant organisms colonizing the epithelia and coaggregating with other Gram-positive and Gram-negative bacteria. In vitro light and electron microscopic studies confirmed that members of the urogenital flora could coaggregate. An examination of specimens from 9 women with urinary tract infection showed the presence of autoaggregated uropathogens free-floating in the urine and attached to epithelial cells. The phenomenon of autoaggregation was also noted in vitro for various uropathogens, suggestive that this may represent a virulence factor. It is evident that bacterial cell-to-cell binding within a strain and among different genera occurs in the urogenital tract. Further studies of the mechanisms that maintain and disrupt these microbial interactions will help to improve our understanding of disease initiation.     

Suppression of polyclonal antibody production in Trypanosoma cruzi-infected mice by treatment with anti-L3T4 antibodies

Acute Trypanosoma cruzi infection of mice results in a very marked polyclonal activation of B and T lymphocytes, accompanied by high numbers of Ig-secreting PFC and lectin-dependent effector CTL. Treatment of mice with monoclonal anti-L3T4 antibodies from the time of infection (days 0, 4, and 8) completely suppresses the polyclonal PFC response and CTL generation. Treatment of nude mice with antibody does not alter the lipopolysaccharide-induced polyclonal PFC response, and it only modulates the isotypic profile of the PFC response to T. cruzi infection, without reducing its magnitude. Furthermore, antibody-treated, T. cruzi-infected euthymic mice do not develop the typical B cell blastogenic response, but show high numbers of activated Lyt-2+ lymphoblasts in the spleen. These results indicate that effector cell generation in T. cruzi-infected mice is predominantly helper T cell-dependent.     

Persistence of polyclonal B cell activation with undetectable parasitemia in late stages of experimental Chagas' disease

The polyclonal B cell responses induced by Trypanosoma cruzi infection last for at least 6 mo after the inoculation of the parasites. In the acute phase of the disease, B cells from spleen and lymph nodes are largely stimulated, whereas a decrease in bone marrow PFC is observed. As the disease progresses, the numbers of Ig-secreting cells in the spleen, lymph nodes, and bone marrow are all enhanced. The isotype distribution of PFC, however, remains unvariable along the course of the infection, and it is characterized by the predominance of IgG2a- and IgG2b-secreting cells. No striking difference in the isotype pattern of resistant and susceptible strains of mice was observed. The continuous and long-lasting B cell stimulation generated during the infection may have important consequences in the pathology of Chagas' disease.     

Cell cycle regulation of histone H1O in CHO cells: a flow cytofluorimetric study after double staining of the cells

The regulation of histone H1O content throughout the cell cycle of non-synchronized Chinese hamster ovary (CHO) cells has been studied using double fluorescent staining and flow cytofluorometry. In exponentially growing cells, the amount of H1O was found to be proportional to the DNA content of the cells, indicating that the protein is synthesized throughout the cell cycle. However, when cells were arrested in G1 at saturation density the amount of H1O was greater than that found in G1 cells of the exponentially growing population. In contrast, the levels of H1-1 were the same for G1 cells of both populations. These results show that the regulation of H1O accumulation differs from that of other histones.     

Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells.

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.     

DNA rearrangements and antigenic variation in Trypanosoma equiperdum: multiple expression-linked sites in independent isolates of trypanosomes expressing the same antigen.

African trypanosomes resist the immune response of their mammalian hosts by varying the surface glycoprotein which constitutes their antigenic identity. The molecular mechanism of this antigenic variation involves the successive activation of a series of genes which code for different variant surface glycoproteins (VSGs). We have studied the expression of two VSG genes (those of VSG-1 and VSG-28) in Trypanosoma equiperdum, and we report the following findings. (i) The expression of both VSG genes is associated with the duplication and transposition of corresponding basic copy genes. (ii) The duplicated transposed copy appears to be the expressed copy. (iii) Although there are multiple genes which cross-hybridize with the VSG-1 cDNA probe, only one of these appears to be used as a template for the expression-linked copy in four independent BoTat-1 clones. (iv) Analysis of the genomic environments of the expressed VSG-1 genes from each of four independently derived BoTat-1 trypanosome clones revealed that there are at least three different sites into which the expression-linked copy can be inserted.