Chemical modification of Vibrio vulnificus metalloprotease with activated polyethylene glycol

Abstract Vibrio vulnificus , an opportunistic human pathogen causing septicemia, produces a metalloprotease which is suspected to be a virulence determinant, but which is labile in vivo due to inactivation by α -macroglobulin. To obtain a derivative which is stable in vivo, the metalloprotease was modified with activated monomethoxy polyethylene glycol. The modified protease retained full activity to a peptide substrate and 10–20% activity to protein substrates, and was resistant to entrapment by α -macroglobulin because of the increased molecular size (approx. 90 kDa). These findings suggest that the modified protease is stable in vivo and may be used to investigate the pathological actions of the protease in the bloodstream.     

Hydrostatic pressure is like high temperature and oxidative stress in the damage it causes to yeast

Abstract A comparison of barotolerance, thermotolerance and oxygen tolerance was made under different physiological conditions, such as heat shocked and recovered state, different growth phases and changes of physiological conditions by mutations. The three kinds of tolerance showed similar features under different physiological conditions. We suggest that the damage caused by hydrostatic pressure may be essentially the same as that due to high temperature and oxidative stress in yeast.     

Preparation and biodegradability of chitin derivatives having mercapto groups

Cell walls of glasswort (Salicornia ramosissima Woods), a halophytic Chenopodiaceae, prepared as alcohol-insoluble solids, were found to be rich in arabinose, galacturonic acid, glucose and proteins, and contained 0·7% ferulic acid and 3·8% acetic acid. Pectic and hemicellulosic polysaccharides were extracted by cyclohexanediaminotetraacetic acid, hot dilute acid, cold dilute alkali and concentrated alkali (twice), with yields of 2·9, 19·1, 4·7, 7·4 and 1·9% of the alcohol-insoluble solids, respectively. Protein-rich material precipitated upon dialysis. The dialysed fractions were fractionated by ion-exchange chromatography, and the main fractions were analysed by gel-filtration and glycosyl linkage analysis. The hot acid extract contained 46·2% arabinose and 28·9% galacturonic acid, with high degrees of methylation and acetylation (65 and 45, respectively). It could be fractionated into a low-molecular-weight arabinan rich in ferulic acid, and a pectic fraction still relatively rich in neutral sugars. The concentrated alkali extracts were rich in xylose (33·4 and 23·6%, respectively). They were separated by ion-exchange chromatography into a fucogalactoxyloglucan and a glucuronoarabinoxylan.     

Intercellular Localization of Acid Invertase in Tomato Fruit and Molecular Cloning of a cDNA for the Enzyme

The localization of acid invertase (AI, EC 3.2.1.26 [EC] ) in tomatofruits was studied. AI was localized in the intercellular fraction(cell wall fraction). A cDNA encoding a wall-bound form of AIfrom tomato fruits was cloned and its nucleotide sequence wasdetermined. The cloned cDNA was 2363 base pairs long and containedan open reading frame of 1908 base pairs which encoded a polypeptideof 636 amino acids. RNA blot analysis indicated that the mRNAfor the acid invertase was about 2.5 kb in length. The levelsof the mRNA were low at the mature green stage but increasedduring ripening of fruit. (Received July 13, 1992; Accepted December 3, 1992)     

Five coordinated nitrosylhemoprotein in the whole cells of denitrifying bacterium,Achromobacter xylosoxidans NCIB 11015

The denitrifying bacterium, Achromobacter xylosoxidans NCIB 11015, was cultivated in meat extract-peptone medium and in Mn-free synthetic medium under denitrifying or non-denitrifying conditions. Electron paramagnetic resonance (EPR) spectra for the whole cells of the bacteria thus obtained were measured at 77K. The characteristic three-line signal was observed in the whole cells of the bacteria under denitrifying conditions, but not under non-denitrifying conditions. The three-line signal was more distinctly observed in the cells cultured in Mn-free medium. This signal could be assigned to nitrosylcytochrome c containing a five-coordinated nitrosylheme. The elemental composition in these cells is also discussed.     

The presequence of the precursor to the nucleus-encoded 30 kDa protein of photosystem II in Euglena gracilis Z includes two hydrophobic domains

A cDNA clone for the extrinsic 30 kDa protein (OEC30) of photosystem II in Euglena gracilis Z was isolated and characterized. The open reading frame of the cDNA encoded a polypeptide of 338 amino acids, which consisted of a long presequence of 93 amino acids and a mature polypeptide of 245 amino acids. Two hydrophobic domains were identified in the presequence, in contrast to the presence of a single hydrophobic domain in the presequence of the corresponding proteins from higher plants. At the N- and C-terminal regions, respectively, of the presequence, a signal-peptide-like sequence and a thylakoid-transfer domain were identified. The presence of a long and unique presequence in the precursor to OEC30 is probably related to the complexity of the intracellular processes required for the synthesis and/or transport of the protein in Euglena.Abbreviations ER endoplasmic reticulum - cDNA complementary DNA - SSU small subunit; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase - Rubico, ribulose 1,5 bisphosphate carboxylase/oxygenase - LHC II light-harvesting chlorophyll protein of photosystem II - PS II photosystem II - OEC30 the extrinsic 30 kDa protein of photosystem II in Euglena - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - TE a solution containing 10 mM Tris-HCl and 1 mM EDTA pH 8.0 - SSPE a solution containing 0.15 M NaCl, 10 mM NaH₂PO₄ and 1 mM EDTA pH 7.4 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PVDF poly(vinylidene difluoride)     

