Random replication and random assortment model for plasmid incompatibility in bacteria

To understand the incompatibility between two related plasmids, both of which replicate in an autonomous state under a common control mechanism, we have developed a model that assumes a random choice mechanism for replication of plasmid copies and their random assortment into daughter cells upon cell division. Segregation kinetics by this model is analyzed mathematically and the number of generations required for segregation is calculated as a function of plasmid copy number per cell. The results obtained offer enough quantitative data to make our model reasonably realistic.     

The role of virulence antigens (VW) in the protection of mice againstYersinia pestis infection

Subcutaneous infection withYersinia enterocolitica harboring plasmid responsible for Ca²⁺ dependence at 37°C induced cell-mediated protective immunity against a lethal challenge withYersinia pestis; the isogenic derivative strain cured from this plasmid subverted the immunity in mice. This is the first identification of the antigen(s) responsible for the induction of cell-mediated protective immunity against the facultatively intracellular bacteria.     

Requirement of the core structure of a complex-type glycopeptide for the binding to immobilized lentil- and pea-lectins

Structural requirements for the binding of oligosaccharides and glycopeptides to immobilized lentil- and pea-lectins were investigated by use of radioactively-labeled glycopeptides and oligosaccharides. The results indicate that an intact 2- acetamido-2-deoxy-β-d-glucopyranosyl residue at the reducing end of a complex-type oligosaccharide is essential for high-affinity binding to lentil lectin-Sepharose but not to concanavalin A-Sepharose and that an asparagine residue is required for the binding of a complex-type glycopeptide to pea lectin-Sepharose. In addition, interaction of a complex-type oligosaccharide with lentil lectin-Sepharose was enhanced by exposure of nonreducing, terminal 2-acetamido-2-deoxy-β-d-glucopyranosyl groups, whereas interaction with pea lectin-Sepharose was enhanced only after exposure of nonreducing, terminal α-d-mannopyranosyl groups.     

Role of glycosaminoglycans in the regulation of T cell proliferation induced by thymic stroma-derived T cell growth factor

The present study investigates the regulatory effects of glycosaminoglycans such as heparin and heparan sulfate on T cell proliferation induced by thymic stromal cell monolayer or its derived T cell growth factor (TCGF). A thymic stromal cell clone (MRL104.8a) supported the growth of Ag-specific, IL-2-dependent Th cell clone (9-16) in the absence of Ag and IL-2 by producing a unique TCGF designated as thymic stroma-derived T cell growth factor (TSTGF). The addition of heparin to cultures in which the growth of 9-16 Th cells was otherwise stimulated by the MRL104.8a monolayer or a semipurified sample of the TSTGF resulted in heparin dose-dependent inhibition of 9-16 Th proliferation. The dose of heparin required for inducing 50% reduction of TSTGF-induced proliferation of Th at a given cell number was found to be proportional to the magnitude of the TSTGF added to cultures, suggesting that heparin exerted its inhibitory effect by binding to the TSTGF rather than by acting on Th cells. A similar growth-inhibiting effect of heparin was observed in IL-7-dependent proliferation of pre-B cell line or Th, but not in IL-2-dependent T cell proliferation or IL-3-dependent myeloid cell proliferation. A strong affinity of TSTGF and IL-7 for heparin was confirmed by the fact that both TSTGF and IL-7 adhered to columns of heparin-agarose and were eluted by salt. When various glycosaminoglycans were tested for the heparin-like Th growth-regulatory capacity, heparan sulfate exhibited Th growth-inhibiting ability comparable to that observed for heparin. These results indicate that the activity of thymic and/or bone marrow stroma-derived lymphocyte growth factor (TSTGF/IL-7) but not of Th-producing TCGF (IL-2) is negatively regulated by heparin or heparan sulfate, which would represent major glycosaminoglycans in the extra-cellular matrix of stromal cells.     

Primary structure of a 15-kDa protein from rat intestinal epithelium. Sequence similarity to fatty-acid-binding proteins

An abundant and novel cytosolic protein was purified from the rat intestinal epithelium by gel filtration, ion-exchange and hydroxylapatite chromatography. The protein was eluted into two different positions (fractions 1 and 2) on DEAE-cellulose chromatography. We have completed the primary structure of the protein of fraction 1 by Edman degradation. The protein (144565 Da) contains 127 amino acid residues and has an acetylated alanine at its NH2-terminus. Comparison of the primary structure of the protein with porcine gastrotropin [Walz, A. D., Wider, M. D., Snow, J. W., Dass, C. & Desiderio, D. M. (1988) J. Biol. Chem. 263, 14189-14195] and rat hepatic fatty-acid-binding protein revealed that identical residues within these proteins are found in 90 and 54 out of a total of 127 positions, respectively. Bioactivity studies demonstrated that neither the protein nor liver and intestinal fatty-acid-binding proteins influence gastric acid secretory activity in rats with gastric fistulas compared to pentagastrin. The protein showed very low affinity for palmitic-acid-binding in vitro assay system and only trace amounts of endogenous fatty acids were detected from the protein. The protein, rat intestinal 15-kDa protein is considered to be a new member of the fatty-acid-binding protein family based on its structural features.     

