Upstream Regulators and Downstream Effectors of NADPH Oxidases as Novel Therapeutic Targets for Diabetic Kidney Disease

Oxidative stress has been linked to the pathogenesis of diabetic nephropathy, the complication of diabetes in the kidney. NADPH oxidases of the Nox family, and in particular the homologue Nox4, are a major source of reactive oxygen species in the diabetic kidney and are critical mediators of redox signaling in glomerular and tubulointerstitial cells exposed to the diabetic milieu. Here, we present an overview of the current knowledge related to the understanding of the role of Nox enzymes in the processes that control mesangial cell, podocyte and tubulointerstitial cell injury induced by hyperglycemia and other predominant factors enhanced in the diabetic milieu, including the renin-angiotensin system and transforming growth factor-β. The nature of the upstream modulators of Nox enzymes as well as the downstream targets of the Nox NADPH oxidases implicated in the propagation of the redox processes that alter renal biology in diabetes will be highlighted.     

Chemical structures of a galactose-rich glycoprotein of Leishmania tarentolae

1. Aqueous phenol treatment of water extracted disrupted cells of Leishmania tarentolae (LV-414) provided a glycoprotein mixture which was purified by gel filtration chromatography, and Concanavalin A-Sepharose column. 2. The bound fraction on Concanavalin A-Sepharose column (protein 74%, and carbohydrate, 26%) had [alpha]D + 9 degrees and contained mannose (18%), galactose (60%), and glucose (22%), and some of the galactose residues were resistant to periodate oxidation. 3. Treatment of the phenol extract with hot aqueous NaBH4 containing NaOH gave a preparation having mannose (12%), galactose (82%), and glucose (6%). 4. Methylation analysis showed the presence of a mainly linear structure with non-reducing end-units of mannopyranose (6%), 3-O-substituted galactopyranosyl (64%), 2-O- (11%), and 6-O- (5%) substituted mannopyranosyl, and 4-O- (9%), and 4,6-di-O- (3%) substituted glycopyranosyl units. 5. The specific rotation of the preparation, +20 degrees, indicated beta-linked galactopyranosyl units.     

Leptomonas samueli glycoconjugates. Comparison with Herpetomonas samuelpessoai

Xylose and mannose are the main manosaccharide components of Herpetomonas samuelpessoai and Leptomonas samueli promastigotes. Variations in the xylose/mannose ratios are related to the age of cultures. Phenol-aqueous extraction disclosed in both species the presence of carbohydrate-containing fractions which were both soluble and insoluble in chloroform/methanol/water (10:10.3). The xylose enriched, uronic acid-containing glycoconjugates of L. samueli were mainly composed of (1----4)-linked xylose units (Methylation-mass spectrometry and 13C NMR), similar to the glucuronoxylan of H. samuelpessoai (Mendon?a-Previato et al., 1979 Biochem 18 149-154). SDS-PAGE and sugar composition analysis disclosed similarities between glycoconjugates of H. samuelpessoai and L. samueli, two species dwelling in the same host, therefore an example of convergent adaptation.     

Location of O-acetyl groups in the acidic d-xylan of mimosa scabrella (bracatinga). A study of O-acetyl group migration

Extraction with dimethyl sulfoxide of wood-meal of the stem of bracatinga (Mimosa scabrella), a south Brazilian hardwood, that was defatted and delignified by treatment with aqueous chlorine at 0–5° followed by extraction with cold ethanol, gave a soluble O-acetylated 4-O-methyl-d-glucurono-d-xylan having (1→4)-linked β-d-xylopyranosyl residues that were unsubstituted (65%) and 2-O-(14%), 3-O- (16%), and 2,3-di-O-acetylated (5%), as determined by methylation analysis. Another preparation obtained by use of refluxing ethanol in the delignification process showed neither removal nor migration of acetyl groups. By comparison with synthetic, partly O-acetylated d-xylans of known composition, ¹³C-n.m.r. spectroscopy indicated that O-acetyl group migration does not occur during treatment with cold aqueous chlorine, refluxing ethanol, or water at 70°. Methyl 2-O-acetyl-4-O-methyl-β-d-xylopyranoside (6) was also unaffected by aqueous chlorine. O-Acetyl group migration took place more readily in aqueous and dimethyl sulfoxide solutions of 6 than of O-acetyl-d-xylans. The lowest temperatures at which migration was observed in monosaccharides was at 50 and 70° for solutions in D₂O and (CD₃)₂SO, respectively.     

Myosin VIIA mutation screening in 189 Usher syndrome type 1 patients.

