Integral Membrane Components Research Articles

Comparative transcriptome analysis provides clues to molecular mechanisms underlying blue-green eggshell color in the Jinding duck (Anas platyrhynchos)

BackgroundIn birds, blue-green eggshell color (BGEC) is caused by biliverdin, a bile pigment derived from the degradation of heme and secreted in the eggshell by the shell gland. Functionally, BGEC might promote the paternal investment of males in the nest and eggs. However, little is known about its formation mechanisms. Jinding ducks (Anas platyrhynchos) are an ideal breed for research into the mechanisms, in which major birds lay BGEC eggs with minor individuals laying white eggs. Using this breed, this study aimed to provide insight into the mechanisms via comparative transcriptome analysis.ResultsBlue-shelled ducks (BSD) and white-shelled ducks (WSD) were selected from two populations, forming 4 groups (3 ducks/group): BSD1 and WSD1 from population 1 and BSD2 and WSD2 from population 2. Twelve libraries from shell glands were sequenced using the Illumina RNA-seq platform, generating an average of 41 million clean reads per library, of which 55.9% were mapped to the duck reference genome and assembled into 31,542 transcripts. Expression levels of 11,698 genes were successfully compared between all pairs of 4 groups. Of these, 464 candidate genes were differentially expressed between cross-phenotype groups, but not for between same-phenotype groups. Gene Ontology (GO) annotation showed that 390 candidate genes were annotated with 2234 GO terms. No candidate genes were directly involved in biosynthesis or transport of biliverdin. However, the integral components of membrane, metal ion transport, cholesterol biosynthesis, signal transduction, skeletal system development, and chemotaxis were significantly (P < 0.05) overrepresented by candidate genes.ConclusionsThis study identified 464 candidate genes associated with duck BGEC, providing valuable information for a better understanding of the mechanisms underlying this trait. Given the involvement of membrane cholesterol contents, ions and ATP levels in modulating the transport activity of bile pigment transporters, the data suggest a potential association between duck BGEC and the transport activity of the related transporters.

Effects of proteasome inhibitor MG-132 on the parasite Schistosoma mansoni.

Proteasome is a proteolytic complex responsible for intracellular protein turnover in eukaryotes, archaea and in some actinobacteria species. Previous work has demonstrated that in Schistosoma mansoni parasites, the proteasome inhibitor MG-132 affects parasite development. However, the molecular targets affected by MG-132 in S. mansoni are not entirely known. Here, we used expression microarrays to measure the genome-wide changes in gene expression of S. mansoni adult worms exposed in vitro to MG-132, followed by in silico functional analyses of the affected genes using Ingenuity Pathway Analysis (IPA). Scanning electron microscopy was used to document changes in the parasites’ tegument. We identified 1,919 genes with a statistically significant (q-value ≤ 0.025) differential expression in parasites treated for 24 h with MG-132, when compared with control. Of these, a total of 1,130 genes were up-regulated and 790 genes were down-regulated. A functional gene interaction network comprised of MG-132 and its target genes, known from the literature to be affected by the compound in humans, was identified here as affected by MG-132. While MG-132 activated the expression of the 26S proteasome genes, it also decreased the expression of 19S chaperones assembly, 20S proteasome maturation, ubiquitin-like NEDD8 and its partner cullin-3 ubiquitin ligase genes. Interestingly, genes that encode proteins related to potassium ion binding, integral membrane component, ATPase and potassium channel activities were significantly down-regulated, whereas genes encoding proteins related to actin binding and microtubule motor activity were significantly up-regulated. MG-132 caused important changes in the worm tegument; peeling, outbreaks and swelling in the tegument tubercles could be observed, which is consistent with interference on the ionic homeostasis in S. mansoni. Finally, we showed the down-regulation of Bax pro-apoptotic gene, as well as up-regulation of two apoptosis inhibitor genes, IAP1 and BRE1, and in contrast, down-regulation of Apaf-1 apoptotic activator, thus suggesting that apoptosis is deregulated in S. mansoni exposed to MG-132. A considerable insight has been gained concerning the potential of MG-132 as a gene expression modulator, and overall the data suggest that the proteasome might be an important molecular target for the design of new drugs against schistosomiasis.

