The role of the N terminus and transmembrane domain of TRPM8 in channel localization and tetramerization

Transient receptor potential (TRP) channels are a family of cation channels involved in diverse cellular functions. They are composed of a transmembrane domain of six putative transmembrane segments flanked by large N- and C-terminal cytoplasmic domains. The melastatin subfamily (TRPM) channels have N-terminal domains of approximately 700 amino acids with four regions of shared homology and C-terminal domains containing the conserved TRP domain followed by a coiled-coil region. Here we investigated the effects of N- and C-terminal deletions on the cold and menthol receptor, TRPM8, expressed heterologously in Sf21 insect cells. Patch-clamp electrophysiology was used to study channel activity and revealed that only deletion of the first 39 amino acids was tolerated by the channel. Further N-terminal truncation or any C-terminal deletions prevented proper TRPM8 function. Confocal microscopy with immunofluorescence revealed that amino acids 40-86 are required for localization to the plasma membrane. Furthermore, analysis of deletion mutant oligomerization shows that the transmembrane domain is sufficient for TPRM8 assembly into tetramers. TRPM8 channels with C-terminal deletions tetramerize and localize properly but are inactive, indicating that although not essential for tetramerization and localization, the C terminus is critical for proper function of the channel sensor and/or gate.     

The plasma binding protein for vitamin D is a site of discrimination against vitamin D-2 compounds by the chick

The binding of 25-hydroxy-[26,27-3H]vitamin D-3 and 25-hydroxy-[26,27-3H]vitamin D-2 to the vitamin D binding protein in the plasma of both rats and chicks has been studied. In the case of rats, sucrose density gradient analysis, competitive displacement, and Scatchard analysis demonstrate that 25-hydroxyvitamin D-3 and 25-hydroxyvitamin D-2 are bound equally well to the vitamin D binding protein. In contrast, 25-hydroxyvitamin D-2 is poorly bound, while 25-hydroxyvitamin D-3 is tightly bound to the vitamin D binding protein in chick plasma. On the other hand, the chick intestinal receptor binds 1,25-dihydroxyvitamin D-2 and 1,25-dihydroxyvitamin D-3 equally well with a KD of 7.10(-11) M for both compounds. These results strongly suggest that the failure of the plasma transport protein in chicks to bind the vitamin D-2 compounds may be responsible for their relative ineffectiveness in these animals.     

Influence of follicular components on oocyte meiosis in vitro

Oocytes and follicular components obtained from ovaries recovered from mature Hereford cows at slaughter were used to determine follicular influence on oocyte maturation. Some oocytes were fixed immediately to determine the stage of maturation. The remaining oocytes were cultured for 32 to 34 hr in various environments to determine the influences of the granulosum and follicular fluids on meiotic changes. All noncultured oocytes had dictyate nuclei except one in premetaphase. Oocytes cultured in 50 or 100% follicular fluid or in contact with stratum granulosum cells showed some meiotic inhibition both before and after germinal vesicle breakdown (GVB). The least resumption of meiosis occurred in oocytes cultured in their intact follicles.     

Resonance Raman-spectroscopic studies of the hapten features involved in the binding of 2,4-dinitrophenyl haptens by the mouse myeloma proteins MOPC 315 and MOPC 460.

The binding of four dinitrophenyl haptens to the mouse myeloma proteins MOPC 315 IgA (immunoglobulin A) and MOPC 460IgA was studied by resonance Raman spectroscopy. Isotopic substitution with 15N and 2H was used to assign features in the resonance Raman spectra of the free haptens. Changes in each of these features on binding to the proteins could then be attributed to interactions of the proteins' binding sites with either the p-NO2 or the o-NO2/amine regions of the haptens. The interactions between a given hapten and MOPC 315 IgA are often quite distinct from those between the same hapten and MOPC 460 IgA. Moreover, for both antibodies the nature of the R side chain in a Dnp-NHR (Dnp, 2,4-dinitrophenyl) compound appears to modify the interactions between the Dnp chromophore and the protein. Thus, with the haptens studied, there is no unique set of contacts between the Dnp group and the binding site. The contacts expected between epsilon-2,4-dinitrophenyl-L-lysine and the site on MOPC 315 IgA, on the basis of a recent model for this site [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977) Nature (London) 266, 31--37] were not detected. However, the contacts between this hapten and the site on MOPC 460 IgA were closer to those predicted by the model for MOPC 315 IgA.     

Correlation of genomic and physiological traits of thermoanaerobacter species with biofuel yields

Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B₁₂ biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.     

Bovine aortic chondroitin sulphate- and dermatan sulphate-containing proteoglycans. Isolation, fractionation and chemical characterization.

1. Guanidinium chloride (4M) in the presence of proteinase inhibitors extracted 90% of bovine aorta galactosaminoglycans as proteoglycans that were subsequently purified by ion-exchange and gel chromatography. 2. Fractionation of the calcium salts of the purified proteoglycans with increasing concentration of ethanol yielded fractions PG-25 (28%), PG-35 (45%) and PG-50 (37%). 3. Fraction PG-50 contained proteochondroitin 6-sulphate, whereas fractions PG-25 and PG-35 were proteodermatan sulphates of greatly different carbohydrate composition; the molar proportions of L-iduronate-N-acetylgalactosamine 4-sulphate, D-glucuronate-N-acetyl-galactosamine 4-sulphate and D-glucuronate-N-acetylgalactosamine 6-sulphate were 75: 18 :7 in fraction PG-25 and 14 :46 :40 in fraction PG-35. 4. The presence of alternating or mixed sequences with L-iduronate- and D-glucuronate-containing repeating disaccharides was indicated by the formation of tetrasaccharides after chondroitinase AC digestion (single L-iduronate residues) and by the release of fragments containing four or five consecutive D-glucuronate-N-acetylgalactosamine repeats after periodate oxidation and alkaline elimination. 5. The amino acid compositions of fractions PG-25 and PG-35 were similar and markedly different from that of fraction PG-50, which also contained more side chains.     

Preliminary Assessment of Gastrointestinal Parasites in Two Wild Groups of Endangered Moor Macaques (Macaca maura) from Sulawesi

International Journal of Primatology - Information on parasite biodiversity and abundance can improve our understanding of parasitic infections on endangered wildlife, as parasites can affect host...     

Promotion as Prevention: Positive Youth Development as Protective against Tobacco,Alcohol, Illicit Drug,and Sex Initiation

The present study was designed to examine the association of positive youth development with the likelihood of tobacco, alcohol, marijuana, hard drug, and sex initiation between 5th and 10th grades. A national, largely middle-class sample of 5,305 adolescents, participating in a longitudinal study funded by the National 4-H Council (although not all participants were enrolled in 4-H or other after-school programs), completed measures of positive youth development (PYD) constructs and of tobacco, alcohol, marijuana, and hard drug use once per year between 5th and 10th grades. At the 9th and 10th grade assessments, adolescents were asked whether they had initiated sexual intercourse and, if so, at what age they had first engaged in intercourse. Although the present sample was somewhat lower risk compared to national averages, survival analysis models indicated that PYD was significantly and negatively associated with the initiation hazards for tobacco use, marijuana use, and sex initiation for girls only, and with hard drug use for both genders. PYD was also positively associated with the odds of condom use across genders. Results are discussed with regard to PYD as a preventive process.