Biomedical subjects
Publications and source records attributed to K Horikoshi.
Two plasmids containing the penicillinase gene of alkalophilic Bacillus sp. strain 170, pEAP1 and pEAP2, were constructed. Most of the penicillinase produced by Escherichia coli, which carried these plasmids, was found in the culture medium. This excretion is caused by the cloned DNA fragment which contains some component that changes the outer membrane of E. coli.
Using the solid phase phosphotriester method, a gene coding for human fibroblast interferon consisting of 166 amino acid residues was chemically synthesized. The gene obtained by ligation of 61 synthesized oligodeoxyribonucleotide fragments was inserted into the downstream of tryptophan promoter, and expressed in E. coli. Lysate of this E. coli showed the antiviral activity which was specifically neutralized by anti-fibroblast interferon antibody. No particular advantage was observed in the expression efficiency by the synthetic gene over that by the native gene.
An extracellular polyamine oxidase produced by Penicillium sp. No. PO-1 was completely purified using the chromatofocusing method with a very high yield (93%) of the activity. The enzyme was composed of two identical subunits (Mr 64 000) and contained FAD. The optimal pH for activity was approx. 4.0. The enzyme oxidized spermidine and spermine. Km and Vmax values for spermidine were respectively 8.2 microM and 16.4 mumol H2O2/mg protein per min. Corresponding values for spermine were 5.3 microM and 13.3 mumol H2O2/mg protein per min. The enzyme attacked the secondary amino group of spermidine and spermine, and produced putrescine, 3-aminopropionaldehyde and H2O2. The enzyme activity was completely inhibited by phenylhydrazine. However, sulfhydryl reagents showed no effect on the activity. It is expected that the enzyme will be useful in determining the amount of polyamine in body fluids.
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NAD+-dependent maltose dehydrogenase was purified about 250-fold from the cell free extract of an alkalophilic Corynebacterium sp. No. 93-1. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight of the enzyme was determined to be 40 000 +/- 2000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme appeared to be a single peptide chain. The isoelectric point was pH 4.50. The optimal pH was 10.2. The enzyme was stable over the range of pH 6 to 10. NAD+-dependent maltose dehydrogenase showed very wide substrate specificity on monosaccharides, disaccharides and trisaccharides. Among these substrates, maltose was the most reactive. Also, the enzyme showed oxidative activity on maltotetraose and maltopentaose. The Km values at pH 10 were 2.1 mM for maltose and 0.15 mM for NAD+. It was conjectured that the primary product of this reaction was maltono-delta-lactone and its was hydrolyzed non-enzymatically to maltobionic acid. p-Chloromercuribenzoic acid, Hg2+ and Ag2+ completely inhibited the activity, and HADH also showed competitive inhibition on the activity.
Alkalophilic Bacillus No. 8-1 and No. 10A-2, isolated on alkaline media containing NaHCO3 and K2CO3, respectively, required Na+ specifically for the uptake of alpha-aminoisobutyric acid (AIB) into the cells. The K+ added to the uptake medium inhibited the accumulation of AIB in the cells of Bacillus No, 8-1, acting as a competitive inhibitor of the system. Sodium ion prevented the release of AIB from pre-loaded cells. Both potassium and lithium ions had the effect of retaining the AIB pool. The Vmax value for transport increased about five-fold when the pH value of medium was raised from 7 to 9, whereas the Km value decreased with the increase of concentration of sodium ion in the medium of pH 7 or 9. Sodium ion gradient across the membrane caused transient uptake by AIB by membrane vesicles of Bacillus No. 8-1.
Membrane vesicles were isolated from alkalophilic Bacillus No. 8-1, and the active transport of amino acids was studied. The transport of amino acids was dependent upon substrate oxidation and the presence of Na+. Concentrative uptake of amino acids was stimulated by the addition of an artificial electron donor system, ascorbate-phenazine methosulfate (PMS), and to a lesser extent by NADH, while succinate, L-lactate, and alpha-glycerol-phosphate did not stimulate the uptake. N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD) and cytochrome c were able to replace PMS, and reduced forms of these compounds were also very efficient electron donors. Amino acid transport was dependent on electron transfer, and inhibition of NADH oxidation by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), and sodium azide directly prohibited serine transport. The pH optima for serine transport lay between pH 8 and 9 for all energy sources. Sodium ion stimulated serine transport in the presence of NADH, NADH plus cytochrome c or succinate plus PMS, but had no stimulatory effect on the corresponding dehydrogenase activities. Sodium ion was also required for accumulation of serine in response to an artificial membrane potential where the respiratory chain was not operative. These results indicated that the stimulatory effect of Na+ on amino acid uptake was on the transport process itself.
