Filamentous Cells of Escherichia coli Formed in the Presence of Mitomycin

The effects of mitomycin C on cell elongation of Escherichia coli B were studied. Filament formation was most marked in cultures treated with a moderate level (1 mug/ml) of the antibiotic, becoming less obvious at higher levels (10 mug/ml). Cells treated with a bacteriostatic concentration (0.1 mug/ml or less) of mitomycin C were also significantly elongated. The filamentous or elongated cells appeared to lack septa, since their spheroplasts were considerably larger than those formed from normal cells. The appearance of empty spheres also indicated some defects in the surfaces of the filamentous cells. Electron micrographs of the filaments revealed a characteristic difference in the arrangement of the nuclei in the filaments formed in the presence of low (0.1 mug/ml) and high (5 mug/ml) concentrations of mitomycin C. The filaments formed by the low level of mitomycin C had normal well-defined nuclear bodies distributed along the long axis, whereas those formed by the elevated level of the antibiotic contained smaller nuclei. The latter were characteristically confined to the center of the cells and did not extend out to the tips of the filaments.     

Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.     

Lineage, migration, and morphogenesis of longitudinal glia in the Drosophila CNS as revealed by a molecular lineage marker

Previous studies described three different classes of glial cells in the developing CNS of the early Drosophila embryo that prefigure and ensheath the major CNS axon tracts. Among these are 6 longitudinal glial cells on each side of each segment that overlie the longitudinal axon tracts. Here we use transformant lines carrying a P element containing a 130 bp sequence from the fushi tarazu gene in front of the lacZ reporter gene to direct beta-galactosidase expression in the longitudinal glia. Using this molecular lineage marker, we show that 1 of the "neuroblasts" in each hemisegment is actually a glioblast, which divides once symmetrically, in contrast to the typical asymmetric neuroblast divisions, producing 2 glial cells, which migrate medially and divide to generate the 6 longitudinal glial cells. As with neuroblasts, mutations in Notch and other neurogenic genes lead to supernumerary glioblasts. The results indicate that the glioblast is similar to other neuroblasts; however, the positionally specified fate of this blast cell is to generate a specific lineage of glia rather than a specific family of neurons.     

Structure of chicken 16-kDa beta-galactoside-binding lectin. Complete amino acid sequence, cloning of cDNA, and production of recombinant lectin

The complete primary structure of chicken 16-kDa beta-galactoside-binding lectin (C-16) was determined. It was composed of 134 amino acid residues and has an acetylated NH2 terminus. A cDNA was also cloned, but no signal sequence was found in the initiator region. The initiator methionine remained as the NH2 terminus of the mature lectin. Although C-16 is distinct from chicken 14-kDa beta-galactoside-binding lectin (C-14), it proved to be a member of the vertebrate 14-kDa-type lectin family. Comparison of the primary structures between the vertebrate 14-kDa-type lectins suggests that C-14 and C-16 were produced by gene duplication of an ancestral lectin gene at a time close to the divergence of birds and mammals. Northern and Southern blot analysis indicated that these isolectins are encoded by individual genes which are differently regulated during the development of the embryo. A recombinant C-16 lectin was produced in Escherichia coli. The product was indistinguishable from the authentic C-16 lectin except that the NH2 terminus of the former was found to begin with free methionine.     

Esterified and total 7 alpha-hydroxycholesterol in human serum as an indicator for hepatic bile acid synthesis

Serum levels of 7 alpha-hydroxycholesterol and activities of hepatic microsomal cholesterol 7 alpha-hydroxylase in surgical patients were analyzed by capillary gas-liquid chromatography-selected ion monitoring technique using a new internal standard, 5 alpha-cholestane-3 beta, 7 beta-diol. We found that concentrations of 7 alpha-hydroxycholesterol obtained after alkaline hydrolysis were higher than those without alkaline hydrolysis, indicating that a considerable amount of 7 alpha-hydroxycholesterol in human serum is present in esterified form. Esterified 7 alpha-hydroxycholesterol could also be quantitatively hydrolyzed with cholesterol esterase, suggesting that fatty acid is bound at the 3 beta-position of the cholestenediol. The serum levels of esterified and free 7 alpha-hydroxycholesterol in patients with cholelithiasis were 198.0 +/- 90.3 and 48.3 +/- 19.8 pmol/ml (mean +/- SD), respectively, and were similar to those in patients without hepatobiliary diseases. After treatment with chenodeoxycholic acid (300 mg per day) for 7 to 10 days, esterified and free 7 alpha-hydroxycholesterol levels decreased to 64.9 +/- 33.6 and 20.5 +/- 11.1 pmol/ml, respectively. Activity of cholesterol 7 alpha-hydroxylase was also inhibited. Treatment with ursodeoxycholic acid (600 mg per day) for 7 to 10 days had no inhibitory effect on serum 7 alpha-hydroxycholesterol levels and the enzyme activity. In all groups, high correlations were found between the activity of cholesterol 7 alpha-hydroxylase and serum levels of 7 alpha-hydroxycholesterol: free (r = 0.71, n = 38, P less than 0.001); esterified (r = 0.87, n = 38, P less than 0.001); total (r = 0.87, n = 38, P less than 0.001). Esterified and total 7 alpha-hydroxycholesterol was more highly correlated with the enzyme activity than the free form. We conclude that a significant amount of 3 beta-acyl esters of 7 alpha-hydroxycholesterol is present in human serum and that serum levels of esterified and/or total 7 alpha-hydroxycholesterol are likely to reflect the activity of hepatic cholesterol 7 alpha-hydroxylase and thus the amount of primary bile acids synthesized in the liver.     

Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B

The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5). The crystals are hexagonal, space group P6(2)22 or P6(4)22. The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees. The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600). The crystals diffract to at least 2.5 A resolution.     

Purification and characterization of the active serine: pyruvate aminotransferase of rat liver mitochondria expressed in Escherichia coli

In the previous study (Oda, T., et al. (1985) Eur. J. Biochem. 150, 415-421), we isolated a cDNA clone which expressed in Escherichia coli a specific size of product having the activity of rat liver serine:pyruvate aminotransferase (SPTm). This specific product (SPT10) was purified to homogeneity through three different column chromatographies. The amino acid composition and N-terminal amino acid sequence of the purified enzyme agreed with those predicted from the nucleotide sequence of cDNA and showed that SPT10 consists of the whole amino acid sequence of mature SPTm and several extra amino acid residues at the N-terminus. The catalytic and physical properties of SPT10, such as substrate specificity, Km for alpha-keto acids, electric charge, and quaternary structure, were all very similar to those of SPTm. Using several cDNA clones which lack a 5'-terminal sequence corresponding to a portion of the N-terminal amino acid sequence of SPTm, we examined the expression profile of the specific product in bacteria transformed with each cDNA clone. The products encoded by these cDNAs were segregated into inclusion bodies and were neither catalytically active nor easily solubilized by sonication. In contrast, the inclusion bodies were not formed in the bacteria transformed with the cDNA clone for SPT10.     

Establishment and characterization of monoclonal antibody against androgen receptor

Hybrid cell lines were prepared by the fusion of BALB/c myeloma NS-1 cells with the lymphocytes of BALB/c mice that were immunized with partially purified androgen receptor (AR) from human prostates. Nine clones of the hybrid progeny were determined for the production of antibodies against AR by immunoprecipitation assay. One of the clones, referred to as "5F4", was chosen for analysis of the detailed specificity. The clone "5F4" secreted IgM class antibodies against AR. Competition study demonstrated that "5F4" antibody inhibited androgen binding of AR, suggesting that the antibody identifies androgen binding site of AR. Immunoblotting analysis showed that the antibody identified the ARs as two proteins, 95 kD and 41 kD proteins, on a sodium dodecyl sulfate polyacrylamide gel. It is suspected that a 95 kD protein should be a monomeric AR and a 41 kD protein is a proteolytic fragment of AR. Immunohistochemical analysis demonstrated that androgen-dependent tissues--human prostatic hypertrophy tissues, an AR abundant prostatic cancer tissue and fibroblast cells from human genital skin--were stained intensely with "5F4" monoclonal antibody, while androgen-independent tissues--fibroblast cells from lymph nodes, an AR deficient prostatic cancer tissue and human prostatic cancer cell line, PC-3--showed no staining. These results also support the specificity of the antibody for AR.     

High-performance capillary electrophoresis of glycoproteins: the use of modifiers of electroosmotic flow for analysis of microheterogeneity.

High-performance capillary electrophoresis (HPCE) is rapidly gaining acceptance as an analytical tool for the study of biological macromolecules. In the present study, the utility of HPCE for separation of glycoproteins is highlighted using a pure ovalbumin preparation. Ovalbumin, the 43-kDa glycoprotein of avian egg white, is known to be heterogeneous in nature with at least nine different carbohydrate structures having been identified on the single Asn residue. HPCE separation in an 87-cm capillary containing borate buffer and 1 mM putrescine resolves five major protein peaks in less than 30 min under nondenaturing conditions. This effect appears to be specific to glycoproteins since analysis of the nonglycosylated protein carbonic anhydrase under the same conditions showed no enhanced separation. The sodium borate buffer is proposed to play a key role in the separation by preferentially complexing with the diols of specific carbohydrate moieties on ovalbumin. Addition of putrescine enhances resolution by slowing bulk flow through the capillary and allowing electrophoretic separation of what is deduced to be closely related glycoforms of ovalbumin. Dephosphorylation of the ovalbumin with either calf intestinal alkaline phosphatase or potato acid phosphatase results in a shift of all peaks to a more rapid migration time and is consistent with a loss of negative charge. This suggests that all major ovalbumin isoforms are phosphorylated to the same degree and that heterology among ovalbumin isoforms resides solely in the carbohydrate structure. The enhanced resolution obtained with the employment of longer capillaries and modifiers of endo-osmotic flow was not restricted to ovalbumin since partial resolution of pepsin isoforms was observed under the same conditions.