Conformational changes of recombinant human granulocyte-colony stimulating factor induced bypH and guanidine hydrochloride

Fluorescence and circular dichroism were used to follow thepH-dependent conformational changes of granulocyte colony stimulating factor (G-CSF). Tryptophan fluorescence of the spectra monitored at 344 nm, or after deconvolution of the emission spectra, at 345 nm, showed a decrease in intensity on going frompH 7 to 4, with a midtransitionpH of 5.8. On the other hand, tyrosine fluorescence measured either by the ratio of intensity at 308 nm to that at 344 nm, or by the fluorescence intensity at 303 nm after deconvolution of the spectra, increased in intensity as thepH was changed from 6 to 2.5, with a midtransitionpH of 4.5. Near UV circular dichroic spectra also showed changes betweenpH 7.5 and 4.5, which correlated with the transition monitored by the tryptophan fluorescence. The guanidine hydrochloride-induced conformational changes of G-CSF at fivepH values from 2.5 to 7.5 were also studied. Circular dichroic and fluorescence spectra revealed minor conformational changes by the addition of 1 or 2 M guanidine HCl at allpH values examined, while the major conformational transition occurred between 2 and 4 M guanidine hydrochloride. The secondary structure of the protein was most stable betweenpH 3.3 and 4.5. The guanidine HCl-induced denaturation of G-CSF involved more than a two-state transition, with detectable intermediate(s) present, and the structure of the intermediate(s) appeared to depend on thepH used. These results are consistent with thepH dependence of the structure described above, and demonstrate the complex conformational properties of G-CSF.     

Kinetic mechanism of beef heart ubiquinol:cytochrome c oxidoreductase.

The electron transfer from ubiquinol-2 to ferricytochrome c mediated by ubiquinol:cytochrome c oxidoreductase [E.C. 1.10.2.2] purified from beef heart mitochondria, which contained one equivalent of ubiquinone-10 (Q10), was investigated under initial steady-state conditions. The Q10-depleted enzyme was as active as the Q10-containing one. Double reciprocal plots for the initial steady-state rate versus one of the two substrates at various fixed levels of the other substrate gave parallel straight lines in the absence of any product. Intersecting straight lines were obtained in the presence of a constant level of one of the products, ferrocytochrome c. The other product, ubiquinone-2, did not show any significant effect on the enzymic reaction. Ferrocytochrome c non-competitively inhibited the enzymic reaction against either ubiquinol-2 or ferricytochrome c. These results indicate a Hexa-Uni ping-pong mechanism with one ubiquinol-2 and two ferricytochrome c molecules as the substrates, which involves the irreversible release of ubiquinone-2 as the first product and the irreversible isomerization between the release of the first ferrocytochrome c and the binding of the second ferricytochrome c. Considering the cyclic electron transfer reaction mechanism, this scheme suggests that the binding of quinone or quinol to the enzyme and electron transfer between the iron-sulfur center and cytochrome c1 are rigorously controlled by the electron distribution within the enzyme.     

Factor XIII-dependent generation of 5th complement component(C5)-derived monocyte chemotactic factor coinciding with plasma clotting.

Blood coagulation or plasma clotting caused generation of a monocyte chemotactic factor(s) in vitro. The chemotactic factor, of which the apparent molecular mass was 75 kDa, shared antigenicity with complement C5 and possessed the affinity to monocytes, but not to polymorphonuclear leukocytes. The generation of the chemotactic factor was hindered in the presence of a thiol enzyme inhibitor, p-chloromercuriphenyl sulfonic acid, at the concentration of 1 mmol/l, although the gelation of plasma was apparently completed. Furthermore, the generation of chemotactic factor was not observed when a plasma deficient in blood coagulation factor XIII, which is a precursor of a thiol enzyme, plasma transglutaminase, was used; and the activity normally appeared when the deficient plasma was reconstituted with purified factor XIII or with a tissue transglutaminase prior to clotting. When the human sera were injected into guinea pig skin, the serum derived from normal plasma or from the reconstituted factor XIII deficient one caused mononuclear cell infiltration, however, the serum from the deficient plasma without reconstitution infiltrated to a significantly smaller extent. These results indicated that the complement system was initiated somehow during the clotting process resulting in the generation of the C5-derived monocyte chemotactic factor in cooperation with factor XIIIa (activated factor XIII).     

Specific and abundant secretion of a novel hydroxyproline-rich glycoprotein from salt-adapted winged bean cells

Winged bean callus was adapted to increasing concentrations of NaCl by sequential transfer to medium with 0, 0.5, 1.0, 1.5, and 2.0% (w/v) NaCl. When the culture media, after cell suspension cultures of callus adapted to 0.5 (SA-0.5), 1.0 (SA-1.0), 1.5 (SA-1.5), or 2.0% (w/v) NaCl (SA-2.0), were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis, six specific or enhanced polypeptide bands (SAP1, -2, -3, -4, -5, and -6) were observed. SAP1, with a molecular weight of 84,000, was abundantly secreted in suspension cultures of SA-1.0 and SA-1.5, and was observed as the most striking polypeptide band. The SAP1 yield was about 4 mg/g cells fresh weight. SAP1 was abundantly secreted after the suspension culture of SA-1.0 in the presence of AlCl₃, but little was secreted in the presence of KCl, LiCl, CaCl₂, MgCl₂, mannitol, sucrose, or abscisic acid. SAP1 was purified from the culture medium after suspension culture of SA-1.0 in the presence of 1.0% (w/v) NaCl. Two steps, ammonium sulfate fractionation and CM-cellulose chromatography, were sufficient for purification to homogeneity. Finally, about 5 mg of SAP1 could be isolated from 7 g of fresh callus cells. Of the amino-terminal 32 amino acid residues of SAP1, 10 and 5 were found to be hydroxyproline and proline, respectively. SAP1 on an acrylamide gel was stained by the periodic acid-Schiff method. It is interesting that SAP1 has pentahydroxyproline blocks (Hyp₅) instead of tetrahydroxyproline blocks (Hyp₄) common to many hydroxyproline-rich glycoproteins in dicotyledons. Thus, this novel hydroxyproline-rich glycoprotein was shown to be abundantly secreted from NaCl-adapted winged bean cells.