Single point mutations of aromatic residues in transmembrane helices 5 and -6 differentially affect TRPV4 activation by 4α-PDD and hypotonicity: Implications for the role of the pore region in regulating TRPV4 activity

The importance of the TRPV4 channel for human physiology has been highlighted in recent years with the identification of an increasing number of hereditary diseases associated with mutations of this channel. However, the functional understanding of TRPV4 associated pathologies remains a puzzle due to incomplete understanding of the polymodal regulation of TRPV4 channels and lack of insight into the structure–function relationship of the channel. In this work, we identified a series of highly conserved aromatic residues in transmembrane (TM) helices 5–6 with profound importance for TRPV4 activity. Substituting F617, Y621 or F624 in TM5 with leucine reduced channel sensitivity to the agonist 4α-PDD and heat, yet two of these mutants – F617L and Y621L – showed increased activation in response to cell swelling. In TM6, a Y702L mutation significantly reduced sensitivity to all of the above stimuli. In conclusion, we have identified residues in TM5-6 which differentially affect heat and agonist activation, and we have demonstrated distinct activation pathways for 4α-PDD and osmolarity.     

Considerations on the performance of immunocastrated male pigs

With the ongoing social pressure on surgical castration of pigs, an increase in the population of pigs that are either not castrated or immunocastrated (IC) can be expected. In both cases, their nutrient requirements and performance will differ from surgically castrated pigs and will require changes in their management. Immunocastration is performed by giving two injections of a modified gonadotrophin-releasing hormone component along with an adjuvant, at least 4 weeks apart. This paper describes the reported differences in growth performance and carcass quality of IC male pigs in comparison with boars (BO) and barrows (BA). Theoretically, IC pigs remain physiologically boar until the second vaccination and therefore, growth may be comparable with BO until this second vaccination. From then on, IC male pigs consume more feed than BO and grow faster when fed ad libitum. IC showed a faster growth and better feed conversion ratio than BA. When fed restrictedly, BO grow faster and more efficiently than BA and IC. IC have a lower carcass yield than BA and BO, whereas meat percentage is intermediate.     

Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences

Background

All viruses in the family Bunyaviridae possess a tripartite genome, consisting of a small, a medium, and a large RNA segment. Bunyaviruses therefore possess considerable evolutionary potential, attributable to both intramolecular changes and to genome segment reassortment. Hantaviruses (family Bunyaviridae, genus Hantavirus) are known to cause human hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. The primary reservoir host of Sin Nombre virus is the deer mouse (Peromyscus maniculatus), which is widely distributed in North America. We investigated the prevalence of intramolecular changes and of genomic reassortment among Sin Nombre viruses detected in deer mice in three western states.

Methods

Portions of the Sin Nombre virus small (S) and medium (M) RNA segments were amplified by RT-PCR from kidney, lung, liver and spleen of seropositive peromyscine rodents, principally deer mice, collected in Colorado, New Mexico and Montana from 1995 to 2007. Both a 142 nucleotide (nt) amplicon of the M segment, encoding a portion of the G2 transmembrane glycoprotein, and a 751 nt amplicon of the S segment, encoding part of the nucleocapsid protein, were cloned and sequenced from 19 deer mice and from one brush mouse (P. boylii), S RNA but not M RNA from one deer mouse, and M RNA but not S RNA from another deer mouse.

Results

Two of 20 viruses were found to be reassortants. Within virus sequences from different rodents, the average rate of synonymous substitutions among all pair-wise comparisons (π_s) was 0.378 in the M segment and 0.312 in the S segment sequences. The replacement substitution rate (π_a) was 7.0 × 10^-4 in the M segment and 17.3 × 10^-4 in the S segment sequences. The low π_a relative to π_s suggests strong purifying selection and this was confirmed by a Fu and Li analysis. The absolute rate of molecular evolution of the M segment was 6.76 × 10^-3 substitutions/site/year. The absolute age of the M segment tree was estimated to be 37 years. In the S segment the rate of molecular evolution was 1.93 × 10^-3 substitutions/site/year and the absolute age of the tree was 106 years. Assuming that mice were infected with a single Sin Nombre virus genotype, phylogenetic analyses revealed that 10% (2/20) of viruses were reassortants, similar to the 14% (6/43) found in a previous report.

Conclusion

Age estimates from both segments suggest that Sin Nombre virus has evolved within the past 37–106 years. The rates of evolutionary changes reported here suggest that Sin Nombre virus M and S segment reassortment occurs frequently in nature.     

Rapid leaf development drives the seasonal pattern of volatile organic compound (VOC) fluxes in a ‘coppiced’ bioenergy poplar plantation

Leaves of fast‐growing, woody bioenergy crops often emit volatile organic compounds (VOC). Some reactive VOC (especially isoprene) play a key role in climate forcing and may negatively affect local air quality. We monitored the seasonal exchange of VOC using the eddy covariance technique in a ‘coppiced’ poplar plantation. The complex interactions of VOC fluxes with climatic and physiological variables were also explored by using an artificial neural network (Self Organizing Map). Isoprene and methanol were the most abundant VOC emitted by the plantation. Rapid development of the canopy (and thus of the leaf area index, LAI) was associated with high methanol emissions and high rates of gross primary production (GPP) since the beginning of the growing season, while the onset of isoprene emission was delayed. The highest emissions of isoprene, and of isoprene photo‐oxidation products (Methyl Vinyl Ketone and Methacrolein, i_ox), occurred on the hottest and sunniest days, when GPP and evapotranspiration were highest, and formaldehyde was significantly deposited. Canopy senescence enhanced the exchange of oxygenated VOC. The accuracy of methanol and isoprene emission simulations with the Model of Emissions of Gases and Aerosols from Nature increased by applying a function to modify their basal emission factors, accounting for seasonality of GPP or LAI.     

