Characterization of low dose streptozotocin-induced progressive diabetes in mice.

Our previous study indicated that a single i.p. injection of 100 mg/kg streptozotocin (STZ) is able to induce slowly progressive diabetes mellitus in adult ICR mice. In the present study, the non-fasting serum insulin levels of the mice administered 100 mg/kg STZ were normal throughout the 24-week-observation after STZ injection. In the STZ-administered mice, the area of islets and the number of insulin-immunoreactive cells (beta-cells) were normal at 1 week and then continued to decrease gradually as the time went on. In contrast, there was a relative increase in the number of glucagon-immunoreactive cells (alpha-cells) in these mice. In addition, in the STZ-administered mice, the degree of glucose tolerance continued to reduce from 2 weeks till 12 weeks when the experiment terminated. The rise in serum insulin levels stimulated by glucose in the STZ-administered mice began to subside from about 2 weeks and had completely ceased by 12 weeks. These results indicate that 100 mg/kg STZ-induced diabetic mouse model is non-insulin-dependent diabetes, which is characterized by impaired insulin response to glucose stimulation.

New model of progressive non-insulin-dependent diabetes mellitus in mice induced by streptozotocin.

This study was designed to clarify the relationship between streptozotocin (STZ) dosage (100, 150 and 200 mg/kg i.p.) and the resulting diabetogenic response in mice (8-week-old male ICR). In this experiment, we found that a single i.p. injection of 100 mg/kg STZ is able to induce progressive diabetes mellitus, in which non-fasting serum glucose levels begin to rise from 3 weeks and continue to rise throughout the experimental period until 9 weeks. The non-fasting serum insulin levels of 100 mg/kg STZ-treated mice were normal during the experimental period. In addition, the population of insulin-immunoreactive cells (beta cells) in the islets of pancreata was slightly less than in normal mice at 9 weeks. In 200 mg/kg STZ-treated mice, on the other hand, the insulin levels were below measurable values and insulin-immunoreactive cells were not observed. It is concluded from these results that the progressive diabetic mouse model induced by a single i.p. injection of 100 mg/kg STZ, unlike 200 mg/kg STZ-induced diabetes which is insulin-dependent, is non-insulin-dependent.

Impaired relaxing response to isoprenaline in isolated thoracic aorta of nephrotic rats: decrease in release of EDRF from endothelial cells.

Isoprenaline-induced relaxation was investigated in aortas from control and daunomycin-induced nephrotic rats. In the endothelium-intact aortas precontracted with phenylephrine, the isoprenaline-induced relaxation and cyclic adenosine monophosphate (AMP) accumulation were significantly less in nephrotic rats than in control animals. Removal of the endothelium, pretreatment with methylene blue (MB), a guanylate cyclase inhibitor, or NW-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, markedly reduced the relaxation induced by isoprenaline in nephrotic and control animals. The increase in cyclic AMP content induced by isoprenaline also was inhibited by these treatments. In addition, the difference in the isoprenaline-induced relaxation and cyclic AMP accumulation between nephrotic and control preparations was abolished by these treatments. The tissue cyclic guanosine monophosphate (GMP) level was not affected by isoprenaline. In the presence of zaprinast (Zap), a cyclic GMP phosphodiesterase inhibitor, the cyclic GMP level in the endothelium-intact tissues was significantly lower in nephrotic rats than in control animals. Removal of endothelium or pretreatment with MB or L-NAME markedly reduced cyclic GMP content in nephrotic and control animals. In the endothelium-denuded tissues, the isoprenaline-induced relaxation and cyclic AMP accumulation were markedly potentiated by a low concentration of nitroprusside (NP). In the endothelium-intact aortas precontracted with phenylephrine, relaxations induced by dobutamine, salbutamol, and forskolin in nephrotic rats were not significantly different from those in control animals. In the endothelium-intact aortas precontracted with KCl, the isoprenaline-induced relaxation also was significantly less in nephrotic rats than in control animals. Pretreatment with prazosin, but not yohimbine, abolished this difference. These results indicate that nephrosis decreases the relaxing response of the endothelium-intact aortas to isoprenaline. In addition, these results suggest that the endothelium-derived relaxing factor (EDRF) released from the endothelial cells markedly enhances isoprenaline-induced increase in the tissue level of cyclic AMP. The decreased relaxing response to isoprenaline in nephrotic rats may be caused by the decrease in the endothelial-dependent cyclic GMP release caused by alpha 1-adrenoceptor activation by isoprenaline.

