Pharmacological characterization of TMEM16A currents

Recent studies have shown that transmembrane protein 16 A (TMEM16A) is a subunit of calcium-activated chloride channels (CACCs). Pharmacological agents have been used to probe the functional role of CACCs, however their effect on TMEM16A currents has not been systematically investigated. In the present study, we characterized the voltage and concentration-dependent effects of 2 traditional CACC inhibitors (niflumic acid and anthracene-9-carboxcylic acid) and 2 novel CACC / TMEM16A inhibitors (CACC_inhA01 and T16A_inhA01) on TMEM16A currents. The whole cell patch clamp technique was used to record TMEM16A currents from HEK 293 cells that stably expressed human TMEM16A. Niflumic acid, A-9-C, CACC_inhA01 and T16A_inhA01 inhibited TMEM16A currents with IC₅₀ values of 12, 58, 1.7 and 1.5 µM, respectively, however, A-9-C and niflumic acid were less efficacious at negative membrane potentials. A-9-C and niflumic acid reduced the rate of TMEM16A tail current deactivation at negative membrane potentials and A-9-C (1 mM) enhanced peak TMEM16A tail current amplitude. In contrast, the inhibitory effects of CACC_inhA01 and T16A_inhA01 were independent of voltage and they did not prolong the rate of TMEM16A tail current deactivation. The effects of niflumic acid and A-9-C on TMEM16A currents were similar to previous observations on CACCs in vascular smooth muscle, strengthening the hypothesis that they are encoded by TMEM16A. However, CACC_inhA01 and T16A_inhA01 were more potent inhibitors of TMEM16A channels and their effects were not diminished at negative membrane potentials making them attractive candidates to interrogate the functional role of TMEM16A channels in future studies.     

Studies of feedback suppression of bile salt synthesis in the bile-fistula rat

We have previously reported that intravenous infusion of taurocholate at 10 mumol (100 g.hr) into bile-fistula rats suppressed bile salt synthesis by 85% (Pries et al. 1983. J. Lipid Res. 24: 141-146). Recently, however, infusion rates twice this high have been reported not to suppress synthesis (Davis et al. 1984. Falk Symposium 42. MTP Press Ltd., Boston. 37-45). Because the only major difference in design of these two studies was supplementation with sodium bicarbonate to replace biliary losses induced by bile salt choleresis, we have repeated our studies with and without bicarbonate supplementation. Without bicarbonate, as before, we found suppression of synthesis during infusion of taurocholate at 10 mumol/(100 g.hr). With bicarbonate, no suppression of synthesis occurred at these infusion rates. These data indicate that bicarbonate supplementation is essential when testing physiological effects of infused bile salt in the bile-fistula rat.     

MicroRNA-directed cleavage of Nicotiana sylvestris PHAVOLUTA mRNA regulates the vascular cambium and structure of apical meristems

Leaf initiation in the peripheral zone of the shoot apical meristem involves a transition to determinate cell fate, but indeterminacy is maintained in the vascular cambium, a tissue critical to the continuous growth of vascular tissue in leaves and stems. We show that the orientation of cambial growth is regulated by microRNA (miRNA)-directed cleavage of mRNA from the Nicotiana sylvestris ortholog of PHAVOLUTA (NsPHAV). Loss of miRNA regulation in semidominant phv1 mutants misdirects lateral growth of leaf midveins and stem vasculature away from the shoot, disrupting vascular connections in stem nodes. The phv1 mutation also expands the central zone in vegetative and inflorescence meristems, implicating miRNA and NsPHAV in regulation of meristem structure. In flowers, phv1 causes reiteration of carpel initiation, a phenocopy for loss of CARPEL FACTORY/DICER LIKE1, indicating that miRNA is critical to the termination of indeterminacy in floral meristems. Results point to a common role for miRNA in spatial and temporal restriction of HD-ZIPIII mediated indeterminacy in apical and vascular meristems.     

