Hiroto Akiyoshi

Alteration of Cell Wall Composition Leads to Amphotericin B Resistance in Aspergillus flavus

An amphotericin B (AmB)‐resistant mutant was isolated from a wild‐type AmB‐susceptible strain of Aspergillus flavus by serial transfer of conidia on agar plates containing stepwise increased concentrations of AmB up to 100 μtg ml−1. The acquired resistance of my celia was specific for polyene‐antibiotics AmB, nystatin and trichomycin. Spheroplasts derived from the resistant mycelia were as susceptible to AmB as the wild‐type. Chemical analysis of the cell wall revealed that levels of alkali‐soluble and ‐insoluble glucans were significantly higher in the resistant mycelia as compared to those in the wild‐type. When resistant mycelia were treated with SDS, they adsorbed as much AmB as wild‐type mycelia. These results suggest that alterations in the cell wall components of mycelia, especially 1,3‐α‐glucan and protein complex in the outermost wall layer, lead to AmB resistance in A. flavus.

Effects of somatostatin on gastrointestinal hormone-induced glycogenolysis and gluconeogenesis in cultured liver cells

SummaryWhen isolated rat liver cells were incubated for 15 min in the presence of vasoactive intestinal peptide, gastric inhibitory polypeptide, secretin or glucagon at a concentration of 2.0μg/ml, glycogenolysis was stimulated by 30%–67% above the control. Slight but significant increase on gluconeogenesis was also observed by the addition vasoactive intestinal peptide, gastric inhibitory polypeptide or secretin. Somatostatin inhibited both glycogenolysis and gluconeogenesis induced by these hormones, but the degrees of inhibition are clearly much higher in the hormone-induced gluconeogenesis than glycogenolysis, and no significant inhibition of glycogenolysis was observed in case of glucagon and VIP.These results suggest the possibility that the so-called enterohepatic axis may play a part of roles in the regulation of serum glucose levels through gastrointestinal hormones belonging to the secretin family, and that it may be further regulated by somatostatin through gluconeogenesis.

Conversion of dNTP to dNMP dependent on DNA synthesis in isolated Yoshida sarcoma nuclei

Nuclei isolated from Yoshida sarcoma cells had activity for conversion of dGTP dependent on DNA synthesis. The ratio of nucleotide generation/generation + incorporation was 0.4 +/0- 0.1, indicating that approx. 40% of the incorporated dGMP was excised. Two lines of evidence indicated the dependence of this activity on DNA synthesis. (1) The activity was observed only in the presence of ATP, which is essential for nuclear DNA synthesis. (2) Inhibitors of DNA synthesis, such as N-ethylmaleimide, aphidicolin, spermine and KCl, also inhibited ATP- or DNA synthesis-dependent dGMP generation. Although nuclei contain nucleoside triphosphatase (N-nucleotidase), this enzyme was not involved appreciably in DNA synthesis-dependent dGMP generation. The reason for this was explained by the following findings. (a) Inhibitors did not decrease dGMP production in the complete absence of DNA synthesis. (b) Inhibitors did not inactivate N-nucleotidase to the same degree as they inhibited DNA synthesis-dependent dGMP generation. (c) Addition of ATP reduced dGMP hydrolysis catalyzed by N-nucleotidase. (d) GDP has no appreciable effect on DNA synthesis-dependent dGMP generation, but had a diluting effect on dGMP production catalyzed by N-nucleotidase. These results show that the pathway of dGMP generation in isolated nuclei was switched on addition of ATP from a N-nucleotidase-catalyzed one to a DNA polymerase-exonuclease-catalyzed one.

Differential effects of propranolol on vasoactive intestinal peptide-, and epinephrine-induced glycogenolysis in cultured liver cells

SummaryWhen isolated rat liver cells were incubated in the presence of epinephrine and dopamine at concentrations ranging from 0.2 μg to 2.0μg per ml, glycogenolysis was maximally stimulated within 15 min. The combined addition to incubation medium showed that somatostatin did not inhibit the stimulated glycogenolysis induced by epinephrine and dopamine. On the other hand, the combined addition to the incubation medium showed that propranolol inhibited the stimulated glycogenolysis induced by epinephrine, while propranolol did not inhibit the stimulated glycogenolysis induced by vasoactive intestinal peptide. These results suggest that the receptor site for vasoactive intestinal peptide may be different from that of epinephrine.

Effects of secretin on glycogenolysis in cultured liver cells

SummaryUpon incubation of hepatocyte isolated from rats with secretin at concentration ranging from 0.1 unit to 1.0 unit per ml, glycogenolysis was maximally stimulated after 15 min. Somatostatin inhibited liver glycogenolysis. The combined addition to the incubation medium showed that somatostatin inhibited the stimulated glycogenolysis induced by secretin, while secretin plus vasoactive intestinal peptide showed no additive effects on glycogenolysis, as compared with single addition of secretin. However, the addition of glucagon to secretin showed additive effects on glycogenolysis.These results suggest that the receptor site for secretin might be different from that for glucagon, but the same as that for vasoactive intestinal peptide. Moreover, the calcium ion was shown to be a prerequisite for the inducible effects of the hormones so far tested.

Evidence for the presence of chromatin-bound deoxyribonucleic acid phosphatase-exonuclease in Yoshida sarcoma ascites cells.

Abstract An enzyme which cleaves the phosphoester bond of 3′-phosphoryl termini of DNA was isolated and purified from the chromatin of Yoshida sarcoma cells. The DNA phosphatase is specific for only 3′-phosphorylated DNA with a lesser activity for its single stranded form. Phosphoester bonds of various nucleotides, 3′-phosphorylated RNA and 5′-phosphorylated DNA were not hydrolysed by the enzyme. The DNA phosphatase required 10 mM MgCl2, and was inactivated by 70 % with 1 mM ϱ-chloromercuribenzoate and completely by heat treatment at 70° for 5 min. Furthermore, an exonuclease activity could not be separated from the purified DNA phosphatase.