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Biomedical subjects

M Fukagawa

Publications and source records attributed to M Fukagawa.

At least 73 records · Page 4Linked to original sources
PubMed1991

Regulation of parathyroid hormone synthesis in chronic renal failure in rats.

To clarify the mechanism of secondary hyperparathyroidism in chronic renal failure at the parathyroid hormone (PTH) synthesis level, we measured PTH messenger RNA (mRNA) levels in parathyroid glands in a rat model of chronic renal failure. Four weeks after 7/8 nephrectomy, hyperplasia of parathyroid glands was evident and serum PTH levels were elevated. Serum concentration of calcium, inorganic phosphate, and 1,25-dihydroxyvitamin D (1,25(OH)2D) of rats with chronic renal failure were not detectably different from those of sham-operated rats. In chronic renal failure rats, PTH mRNA levels were elevated both per RNA and per DNA of parathyroid cells, suggesting increased PTH mRNA levels per cell. The elevated levels of PTH mRNA were returned to normal levels by achieving supraphysiological concentrations of 1,25(OH)2D3 given i.p. twice at 24 and 48 hours before sacrifice, although this was attended by slight hypercalcemia. A synthetic analogue of vitamin D, 22-oxa-1,25(OH)2D3, also suppressed PTH mRNA to normal levels, but without hypercalcemia. These data suggest that secondary hyperparathyroidism in early chronic renal failure may be due in part to the resistance of parathyroid cells to the physiological concentration of 1,25(OH)2D in circulation on PTH synthesis and that 22-oxa-1,25(OH)2D3 may be useful in the management of secondary hyperparathyroidism of chronic renal failure.

Animals↗
PubMed1991

Construction of a 7-aminocephalosporanic acid (7ACA) biosynthetic operon and direct production of 7ACA in Acremonium chrysogenum.

We have used cDNA encoding D-amino acid oxidase, and genomic DNA encoding cephalosporin acylase from Fusarium solani and Pseudomonas diminuta, respectively, to construct a novel hybrid 7-aminocephalosporanic acid (7ACA) biosynthetic operon under the control of regulatory elements from the alkaline protease gene of Acremonium chrysogenum. Transformants of A. chrysogenum BC2116, a high cephalosporin-producing strain, containing this operon, synthesized and secreted low levels of 7ACA. Although the amounts are not yet commercially significant, this represents the first microbial production of 7ACA and demonstrates the feasibility of introducing new biosynthetic capabilities into industrial microorganisms by combining fungal and bacterial genes.

Acremonium↗
PubMed1991

Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis.

A strain of Bacillus species which produced an enzyme named glutaryl 7-ACA acylase which converts 7 beta-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) to 7-amino cephalosporanic acid (7-ACA) was isolated from soil. The gene for the glutaryl 7-ACA acylase was cloned with pHSG298 in Escherichia coli JM109, and the nucleotide sequence was determined by the M13 dideoxy chain termination method. The DNA sequence revealed only one large open reading frame composed of 1,902 bp corresponding to 634 amino acid residues. The deduced amino acid sequence contained a potential signal sequence in its amino-terminal region. Expression of the gene for glutaryl 7-ACA acylase was performed in both E. coli and Bacillus subtilis. The enzyme preparations purified from either recombinant strain of E. coli or B. subtilis were shown to be identical with each other as regards the profile of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were composed of a single peptide with the molecular size of 70 kDa. Determination of the amino-terminal sequence of the two enzyme preparations revealed that both amino-terminal sequences (the first nine amino acids) were identical and completely coincided with residues 28 to 36 of the open reading frame. Extracellular excretion of the enzyme was observed in a recombinant strain of B. subtilis.

Amidohydrolases↗
PubMed1991

Cloning and nucleotide sequences of the complementary and genomic DNAs for the alkaline protease from Acremonium chrysogenum.

Complementary DNA encoding Ac. chrysogenum alkaline protease (Alp) was isolated from the Ac. chrysogenum ATCC11550 cDNA library by express-blot assay. The genomic DNAs encoding Ac. chrysogenum Alp were isolated from the Ac. chrysogenum genomic DNA library using the cloned cDNA as a probe. The 3150 nucleotides of the gene were sequenced. The prepro-Alp consists of 402 amino acids and two intervening sequences are found within the coding region. The amino acid sequence of Ac. chrysogenum Alp has 57% homology to that of Aspergillus oryzae Alp. The entire cDNA, encoding Ac. chrysogenum Alp, when introducing into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium.

