Evaluation of two Japanese regulatory actions using medical information databases: a 'Dear Doctor' letter to restrict oseltamivir use in teenagers, and label change caution against co-administration of omeprazole with clopidogrel.

WHAT IS KNOWN AND OBJECTIVE: The implementation of appropriate epidemiological methodology using medical information databases (MIDs) to evaluate the effects of regulatory actions has been highly anticipated. To assess scientific methods for active pharmacovigilance using MIDs, we conducted a quantitative assessment of the impact of two regulatory actions by the Japanese government: (i) restriction of use of oseltamivir in teenagers in March 2007 and (ii) caution against the co-administration of omeprazole (OPZ) with clopidogrel (CPG) in April 2010. METHODS: Data were obtained from four hub hospitals in Japan. We measured the seasonal proportion of patients prescribed oseltamivir to those prescribed neuraminidase inhibitors for the 2002/2003 to 2010/2011 seasons. The monthly proportion of patients co-administered OPZ and CPG (OPZ+CPG) to those prescribed CPG was measured from May 2009 to April 2011. We evaluated the changes observed with implementation of the regulatory actions. To estimate the impact of the actions, we conducted segmented regression analysis using interrupted time series data. The impact was assessed by two parameter estimates of the regression model: the change in level for short-term effects and change in trend for long-term effects. RESULTS AND DISCUSSION: The use of oseltamivir in the target 10-19 years age group showed a significant and large decline (63·16%) immediately after the intervention (P = 0·0008). No change was observed in OPZ+CPG, although there was a relative inhibitory trend for OPZ+CPG compared with co-administration of lansoprazole or rabeprazole with CPG as the control group. When restricted to new users of CPG, the stratified results were consistent with the overall results. WHAT IS NEW AND CONCLUSION: The current analysis demonstrates the effectiveness of two regulatory actions. The results of the current study indicate that MID research can contribute to assessing and improving pharmacovigilance activities.

Nucleotide sequence of an RNA from cells infected with adenovirus 2.

The principal nucleotide sequence of an RNA in KB cells infectedwith adenovirus 2 has been determined. As isolated, the molecule shows some terminal and internal heterogeneity. The sequence permits extensive base pairing and contains prominent repeating sequences. A portion of the sequence resembles a sequence found in several transfer RNA's.

A case of nivolumab-induced acute-onset type 1 diabetes mellitus in melanoma.

Nivolumab, an anti-PD-1 antibody, is now considered an important therapeutic agent in several advanced malignancies. However, immune-related adverse events such as endocrinopathies have been reported with its use. Thyroid disorder and isolated adrenocorticotropic hormone deficiency have frequently been reported as nivolumab-induced immune-related adverse events. Another endocrinopathy is nivolumab-induced type 1 diabetes mellitus (t1dm), described as diabetes mellitus with rapid onset and complete insulin insufficiency, at times leading to fulminant t1dm. We report the case of a 68-year-old woman who developed pancreatic islet-related autoantibody-negative t1dm, possibly induced by nivolumab, under continuous glucocorticoid administration. She was treated with nivolumab for advanced malignant melanoma, concomitant with 10 mg prednisolone daily for thrombophlebitis tapered to 5 mg after 13 courses of nivolumab therapy. At approximately the 27th course of nivolumab therapy, she showed elevated plasma glucose levels despite preserved insulin secretion. A month later, she developed diabetic ketoacidosis. Her insulin secretion decreased and finally was exhausted. She was diagnosed with acute-onset rather than fulminant t1dm because of a rapidly progressive course to diabetic ketoacidosis during just more than 1 week. She is currently receiving insulin replacement. There has been no recurrence of the melanoma. Thus, nivolumab might induce autoimmune diabetes mellitus, with patients having t1dm-sensitive human leucocyte antigen being more susceptible even when receiving glucocorticoids. Physicians should be aware that nivolumab could potentially induce t1dm as a critical immune-related adverse event.

An ontology-based mediator of clinical information for decision support systems: a prototype of a clinical alert system for prescription.