Nucleotide sequence and characterization of the large mitochondrial rRNA gene of Penicillium urticae, and its comparison with those of other filamentous fungi.

The nucleotide sequence of a large rRNA gene and its flanking regions in cloned fragments of mitochondrial DNA from a patulin producer, Penicillium urticae NRRL2159A, was determined by dideoxy sequencing, and the 5' end and intron-exon border of the 1-rRNA gene were determined by primer extension analysis and RNA sequencing, respectively. In addition to the extensive sequence homology of the 3' end of the P. urticae mt 1-rRNA gene with those of Aspergillus nidulans and Neurospora crassa, the P. urticae gene had a 1,685 bp intron which separates a 3,307 bp 5' exon and a 583 bp 3' exon. In spite of being closely related Penicillium species, the size of the 5' exon of the P. urticae mt 1-rRNA is 472 bp larger than that of P. chrysogenum, whereas the sizes of the 3' exon and intron of P. urticae are very similar to those of P. chrysogenum (581 bp for the 3' exon and 1,678 bp for the intron). The intron of P. urticae contains a structure similar to the consensus one of the self splicing group IA intron and a large open reading frame suggested to be a gene for ribosomal protein S5. A sequence similar to the I-SceI recognition sequence was found at the exon-intron border. Extensive sequence homology was observed between P. urticae and P. chrysogenum, exceptions being in four regions in the 5' exon. These non-homologous regions were located in the hairpin and variable regions outside of the core structures. Comparison of the mt 1-rRNA sequences of several filamentous fungi revealed that the above four non-homologous regions are greatly expanded, and two other non-homologous regions appear at the 3' ends of the 5' exon and 3' exon.

Nephrosis augments contractile response to adrenoceptor agonists by the decrease in release of endothelium-derived relaxing factor from the endothelial cells.

The thoracic aorta was taken from nephrotic rats on the 40th day after a single i.v. injection of daunomycin (10 mg/kg). In the endothelium-intact aorta, the contractions induced by norepinephrine or B-HT 933, an alpha-2 adrenoceptor agonist, were significantly greater in nephrotic rats than in normal animals. However, such a difference was not observed in the KCl- or U46619-induced contractions. The difference in norepinephrine-induced contraction between nephrotic and normal preparations was enhanced by zaprinast, a cyclic GMP phosphodiesterase inhibitor. The contractions elicited by norepinephrine and B-HT 933 were potentiated by either removal of the endothelium or pretreatment with methylene blue, a guanylate cyclase inhibitor. The difference in the contractile response to these agonists between nephrotic and normal preparations was eliminated completely by either treatment. The cyclic GMP level in the endothelium-intact aorta in the presence of zaprinast was significantly lower in nephrotic rats than in normal animals. In the presence of zaprinast, norepinephrine, but not B-HT 933, caused an increase in the cyclic GMP level, which was abolished completely by pretreatment with prazosin, but not by yohimbine. These results suggest that the augmented contractile response to norepinephrine observed in nephrotic aorta may be due to the decrease in the stimulated release of endothelium-derived relaxing factor from the endothelial cells via the stimulation of endothelial alpha-1 adrenoceptors, whereas the augmented response to B-HT 933 may be due, at least in part, to the decrease in spontaneously released endothelium-derived relaxing factor.

Nucleotide sequence of the large mitochondrial rRNA gene of Penicillium chrysogenum.

