ESR evidence of superoxide radical dismutation by human ceruloplasmin

The formation of the paramagnetic complex between human ceruloplasmin and radiation produced superoxide radicals was observed by the ESR method at low temperatures. The disappearance of the complex without changes in the oxidation state of copper give the direct evidence that ceruloplasmin, the major antioxidant in serum, is able to dismutate superoxide radicals.     

Some rumen bacteria degrading fructan

Degradation of fructan obtained from timothy ( Phleum pratense L.) by the following six species of bacteria isolated from sheep rumen was studied: Streptococcus bovis, Bacteroides ruminicola, Selenomonas ruminantium, Butyrivibrio fibrisolvens, Treponema bryantii and Treponema saccharophilum. The enzymatic activity of the bacteria was analysed by TLC. The highest activity was found in whole cells and in the strains B. fibrisolvens No. 3 and T. saccharophilum S.     

Swimming response of goldfish,Carassius auratus,and the tetra,Hemigrammus caudovittatus,larvae to individual food items and patches

Synopsis Swimming speed and swimming path of goldfish and tetra larvae were studied in aquaria containing food patches composed of decapsulated cysts and immobilized nauplii of Artemia salina or sparsely distributed prey. The mean swimming speed of starved larvae in the medium without food was about four times higher than the speed of larvae feeding in a patch. Satiated larvae swam about 1.5 times slower than hungry fish. Consumption of single prey items by starved larvae caused the following sequence of swimming responses: handling pause (cessation of swimming), slow swimming in a restricted area, and fast swimming (approximately twice as fast as hungry larvae before encountering food) accompanied by a widening of the area searched (area increased searching). Mean swimming speed was constant over a broad range (10¹–10³ ind·1^–1 of food density, although at extreme (high or low) values of food density it depended on swimming responses of the predator. Frequency of visits to the different parts of the aquarium strongly depended on encounters of hungry fish with food particles or patches.     

Attempts to Freeze Some Bacteriophages to Ultralow Temperatures

A variety of bacteriophages specific for different hosts, including Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Shigella dysenteriae, Staphylococcus aureus, and Vibrio comma, were frozen at a controlled rate to liquid nitrogen temperatures, and then quick-thawed. Glycerol (10%) was used as a protective additive. Quantitative determinations showed from 10% to virtually 100% recovery in the various cases. Host specificity, plaque morphology, and, in one case, rate of inactivation of phage by homologous antiserum remained unchanged.     

Microbial transformation of azacarbazoles X: Regioselective hydroxylation of 5,11-dimethyl-5H-indolo [2,3-b]quinoline, a novel DNA topoisomerase II inhibitor, by Rhizopus arrhizus

Summary Microbial transformation of cytotoxic 5,11-dimethyl-5H-indolo[2,3-b]quinoline (a compound displaying antitumor activity and affecting the activity of calf thymus DNA topoisomerase II) was performed by the Rhizopus arrhizus strain and yielded a 9-hydroxy derivative. The metabolite obtained displayed a stronger cytotoxity against KB cells than the parent compound (ID₅₀=0.001 mol/mL), and stimulated also the formation of calf thymus topoisomerase II mediated pSP65 DNA cleavage in vitro at the concentration of 3 M. Being analogous to 9-hydroxyellipticine (which is an antitumor alkaloid), this novel indolo[2,3-b] quinoline derivative can be regarded as a novel potential antitumor agent.     

Purification and characterization of Streptococcus sobrinus dextranase produced in recombinant Escherichia coli and sequence analysis of the dextranase gene.

The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 U/mg of protein. Potential immunological cross-reactivity between dextranase and the SpaA protein specified by various recombinant clones was studied by using various antisera and Western blot (immunoblot) analysis. No cross-reactivity was observed. Optimal pH (5.3) and temperature (39 degrees C) and the isoelectric points (3.56, 3.6, and 3.7) were determined and found to be similar to those for dextranase purified from S. sobrinus. The dex DNA restriction map was determined, and several subclones were obtained. The nucleotide sequence of the dex gene was determined by using subclones pYA993 and pYA3009 and UAB66 chromosomal DNA. The open reading frame for dex was 4,011 bp, ending with a stop codon TAA. A ribosome-binding site and putative promoter preceding the start codon were identified. The deduced amino acid sequence of Dex revealed the presence of a signal peptide of 30 amino acids. The cleavage site for the signal sequence was determined by N-terminal amino acid sequence analysis for Dex produced in E. coli chi 2831(pYA902). The C terminus consists of a serine- and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci.     

