Streptozocin- and alloxan-induced H2O2 generation and DNA fragmentation in pancreatic islets. H2O2 as mediator for DNA fragmentation.

Streptozocin (STZ) and alloxan (ALX) exhibit the most potent diabetogenicity and are used for induction of experimental diabetes mellitus. An understanding of the mechanisms of action of the typical diabetogenic agents is important for elucidating the causes of diabetes. Okamoto proposed a model in which DNA fragmentation plays an important role in the development of diabetes. DNA fragmentation supposedly results from the accumulation of superoxide or hydroxyl radicals. However, direct evidence for this accumulation is lacking. With isolated rat pancreatic islets in vitro, we demonstrated that STZ and ALX stimulated H2O2 generation and caused DNA fragmentation. Addition of STZ or ALX resulted in an increase in H2O2 generation. On DNA analysis, when incubated without STZ or ALX, DNA sedimented as a single peak; when incubated with STZ or ALX, DNA sedimented slower as a broad peak and was fragmented. Graded doses of STZ and ALX stimulated H2O2 generation and induced DNA fragmentation; their effects on H2O2 generation and DNA fragmentation were evident at a concentration of 0.1 mM and were maximal at 1 mM. Administration of STX or ALX to rats in vivo stimulated H2O2 generation and caused DNA fragmentation in pancreatic islets. H2O2 itself also induced DNA fragmentation. These findings may support Okamoto's proposal that STZ and ALX induce diabetes through the following biochemical events: STZ and ALX----H2O2 generation----DNA fragmentation----beta-cell destruction. This study may constitute the first demonstration of STZ- and ALX-stimulated H2O2 generation, which probably acts as a mediator of STZ- and ALX-induced DNA fragmentation.

Naturally occurring mutations in human steroid 21-hydroxylase influence adrenal autoantibody binding.

Human 21-hydroxylase (21-OH) genes containing various mutations, truncations, and deletions were expressed in yeast, and autoantibody binding was studied by Western blotting using patient sera and rabbit antibodies to 21-OH. 21-OH autoantibodies in 13 Addisonian sera showed a marked reduction in their ability to recognize 21-OH mutated at Pro453-->Ser (mean +/- SD, 31 +/- 9% of binding to wild type), whereas the effect on rabbit antibody binding was small (88 +/- 11% of binding to wild type; n = 7). Mutation at Arg339-->His had a less pronounced effect on autoantibody binding (85 +/- 11% of binding to wild type; n = 13) and caused a small enhancement of rabbit antibody binding (124 +/- 16% of binding to wild type; n = 7). These studies indicate that Pro453 has a key role in forming an autoantigenic epitope on 21-OH. It is important to note, however, that the Pro453 mutation caused only partial loss of autoantibody binding, i.e. all Addisonian sera studied still reacted with the mutated protein. This may indicate that each serum sample contains at least two different populations of 21-OH autoantibodies, only one of which recognizes a site dependent on Pro453. A series of more extensive modifications of the 21-OH sequence, including truncations (amino acids 460-494, 448-494, and 418-494) and deletions (amino acids 165-379, 142-240, and 142-280) indicated that most of the sequence of amino acids from 241-494 is important for autoantibody binding. The involvement of such an extensive region of the molecule suggests that the binding sites are generated by three-dimensional folding, with Pro453 having a critical role in forming at least one major autoantigenic epitope.

Autoimmune Addison's disease--evidence for a role of steroid 21-hydroxylase autoantibodies in adrenal insufficiency.

Autoantibodies to steroid 21-hydroxylase (21-OH) are characteristic of adult onset Addison's disease and we have investigated the effects of these autoantibodies on recombinant human 21-OH enzyme activity. Antibody preparations from 11/11 Addison sera inhibited the ability of 21-OH to convert progesterone to deoxycorticosterone with 8 IgGs showing almost complete inhibition, 2 partial inhibition and 1 weak inhibition. Control IgGs from patients with autoimmune thyroid disease and normal blood donors had little or no effect on 21-OH activity. Our results suggest that 21-OH autoantibodies have the potential to contribute to adrenal failure in Addison's disease by inhibiting the 21-OH enzyme.

Steroid 21-hydroxylase autoantibodies: measurements with a new immunoprecipitation assay.

