Function,diversity, and evolution of signal transduction in prokaryotes

Major areas covered at the Bacterial Locomotion and Signal Transduction (BLAST) meeting included the clustering of chemoreceptors and its significance to signal amplification, organelle biogenesis, motility, developmental responses mediated by "chemotaxis" operons, and advances in two-component signaling mechanisms.     

Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid

Arbuscular mycorrhizal (AM) fungi take up photosynthetically fixed carbon from plant roots and translocate it to their external mycelium. Previous experiments have shown that fungal lipid synthesized from carbohydrate in the root is one form of exported carbon. In this study, an analysis of the labeling in storage and structural carbohydrates after (13)C(1) glucose was provided to AM roots shows that this is not the only pathway for the flow of carbon from the intraradical to the extraradical mycelium (ERM). Labeling patterns in glycogen, chitin, and trehalose during the development of the symbiosis are consistent with a significant flux of exported glycogen. The identification, among expressed genes, of putative sequences for glycogen synthase, glycogen branching enzyme, chitin synthase, and for the first enzyme in chitin synthesis (glutamine fructose-6-phosphate aminotransferase) is reported. The results of quantifying glycogen synthase gene expression within mycorrhizal roots, germinating spores, and ERM are consistent with labeling observations using (13)C-labeled acetate and glycerol, both of which indicate that glycogen is synthesized by the fungus in germinating spores and during symbiosis. Implications of the labeling analyses and gene sequences for the regulation of carbohydrate metabolism are discussed, and a 4-fold role for glycogen in the AM symbiosis is proposed: sequestration of hexose taken from the host, long-term storage in spores, translocation from intraradical mycelium to ERM, and buffering of intracellular hexose levels throughout the life cycle.     

Characterization of sterol uptake in leaf tissues of sugar beet

The uptake of cholesterol has been characterized in leaf discs from mature leaves of sugar beet (Beta vulgaris L.). This transport system exhibited a simple saturable phase with an apparent Michaelis constant ranging from 30 to 190 M depending on the sample. When present at 10 M excess, other sterols were able to inhibit cholesterol uptake. Moreover, binding assays demonstrated the presence of high-affinity binding sites for cholesterol in purified plasma membrane vesicles. In the range 1–60 M, cholesterol uptake showed an active component evidenced by action of the protonophore carbonyl cyanide m-chlorophenylhydrazone. Energy was required as shown by the inhibition of uptake induced by respiration inhibitors (NaN₃), darkness and photosynthesis inhibitors [3-(3,4-dichlorophenyl)-1,1-dimethylurea, methyl viologen]. Moreover, the process was strongly dependent on the experimental temperature. Uptake was optimal at acidic pH (4.0), sensitive to ATPase modulators, inhibited by thiol reagents (N-ethylmaleimide, p-chloromercuribenzenesulfonic acid, Mersalyl) and by the histidyl-group reagent diethyl pyrocarbonate. The addition of cholesterol did not modify H⁺ flux from tissues, indicating that H⁺-co-transport was unlikely to be involved. MgATP did not increase the uptake, arguing against involvement of an ABC cassette-type transporter. By contrast, cryptogein, a sterol carrier protein from the Oomycete Phytophtora cryptogea, greatly increased absorption. Taken together, the results reported in this work suggest that plant cells contain a specific plasma membrane transport system for sterols.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - PCMBS p-chloromercuribenzenesulfonic acid - PMV plasma membrane vesicle - TLC thin-layer chromatography     

Characterization of two phosphate transporters from barley; evidence for diverse function and kinetic properties among members of the Pht1 family

