Response of Peyer's patch lymphocyte subsets to Giardia muris infection in BALB/c mice. II. B-cell subsets: enteric antigen exposure is associated with immunoglobulin isotype switching by Peyer's patch B cells

The aim of this study was to quantify the response of Peyer's patch B cells, surface IgA-bearing (sIgA) B cells, and surface IgM-bearing (sIgM) B cells to Giardia muris infection. Following infection of a cohort of immunocompetent BALB/c mice with G. muris cysts, Peyer's patch cell suspensions were prepared at serial time points during the infection, incubated with fluorescein-conjugated monoclonal antibodies directed against murine leukocytes, B cells, sIgA B cells, sIgM B cells, or T cells, and analyzed by flow cytometry. Of total Peyer's patch leukocytes, the percentages of B cells, sIgA B cells, and sIgM B cells in uninfected BALB/c mice were 64.7 +/- 2.0% (mean +/- SEM), 30.3 +/- 1.5%, and 52.5 +/- 2.4%, respectively. The total number of Peyer's patch leukocytes increased significantly (1.8 X) during G. muris infection, and returned to control levels as the infection was cleared. The percentages of Peyer's patch T and total B cells did not change significantly during Giardia infection. However, sequential changes were observed in the percentages and numbers of sIgM and sIgA B cells during the infection. Peyer's patch sIgM B cells rapidly increased in percentage and number, reaching maximum levels 1 week after cyst inoculation. After remaining constant the first week, the number of Peyer's patch sIgA B cells increased during the second week of G. muris infection, reaching a maximum level 11-14 days after cyst inoculation. The data support the hypothesis that immunoglobulin isotype switching in Peyer's patches is induced by antigen exposure.     

Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity

Intestinal absorptive cells may modulate both the structure and function of occluding junctions by a cytoskeleton dependent mechanism (Madara, J. L., 1983, J. Cell Biol., 97:125-136). To further examine the putative relationship between absorptive cell occluding junctions and the cytoskeleton, we assessed the effects of cytochalasin D (CD) on occluding junction function and structure in guinea pig ileum using ultrastructural and Ussing chamber techniques. Maximal decrements in transepithelial resistance and junctional charge selectivity were obtained with 10 micrograms/ml CD and the dose-response curves for these two functional parameters were highly similar. Analysis of simultaneous flux studies of sodium and the nonabsorbable extracellular tracer mannitol suggested that CD opened a transjunctional shunt and that this shunt could fully account for the increase in sodium permeability and thus the decrease in resistance. Structural studies including electron microscopy of detergent-extracted cytoskeletal preparations revealed that 10 micrograms/ml CD produced condensation of filamentous elements of the peri-junctional contractile ring and that this was associated with brush border contraction as assessed by scanning electron microscopy. Quantitative freeze-fracture studies revealed marked aberrations in absorptive cell occluding junction structure including diminished strand number, reduced strand-strand cross-linking, and failure of strands to impede the movement of intramembrane particles across them. In aggregate these studies show that CD-induced perturbation of the absorptive cell cytoskeleton results in production of a transepithelial shunt which is fully explained by a defect in the transjunctional pathway. Furthermore, substantial structural abnormalities in occluding junction structure accompany this response. Lastly, the abnormalities in occluding junction structure and function coincide with structural changes in and contraction of the peri-junctional actin-myosin ring. These data suggest that a functionally relevant association may exist between the cytoskeleton and the occluding junction of absorptive cells. We speculate that such an association may serve as a mechanism by which absorptive cells regulate paracellular transport.     

A Method for Combined Gross Skeletal Staining and Feulgen Staining of Embryonic Chick Tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

Established cell lines from nonmammalian vertebrates: Models for DNA repair studies

Summary Several established cell lines from different classes of vertebrates were assayed for the presence of O⁶-methylguanine acceptor protein. This protein is instrumental in removing adducts from DNA caused by exposure to alkylating agents. Cultured cells had levels of acceptor protein activity within the range found in fresh tissues from animals in the same class. We suggest that cells from lower vertebrates are satisfactory in vitro models for studies of this DNA repair function.     

