Evidence that a pertussis-toxin-sensitive substrate is involved in the stimulation by epidermal growth factor and vasopressin of plasma-membrane Ca2+ inflow in hepatocytes.

1. In hepatocytes, epidermal growth factor (EFG) (a) increased the rate of 45Ca2+ exchange in cells incubated at 1.3 mM extracellular Ca2+, (b) increased the activity of glycogen phosphorylase a and the intracellular free Ca2+ concentration (measured with quin2) in a process dependent on the concentration of extracellular Ca2+, and (c) enhanced the increase in glycogen phosphorylase activity which follows the addition of Ca2+ to cells previously incubated in the absence of Ca2+. It is concluded that EGF stimulates plasma-membrane Ca2+ inflow. 2. The effects of the combination of EGF and vasopressin on the rate of 45Ca2+ exchange and on the rate of increase in glycogen phosphorylase activity were the same as those of vasopressin alone. 3. The amount of 45Ca2+ released by EGF from internal stores was about 30% of that released by vasopressin. No detectable increase in [3H]inositol mono-, bis- or tris-phosphate was observed after the addition of EGF to cells labelled with myo-[3H]inositol. 4. In hepatocytes isolated from rats treated with pertussis toxin, the effects of EGF and vasopressin on phosphorylase activity (measured at 1.3 mM-Ca2+) and on the rate of Ca2+ inflow (measured with quin2) were markedly decreased compared with those in normal cells. 5. Treatment with pertussis toxin did not impair the ability of vasopressin to release Ca2+ from internal stores, but decreased vasopressin-stimulated [3H]inositol polyphosphate formation by 50%. 6. It is concluded that the mechanism(s) by which vasopressin and EGF stimulate plasma-membrane Ca2+-inflow transporters in hepatocytes involves a GTP-binding regulatory protein sensitive to pertussis toxin, and does not require an increase in the concentration of inositol trisphosphate comparable with that which induces the release of Ca2+ from the endoplasmic reticulum.     

The importance of biotic filtering on boreal conifer recruitment at alpine treeline

Treeline, the ecotone where forest transitions to alpine or tundra ecosystems, is considered the thermal limit to tree growth and survival. Despite temperature increases across mountainous areas and high latitudes globally, there has been no ubiquitous change in treeline position. The process of range expansion must initially depend on increased recruitment at, or beyond current range limits and recruitment limitations have been hypothesized as a mechanism for the variable response of treeline position to climate warming. We conducted a unique series of observational and experimental studies to quantify early-life stage constraints, from seed production to seedling establishment, on black spruce Picea mariana and tamarack Larix laricina recruitment at a model alpine treeline in Newfoundland, Canada. We found recruitment at treeline to be simultaneously seed and establishment limited. The treeline population produced fewer seeds than the forest population and black spruce seeds produced at treeline were less viable. Tamarack was more seed limited than black spruce where seed viability was low regardless of altitudinal position. Post-dispersal seed predation greatly constrained recruitment across the altitudinal gradient; however, black spruce seeds experienced the lowest levels of invertebrate seed predation on the lichen mat at treeline. If seeds were not consumed, individuals at treeline were establishment limited where germination and seedling establishment was both less abundant and delayed on lichen substrate. Our study highlights the need for multiple factors to align temporally for significant recruitment at treeline to occur.     

Medicinal use of fauna by a traditional community in the Brazilian Amazonia

Background

Zootherapy inventories are important as they contribute to the world documentation of the prevalence, importance and diversity of the medicinal use of animals in traditional human communities. The present study aims to contribute with a more valuable example of the zootherapy practices of a traditional community in the Brazilian Amazonia ?C the ??Riozinho do Anfrísio?? Extractive Reserve, in Northern Brazil.

Methods

We used the methods of participant observation and semi-structured interviews, applied to 25 informants. We employed the combined properties of two indices to measure the medicinal importance of each cited species to the studied community, as well as their versatility in the treatment of diseases: the well known Use Value (UV) and the Medicinal Applications Value (MAV) that we developed.

Results

We recorded 31 species of medicinal animals from six taxonomic categories, seven of which are new to science. The species are used for the treatment of 28 diseases and one species is used as an amulet against snakebites. The five species with the highest UV indices are the most popular and valued by the studied community. Their contrasting MAV indices indicate that they have different therapeutic properties: specific (used for the treatment of few diseases; low versatility) and all-purpose (several diseases; high versatility). Similarly, the most cited diseases were also those that could be treated with a larger number of animal species. Ten species are listed in the CITES appendices and 21 are present in the IUCN Red List. The knowledge about the medicinal use of the local fauna is distributed evenly among the different age groups of the informants.