Cytological studies on rust fungi. (vi) fine structures of infection process of Kuehneola japonica (diet.) dietel

The behavior of rust fungi in their host plants has been elucidated by electron microscopy. However, most of the ultrastructural studies on rust fungi have focused on the uredial stage. In order to elucidate the features of the sporidial stage, we studied the fine structure of Kuehneola japonica, a short-cycle rust, in rose leaves. Infection pegs arising from appressoria penetrated the host walls. Papillae formed at the time of penetration against the outer epidermal cell walls. The papillae which had formed at the penetration sites grew extensively and partially surrounded the intracellular hyphae which were connected with the infection pegs. The intracellular hyphae in the epidermal cells developed further and entered adjacent parenchyma cells. Walls of parenchyma cells either invaginated or thin papillae formed at penetration sites and the invaginated walls or papillae surrounded the necks of the intracellular hyphae. Intracellular hyphae in both epidermal and parenchyma cells were not enveloped by the sheath before 20 days after inoculation. In specimens prepared 20 days after inoculation, some of the intracellular hyphae were enveloped by a sheath in both palisade and spongy parenchyma cells. The sheathed hyphae resembled haustoria of other rust fungi which had been described previously. Teliospore initials were formed in mycelial masses in intercellular spaces between the epidermal cells and palisade parenchyma cells 20 days after inoculation. Uninucleate teliospores developed from teliospore initials 30 days after inoculation.Contribution No. 32.     

Interaction of amylose and other α-glucans with hydrophobic fluorescent probe (2-p-toluidinylnaphthalene-6-sulfonate)

Fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was enhanced in the presence of maltooligosaccharides, amylose, and other α-glucans. The dependence of relative TNS fluorescence intensity per glucose unit on chain length of oligosaccharides was examined. The values of binding constant and thermodynamic parameters, assuming the 1:1 complex for TNS-amylose (number-average degree of polymerization, DP_N = 17), were determined by the fluorescence titration. The values of thermodynamic parameters for 1:1 complex formation of TNS-α- and β-cyclodextrins were also determined and compared with those of TNS-amylose (DP_N = 17). The fluorescence intensity of TNS in the presence of amylose (DP_N = 600) decreased by the action of glucoamylase and taka-amylase A. The fluorescence of TNS-amylose (DP_N = 17) system increased with the increased ionic strength. In the presence of pullulan, TNS fluorescence was also enhanced and decreased by the action of pullulanase. Amylopectin enhanced TNS fluorescence rather more strongly than amylose (DP_N = 17) at the same concentration. In the presence of dextran, the fluorescence of TNS was scarcely enhanced. The degree of fluorescence enhancement of TNS in the presence of α-glucans seems to reflect the structures of α-glucans in solution, since TNS fluorescence is enhanced in the hydrophobic environment or by the disturbance of free intramolecular rotation.     

A case of trisomy 3q21→qter syndrome

Summary An infant with karyotype 46,XY,der(8),t(3;8)(q21;p23) is presented. The presence of trisomy 3q21qter syndrome is suspected on the basis of comparison of the clinical and laboratory findings of this patient with those of cases that have been reported as partial 3q trisomy. The common phenotypic features of this syndrome include growth failure and mental or developmental retardation, hypotonia, persistent lanugo, distorted head, congenital glaucoma, short and upturned nose, prominent maxilla, micrognathia, short, webbed neck, short limbs, retroflexed third and fourth toes, cutaneous syndactyly of the second, third and fourth toes, and elevated galactose-1-phosphate uridyl transferase activity in the red blood cells.     

Simultaneous analysis of pipecolic acid with proline in the brain by selected ion-monitoring technique

A method for the simultaneous analysis of pipecolic acid and proline in the brain is developed. The qualification and quantification of pipecolic acid and proline are accomplished with gas chromatography/mass spectrometry including a selected ion-monitoring technique by using deuterium-labeled proline as an internal standard, after the amino and carboxylic groups of these cyclic amino acids are derivatized with boron trifluoride methanol complex and heptafluorobutyric anhydride. The lower limit of quantification for the method is picomole levels and the concentration of pipecolic acid and proline in rat whole brain is determined to be 1.05 and 71.50 nmol/g of tissue, respectively.