Streptozotocin Stimulates the Ion Channel TRPA1 Directly: INVOLVEMENT OF PEROXYNITRITE*

Streptozotocin (STZ)-induced diabetes is the most commonly used animal model of diabetes. Here, we have demonstrated that intraplantar injections of low dose STZ evoked acute polymodal hypersensitivities in mice. These hypersensitivities were inhibited by a TRPA1 antagonist and were absent in TRPA1-null mice. In wild type mice, systemic STZ treatment (180 mg/kg) evoked a loss of cold and mechanical sensitivity within an hour of injection, which lasted for at least 10 days. In contrast, Trpa1^−/− mice developed mechanical, cold, and heat hypersensitivity 24 h after STZ. The TRPA1-dependent sensory loss produced by STZ occurs before the onset of diabetes and may thus not be readily distinguished from the similar sensory abnormalities produced by the ensuing diabetic neuropathy. In vitro, STZ activated TRPA1 in isolated sensory neurons, TRPA1 cell lines, and membrane patches. Mass spectrometry studies revealed that STZ oxidizes TRPA1 cysteines to disulfides and sulfenic acids. Furthermore, incubation of tyrosine with STZ resulted in formation of dityrosine, suggesting formation of peroxynitrite. Functional analysis of TRPA1 mutants showed that cysteine residues that were oxidized by STZ were important for TRPA1 responsiveness to STZ. Our results have identified oxidation of TRPA1 cysteine residues, most likely by peroxynitrite, as a novel mechanism of action of STZ. Direct stimulation of TRPA1 complicates the interpretation of results from STZ models of diabetic sensory neuropathy and strongly argues that more refined models of diabetic neuropathy should replace the use of STZ.     

The crystal structure of <Emphasis Type="Italic">Escherichia coli</Emphasis> TdcF,a member of the highly conserved YjgF/YER057c/UK114 family

Background  

The YjgF/YER057c/UK114 family of proteins is widespread in nature, but has as yet no clearly defined biological role. Members of the family exist as homotrimers and are characterised by intersubunit clefts that are delineated by well-conserved residues; these sites are likely to be of functional significance, yet catalytic activity has never been detected for any member of this family. The gene encoding the TdcF protein of E. coli, a YjgF/YER057c/UK114 family member, resides in an operon that strongly suggests a role in the metabolism of 2-ketobutyrate for this protein.     

A novel sulfoglycosphingolipid of mouse small intestine, IV3-sulfogangliotetraosylceramide, demonstrated by negative ion fast atom bombardment mass spectrometry

In a comparative study of acidic glycosphingolipids in the small intestine of several species it was found that the mouse contained a complex sulfoglycolipid as a major component (Breimer, M.E., Hansson, G.C., Karlsson, K.-A., and Leffler, H. (1983) J. Biochem. (Tokyo) 93, 1473-1485). Fast atom bombardment negative ion mass spectrometry proved the presence and location of the sulfate group and also showed the saccharide sequence and ceramide composition. Combined with NMR spectroscopy of the intact structure and degradative studies the structure was shown to be -O3SO----3Galp beta 1----3GalNAcp beta 1----4Galp beta 1----4Glcp beta 1----1Cer. The sulfoglycolipid was enriched in epithelial cells of mouse small intestine where it constituted at least 90% of the acidic glycolipids and 4-8 mol% of the total glycosphingolipids.     

Leukotrienes increase levels of prostanoids in cerebrospinal fluid in piglets

We investigated effects of exogenous leukotrienes (C4, D4, or E4) on levels of prostanoids in cerebrospinal fluid in newborn pigs (1-5 days). A "closed" cranial window was placed over the parietal cortex. Pial arterial diameter was measured with a microscope and electronic micrometer system. Levels in cerebrospinal fluid (CSF) of 6-keto-Prostaglandin F1 alpha (6-keto-PGF1 alpha), Thromboxane B2 (TXB2), and Prostaglandin E2 (PGE2) were measured by radioimmunoassay. Topical application of leukotrienes C4, D4, or E4 (5,000 ng/ml) similarly constricted pial arteries by 15 +/- 2% (n = 14) (mean +/- SEM). In addition, leukotrienes increased levels of 6-keto-PGF1 alpha from 806 +/- 136 to 1,612 +/- 304 pg/ml (n = 13), TXB2 from 161 +/- 31 to 392 +/- 81 pg/ml (n = 10), and PGE2 from 2,271 +/- 342 to 4,636 +/- 740 pg/ml (n = 13). Each type of leukotriene had similar effects on prostanoid synthesis. In other experiments (n = 5), we found that 2.0 ng/ml PGE2 in CSF dilated pial arteries by 24 +/- 8% and that 1.0 ng/ml PGI2 dilated pial arteries by 15 +/- 6%. These results indicate that leukotrienes are able to increase levels of prostanoids in cerebral cortex.     

Molecular phylogenetic evidence that the phylum Haplosporidia has an alveolate ancestry

The phylogenetic position of the phylum Haplosporidia among other protists was investigated with the complete 16S-like rRNA gene sequences from two species in the phylum: Haplosporidium nelsoni, a parasite of oysters, and Minchinia teredinis, a parasite of shipworms. Because the lack of obvious morphological homologies with other protists hampered decisions regarding taxonomic composition for sequence alignment and phylogenetic analysis, the complete sequences for these two haplosporidians were directed as search queries to the blast/ncbi.nlm.nih.gov electronic mail server. The results of this heuristic similarity search provided a basis for constructing a preliminary higher-taxonomic-level analysis comparing the haplosporidians with species from the slime molds, fungi, algae, amoebae, ciliates, dinoflagellates, and apicomplexans. Maximum parsimony yielded equivocal results, whereas transversionally weighted parsimony suggested an affinity with the alveolates (i.e., the ciliates, dinoflagellates, and apicomplexans). Multiple alignment of the two haplosporidian sequences against 17 taxa in a secondary analysis focusing on the alveolates and subsequent parsimony analysis placed the phylum Haplosporidia as a monophyletic group within the Alveolata and as a taxon of equal rank with the other three alveolate phyla. The precise placement within the Alveolata was sensitive to weighting.      