Autoantibodies (Abs) to steroid 21-hydroxylase (21-OH) are a major component of adrenal cortex Abs and are characteristic of autoimmune Addison's disease. We have developed a new method for measuring Abs to 21-OH based on 125I-labeled recombinant human 21-OH produced in yeast. With this assay, 21-OH Abs were detected in 43 of 60 (72%) sera from patients with isolated Addison's disease, 11 of 12 (92%) autoimmune polyglandular syndrome type I sera, 27 of 27 (100%) autoimmune polyglandular syndrome type II sera, and 24 of 30 (80%) sera from patients who were positive for adrenal cortex antibodies by immunofluorescence but had no overt Addison's disease. 21-OH Abs were found by 125I assay in 4 of 150 (2.7%) sera from patients with insulin-dependent diabetes mellitus, 1 of 77 (1.3%) Graves' sera, 1 of 67 (1.5%) Hashimoto's sera, and 6 of 243 (2.5%) sera from healthy blood donors. 21-OH Abs were not detected in 9 sera from patients with Addison's disease due to tuberculosis, 32 sera from patients with noninsulin-dependent diabetes mellitus, 35 sera from patients with myasthenia gravis, or 17 sera from patients with premature ovarian failure. There was good agreement between the 125I-labeled 21-OH assay and an assay based on 35S-labeled 21-OH produced in an in vitro transcription/translation system (r = 0.86; n = 129; P < 0.001). In the case of sera from patients with Addison's disease, insulin-dependent diabetes mellitus, Graves' disease, and Hashimoto's disease and from healthy blood donors that were low positive in the 125I assay, neutralization studies with unlabeled 21-OH confirmed the presence of specific 21-OH Abs. Overall, the 21-OH Ab assay based on 125I-labeled 21-OH showed good sensitivity, precision, and disease group specificity. This, combined with a simple assay protocol and the convenience of 125I handling and counting, make it attractive for routine use. Further investigations with the new assay should allow wider assessment of the prevalence and pattern of inheritance of adrenal autoimmunity. In addition, studies of the effect of treatment or possible preventative measures on 21-OH Ab levels in individuals without overt adrenal failure may suggest ways of delaying the onset of autoimmune Addison's disease.

Autoimmune Addison's disease. Analysis of autoantibody binding sites on human steroid 21-hydroxylase.

Human steroid 21-hydroxylase (21-OH) expressed in an in vitro translation system was found to react specifically with adrenal autoantibodies from patients with Addison's disease. The epitopes on 21-OH which reacted with autoantibodies were studied by incorporating a series of terminal and internal deletions into the 21-OH gene and analysing the expressed proteins by Western blotting. N-Terminal deletions up to amino acid 280 had no effect on autoantibody binding whereas a series of C-terminal deletions and truncations (amino acids 281-494) showed marked effects. Our results indicate that a central segment (281-379) and a C-terminal segment (380-494) of 21-OH interact to form at least one major autoantibody binding site.

Cytoplasmic pH in cultured porcine thyroid cells: alkalinization by Ca2+.

The role of calcium in cytoplasmic pH (pHi) changes was studied using 2',7'-bis(2-carboxyethyl)-5-(and 6-)carboxyfluorescein, an internalized fluorescent pH indicator, in cultured porcine thyroid cells. The Ca2+ ionophores A23187 and ionomycin stimulated thyroid cell alkalinization. An increase in cytoplasmic free calcium resulted in activation of Na+/H+ exchange which alkalinized the cells.

An abnormal sodium metabolism in Japanese patients with essential hypertension, judged by serum sodium distribution, renal function and the renin-aldosterone system.

OBJECTIVE: The role of the renin-aldosterone system and the ability of renal sodium reabsorption to facilitate pressure natriuresis were analyzed by using a sufficient number of Japanese patients with essential hypertension. METHODS: We studied 3222 normal Japanese subjects (610 in Kashiwa City Hospital and 2612 in Shinshu University Hospital), 741 Japanese patients with essential hypertension (256 in Kashiwa City Hospital and 485 in Shinshu University Hospital), 20 patients with aldosterone-producing adenomas and 11 patients with idiopathic hyperaldosteronism to determine the possible roles of sodium, renal function, and plasma aldosterone concentration (PAC) on blood pressure elevation. Inappropriate elevation of aldosterone levels [elevation of the aldosterone:plasma renin activity (PRA) ratio] was used to assess aldosterone action. RESULTS: The peak of the serum sodium distribution curve was approximately 2 mmol/l higher in the patients with essential hypertension than it was in controls. The prevalence of higher serum sodium concentrations (> or = 147 mmol/l) also was increased significantly hypertensive patients. Age-related deterioration of renal function did not explain the hypertension and abnormal sodium metabolism in the hypertensive patients. In stepwise regression analysis, the serum sodium concentration was related inversely to the PRA and positively to the PAC:PRA ratio. Although there was an inverse relationship between urinary sodium excretion (representing sodium intake) and the PRA, urinary sodium excretion proved not to be significant as a source of variation in the PAC or in the PAC:PRA ratio in the hypertensive patients. Although the PAC was within the normal range in patients with serum sodium concentrations of 147 mmol/l or more and an elevated PAC:PRA ratio, it was inappropriately high for the stimulus applied, as indicated by the PRA; this is similar to the situation with aldosterone-producing adenomas or idiopathic hyperaldosteronism. CONCLUSION: Serum sodium distribution patterns differed between normal subjects and patients with essential hypertension in this Japanese population. The deterioration of renal function and increased sodium intake did not explain this abnormal sodium metabolism. A higher serum sodium concentration is related to an elevated blood pressure, and, in some patients, an inappropriate elevation of plasma aldosterone levels. Of the Japanese hypertensive patients, 10-14% exhibited serum sodium concentrations of 147 mmol/l or more and inappropriate elevations of aldosterone level (suppressed PRA and normal aldosterone level). The defect in these patients presumably lies in the inappropriately high secretion of aldosterone.

