Trypsin-like Hatching Enzyme of Mouse Blastocysts: Evidence for Its Participation in Hatching Process before Zona Shedding of Embryos6

Effects of twelve protease inhibitors on hatching of mouse embryos were investigated. Mouse hatching was strongly or moderately inhibited by trypsin inhibitors including p-toluenesulfonyl-Lys-CH₂Cl (TLCK) and chicken ovomucoid, while inhibitors for chymotrypsin and elastase showed weak or no inhibition. These results indicate the participation of a trypsin-like protease in the hatching of mouse embryos as a hatching enzyme., Since TLCK is the strongest and an irreversible inhibitor for the enzyme, timing of the participation of the hatching enzyme in the hatching process was examined by pulse treatment of embryos with TLCK before and during the zona shedding. The results indicated that a trypsin-like hatching enzyme functions before, but not during, the zona shedding of embryos, especially during a 15 h period immediately before the beginning of the shedding.     

Topographic pattern of the rearrangement of the dystrophin gene in Japanese Duchenne muscular dystrophy

To compare the frequency and distribution of rearrangements in the dystrophin gene in Duchenne muscular dystrophy (DMD) between Japanese DMD patients and those in North America and Europe, Southern blot analyses of the dystrophin gene were carried out in 88 probands classified as DMD. Gene rearrangements were found in 61 (69%) subjects, and they were composed of partial gene deletions in 53 (60%) probands and partial duplications in 7 (8%) probands. A total deletion of the gene was found in 1 (1%) patient. Among 53 patients with deletions, 34 (64%) had breakpoints between introns 44 and 52 and 7 (13%) had breakpoints between introns 2 and 11. Both the frequency and the distribution of gene rearrangements found in this study were similar to those reported in North America and Europe. These data suggest that there are no ethnic or racial differences in the frequency and distribution of rearrangements thought to be caused by similar mechanisms in the dystrophin gene in all human racial groupings.     

Duplication detection in Japanese Duchenne muscular dystrophy patients and identification of carriers with partial gene deletions using pulsed-field gel electrophoresis

DNA samples from 21 unrelated Japanese patients with Duchenne muscular dystrophy (DMD) with nondeletion-type abnormality in the dystrophin gene and three samples from possible deletion carriers were analyzed using pulsed-field gel electrophoresis (PFGE). Among the 21 patients, 7 were found to carry partial duplications of the dystrophin gene spanning 50–400 kb. Of these 7 patients, 4 carried duplications corresponding to the major hot-spot regions for deletions (7.5–8.5 kb from the 5 end of cDNA), whereas two cases contained duplications in a region about 10 kb from the 5 end of cDNA, where causative mutations are reported to be rare. Only 1 case was found to contain a duplication of a region about 1 kb from the 5 end of cDNA, which is the reported duplication prone region. A combination of Southern blot analyses of conventional agarose gel electrophoresis and PFGE was confirmed to be useful, not only for detecting duplications and deletions, per se, but also for identifying carriers in the affected family.     

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)