Conclusions

This study shows that the local fauna represents an important medicinal resource for the inhabitants of the protected area. The combined use of the UV and MAV indices allowed identifying the species with the highest therapeutic potential. This type of information about a species may be of interest to pharmacological research, and is crucial to its conservation, since it helps signaling the species that may undergo higher hunting pressures. Data on zootherapy can also be of interesting to ecologists by contributing to indicators of local biodiversity richness.     

Plasma neurotensin release and gastric emptying in the dumping syndrome

Seventy-three patients were studied after ingesting a liquid glucose meal, tagged with ¹¹³Indium. Nineteen of these patients were awaiting surgery for their duodenal ulcer, while 54 were studied postoperatively, 25 of whom experienced troublesome postprandial (dumping) symptoms in their daily lives. The radioactive marker emptied significantly faster in the symptomatic patients than in the symptomfree, pre and post-operative groups (initial emptying rate 3.45 ± 0.23, compared with 1.16 ± 0.19 and 1.27 ± 0.15% fall in counts/min respectively; p < 0.01). Initial (20 min) rises in the plasma concentrations of neurotensin-like immunoreactivity measured during the test correlated significantly with the rate of gastric emptying in all patients, being greatest in patients with dumping symptoms. Physiological concentrations of neurotensin have been shown to delay gastric emptying. The excessive rise in plasma neurotensin-like immunoreactivity in patients with dumping symptoms, presumably occuring as a result of the rapid passage of nutrients to the neurotensin-rich ileum, may possibly have a compensatory role in slowing further emptying from the stomach.     

Exploration of ligands to the Qi site semiquinone in the bc1 complex using high-resolution EPR

Pulsed EPR spectroscopy was used to explore the structural neighborhood of the semiquinone (SQ) stabilized at the Qi site of the bc1 complex of Rhodobacter sphaeroides (EC 1.10.2.2) and to demonstrate that the nitrogen atom of a histidine imidazole group donates an H-bond to the SQ. Crystallographic structures show two different configurations for the binding of ubiquinone at the Qi site of mitochondrial bc1 complexes in which histidine (His-201 in bovine sequence) is either a direct H-bond donor or separated by a bridging water. The paramagnetic properties of the SQ formed at the site provide an independent method for studying the liganding of this intermediate species. The antimycin-sensitive SQ formed at the Qi site by either equilibrium redox titration, reduction of the oxidized complex by ascorbate, or addition of decylubihydroquinone to the oxidized complex in the presence of myxothiazol all showed similar properties. The electron spin echo envelope modulation spectra in the 14N region were dominated by lines with frequencies at 1.7 and 3.1 MHz. Hyperfine sublevel correlation spectroscopy spectra showed that these were contributed by a single nitrogen. Further analysis showed that the 14N nucleus was characterized by an isotropic hyperfine coupling of approximately 0.8 MHz and a quadrupole coupling constant of approximately 0.35 MHz. The nitrogen was identified as the N-epsilon or N-delta imidazole nitrogen of a histidine (it is likely to be His-217, or His-201 in bovine sequence). A distance of 2.5-3.1 A for the O-N distance between the carbonyl of SQ and the nitrogen was estimated. The mechanistic significance is discussed in the context of a dynamic role for the movement of His-217 in proton transfer to the site.     

On the relationship between an Asian haplotype on chromosome 6 that reduces androstenone levels in boars and the differential expression of SULT2A1 in the testis

Background

Androstenone is one of the major compounds responsible for boar taint, a pronounced urine-like odor produced when cooking boar meat. Several studies have identified quantitative trait loci (QTL) for androstenone level on Sus scrofa chromosome (SSC) 6. For one of the candidate genes in the region SULT2A1, a difference in expression levels in the testis has been shown at the protein and RNA level.

Results

Haplotypes were predicted for the QTL region and their effects were estimated showing that haplotype 1 was consistently related with a lower level, and haplotype 2 with a higher level of androstenone. A recombinant haplotype allowed us to narrow down the QTL region from 3.75 Mbp to 1.94 Mbp. An RNA-seq analysis of the liver and testis revealed six genes that were differentially expressed between homozygotes of haplotypes 1 and 2. Genomic sequences of these differentially expressed genes were checked for variations within potential regulatory regions. We identified one variant located within a CpG island that could affect expression of SULT2A1 gene. An allele-specific expression analysis in the testis did not show differential expression between the alleles of SULT2A1 located on the different haplotypes in heterozygous animals. However a synonymous mutation C166T (SSC6: 49,117,861 bp in Sscrofa 10.2; C/T) was identified within the exon 2 of SULT2A1 for which the haplotype 2 only had the C allele which was higher expressed than the T allele, indicating haplotype-independent allelic-imbalanced expression between the two alleles. A phylogenetic analysis for the 1.94 Mbp region revealed that haplotype 1, associated with low androstenone level, originated from Asia.