Cytoplasmic pH in the action of insulin-like growth factor-I in cultured porcine thyroid cells.

The effects of insulin-like growth factor-I (IGF-I) on cytoplasmic pH (pHi) and [3H]thymidine incorporation were studied in primary cultures of porcine thyroid cells. IGF-I alkalinized thyroid cells and stimulated thymidine incorporation in a dose-dependent manner; the effects of IGF-I on alkalinization (the maximal rates of change of cytoplasmic pHi/min ((dpHi/dt)max)) and thymidine incorporation were observed at 2 ng/ml and were maximal at 100 ng/ml, with half-maximal stimulation at approximately 10 ng/ml. The results indicate that Na+/H+ exchange or cell alkalinization may function as a transmembrane signal transducer in the action of IGF-I on thyroid cell proliferation. Several mitogens and comitogens which activate sodium hydrogen exchange, including epidermal growth factor, platelet-derived growth factor and nerve growth factor, have been listed. Activation with IGF-I has not, however, been presented before. Thus the present study constitutes the first demonstration of IGF-I-stimulated activation of Na+/H+ exchange or cell alkalinization.

Autoantibody binding to steroid 21-hydroxylase--effect of five mutations.

Steroid 21-hydroxylase (21-OH) is a key haem containing steroidogenic enzyme and a major adrenal specific autoantigen. Cys 428 in 21-OH is thought to have an important role in haem binding and we now describe the effects of mutations at Cys 428 (to Ser, Arg and Phe) on 21-OH autoantibody binding. Expression of wild type and mutated 21-OH was carried out using an in vitro transcription/translation (TnT) system and reactivity of 21-OH autoantibodies with mutated 21-OH analysed by western blotting (in the case of unlabelled proteins) or immunoprecipitation assay (IPA) (in the case of 35S-labelled proteins). All 3 substitutions at Cys 428 had similar effects on 21-OH autoantibody binding and each one caused a reduction in autoantibody binding to about 50% of wild type in the case of IPA and to about 70% of wild type in the case of western blotting analysis. In addition to mutations at Cys 428, we studied 2 naturally occurring mutations at Pro 30 to Leu and Ile 172 to Asn which are associated with diminished 21-OH enzyme activity. The Pro 30 mutation had no effect, but the Ile 172 mutation caused a reduction in 21-OH autoantibody binding in the IPA to about 80% of wild type. Overall, our studies emphasise the close relationship between the 21-OH aminoacid sequences important for 21-OH enzyme activity and 21-OH autoantibody binding.

Glutamic acid decarboxylase autoantibody assay using 125I-labelled recombinant GAD65 produced in yeast.

We describe a new method for measuring autoantibodies (Ab) to the 65 kDa isoform of glutamic acid carboxylase (GAD65). In particular, GAD65 without the hydrophobic N-terminal region has been produced in yeast, purified, labelled with 125I and reacted with GAD65 Ab. Antibody bound 125I-GAD65 is then precipitated by the addition of solid phase protein A. With the assay, GAD65 Ab were detected in 59 of 71 (83%) islet cell antibody (ICA) positive IDDM patients and in 8 of 23 (35%) ICA negative IDDM patients (overall 67 of 94 (71%) of IDDM patients). Low concentrations of GAD65 Ab were also detected in 2/98 (2%) healthy blood donors and 1/27 (4%) Graves' disease patients had a high level of antibody. GAD65 Ab were not detected in any of 10 Hashimoto's thyroiditis, 20 Addison's disease or 19 myasthenia gravis sera. There was good agreement between the 125I assay and the current reference method based on 35S-labelled full-length GAD65 (produced by in vitro transcription/translation reaction) and solid phase protein A (r = 0.91, n = 108). Overall, our 125I assay showed sensitivity, precision and disease group specificity at least as good as any assay so far described. These features, combined with a simple assay protocol and the convenience of 125I counting and handling indicate that the method is suitable for routine GAD65 Ab measurements.

