Kidney enlargement and multiple liver cyst formation implicate mutations in PKD1/2 in adult sporadic polycystic kidney disease.

Distinguishing autosomal-dominant polycystic kidney disease (ADPKD) from other inherited renal cystic diseases in patients with adult polycystic kidney disease and no family history is critical for correct treatment and appropriate genetic counseling. However, for patients with no family history, there are no definitive imaging findings that provide an unequivocal ADPKD diagnosis. We analyzed 53 adult polycystic kidney disease patients with no family history. Comprehensive genetic testing was performed using capture-based next-generation sequencing for 69 genes currently known to cause hereditary renal cystic diseases including ADPKD. Through our analysis, 32 patients had PKD1 or PKD2 mutations. Additionally, 3 patients with disease-causing mutations in NPHP4, PKHD1, and OFD1 were diagnosed with an inherited renal cystic disease other than ADPKD. In patients with PKD1 or PKD2 mutations, the prevalence of polycystic liver disease, defined as more than 20 liver cysts, was significantly higher (71.9% vs 33.3%, P = .006), total kidney volume was significantly increased (median, 1580.7 mL vs 791.0 mL, P = .027) and mean arterial pressure was significantly higher (median, 98 mm Hg vs 91 mm Hg, P = .012). The genetic screening approach and clinical features described here are potentially beneficial for optimal management of adult sporadic polycystic kidney disease patients.

Expression and function of the Delta-1/Notch-2/Hes-1 pathway during experimental acute kidney injury.

The Notch signaling pathway consists of several receptors and their ligands Delta and Jagged and is important for embryogenesis, cellular differentiation and proliferation. Activation of Notch receptors causes their cleavage yielding cytoplastic domains that translocate into the nucleus to induce target proteins such as the basic-loop-helix proteins Hes and Hey. Here we sought to clarify the significance of the Notch signaling pathway in acute kidney injury using a rat ischemia-reperfusion injury model and cultured NRK-52E cells. Analysis of the whole kidney after injury showed increased expression of Delta-1 and Hes-1 mRNA and protein along with processed Notch-2. Confocal microscopy, using specific antibodies, showed that Delta-1, cleaved Notch-2 and Hes-1 colocalized in the same segments of the injured renal proximal tubules. Recombinant Delta-1 significantly stimulated NRK-52E cell proliferation. Our study suggests that the Delta-1/Notch-2/Hes-1 signaling pathway may regulate the regeneration and proliferation of renal tubules during acute kidney injury.

The phosphoinositide-3 kinase gamma-Akt pathway mediates renal tubular injury in cisplatin nephrotoxicity.

Nephrotoxicity is a frequent complication of cisplatin-based chemotherapy often limiting its use. In this study, we attempted to the role of the phosphoinositide-3 kinase (PI3K)-gamma-Akt pathway in this form of acute kidney injury. Using PI3K-gamma knockout mice, we found that a conventional dose of cisplatin was more lethal in the knockout mice where the blood urea nitrogen and serum creatinine were significantly higher in them than in wild-type mice. Phosphorylation of Akt in the renal tubules was abrogated in the knockout mice with the severity of renal dysfunction and numbers of TUNEL (terminal deoxynucleotidyl transferase (TdT) mediated nick-end labeling)-positive renal tubule cells being higher in the knockout than in wild-type mice. Cisplatin treatment significantly increased. Caspase-3 activity, histone-associated DNA fragments, and number of annexin V-positive cells was significantly higher in cisplatin-treated primary cultured renal tubular epithelial cells of knockout mice. Transfection of dominant-active forms of Akt and PI3K-gamma ameliorated apoptosis of the tubule epithelial cells derived from the knockout mice. Our results suggest that the PI3K-gamma-Akt pathway lessens apoptosis and plays a critical role in the maintenance of renal function in cisplatin-induced acute kidney injury.

Efficient and ligand-dependent regulated erythropoietin production by naked dna injection and in vivo electroporation.