OBJECTIVE: We have been developing a decision support system that uses electronic clinical data and provides alerts to clinicians. However, the inference rules for such a system are difficult to write in terms of representing domain concepts and temporal reasoning. To address this problem, we have developed an ontology-based mediator of clinical information for the decision support system. METHODS: Our approach consists of three steps: 1) development of an ontology-based mediator that represents domain concepts and temporal information; 2) mapping of clinical data to corresponding concepts in the mediator; 3) temporal abstraction that creates high-level, interval-based concepts from time-stamped clinical data. As a result, we can write a concept-based rule expression that is available for use in domain concepts and interval-based temporal information. The proposed approach was applied to a prototype of clinical alert system, and the rules for adverse drug events were executed on data gathered over a 3-month period. RESULTS: The system generated 615 alerts. 346 cases (56%) were considered appropriate and 269 cases (44%) were inappropriate. Of the false alerts, 192 cases were due to data inaccuracy and 77 cases were due to insufficiency of the temporal abstraction. CONCLUSION: Our approach enabled to represent a concept-based rule expression that was available for the prototype of a clinical alert system. We believe our approach will contribute to narrow the gaps of information model between domain concepts and clinical data repositories.

Accuracy of economic studies on surgical site infection.

Estimating the cost of hospital infection has become a matter of increasing interest in terms of health economics. This study aimed to assess the accuracy of economic studies on hospital infections, using surgical site infection (SSI) as an example. A search was performed for original articles reporting the cost of SSI, published in the English language between 1996 and 2005. For the critical review, the period of cost tracking, classification of costs and cost counting methods were noted. Fifteen articles met the inclusion criteria. The costs of SSI vary according to surgical procedures, country, publication year, study design and accounting method. Only two studies estimated the additional cost of SSI after discharge. All 15 studies included healthcare costs and none measured patient/family resources. In 10 studies, the costs were calculated based on accounting. Three studies used estimated costs from the ratio of costs to charges and two studies used charge data in place of cost data. It will become increasingly important for future studies to perform multi-centre prospective surveys, establish a standard method for cost accounting, include the cost of healthcare services following hospitalization and consider the morbidity cost to patients themselves from a societal perspective.

Orphan receptor DAX-1 is a shuttling RNA binding protein associated with polyribosomes via mRNA.

The DAX-1 (NR0B1) gene encodes an unusual member of the nuclear hormone receptor superfamily which acts as a transcriptional repressor. Mutations in the human DAX-1 gene cause X-linked adrenal hypoplasia congenita (AHC) associated with hypogonadotropic hypogonadism (HHG). We have studied the intracellular localization of the DAX-1 protein in human adrenal cortex and mouse Leydig tumor cells and found it to be both nuclear and cytoplasmic. A significant proportion of DAX-1 is associated with polyribosomes and is found complexed with polyadenylated RNA. DAX-1 directly binds to RNA, two domains within the protein being responsible for cooperative binding activity and specificity. Mutations in DAX-1 found in AHC-HHG patients significantly impair RNA binding. These findings reveal that DAX-1 plays multiple regulatory roles at the transcriptional and posttranscriptional levels.

Genogrouping of vaccine breakdown strains (VBS) of feline calicivirus in Japan.

