Glutathione ester protects against hydroxynonenal-induced loss of auditory hair cells.

OBJECTIVE: Test the ability of glutathione monoethyl ester (GSH(e)) to protect auditory hair cells against the ototoxic effects of 4-hydroxy-2,3-nonenal (HNE). STUDY DESIGN AND SETTING: Organ of Corti explants were either untreated or treated with one of a series of four concentrations of GSH(e) for one day, then exposed to HNE. Counts of FITC-phalloidin-labeled hair cells determined both HNE ototoxicity and GSH(e) otoprotection. RESULTS: HNE was toxic to hair cells at physiologically relevant levels, eg, 400 muM, and GSH(e) provided a significant level of protection against HNE ototoxicity (P < 0.05) at all levels tested, ie, 1.16 to 9.3 mM. CONCLUSION: GSH(e) protects auditory hair cells from damage and loss initiated by a naturally occurring ototoxic molecule, ie, HNE (a by-product of oxidative stress). SIGNIFICANCE: Treatment with GSH(e) may be an effective therapy to protect the cochlea against the adverse effects of traumas (eg, electrode insertion) that generate oxidative stress.     

Virulence of Candida albicans mutants

Mutant strains of the fungal pathogen Candida albicans blocked in pyrimidine transport and salvage metabolism were tested for virulence in various animal models. The growth rate, germination and proteolytic enzyme production did not correlate with the virulence of the strains. However, a defect in the uridine transport system significantly decreased virulence in murine candidosis, although it had no effect in vaginal candidosis or in a Candida cyst model. It remains unclear whether this is due to the differing host defence mechanisms involved in systemic and superficial mycoses, or to the different requirements of the fungal systems for adherence and tissue invasion in the two types of infection.     

Cloning and expression analysis of the chick DAN gene, an antagonist of the BMP family of growth factors.

Differential screening-selected gene aberrative in neuroblastoma (DAN) is a member of a cystine knot protein family that includes Cerberus and Gremlin. First isolated in a screen to identify genes down-regulated in transformed rat fibroblasts, DAN has subsequently been cloned in Xenopus, mouse, and human. Overexpression of DAN suppresses the transformed phenotype and retards the cell's entry into S phase. Biochemical analyses have demonstrated DAN's ability to bind bone morphogenetic proteins and antagonize their signaling activity. In this study, chick DAN was cloned and sequenced, revealing a conserved cystine knot region as well as an N-glycosylation site. A riboprobe was designed from the 3' chick DAN coding sequence and used for analysis of DAN in the developing chick embryo by in situ hybridization. Chick DAN was expressed beginning at stage 10 in the developing somites and the medial otic epithelium. Expression in the neural layer of the eye became apparent at stage 14. By stage 17, expression had expanded to the base of the hindbrain. Limb bud labeling began at stage 20, whereas expression in the branchial arches appeared at stage 25. Chick DAN expression generally corresponded to that of mouse DAN expression as shown by comparative in situ hybridization. However, chick DAN was found in the otic epithelium and notochord, whereas mouse DAN was restricted to the overlying otic ectomesenchyme and was absent from the notochord. This observation suggests that DAN may play different roles in chick and mouse otic and notochord development.     

Views of problem drinking among Native American,Hispanic, and Anglo children

Native American, Hispanic, and Anglo sixth graders reacting to an example of teenage problem drinking expressed similar beliefs and attitudes in many respects. However, Native American children viewed the problem as less serious, subscribed more to a disease theory of alcoholism, attributed less causal responsibility to the individual, and adopted a less aggressive approach toward treatment than did Hispanic, and especially Anglo, children. Their less conventional value orientations accounted for all these differences except their stronger endorsement of a disease theory of problem drinking.     

Identification of a parathyroid hormone in the fish Fugu rubripes.

