Existence of a Novel Hemagglutinin Having No Protease Activity in Vibrio mimicus

The protease elaborated by Vibrio mimicus is known to possess hemagglutinating ability to chicken erythrocytes, the well-known HA/protease. A non-protease hemagglutinin (HA) with strong agglutinating ability towards rabbit erythrocytes was obtained from 32 hr culture supernatant of a pathogenic environmental strain of V. mimicus. This HA (V. mimicus HA: VMHA) appeared stable at relatively higher temperature and agglutinated the erythrocytes from rabbit, guinea pig and mouse but not the erythrocytes from chicken, bovine, horse and sheep. Simple sugars, metal ions and chelating agents failed to inhibit the activity of VMHA. The activity of VMHA was found to be sensitive to digestion by proteolytic enzymes including HA/protease. These results provide evidence for the existence of novel HA other than HA/protease in V. mimicus.     

A novel and efficient method for enzymatic synthesis of high purity maltose using moranoline (1-deoxynojirimycin).

A transglycosylation reaction with moranoline (1-deoxynojirimycin) was done with soluble starch as the glucosyl donor and Bacillus macerans amylase as a cyclodextrin glycosyltransferase [EC 2.4.1.19]. The resultant transglycosylation products with moranoline, obtained by treating the reaction mixture with a strong cation exchange resin, were hydrolyzed by beta-amylase [EC 3.2.1.2] from sweet potatoes. The hydrolysate was treated with a strong cation exchange resin, and high purity maltose was obtained.     

Histochemical study on the maturation of human megakaryocytes using microfluorometry

Summary A microcytofluorometrical DNA measurement was basically studied and was applied to single megakaryocytes previously identified on a Wright-Giemsa stained smear. The smear was first photographed and the location of each megakaryocyte was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained with 4,6-diamidino-2-phenylindole (DAPI) reagent (pH 7.4) at 4° C. Nuclear blue fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA with the coefficient of variation (CV) of 3.6% when stained for 30 min. After 30 min DAPI staining, the DNA measurement was microcytofluorometrically performed in single megakaryocytes which had been morphologically classified into 4 groups on the basis of cytoplasmic maturation, Bessis' classification, assessed on Wright-Giemsa-stained bone-marrow smears from normal human beings. The histograms of the cells did not show any difference in DNA ploidy distribution among the classes: that is, the DNA histograms disclosed ploidy distribution from 4 N to 64 N with the largest population of 16 N. These findings suggest that nuclear DNA synthesis is completed before platelet production starts. This method is useful for comparing the morphological features and DNA content of single megakaryocytes.     

Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically

 We devised a new microfluorometric method for determining the ploidy of megakaryocytes identified immunologically in bone marrow smears. The smears were immunostained by incubation with mouse monoclonal anti-glycoproteins (GP) IIb antibodies, followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies. They were then stained with 4′,6-diamidino-2-phenylindole (DAPI). Megakaryocytes were identified by their GPIIb immunofluorescence using a microfluorometer and, after the filters were changed, their DNA content was assayed by measuring the intensity of DAPI fluorescence. This intensity was shown to be proportional to the DNA content when the aperture of the objective lens was reduced. We compared these results with those obtained when megakaryocytes were identified morphologically, using DAPI staining after Wright-Giemsa destaining. In all 12 normal controls, the ploidy peaks were shown to be 16N by both methods, and the mean ploidy detected by the immunological method was only reduced 0.961 times relative to the estimate from the morphological method. In contrast, in eight myelodysplastic syndrome (MDS) patients, the ploidy peaks were either 8N or 4N and the mean was reduced by 0.906 times (P=0.018). Thus we could immunologically identify small megakaryocytes which we could not identify morphologically. Therefore, this method is useful for measuring megakaryocytic ploidy, especially in the pathological megakaryocytes of MDS patients. Accepted: 29 April 1997     

DNA polymerases and DNA topoisomerases solubilized from nuclear matrices of regenerating rat livers