Although prevention of feline calcivirus (FCV) infection by vaccination has been attempted, and isolation of FCV, development of the disease, and a few fatal cases in vaccinated cats have been reported. Fifteen FCV strains isolated from cats that had been vaccinated with commercially available FCV vaccines (F9, FCV-255, and FC-7) were genogrouped. Molecular analysis of viral genomes involved the construction of a phylogenetic tree of capsid genes using the NJ method. Cat anti-F9 serum and rabbit anti-FCV-255 serum were used for virus neutralization tests. Molecular phylogenetic analysis of the amino acid sequences of 15 virus isolates and those of the previously published and GenBank-deposited 9 global and 14 Japanese strains showed that 8 (53%) of the 15 virus isolates as well as the vaccine strains F9 and FCV-255 belonged to genogroup I (G(A)I), and 7 (47%) belonged to genogroup II (G(A)II). Of the 8 G(A)I strains, 2 were isolated from cats that had been vaccinated with an F9 strain live vaccine, 5 from cats vaccinated with an FCV-255-derived vaccine, and 1 from a cat vaccinated with an FC-7-derived vaccine. Of the 7 GAll strains, 5 were isolated from cats that had been vaccinated with the F9 strain live vaccine, 1 from a cat vaccinated with the FCV-255-derived vaccine, and 1 from a cat vaccinated with the FC-7-derived vaccine. These results indicate that more vaccine breakdown strains isolated from the cats vaccinated with the F9 strain-derived vaccine belong to G(A)II than to G(A)I, whereas more vaccine breakdown strains isolated from the cats vaccinated with the FCV-255 strain-derived vaccine belong to G(A)I than to G(A)II, and that when the FC-7 strain-derived vaccine is used, the vaccine breakdown strains belong almost equally to G(A)I and G(A)II. Thus, the genogroups of virus isolates varied with the vaccine strain used (p < 0.05). On the other hand, the neutralizing titres of feline anti-F9 serum and rabbit anti-FCV-255 serum against the 15 isolates were very low, showing no relationships between neutralizing antibody titres and genogroups. The DNA sequence identities between the virus isolates and the vaccine strains were low, at 70.6-82.9%, and no strains were found to have sequences derived from the vaccine strains. Alignment of amino acid sequences showed that the G(A)I or G(A)II virus isolates from the F9-vaccinated cats differed at position 428 of the 5' hypervariable region (HVR) of capsid region of the F9 strain, whereas those from the FCV-255-vaccinated cats differed at positions 438, 453, and 460 of the 5'HVR of capsid region E of the F9 strain. We speculate that these differences influence genogrouping. The amino acid changes within the F9 linear epitopes common to G(A)I and G(A)II were noted at positions 450, 451, 457 of 5'HVR of the capsid region E in the isolates from F9-derived vaccine-treated cats, and 449, 450, and 451 of 5'HVR of capsid region E in the isolates from FCV-255-derived vaccine-treated cats, suggesting that these amino acid changes are involved in escapes. These results suggest that alternate vaccination with the F9 and FCV-255 strains or the use of a polyvalent vaccine containing GAll strains serves to inhibit development.

Malignant and benign ovarian neoplasms among atomic bomb survivors, Hiroshima and Nagasaki, 1950-80.

For 1950-80, 194 ovarian cancer cases were ascertained among the 70,030 females of the Radiation Effects Research Foundation's Life-Span Study E-85 sample, and 106 autopsied cases with benign ovarian neoplasms were ascertained among all 3,046 autopsies performed in the same sample. On the basis of microscopic review, 66% of the cancer and 84% of the benign tumor cases were classified by histologic type. The age-adjusted ovarian cancer incidence rates showed a statistically significant increase with increased exposure dose, both in the entire exposed group (P less than .01) and in the microscopically reviewed subset (P less than .01). This dose response was only significant (P less than .01) in the latter half of the study period, 1965-80. The radiation effect was higher in the younger age group at the time of the bomb (ATB) for the specific attained age or was adjusted for attained age. In general, relative risk (greater than or equal to 100 rad vs. 0 rad) did not differ by attained age, except for the youngest age group, less than 20 years old ATB, where the relative risk tended to decrease with increased attained age, although cases were few in number and follow-up study was necessary. Estimated minimum latent period for radiation-induced ovarian cancer seemed to be 15-20 years. The proportion of autopsied cases with benign ovarian tumor increased with increasing exposure dose, both in the entire series of cases (P less than .05) and in the microscopically reviewed subset. Statistical significance, however, was not achieved in the latter group (P greater than .10). The distribution of histologic types of both cancer and benign tumor of the ovary did not vary significantly with radiation dose. The data are consistent with the hypothesis that radiation injury of the ovaries and secondary excess of gonadotropic hormones are important causative factors in the development of ovarian neoplasms.

