A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors

ATP-gated P2X4 receptors (P2X4R) are abundantly expressed in the CNS. However, little is known about the molecular targets for ethanol action in P2X4Rs. The current investigation tested the hypothesis that the ectodomain-transmembrane (TM) interface contains residues that are important for the action of ethanol in P2X4Rs. Wild type (WT) and mutant P2X4R were expressed in Xenopus oocytes. ATP concentration–response curves and ethanol (10–200 mM)-induced changes in ATP EC₁₀-gated currents were determined using two-electrode voltage clamp (−70 mV). Alanine substitution at the ectodomain-TM1 interface (positions 50–61) resulted in minimal changes in ethanol response. On the other hand, alanine substitution at the ectodomain-TM2 interface (positions 321–337) identified two key residues (D331 and M336) that significantly reduced ethanol inhibition of ATP-gated currents without causing marked changes in ATP I _max, EC₅₀, or Hill's slope. Other amino acid substitutions at positions 331 and 336 significantly altered or eliminated the modulatory effects of ethanol. Linear regression analyses revealed a significant relationship between hydropathy and polarity, but not molecular volume/molecular weight of the residues at these two positions. The results support the proposed hypothesis and represent an important step toward developing ethanol-insensitive receptors for investigating the role of P2X4Rs in mediating behavioral effects of ethanol.     

Using HIV Surveillance Registry Data to Re-Link Persons to Care: The RSVP Project in San Francisco

Background

Persons with unsuppressed HIV viral load (VL) who disengage from care may experience poor clinical outcomes and potentially transmit HIV. We assessed the feasibility and yield of using the San Francisco Department of Public Health (SFDPH) enhanced HIV surveillance system (eHARS) to identify and re-engage such persons in care.

Methods

Using SFDPH eHARS data as of 4/20/2012 (index date), we selected HIV-infected adults who were alive, had no reported VL or CD4 cell count results in the past nine months (proxy for “out-of-care”) and a VL >200 copies/mL drawn nine to 15 months earlier. We prioritized cases residing locally for investigation, and used information from eHARS and medical and public health databases to contact them for interview and referral to the SFDPH linkage services (LINCS). Twelve months later, we matched-back to eHARS data to assess how HIV laboratory reporting delays affected original eligibility, and if persons had any HIV laboratory results performed and reported within 12 months after index date (‘new labs’).

Results

Among 434 eligible persons, 282 were prioritized for investigation, of whom 75 (27%) were interviewed, 79 (28%) could not be located, and 48 (17%) were located out of the area. Among the interviewed, 54 (72%) persons accepted referral to LINCS. Upon match-back to eHARS data, 324 (75%) in total were confirmed as eligible, including 221 (78%) of the investigated; most had new labs.

Conclusions

Among the investigated persons presumed out-of-care, we interviewed and offered LINCS referral to about one-quarter, demonstrating the feasibility but limited yield of our project. Matching to updated surveillance data revealed that a substantial minority did not disengage from care and that most re-engaged in HIV care. Verifying persons’ HIV care status with medical providers and improving timeliness of transfer and cross-jurisdictional sharing of HIV laboratory data may aid future efforts.     

Genetic diversity and population structure identify the potential source of the invasive red clover casebearer moth, <Emphasis Type="Italic">Coleophora deauratella</Emphasis>, in North America

The red clover casebearer, Coleophora deauratella, is an invasive pest of red clover grown for seed in North America. In 2006, an outbreak in Alberta, Canada was discovered that resulted in significant seed losses, while further invasion threatens the world’s largest red clover forage seed production region in Oregon, USA. Prior to the recent outbreak, C. deauratella was thought to be restricted to eastern North America in its invasive range. We sequenced a 615-bp fragment of the mitochondrial cytochrome c oxidase subunit 1 gene, and developed three microsatellite markers to assess the genetic diversity and population structure of C. deauratella in North America and its native range in Europe. We observed signatures of a founder effect in North American populations and a further loss of genetic diversity within Alberta populations. Most genetic differentiation was found between continents, with no evidence of isolation-by-distance within each continent. From the limited number of European populations sampled, a single introduction from Switzerland is the most probable source of North American populations based on similar mitochondrial diversity and lack of population differentiation. Within North America, based on increased genetic diversity compared to the rest of the continent, the first North American record from Ithaca, NY, and the first documented outbreak in southern Ontario in 1989, the initial C. deauratella invasion most likely occurred in southern Ontario, Canada or adjacent states in the USA, followed by transport throughout the continent. This study provides insight into the phylogeographic history of C. deauratella in North America and Europe and may help to identify a regional source of future classical biological control agents.     