Evaluation of α-linolenic acid for freezability and in vivo fertility of Nili Ravi (Bubalus bubalis) buffalo semen

Alpha linolenic acid (ALA) is integral component of cell membrane that protects the cell in stressful events and involves in many metabolic pathways. It was hypothesized that ALA have the ability to protect the structural and functional integrity of buffalo spermatozoa during freeze-thawing. Therefore, study was designed to evaluate ALA supplementation (0, 5, 10 and 20 ng/mL) in extender on freezability and in vivo fertility of buffalo bull spermatozoa. Semen from three adult Nili-Ravi buffalo bulls of similar age was collected with artificial vagina (42 °C) for five weeks (replicates; N = 30). Qualified semen ejaculates (>1 mL volume, >60% motility; >0.5 billion/mL concentration) were diluted with tris-citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/mL ALA at 37 °C and cryopreserved following established protocol. Sperm motility and plasma membrane integrity were recorded higher (P < 0.05) in extender containing 5.0 ng/mL of ALA compared to control. Nevertheless, sperm viability, live dead ratio and chromatin integrity were observed higher (P < 0.05) in all experimental extenders with ALA compared to control. The number of abnormal sperm reduced significantly in all experimental extenders having ALA. A total of 539 artificial inseminations were performed with the best evolved extender having ALA (5.0 ng/mL; 272 inseminations) and control (267 inseminations). In vivo fertility rates of buffalo semen were recorded higher (P < 0.05) with extender containing ALA (5.0 ng/mL) (58%) compared to control (46%). In conclusion, supplementing 5.0 ng/mL ALA in extender improved the post-thaw quality and in vivo fertility of cryopreserved Nili-Ravi buffalo bull semen.

Transcriptome changes of blue-green algae, Arthrospira sp. in response to sulfate stress

Arthrospira platensis, a protein-rich blue-green alga is utilized as feed supplements for human and animals, which primarily depend upon nutrients such as carbon, nitrogen, phosphorus and sulfur in the presence of sunlight for their growth and survival. Cyanobacteria depend on sulfur for the synthesis and modification of a variety of biomolecules but, the exact mechanism of sulfur metabolism and impact of sulfate deprivation on A. platensis remain unclear. In this study, we analysed the change in growth and difference in the expression of key molecules of A. platensis during sulfate deprivation by comparing the transcriptome profiles of A. platensis grown at normal and sulfur deprived culture conditions. Observations showed that sulfate stress affected the levels of pigments in spirulina with slightly reduced growth. Transcriptome profile showed that expression profiles of different genes involved in various sulfur-dependent pathways were altered due to sulfur depletion. Major genes that were down-regulated include iron-sulfur clusters biosynthesis due to the absence of sulfur. Expression of genes involved in pathways such as translation, amino acid biosynthesis, protein folding, rRNA binding, were down-regulated, whereas genes involved in carbohydrate metabolism, phosphor relay sensor kinase activity, integral components of membrane, plasma membrane, thylakoid membrane, metal ion binding and DNA repair, were up-regulated during sulfur stress. Genes involved in transcription were up-regulated and hence there is a higher number of transcripts in A. platensis grown in sulfur depleted state. However, genes involved in translation were down-regulated; thus, there was a reduction in total protein content. Based on this analysis, it could be concluded that A. platensis was able to sustain sulfate stress, by its ability to alter the expression of genes that are specifically involved in sulfur metabolism and various other sulfur-dependent pathways.

Differential Effects of Omega-3 Fatty Acid, Docosahexaenoic Acid (DHA), In Animals and Humans

Lipids are essential biomolecules that make up most structural components of the cell. Omega-3 polyunsaturated fatty acids, in particular the Docosahexaenoic Acid (DHA), are probably the most important structural component. Being an integral membrane component, DHA can mediate many beneficial functions and studies in animal models have indeed witnessed these beneficial effects. However, their effects in humans have been controversial and dependent on a number of factors. Although many clinical trials have been conducted to ascertain these effects the results obtained are still inconclusive.

Oxidized phagosomal NOX2 complex is replenished from lysosomes.

ABSTRACTIn dendritic cells, the NADPH oxidase 2 complex (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome that kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome b558 (which comprises CYBB and CYBA), traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome b558 is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome b558 also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake that is required to initiate T cell responses.