The development of an in vivo gene transfer approach to deliver physiologic levels of recombinant proteins to the systemic circulation would represent a significant advance in the treatment of protein deficiencies-disorders. However, the ability to regulate transgene expression will become paramount for safety and efficacy in gene transfer therapy. We have described the construction of an efficient and ligand-dependently regulated erythropoietin (EPO) production system using naked plasmid and in vivo electroporation. Two plasmids, one encoding the chimeric GeneSwitch protein and the other encoding an inducible transgene for human EPO, were developed. Modulation of the level of secretion of EPO into the serum was achieved by intraperitoneal administration of mifepristone (MFP). Rats were divided into 4 groups: one group received EPO plasmid with MFP for 30 days, a second group received with EPO plasmid MFP for 9 days, a third group received EPO plasmid without MFP, and a fourth group received control plasmid. A pair of electrodes was inserted into the muscle of the right thigh and 100 micrograms of each plasmid was injected. In vivo electrporation (8 times at 100 V for 50 milliseconds) was performed. The presence of vector-derived EPO mRNA was detected by reverse transcriptase-polymerase chain reaction only in the EPO and MFP(+) group. The hematocrit levels increased continuously from the preinjection level of 42.7% to 53.8% on day 28 in the EPO and MFP(+) group. The serum EPO levels increased only in the EPO and MFP(+) group. There was no significant change in hematocrit levels and EPO levels in the EPO and MFP(-) group. These results demonstrate that EPO gene transfer with the GeneSwitch system by in vivo electroporation is a useful procedure for efficient and drug-dependent regulated delivery of EPO.

Glucocorticoids stimulate p21(CIP1) in mesangial cells and in anti-GBM glomerulonephritis.

BACKGROUND: Glucocorticoids are widely used for the treatment of glomerulonephritis, but the mechanism of cell cycle inhibition by glucocorticoids is poorly understood at a molecular level. METHODS: The effects of dexamethasone on cell cycle progression were examined in rat mesangial cells. To investigate the mechanisms of cell cycle inhibition by dexamethasone, we transfected the -2.3 kb p21(CIP1) promoter-CAT construct to mesangial cells using an electroporation METHOD: We also examined whether glucocorticoids stimulate the expression of p21(CIP1) and inhibit cell proliferation in glomeruli of anti-glomerular basement membrane (GBM) glomerulonephritis in rats. RESULTS: Dexamethasone inhibited 3H-thymidine uptake and the percentages of S and G2/M phases in rat mesangial cells. Dexamethasone stimulated CAT activity of the p21(CIP1) promoter 4.5-fold. Deletion analysis of the p21(CIP1) promoter revealed that the glucocorticoid-responsive region (GRE) is present between -1.4 and -1.1 kb upstream of the transcription initiation site. Dexamethasone inducibility of p21(CIP1) promoter activity requires the presence of the C/EBP alpha DNA binding site in the GRE of the p21(CIP1) promoter and C/EBP alpha protein. Intravenous injection of anti-GBM antibody caused mesangial proliferation, crescent formation, and proteinuria in rats. Ten days of administration of prednisolone (1 mg/kg/day) reduced proteinuria and inhibited mesangial cell proliferation and crescent formation. The glomerular-sieving method revealed that prednisolone increased p21(CIP1) expression in glomeruli. CONCLUSION: These data suggest that the cell cycle arrest of mesangial cells is mediated by a functional link between the glucocorticoid receptor and the transcriptional control of p21(CIP1) not only in vitro but also in vivo. Our observations provide new insights into the molecular mechanisms of glucocorticoid action in glomerulonephritis.

Seasonal changes in human sleep-wake rhythm in Antarctica and Japan.

The subjects were eight men of the Japanese Antarctic Research Expedition (average age 35.8 years), and 10 healthy people living around Kofu, Japan (28.9 years). They completed a sleep log for 12 to 18 months, and the sleep-wake state was scored in 10-min epochs. Q24 values calculated by chi2 periodgram were low in the Antarctic midwinter. This means that there was difficulty in synchronizing to a 24-h period in the Antarctic midwinter. In Antarctica, sleep onset and offset times were delayed mostly in the midwinter. In Japan, sleep offset time was delayed mostly around the winter solstice.

Validity of sleep log compared with actigraphic sleep-wake state II.