Usher syndrome type 1b (USH1B) is an autosomal recessive disorder characterized by congenital profound hearing loss, vestibular abnormalities, and retinitis pigmentosa. The disorder has recently been shown to be caused by mutations in the myosin VIIa gene (MYO7A) located on 11q14. In the current study, a panel of 189 genetically independent Usher I cases were screened for the presence of mutations in the N-terminal coding portion of the motor domain of MYO7A by heteroduplex analysis of 14 exons. Twenty-three mutations were found segregating with the disease in 20 families. Of the 23 mutations, 13 were unique, and 2 of the 13 unique mutations (Arg212His and Arg212Cys) accounted for the greatest percentage of observed mutant alleles (8/23, 31%). Six of the 13 mutations caused premature stop codons, 6 caused changes in the amino acid sequence of the myosin VIIa protein, and 1 resulted in a splicing defect. Three patients were homozygotes or compound heterozygotes for mutant alleles; these three cases were Tyr333Stop/Tyr333Stop, Arg212His-Arg302His/Arg212His-Arg302His, and IVS13nt-8c-->g/Glu450Gln. All the other USH1B mutations observed were simple heterozygotes, and it is presumed that the mutation on the other allele is present in the unscreened regions of the gene. None of the mutations reported here were observed in 96 unrelated control samples, although several polymorphisms were detected. These results add three patients to single case reported previously where mutations have been found in both alleles and raises the total number of unique mutations in MYO7A to 16.     

Efficient strategies for genomic searching using the affected-pedigree-member method of linkage analysis.

The affected-pedigree-member (APM) method of linkage analysis is a nonparametric statistic that tests for nonrandom cosegregation of a disease and marker loci. The APM statistic is based on the observation that if a marker locus is near a disease-susceptibility locus, then affected individuals within a family should be more similar at the marker locus than is expected by chance. The APM statistic measures marker similarity in terms of identity by state (IBS) of marker alleles; that is, two alleles are IBS if they are the same, regardless of their ancestral origin. Since the APM statistic measures increased marker similarity, it makes no assumptions concerning how the disease is inherited; this can be an advantage when dealing with complex diseases for which the mode of inheritance is difficult to determine. We investigate here the power of the APM statistic to detect linkage in the context of a genomewide search. In such a search, the APM statistic is evaluated at a grid of markers. Then regions with high APM statistics are investigated more thoroughly by typing more markers in the region. Using simulated data, we investigate various search strategies and recommend an optimal search strategy that maximizes the power to detect linkage while minimizing the false-positive rate and number of markers. We determine an optimal series of three increasing cut-points and an independent criterion for significance.     

New polymorphic markers in the vicinity of the pearl locus on mouse Chromosome 13

We have used a Mus domesticus/-Mus spretus congenic animal that was selected for retention of Mus spretus DNA around the pearl locus to create a highly polymorphic region suitable for screening new markers. Representation difference analysis (RDA) was performed with either DNA from the congenic animal or C57BL/6J as the driver for subtraction. Four clones were identified, characterized, and converted to PCR-based polymorphic markers. Three of the four markers equally subdivide a 10-cM interval containing the pearl locus, with the fourth located centromeric to it. These markers have been placed on the mouse genetic map by use of an interspecific backcross panel between Mus domesticus (C57BL/6J) and Mus spretus generated by The Jackson Laboratory. Received: 12 June 1995 / Accepted: 17 August 1995     

Preparation of α and β anomers of various isomeric methyl O-d-galactopyranosyl-d-galactopyranosides. Standards for interpretation of 13C-n.m.r. spectra of d-galactopyranans

The four isomers of methyl O-β-d-galactopyranosyl-β-d-galactopyranoside were prepared by condensation of 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl bromide with appropriate, partially O-substituted derivatives of methyl β-d-galactopyranoside. Reaction of 3,4,6-tri-O-acetyl-1,2-O-(1-ethoxyethylidene)-α-d-galactopyranose with the same acceptors, in the presence of mercuric bromide, led to the formation of α and β linkages. Thus, it was possible to assign ¹³C-n.m.r. resonances of α and β anomers of methyl O-d-galactopyranosyl-β-d-galactopyranosides. In terms of application of these shift values and those of related d-galactobioses to the structural analysis of d-galactopyranans by shift comparisons, some generalizations can be made. For β-d-galactopyranans, the resonances of glycosyloxylated carbon atoms of methyl O-β-d-galactopyranosyl-β-d-galactopyranosides are sensitive to structure and appear to have typical values, whereas limited variation was observed with shifts of C-1′ signals. On the other hand, for assigning structures to d-galactopyranans containing α linkages, the C-1′ shifts (at higher field) of methyl O-α-d-galactopyranosyl-β-d-galactopyranosides are sensitive to linkage position, whereas those of glycosyloxylated carbon atoms vary only a little.