Gene organization in the region containing a new gene involved in chromosome partition in Escherichia coli.

A new mutation, parC, causing abnormal chromosome segregation was identified in two thermosensitive mutants of Escherichia coli. The thermosensitive growth of the mutants was corrected by pLC4-14 in the Clarke-Carbon collection. This plasmid carries a putative gene which can suppress the cell division defect due to ftsI (pbpB) and has hence been termed sufI (sui). The nearness of parC to metC was confirmed, and cotransduction frequency of parC was 59% with metC and 20% with glc. The parC-sufI region was analyzed by subcloning the chromosome region of pLC4-14. The parC and the sufI gene products were electrophoretically identified as proteins of 75 and 55 kilodaltons (kDa), respectively. The allelism of parC+ on pLC4-14 to parC1215 was confirmed by cloning parC1215. The sufI gene appeared to be dispensable for cell viability, and overproduction of its product caused suppression of ftsI. An essential gene coding for a 25-kDa protein was found between the parC and the sufI gene. These three genes were transcribed in the same direction and may be organized into an operon, with parC to the proximal side and with internal promoters at least for the distal genes. The localization of the gene products was examined in maxicells. The sufI protein was synthesized as a precursor which could be chased into a mature form. The major part of the mature form was found in the soluble fraction. The 25-kDa protein was found almost exclusively in the membrane fraction. The parC protein was associated with the membrane fraction in the presence of Mg2+ but found in the soluble fraction when Mg2+ was sequestered with EDTA.     

Transport of 1,5-anhydro-D-glucitol across plasma membranes in rat hepatoma cells

The transport of 1,5-anhydro-D-glucitol (AG) across plasma membranes was investigated in rat hepatoma cells, Reuber H-35. The AG uptake by the cells showed a concentration gradient dependency: the uptake was saturated within 40 s, which was less than one-third of the saturation time for 2-deoxy-D-glucose (DG) uptake. Furthermore, the Km value of the transport system for AG was higher than 100 mM. Though AG has a pyranoid structure resembling that of glucose, AG did not compete for cellular uptake with DG, D-glucose or 3-O-methyl-D-glucose, which are taken into cells through the glucose transporters. Conversely, the DG transport was not inhibited by AG at concentrations up to 50 mM. AG transport was hardly inhibited by 10 microM cytochalasin B, which strongly inhibits glucose transporters. In contrast, the AG transport was inhibited by 100 microM phloretin much more strongly than the DG transport when cells were preincubated with the inhibitor; the inhibition constant was 28.0 microM. The AG transport was not inhibited by 100 microM phloridzin, while the DG uptake was slightly inhibited by phloridzin. On the basis of these observations we propose that the AG uptake into rat hepatoma cells is mediated by a carrier distinct from glucose transporters.     

1,25-Dihydroxyvitamin D stimulates sodium-dependent phosphate transport by renal outer cortical brush-border membrane vesicles by directly affecting membrane fluidity

We examined the effects of several forms of vitamin D added to renal brush-border membrane suspensions on phosphate and glucose transport and on membrane fluidity. The 1,25-D stimulated and the other vitamin D decreased phosphate uptake. In contrast, glucose uptake was not affected by the treatment of vitamin D. The 1,25-D resulted in a significant shift of the lower transition temperature in Arrhenius plots for phosphate, but not for glucose uptakes, from 15 degrees C to 11.5 degrees C. These data indicate that the 1,25-D may alter membrane fluidity, limited to the phosphate transporter, thus affecting the phosphate uptake.     

Amino acid sequence of myoglobin from the mollusc Dolabella auricularia

The complete amino acid sequence of the myoglobin from Dolabella auricularia, a common gastropodic mollusc on the Japanese coast, has been determined. The myoglobin is composed of 146 amino acid residues, is acetylated at the NH2 terminus, and contains a single histidine residue at position 95 which most likely corresponds to the heme-binding proximal histidine. The sequence of Dolabella myoglobin shows strong homology (72-77%) with those of Aplysia myoglobins. The autoxidation rate of Dolabella oxymyoglobin (MbO2) was examined in 0.1 M buffer at 25 degrees C over pH range 4.8-12. Dolabella MbO2 was extremely unstable between pH 7 and 11, and the pH dependence of the stability was quite different from that of sperm whale MbO2. This property may be partly due to the absence of a distal (E7) histidine in Dolabella myoglobin.     

Generation of activated killer (AK) cells by recombinant interleukin 2 (rIL 2) in collaboration with interferon-gamma (IFN-gamma)