Lack of increased genetic damage in 1,3-butadiene-exposed Chinese workers studied in relation to EPHX1 and GST genotypes

1,3-Butadiene (BD) is an important industrial chemical and pollutant. Its ability to induce genetic damage and cause hematological malignancies in humans is controversial. We have examined chromosome damage by fluorescence in situ hybridization (FISH) and mutations in the HPRT gene in the blood of Chinese workers exposed to BD. Peripheral blood samples were collected and cultured from 39 workers exposed to BD (median level 2 ppm, 6 h time-weighted average) and 38 matched controls in Yanshan, China. No difference in the level of aneuploidy or structural changes in chromosomes 1, 7, 8, and 12 was detected in metaphase cells from exposed subjects in comparison with matched controls, nor was there an increase in the frequency of HPRT mutations in the BD-exposed workers. Because genetic polymorphisms in glutathione S-transferase (GST) enzymes and microsomal epoxide hydrolase (EPHX1) may affect the genotoxic effects of BD and its metabolites, we also related chromosome alterations and gene mutations to GSTT1, GSTM1 and EPHX1 genotypes. Overall, there was no effect of variants in these genotypes on numerical or structural changes in chromosomes 1, 7, 8 and 12 or on HPRT mutant frequency in relation to BD exposure, but the GST genotypes did influence background levels of both hyperdiploidy and HPRT mutant frequency. In conclusion, our data show no increase in chromosomal aberrations or HPRT mutations among workers exposed to BD, even in potentially susceptible genetic subgroups. The study is, however, quite small and the levels of BD exposure are not extremely high, but our findings in China do support those from a similar study conducted in the Czech Republic. Together, these studies suggest that low levels of occupational BD exposure do not pose a significant risk of genetic damage.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Effects of CO(2) and O(2) on Photosynthesis and Growth of Autotrophic Tobacco Callus

Gas exchange measurements were made on plants from two natural populations differing in salt tolerance of Andropogon glomeratus, a C₄ nonhalophyte, to examine the effect of salinity on components responsible for differences in photosynthetic capacity. Net CO₂ uptake and stomatal conductance decreased with increasing salinity in both populations, but to a greater extent in the inland (nontolerant) population. The intercellular CO₂ concentrations increased with increasing salinity in the inland population, but decreased in the marsh (tolerant) population. Water use efficiency decreased as salinity increased in the inland population, and remained unchanged in the marsh population. Carboxylation efficiency decreased and CO₂ compensation points increased with increasing salinity in both populations, but to a lesser extent in the marsh population. Carboxylation efficiencies were higher with 2% relative to 21% atmospheric O₂ in salt stressed plants, suggesting that a decrease in the carboxylation:oxygenation ratio of ribulose 1,5-bisphosphate carboxylase/oxygenase was partly responsible for the decrease in photosynthetic capacity. Populational differences in photosynthetic capacity were the result of greater salinity-induced changes in carboxylation efficiency in the inland population, and not due to differences in the stomatal limitation to CO₂ diffusion.     

Ant genomics sheds light on the molecular regulation of social organization

Ants are powerful model systems for the study of cooperation and sociality. In this review, we discuss how recent advances in ant genomics have contributed to our understanding of the evolution and organization of insect societies at the molecular level.     

Resynchronization of ovulation and timed insemination in lactating dairy cows III. Administration of GnRH 23 days post AI and ultrasonography for nonpregnancy diagnosis on day 30