Heat stress-induced effects of photosystem I: an overview of structural and functional responses

Temperature is one of the main factors controlling the formation, development, and functional performance of the photosynthetic apparatus in all photoautotrophs (green plants, algae, and cyanobacteria) on Earth. The projected climate change scenarios predict increases in air temperature across Earth’s biomes ranging from moderate (3–4?°C) to extreme (6–8?°C) by the year 2100 (IPCC in Climate change 2007: The physical science basis: summery for policymakers, IPCC WG1 Fourth Assessment Report 2007; Climate change 2014: Mitigation of Climate Change, IPCC WG3 Fifth Assessment Report 2014). In some areas, especially of the Northern hemisphere, even more extreme warm seasonal temperatures may occur, which possibly will cause significant negative effects on the development, growth, and yield of important agricultural crops. It is well documented that high temperatures can cause direct damages of the photosynthetic apparatus and photosystem II (PSII) is generally considered to be the primary target of heat-induced inactivation of photosynthesis. However, since photosystem I (PSI) is considered to determine the global amount of enthalpy in living systems (Nelson in Biochim Biophys Acta 1807:856–863, 2011; Photosynth Res 116:145–151, 2013), the effects of elevated temperatures on PSI might be of vital importance for regulating the photosynthetic response of all photoautotrophs in the changing environment. In this review, we summarize the experimental data that demonstrate the critical impact of heat-induced alterations on the structure, composition, and functional performance of PSI and their significant implications on photosynthesis under future climate change scenarios.     

A comparison of genetic stability in tea [Camellia sinensis (L.) Kuntze] plantlets derived from callus with plantlets from long-term in vitro propagation

Plant Cell, Tissue and Organ Culture (PCTOC) - The development of tissue-specific or inducible promoters is important for plant genetic engineering. In this study, we isolated two novel promoters...     

Demonstration and ontogenesis in the rat of a serum protein analogous to human thyroxine binding globulin

It has been reported evidence based on equilibrium binding, electrophoretic, immunoelectrophoretic studies, that the rat possesses a major high affinity thyroid hormone binding protein, with an electrophoretic mobility and binding properties similar to those of the human thyroxine binding globulin (TBG). It is shown that in the sera of postnatal developing animals, between 3 and 21 days, the thyroxine (T4) and the triiodothyronine (T3) binding activities increase up to 10 times over adult or foetal levels, due to a high transient post-natal surge of the rat TBG. In the adult serum, the TBG persists in decreased amounts: it then yields the predominant role as T4 carrier to the thyroid binding prealbumin (TBPA), but retains the major role as binder of T3, i.e. of the biologically active thyroid hormone.     

GC/MS analysis of some bioactive constituents from Carthamus lanatus L

Sterols, triterpenes, volatiles, polar and other constituents in aerial parts of Carthamus lanatus were analyzed by gas chromatography-mass spectrometry. Over 90 compounds were identified most of them new for the species. Sitosterol and stigmasterol were the most abundant of 10 sterols identified in the sterol fraction. Taraxasterol, alpha- and beta-amyrine prevailed in the triterpene fraction. Volatiles, sterols and a fraction of the dichloromethane extract showed strong cytotoxicity (Artemia salina assay).     

Incorporation of anthraquinonyl group into lambda-Cro repressor protein for strand- and position-specific photocleavage of double-stranded DNA

lambda-Cro repressor protein incorporated with a 2-anthraquinonylalanine (anqAla) at the 64th position was chemically synthesized by solid-phase method. The 64th position was selected according to previous information on various mutants of Cro incorporated with a single anqAla unit, that were synthesized through an Escherichia coli in vitro protein-synthesizing system. The 64anqAla mutant bound to a dsDNA of consensus operator sequence and underwent strand- and position-specific photocleavage of the dsDNA at the GG sequence after treatment with piperidine. The mutant also underwent position-specific self-photoscission. The self-photoscission was retarded in the presence of the dsDNA.     

Genome-Wide Screens in Saccharomyces cerevisiae Highlight a Role for Cardiolipin in Biogenesis of Mitochondrial Outer Membrane Multispan Proteins

A special group of mitochondrial outer membrane (MOM) proteins spans the membrane several times via multiple helical segments. Such multispan proteins are synthesized on cytosolic ribosomes before their targeting to mitochondria and insertion into the MOM. Previous work recognized the import receptor Tom70 and the mitochondrial import (MIM) complex, both residents of the MOM, as required for optimal biogenesis of these proteins. However, their involvement is not sufficient to explain either the entire import pathway or its regulation. To identify additional factors that are involved in the biogenesis of MOM multispan proteins, we performed complementary high-throughput visual and growth screens in Saccharomyces cerevisiae. Cardiolipin (CL) synthase (Crd1) appeared as a candidate in both screens. Our results indeed demonstrate lower steady-state levels of the multispan proteins Ugo1, Scm4, and Om14 in mitochondria from crd1Δ cells. Importantly, MOM single-span proteins were not affected by this mutation. Furthermore, organelles lacking Crd1 had a lower in vitro capacity to import newly synthesized Ugo1 and Scm4 molecules. Crd1, which is located in the mitochondrial inner membrane, condenses phosphatidylglycerol together with CDP-diacylglycerol to obtain de novo synthesized CL molecules. Hence, our findings suggest that CL is an important component in the biogenesis of MOM multispan proteins.     

Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)-MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.