Block of acid secretion by amytal and its partial reversal by menadione with ascorbate in the gastric mucosa of the guinea-pig.

The effect of amytal on energy metabolism and acid secretion in an isolated gastric mucosa of the guinea-pig were studied. Determination of adenine nucleotides, creatine phosphate, pyruvate and lactate in the gastric mucosa showed that amytal depressed the levels of ATP, creatine phosphate and energy charge with elevation of the AMP and pyruvate levels. This treatment inhibited concomitantly acid secretion and active chloride transport detected by short circuit current. The addition of menadione with ascorbate to the medium in the presence of amytal partially restored ATP and energy charge levels and also induced a partial recovery of acid secretion and activ chloride transport. These results suggest that ATP is a direct energy donor for acid secretion in the gastric mucosa of the guinea-pig.

Na+-gradient-dependent transport of L-proline and analysis of its carrier system in brush-border membrane vesicles of the guinea-pig ileum.

Transport of L-proline was studied with membrane vesicles prepared from the brush borders of the guinea-pig ileum. The presence of an Na+ gradient from outside to inside of the vesicles stimulated L-proline uptake. Accumulation of amino acid in the vesicles reached a maximum 30 s after incubation, then decreased due to efflux and finally equilibrated at a level nearly identical to that shown in the absence of an Na+ gradient in 30 min. The peak level of the uptake was 3.5-times greater than the final equilibrium level. The equilibrium level of L-proline uptake decreased with increasing medium osmolarity. Extrapolation to infinite medium osmolarity, that is, under the condition of zero intravesicular space, showed no uptake, indicating transport of L-proline into membrane vesicles. The initial rate of uptake for 15 s was enhanced with increasing concentrations of Na+ in the external medium. A small part of the L-proline transport occurred by simple diffusion in addition to Na+-gradient-dependent transport. When L-proline concentrations were varied and transport due to diffusion was subtracted, the initial rate of uptake dependent on Na+ gradient (out greater than in) obeyed Michaelis-Menten kinetics with Km and V values of 0.67 mM and 2.73 nmol/15 s per mg protein, respectively. Evidence was obtained which indicates that L-cysteine is a substract specific for transport through system ASC (alanine-, serine-, and cysteine-preferring) and that transport in the presence of an Li+ gradient (out > in) also takes palce by the ASC system. The uptake of L-proline in the presence of an Na+ gradient (out > in) was inhibited 90% by a large excess of alpha-(methylamino)-isobutyrate, the model substrate specific for the A system (alanine-preferring). This indicates than 90% of Na+-gradient-dependent L-proline uptake is supported by the A system. The remaining 10% of L-proline uptake was found to be catalyzed by the ASC system, since L-proline uptake equivalent to this alpha-(methylamino)-isobutyrate-uninhibited part was demonstrated in the presence of Li+ gradient.

Interferon-gamma suppresses thyrotropin receptor promoter activity by reducing thyroid transcription factor-1 (TTF-1) binding to its recognition site.

Interferon-gamma (IFN gamma) is known to suppress the expression of thyroid-specific genes, such as thyroglobulin, thyroid peroxidase, and the TSH receptor (TSHR). In the present study, we show that this reflects, in part, a transcriptional action mediated by thyroid transcription factor-1 (TTF-1). Thus, transfected into rat FRTL-5 cells, the activity of reporter plasmids, containing rat TSHR promoter ligated to a chloramphenicol acetyltransferase gene, was significantly suppressed in the presence of rat IFN gamma. A -199-bp promoter construct showed the greatest suppression by IFN gamma whereas a -177-bp construct, in which the TTF-1 binding site was deleted, showed less suppressibility. The suppressive effect was rat IFN gamma-specific, since human IFN alpha, -beta, and -gamma exhibited no significant effects. The effect was concentration-dependent from 3-50 U/ml. In FRT rat thyroid cells that do not express TTF-1, IFN gamma-induced suppression on the promoter activity was not observed. In addition, when the TTF-1 binding site was mutated so that TTF-1 can not bind, IFN gamma-induced suppression was significantly reduced. In gel mobility shift analyses, a protein-DNA complex formed by TTF-1 was reduced when the nuclear extract prepared from IFN gamma-treated FRTL-5 cells was used; however, expression of TTF-1 mRNA and TTF-1 protein, which were assessed by Northern blot analysis and Western blot analysis, respectively, were not affected by IFN gamma treatment of FRTL-5 cells. Instead, reduction of DNA-binding affinity of TTF-1 was evident when competition analysis was performed in gel mobility shift analysis. From these results, we conclude that IFN gamma suppresses TSHR promoter activity, in part, by reducing TTF-1 binding to its recognition site. We also raise the possibility that the suppressive effect of IFN gamma on promoter activity is mediated by additional element(s) and factor(s) downstream of the TTF-1 site.