The objective was to compare pregnancy rates to resynchronization and timed AI (TAI) protocols in lactating dairy cows that received GnRH at 23 d and were diagnosed not pregnant at 30 d after the pre-enrollment AI. Nonpregnant cows (624) at ultrasonography on day 30 (study day 0) were classified as diestrus (74.8%), metestrus (5.6%) and without a CL (19.5%). Cows in diestrus were assigned either to the GnRH group (PGF2alpha on day 0, GnRH on day 2 and TAI 16 h later, n = 238) or the estradiol cypionate (ECP) group (PGF2alpha on day 0, ECP on day 1, and TAI 36 h later, n = 229). Cows in metestrus were assigned to the Modified Heatsynch Group (GnRH on day 0, PGF(2alpha) on day 7, ECP on day 8 and TAI on day 9, n = 35). Cows without a CL (n = 122) were classified either as proestrus (10.6%), ovarian cysts (7.5%) or anestrus (1.4%), and assigned to factorial treatments (i.e., use of GnRH versus CIDR) to either the GnRH group (GnRH on day 0, PGF2alpha on day 7, GnRH on day 9 and TAI 16 h later, n = 28), the CIDR group (CIDR insert from days 0 to 7, PGF2alpha on day 7, GnRH on day 9 and TAI 16 h later, n = 34), the GnRH + CIDR group (GnRH on day 0, CIDR insert from days 0 to 7, PGF2alpha on day 7, GnRH on day 9 and TAI 16h later, n = 32), and the control group (PGF2alpha on day 7, GnRH on day 9 and TAI 16 h later, n = 28). For cows without a CL, plasma P4 concentrations were determined on days 0, 7, 10 and 17 and ovarian structures determined on days 0, 7 and 17. Pregnancy rates were evaluated at 30, 55 and 90 d after the resynchronized AI. For cows in diestrus, there were no differences in pregnancy rates on days 30, 55 and 90 for cows in the GnRH (27.5, 26.5 and 24.2%) or ECP (29.1, 25.5 and 24.1%) groups. In addition, there were no differences in pregnancy losses between days 30 and 55 and 55 and 90 between the GnRH (7.0 and 8.6%) and ECP (9.8 and 5.4%) groups. For cows without a CL, GnRH on day 0 increased the proportion of cows with a CL on days 7 and 17 and plasma P4 concentration on day 17 in cows with ovarian cysts but not for cows in proestrus. The CIDR insert increased pregnancy rate in cows with ovarian cysts but reduced pregnancy rate for cows in proestrus.     

Ethanol production at 45 °C by Kluyveromyces marxianus IMB3 during growth on molasses pre-treated with Amberlite® and non-living biomass

The use of high concentrations of molasses as a fermentation feed-stock for ethanol production is normally precluded by the presence of inhibitory compounds. Use of the thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 in fermentations containing high concentrations of molasses resulted in sub-optimal production of ethanol. The results suggested that this was caused by the presence of inhibitory materials rather than an intolerance to increased concentrations of ethanol. In the current study we describe the pretreatment of molasses preparations with either an Amberlite^® monobed mixed ion-exchange resin or non-living microbial biomass from a local distillery. In the study molasses samples diluted to yield a final sugar concentration of 160?g/l were used as the substrate. Control fermentations using the untreated molasses dilutions yielded a maximum ethanol concentration of 40?g/l, representing 49% of the maximum theoretical yield. Fermentations using molasses samples pre-treated with Amberlite^® or non-living biomass yielded maximum ethanol concentrations of 58 and 54?g/l, representing 71 and 66% of the maximum theoretical yield, respectively. The results suggest that pre-treatment brings about removal of toxic or inhibitory materials from the fermentation feed-stock and we believe that such pre-treatments, particularly using the less expensive non-living biomass preparations may find a role in processes concerned with the commercial production of ethanol from molasses using this microorganism.     

Vitamin K and oxidative phosphorylation

1. Oxidative phosphorylation was studied in a cell-free preparation of Mycobacterium phlei and in rat-liver mitochondria. Phosphorylation was destroyed in both systems by long-wave ultraviolet radiation and restored by the addition of small amounts of [2-Me-(14)C,(3)H]phylloquinone. When the radioactive quinones were recovered from the phosphorylating system and chromatographed with carrier phylloquinone and menaquinone-4 in adsorption and partition systems, only the phylloquinone band was labelled, and its isotopic ratio was identical with that of the original [2-Me-(14)C,(3)H]phylloquinone. This result does not support the contention that the role of vitamin K in oxidative phosphorylation involves a cyclic mechanism with intermediate formation of a quinone methide. 2. When the [2-Me-(14)C,(3)H]phylloquinone was given intravenously to rats and radioactive phylloquinone isolated from their liver mitochondria and microsomes 20hr. later, its isotopic ratio was unchanged. There was thus no evidence for quinone methide formation in vivo. No measurable conversion of phylloquinone into menaquinone-4 was observed. 3. When [(14)C]menadione was given intraperitoneally to rats whose alimentary tract had been treated with neomycin, conversion into menaquinone-4 was found in the liver mitochondria and microsomes, but there was also some indication that there had been synthesis of phylloquinone.