An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase

A new alginate lyase-producing micro-organism, designated as Bacillus sp. strain ATB-1015, was effectively isolated from soil samples pretreated for 3 months with a substrate of the enzyme, sodium alginate. Alginate lyase activity was assayed by the degrading activity of biofilm on Teflon sheet discs, which was formed by a mucoid strain of Pseudomonas aeruginosa PAM3 selected from clinical isolates. The extracellular alginate lyase was precipitated with ammonium sulphate from the culture broth, and purified by gel filtration and anion exchange chromatography. The molecular weight of the lyase was estimated to be 41 kDa by SDS polyacrylamide gel electrophoresis and Sephacryl S-200 HR column chromatography. The optimum pH and temperature for the enzyme activity were around 7·5 and 37 °C, respectively, and the Km value was 0·17% with the substrate, sodium alginate. The lyase activity was completely inhibited by treatment with 1 mmol l⁻¹ of EDTA and the decreased activity was almost completely recovered by the addition of 2 mmol l⁻¹ of CaCl₂. The activity was not affected by treatment with the protein denaturants, 0·01 mol l⁻¹ of SDS or 1 mmol l⁻¹ of urea. The lyase had substrate specificity for both the poly-guluronate and poly-mannuronate units in the alginate molecule.     

Threshold-dependent DNA synthesis by pure pressure in human aortic smooth muscle cells: Gialpha-dependent and -independent pathways

Mechanical forces related to pressure and flow are important for the etiology of atherosclerosis and hypertension. We hypothesized the presence of mechanosensors that were solely sensitive to pure atmospheric pressure in the absence of shear and tensile stresses. A pressure-loading apparatus was set up to examine the effects of atmospheric pressure on human aortic smooth muscle cells (HASMC). Pressure application of 140 to 180 mmHg produced DNA synthesis in a pressure-dependent manner. In contrast, pressure of 120 mmHg or less produced no significant change. Both extracellular signal-regulated kinase and c-Jun N-terminal kinase activities, but not p38 activity, were stimulated by pressures of more than 160 mmHg. Pertussis toxin (PTx) completely inhibited the pressure-induced increase of DNA synthesis under the high pressure of 200 mmHg. These data suggest that HASMC have a mechanosensing cellular switch for DNA synthesis which is sensitive to pure atmospheric pressure, and that the molecular switch is activated by pressure of more than 140 mmHg. The activation mechanism consists of PTx-sensitive and -insensitive pathways, and the former is activated by high pure pressure.     

The different effects of recombinant human tumor necrosis factor on rat fibrosarcoma sublines

Summary The antitumor effect of recombinant human tumor necrosis factor (rH-TNF) on two clones of rat fibrosarcoma with different metastatic potential to lymph nodes was examined. The colony formation of clone A, which has high metastatic potential, was completely inhibited by continuous exposure to rH-TNF at 50 U/ml. In contrast, colony formation of clone G, which has low metastatic potential, was not inhibited by high concentrations of rH-TNF (10,000 U/ml). The inhibitory effect of rH-TNF on colony formation by clone A was also observed with a 1-h exposure to rH-TNF. This effect was time and concentration dependent, as determined by the colony assay, ³H-thymidine uptake assay, and ⁵¹Cr-release assay. ³H-thymidine and ³H-uridine uptake per cell of clone A exposed to rH-TNF was not decreased. This suggests that the mechanisms of the antitumor effect of rH-TNF were not due to inhibition of DNA and RNA synthesis of tumor cells. In vivo growth and lymph node metastases of clone A inoculated i.p. to Donryu strain rats were completely suppressed by 14 consecutive i.p. injections of 10⁵ or 10⁶ U/kg per day of rH-TNF. On the other hand the growth of clone G was not influenced by rH-TNF administration.     

Angiopoietin 2 expression in the retina: upregulation during physiologic and pathologic neovascularization

Vascular development in the embryo requires coordinated signaling through several endothelial cell-specific receptors; however, it is not known whether this is also required later during retinal vascular development or as part of retinal neovascularization in adults. The Tie2 receptor has been implicated in stabilization and maturation of vessels through action of an agonist ligand, angiopoietin 1 (Ang1) and an antagonistic ligand, Ang2. In this study, we have demonstrated that ang2 mRNA levels are increased in the retina during development of the deep retinal capillaries by angiogenesis and during pathologic angiogenesis in a model of ischemic retinopathy. Mice with hemizygous disruption of the ang2 gene by insertion of a promoterless beta-galactosidase (beta gal) gene behind the ang2 promoter, show constitutive beta gal staining primarily in cells along the outer border of the inner nuclear layer identified as horizontal cells by colocalization of calbindin. During development of the deep capillary bed or retinal neovascularization, other cells in the inner nuclear layer and ganglion cell layer, in regions of neovascularization, stain for beta gal. Thus, there is temporal and spatial correlation of Ang2 expression with developmental and pathologic angiogenesis in the retina, suggesting that it may play a role.     

Comparative studies of genes encoding thermostable L-2-halo acid dehalogenase from Pseudomonas sp. strain YL, other dehalogenases, and two related hypothetical proteins from Escherichia coli.