Acremonium↗
PubMed1989

Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messenger RNA in the rat.

To examine the effects of serum calcium concentrations on PTH biosynthesis, rats were made hyper- (serum total calcium, approximately 3.5 mM) or hypocalcemic (approximately 1.25 mM) and steady-state levels of PTH mRNA in parathyroid cells were measured by the primer extension method using a 32P-labeled synthetic oligomer. PTH mRNA levels increased about twofold in the rats made slightly hypocalcemic by infusion of calcium-free solution and decreased slightly in those made hypercalcemic by CaCl2 infusion (120-150 mumol/h) compared with the levels present in nonfasting control rats. Infusion of calcitonin (0.5 U/h) or EGTA (90 mumol/h) with calcium-free solution increased PTH mRNA levels further (two- to sevenfold) above the levels present in animals infused with calcium-free solution alone. These changes in PTH mRNA levels were observed after 48- but not 24-h infusion, and there was an inverse correlation between PTH mRNA levels and serum calcium concentrations. The results suggest that changes in serum calcium concentrations in the near physiological range regulate the biosynthesis of PTH by affecting steady-state levels of PTH mRNA when hypercalcemia or hypocalcemia continues for a relatively long period.

Actins↗
PubMed1989

A case of the watery diarrhea-hypokalemia-achlorhydria syndrome: successful preoperative treatment of watery diarrhea with a somatostatin analogue.

A 35-year-old man presenting with severe watery diarrhea was diagnosed as having the watery diarrhea, hypokalemia and achlorhydria (WDHA) syndrome with the elevation of plasma vasoactive intestinal peptide (VIP) level. Imaging diagnostic techniques revealed a hypervascular tumor at the tail of the pancreas as well as a solitary liver metastasis. During the patient's stay in hospital, he developed acute renal failure probably due to persistent dehydration and severe hypokalemia. Although these complications improved with artificial dialyses, severe watery diarrhea continued, which made it difficult to achieve surgical resection of the tumor. A new long-acting and potent somatostatin analogue, SMS 201-995 (Sandoz Ltd, Basel, Switzerland), was tested and was shown to be effective; after a few hours of subcutaneous injection of this agent, the watery diarrhea disappeared, which in turn improved the patient's hypokalemia, hypercalcemia and metabolic acidosis. Three weeks later, distal pancreatectomy with splenectomy and hepatic lobectomy were successfully performed, and the patient resumed his normal life. The somatostatin analogue has been reported to be useful in the long-term treatment of patients with inoperable WDHA syndrome. The present case demonstrated that short-term administration of this agent is also useful for improving the condition of WDHA patients at the preoperative stage.

Adenoma, Islet Cell↗
PubMed1984

Restoration of antigenicity of tissue antigens, cell-bound immunoglobulins and immune deposits in paraffin-embedded tissue. The influence of fixation and proteolytic enzymatic digestion.

The possibility of preservation and restoration of antigenicity of some antigens in paraffin-embedded tissue was evaluated by direct immunofluorescent technique on deparaffinized sections. Fixation with 96% ethanol-1% acetic acid, 10% neutral buffered formalin and p-formaldehyde was useful for the preservation of tissue antigens and immune deposits, whose antigenicity could be easily restored by trypsin digestion. Neutral buffered formalin was also a satisfactory fixative in immunofluorescent staining on lymphocyte/plasma cell-bound immunoglobulins. Fixation with alcohol-Bouin's fluid showed contrast results; feasible for staining of cell-bound immunoglobulins, but poor for that of glomerular immune deposits. After papain digestion, BSA and lysozyme, antigens of immune complexes, were easily detected in experimental chronic serum sickness glomerulonephritis. Pepsin was more efficient than trypsin in restoring the antigenicity of renal tissue antigens such as fibronectin and polyantigenic basement membrane, but the brush border antigen of the proximal renal tubules was frail to the pepsin digestion. In general, the enzymatic digestion time necessary for the restoration of antigenicity was in parallel with fixation time. Results obtained have shown that deparaffinized sections could be used as satisfactory substrate for immunohistochemistry when proper fixation and efficient proteolytic enzymatic pretreatments were performed.

Animals↗