Cyanothece sp. ATCC 51142 possesses six host restriction-modification systems (HRMS), and these HRMS were designated as Csp68KI to Csp68KVI. So far, four restriction enzymes have been characterized biochemically, and the other two were deduced based on the cloning of corresponding DNA methyltransferase genes M. Csp68KIV and M. Csp68KV from the Cyanothece genome. Csp68KI, Csp68KII, Csp68KIII, Csp68KIV, Csp68KV, and Csp68KVI are isoschizomers of AvaII, AsuII, AvaIII, MspI, HaeIII, and FnuDII, respectively. The cleavage specificities for Csp68KI, Csp68KII, Csp68KIII, and Csp68KVI were characterized. The Cyanothece restriction enzymes showed different temperature and salt requirements for their optimal activity. For example, the restriction enzyme Csp68KII functions optimally at 50 degreesC and Csp68KIII requires higher salt for its activity.
The potent part of the angiotensin I-converting enzyme (ACE) inhibitory activity from Porphyra yezoensis hydrolysate was fractionated by using ion-exchange and gel-filtration techniques. Oral administration of the most potent inhibitory fraction (SP-I fraction, 200 mg/kg) to spontaneously hypertensive rats (SHR) showed a hypotensive effect. Using octadecylsilano column chromatography, the SP-I fraction was further separated into several peptides with potent inhibitory activities. The amino acid sequences of ACE inhibitory peptides derived from Porphyra yezoensis were Ile-Tyr (IC50: 2.69 µM), Met-Lys-Tyr (7.26 µM), Ala-Lys-Tyr-Ser-Tyr (1.52 µM), and Leu-Arg-Tyr (5.06 µM).
Although carp muscular and intestinal dipeptidases are metalloenzymes acting only on dipeptides, some structural and enzymatic differences occur between them. The present study verifies distinctive actions during dipeptide hydrolysis by these enzymes in terms of their kinetic characterization. The intestinal enzyme was differentially inhibited by EDTA and 1,10-phenanthroline, whereas these compounds induced a similar level inhibition of the muscular enzyme. The Km and Vmax values of both enzymes for l-leucine-glycine hydrolysis varied during incubations with 1,10-phenanthroline and EDTA, and the Km or Vmax values of the intestinal enzyme increased or remained the same with increasing concentrations of 1,10-phenanthroline, respectively. Analysis of the kinetic parameters indicated that Co2+ and Mn2+ had noncompetitive effects on the muscular enzyme and that a noncompetitive activation on the intestinal enzyme was stimulated by 1.5 mM of Mg2+ and with increasing concentrations of Mn2+. The muscular enzyme acted on a wide range of l-configuration dipeptides, whereas the intestinal enzyme acted only on a select range of dipeptides with a hydrophobic amino acid at the N-terminal position. The Kcat/Km values of both enzymes for dipeptide hydrolysis showed that highly hydrophobic dipeptides served as their preferential substrates. Other kinetic parameters demonstrated distinctive hydrolytic action of the two enzymes on these dipeptides: a strong affinity of the low catalytic rate muscular enzyme, and a weak affinity of the high catalytic rate intestinal enzyme.
The random amplified polymorphic DNA (RAPD) technique tvas used to characterize six isolates of Hizikia fusiformis collected from Chungmu, Haenam, Ki jang, Pusan, Wando, and Yosu in Korea. Total DNA was extracted by the LiCl extraction method from branches of H. fusiformis. The extracted DNA (0.35 ng) in 25 mu l reaction volume was amplified by 45 cycles of the polymerase chain reaction with arbitrary primers. Thirty-one primers resulted in 715 PCR products of both conserved and polymorphic bands, The genetic similarity for the six isolates ranged from 23% to 59%. Among them, Hizikia isolates collected from Pusan, Haenam, and Wando shared 59% of their major bands; isolates collected From Kijang and Yosu were shown to bt: most distantly related with the others based on genetic similarity.
