Catalytic function of a tyrosyl residue in tryptophanase

Tryptophanase has an essential tyrosyl residue/active site which can be modified by tetranitromethane. Pyridoxal 5'-phosphate can prevent this modification efficiently, whereas pyridoxal 5'-phosphate N-oxide cannot, indicating that the free pyridinium N is required for the interaction of the coenzyme with the tyrosyl residue, probably via a hydrogen bond. The weakened binding of the coenzyme to the modified enzyme was confirmed on gel filtration, the modified enzyme being dissociated from the coenzyme seven-fold faster than the native enzyme. Furthermore, absorption spectral analyses demonstrated that the modified enzyme can catalyze the transaldimination step, but fails to abstract the alpha-H of substrates. The tyrosyl residue, therefore, not only participates in coenzyme binding, but also contributes to alpha-H labilization.     

The APC/C activator Cdh1 regulates the G2/M transition during differentiation of placental trophoblast stem cells

Differentiation of placental trophoblast stem (TS) cells to trophoblast giant (TG) cells is accompanied by transition from a mitotic cell cycle to an endocycle. Here, we report that Cdh1, a regulator of the anaphase-promoting complex/cyclosome (APC/C), negatively regulates mitotic entry upon the mitotic/endocycle transition. TS cells derived from homozygous Cdh1 gene-trapped (Cdh1^GT/GT) murine embryos accumulated mitotic cyclins and precociously entered mitosis after induction of TS cell differentiation, indicating that Cdh1 is required for the switch from mitosis to the endocycle. Furthermore, the Cdh1^GT/GT TS cells and placenta showed aberrant expression of placental differentiation markers. These data highlight an important role of Cdh1 in the G2/M transition during placental differentiation.     

Gene-specific transposon mutagenesis of the biphenyl/polychlorinated biphenyl-degradation-controlling bph operon in soil bacteria.

A transposon, Tn5-B21, was gene-specifically inserted into the chromosomal biphenyl/polychlorinated biphenyl-catabolic operon (bph operon) of soil bacteria. The cloned bphA, bphB and bphC genes of Pseudomonas pseudoalcaligenes KF707, coding for conversion of biphenyl into a ring meta-cleavage product (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid), carried random insertions of Tn5-B21. The mutagenized bphABC DNA, carried by a suicide plasmid, was introduced back into the parent strain KF707, resulting in the appearance of gene-specific transposon mutants by double crossover homologous recombination: the bphA::Tn5-B21 mutant did not attack 4-chlorobiphenyl, the bphB::Tn5-B21 mutant accumulated dihydrodiol, and the bphC::Tn5-B21 mutant produced dihydroxy compound. Gene-specific transposon mutants of the bph operon were also obtained for some other biphenyl-utilizing strains which possess bph operons nearly identical to that of KF707.     

Secretion and role of interleukin-1-like factor in the antibody response to dinitrophenyl dextran and other type 2 T-independent antigens

Splenic adherent cells (SAC) were found to produce a humoral factor when they were cultured with dinitrophenyl-dextran or some other type 2 T-independent (TI-2) antigens. The factor substituted adherent cells in in vitro antibody responses to TI-2 antigens, and acted in an antigen-nonspecific and H-2-nonrestricted manner. T cells were indicated not to participate in the production of the factor. The factor was eluted from a Sephadex G-75 column with interleukin-1 (IL-1) activity to promote a thymocyte proliferation response to phytohemagglutinin. The molecular weight of the factor was estimated to be 16,000 Da. Both activities of the factor were absorbed by LBRM-33-1A5 cells. These results indicate that SAC secrete IL-1-like factor on direct stimulation by TI-2 antigens and that the secretion of the factor represents a major function of SAC in the antibody response to these antigens.     

B cells as antigen-presenting cells: antibody production in vitro against a T-dependent antigen

It was examined whether B cells can serve as antigen-presenting cells (APC) in the antibody response to a T-dependent antigen, trinitrophenyl-ovalbumin (TNP-OVA). B cells purified from mice primed with TNP (TNP-B cells) responded to TNP-OVA in the presence of purified T cells sensitized with OVA (OVA-T cells). OVA-T cells required the addition of APC to proliferate in response to TNP-OVA. APC activity of TNP-B cells in the T-cell proliferation was abolished by 4000 R irradiation. Our experiments also revealed that an antibody response requires more adherent cells than the T-cell proliferation. These results indicate that adherent cells possibly accompanying the T- and B-cell preparations were at a less than functional level. There was genetic restriction between T and B cells for the antibody response. B cells in the pellet fraction of 70% Percoll density sedimentation behaved similarly to the unfractionated TNP-B cells in the antibody response. A T-cell clone specific for human gamma-globulin (HGG) also induced an anti-TNP antibody response in B cells from unprimed mice in the presence of TNP-HGG. These results suggest that B cells are able to elicit an antibody response to a T-dependent antigen in the presence of carrier-primed T cells without the participation of macrophages.     

