A reactive hydroxymethyl sulfate ester formed regioselectively from the carcinogen, 7,12-dihydroxymethylbenz[alpha]anthracene, by rat liver sulfotransferase

The carcinogen, 7,12-dihydroxymethylbenz[alpha]anthracene (DHBA), was regioselectively conjugated in the presence of 3'-phosphoadenosine 5'-phosphosulfate by male rat liver cytosolic sulfotransferase to DHBA 7-sulfate. The sulfate ester was highly reactive and showed a potent, intrinsic mutagenicity toward Salmonella typhimurium TA 98.     

Increase of translatable mRNA for major microsomal proteins in n-alkane-grown Candida maltosa.

In an n-alkane-assimilating Candida sp., transfer from glucose- to n-alkane-containing medium induced changes in the microsomal proteins, and several distinctive polypeptides were demonstrated in the solubilized microsomal fraction derived from n-alkane-grown cells. Long-term-labeling and pulse-labeling experiments in vivo demonstrated the synthesis of the specific microsomal polypeptides. The polypeptides were synthesized as in vitro translation products directed by polyadenylated RNA extracted from n-alkane-grown cells. Two major polypeptides were partially purified from the microsomal fraction from n-alkane-grown cells, and antiserum was prepared in a rabbit. Immunoprecipitation of these two polypeptides was accompanied by an increase in the amount of translatable mRNA. The molecular weights of the polypeptides derived from long-term-labeling, pulse-labeling and in vitro translation experiments appeared to be identical.     

Scleroblast cultures from the gorgonianLeptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary Scleroblasts were separated from fragmented tissue of growing tips ofLeptogorgia virgulata and cultured using a modification of the technique of Rannou. Replacement of fetal bovine serum with horse serum seemed to increase scleroblast viability. Cell adhesion occurred from 14 to 43 d. Cultured scleroblasts demonstrated cell aggregation, spicule formation, and extrusion of spicules into the external medium. Cells showing spicules in the process of being extruded appeared on the average after 24 d of culture. Variability among cultures was marked with respect to both division and spicule formation. Healthy cultures were maintained for more than 4 mo. This work was supported by National Science Foundation grants PCM8201389 and DCB8502698. This is contribution No. 674 of Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina.     

A new class of site-specific endodeoxyribonucleases. Endo.Sce I isolated from a eukaryote, Saccharomyces cerevisiae

We had found that yeasts had intracellular endodeoxyribonucleases that cut phage DNA into a set of double-stranded fragments with discrete chain lengths. We purified one of them to apparent homogeneity from Saccharomyces cerevisiae and designated it Endo.Sce I. Sequence analysis around 5 cleavage sites in plasmid DNA and phage DNA revealed that Endo.Sce I cuts a defined phosphodiester bond in each strand of double helix at the cleavage sites and produces free cohesive ends consisting of 4 nucleotides protruding at 3'-termini. However, unlike in the case of prokaryotic type II-restriction endonucleases, (i) Endo.Sce I seems to consist of two nonidentical subunits, (ii) no common palindrome or consensus sequence including more than 5 base pairs is detected at or near these cleavage sites, and (iii) Endo.Sce I can cut the DNA isolated from the cells that produced Endo.Sce I. All of the 5 cleavage sites are included in inverted repeats, but these inverted repeats are variable in size, nucleotide sequence, and distance between repeating units. An inverted repeat itself is not a structure recognized by Endo.Sce I. This study shows that Endo.Sce I is the first example of eukaryotic site-specific endonuclease and has properties, as described above, which distinguish it from prokaryotic restriction endonucleases.     