Overproduction of a dextranase inhibitor by Streptococcus sobrinus mutants.

An inhibitor of Streptococcus sobrinus endodextranase was detected in the extracellular fractions of UAB66 mutants identified following ethyl methanesulfonate mutagenesis as either devoid of dextranase activity (Dex-) or overproducing water-soluble glucan. The two groups of mutants had the same phenotype and displayed no dextranase activity in assays of extracellular fractions (H. Murchison, S. Larrimore, and R. Curtiss III, Infect. Immun. 34:1044-1055, 1981) and had been shown to be defective in adherence (Adh-) and capable of inhibiting adherence of wild-type strains during cocultivation in vitro (H. Murchison, S. Larrimore, and R. Curtiss III, Infect. Immun. 50:826-832, 1985) and in vivo in gnotobiotic rats (K. Takada, T. Shiota, R. Curtiss III, and S. M. Michalek, Infect. Immun. 50:833-843, 1985). By analysis of proteins in Western blots (immunoblots) and following blue dextran-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDS-PAGE), it was demonstrated that these Dex- mutants did synthesize enzymatically active dextranase. From the results of mixing experiments, it was determined that these Dex- Adh- mutants produced enhanced amounts of a cell surface-localized or a cell-associated dextranase inhibitor (Dei). Dei was heat stable but trypsin sensitive. By adding excess dextranase following BD-SDS-PAGE, Dei was detected as blue bands with apparent molecular masses of 43, 40, 37, 27, and 23 kDa. Dei competitively inhibits dextranase activity and is synthesized by wild-type S. sobrinus strains, with the amount varying depending upon growth medium and stage in the growth cycle. R. M. Hamelik and M. M. McCabe (Biochem. Biophys. Res. Commun. 106:875-880, 1982) previously described a Dei in a wild-type S. sobrinus strain.     

Cloning and DNA sequencing of the dextranase inhibitor gene (dei) from Streptococcus sobrinus.

Some dextranase-deficient (Dex-) mutants of Streptococcus sobrinus UAB66 (serotype g) synthesize a substance which inhibits dextranase activity (S.-Y. Wanda, A. Camilli, H. M. Murchison, and R. Curtiss III, J. Bacteriol. 176:7206-7212, 1994). This substance produced by the Dex- mutant UAB108 was designated dextranase inhibitor (Dei) and identified as a protein. The Dei gene (dei) from UAB108 has been cloned into pACYC184 to yield pYA2651, which was then used to generate several subclones (pYA2653 to pYA2657). The DNA sequence of dei was determined by using Tn5seq1 transposon mutagenesis of pYA2653. The open reading frame of dei is 990 bp long. It encodes a signal peptide of 38 amino acids and a mature Dei protein of 292 amino acids with a molecular weight of 31,372. The deduced amino acid sequence of Dei shows various degrees of similarity with glucosyltransferases and glucan-binding protein and contains A and C repeating units probably involved in glucan binding. Southern hybridization results showed that the dei probe from UAB108 hybridized to the same-size fragment in S. sobrinus (serotype d and g) DNA, to a different-size fragment in S. downei (serotype h) and S. cricetus (serotype a), and not at all to DNAs from other mutans group of streptococci.     

REVIEW ARTICLE: Cyanogenesis in cassava (Manihot esculenta Crantz)

Cassava is the most agronomically important of the cyanogeniccrops. Linamarin, the predominant cyanogenic glycoside in cassava,can accumulate to concentrations as high as 500 mg kg^–1fresh weight in roots and to higher levels in leaves. Recently,the pathway of linamarin synthesis and the cellular site oflinamarin storage have been determined. In addition, the cyanogenicenzymes, linamarase and hydroxynitrile lyase, have been characterizedand their genes cloned. These results, as well as studies onthe organ- and tissue-specific localization of linamarase andhydroxy-nitrile lyase, allow us to propose models for the regulationof cyanogenesis in cassava. There remain, however, many unansweredquestions regarding the tissue-specific synthesis, transport,and accumulation of cyanogenic glycosides. The resolution ofthe sequestions will facilitate the development of food processing,biochemical and transgenic plant approaches to reducing thecyanogen content of cassava foods. Key words: Cyanide, cyanogenic glycosides, linamarin, cyanogens