Putative phosphate transporters have been identified in a barley (Hordeum vulgare L.) genomic library by their homology to known phosphate transporters from dicot species. The genes designated HORvu;Pht1;1 and HORvu;Pht1;6 encode proteins of 521 and 535 amino acids respectively with 12 predicted membrane-spanning domains and other motifs common to the Phtl family of phosphate transporters. HORvu;Pht1;1 is expressed exclusively in roots and is strongly induced by phosphate deprivation. HORvu;Pht1;6 is expressed in the aerial parts of the plant with strongest expression in old leaves and flag leaves. In situ hybridization showed that HORvu;Pht1;6 is expressed in the phloem of vascular bundles in leaves and ears. In order to study the biochemical properties of HORvu;Pht1;1 and HORvu;Pht1;6, the genes were expressed in transgenic rice (Oryza sativa L.) plants under the control of the rice actin promoter and suspension cell cultures were generated. Cells derived from transgenic plants were able to take up phosphate at a much higher rate than control cells, demonstrating that both genes encode functional phosphate transporters. The estimated Km for phosphate for cells expressing HORvu;Pht1;1 was 9.06 +/- 0.82 microM, which is characteristic of a high-affinity transporter. The rate of phosphate uptake decreased with increasing pH, suggesting that HORvu;Pht1;1 operates as a H+/H2PO4(-) symporter. In contrast, the estimated Km for phosphate for cells expressing HORvu;Pht1;6 was 385 +/- 61 microM, which is characteristic of a low-affinity transporter. Taken together, the results suggest that HORvu;Pht1;1 functions in uptake of phosphate at the root surface, while HORvu;Pht1;6 probably functions in remobilization of stored phosphate from leaves.     

The monotreme genome: a patchwork of reptile, mammal and unique features?

The first specimen of platypus (Ornithorhynchus anatinus) that reached Britain in the late 18th century was regarded a scientific hoax. Over decades the anatomical characteristics of these unique mammals, such as egg laying and the existence of mammary glands, were hotly debated before they were accepted. Within the last 40 years, more and more details of monotreme physiology, histology, reproduction and genetics have been revealed. Some show similarities with birds or reptiles, some with therian mammals, but many are very specific to monotremes. The genome is no exception to monotreme uniqueness. An early opinion was that the karyotype, composed of a few large chromosomes and many small ones, resembled bird and reptile macro- and micro-chromosomes. However, the platypus genome also features characteristics that are not present in other mammals, such as a complex translocation system. The sex chromosome system is still not resolved. Nothing is known about dosage compensation and, unlike in therian mammals, there seems to be no genomic imprinting. In this article we will recount the mysteries of the monotreme genome and describe how we are using recently developed technology to identify chromosomes in mitosis, meiosis and sperm, to map genes to chromosomes, to unravel the sex chromosome system and the translocation chain and investigate X inactivation and genomic imprinting in monotremes.     

Gamma S-crystallin of bovine and human eye lens: solution structure, stability and folding of the intact two-domain protein and its separate domains

Human and bovine gammaS-crystallin (HgammaS and BgammaS) and their isolated N- and C-terminal domains were cloned and expressed as recombinant proteins in E. coli. HgammaS and BgammaS are found to be authentic according to their spectral and hydrodynamic properties. Both full-length proteins and isolated domains are monomeric and exhibit high thermal and pH stabilities. The thermodynamic characterization made use of chemically and thermally-induced equilibrium unfolding transitions at varying pH. In spite of its exemplary two-domain structure, gammaS-crystallin does not show bimodal unfolding characteristics. In the case of BgammaS, at pH 7.0, the C-terminal domain is less stable than the N-terminal one, whereas for HgammaS the opposite holds true. Differential scanning calorimetry confirms the results of chemically-induced equilibrium unfolding transitions. Over the whole pH range between 2.0 and 11.5, HgammaS-crystallin and its isolated domains (HgammaS-N and HgammaS-C) follow the two-state model. The two-state unfolding of the intact two-domain protein points to the close similarity of the stabilities of the constituent domains. Obviously, interactions between the domains do not contribute significantly to the overall stability of gammaS-crystallin. In contrast, the structurally closely related gammaB-crystallin owes much of its extreme stability to domain interactions.     

Apolipoprotein A-I induced amyloidosis

Amyloidosis is characterized by extracellular deposits of protein fibrils with a high content of β-sheets in secondary structure. The protein forms together with proteoglycans amyloid fibrils causing organ damage and serious morbidity. Intact apolipoprotein A-I (apoA-I) is an important protein in lipid metabolism regulating the synthesis and catabolism of high density lipoproteins (HDL). Usually, apoA-I is not associated with amyloidosis. However, four naturally occuring mutant forms of apoA-I are known so far resulting in amyloidosis. The most important feature of all variants is the very similar formation of N-terminal fragments which were found in the amyloid deposits (residues 1–83 to 1–94). The new insights in the understanding of the association of apoA-I with HDL, its metabolism, and its hypothesized structural findings may explain a common mechanism for the genesis of apoA-I induced amyloidosis. Here we summarized the specific features of all known amyloidogenic variants of apoA-I and speculate about its metabolic pathway, which may have general implications for the metabolism of apoA-I.     