Capsid protein genetic analysis and viral spread to the spinal cord in cats experimentally infected with feline calicivirus (FCV).

We investigated primitively the molecular basis of the neural spread of a feline calcivirus isolate (FCV-S) from the spinal cord of a cat that died after manifesting excitation. Experimental infections of cats with three clones from parent virus isolate FCV-S, isolated based on plaque size, were performed, and virus recovery from the spinal cord and the nucleotide and predicted amino acid sequences of the viral capsid protein region (ORF2) were compared. In the experimental infection with the one-time cloned virus (C1L1) isolated from a large plaque, the C1L1 was recovered from the spinal cord. In contrast, seven-times cloned C6L7 (from large plaque) and five-times cloned C5S2 (isolated from small plaque) were not recovered from the spinal cord. Genetic analysis of the capsid protein gene of the three viral clones revealed that four bases were different and two amino acids were different at positions 34 (Val in C6L7 and Ala in C1L1 and C5S2) and 46 (Leu in C6L7 and Pro in C1L1 and C5S2) between C6L7 (with large plaque) and C5S2 (with small plaque). The amino acid at position 434 of C1L1 was different from those of C6L7 and C5S2 (Gly in C1L1, D (Asp) in C6L7 and C5S2). From these results, the plaque size seemed not to be related to the spread of virus to the spinal cord. Clone C1L1, which spread to the spinal cord, had a difference of one amino acid from the other two clones, which may be related to the ability to spread to the spinal cord.

Evaluating multiplexed next-generation sequencing as a method in palynology for mixed pollen samples.

The identification of pollen plays an important role in ecology, palaeo-climatology, honey quality control and other areas. Currently, expert knowledge and reference collections are essential to identify pollen origin through light microscopy. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach, but the assessment of mixed pollen samples originating from multiple plant species is still a tedious and error-prone task. Next-generation sequencing has been proposed to avoid this hindrance. In this study we assessed mixed pollen probes through next-generation sequencing of amplicons from the highly variable, species-specific internal transcribed spacer 2 region of nuclear ribosomal DNA. Further, we developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. We assessed in total 16 mixed pollen samples, 14 originated from honeybee colonies and two from solitary bee nests. The sequencing technique resulted in higher taxon richness (deeper assignments and more identified taxa) compared to light microscopy. Abundance estimations from sequencing data were significantly correlated with counted abundances through light microscopy. Simulation analyses of taxon specificity and sensitivity indicate that 96% of taxa present in the database are correctly identifiable at the genus level and 70% at the species level. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialised palynological expert knowledge.

New mutations of DAX-1 genes in two Japanese patients with X-linked congenital adrenal hypoplasia and hypogonadotropic hypogonadism.

