Developmental changes in passive stiffness and myofilament Ca2+ sensitivity due to titin and troponin-I isoform switching are not critically triggered by birth

The giant protein titin, a major contributor to myocardial mechanics, is expressed in two main cardiac isoforms: stiff N2B (3.0 MDa) and more compliant N2BA (>3.2 MDa). Fetal hearts of mice, rats, and pigs express a unique N2BA isoform ( approximately 3.7 MDa) but no N2B. Around birth the fetal N2BA titin is replaced by smaller-size N2BA isoforms and N2B, which predominates in adult hearts, stiffening their sarcomeres. Here we show that perinatal titin-isoform switching and corresponding passive stiffness (STp) changes do not occur in the hearts of guinea pig and sheep. In these species the shift toward "adult" proportions of N2B isoform is almost completed by midgestation. The relative contributions of titin and collagen to STp were estimated in force measurements on skinned cardiac muscle strips by selective titin proteolysis, leaving the collagen matrix unaffected. Titin-based STp contributed between 42% and 58% to total STp in late-fetal and adult sheep/guinea pigs and adult rats. However, only approximately 20% of total STp was titin based in late-fetal rat. Titin-borne passive tension and the proportion of titin-based STp generally scaled with the N2B isoform percentage. The titin isoform transitions were correlated to a switch in troponin-I (TnI) isoform expression. In rats, fetal slow skeletal TnI (ssTnI) was replaced by adult carciac TnI (cTnI) shortly after birth, thereby reducing the Ca2+ sensitivity of force development. In contrast, guinea pig and sheep coexpressed ssTnI and cTnI in fetal hearts, and skinned fibers from guinea pig showed almost no perinatal shift in Ca2+ sensitivity. We conclude that TnI-isoform and titin-isoform switching and corresponding functional changes during heart development are not initiated by birth but are genetically programmed, species-specific regulated events.     

Simultaneous freeze tolerance and avoidance in individual fungus gnats, <Emphasis Type="Italic">Exechia nugatoria</Emphasis>

Freeze tolerance and freeze avoidance are typically described as mutually exclusive strategies for overwintering in animals. Here we show an insect species that combines both strategies. Individual fungus gnats, collected in Fairbanks, Alaska, display two freezing events when experimentally cooled and different rates of survival after each event (mean ± SEM: −31.5 ± 0.2°C, 70% survival and −50.7 ± 0.4°C, 0% survival). To determine which body compartments froze at each event, we dissected the abdomen from the head/thorax and cooled each part separately. There was a significant difference between temperature levels of abdominal freezing (−30.1 ± 1.1°C) and head/thorax freezing (−48.7 ± 1.3°C). We suggest that freezing is initially restricted to one body compartment by regional dehydration in the head/thorax that prevents inoculative freezing between the freeze-tolerant abdomen (71.0 ± 0.8% water) and the supercooled, freeze-sensitive head/thorax (46.6 ± 0.8% water).     

Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception

The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects’ genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen, e.g. for absinthin, the bitter principle of the beverage absinth. These findings illustrate the extent to which genetic influences on taste are complex, yet arise from both receptor activation patterns and linkage structure among receptor genes.     

Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton.

A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.     

Experience matters: prior exposure to plant toxins enhances diversity of gut microbes in herbivores

For decades, ecologists have hypothesised that exposure to plant secondary compounds (PSCs) modifies herbivore‐associated microbial community composition. This notion has not been critically evaluated in wild mammalian herbivores on evolutionary timescales. We investigated responses of the microbial communities of two woodrat species (Neotoma bryanti and N. lepida). For each species, we compared experienced populations that independently converged to feed on the same toxic plant (creosote bush, Larrea tridentata) to naïve populations with no exposure to creosote toxins. The addition of dietary PSCs significantly altered gut microbial community structure, and the response was dependent on previous experience. Microbial diversity and relative abundances of several dominant phyla increased in experienced woodrats in response to PSCs; however, opposite effects were observed in naïve woodrats. These differential responses were convergent in experienced populations of both species. We hypothesise that adaptation of the foregut microbiota to creosote PSCs in experienced woodrats drives this differential response.     