DNA topoisomerase activity together with the activities of DNA polymerase were detected in a form tightly associated with rat liver nuclear matrices. DNA polymerase activities were solubilized from the nuclear matrices of regenerating rat livers by sonic treatment followed by extraction of these activities with detergent and salt. The predominant activity was mainly α-polymerase as judged from the size determined by sucrose density gradient centrifugation. However, only β-polymerase activity was detected in the matrix of normal rat livers. DNA topoisomerase activity, detected in both regenerating and normal liver nuclear matrices, showed a molecular size of about 4 S in sucrose gradient, and was active in the presence of EDTA. These results suggest that this enzyme belongs to type I topoisomerase.     

Purification and characterization of extracellular soluble and membrane-bound insoluble alkaline phosphatases possessing phosphodiesterase activities in Bacillus subtilis.

A membrane-bound insoluble alkaline phosphatase (APase) and an extracellular soluble APase were purified, respectively, from a membrane preparation of Bacillus subtilis 6160-BC6, which carries a mutation to produce APase constitutively, and from a culture fluid of a mutant strain. RAN 1, isolated from strain 6160-BC6, which produces an extracellular soluble APase. The two preparations were homogeneous, as judged by sodium dodecyl sulfate discontinuous gel electrophoresis and by gel electrophoreses in the presence of 8 M urea at pH 9.3 and 4.3. RAN 1 APase was crystallized. Both preparations possessed phosphatase and phosphodiesterase activities, and their pH optima were both at 9.5. They were competitively inhibited by phosphate or arsenate and were activated by the addition of Ca2+ but not by Zn2+. The APase and alkaline phosphodiesterase activities seemed to be contained in the same protein molecule. The molecular weight of 6160-BC6 APase was estimated to be 46,000 +/- 1,000, and that of RAN 1 APase was estimated to be 45,000 +/- 1,000. The largest difference between the 6160-BC6 and RAN 1 APase's was in solubility in low-ionic-strength solutions. Present results suggest that each enzyme is composed of a single polypeptide chain and that 6160-BC6 APase aggregates in solutions of low ionic strength.     

Kinetic study of denaturation and subsequent reduction of disulfide bonds of lysozyme by the rapid ultrasonic absorption measurement

A New technique for the rapid measurement of ultrasonic absorption with a sampling interval of 5 msec has been developed and applied to the kinetic study of denaturation and subsequant redution of hen egg-white lysozyme. The lysozyme is denatured by guanidine hydrochloride (GuHCl) orLiBr, and afetr denaturation by GuHCl, its disulfide bonds are reduced by dithiothreitol (DTT). The ultrasonic adsorption coefficient at 9 MHz increases with denaturation but decreases with reduction. The rate constant of denaturation by GuHCl obtained from the rime variance of ultasonic agrees well with that from uv absorption and optical rotation. The time variance if absorption after GuHCl and Dtt have been simultaneously added exhibits two rate constants. Analysis of the constants as functions of regeant concentrations indicates that the intermediates state between native and reduced states is not necessarily the completely denatured state but depends on the concentartions of GuHCl and DTT.     

Action of sweet-potato beta-amylase on phosphodextrin of potato starch

The specificity of sweet-potato beta-amylase in the vicinity of the phosphate ester groups was studied by determining the structures of the phosphorylated oligosaccharides (alpha-phosphodextrin and beta-limit-alpha-phosphodextrin) formed by its action on potato starch. The beta-limit-alpha-phosphodextrin was separated by chromatography on Dowex-1 (HCOO^?) resin into three fractions that were distinguishable by the d.p. and by the ratio of d-glucose 6-phosphate residues to total organic phosphate. Each fraction contained linear molecules having one phosphate ester group that was not located at the reducing or non-reducing terminals. The smallest phosphodextrin was 6²-phosphorylmaltotriose. It was deduced that beta-amylase hydrolysed (1→4)-α-d linkages from the non-reducing end until one or two d-glucosyl residues remained attached to the phosphorylated residue, depending on whether there was originally an odd or even number of glucosyl residues on the non-reducing side of the phosphorylated residue.