This study was designed to investigate the antioxidative activity of a substance isolated and purified from culture broth of Bacillus sp. The antioxidative substance was identified as 2,6-bis(1,1-dimethylethyl)phenol (Phen-251). The antioxidative activity of Phen-251 was higher than that of dl-tocopherol and BHA against low-density lipoprotein (LDL) oxidation by thiobarbituric acid reactive substance (TBARS). Phen-251 inhibited the copper-mediated oxidation of human LDL in a dose-dependent manner with almost complete inhibition at 40 µg/ml. Phen-251 at a concentration of 60 µg/ml also inhibited oxidation of LDL induced by J774. LDL oxidized by copper-mediated or cell-induced oxidation was degraded at a much greater rate than native LDL. These results suggested the importance of further research on Phen-251 in the investigation of atherosclerosis and free radical-induced injury.
The world's expanding human population continually needs new sources of food, energy, materials and fine chemicals. It has been realized by many that the marine realm is a rich and largely untapped resource of products that are of potential interest to mankind. It is therefore striking that (except for marine food) only a few of these marine natural products have reached the stage of commercial production.
A marine bacterium, identified as a pseudomonad, isolated from Suberea creba Bergquist, 1995 (Porifera, Dictyoceratida, Verongida, Aplysinellidae) collected along the eastern coast of New Caledonia, gave in culture phenazine-alpha-carboxamide, 2-n-heptylquinol-4-one, 2-n-nonylquinol-4-one, 2-n-(1'E-nonenyl)quinol-4-one, 3-n-heptyl-3-hydroxyquinolin-2,4-dione, a N-oxide-2-n-heptylquinoline derivative, and a benzyldiketopiperazine. None of these products could be detected, at the HPLC-UV sensitivity level, in the sponge extracts, which contained instead (+)-aerothionin, homoaerothionin, (+)-aeroplysinin-1, dibromo-, bromochloro-, and dichloroverongiaquinol. 2-n-Heptylquinol-4-one, (+)-aeroplysinin-1, and dibromoverongiaquinol showed strong antibacterial activity in vitro. The latter also proved promising for mariculture, rivaling chloramphenicol as an antibacterial agent in cultures of Pecten maximus larvae, while being nontoxic according to the Artemia salina test.
A cell-bound polysaccharide (CBP) produced by the marine bacterium Zoogloea sp. (KCCM 10036) was used as the adsorbent of metal ions and as a new support for enzyme immobilization, The CBP gel beads showed highly effective adsorbing in Cr, Pb, and Fe ion in solutions. The adsorption rates were above 95% at pH 5.0, 25 degrees C, in 10 mg/liter of each metal solution. The gel beads formed by the CBP were stable within the range of pH 4.0-7.0 and at a temperature of 40-55 degrees C, The optimum pH and temperature of the immobilized glucoamylase by the CBP gel beads (poly-G) were 5.0 and 45 degrees C, respectively. The immobilized glucoamylase produced 10.5 mg/liter of glucose from 10 mg/ml of soluble starch.
Although carp muscular and intestinal dipeptidases are metalloenzymes acting only on dipeptides, some structural and enzymatic differences occur between them. The present study verifies distinctive actions during dipeptide hydrolysis by these enzymes in terms of their kinetic characterization. The intestinal enzyme was differentially inhibited by EDTA and 1,10-phenanthroline, whereas these compounds induced a similar level inhibition of the muscular enzyme. The Km and Vmax values of both enzymes for l-leucine-glycine hydrolysis varied during incubations with 1,10-phenanthroline and EDTA, and the Km or Vmax values of the intestinal enzyme increased or remained the same with increasing concentrations of 1,10-phenanthroline, respectively. Analysis of the kinetic parameters indicated that Co2+ and Mn2+ had noncompetitive effects on the muscular enzyme and that a noncompetitive activation on the intestinal enzyme was stimulated by 1.5 mM of Mg2+ and with increasing concentrations of Mn2+. The muscular enzyme acted on a wide range of l-configuration dipeptides, whereas the intestinal enzyme acted only on a select range of dipeptides with a hydrophobic amino acid at the N-terminal position. The Kcat/Km values of both enzymes for dipeptide hydrolysis showed that highly hydrophobic dipeptides served as their preferential substrates. Other kinetic parameters demonstrated distinctive hydrolytic action of the two enzymes on these dipeptides: a strong affinity of the low catalytic rate muscular enzyme, and a weak affinity of the high catalytic rate intestinal enzyme.