Preparation of biological fluids for simultaneous analysis of prostaglandin cyclo-oxygenase synthesized compounds by gas chromatography with electron capture detection

A method for isolating climax products of the arachidonic acid cascade from biological fluids is described which allows simultaneous measurement of PGs (PGE2, PGD2, 6-keto-PGF1 alpha, TXB2, 6-keto-PGE1, 6, 15-diketo-13,14-dihydro-PGF1 alpha) by electron capture detection of pentafluorobenzyloxime methyl ester trimethylsilyl (TMS) ether derivatives. PGs are adsorbed onto Amberlite XAD-2 from acidified solution and nonadhering substances removed by sequential administration of water, then petroleum ether. PGs are extracted into methanol. Following evaporation and reconstitution in water, the PGs are extracted into ethyl acetate at pH 3 and the ethyl acetate extracts are purified by lipidex column chromatography. Derivatization to pentafluorobenzyloxime methyl ester TMS ethers of PGs in the sample is followed by separation on either glass packed-columns or SCOT capillary columns, and detection by an electron capture detector. PGA2, added to the unpurified sample, is used as an internal standard for quantification. The methods have performed well on all biological fluids thus far examined. Examples of chromatographs from urine, Krebs-perfused lung effluents, and blood are shown.     

The effects of indomethacin on the pulmonary vascular response to hypoxia in the premature and mature newborn goat.

The effects of indomethacin on the pulmonary circulation and the response of the circulation to hypoxia were investigated in premature and mature newborns using an isolated perfusion technique on otherwise intact left lungs in situ. There was an increase in pulmonary vascular resistance and augmentation of the increase in pulmonary vascular resistance during hypoxia following indomethacin. These effects were greater in the premature than in the mature newborn. Indomethacin effectively removes a dilator influence on the pulmonary circulation. The results are consistent with the concept that prostaglandins are important in regulating pulmonary vascular resistance.     

Actin filaments elongate from their membrane-associated ends

In limulus sperm an actin filament bundle 55 mum in length extends from the acrosomal vacuole membrane through a canal in the nucleus and then coils in a regular fashion around the base of the nucleus. The bundle expands systematically from 15 filaments near the acrosomal vacuole to 85 filaments at the basal end. Thin sections of sperm fixed during stages in spermatid maturation reveal that the filament bundle begins to assemble on dense material attached to the acrosomal vacuole membrane. In micrographs fo these early stages in maturation, short bundles are seen extending posteriorly from the dense material. The significance is that these short, developing bundles have about 85 filaments, suggesting that the 85-filament end of the bundle is assembled first. By using filament bundles isolated and incubated in vitro with G actin from muscle, we can determine the end “preferred” for addition of actin monomers during polymerization. The end that would be associated with the acrosomal vacuole membrane, a membrane destined to be continuous with the plasma membrane, is preferred about 10 times over the other, thicker end. Decoration of the newly polymerized portions of the filament bundle with subfragment 1 of myosin reveals that the arrowheads point away from the acrosomal vacuole membrane, as is true of other actin filament bundles attached to membranes. From these observations we conclude that the bundle is nucleated from the dense material associated with the acrosomal vacuole and that monomers are added to the membrane-associated end. As monomers are added at the dense material, the thick first-made end of the filament bundle is pushed down through the nucleus where, upon reaching the base of the nucleus, it coils up. Tapering is brought about by the capping of the peripheral filaments in the bundle.     

Modification of deoxyribonucleic acid by a diol epoxide of benzo[a]pyrene. Relation to deoxyribonucleic acid structure and conformation and effects on transfectional activity.

The effects of secondary structure on DNA modification by (+/-)-7 beta, 9 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene [(+/-)BPDE I] were investigated. No differences in the total extent of (+/-) BPDE I binding to double- and single-stranded calf thymus DNA were found. High-performance liquid chromatography (LC) of the nucleoside adducts obtained from hydrolysates of native and denatured calf thymus, as well as from superhelical and linear plasmid DNA, indicated that in all cases the major adduct (60--80% of total adducts) was formed by reaction of the (+) enantiomer of BPDE I with the N-2 position of dG residues in the DNA. A minor adduct formed from the reaction of the (-) enantiomer with dG residues was also detected and was present in greater amounts in denautred DNA than in native DNA. Small amounts of BPDE I--dA and BPDE I--dC adducts were also detected in both the single- and double-stranded DNAs. Restriction enzyme analysis of BPDE I modified SV40 and phage lambda DNA provided evidence that the modification of DNA by this carcinogen is fairly random with respect to nucleotide sequence. Partial hydrolysis of modified plasmid DNA by the single-strand-specific S1 nuclease and LC analysis of the nucleoside adducts in the digested and undigested fractions of the DNA revealed no preferential excision by the S1 nuclease of the different BPDE I--deoxynucleoside adducts. Functional changes in BPDE I modified DNA were demonstrated. With increasing extents of modification, there was a decrease in the ability of plasmid DNA to transfect a receptive Escherichia coli strain to antibiotic resistance.