On screening for microorganisms in soil obtained in Japan that produce large amounts of gamma-cyclodextrin (gamma-CD), we identified a novel alkalophilic bacterium, Bacillus clarkii 7364. The cyclodextrin glucanotransferase (CGTase) secreted into the culture medium by this bacterium was purified by affinity chromatography on a gamma-CD-immobilized column, followed by chromatography on a gel filtration column. The enzyme converted 13.7% of pre-gelatinized potato starch (10% w/w per reaction mixture) into CDs, and the majority (79%) of the product CDs was of the gamma form. This property is quite unique among known CGTases and thus we named this enzyme gamma-CGTase. The N-terminal and internal amino acid sequences of gamma-CGTase were determined and used to design PCR primers for amplification of the nucleotide sequence that encodes the gamma-CGTase gene. The entire gene sequence amplified by PCR was determined and then cloned into E. coli. The recombinant enzyme synthesized by E. coli retained biochemical properties quite similar to those of the original one. Comparison of the deduced amino acid sequence of gamma-CGTase with those of other known CGTases that have different product specificities revealed the importance of subsites -3 and -7 for the preferential gamma-cyclization activity.
The clinical course and computed tomographic (CT) findings of 23 patients with brain tumors manifesting as tumoral hemorrhage were reviewed. The most common symptoms were headache and clouding of consciousness. A CT finding of a lesion located next to a solid or irregular clot indi cated intratumoral hemorrhage. Precontrast CT demonstrating an indent on the hematoma surface was a valuable indicator of tumoral hemorrhage.A CT finding of accumulated levels of blood⁄ fluid or a hyperdense mass containing small hematoma indicated intratumoral hemorrhage, and ob scure hyperdensity indicated intratumoral hemorrhagic infarction. Such findings were often difficult to distinguish from spontaneous intracerebral hemorrhage due to other factors. The incidence of rebleeding from residual tumors was high, carrying a very poor prognosis, so radical removal of brain tumors with hemorrhage is very important.
✓ Subarachnoid hemorrhage (SAH) was produced experimentally by injecting normal dog's blood as well as reserpinized dog's blood into the chiasmal cistern of the dog. The following observations were made: 1) After SAH with normal dog's blood, the intima of the basal truncal arterial wall showed some or all of such ultrastructural changes as appearance of vacuoles and dense bodies in endothelial cells, detachment of endothelial cells, appearance of intimal cells, and intimal thickening. The changes first appeared 2 hours after SAH, culminated at 3 to 7 days after SAH, and persisted up to 1 month after SAH. 2) After SAH with normal dog's blood, the media of the basal truncal arterial wall showed some or all of such ultrastructural changes as moth-eaten contour of muscle cells, appearance of intracytoplasmic vacuoles and dense bodies, appearance of cell debris, enlargement of interstitial space, and appearance therein of dense particles. These findings, which, in short, are to be expressed as myonecrosis and its repairing process, first appeared 2 hours after SAH, culminated at 1 to 4 months after SAH, and persisted up to 1 year after SAH. 3) Three and 5 days following SAH with reserpinized dog's blood, ultrastructural findings of the intima and media of the basal truncal arterial wall were entirely normal. On the basis of the above findings, it was concluded that the ultrastructural changes in the cerebral arterial wall observed after SAH with normal dog's blood occurred as a consequence of vasospasm. The possibility that late spasm, in turn, might be facilitated by myonecrosis, could not be denied.
In the present study, we identified cDNA clones of mouse acrosin from a testis lambda gt11 library. The deduced amino acid sequence indicates that mouse acrosin is initially synthesized as a single-chain polypeptide with a 16-residue signal peptide followed by a 23-residue light chain and then a 394-residue heavy chain; mouse acrosin zymogen contains 417 amino acid residues with a calculated molecular mass of 46,993 Da. The cDNA-derived sequence of mouse proacrosin shows a high degree of similarity with human and porcine proacrosins and major portions of bovine trypsin, including the active site residues, the recognition site for substrate, the location of 12 cysteine residues, and two potential N-glycosylation sites. The sequence homology suggests that mouse proacrosin is converted to a mature acrosin, which consists of the light and heavy chains with a combined molecular mass of 35,587 Da, by cleavage of the peptide bond between Arg23 and IIe24, and sequential removal of 23-, 26-, and 50-residue COOH-terminal segments. Using Northern blot analysis of RNAs from various mouse tissues, the acrosin gene transcript was present only in testis. The 1,800-base acrosin message was first detectable in 18-day-old testis. At the same time of testicular development, some of the acrosin mRNA was actually associated with polysomes. Also, in situ hybridization analysis suggests that the acrosin gene is expressed only in the round spermatid. Therefore, it is most likely that transcription of the mouse acrosin gene and subsequent translation of its mRNA first occur in the early stages of the round spermatid, and that the acrosin message is not under translational control.
beta-N-Acetylhexosaminidase (beta-Hex, EC, 3.2.1.52) was released from cauda epididymal boar sperm by treatment with ionophore A23187, indicating that this enzyme is localized in the acrosome. beta-Hex was extracted on a large scale, with 2% acetic acid containing 0.2% Brij 35, from washed ejaculated sperm. By gel filtration chromatography, beta-Hex was separated into a high-molecular-weight fraction (beta-Hex I) and a low-molecular-weight fraction (beta-Hex I). beta-Hex I, which is predominant under acidic conditions (pH 6.5), dissociated into beta-Hex II under alkaline conditions (pH 7.4). beta-Hex II, converted from beta-Hex I, associated again to form beta-Hex I under acidic conditions. By sequential chromatography on ion-exchange, lectin, gel filtration, and ion-exchange HPLC columns, beta-Hex I and II were purified 1200-fold and 4000-fold, respectively, with a combined recovery of 23% as measured with synthetic substrate. An inhibitor of beta-Hex, O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl-carbamate (PUGNAC), reduced the in vitro fertilization rate in porcine cumulus-enclosed eggs, but barely changed the rate when cumulus-free eggs were used. beta-Hex I was shown to possess cumulus dispersion activity, suggesting that beta-Hex plays a role in the passing by sperm through cumulus cells before they bind to the zona pellucida.
Water-soluble dietary fiber provides numerous health benefits. A novel procedure to efficiently manufacture water-soluble indigestible polysaccharides was developed by heating glucose at 180 C in the presence of activated carbon. Aside from its ability to catalytically assist the polycondensation of saccharides, activated carbon provides the added benefits of being easily separable from the reactants and suppressing coloration of the product. Prior to purification, the indigestible fraction made up over 80% of the reaction mixture. After hydrolysis catalyzed by α-amylase and glucoamylase, and fractionation by ionexchange chromatography, a total of 99.7% dietary fiber content was attained. This indigestible fraction, termed resistant glucan, was only minimally degraded by upper digestive tract enzymes, similar to the digestibility of polydextrose. Structural analysis by methylation and NMR indicated that the resistant glucan formed a highly branched structure containing α-and β-1,2-, 1,3-, 1,4-, and 1,6-linkages. On an industrial scale, the resistant glucan was obtained from glucose syrup (DE 86) by heating with activated carbon, enzymatic hydrolysis, refining, fractionating, and drying. Our facile method is an efficient means to obtain water-soluble dietary fiber.
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