A marine cyanobacterial strain with enhanced heavy metal ion tolerance was constructed by introducing an exogenous cyanobacterial metallothionein gene, smtA, from a freshwater unicellular cyanobacterium, Synechococcus sp. PCC 7942. An expression vector harboring the c-phycocyanin (cpc) promoter and cpc N-terminal region was constructed and smtA was inserted into its multiple cloning site. The marine cyanobacterium, Synechococcus sp. NKBG 15041c, was highly sensitive to the heavy metal ions present in the medium. However, the recombinant marine cyanobacteria harboring the expression vector with smtA could grow even in the presence of 4 µM of CdCl2, at which concentration the wild-type strain did not grow.
A novel dipeptidase was purified to homogeneity from the crude extract of carp ordinary muscle with an increase in specific activity of 4041-fold and a 4% recovery rate. The enzyme was determined to have molecular weights of 54,000 after reduction and 106,000 without reduction, indicating that it is composed of two sulfide-linking molecules of subunit peptide chains of identical molar size. The optimum hydrolysis pH and temperature of the enzyme were evaluated by l-leucine-glycine to be pH 8.5 and 40 degreesC, respectively, and it was markedly stable in the weak alkaline region and at temperatures below 30 degreesC. Dipeptide hydrolysis of the enzyme was inhibited by metalloprotease inhibitors, sulfide-specific reagents, metal chelating reagents and reductants. In addition, sulfide-affinity bivalent metals potently inactivated the enzyme, while Mg2+ and Mn2+ activated it to different extents, and Mn2+ was also effective on the restoration of the nearly completely inactivated enzyme. The enzyme had a broad range of action on dipeptides that are composed of only l-amino acids, such as l-leucine, l-methionine, l-phenylalanine, and l-valine at the C-terminal and l-alanine, l-leucine, l-methionine, and l-valine at the N-terminal, but it had no catalytic action on dipeptides containing l-proline and d-amino acids, tripeptides, and peptide derivatives.
The preliminary study of rDNA polymorphisms in P. monodon showed that inter- and intraindividual polymorphisms of rDNA were clearly observed in this species. Individual-specific rDNA restriction patterns were observed when digested with BamHI and SacI. The intergenic spacers (IGS) region of P. monodon rDNA plays an important role in length heteroplasmy at both between- and within-individual levels in this species.
Ammonium nitrogen, phosphate, and silicate contents in Noctiluca scintillans cell fluid were estimated at 2470, 183, and 54 pmol per individual, respectively. Ammonium nitrogen and phosphate concentrations at the surface layer of the water column seemed to be related to cell abundance of N. scintillans at the sampling location. Patches of N. scintillans provided 16 and 25 times greater concentrations of ammonium nitrogen and phosphate in the uppermost layer (0-10 cm depth) of the water column than those in the ambient seawater, respectively. Silicate concentration within patches, however, was close to that in surrounding water. The morphology of fecal pellets of N. scintillans fed on the diatom, Thalassiosira sp., was examined with a scanning electron microscope. The fecal pellet was composed entirely of visible structural diatom cells. It could be inferred that N. scintillans excretes silica from the diatom undigested in its fecal pellets and thus no remarkably high silicate concentration within red tide patches was observed.
A novel putative SINE sequence was found in a randomly selected cosmid clone of Salmo salar. The sequence (cSSML032Alu, GenBank accession number: L77085) was found to have 78% identity in 212 bp, with one Salvelinus namaycush AluI satellite sequence (GenBank accession number: U27096) and 69% identity in 197 bp, with another AluI satellite sequence from S. namaycush (GenBank accession number: U27091). Colony hybridization of cSSML032Alu to a salmon cosmid library indicated its dispersed presence in the genome, with a copy number in the range of a few thousand. Direct terminal repeats of nine bases and a potential poly(A) tail remnant identified in the S. salar cSSML032Alu sequence indicate a possible ancient retroposon unit.