POSSIBILITIES OF DE NOVO TRANSCRIPTOME SEQUENCING IN PYLOGENETIC RESEARCH ON AN EXAMPLE OF TARAXACUM OFFICINALE (ASTERACEAE)

&lt;p class="Default"&gt; &lt;/p&gt;&lt;p&gt;As a result of the research we obtained a cDNA library, sequences and carried out &lt;em&gt;de novo &lt;/em&gt;assembly and functional annotation of the transcriptome of systematically complex, almost cosmopolitan &lt;em&gt;Taraxacum officinale&lt;/em&gt;. Systematics of the species is complicated because of separation of many microspecies and intraspecific taxa, which may be explained by the presence of ploidy races, the phenomenon of apomixis and significant polymorphism. Attempts of separation of microspecies and closely related species based on sequencing of chloroplast and nuclear DNA fragments, did not give acceptable results. We have made the first attempt to analyze the transcriptome for understanding the genome evolution of agamospermous-sexual complex of the species. A total of 84440 reads were obtained with a total 31,540,710 bp. As a result of the &lt;em&gt;de novo &lt;/em&gt;assembly, we obtained 13902 contigs, with an average GC content equal to 38.1% and a maximum length of 5255 bp. In total, we received 3798 annotated genes. According to the functional annotation based on sequence homology, 2687 contigs were attributed to biological processes (19.32%), 3299 – to molecular functions (23.7%), 2157 – to the cellular component (15.51%) and 7497 contigs – with unknown functions. In the first category “single-organism cellular process”, “response to stimulus”, “photosynthesis-light reaction”, “oxidation-reduction” and “translation” have dominated. In the category of molecular function “nucleic acid binding”, “hydrolase activity”, “transferase” and “oxidoreductase” activities have dominated. In the cellular component category “integral component of membrane”, “chloroplast thylakoid membrane”, “photosystems” and “nucleus” have dominated.&lt;/p&gt;

Endothelial basement membrane laminin 511 is essential for shear stress response

Shear detection and mechanotransduction by arterial endothelium requires junctional complexes containing PECAM‐1 and VE‐cadherin, as well as firm anchorage to the underlying basement membrane. While considerable information is available for junctional complexes in these processes, gained largely from in vitro studies, little is known about the contribution of the endothelial basement membrane. Using resistance artery explants, we show that the integral endothelial basement membrane component, laminin 511 (laminin α5), is central to shear detection and mechanotransduction and its elimination at this site results in ablation of dilation in response to increased shear stress. Loss of endothelial laminin 511 correlates with reduced cortical stiffness of arterial endothelium in vivo, smaller integrin β1‐positive/vinculin‐positive focal adhesions, and reduced junctional association of actin–myosin II. In vitro assays reveal that β1 integrin‐mediated interaction with laminin 511 results in high strengths of adhesion, which promotes p120 catenin association with VE‐cadherin, stabilizing it at cell junctions and increasing cell–cell adhesion strength. This highlights the importance of endothelial laminin 511 in shear response in the physiologically relevant context of resistance arteries.

Highly sensitive detection of lipopolysaccharides using an aptasensor based on hybridization chain reaction.

Lipopolysaccharides (LPS), integral components of the outer membrane of all gram-negative bacteria, are closely associated with foodborne diseases such as fever, diarrhea and hypotension, and thus, the early and sensitive detection of LPS is necessary. In this study, an aptasensor assay based on hybridization chain reaction (HCR) was developed to detect LPS. Briefly, two complementary stable species of biotinylated DNA hairpins coexisted in solution until the introduction of a detection probe triggered a hybridization chain reaction cascade. The DNA conjugates specifically reacted with the LPS, which were captured by the ethanolamine aptamer attached to the reaction well surface. After optimizing the key reaction conditions, such as the reaction time of HCR, the amount of the capture probe and detection probes, the increase in the LPS concentration was readily measured by the optical density value, and a relatively low detection limit (1.73 ng/mL) was obtained, with a linear response range of 1–105 ng/mL. The approach presented herein introduced the use of an aptasensor for LPS discrimination and HCR for signal amplification, offering a promising option for detecting LPS.

Characterization and differential expression analysis of Toxocara canis aquaporin-1 gene.