We have determined the nucleotide sequence of the gene encoding thermostable L-2-halo acid dehalogenase (L-DEX) from the 2-chloroacrylate-utilizable bacterium Pseudomonas sp. strain YL. The open reading frame consists of 696 nucleotides corresponding to 232 amino acid residues. The protein molecular weight was estimated to be 26,179, which was in good agreement with the subunit molecular weight of the enzyme. The gene was efficiently expressed in the recombinant Escherichia coli cells: the amount of L-DEX corresponds to about 49% of the total soluble proteins. The predicted amino acid sequence showed a high level of similarity to those of L-DEXs from other bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp. strain B (38 to 57% identity) but a very low level of similarity to those of haloacetate dehalogenase H-1 from Moraxella sp. strain B (10%) and haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (12%). By searching the protein amino acid sequence database, we found two E. coli hypothetical proteins similar to the Pseudomonas sp. strain YL L-DEX (21 to 22%).     

Competitive inhibition and selectivity enhancement by Ca in the uptake of inorganic elements (Be, Na, Mg, K, Ca, Sc, Mn, Co, Zn, Se, Rb, Sr, Y, Zr, Ce, Pm, Gd, Hf) by carrot (Daucus carota cv. U.S. harumakigosun)

We investigated the uptake of inorganic elements (Be, Na, Mg, K, Ca, Sc, Mn, Co, Zn, Se, Rb, Sr, Y, Zr, Ce, Pm, Gd, and Hf) and the effect of Ca on their uptake in carrots (Daucus carota cv. U.S. harumakigosun) by the radioactive multitracer technique. The experimental results suggested that Na, Mg, K, and Rb competed for the functional groups outside the cells in roots with Ca but not for the transporter-binding sites on the plasma membrrane of the root cortex cells. In contrast, Y, Ce, Pm, and Gd competed with Ca for the transporters on the plasma membrane. The selectivity, which was defined as the value obtained by dividing the concentration ratio of an elemental pair, K/Na, Rb/Na, Be/Sr, and Mg/Sr, in the presence of 0.2 and 2 ppm Ca by that of the corresponding elemental pair in the absence of Ca in the solution was estimated. The selectivity of K and Rb in roots was increased in the presence of Ca. The selectivity of Be in roots was not affected, whereas the selectivity of Mg was increased by Ca. These observations suggest that the presence of Ca in the uptake solution enhances the selectivity in the uptake of metabolically important elements against unwanted elements.     

Identification and characterization of a novel polysaccharide deacetylase C (PdaC) from Bacillus subtilis

Cell wall metabolism and cell wall modification are very important processes that bacteria use to adjust to various environmental conditions. One of the main modifications is deacetylation of peptidoglycan. The polysaccharide deacetylase homologue, Bacillus subtilis YjeA (renamed PdaC), was characterized and found to be a unique deacetylase. The pdaC deletion mutant was sensitive to lysozyme treatment, indicating that PdaC acts as a deacetylase. The purified recombinant and truncated PdaC from Escherichia coli deacetylated B. subtilis peptidoglycan and its polymer, (-GlcNAc-MurNAc[-L-Ala-D-Glu]-)(n). Surprisingly, RP-HPLC and ESI-MS/MS analyses showed that the enzyme deacetylates N-acetylmuramic acid (MurNAc) not GlcNAc from the polymer. Contrary to Streptococcus pneumoniae PgdA, which shows high amino acid sequence similarity with PdaC and is a zinc-dependent GlcNAc deacetylase toward peptidoglycan, there was less dependence on zinc ion for deacetylation of peptidoglycan by PdaC than other metal ions (Mn(2+), Mg(2+), Ca(2+)). The kinetic values of the activity toward B. subtilis peptidoglycan were K(m) = 4.8 mM and k(cat) = 0.32 s(-1). PdaC also deacetylated N-acetylglucosamine (GlcNAc) oligomers with a K(m) = 12.3 mM and k(cat) = 0.24 s(-1) toward GlcNAc(4). Therefore, PdaC has GlcNAc deacetylase activity toward GlcNAc oligomers and MurNAc deacetylase activity toward B. subtilis peptidoglycan.     

The 4′-hydroxy group is responsible for the in vitro cytogenetic activity of resveratrol

We previously reported that 3,5,4′-trihydroxy-trans-stilbene (resveratrol), a polyphenolic phytoalexin found in grapes, induces a high frequency of sister chromatid exchanges (SCEs) in vitro. In this study, to investigate structure activity relationships, we synthesized six analogues of resveratrol differing in number and position of hydroxy groups, and we investigated their activity in chromosomal aberration (CA), micronucleus (MN) and sister chromatid exchange (SCE) tests in a Chinese hamster cell line (CHL). Two of the six analogues (3,4′-dihydroxy-trans-stilbene and 4-hydroxy-trans-stilbene) showed clear positive responses in a concentration-dependent manner in all three tests. Both were equal to or stronger than resveratrol in genotoxicity. The 4′-hydroxy (OH) analogue had the simplest chemical structure and was the most genotoxic. The other analogues did not have a 4′-hydroxy group. These results suggested that a 4′-hydroxy group is essential to the genotoxicity of stilbenes.