Activation of human peripheral blood mononuclear cells (PBMC) by interleukin 2 (IL 2) and the role of interferon-gamma (IFN-gamma) in the IL 2-induced activation were investigated. Activated killer (AK) cells against NK-resistant tumor cell lines were induced in the medium containing recombinant IL 2 (rIL 2) and autologous serum without any other stimulating agents. AK activity was induced by doses of rIL 2 as low as 3 U/ml, and reached a maximum at 10(3) U/ml. Incubation of PBMC with rIL 2 resulted in IFN-gamma production and augmented NK activity after 1 day of culture, and in induction of AK cells and proliferative response after 2 days of culture. These results suggested that endogenous IFN-gamma was required for rIL 2-induction of AK cells and proliferative response. To prove this, PBMC were cultured with rIL 2 and rIFN-gamma or were pretreated with rIFN-gamma before culture with rIL 2. Both rIFN-gamma treatments of PBMC augmented rIL 2-induced AK activity and proliferative response. rIL 2-induced IFN-gamma production was also enhanced by the rIFN-gamma pretreatment of PBMC. The addition of anti-IFN-gamma antibody to rIL 2 cultures abrogated the rIL 2-induced NK augmentation, AK generation, and proliferative response in proportion to the decreased amounts of endogenous IFN-gamma detectable in culture. rIFN-gamma and/or rIL 2 cultures of PBMC increased Tac antigen expression on cell surfaces as measured by flow cytometry. Enhanced Tac expression by rIL 2 was abrogated by adding anti-IFN-gamma antibody. These data indicate that: 1) AK generation and IFN-gamma production are mediated by IL 2, and 2) IFN-gamma production may be required for IL 2 induction of AK cells and proliferative response. These finding are consistent with the hypothesis that AK generation involves a collaboration between IL 2 and IFN-gamma, in which IL 2 stimulates PBMC to produce IFN-gamma, which in turn acts as a differentiation signal that may be involved in the IL 2-initiated AK generation and proliferative response.     

Amino acid sequence of polypeptide chain IIB of extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus

The giant extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus consists of two types of subunits: a "monomeric" chain (chain I) and a disulfide-bonded trimer of chains IIA, IIB, and IIC. The complete amino acid sequence of chain IIB was determined. This chain has 148 amino acid residues and a molecular weight of 17,236 including a heme group. Of the residues in chain IIB, 74 (50%) and 34 (30%) were found to be identical with those in the corresponding positions in Tylorrhynchus chains IIC and I, respectively (Suzuki, T., Furukohri, T., and Gotoh, T. (1985) J. Biol. Chem. 260, 3145-3154). Marked differences were found between the chains of Tylorrhynchus and Lumbricus in the COOH-terminal regions. Significant differences were predicted between the monomeric chain I and the "trimeric" chains (IIB and IIC) in the hydropathy profiles and alpha-helical contents.     

HPLC-studies on nonmercapt-mercapt conversion of human serum albumin

Human mercaptalbumin (HMA) and nonmercaptalbumin (HNA) could be separated by high-performance liquid chromatography (HPLC) at neutral pH. Using HPLC, the present authors found the nonmercapt-mercapt conversion (HNA----HMA) during hemodialysis and the mercapt-nonmercapt conversion (HMA----HNA) after hemodialysis in chronic renal failure, indicating HMA as the covalent carrier protein for sulfur-containing amino acids.     

Purification and characterization of a lectin from the beetle, Allomyrina dichotoma

A lectin was purified from the hemolymph of Allomyrina dichotoma larvae by affinity chromatography on acid-treated Sepharose 4B. The purified lectin showed two protein bands on polyacrylamide gel electrophoresis. These two lectin bands (allo A-I and -II) were separated by DEAE-Cellulofine column chromatography. By gel filtration on Sephadex G-100, the molecular weights of allo A-I and -II were estimated to be 65,000 and 66,500, respectively. On the other hand, by SDS-polyacrylamide gel electrophoresis after cross-linking of subunits with glutaraldehyde, they are estimated to be 38,000 and 39,000, respectively. On SDS-polyacrylamide gel electrophoresis, it was proved that both allo A-I and -II lectin consisted of two subunits, respectively. The molecular weights were 17,500 and 20,000 for allo A-I, and 19,000 and 20,000 for allo A-II. The isoelectric points of allo A-I and -II were estimated to be 6.4 and 5.9, respectively. On double immunodiffusion, allo A-I and -II gave single precipitin lines, which fused completely with each other, against the antibody to crude allo A. The hemagglutinating activity of allo A-I and -II was inhibited only by beta-linked D-galactose such as lactose and lactulose.     

Overlapping of the coding regions for alpha and gamma components of penicillin-binding protein 1 b in Escherichia coli

Summary The mode of biosynthesis of penicillin-binding protein(PBP)-1 b in Escherichia coli was investigated by use of the plasmid carrying the ponB(PBP-1 b) gene region. Analyses of the products synthesized in minicells and in vitro showed that PBP-1 b was synthesized as two molecular species corresponding to the and components of PBP-1 b. The coding regions for the and components were located within the ca. 3.7 kb MluI-HincII fragment and transcribed in the direction from the HincII to the MluI site. The capacity for producing the component was abolished by a deletion extending to the MluI site ca. 0.7 kb inward from the HincII end of the ca. 3.7 kb fragment; the remaining 3.0 kb region with the MluI site at both ends directed the production of the component alone. The production of the component was enough to correct all the known defects caused by a ponB mutation. In addition to these results, the analyses for cross-reacting materials produced in correspondence to the various deletions indicated that the coding regions for the and components overlapped and that the N-terminal portion was responsible for the difference between the two components. The distal region about 0.7 kb long inward from the MluI end of the MluI-HincII fragment was dispensable for producing the functional PBP-1 b, although the PBP-1 b produced was curtailed. By a larger distal deletion reaching almost to the middle of the MluI-HincII fragment, the polypeptide produced for PBP-1 b lost the ability to bind penicillin and still retained a low but significant activity for glycan synthesis. We suggest, therefore, that the polypeptide portion required for transglycosylase activity resides on the N-terminal half of PBP-1 b, followed by the middle portion necessary for penicillin-binding and the C-terminal part dispensable for the function of PBP-1 b.