Dipyridamole reduces urinary albumin excretion in diabetic patients with normo- or microalbuminuria.

The effect of 150 mg/day dipyridamole p.o. on urinary albumin excretion (UAE) was studied in 48 patients with diabetes mellitus without clinically discernible nephropathy. In 42 patients who were followed at an outpatient clinic, albumin/creatinine ratio (Ualb/Ucreat: mg/mmol) of untimed urine obtained from the same subjects repetitively was employed as an index of UAE. In 6 hospitalized patients, albumin excretion rate (AER) (micrograms/min) of 24-h-collected urine was determined. When followed without dipyridamole for 10.8 (the mean) months (N = 27, outpatients), the mean Ualb/Ucreat increased from 8.1 to 20.5. Of these, 11 patients with Ualb/Ucreat greater than 1.0 at the end of the observation period subsequently received dipyridamole for 4.2 months, and the ratio decreased from 49.0 to 7.3. When treated with dipyridamole for 9.0 months without a pre-treatment observation period (N = 15, outpatients), the ratio decreased from 9.8 to 5.6. AER of hospitalized patients who received dipyridamole for 10.0 days reduced from 68.0 to 21.9. All of these changes were statistically significant. Urinary beta 2 MG, blood pressure, serum creatinine and glycemic control were unaffected by the dipyridamole treatment. We conclude that dipyridamole reduces UAE in diabetic patients with subclinical level of albuminuria.

DNA sequence of type 1 fimbrin, Fpul1, gene from a chicken pathogenic Escherichia coli serotype O78.

The gene encoding type 1 fimbriae of chicken pathogenic Escherichia coli serotype O78 (designated Fpul1) was cloned and the genetic region encoding fimbrial subunit was sequenced. The nucleotide sequence and its deduced amino acid sequence demonstrated that the Fpul1 was a novel variation among E. coli type 1 fimbriae and showed an extensive homology to previously reported Klebsiella pneumoniae type 1 fimbriae. The E. coli K-12 strains carrying the Fpul1 genes did not show the acid-induced autoagglutination, suggesting that the Fpul1 was genetically distinct from the acid-induced autoagglutination.

Serotypes and antimicrobial susceptibility of Actinobacillus pleuropneumoniae isolated from piglets with pleuropneumonia.

Minimal inhibitory concentrations (MICs) of 20 antimicrobial agents were determined for 67 field strains of Actinobacillus pleuropneumoniae isolated from the lungs of piglets with pleuropneumonia during the period 1992 to 1994, in Japan. Of these 67 strains, all 29 strains that were identified as serotypes 1 and 7 were resistant to chloramphenicol and tiamphenicol, whereas all of those identified as serotypes 2, 5 and 8 were susceptible to these antibiotics. Furthermore, 20 of 23 streptomycin-resistant strains were serotype 1, while, only 3 strains were serotype 2. These results suggest that the different serotypes of A. pleuropneumoniae vary in terms of antimicrobial-resistance.

Isolation and characterization of type 1 fimbriae from a chicken pathogenic Escherichia coli serotype O78.

Type 1 fimbriae from chicken pathogenic Escherichia coli strain PDI-386 (serotype O78) was purified and characterized. Because of the acid-induced autoagglutination (T. Sekizaki, Y. Nakasato, and I. Nonomura, J. Vet. Med. Sci. 54, 493-499, 1992), the fimbriae could be easily purified by repeating acid sedimentation, washing, and dissolving in buffer (pH 8.0). In electron microscopy, the purified fimbriae showed a filament of 8 nm in diameter and 10 microns in average length. The molecular mass of the protein subunit of the purified fimbriae estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 19,000 daltons. The amino acid composition and its NH2-terminal sequence were similar to the previously described one of the Klebsiella pneumoniae type 1 fimbriae. Moreover, there was an immunological relatedness between them. These results indicated that a molecular diversity found between the fimbriae of E. coli and that of K. pneumoniae has already been existed among chicken pathogenic E. coli strains.

Alloxan-induced DNA strand breaks in pancreatic islets. Evidence for H2O2 as an intermediate.