Optimum conditions for ursodeoxycholic acid production from lithocholic acid by Fusarium equiseti M41.

Ursodeoxycholic acid dissolves cholesterol gallstones in humans. In the present study optimum conditions for ursodeoxycholic acid production by Fusarium equiseti M41 were studied. Resting mycelia of F. equiseti M41 showed maximum conversion at 28 degrees C, pH 8.0, and dissolved oxygen tension of higher than 60% saturation. Monovalent cations, such as Na+, K+, and Rb+, stimulated the conversion rate more than twofold. In the presence of 0.5 M KCl, the initial uptake rate and equilibrium concentration of lithocholic acid (substrate) were enhanced by 5.7- and 1.7-fold, respectively. We confirmed that enzyme activity catalyzing 7 beta-hydroxylation of lithocholic acid was induced by substrate lithocholic acid. The activity in the mycelium was controlled by dissolved oxygen tension during cultivation: with a dissolved oxygen tension of 15% and over, the activity peak appeared at 25 h of cultivation, whereas the peak was delayed to 34 and 50 h with 5 and 0% dissolved oxygen tension, respectively. After reaching the maximum, the 7 beta-hydroxylation activity in the mycelium declined rapidly at pH 7.0, but the decline was retarded by increasing the pH to 8.0. Several combinations of operations, such as pH shift (from pH 7 to 8), addition of 0.5 M KCl, and dissolved oxygen control, were applied to the production of ursodeoxycholic acid in a jar fermentor, and a much larger amount of ursodeoxycholic acid (1.2 g/liter) was produced within 96 h of cultivation.     

Alloantigen presentation by B cells: two types of alloreactive T cell hybridomas, B cell-reactive and B cell-nonreactive

T cell-depleted, Sephadex G-10-passed unstimulated splenic B cells from C57BL/6 mice stimulated splenic T cells from CKB mice to produce IL 2 and to proliferate. The stimulatory ability of the unstimulated B cells was eliminated by 4000 rad irradiation of the unstimulated stimulator B cells. LPS-activated B cells could stimulate responder T cells more efficiently than unstimulated B cells. For further analysis of allostimulation by B cells, we established a series of alloreactive T cell hybridomas. Forty-five percent of these alloreactive T cell hybridomas could be stimulated to produce IL 2 by either macrophage-dendritic cells or unstimulated B cells. Fifty-five percent of these alloreactive T cell hybridomas could be stimulated by macrophage-dendritic cells but not by unstimulated B cells. T cell hybridomas that were not reactive with unstimulated B cells were also nonreactive to LPS-activated B cells. Analysis of two representative I-Ab-reactive T cell hybridoma clones, B cell-reactive clone CB-11.4 and B cell-nonreactive clone HTB-9.3, revealed again that the stimulatory ability of unstimulated B cells was sensitive to 4000 rad irradiation in the activation of CB-11.4 clone and that CB-11.4 could be stimulated more efficiently by LPS-activated B cells than by unstimulated B cells, but HTB-9.3 could not be stimulated by LPS-activated B cells. Thus, there may be two distinct types of T cells in the alloreaction: B-cell-reactive and B cell-nonreactive.     

Genetic analysis of three additional fla genes in Salmonella typhimurium

In Salmonella typhimurium, 27 fla genes responsible for formation of flagella have been identified and assigned to three regions on the genetic map, termed fla regions I to III. By genetic analysis of 1984 non-flagellate mutants obtained from a phase-1 stable strain of S. typhimurium, SJW1103, three additional fla genes were identified; one, termed flaW, was assigned to fla region I and the other two, termed flaV and flaX, to fla region III. By intergeneric complementation tests, the flaW, flaV and flaX genes were shown to be functionally homologous with flaS, flbC and flaP of Escherichia coli, respectively. Electron microscopy showed that flaW and flaV mutants carried hook-basal body structures.     

Genetic analysis of H2, the structural gene for phase-2 flagellin in Salmonella

Non-flagellate H2 mutants were isolated from a phase-2 stable strain, SJW806 H1-gt- H2-enxon vh2-, a derivative of Salmonella typhimurium. By transductional crosses a deletion map and a recombination map of the H2 gene were made. There are three regions especially rich in nonflagellate mutational sites. By the use of the deletion map, mutational sites of 21 flagellar shape mutants were also determined. Most of them were located at two regions which coincide with two of the three regions rich in non-flagellate mutational sites. A gene, vh2, is closely linked to the promoter side of the H2 gene. Three-factor transductional crosses showed that the vh2 gene was on the left of the H2 gene in the present map. The H2 gene forms part of an operon with the distal gene rh1 which specifies the H1 repressor. Thus, a polarity effect of the H2 mutations on the expression of the rh1 gene was examined by observing whether a wild-type H1 allele introduced into the H2 mutants was expressed or not. Many of the H2 mutations were polar, and most of the strongly polar mutations were located in the left (promoter-proximal) half of the H2 gene, while most of the mutations in the right half of the gene were weakly polar or non-polar.