Inability of Pseudomonas stutzeri denitrification mutants with the phenotype of Pseudomonas aeruginosa to grow in nitrous oxide.

Pseudomonas aeruginosa PAO1 reduced nitrous oxide to dinitrogen but did not grow anaerobically in nitrous oxide. Two transposon insertion Nos- mutants of Pseudomonas stutzeri exhibited the P. aeruginosa phenotype. Growth yield studies demonstrated that nitrous oxide produced in vivo was productively respired, but nitrous oxide supplied exogenously was not. The defect may be in electron transport or in nitrous oxide uptake.     

Adenosine 5'-diphosphate as an allosteric effector of phosphorylase kinase from rabbit skeletal muscle

Equilibrium binding and activity studies indicate that adenosine 5'-diphosphate binds to phosphorylase kinase with high affinity at a site, or sites, distinct from the catalytic site. Equilibrium dialysis at pH 6.8 and 8.2, with and without Mg2+, and with phosphorylated and nonphosphorylated enzyme preparations revealed approximately 8 ADP binding sites per alpha 4 beta 4 gamma 4 delta 4 hexadecamer, with Kd values ranging from 0.26 to 17 microM. Decreasing the pH from 8.2 to 6.8 or removing the Mg2+ enhanced the affinity for ADP. At pH 6.8, ADP stimulated the phosphorylase conversion and autophosphorylation activities of the nonactivated enzyme. Analogs of ADP with modifications at the 2'-, 3'-, and 5'-positions allowed determination of structural requirements for the stimulation of activity. ADP seems to alter the conformation of the beta subunit because addition of the nucleotide inhibits its dephosphorylation by phosphoprotein phosphatase and its chemical cross-linking by 1,5-difluoro-2,4-dinitrobenzene. The binding affinities and effects of ADP suggest that it may function physiologically as an allosteric effector of phosphorylase kinase.     

ESCHERICHIA COLI Rho Factor Is Involved in Lysis of Bacteriophage T4-Infected Cells

A Rid (Rho interaction deficient) phenotype of bacteriophage T4 mutants was defined by cold-sensitive restriction (lack of plaque formation) on rho+ hosts carrying additional polar mutations in unrelated genes, coupled to suppression (plaque formation) in otherwise isogenic strains carrying either a polarity-suppressing rho or a multicopy plasmid expressing the rho+ allele. This suggests that the restriction may be due to lower levels of Rho than what is available to T4 in the suppressing strains.--Rid394 X 4 was isolated upon hydroxylamine mutagenesis and mapped in the t gene; other t mutants (and mot, as well as dda dexA double mutants) also showed a Rid phenotype. In liquid culture in strains that restricted plaque formation Rid394 X 4 showed strong lysis inhibition (a known t- phenotype) but no prolonged phage production (another well-known t- phenotype). This implies that when Rho is limiting the t mutant shuts off phage production at the normal time. Lysis inhibition was partially relieved, and phage production prolonged to varying extents depending on growth conditions in strains that allowed plaque formation. No significant effect on early gene expression were found. Apparently, both mutant (polarity-suppressing) and wild-type Rho can function in prolonging phage production and partially relieving lysis inhibition of Rid394 X 4 when present at a sufficiently high level, and Rho may play other role(s) in T4 development than in early gene regulation.     

Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum.

We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe. The rhizobial glnA gene has homology to the E. coli glnA gene throughout the entire length of the gene and can complement an E. coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E. coli lac promoter. High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E. coli. The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation. DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B. japonicum, E. coli, and Anabaena sp. strain 7120 glutamine synthetases. S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 +/- 2 base pairs upstream from the initiation codon. This glnA promoter is active when B. japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources. There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nif promoters of the same B. japonicum strain.