Twenty-five young people (Y group), three elderly people and seven people with various sleep disorders (SD group) kept a sleep log for 2-7 days, and their wrist-activity was monitored simultaneously. The sensitivity and specificity of the sleep log, and the ratio of agreement between the sleep log and actigraphic sleep-wake state were calculated. The sensitivity and specificity in Y group were 87.93+/-6.49% and 96.51+/-2.37%, respectively. The sensitivity in SD group was significantly lower than in Y group. Even in Y group one-hour agreement ratios dropped during the sleep onset period.

An aureobasidin A resistance gene isolated from Aspergillus is a homolog of yeast AUR1, a gene responsible for inositol phosphorylceramide (IPC) synthase activity.

The AUR1 gene of Saccharomyces cerevisiae, mutations in which confer resistance to the antibiotic aureobasidin A, is necessary for inositol phosphorylceramide (IPC) synthase activity. We report the molecular cloning and characterization of the Aspergillus nidulans aurA gene, which is homologous to AUR1. A single point mutation in the aurA gene of A. nidulans confers a high level of resistance to aureobasidin A. The A. nidulans aurA gene was used to identify its homologs in other Aspergillus species, including A. fumigatus, A. niger, and A. oryzae. The deduced amino acid sequence of an aurA homolog from the pathogenic fungus A. fumigatus showed 87% identity to that of A. nidulans. The AurA proteins of A. nidulans and A. fumigatus shared common characteristics in primary structure, including sequence, hydropathy profile, and N-glycosylation sites, with their S. cerevisiae, Schizosaccharomyces pombe, and Candida albicans counterparts. These results suggest that the aureobasidin resistance gene is conserved evolutionarily in various fungi.

Two-dimensional electrophoresis of Malassezia allergens for atopic dermatitis and isolation of Mal f 4 homologs with mitochondrial malate dehydrogenase.

The yeast Malassezia furfur is a natural inhabitant of the human skin microflora that induces an allergic reaction in atopic dermatitis. To identify allergens of M. furfur, we separated a crude preparation of M. furfur antigens as discrete spots by 2-D PAGE and detected IgE-binding proteins using sera of atopic dermatitis patients. We identified the known allergens, Mal f 2 and Mal f 3, and determined N-terminal amino acid sequences of six new IgE-binding proteins including Mal f 4. The cDNA and genomic DNA encoding Mal f 4 were cloned and sequenced. The gene was mitochondrial malate dehydrogenase and encoded Mal f 4 composed of 315 amino acids and a signal sequence of 27 amino acids. We purified Mal f 4, which had a molecular mass of 35 kDa from a membrane fraction of a lysate of cultured cells. Thirty of 36 M. furfur-allergic atopic dermatitis patients (83.3%) had elevated serum levels of IgE to purified Mal f 4, indicating that Mal f 4 is a major allergen. There was a significant correlation of the Phadebas RAST unit values of Mal f 4 and the crude antigen, but not between Mal f 4 and the known allergen Mal f 2.

A novel cell-free system for peptide synthesis driven by pyridine.

Previously we demonstrated that ribosomes can synthesize polypeptides in the presence of high concentrations (40-60%) of pyridine without any protein factors. Here we analyze additional ribosomal parameters in 60% pyridine using Escherichia coli ribosomes. Ribosomal subunits once exposed to pyridine failed to re-associate to 70S ribosomes in aqueous buffer systems even in the presence of 20 mM Mg2+, whereas they formed 70S complexes in the presence of 60% pyridine. Two-dimensional gel electrophoresis of ribosomal proteins revealed that some proteins located at the protuberances of the large subunit, e. g. L7/L12 and L11 forming the elongation factor-binding domain, were released in the pyridine system. The aminoglycoside neomycin, a strong inhibitor of the ribosomal (factor-independent) translocation reaction, completely blocked poly(Phe) synthesis and translocation activities in the pyridine system, whereas these activities were not affected at all by gypsophilin, a ribotoxin that inhibits factor-dependent translocation. Another inhibitor of the ribosomal translocation, thiostrepton, had no effect concerning the two activities, which is consistent with the fact that this antibiotic requires L11 for its binding to the ribosome. These results suggest that the ribosomes can perform a translocation reaction in the pyridine system, but in a factor-independent (spontaneous) manner.