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From 1971 to 1977, a total of 43 cases with ventricular septal defect associated with pulmonary hypertension (VSD + PH), ranging from 3 months to 6 years of age, underwent VSD closure using cardiopulmonary bypass with a prophylactic dose of phenoxybenzamine (POB) 1 mg/Kg. Twenty of these cases are included in the present study of the effect of POB on pulmonary hypertension. The ratios of pulmonary to systemic arterial pressures (Pp/Ps) and vascular resistances (Rp/Rs) were measured before, immediately after and 1 month after VSD closure. In all cases, the Pp/Ps before VSD closure was larger than 0.75. Both Pp/Ps and Rp/Rs markedly decreased immediately after VSD closure but rose again 1 month thereafter. These results suggest the possibility of pulmonary vasodilating effect of POB, which could be potentially useful in relieving the right ventricular load in the early postoperative period. The over all mortality was 3 out of 43 cases including 2 late deaths.
Lung biopsy specimens were taken from 39 infants younger than 12 months of age with congenital heart disease and severe pulmonary hypertension (Pp/Ps greater than or equal to 0.75) accompanied by respiratory distress. The pathological change in lung specimens and clinical courses were compared. These 39 infants underwent surgical treatment of patent ductus arteriosus (PDA), seven patients; ventricular septal defect (VSD), 13 patients; and complex heart anomaly, 19 patients. The common pathological findings of the lung specimens taken from these infants were lymphoid cellular infiltration and thickening of the alveolar septum, which we have called "septitis" in the present study. In most cases pulmonary vascular obstructive change was within Grade 2 of the Health-Edwards criteria. Septitis was classified into three categories: mild, moderate, and severe. Only three of the 19 infants with severe septitis survived postoperatively, whereas 10 of the 12 infants with moderate septitis and all eight with mild septitis could be successfully weaned. The cause of septitis remains unidentified. We have found the patient's age and pulmonary hypertension to be closely related to the grade of septitis in this study. Septitis plays a much more important role than pulmonary vascular obstructive change in the prognosis of pulmonary hypertensive heart disease in early infancy.
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Succinylated cyclodextrin glycosyltransferase (EC 3.2.1.19) of an alkalophilic Bacillus sp. was adsorbed on a vinylpyridine copolymer. The enzyme had about 25% of the activity of soluble enzyme added. No increase of pH or thermal stability of the enzyme was observed by the adsorption, whereas optimum temperature for the enzyme action was shifted from 50 to 55 degrees C. The enzyme converted starch to cyclodextrine without significant loss of activity under the conditions of 4 times reusing of 6 hr conversion by the batch system or 2 weeks continuous reaction by the column system at 55 degrees C and pH 8.0. About 46% of the potato starch solution [15% (w/v)] was converted to cyclodextrins by the enzyme, and 52% was converted by the simultaneous action of the enzyme and alkaline pullulanase of alkalophilic Bacillus sp. (No. 202-1). These values were almost the same as those obtained by the soluble enzyme or enzymes system.
Alkalophilic Bacillus no. 8-1 grows well in alkaline media containing 2.5 to 5% NaCl. The uptake of alpha-aminoisobutyric acid (AIB) into the cells is stimulated by the addition of NaCl (Na+) up to a concentration of 0.2 M, but other monovalent cations such as K+, Li+, or NH4+ cannot substitute for Na+. The kinetic studies reveal that, when the Na+ concentration increases from 0.02 to 0.2 M in alkaline medium, the Km for transport decreases, whereas Vmax remains almost constant. Competition studies indicate that glycine, L-alanine, L-serine, and AIB share common carriers for the transport of the compounds into cells. Other alkalophilic bacteria are also found to require Na+ for the uptake of AIB into the cells.
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