STUDIES ON SHELL FORMATION : IX. An Electron Microscope Study of Crystal Layer Formation in the Oyster

Details of crystal growth in the calcitostracum of Crassostrea virginica have been studied with the purpose of analyzing the formation of the overlapping rows of oriented tabular crystals characteristic of this part of the shell. Crystal elongation, orientation, and dendritic growth suggest the presence of strong concentration gradients in a thin layer of solution in which crystallization occurs. Formation of the overlapping rows can be explained by three processes observed in the shell: a two-dimensional tree-like dendritic growth in which one set of crystal branchings creeps over an adjacent set of branchings; three-dimensional dendritic growth; and growth by dislocation of crystal surfaces. Multilayers of crystals may thus be formed at one time. This is favored by infrequent secretion of a covering organic matrix which would inhibit crystal growth. The transitional zone covering the outer part of the calcitostracum and the inner part of the prismatic region is generally characterized by aggregates of small crystals with definite orientation. Growth in this zone appears to take place in a relatively homogeneous state of solution without strong concentration gradients. Thin membranes and bands of organic matrix were commonly observed in the transitional zone bordering the prismatic region. The membrane showed a very fine oriented network pattern.     

Enzymic conversion of 11,12-leukotriene A4 to 11,12-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid. Purification of an epoxide hydrolase from the guinea pig liver cytosol

(11S,12S)-Epoxy-5,14-cis-7,9-trans-eicosatetraenoic acid (11,12-leukotriene A4) was nonenzymically converted to seven compounds: two diastereomers of (12S)-hydroxyeicosatetraeno-delta-lactones (major products), two diastereomers of (5,12S)-dihydroxyeicosatetraenoic acid and three stereoisomers of (11,12S)-dihydroxyeicosatetraenoic acid. Among these compounds, (11R,12S)-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid proved to be the only enzymic product. This hydrolysis activity was present in the cytosol fractions of various tissues of guinea pig such as liver, adrenal gland, small intestine, and brain. We purified the epoxide hydrolase to an apparent homogeneity from the guinea pig liver. The enzyme had a molecular weight of 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 7.3. The partial amino acid sequence was different from that of the microsomal enzyme. Km and Vmax values for 11,12-leukotriene A4 were 18 microM and 2.4 mumol/min/mg protein, respectively. These results indicate that 11,12-dihydroxyeicosatetraenoic acid is enzymically synthesized from 11,12-leukotriene A4 by the action of the cytosolic epoxide hydrolase in vitro.     

The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish,Carassius auratus

Goldfish Carassius auratus were acclimated to either 10 or 30°C for a minimum of 5 weeks. A 65-kDa protein specific to warm-temperature-acclimated fish was extracted from the gel with 70% formic acid after two-dimensional electrophoresis of the muscle cytoplasmic protein fraction. The 65-kDa protein thus prepared to homogeneity was used to raise specific antibodies in rabbit by conventional methods. The antibody produced exhibited specific reaction with a protein having the same molecular weight from brain and liver tissue, suggesting that the 65-kDa protein is a ubiquitous cytosolic component in warm-acclimated goldfish. When water temperature was increased from 20 to 30°C over a 20-h period, a prominent amount of the 65-kDa protein was observed in muscle tissue extracts within 5 days of additional rearing; this was demonstrated by immunoblotting with the specific antibody. The N-terminal amino acid sequence of the 65-kDa protein was determined as Asp-Glu-Pro-Gln-Gly-His-Gln-His (or Asp)-Glu-Leu, differing from that of a family of known heat-shock proteins having about 70 kDa in molecular mass (hsp 70). No interaction between ATP and the 65-kDa protein revealed by ATP-agarose affinity chromatography further confirmed the different properties of the 65-kDa protein from those of hsp 70.Abbreviations ATP adenosine 5-triphosphate - hsp heat-shock protein(s) - IgG immunoglobulin G - mRNA messenger ribonucleic acid - PMSF phenylmethylsulphonyl fluoride - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Variations in concentrations of bacterial metabolites, enzyme activities, moisture, pH and bacterial composition between and within individuals in faeces of seven healthy adults