The gene for a novel member of the whey acidic protein family encodes three four-disulfide core domains and is asynchronously expressed during lactation

Secretion of whey acidic protein (WAP) in milk throughout lactation has previously been reported for a limited number of species, including the mouse, rat, rabbit, camel, and pig. We report here the isolation of WAP from the milk of a marsupial, the tammar wallaby (Macropus eugenii). Tammar WAP (tWAP) was isolated by reverse-phase HPLC and migrates in SDS-polyacrylamide gel electrophoresis at 29.9 kDa. tWAP is the major whey protein, but in contrast to eutherians, secretion is asynchronous and occurs only from approximately days 130 through 240 of lactation. The full-length cDNA codes for a mature protein of 191 amino acids, which is comprised of three four-disulfide core domains, contrasting with the two four-disulfide core domain arrangement in all other known WAPs. A three-dimensional model for tWAP has been constructed and suggests that the three domains have little interaction and could function independently. Analysis of the amino acid sequence suggests the protein belongs to a family of protease inhibitors; however, the predicted active site of these domains is dissimilar to the confirmed active site for known protease inhibitors. This suggests that any putative protease ligand may be unique to either the mammary gland, milk, or gut of the pouch young. Examination of the endocrine regulation of the tWAP gene showed consistently that the gene is prolactin-responsive but that the endocrine requirements for induction and maintenance of tWAP gene expression are different during lactation.     

Synthesis and biodistribution of [(11)C]R116301, a promising PET ligand for central NK(1) receptors

N1-(2,6-Dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-di(trifluoromethyl)[carbonyl-(11)C]benzoyl]hexahydro-4-pyridinyl}piperazino)acetamide ([(11)C]R116301) was prepared and evaluated as a potential positron emission tomography (PET) ligand for investigation of central neurokinin(1) (NK(1)) receptors. 1-Bromo-3,5-di(trifluoromethyl)benzene was converted in three steps into 3,5-di(trifluoromethyl)[carbonyl-(11)C]benzoyl chloride, which was reacted with N1-(2,6-dimethylphenyl)-2-{4-[(2R,4S)-2-benzylhexahydro-4-pyridinyl]piperazino}acetamide providing [(11)C]R116301 in 45-57% decay-corrected radiochemical yield. The total synthesis time, from end of bombardment (EOB) to the formulated product, was 35 min. Specific activity (SA) was 82-172 GBq/micromol (n=10) at the end of synthesis. N1-([4-(3)H]-2,6-Dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-di(trifluoromethyl)benzoyl]hexahydro-4-pyridinyl}piperazino)acetamide ([(3)H]R116301) was also synthesized (SA: 467 GBq/mmol). The B(max) for [(3)H]R116301 measured in vitro on Chinese hamster ovary cell membranes stably transfected with the human NK(1) receptor was 19.10+/-1.02 pmol/mg protein with an apparent dissociation constant of 0.08+/-0.01 nM. Ex vivo, in vivo and in vitro autoradiography studies with [(3)H]R116301 in gerbils demonstrated a preferential accumulation of the radioactivity in the striatum, olfactory tubercule, olfactory bulb and locus coeruleus. In vivo, the biodistribution of [(11)C]R116301 in gerbils revealed that the highest initial uptake is in the lung, followed by the liver and kidney. In the brain, maximum accumulation was found in the olfactory tubercules (1.10+/-0.08 injected dose (ID)/g 20 min post injection (p.i.)) and the nucleus accumbens (1.00+/-0.12ID/g 10 min p.i.). Tissue/cerebellum concentration ratios for striatum and nucleus accumbens increased with time due to rapid uptake followed by a slow wash out (1.29 and 1.64, respectively, 30 min p.i.). A tissue to cerebellum ratio of 1.33 and 1.62 was also observed for olfactory bulb and olfactory tubercules, respectively (20 min p.i.). In summary, [(11)C]R116301 appears to be a promising radioligand suitable for the visualization of NK(1) receptors in vivo using PET.     

Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase.

Nitric oxide, which accounts for the biological activity of endothelium-derived relaxing factor (EDRF), is synthesized in endothelial cells from L-arginine by nitric oxide synthase (NOS). We report here the cloning and functional expression of a cDNA encoding human endothelial NOS. Oligonucleotides corresponding to amino acid sequences shared by cytochrome P450 reductase and the recently identified brain NOS were synthesized to amplify a partial cDNA encoding a bovine endothelial cell NOS-related protein. This partial cDNA was used to isolate a cDNA encoding a human vascular endothelial NOS. The translated human protein is 1294 amino acids long and shares 52% of its amino acid sequence with brain NOS. Using RNA blot hybridization, abundant endothelial NOS mRNA was detected in unstimulated human umbilical vein endothelial cells. To determine the functional activity of the endothelial protein, we ligated the cDNA into an expression vector and transfected it into NIH3T3 cells. Cells expressing this cDNA contained abundant NADPH diaphorase activity, a histochemical marker for NOS. In co-culture assays, nitric oxide production by transfected cells increased guanylate cyclase activity in reporter rat fetal lung fibroblasts. In addition, NOS-catalyzed conversion of arginine to citrulline in transfected cells was significantly increased by A23187, a calcium ionophore. Isolation of a cDNA encoding a calcium-regulated, constitutively expressed human endothelial NOS, capable of producing EDRF in blood vessels, will accelerate the characterization of the role of this enzyme in normal and abnormal endothelial regulation of vascular tone.     

Apparent cooperativity of cation binding to protonated transfer RNA measured by quantitation of released protons

Magnesium, spermidine and spermine are bound in a cooperative way to protonated tRNA. Experimental evidence suggests that tRNA may be considered as an allosteric molecule.