Identification and cloning of two novel allergens from the lipophilic yeast, Malassezia furfur.

Two novel allergens, designated Mal f 2 and Mal f 3 according to the WHO/IUIS Allergen Nomenclature Subcommittee recommendation, were isolated from the lipophilic yeast Malassezia furfur cell extracts and the genes coding for those were cloned. Mal f 2 and Mal f 3 had apparent molecular weights of 21 kDa and 20 kDa, respectively, on SDS-PAGE under reducing conditions. The identified cDNA clone of Mal f 2 encoded an open reading frame of 177 amino acid residues. Fifty-one percent identity was found between the Mal f 2 and Mal f 3 sequences. Comparison of the Mal f 2 and Mal f 3 sequences with known protein sequences revealed that they had sequence homology with two peroxisomal membrane proteins of Candida boidinii and an Aspergillus fumigatus allergen, Asp f 3. In RAST, both Mal f 2- and Mal f 3-specific IgE antibodies could be detected in approximately 70 % of sera from M. furfur sensitized patients with atopic dermatitis.

Validity of sleep log compared with actigraphic sleep-wake state.

Seven women and 11 men, mean age 30.1 years, kept a sleep log for 5-7 days, and their wrist activity was monitored each minute. Sleep-wake state in the sleep log and actigraphic sleep-wake state were compared, and the sensitivity and specificity of the sleep log were calculated for each subject. The ratio of agreement between these two parameters was computed for each subject. The sensitivity and specificity of the sleep log were 72.73-97.56% (mean 86.71%) and 92.85-99.68% (mean 97.04%), respectively. The agreement ratio was 87.30-97.85% (93.48%), but 1-h agreement ratios from midnight dropped during the sleep onset period.

Role of ABC transporters in aureobasidin A resistance.

Aureobasidin A (AbA) has strong antifungal effects arising from an unusual mechanism. We show that AbA interacts with ATP-binding cassette (ABC) transporters in yeast and mammalian cells. We isolated a gene of Saccharomyces cerevisiae that conferred resistance to AbA when the gene was present in multiple copies. The gene was identical to YOR1/YRS1, which confers resistance to oligomycin, reveromycin, and organic anions, none of which have structures similar to that of AbA. We also isolated an aur3R recessive mutant of S. cerevisiae with increased resistance to AbA. Northern hybridization showed that the aur3R mutant expressed not only YOR1 but also the ABC transporter-encoding gene PDR5 at high levels. Genetic studies showed that the aur3R mutant had a mutation in the PDR1 gene, which encodes a transcriptional regulator of PDR5 and YOR1. Analysis of a yor1 disruptant of the aur3/pdr1 mutant showed that both the functional YOR1 gene and the mutation in PDR1 were necessary for AbA resistance. These results suggest that YOR1 is more important than PDR5 for AbA resistance. We found in Candida albicans a novel gene whose sequence was similar to the sequence of YOR1 in S. cerevisiae. The amino acid sequence of the C. albicans YOR1 homolog showed no significant similarity to the sequences of CDR1 and CDR2, which are ABC transporters of C. albicans. Furthermore, AbA inhibited the efflux of the anticancer agent vincristine through P glycoproteins in cancer cells with multidrug resistance.

Isolation and characterization of the aureobasidin A-resistant gene, aur1R, on Schizosaccharomyces pombe: roles of Aur1p+ in cell morphogenesis.