Toxocara canis is an intestinal nematode of canids with a worldwide distribution, causing an important but neglected parasitic zoonosis in humans. Aquaporins (AQP) are a family of water channel proteins, which function as membrane channels to regulate water homeostasis. In this study, the coding sequence of aquaporin-1 gene of T. canis (Tc-aqp-1) was cloned and characterized. The obtained Tc-aqp-1 coding sequence was 933bp in length, which predicted to encode 311 amino acids. Two conserved asparagine-proline-alanine (NPA) motifs were identified in the multiple sequence alignments. Phylogenetic analysis revealed the closest relationship between T. canis and Opisthorchis viverrini based on aquaporin-1 amino acid sequence. A structure was predicted with ligand binding sites predicted at H93, N95, N226, L94, I79, and I210 and with active sites predicted at I256 and G207. Gene Ontology (GO) annotations predicted its cellular component term of integral component of plasma membrane (GO: 0005887), molecular function term of channel activity (GO: 0015250), and biological process term of water transport (GO: 0006833). Tissue expression analysis revealed that the Tc-aqp-1 was highly expressed in the intestine of adult male. The findings of the present study provide the basis for further functional studies of T. canis aquaporin-1.

Cholesterol influences potassium currents in inner hair cells isolated from guinea pig cochlea.

There is a correlation between serum hyperlipidemia and hearing loss. Cholesterol is an integral component of the cell membrane and regulates the activity of ion channels in the lipid bilayer. The aim of this study was to investigate the effects of cholesterol on the potassium currents in IHCs by using the cholesterol-depleting drug, MβCD, and water-soluble cholesterol. IHCs were acutely isolated from a mature guinea-pig cochlea and potassium currents were recorded. MβCD and water-soluble cholesterol were applied to IHCs under pressure puff pipettes. IHCs showed outwardly rectifying currents (IK,f and IK,s) in response to depolarizing voltage pulses, with only a slight inward current (IK,n) when hyperpolarized. In 10mM MβCD solutions, the amplitude of outward K currents reversely decreased; however, fast activation kinetics was preserved. In contrast, in solution of 1mM water-soluble cholesterol, the amplitude of outward K currents reversely increased. At the membrane potential of +110mV, relative conductances were 0.87±0.07 and 1.18±0.11 in MβCD solutions and cholesterol solutions, respectively. The amplitude of K currents in isolated IHCs was reversely changed by cholesterol-depleting drug and water-soluble cholesterol. These results demonstrated the possibility of the involvement of IHC function in hyperlipidemia-induced inner ear disorders.

Molecular Assembly of the Mitochondrial Calcium Uniporter Complex

Mitochondrial Ca2+ regulates a wide variety of physiological processes, including ATP production and cell death. Rapid Ca2+ uptake into mitochondria is mediated by the mitochondrial calcium uniporter (MCU) complex, which is composed of the pore forming MCU protein, and the regulatory proteins EMRE, and MICU1/MICU2. Currently, the submitochondrial localization and transmembrane orientation of these proteins are either unknown or under debate. We attack these issues using a classical thiol-modification approach. We removed native MCU complex proteins in HEK293 cells using CRISPR/Cas9, and reintroduced mutants containing a single cysteine at defined positions. Treatments of the mitoplast with a bulky, thiol-reactive compound polyethylene glycol (PEG) maleimide would alter the molecular weight of the protein if the engineered Cys is exposed to the intermembrane space. Using this strategy along with other biochemical methods, we demonstrated that (1) MCU adopts an orientation with the signature DIME motif facing intermembrane space (IMS), (2) the conserved C-terminal polyaspartic tail of EMRE is in the IMS, (3) both MICU1 and MICU2 are associated with the outer leaflet of the inner membrane, and (4) although MICU1 is a peripheral membrane protein, MICU2 is an integral component of the inner membrane. Then, applying domain interaction analysis and mutagenesis screening, we identified molecular contacts that govern the Ca2+ transport behavior of the MCU complex. In particular, we demonstrate that EMRE interacts with MCU through the transmembrane helices to activate the Ca2+ pore, while using the polyaspartic tail to recruit MICU1/2 to gate the pore. This dual functionality of EMRE ensures that all functional MCU complexes respond appropriately to Ca2+ stimuli from the cytosol, safeguarding against dangerous Ca2+ leakage, which could diminish mitochondrial energy output and potentially trigger apoptotic cell death.