Congenital adrenal hypoplasia, an X-linked disorder, is characterized by primary adrenal insufficiency and frequent association with hypogonadotropic hypogonadism. The X-chromosome gene DAX-1 has been most recently identified and shown to be responsible for this disorder. We analyzed the DAX-1 genes of two unrelated Japanese patients with congenital adrenal hypoplasia and hypogonadotropic hypogonadism by using PCR amplification of genomic DNA and its complete exonic sequencing. In a family containing several affected individuals, the proband male patient had a stop codon (TGA) in place of tryptophan (TGG) at amino acid position 171. As expected, his mother was a heterozygous carrier for the mutation, whereas his father and unaffected brother did not carry this mutation. In another male patient with noncontributory family history, sequencing revealed a 1-bp (T) deletion at amino acid position 280, leading to a frame shift and, subsequently a premature stop codon at amino acid position 371. The presence of this mutation in the patients' genome was further confirmed by digestion of genomic PCR product with MspI created by this mutation. Family studies using MspI digestion of genomic PCR products revealed that neither parent of this individual carried the mutation. These results clearly indicate that congenital adrenal hypoplasia and hypogonadotropic hypogonadism result from not only inherited but also de novo mutation in the DAX-1 gene.

Nicotinamide potentiates TSHR and MHC class II promoter activity in FRTL-5 cells.

Here we show that nicotinamide modulates the promoter activity of rat thyrotropin (TSHR) and major histocompatibility complex (MHC) class II genes in rat FRTL-5 thyroid cells, and have identified a novel mechanism for its action. TSHR and MHC class II, are potentiated through reduced expression of a common repressor of these two genes, TSEP-1 (TSHR suppressor element binding protein-1)/YB-1. Thus we show that TSHR mRNA is increased and TSHR promoter activity was concentration-dependently activated from 0 to 40 mM nicotinamide. The promoter lengths of TSHR and MHC class II containing TSEP/YB-1 binding sites were enhanced by 40 mM nicotinamide, but not the ones deleted of these binding sites. TSEP-1/YB-1 binding to the recognition sites in both TSHR and MHC class II promoters was reduced in nicotinamide-treated FRTL-5 nuclear extracts. Nicotinamide reduced the expression of TSEP-1/YB-1 mRNA and TSEP-1/YB-1 protein in the nucleus.

A novel amphiphilic chiral ligand derived from D-glucosamine. Application to palladium-catalyzed asymmetric allylic substitution reaction in an aqueous or an organic medium, allowing for catalyst recycling

A novel amphiphilic phosphinite-oxazoline chiral compound, 2-methyl-4,5-[4,6-O-benzylidene-3-O-bis[4-((diethylamino)methyl)phenyl]phosphino-1,2-dideoxy-alpha-D-glucopyranosyl]-[2,1-d]-2-oxazoline (1), has been prepared from natural D-glucosamine hydrochloride. The newly prepared complex, [Pd(2-methyl-4,5-[4,6-O-benzylidene-3-O-bis[(4-((diethylmethylammonium)methyl)phenyl)]phosphino-1,2-dideoxy-alpha-D-glucopyranosyl]-[2,1-d]-2-oxazoline)(eta3-C3H5)]3+ x 3BF4- (3), is soluble in water and an efficient catalyst for asymmetric allylic substitution reaction in water or an aqueous/organic biphasic medium (up to 85% ee). This catalytic system offers an easy separation of the aqueous catalyst phase from the product phase and allows recycling of the catalyst phase. In addition, compound 1 also works as an effective ligand for the palladium-catalyzed reaction under conventional homogeneous conditions in an organic medium, in which the catalyst (Pd-1 complex) can be recovered by simple acid/base extraction and reused in the second reaction.

Hepatic actinomycosis: case report and review of the literature in Japan.

Hepatic actinomycosis is rare. We report an 86-year-old Japanese man with a 3-day history of high fever and anorexia who had an actinomycotic liver abscess complicated by disseminated intravascular coagulation (DIC). A definitive diagnosis was made when an Actinomyces species was cultured from aspirated pus. The clinical course was satisfactory. Treatment included prompt percutaneous drainage coupled with long-term intravenous administration of high-dose minocycline and piperacillin, combined with therapy for DIC. We reviewed 11 cases in Japan of Actinomyces involving the liver, including the case reported here. In most patients, there were no predisposing factors. Common symptoms and laboratory findings included fever, abdominal pain, leukocytosis, and elevated C-reactive protein. In 6 of the 11 patients a partial hepatectomy was performed because hepatic tumor was suspected. Five patients presented with a liver abscess. Hepatic actinomycosis should be considered in the differential diagnoses of pyogenic liver abscess and space-occupying lesions of the liver.