The effect of aeration, pH, stirring rate, and temperature on the biomass production and superoxide dismutase (SOD) activity of the marine yeast Debaryomyces hansenii strain C-11 was determined. The cell biomass yield was approximately 50% in a seawater-formulated medium using glucose as the carbon source. The SOD activity increased by application of a pulse of oxygen or 0.8 mM sulfate copper into the chemical reactor. The SOD enzyme had an activity of 400 units/mg of protein in a crude extract produced under such conditions, the best activity ever reported for this enzyme in a crude preparation.
Gametophytic cells of Eisenia bicyclis (Kjellman) Setchell were cryopreserved in liquid nitrogen (LN). Ethylene glycol was the most effective cryoprotectant when used alone, and in combination with 10% (w/v) proline improved the survival. Viability of female and male cryopreserved cells after thawing reached 62.0% and 52.6% immediately after thawing, but these levels decreased to 31.1% and 27.2% after 4 days postthawing culture. The optimal prefreezing temperature was -40 degreesC, and cells prefrozen to temperatures >-40 degreesC were damaged mainly by intracellular ice crystal formation during immersion in LN, while those prefrozen to temperatures <-40 degreesC were damaged mainly by excessive dehydration during prefreezing. The survival rates were not affected after storage in LN for at least 200 days.
An extracellular polysaccharide produced by a marine microalga Gymnodinium A3 OKU-1 strain showed a strong cytotoxicity for the human leukemia K-562 cells (CC50 = 11.6 µg/ml). We revealed that this polysaccharide induced apoptosis of K-562 cells, as shown by cell morphology, nuclear fragmentation, in situ end-labeling of fragmented DNA, and internucleosomal DNA fragmentation, suggesting that the mechanism of cytotoxicity of this polymer was based on the induction of apoptosis.
Thermostable dipeptidase was purified to homogeneity from soluble cytoplasmic extract of carp intestine with an increase in specific activity of 372-fold and a 2% recovery rate. The enzyme was determined to have molecular weights of 64,000 after reduction and 320,000 without reduction, indicating that it is composed of five sulfide-linking molecules of subunit peptide chains of identical molar size. The optimum hydrolysis pH and temperature of the enzyme were evaluated by L-leucine-glycine to be pH 9.0 and 60 degrees C, respectively, and it was markedly stable in the alkaline region and at temperatures below 70 degrees C. Dipeptide hydrolysis of the enzyme was inhibited by sulfide-specific and tryptophan-specific inhibitors, reductant and metal chelating reagents. In addition, varying concentrations of EDTA and 1,10-phenanthroline inactivated the enzyme. Sulfide-affinity bivalent metals potently inactivated the enzyme, while Mg2+ and Mn2+ activated it linearly and stepwise, respectively. The enzyme had a narrow range of action on dipeptides of X-glycine and L-leucine-X, but it had marginal or no effect on other dipeptides, tripeptides, and peptide derivatives. Its maximum hydrolysis occurred with L-leucine-L-leucine, and both L-leucine-glycine and L-leucine-L-serine served as preferential substrates.
A gram-positive bacterium designed KP7 that was capable of growing on phenanthrene at 42 degrees C was isolated. In this strain, 1-hydoxy-2-naphthoate, a common intermediate in phenanthrene-degradative pathways, was transformed to 2-carboxybenzaldehyde and o-phthalate. The activities of 1-hydroxy-2-naphthoate dioxygenase and 2-carboxybenzaldehyde dehydrogenase in cells of strain KP7 were induced after their growth on phenanthrene. Thus, phenanthrene was degraded via 1-hydroxy-2-naphthoate and o-phthalate in strain KP7. Chemotaxonomic and 16S rRNA sequence analysis classified this strain as Nocardioides sp.
An extracellular polysaccharide from the marine microalga dinoflagellate Gymnodinium sp, A3, which was a D-galactan sulfate associated with L(+)-lactic acid, showed cytotoxicities to various human lymphoid cells, especially to MT-4 cells (CC50 2.67 mu g/ml). Close observations of morphological change, flow cytometry, and in situ end-labeling of fragmentated DNA revealed the mechanism of cytotoxicity of this polymer to be based on the induction of apoptosis.