Isolation and characterization of three Mammalian orthoreoviruses from European bats

In recent years novel human respiratory disease agents have been described in South East Asia and Australia. The causative pathogens were classified as pteropine orthoreoviruses with strong phylogenetic relationship to orthoreoviruses of flying foxes inhabiting these regions. Subsequently, a zoonotic bat-to-human transmission has been assumed. We report the isolation of three novel mammalian orthoreoviruses (MRVs) from European bats, comprising bat-borne orthoreovirus outside of South East Asia and Australia and moreover detected in insectivorous bats (Microchiroptera). MRVs are well known to infect a broad range of mammals including man. Although they are associated with rather mild and clinically unapparent infections in their hosts, there is growing evidence of their ability to also induce more severe illness in dogs and man. In this study, eight out of 120 vespertilionid bats proved to be infected with one out of three novel MRV isolates, with a distinct organ tropism for the intestine. One isolate was analyzed by 454 genome sequencing. The obtained strain T3/Bat/Germany/342/08 had closest phylogenetic relationship to MRV strain T3D/04, isolated from a dog. These novel reoviruses provide a rare chance of gaining insight into possible transmission events and of tracing the evolution of bat viruses.     

Proline fed to intact soybean plants influences acetylene reducing activity and content and metabolism of proline in bacteroids

Supplying l-proline to the root system of intact soybean (Glycine max [L.] Merr.) plants stimulated acetylene reducing activity to the same extent as did supplying succinate. Feeding l-proline also caused an increase in bacteroid proline dehydrogenase activity that was highly correlated with the increase in acetylene-reducing activity. Twenty-four hours after irrigating with l-proline, endogenous proline content had increased in host cell cytoplasm and bacteroids, about three- and eightfold, respectively. In bacteroids, proline concentration was calculated to be at least 3.5 millimolar. In experiments in which [U-¹⁴C]l-proline was supplied to uprooted, intact plants incubated in aerated solution, ¹⁴C-labeled products of proline metabolism, as well as [¹⁴C]proline itself, accumulated in both host cells and bacteroids. When plants were incubated in aerated solutions containing [5-³H]l-proline, ³H-labeled proline was found in host cells and bacteroids. [³H] Pyrroline-5-carboxylate was found in bacteroids, but not host cells, after a 2-hour incubation in [5-³H]l-proline. When [U-¹⁴C]l-proline was supplied for 24 hours, a significant amount of [¹⁴C] pyrroline-5-carboxylate was found in the host cells, in contrast with the results from the shorter incubation in [5-³H]proline, although the amount in the host cells was only about half the quantity found in the bacteroids. Taken as a whole, these results indicate that proline crosses both plant and bacterial membranes under the in vivo experimental conditions utilized and are consistent with a significant role for proline as an energy source in support of bacteroid functioning. In spite of the increase in acetylene-reducing activity when proline was supplied to the root system of intact plants, proline application did not rescue stemgirdled plants from loss of acetylene-reducing activity, although succinate application did. This suggests a nonphloem route for succinate, but not proline, from roots to nodules.     

The role of an intracellular cysteine stretch in the sorting of the type II Na/phosphate cotransporter

The type II Na/phosphate cotransporters (NaPi-II) are critical for the control of plasma phosphate levels in vertebrates. NaPi-IIb mediates phosphate uptake from the small intestine followed by glomerular filtration and selective reabsorption from the renal proximal tubule by NaPi-IIa and NaPi-IIc. A C-terminal stretch of cysteine residues represents the hallmark of the NaPi-IIb isoforms. This motif is well conserved among NaPi-IIb type transporters but not found in other membrane proteins. To investigate the role of this motif we analyzed NaPi-II constructs in transiently and stably transfected MDCK cells. This cell line targets the NaPi-IIb isoforms from flounder and mouse to the apical membrane whereas the mouse IIa isoform shows no plasma membrane preference. Different parts of mouse NaPi-IIa and NaPi-IIb C-termini were fused to GFP-tagged flounder NaPi-II. The constructs showed strong staining of the plasma membrane with NaPi-IIb related constructs sorted predominantly apically, the IIa constructs localized apically and basolaterally with slight intracellular retention. When the cysteine stretch was inserted into the NaPi-IIa C-terminus, the construct was retained in a cytoplasmic compartment. 2-bromopalmitate, a specific palmitoylation inhibitor, released the transporter to apical and basolateral membranes. The drug also leads to a redistribution of the NaPi-IIb construct to both plasma membrane compartments. Immunoprecipitation of tagged NaPi-II constructs from [³H]-palmitate labeled MDCK cells indicated that the cysteine stretch is palmitoylated. Our results suggest that the modified cysteine motif prevents the constructs from basolateral sorting. Additional sorting determinants located downstream of the cysteine stretch may release the cargo to the apical compartment.