A PTH gene has been isolated from the fish Fugu rubripes. The encoded protein of 80 amino acid has the lowest homology with any of the PTH family members. Fugu PTH(1-34) had 5-fold lower potency than human PTH(1-34) in a mammalian cell system. INTRODUCTION: Parathyroid hormone (PTH) is the major hypercalcemic hormone in higher vertebrates. Fish lack parathyroid glands, but there have numerous attempts to identify and isolate PTH from fish. MATERIALS AND METHODS: Polymerase chain reaction (PCR) was performed with primers based on preliminary data from the Joint Genome Institute database. PCR amplification was performed on genomic DNA isolated from Fugu rubripes. PCR products were purified and DNA was sequenced. All sequence was confirmed from more than one independently amplified PCR product. Multiple sequence alignments were carried out, and the percentage of identities and similarities were calculated. An unrooted phylogenetic tree, using all the known PTH and PTH-related protein (PTHrP) amino acid sequences, was determined. Synthetic peptides were tested in a biological assay that measured cyclic adenosine 3',5'-monophosphate formation in UMR106.1 cells. Rabbit polyclonal antisera specific for N-terminal human PTHrP and one rabbit polyclonal antiserum specific for N terminus hPTH were used to test the cross-reactivity with fPTH(1-34) in immunoblots.     

Subcellular localization of the inositol 1,4,5-triphosphate receptor, P400, in the vestibular complex and dorsal cochlear nucleus of the rat

The subcellular localization of the inositol 1,4,5-trisphosphate receptor protein, P₄₀₀, was studied in the vestibular complex, an area to which Purkinje cells project, as well as in neurons of the dorsal cochlear nucleus and in ectopic Purkinje cells of adult rat brain. The receptor was demonstrated by electron microscopical immunocytochemistry using the avidin-biotin peroxidase complex procedure, with the monoclonal antibody 4C11 raised against mouse cerebellar inositol 1,4,5-trisphosphate receptor protein. Immunoreactivity was found in preterminal fibres and terminal boutons in the nuclei of the vestibular complex, generally associated with the subsurface systems and stacks or fragments of smooth endoplasmic reticulum. Ectopic Purkinje cells and cartwheel cells of the dorsal cochlear nucleus also displayed immunoreactivity, but this was much less intense in the latter. The results of the present study suggest that this receptor protein, involved in the release of Ca²⁺, is located in sites that enable it to influence the synthesis, transport and release of neurotransmitters.     

KCNJ2 mutations in arrhythmia patients referred for LQT testing: A mutation T305A with novel effect on rectification properties

BACKGROUND: Loss-of-function mutations in the KCNJ2 cause approximately 50% of Andersen-Tawil Syndrome (ATS) characterized by a classic triad of periodic paralysis, ventricular arrhythmia, and dysmorphic features. Do KCNJ2 mutations occur in patients lacking this triad and lacking a family history of ATS? OBJECTIVES: The purpose of this study was to identify and characterize mutations in the KCNJ2-encoded inward rectifier potassium channel Kir2.1 from patients referred for genetic arrhythmia testing. METHODS: Mutational analysis of KCNJ2 was performed for 541 unrelated patients. The mutations were made in wild type (WT) and expressed in COS-1 cells and voltage clamped for ion currents. RESULTS: Three novel missense mutations (R67Q, R85W, and T305A) and one known mutation (T75M) were identified in 4/249 (1.6%) patients genotype-negative for other known arrhythmia genes with overall incidence 4/541 (0.74%). They had prominent U-waves, marked ventricular ectopy, and polymorphic ventricular tachycardia but no facial/skeletal abnormalities. Periodic paralysis was present in only one case. Outward current was decreased to less than 5% of WT for all mutants expressed alone. Co-expression with WT (simulating heterozygosity) caused a marked dominant negative effect for T75M and R82W, no dominant negative effect for R67Q, and a novel selective enhancement of inward rectification for T305A. CONCLUSIONS: KCNJ2 loss of function mutations were found in approximately 1% of patients referred for genetic arrhythmia testing that lacked criteria for ATS. Characterization of three new mutations identified a novel dominant negative effect selectively reducing outward current for T305A. These results extend the range of clinical phenotype and molecular phenotype associated with KCNJ2 mutations.