Alloxan exhibits the most potent diabetogenicity and has been used for induction of experimental diabetes mellitus. Understanding the mechanisms of action of the typical diabetogenic agent is important for elucidating the causes of diabetes. Okamoto (Okamoto, H. (1985) BioEssays 2, 15-21) proposed a model in which DNA fragmentation plays an important role for the development of diabetes. This DNA fragmentation is supposed to result from the accumulation of superoxide or hydroxyl radicals. However, direct evidence for this accumulation is lacking. Using rat pancreatic islets, we demonstrated that alloxan stimulated H2O2 generation, which induced DNA strand breaks. These findings support Okamoto's proposal that alloxan induces diabetes through the following biochemical events: alloxan----H2O2 generation----DNA strand breaks----diabetes mellitus. Perhaps this report constitutes the first demonstration of alloxan-stimulated H2O2 generation which could conceivably act as an intermediate for alloxan-induced DNA strand breaks.

Restriction map of a virulence-associated plasmid of Rhodococcus equi.

The restriction cleavage map of a virulence-associated plasmid pREAT701 of Rhodococcus equi was constructed with EcoRI and HindIII by means of cloning the restriction fragments, cross Southern hybridization with each fragment, and hybridization with probes generated by modified inverse PCR. The genetic region responsible for expression of virulence-associated 15- to 17-kilodalton antigens was determined.

Association of annexin V with prolactin in the rat anterior pituitary gland.

When pituitary extracts were subjected to non denaturing polyacrylamide gel electrophoresis, an unknown protein was found to associate with a proportion of the prolactin. This protein was dissociated from prolactin by sodium dodecyl sulfate. The protein was purified and sequenced. As the amino terminus was blocked, the amino acid sequences of three peptide fragments were determined. The obtained sequences of 41 amino acids were identical to partial sequences of a known protein, rat Annexin V. The molecular mass, 36 kDa, was also the same as the molecular weight of Annexin V. The existence of Annexin V mRNA in rat pituitary glands was also confirmed by polymerase chain reaction. These results show that Annexin V, a member of the calcium-dependent phospholipid binding proteins, is synthesized in the rat pituitary gland, and suggest its association with prolatin in the gland.

[Forty-one cases of Cushing's syndrome: a comparison between Cushing's syndrome (adrenal adenoma) and Cushing's disease (adrenal hyperplasia)].

We experienced 41 cases of Cushing's syndrome (12 males and 29 females, 15 years old - 65 years old) during the last 20 years. These included 20 patients with unilateral adrenal adenoma (Cushing's syndrome), 19 patients with bilateral adrenal hyperplasia (Cushing's disease), one patient with adrenal carcinoma and one patient with primary adrenocortical nodular dysplasia (PAND). Moreover, these cases included some special ones, i.e. 5 cases with destructive thyroiditis after treatment, 2 cases with aggravation of arthritis after treatment, a case of Carney's complex with PAND, one case with paradoxical response to dexamethasone, and one case combined with empty sella syndrome. The most specific clinical signs were moon face (95% occurrence), hypertension (95%) and subcutaneous bruising (80%). Other significant signs were eye edema (66%), buffalo hump (68%), subcutaneous purpura (63%) and osteoporosis (49%). Skin striae was not a common sign in our cases (41%). Renal stone was observed in only 20% of our patients but was a significant sign in this syndrome. There was no difference in the occurrence of each clinical sign between Cushing's syndrome and Cushing's disease. The elevation of white blood cell count (WBC) and serum sodium, a decrease of serum potassium, and a decrease of reabsorption of phosphate (%TRP) were observed. Thyroid-stimulating hormone (TSH) and human growth hormone (HGH) were suppressed in patients with Cushing's syndrome and patients with Cushing's disease. These results were consistent with those of previous reports. However, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) were high in those patients with Cushing's syndrome and those with Cushing's disease. Oral glucose tolerance test was carried out in 34 patients before and after treatment. Thirty-one percent of those had diabetes mellitus and 26% had impaired glucose tolerance (IGT). The response of IRI in this test was high in patients with Cushing's syndrome and patients with Cushing's disease, and decreased 4 weeks after treatment in those with Cushing's syndrome but remained high in those with Cushing's disease. Plasma ACTH level and urinary 17-OHCS excretion were significantly higher in Cushing's disease than in Cushing's syndrome. During an 8mg-high-dose dexamethasone suppression test, urinary 17-OHCS excretion in 13 of 14 patients with Cushing's disease (93%) was suppressed by more than 50% of baseline on the second day of testing. However, all of 18 patients with Cushing's syndrome, who had an 8mg-dexamethasone suppression test, failed to suppress urinary 17-OHCS by 50% of baseline.(ABSTRACT TRUNCATED AT 400 WORDS)