Conclusions

Differential expression could be observed for six genes by RNA-seq analysis. No difference in the ratio of C:T expression of SULT2A1 for the haplotypes was found by the allele-specific expression analysis, however, a difference in expression between the C over T allele was found for a variation within SULT2A1, showing that the difference in androstenone levels between the haplotypes is not caused by the SNP in exon 2.     

Deficiency of Starch Synthase IIIa and IVb Alters Starch Granule Morphology from Polyhedral to Spherical in Rice Endosperm

Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production.Starch is the most important carbohydrate storage material and contains the Glc polymers amylose and amylopectin. At least four classes of enzymes, ADP-Glc pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE), are necessary for efficient starch biosynthesis in storage tissues.SSs (EC 2.4.1.21) play a central role in starch synthesis during α-glucan elongation by adding Glc residues from ADP-Glc to the nonreducing ends via α-1,4-glucosidic linkages. Rice (Oryza sativa) contains 11 SS genes that are grouped into six classes, SSI to SSV and granule-bound starch synthase (GBSS; Supplemental Fig. S1; Hirose and Terao, 2004; Ohdan et al., 2005). Every class contains multiple isozymes, except for SSI and SSV; SSI, SSIIa, SSIIIa, and GBSSI are highly expressed in developing rice endosperm (Hirose and Terao, 2004; Ohdan et al., 2005). SSI elongates short amylopectin chains with degree of polymerization (DP) from 6 or 7 to DP 8 to 12 (Fujita et al., 2006). SSIIa elongates amylopectin from DP 6 to 12 to DP 13 to 24 (Umemoto et al., 2002; Nakamura et al., 2005), and SSIIIa elongates long amylopectin chains with DP 33 or greater (Fujita et al., 2007). GBSSI synthesizes amylose and extra-long amylopectin chains (Sano, 1984; Takeda et al., 1987; Hizukuri, 1995). The functions of other SS isozymes, such as SSIIb, SSIIc, SSIIIb, SSIVa, SSIVb, SSV, and GBSSII, remain largely unknown due to the lack of respective mutant lines. It is not clear how SS isozymes contribute to starch granule formation.Rice endosperm amyloplasts produce characteristic compound-type starch granules, which consist of dozens of polyhedral, sharp-edged granules (Matsushima et al., 2010). Compound-type starch granules are the most common type in endosperm of Poaceae species (Tateoka, 1962; Grass Phylogeny Working Group, 2001; Prasad et al., 2011; Matsushima et al., 2013). Simple-type starch granules (one starch granule per amyloplast) are produced in some species of the Bambusoideae, Pooideae, Micrairoideae, Chloridoideae, and Panicoideae subfamilies. The taxonomic relationships in the Poaceae do not enable an accurate prediction of granule morphology (Tateoka 1962; Shapter et al., 2008; Matsushima et al., 2013).Two studies that changed starch granule shape from simple type to compound type have been reported (Suh et al., 2004; Myers et al., 2011). A hull-less cultivar of cv Betzes barley (Hordeum vulgare), cv Nubet, contains simple-type and bimodal starch granules, which are typical of wild-type barley. Chemical mutagenesis of cv Nubet produced a mutant called franubet, which contains compound-type starch granules (Suh et al., 2004). In the maize monogalactosyldiacylglycerol synthase-deficient mutant opaque5, simple-type granules are replaced by compound-type granules separated by a membranous structure (Myers et al., 2011). The molecular mechanisms that control starch granule morphology in cereal endosperm are largely unknown, although an alteration in membrane lipid synthesis may be involved (Myers et al., 2011).A structural model for the compound-type amyloplast is shown Figure 1. The amyloplast envelope contains an outer envelope membrane (OEM), inner envelope membrane (IEM), and intermembrane space (IMS). Each starch granule is enclosed by an IEM, and granules are separated by a septum-like structure (SLS; Yun and Kawagoe, 2010). In this model, the IMS and SLS are directly connected, and fluorescent proteins such as GFP and Cherry can move freely between the two (Fig. 1; Kawagoe, 2013). The chloroplast envelope membrane contains little protein compared with the thylakoid membrane (Heber and Heldt, 1981). The endosperm amyloplast envelope membrane contains even less protein. Low protein content could be a major reason why the amyloplast envelope in rice endosperm is difficult to observe using high-resolution electron microscopy. In transgenic rice, a fluorescent protein fused to an IEM protein, the ADP-Glc transporter BRITTLE1, visualized the amyloplast IEM (Yun and Kawagoe, 2010). Fluorescent proteins