Isolation and chromosomal localization of the human glutathione peroxidase gene

We have isolated cDNA clones for the gene, termed GPX1, encoding the major human selenoprotein, glutathione peroxidase. Sequence analysis confirmed previous findings that the unusual amino acid seleno-cysteine is encoded by the opal terminator codon UGA. Southern blot analysis of human genomic DNA with the GPX1 cDNA showed that restriction endonucleases without sites in the probe sequence produced three hybridizing bands at standard stringency, diminishing to one strongly and one weakly hybridizing band at high stringency. In situ hybridization localized the human GPX1 gene to a single site on chromosome 3, at region 3q11-13.1. Thus, three genomic sites bear sequence homology to the GPX1 cDNA, and the one most homologous maps to 3q11-13.1.     

Conductive chloride flux across amphibian skin: inhibition by indacrinone and cobalt ion.

When amphibian skin was incubated under conditions in which transepithelial sodium transport was abolished, a conductive transepithelial Cl- flux arose when Cl- was removed from one of the compartments. This flux was matched by short-circuit current and it accounted entirely for transepithelial conductance. Cl- influx was larger than efflux; it was linearly related to the magnitude of transepithelial Cl- concentration difference. When applied to the epithelial surface of the tissue, divalent metal cations such as Co2+, and the ethacrynic acid derivative, indacrinone, reduced rapidly and reversibly both transepithelial Cl- (in)flux and short-circuit current. Frog skin proved to be more sensitive to these inhibitors than toad skin. Further characterization of transepithelial Cl- pathway(s) should benefit from the fact that Cl- across amphibian skin can easily be monitored by the short-circuit current method, and from the availability of agents which inhibit this passive flux rapidly and reversibly.     

Chemical behaviour of cytidine 5'-monophospho-N-acetyl-beta-D-neuraminic acid under neutral and alkaline conditions

The chemical behaviour of CMP-N-acetylneuraminic acid under neutral and different alkaline conditions has been investigated. The products formed were isolated by ion-exchange chromatography and gel filtration and analysed by colorimetric methods, thin-layer chromatography, combined gas-liquid chromatography/mass spectrometry and/or 360-MHz 1H-NMR spectroscopy. A maximum stability of CMP-N-acetylneuraminic acid was observed at pH8-11. In the tested pH range of 6-13, CMP and N-acetylneuraminic acid were formed in variable amounts as decomposition products. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid was produced at pH greater than 7; the amount of this substance increased with increasing pH. In anhydrous triethylamine its yield was 50%. A new neuraminic acid derivative, N-acetyl-beta-D-neuraminic acid 2-phosphate, could be isolated from the mixture of alkaline decomposition products of CMP-N-acetylneuraminic acid. The yield of this compound was maximum 22% in anhydrous triethylamine. Because 2-deoxy-2,3-dehydro-N-acetylneuraminic acid was formed under simulated physiological conditions, it is assumed that this compound, which occurs in tissues and fluids of man and animals, is derived from CMP-N-acetylneuraminic acid non-enzymically also under conditions in vivo.     

Purification and characterization of catalytic fragments of phosphorylase kinase gamma subunit missing a calmodulin-binding domain

A catalytic fragment preparation of rabbit muscle phosphorylase kinase produced by limited chymotryptic digestion was isolated and identified as the NH2-terminal region of the gamma subunit by Edman degradation. Mass spectral analysis, gas phase sequence analysis, and amino acid analysis of the active fragment carboxyl-terminal peptides revealed multiple COOH termini generated at residues Tyr290, Arg296, and Phe298 in the gamma subunit sequence. These active fragment species are about 24% smaller than the gamma subunit (Mr 44,673) and range in size from Mr 33,279 to Mr 34,275. The active fragment preparation exhibits a specific activity about 6-fold higher than that of the gamma subunit-calmodulin complex. Calmodulin confers calcium sensitivity to the gamma subunit but has no effect on the enzymatic properties of active fragment. Affinity measurements demonstrated a dissociation constant of 0.7 microM for active fragment binding to dansylcalmodulin, a value about 28-fold weaker than reported for the gamma subunit. These data support the presence of a calmodulin binding domain in the COOH-terminal region of the gamma subunit.     