Effect of an aldose reductase inhibitor on glomerular basement membrane anionic sites in streptozotocin-induced diabetic rats.

The present study was conducted in order to determine whether an aldose reductase inhibitor (ARI), epalrestat, prevents the progression of diabetic nephropathy in rats. Rats were made diabetic by intravenous injection of streptozotocin (STZ 50 mg/kg) and epalrestat (100 mg/kg) was administered orally through a gastric tube once daily for 4 weeks. Examination by electron microscope revealed that the number of anionic sites (AS) in the lamina rara externa per 1000 nm of glomerular basement membrane (GBM) was significantly decreased in diabetic rats compared to control values (17.6 + or - 0.4 vs. 21.9 + or -0.4, P < 0.01), whereas, significant recovery (20.3 + or - 0.7, P < 0.05) was observed after 4 weeks of epalrestat treatment. Urinary albumin excretion (UAE) rate was markedly increased in diabetic rats and the treatment resulted in its significant suppression from diabetic rats. In conclusion, administration of epalrestat to diabetic rats is capable of preventing a reduction in the number of AS in GBM which would ameliorate an increased permeability of the basement membrane leading to albuminuria.

Protective effect of heparin on renal glomerular anionic sites of streptozotocin-injected rats.

Albuminuria at the rate of 500 micrograms/24 h was observed in rats treated with 50 mg/kg body wt. i.v. streptozotocin (STZ). When STZ was combined with heparin, administered twice daily (250 IU/kg subcutaneous injection) after 24 h following STZ injection, the daily urinary albumin excretion (U-AE) was less than half the amount found in non-heparinized rats (280.3 micrograms/24 h). The observation period in both instances was 8 weeks. When animals were permitted to develop albuminuria over the first 4 weeks, subsequent heparin administration for another 4 weeks lowered U-AE from 500 micrograms/24 h to less than half the amount (227.8 micrograms/24 h). A significantly negative correlation (P < 0.001) existed between U-AE and the number of anionic sites (AS) in the lamina rara externa of glomerular basement membranes, as visualized by electron microscopy. The number of AS per 1000 nm in STZ-injected rats (15.5 +/- 0.2) was lower than that in control rats (23.1 +/- 0.6); however, in heparinized animals, regardless of STZ, the values were not significantly different from normal. Heparin by itself had no effect on the number of AS in normal rats. All STZ-injected animals became hyperglycemic (400-550 mg/dl), but received no insulin. Heparin had no effect on plasma glucose levels. From these results, it is concluded that heparin suppressed both of an increase in U-AE and a decrease in AS count of glomerular basement membranes in STZ-injected rats.

Type 1 angiotensin II receptors of adrenal tumors.

The present study was designed to clarify the transcriptional regulation of the human type 1 angiotensin II receptor (AT1) gene and its pathophysiological roles in steroidogenesis by adrenal tumors. A cDNA encoding type 1 angiotensin II receptor (AT1) was isolated from a human liver cDNA library encoding a protein of 359 amino acids with seven transmembrane segments. It is very likely that human has only one type of AT1 receptor, in contrast with rodents. A genomic clone containing 217 bp of exon 1 and 2558 bp of the 5'-flanking region of human AT1 receptor gene was isolated. Its proximal promoter region contained putative TATA and GC boxes, CRE and AP1 sites. Aldosterone-producing adenoma (APA) contained significantly higher levels of mRNA for AT1 and ACTH receptors than normal tissues adjacent to APA. There were no mutations within the cytoplasmic third loops of AT1 and ACTH receptors in APAs examined. APA showed increased expression of the mRNA for P450c11 and decreased expression of the mRNA for P450c17. These results suggest that renin-independent overproduction and clinically observed ACTH-dependent production of aldosterone in APAs may results from the enhanced transcription of P450c11 and ACTH receptor genes. The mechanism of the discordantly increased expression of AT1 receptor in APA remains to be clarified.