fused to the chloroplast OEM protein OEP7 visualized the amyloplast OEM in endosperm (Kawagoe, 2013). These studies revealed that the outermost membranes of rice amyloplasts are OEM and contain intraamyloplast compartments. Starch is synthesized within the amyloplast compartments and is ultimately formed as compound-type granules that are individually wrapped in IEM (Yun and Kawagoe, 2010; Kawagoe, 2013).Open in a separate windowFigure 1.Structural model of the wild-type amyloplast in developing rice endosperm. The OEM is in black, the IEM is in magenta, the IMS is in green, and the SLS is in blue. G, Starch granules.Confocal microscopy analyses of the rice IEM protein, BRITTLE1, revealed that an SLS, or cross wall, divides starch granules in the amyloplast (Yun and Kawagoe, 2010). A model for the synthesis of compound-type starch granules consisting of polyhedral, sharp-edged granules proposed that the SLS functions as a mold that casts growing granules into a characteristic shape (Yun and Kawagoe, 2010; Kawagoe, 2013). The model postulates a central role for the SLS in producing characteristic compound-type granules, although neither the SLS components nor the enzymes affecting its properties have been characterized.Arabidopsis (Arabidopsis thaliana) SS genes are grouped into six classes. Leaf transitory starch biosynthesis has been investigated in single mutants of SSI, SSII, SSIII, and SSIV and in various double and triple SS mutants (Ral et al., 2004; Delvallé et al., 2005; Zhang et al., 2005, 2008; Szydlowski et al., 2009, 2011). Starch granules in leaf chloroplasts are reduced in number but enlarged in the ssIV mutant (Roldán et al., 2007; Crumpton-Taylor et al., 2013) and in the ssIV double and triple mutants (Szydlowski et al., 2009). Immature ssIV leaves have no starch granules but accumulate the starch synthase substrate ADP-Glc at high concentrations. Starch granules are flattened and discoid in wild-type leaves but are rounded in mature leaves of ssIV, suggesting that SSIV is essential for coordinating granule formation with chloroplast division during leaf expansion (Crumpton-Taylor et al., 2013). The ssIII ssIV double mutant does not accumulate measurable amounts of starch in the leaves, despite the presence of SSI and SSII activity (Szydlowski et al., 2009), implying that Arabidopsis SSIII and SSIV are involved in the initiation of starch granule formation and that either SSIII or SSIV is sufficient. Overexpression of AtSSIV increases the starch level in Arabidopsis leaves and potato (Solanum tuberosum) tubers (Gámez-Arjona et al., 2011). In transgenic plants, the AtSSIV-GFP fusion protein is enriched in specific regions at the edge of granules in Arabidopsis chloroplasts and potato tuber amyloplasts. In rice, SSIVa and SSIVb are expressed in the endosperm and other organs at an early developmental stage (Hirose and Terao, 2004; Ohdan et al., 2005).In this study, two rice allelic SSIVb-deficient mutant lines (ss4b) were generated by insertion of the retrotransposon Tos17 and crossed with the SSIIIa null mutant (ss3a). Surprisingly, the ss3a ss4b endosperm produced spherical starch granules that were separated from each other within amyloplasts, whereas the single mutants produced compound-type polyhedral starch granules. The SSIVb and GBSSI enzymes were localized to distinct compartments in developing amyloplasts. We discuss the changes in rice starch structure due to the deficiency of both SSIIIa and SSIVb, the alteration in starch granule morphology, and possible unconventional functions of SSIIIa and SSIVb. We also present a model of how spherical granules are produced in ss3a ss4b rice endosperm.     

Genotypic and phenotypic properties of <Emphasis Type="Italic">Candida parapsilosis</Emphasis><Emphasis Type="Italic">sensu strictu</Emphasis> strains isolated from different geographic regions and body sites

Background  

Candida parapsilosis is known to show limited genetic variability, despite different karyotypes and phenotypes have been described. To further investigate this aspect, a collection of 62 sensu strictu C. parapsilosis independent isolates from 4 geographic regions (Italy, n = 19; New Zealand, n = 15; Argentina, n = 14; and Hungary, n = 14) and different body sites (superficial and deep seated) were analysed for their genetic and phenotypic traits. Amplification fragment length polymorphism (AFLP) analysis was used to confirm species identification and to evaluate intraspecific genetic variability. Phenotypic characterisation included clinically relevant traits, such as drug susceptibility, in vitro biofilm formation and aspartyl protease secretion.