To study the mechanism of action of the antibiotic aureobasidin A (AbA) on yeasts, we isolated a dominant mutant of Schizosaccharomyces pombe which gave high resistance to AbA. From a genomic library of the mutant, an aur1R mutant gene conferring AbA resistance was isolated. One amino-acid mutation, a substitution of glycine with cysteine at residue 240, was responsible for the acquisition of AbA resistance. The wild-type aur1+ gene was essential for viability, and its over-expression enhanced significant resistance to AbA. The predicted protein of S. pombe aur1R was highly homologous in primary structure and hydropathy profile with that of Saccharomyces cerevisiae AUR1R isolated as an AbA-resistance gene. To analyze a role in cell growth of S. pombe aur1+, temperature-sensitive mutants (aur1ts) were obtained by random mutagenesis procedures using a modified PCR. The aur1ts mutation caused a defect in cell elongation at the non-permissive temperature and finally led to cell death. These results suggest that Aur1p was a target of the antibiotic AbA and was required in the cell elongation of cell-end tips and in the viability of S. pombe.

Transformation system for prototrophic industrial yeasts using the AUR1 gene as a dominant selection marker.

We show a new transformation system for prototrophic yeast strains including those of Saccharomyces cerevisiae, Kluyveromyces lactis, K. marxianus, and Candida glabrata. This system is composed of an antibiotic, aureobasidin A (AbA), and its resistance gene AUR1-C as a selection marker. Southern analysis of genomic DNAs of the transformants indicated that the copy number of the plasmid increased from one to more than four, depending on the concentration of AbA used for selection of the transformants. The AUR1-C gene was also effective as a selection marker for gene disruption, and was able to disrupt both copies of the gene on homologous chromosomes of diploid cells by a single round of transformation. This system has a broad application in the transformation and gene disruption of prototrophic strains of a variety of yeast species.

AUR1, a novel gene conferring aureobasidin resistance on Saccharomyces cerevisiae: a study of defective morphologies in Aur1p-depleted cells.

Aureobasidin A (AbA), a cyclic depsipeptide produced by Aureobasidium pullulans R106, is highly toxic to fungi including Saccharomyces cerevisiae. We isolated several dominant mutants of S. cerevisiae which are resistant to more than 25 micrograms/ml of AbA. From a genomic library of one such AUR1 mutant, the AUR1R (for aureobasidin resistant) mutant gene was isolated as a gene that confers resistance to AbA on wild-type cells. Its nucleotide sequence showed that the predicted polypeptide is a hydrophobic protein composed of 401 amino acids, which contains several possible transmembrane domains and at least one predicted N-linked glycosylation site. Comparison of the mutant gene with the wild-type aur1+ gene revealed that the substitution of Phe at position 158 by Tyr is responsible for acquisition of AbA resistance. We suggest that the gene product of the wild-type aur1+ is a target for AbA on the basis of following results. Firstly, cells that overexpress the wild-type aur1+ gene become resistant to AbA, just as cells with an AUR1R mutation do. Secondly, disruption of the aur1+ gene demonstrated that it is essential for growth. Thirdly, in the cells with a disrupted aur1 locus, pleiotropic morphological changes including disappearance of microtubules, degradation of tubulin and abnormal deposition of chitin were observed. Some of these abnormalities are also observed when wild-type cells are treated with AbA. The abnormality in microtubules suggests that the Aur1 protein is involved in microtubule organization and stabilization.

Enzyme-linked immunosorbent assay for human autoantibody to glial fibrillary acidic protein: higher titer of the antibody is detected in serum of patients with Alzheimer's disease.

We have developed an enzyme-linked immunosorbent assay (ELISA) to detect anti-glial fibrillary acidic protein (GFAP) autoantibody in human sera. The ELISA was prepared by coating microtest plates with purified GFAP from bovine spinal cord. The autoantibody activities were assayed in the serum from 219 control subjects, 39 Alzheimer's disease patients and 39 cerebrovascular dementia patients. Higher titer of the antibody was observed in the serum of Alzheimer's disease patients. Since the titer showed no significant change with aging or with sex in the control serum, we could determine a certain normal value of the antibody titer. The percentage of abnormal subjects whose antibody levels were over the normal value was 53.8% in Alzheimer's disease (presenile onset) patients, 30.8% in Alzheimer's disease (senile onset) patients, 10.3% in cerebrovascular dementia patients and 5.5% in control subjects. We discuss the relationship between the anti-GFAP autoantibody and the pathogenesis of Alzheimer's disease and suggest that the evaluation of anti-GFAP autoantibody level may be useful in diagnosing Alzheimer's disease.