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.

Aminopeptidase N from Bombyx mori as a candidate for the receptor of Bacillus thuringiensis Cry1Aa toxin.

Cry1Aa toxin-binding proteins from the midgut brush border membrane vesicles of Bombyx mori, a toxin-susceptible silkworm, were analyzed to find candidates for the toxin receptors. Ligand blotting showed that Cry1Aa toxin bound to a 120-kDa protein. A part of the 120-kDa protein was solubilized from the membrane vesicles with phosphatidylinositol-specific phospholipase C, resulting in a 110-kDa protein which therefore may be linked to a glycosyl-phosphatidylinositol anchor. The 120-kDa and 110-kDa Cry1Aa toxin-binding proteins were solubilized with detergent or pohosphatidylinositol-specific phospholipase C, respectively, and purified using anion-exchange chromatography. Scatchard plot analysis for the specific binding of purified 110-kDa protein to Cry1Aa toxin yielded a Kd value of 7.6 nM, which was similar to that for the binding of intact brush border membrane vesicles to the toxin. N-terminal and internal amino acid sequences of the 120-kDa and 110-kDa proteins showed high degrees of similarity to those of aminopeptidase N, a putative Cry1Ac toxin receptor, reported in Manduca sexta and Heliothis virescens. On this basis, the 120-kDa Cry1Aa toxin-binding protein from B. mori was identified as a member of the aminopeptidase family.

Molecular cloning and characterization of the ABC transporter expressed in Trachea (ATET) gene from Drosophila melanogaster.

A novel member of the ATP-binding cassette (ABC) transporter proteins has been cloned from Drosophila melanogaster. The gene is designated as ABC Transporter Expressed in Trachea (Atet), because the transcript was localized to the respiratory system by in situ hybridization analysis of whole-mount embryos using digoxigenin-labeled RNA probes. The hybridization signal was also observed in amnioserosa. Northern blot analysis identified a single 4.5 kb mRNA expressed in all developmental stages at a relatively constant level. The Atet gene mapped to 24E on the left arm of the second chromosome. The Atet protein shows extensive homology with the Drosophila white gene product, which is reported to form heterodimers with the brown or scarlet gene product to transport guanine or tryptophan into the pigment cells in the compound eye. By analogy, Atet is suggested to be involved in transporting a small molecule after dimerization with a partner protein.

Isolation and characterization of a new acetate-requiring mutant strain, ace-9, of Neurospora crassa.

A new acetate-requiring mutant strain of Neurospora crassa, ace-9, has been isolated. The mutant gene was mapped between nuc-2 and arg-12 on the right arm of the second linkage group. The ace-9 mutant strain shows very weak activity of pyruvate dehydrogenase complex (PDHC). Three strains that show no activity of PDHC had already been found, i.e., ace-2, ace-3, and ace-4. Thus the ace-9 is the fourth gene that causes the deficiency in PDHC activity by a mutation. Deficiency of PDHC activity in ace-9 strain seems to be due to defective E1 component, because (1) the activity of E1 component enzyme is very weak in ace-9 mutant strain, and (2) normal PDHC activity was resumed when a preparation of ace-9 was mixed with E1-E2 fraction of wild type or with E1 component of wild type E. coli. Difference in thermostability of both E1 component enzyme and PDHC between ace-9 and the wild type strains supports this conclusion.

A basal unit of valine-sensitive acetolactate synthase of Neurospora crassa.

Valine-sensitivity as well as activity of acetolactate synthase of Neurospora crassa was stabilized with 1.2 M potassium phosphate buffer during extraction from mitochondria and early stages of purification, and with 20% glycerol plus 5 mM sodium pyruvate during Sephadex G200 gel chromatography. The enzyme was expressed as four molecular species having the molecular weights of about 500,000, 140,000, 68,000 and 51,000, respectively. The first and the third species showed valine-sensitivity, but the second and the fourth did not. The third molecular species with a molecular weight of 68,000 may be the basal unit of valine-sensitive acetolactate synthase of Neurospora crassa.

Localization of pyruvate carboxylase in the cells of Neurospora crassa.

The cell wall of Neurospora crassa was digested enzymatically and the cytosolic and the mitochondrial fractions were separated. The activity of pyruvate carboxylase (EC 6.4.1.1) was detected entirely in the cytosolic fraction. This indicates that the location of pyruvate carboxylase of N. crassa is in the cytosol, but is not in the mitochondria; this is different from the situation in animal tissues.

Solubilization of valine-sensitive acetohydroxy acid synthetase from Neurospora mitochondria.

Acetohydroxy acid synthetase [EC 4.1.3.18] of Neurospora crassa has been solubilized from a mitochondrial pellet fraction by sonic treatment in 1.0 M potassium phosphate buffer at pH 7.5 containing 10 mM MgSO4 and centrifugation at 180,000 X g for 20 min. The soluble enzyme thus obtained has a high specific activity and a high sensitivity to valine comparable to the original mitochondrial pellet suspension when the activity was determined in high concentrations of potassium phosphate.