CEP Biomarkers as Potential Tools for Monitoring Therapeutics

Background

Carboxyethylpyrrole (CEP) adducts are oxidative modifications derived from docosahexaenoate-containing lipids that are elevated in ocular tissues and plasma in age-related macular degeneration (AMD) and in rodents exposed to intense light. The goal of this study was to determine whether light-induced CEP adducts and autoantibodies are modulated by pretreatment with AL-8309A under conditions that prevent photo-oxidative damage of rat retina. AL-8309A is a serotonin 5-HT_1A receptor agonist.

Methods

Albino rats were dark adapted prior to blue light exposure. Control rats were maintained in normal cyclic light. Rats were injected subcutaneously 3x with 10 mg/kg AL-8309A (2 days, 1 day and 0 hours) before light exposure for 6 h (3.1 mW/cm², λ=450 nm). Animals were sacrificed immediately following light exposure and eyes, retinas and plasma were collected. CEP adducts and autoantibodies were quantified by Western analysis or ELISA.

Results

ANOVA supported significant differences in mean amounts of CEP adducts and autoantibodies among the light + vehicle, light + drug and dark control groups from both retina and plasma. Light-induced CEP adducts in retina were reduced ~20% following pretreatment with AL-8309A (n = 62 rats, p = 0.006) and retinal CEP immunoreactivity was less intense by immunohistochemistry. Plasma levels of light-induced CEP adducts were reduced at least 30% (n = 15 rats, p = 0.004) by drug pretreatment. Following drug treatment, average CEP autoantibody titer in light exposed rats (n = 22) was unchanged from dark control levels, and ~20% (p = 0.046) lower than in vehicle-treated rats.

Conclusions

Light-induced CEP adducts in rat retina and plasma were significantly decreased by pretreatment with AL-8309A. These results are consistent with and extend previous studies showing AL-8309A reduces light-induced retinal lesions in rats and support CEP biomarkers as possible tools for monitoring the efficacy of select therapeutics.     

The androgen-dependent rat prostate protein,probasin, is a heparin-binding protein that co-purifies with heparin-binding growth factor-1

Summary Rat prostate extracts contain an abundant 20–22 kilodalton heparin-binding protein with near identical chromatographic properties, but only 0.2–1% of the mitogenic activity, of bovine brain heparin-binding growth factor-1 (acidic fibroblast growth factor). Amino terminal amino acid sequence (met-met-thr-asp-lys-asn-leu-lys-lys-lys-ile-glu-gly-asn-trp-arg-thr-val-tyr-leu-ala-ala-ser-?-val-glu-lys-ile-asn-glu-gly-ser-pro) and immunochemical analysis revealed that the protein is identical to the androgen-dependent protein “probasin”. This work was supported in part by NCI grant CA37589 (W. L. M., J. W. C.) and the Medical Research Council of Canada (R. J. M.).     

A group of type I keratin genes on human chromosome 17: characterization and expression.

The human type I keratins K16 and K14 are coexpressed in a number of epithelial tissues, including esophagus, tongue, and hair follicles. We determined that two genes encoding K16 and three genes encoding K14 were clustered in two distinct segments of chromosome 17. The genes within each cluster were tightly linked, and large parts of the genome containing these genes have been recently duplicated. The sequences of the two K16 genes showed striking homology not only within the coding sequences, but also within the intron positions and sequences and extending at least 400 base pairs 5' upstream and 850 base pairs 3' downstream from these genes. Despite the strong homologies between these two genes, only one of the genes encoded a protein which assembled into keratin filaments when introduced into simple epithelial cells. While there were no obvious abnormalities in the sequence of the other gene, its promoter seemed to be significantly weaker, and even a hybrid gene with the other gene's promoter gave rise to a much reduced mRNA level after gene transfection. To demonstrate that the functional K16 gene that we identified was in fact responsible for the K16 expressed in human tissues, we made a polyclonal antiserum which recognized our functional K16 gene product in both denatured and filamentous form and which was specific for bona fide human K16.