Structure and Switch Cycle of SRβ as Ancestral Eukaryotic GTPase Associated with Secretory Membranes

G proteins of the Ras-family of small GTPases trace the evolution of eukaryotes. The earliest branching involves the closely related Arf, Sar1, and SRβ GTPases associated with secretory membranes. SRβ is an integral membrane component of the signal recognition particle (SRP) receptor that targets ribosome-nascent chain complexes to the ER. How SRβ integrates into the regulation of SRP-dependent membrane protein biogenesis is not known. Here we show that SRβ-GTP interacts with ribosomes only in presence of SRα and present crystal structures of SRβ in complex with the SRX domain of SRα in the GTP-bound state at 3.2 Å, and of GDP- and GDP · Mg(2+)-bound SRβ at 1.9 Å and 2.4 Å, respectively. We define the GTPase switch cycle of SRβ and identify specific differences to the Arf and Sar1 families with implications for GTPase regulation. Our data allow a better integration of SRβ into the scheme of protein targeting.

Chlamydia trachomatis Relies on Autonomous Phospholipid Synthesis for Membrane Biogenesis

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome and is thought to rely on its mammalian host cell for nutrients. Although several lines of evidence suggest C. trachomatis utilizes host phospholipids, the bacterium encodes all the genes necessary for fatty acid and phospholipid synthesis found in free living Gram-negative bacteria. Bacterially derived phospholipids significantly increased in infected HeLa cell cultures. These new phospholipids had a distinct molecular species composition consisting of saturated and branched-chain fatty acids. Biochemical analysis established the role of C. trachomatis-encoded acyltransferases in producing the new disaturated molecular species. There was no evidence for the remodeling of host phospholipids and no change in the size or molecular species composition of the phosphatidylcholine pool in infected HeLa cells. Host sphingomyelin was associated with C. trachomatis isolated by detergent extraction, but it may represent contamination with detergent-insoluble host lipids rather than being an integral bacterial membrane component. C. trachomatis assembles its membrane systems from the unique phospholipid molecular species produced by its own fatty acid and phospholipid biosynthetic machinery utilizing glucose, isoleucine, and serine.

Cholesterol removal from adult skeletal muscle impairs excitation-contraction coupling and aging reduces caveolin-3 and alters the expression of other triadic proteins.

Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation–contraction (E–C) coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca2+ transients without affecting membrane integrity or causing sarcoplasmic reticulum (SR) calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not SR membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na+/K+-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX) activity and protein content of NOX2 subunits (p47phox and gp91phox), implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs E–C coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged animals.

The internal gene duplication and interrupted coding sequences in the MmpL genes of Mycobacterium tuberculosis: Towards understanding the multidrug transport in an evolutionary perspective

The multidrug resistance has emerged as a major problem in the treatment of many of the infectious diseases. Tuberculosis (TB) is one of such disease caused by Mycobacterium tuberculosis. There is short term chemotherapy to treat the infection, but the main hurdle is the development of the resistance to antibiotics. This resistance is primarily due to the impermeable mycolic acid rich cell wall of the bacteria and other factors such as efflux of antibiotics from the bacterial cell. The MmpL (Mycobacterial Membrane Protein Large) proteins of mycobacteria are involved in the lipid transport and antibiotic efflux as indicated by the preliminary reports. We present here, comprehensive comparative sequence and structural analysis, which revealed topological signatures shared by the MmpL proteins and RND (Resistance Nodulation Division) multidrug efflux transporters. This provides evidence in support of the notion that they belong to the extended RND permeases superfamily. In silico modelled tertiary structures are in homology with an integral membrane component present in all of the RND efflux pumps. We document internal gene duplication and gene splitting events happened in the MmpL genes, which further elucidate the molecular functions of these putative transporters in an evolutionary perspective.

Inhibitory effect of piperine on Helicobacter pylori growth and adhesion to gastric adenocarcinoma cells.