Fallback foraging as a way of life: Using dietary toughness to compare the fallback signal among capuchins and implications for interpreting morphological variation

The genus Cebus is one of the best extant models for examining the role of fallback foods in primate evolution. Cebus includes the tufted capuchins, which exhibit skeletal features for the exploitation of hard and tough foods. Paradoxically, these seemingly “specialized” taxa belong to the most ubiquitous group of closely related primates in South America, thriving in a range of different habitats. This appears to be a consequence of their ability to exploit obdurate fallback foods. Here we compare the toughness of foods exploited by two tufted capuchin species at two ecologically distinct sites; C. apella in a tropical rainforest, and C. libidinosus in a cerrado forest. We include dietary data for one untufted species (C. olivaceus) to assess the degree of difference between the tufted species. These data, along with information on skeletal morphology, are used to address whether or not a fallback foraging species exhibits a given suite of morphological and behavioral attributes, regardless of habitat. Both tufted species ingest and masticate a number of exceedingly tough plant tissues that appear to be used as fallback resources, however, C. libidinosus has the toughest diet both in terms of median and maximal values. Morphologically, C. libidinosus is intermediate in absolute symphyseal and mandibular measurements, and in measures of postcranial robusticity, but exhibits a higher intermembral index than C. apella. We propose that this incongruence between dietary toughness and skeletal morphology is the consequence of C. libidinosus' use of tools while on the ground for the exploitation of fallback foods. Am J Phys Anthropol 140:687–699, 2009. © 2009 Wiley-Liss, Inc.     

Broadband Onset Inhibition Can Suppress Spectral Splatter in the Auditory Brainstem

In vivo intracellular responses to auditory stimuli revealed that, in a particular population of cells of the ventral nucleus of the lateral lemniscus (VNLL) of rats, fast inhibition occurred before the first action potential. These experimental data were used to constrain a leaky integrate-and-fire (LIF) model of the neurons in this circuit. The post-synaptic potentials of the VNLL cell population were characterized using a method of triggered averaging. Analysis suggested that these inhibited VNLL cells produce action potentials in response to a particular magnitude of the rate of change of their membrane potential. The LIF model was modified to incorporate the VNLL cells’ distinctive action potential production mechanism. The model was used to explore the response of the population of VNLL cells to simple speech-like sounds. These sounds consisted of a simple tone modulated by a saw tooth with exponential decays, similar to glottal pulses that are the repeated impulses seen in vocalizations. It was found that the harmonic component of the sound was enhanced in the VNLL cell population when compared to a population of auditory nerve fibers. This was because the broadband onset noise, also termed spectral splatter, was suppressed by the fast onset inhibition. This mechanism has the potential to greatly improve the clarity of the representation of the harmonic content of certain kinds of natural sounds.     

Captive Reptile Mortality Rates in the Home and Implications for the Wildlife Trade

The trade in wildlife and keeping of exotic pets is subject to varying levels of national and international regulation and is a topic often attracting controversy. Reptiles are popular exotic pets and comprise a substantial component of the live animal trade. High mortality of traded animals raises welfare concerns, and also has implications for conservation if collection from the wild is required to meet demand. Mortality of reptiles can occur at any stage of the trade chain from collector to consumer. However, there is limited information on mortality rates of reptiles across trade chains, particularly amongst final consumers in the home. We investigated mortality rates of reptiles amongst consumers using a specialised technique for asking sensitive questions, additive Randomised Response Technique (aRRT), as well as direct questioning (DQ). Overall, 3.6% of snakes, chelonians and lizards died within one year of acquisition. Boas and pythons had the lowest reported mortality rates of 1.9% and chameleons had the highest at 28.2%. More than 97% of snakes, 87% of lizards and 69% of chelonians acquired by respondents over five years were reported to be captive bred and results suggest that mortality rates may be lowest for captive bred individuals. Estimates of mortality from aRRT and DQ did not differ significantly which is in line with our findings that respondents did not find questions about reptile mortality to be sensitive. This research suggests that captive reptile mortality in the home is rather low, and identifies those taxa where further effort could be made to reduce mortality rates.