The nucleotide sequence of a large rRNA (1-rRNA) gene and its flanking regions in the cloned fragments of mitochondrial (mt) DNA from Penicillium chrysogenum NRRL1951 (Sekiguchi J., Ohsaki, T., Yamamoto, H., Koichi, K. and Shida, T. (1990) J. Gen. Microbiol. 136, 535-543) was determined and compared with those in Aspergillus nidulans and Neurospora crassa mitochondrial DNAs. The P. chrysogenum mt 1-rRNA gene has a 1678 bp intron which intervenes between a 2835 bp 5' exon and a 581 bp 3' exon, and extensive homology exists between overall sequences of mt 1-rRNA genes of P. chrysogenum and A. nidulans. The P. chrysogenum intron contains a large open reading frame which encodes a polypeptide comprising of 399 amino acids. The intron sequence also suggests that the intron belongs to a self-splicing group IA.

Transformation of Penicillium urticae with plasmids containing the hygromycin B resistance gene.

A transformation system with efficiencies between 6 and 50 stable transformants per micrograms of DNA was developed for Penicillium urticae J1 (ATCC48163) using hygromycin B-resistant plasmids containing or not containing fragments of the P. urticae genome. The tandem repeated integration and/or random integration of vector DNA were observed. Although P. urticae was unable to grow in the presence of 200 micrograms/ml hygromycin B, the transformants were resistant to more than 5 mg/ml of hygromycin B.

Impaired endothelium-dependent relaxation in isolated thoracic aorta of rats with daunomycin-induced nephrosis.

A single i.v. injection of daunomycin (10 mg/kg) into rats produced severe proteinuria and hypercholesterolemia without atherosclerosis on the 20th and 40th days after the treatment. However, these changes were not observed on the 5th day. No change in systolic blood pressure was seen through the 40-day experimental period. Relaxation to acetylcholine, A23187 and nitroprusside was examined in aortic rings precontracted with phenylephrine (3 x 10(-6) M). Acetylcholine-induced relaxation was significantly attenuated in the nephrotic rats on the 20th and 40th days, in comparison to the control animals. In aortic rings taken from control and nephrotic rats on the 40th day, removal of the endothelium or treatment with methylene blue (10(-5) M) completely abolished the relaxation induced by acetylcholine (10(-5) M). In addition, acetylcholine (10(-5) M) induced a transient increase in the aortic cyclic GMP and this increase was completely abolished by removal of the endothelium. In the preparations of nephrotic rats on the 20th and 40th days, the cyclic GMP levels stimulated by acetylcholine (10(-5) M) were decreased to about 50% in comparison to their respective control. A23187 also evoked diminished relaxation in nephrotic rats on the 20th and 40th days. However, on the 40th day after the treatment, the effects of nitroprusside in relaxing the aorta and in elevating the cyclic GMP level in the aorta were not altered by nephrosis. In addition, the nitroprusside-induced relaxation and cyclic GMP accumulation were not affected by removal of the endothelium. These results indicate that endothelium-dependent relaxation is attenuated with the development of nephrosis.

Re-evaluation of protein-bound glutathione in rat liver.

The extent of intracellular glutathione binding to proteins through a disulfide linkage in rat liver was examined quantitatively. The content of glutathione associated with the acid-precipitable fraction and releasable on borohydride treatment was 0.024 +/- 0.016 mumol/g liver, which accounted for less than one per cent of the total glutathione (6-7 mumol/g liver) in the liver of fed rats. Most of the thiol (2-4 mumol/g liver) liberated from liver proteins into the acid-soluble fraction on borohydride reduction in the presence of guanidine hydrochloride was not glutathione but was proteinaceous in nature. The amounts of thiols liberated per g of liver were similar in fed, fasted, and dibutyryl-3',5'-cyclic AMP-treated rats.

Relative contributions of sulfur atoms of dietary cysteine and methionine to rat liver glutathione and proteins.