Variations between and within individuals, and correlations between concentrations of bacterial metabolites, including putrefactive products, ammonia and short chain fatty acids (SCFAs), enzyme activities, moisture and pH, as well as bacterial composition, were studied in faecal samples from seven healthy adults over a period of 7 months. Large variations, both between and within individuals, were observed in concentrations of putrefactive products. Although values for ammonia, SCFAs, enzyme activities, moisture and pH were generally variable, significant person-to-person differences were observed.
While ranges of log viable counts of the predominant bacteria such as eubacteria, bifidobacteria and bacteroides in each subject remained between 0·2 and 1·3, those of enterobacteria, streptococci (including enterococci) and lecithinase-negative clostridia varied between 0·4 and 3·0. Levels of bifidobacteria, enterobacteria, streptococci and total aerobic bacteria showed inter-individual variations. Correlations were found among certain of the parameters: moisture correlated negatively with p -cresol ( r = -0·707), pH ( r = -0–671) and β-glucosidase activity (GS) ( r = -0·608), and positively with acetic acid ( r = 0·621), while negative correlations were observed in pH with acetic and butyric acids ( r = -0·690 and -0·623, respectively).
No significant correlations were found between bacterial compositions, and other faecal factors such as pH, moisture, metabolic enzyme activities and concentrations of putrefactive products.     

Studies on formation and resorption of fish scales

Summary Scale formation in Cyprinodon variegatus was found to be initiated at about 26 to 30 days after hatching. Ultrastructural investigation revealed that within 4 to 6 h in the first-formed scales the marginal cells begin to flatten and differentiate into osteogenic cells, which later change to osteoblasts and fibroblasts. These cells are separated from the surrounding epithelial cells by a basal lamina. The osteoid is formed by the marginal and osteogenic cells; the osseous layer by the osteoblasts; and the fibrillary plate by the fibroblasts.The osteoid is formed within 2 to 3 h after the initiation of the scale, and within 20 to 24 h the osseous layer is formed. Hydroxyapatite crystals are deposited in the matrix of the osseous layer without apparent association with collagen fibers. No matrix vesicles or dense bodies are evident at the sites of calcification. The fibrillary plate arises 18 to 20 h after the initiation of the scale. It is also partially calcified, but not before the third week of scale formation. The crystals develop almost exclusively between the collagen fibers at the extreme edge of the calcifying front, but solid calcification of the fibers results with further growth of the crystals. The fibroblasts appear to participate in calcification of the fibrillary plate.Contribution No. 332, Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, USA     

Purification and characterization of the hydantoin racemase of Pseudomonas sp. strain NS671 expressed in Escherichia coli.

The hydantoin racemase gene of Pseudomonas sp. strain NS671 had been cloned and expressed in Escherichia coli. Hydantoin racemase was purified from the cell extract of the E. coli strain by phenyl-Sepharose, DEAE-Sephacel, and Sephadex G-200 chromatographies. The purified enzyme had an apparent molecular mass of 32 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By gel filtration, a molecular mass of about 190 kDa was found, suggesting that the native enzyme is a hexamer. The optimal conditions for hydantoin racemase activity were pH 9.5 and a temperature of 45 degrees C. The enzyme activity was slightly stimulated by the addition of not only Mn2+ or Co2+ but also metal-chelating agents, indicating that the enzyme is not a metalloenzyme. On the other hand, Cu2+ and Zn2+ strongly inhibited the enzyme activity. Kinetic studies showed substrate inhibition, and the Vmax values for D- and L-5-(2-methylthioethyl)hydantoin were 35.2 and 79.0 mumol/min/mg of protein, respectively. The purified enzyme did not racemize 5-isopropylhydantoin, whereas the cells of E. coli expressing the enzyme are capable of racemizing it. After incubation of the purified enzyme with 5-isopropylhydantoin, the enzyme no longer showed 5-(2-methylthioethyl)hydantoin-racemizing activity. However, in the presence of 5-(2-methylthioethyl)hydantoin, the purified enzyme racemized 5-isopropylhydantoin completely, suggesting that 5-(2-methylthioethyl)hydantoin protects the enzyme from inactivation by 5-isopropylhydratoin. Thus, we examined the protective effect of various compounds and found that divalent-sulfur-containing compounds (R-S-R' and R-SH) have this protective effect.