The relative contributions of sulfur atoms of dietary L-Cys and L-Met to the syntheses of proteins and GSH in rat liver were examined. 1) When the amount of sulfur-containing amino acids in L-Trp-deficient diets was fixed at 0.36%, incorporation of L-[35S]Cys into GSH was proportional to the amount of L-Cys administered, in the presence of L-Met in the diet. Incorporation of 35S from L-Met into GSH was lower than that from the same amount of L-Cys and became negligible in the presence of a large amount of L-Cys. 2) When rats were given L-Trp-deficient diet, more L-Cys was always incorporated into liver GSH than into proteins. But, when rats were given L-Trp-fortified diet containing more L-Met than L-Cys, more L-Cys was incorporated into liver proteins than into GSH. 3) Met-sulfur was preferentially incorporated into liver proteins with or without L-Trp. Its absolute incorporation into proteins was significantly greater in the presence of L-Trp than in its absence. 4) When the amount of L-Met in the diet was increased from 0.18 to 0.36 or 0.54%, incorporation of Met-sulfur into proteins increased proportionally, in the presence of 0.18% L-Cys. Unexpectedly, incorporation of L-Cys formed from L-Met into liver proteins was larger than that from L-[3H]Met itself. L-Cys formed from L-Met was incorporated into proteins more readily than L-Cys given as such. 5) When the amount of L-Met in the diet was increased from 0.18 to 0.54%, incorporation of Met-35S into GSH increased 8-fold. Even with a large excess of L-Met, L-Cys was invariably incorporated into GSH. 6) These results are consistent with the role of liver GSH as a reservoir of cysteine, as proposed by us.

Rat liver glutathione: possible role as a reservoir of cysteine.

Rat liver contains a high concentration (7-8mM) of reduced glutathione and its level changes rapidly when starving or feeding rats. We concluded that one of the functions of liver glutathione was to act as a reservoir of cysteine. When starved rats were fed a protein-free diet, the increase in liver glutathione was dependent on the amount of cysteine added to the diet. A cysteine-dependent increase of glutathione was also observed in rats fed a diet containing gelatin with cysteine, but the increase was relatively lowered compared with rats fed a protein-free diet containing the same amount of cysteine. This suppression of the increase in glutathione was observed much more clearly when the gelatin diet was fortified with tryptophan in addition to cysteine. In the presence of tryptophan, L-[35S]-cysteine in the diet appeared to be incorporated primarily into liver and serum proteins, and degradation of liver glutathione must also have been enhanced. Addition of excess cysteine to the diet masked the effects of gelatin and tryptophan, stimulated glutathione synthesis in the liver as well as incorporation of dietary cysteine into protein fractions. Prolonged starvation of rats or injection of dibutyryl-3',5'-cyclic AMP lowered the glutathione level,but the level did not decrease below 2 to 3 mM. These findings suggest that there may be at least two pools of glutathione. A labile fraction, constituting one-third to one-half the total liver glutathione, probably serves as a reservoir of cysteine which can be released by gamma-glutamyl-transferase when necessary.

Higher transpeptidation activity and broad acceptor specificity of gamma-glutamyltransferases of tumors.

The gamma-glutamyltransferase (EC 2.3.2.2) (=gamma-glutamyltranspeptidase, gamma-GTP) activity in hepatoma induced by 3'-methyl-4-(dimethylamino)azobenzene (3'-Me-DAB) was 120-fold higher than that of normal liver and high activity was also found in bovine hepatocellular carcinoma. gamma-GTPs from these malignant tissues responded more and showed broader specificity to gamma-glutamyl group acceptors than those from normal tissue such as bovine, rat, and mouse liver and bovine kidney. Three species of gamma-GTP were isolated from bovine kidney by DEAE-cellulose chromatography, whereas only two species were isolated from bovine hepatocellular carcinoma. The carcinoma lacked the least acidic enzyme species. Appropriate gamma-glutamyl group acceptors stimulated more-acidic enzyme species more than less-acidic species in both tissues. The fractions separated from the hepatoma were stimulated more than those of kidney by gamma-glutamyl group acceptor. The enzymes from normal tissues responded similarly to a gamma-glutamyl group acceptor irrespective of the difference in their activity. Thus, gamma-GTPs of malignant tissues appear to be more versatile for amino acid transport, both qualitatively and quantitatively. In these properties the enzyme of mouse fetal liver which showed the highest activity in the last period of pregnancy resembled the enzymes of malignant rather than normal tissues. The activity of hepatic gamma-GTP is not parallel with the rate of cell proliferation during normal development.