BackgroundPiperine is a compound comprising 5-9% of black pepper (Piper nigrum), which has a variety of biological roles related to anticancer activities. Helicobacter pylori has been classified as a gastric carcinogen, because it causes gastritis and gastric cancer by injecting the virulent toxin CagA and translocating VacA. The present study investigated the inhibitory action of piperine on H. pylori growth and adhesion.MethodsInhibition of H. pylori growth was determined by the broth macrodilution method, and adhesion to gastric adenocarcinoma cells validated by urease assay. Motility test was performed by motility agar and the expression of adhesion gene and flagellar gene in response to the piperine treatment was assessed by RT-PCR and immunoblotting.ResultsAdministrated piperine suppressed the level of H. pylori adhesion to gastric adenocarcinoma cells in a dose dependent manner and the inhibition was statistically significant as determined by Student’s t-test. In addition, piperine treatment effects on the flagellar hook gene flgE and integral membrane component of the export apparatus gene flhA expression to be suppressed and piperine diminished the H. pylori motility.ConclusionsflhA, encodes an integral membrane component of the export apparatus, which is also one of the regulatory protein in the class 2 genes expression and flgE is one of them that encodes hook part of the flagella. Suppression of both genes, leads to less motility results in the organism attracted less towards to the gastric epithelial cells might be the possible reason in the adhesion inhibition. To our knowledge, this is the first report published on the inhibitory effects of piperine against the adhesion of H. pylori to gastric adenocarcinoma cells.

Relationship of Azole Resistance with the Structural Alteration of the Target Sites: Novel Synthetic Compounds for Better Antifungal Activities

Azoles have consistently been recognized as the mainstays of antifungal drugs, amongst which ergosterol prevails as an integral component of fungal plasma membrane. It is most commonly produced via demethylation of lanosterol by the cytochrome P450-dependent enzyme lanosterol 14α-demethylase. Azoles exert their antifungal activities via binding to lanosterol 14 α-demethylase and thereby preventing the demethylation of lanosterol. This leads to the depletion of demethylated lanosterol (ergosterol) with the concomitant accumulation of methylated sterol precursors (lanosterol, 4,14-dimethylzymosterol, and 24-methylenedihydrolanosterol) and deterioration of the membrane integrity, resulting in fungal growth inhibition. Resistance to azoles is a concern, particularly during the long-term treatment of fungus mediated cellular complications. To combat azole resistance and to extend the spectrum of treatable pathogens, the development of novel and more potent azoles, with alteration in active sites has attracted worldwide scientific attention. With such an insight, this review focuses on antifungal potentials of azole compounds with an emphasis on the corresponding drug resistance episodes complemented with novel strategies for the development of new generation of azole compounds. Keywords: Azole, lanosterol, ergosterol, 14-α-lanosterol-demethylase, structure-function relationship, drug resistance.

The kidney tight junction (Review)

The tight junction is an important subcellular organelle which plays a vital role in epithelial barrier function. Claudin, as the integral membrane component of tight junctions, creates a paracellular transport pathway for various ions to be reabsorbed by the kidneys. This review summarizes advances in claudin structure, function and pathophysiology in kidney diseases. Different claudin species confer selective paracellular permeability to each of three major renal tubular segments: the proximal tubule, the thick ascending limb of Henle’s loop and the distal nephron. Defects in claudin function can cause a wide spectrum of kidney diseases, such as hypomagnesemia, hypercalciuria, kidney stones and hypertension. Studies using transgenic mouse models with claudin mutations have recapitulated several of these renal disease phenotypes and have elucidated the underlying biological mechanisms. Modern recording approaches based upon scanning ion conductance microscopy may resolve the biophysical nature of claudin transport function and provide novel insight into tight junction architecture.

Peptidergic cell-specific synaptotagmins in Drosophila: localization to dense-core granules and regulation by the bHLH protein DIMMED.

Bioactive peptides are packaged in large dense-core secretory vesicles, which mediate regulated secretion by exocytosis. In a variety of tissues, the regulated release of neurotransmitters and hormones is dependent on calcium levels and controlled by vesicle-associated synaptotagmin (SYT) proteins. Drosophila express seven SYT isoforms, of which two (SYT-α and SYT-β) were previously found to be enriched in neuroendocrine cells. Here we show that SYT-α and SYT-β tissue expression patterns are similar, though not identical. Furthermore, both display significant overlap with the bHLH transcription factor DIMM, a known neuroendocrine (NE) regulator. RNAi-mediated knockdown indicates that both SYT-α and SYT-β functions are essential in identified NE cells as these manipulations phenocopy loss-of-function states for the indicated peptide hormones. In Drosophila cell culture, both SYT-α and neuropeptide cargo form DIMM-dependent fluorescent puncta that are coassociated by super-resolution microscopy. DIMM is required to maintain SYT-α and SYT-β protein levels in DIMM-expressing cells in vivo. In neurons normally lacking all three proteins (DIMM(-)/SYT-α(-)/SYT-β(-)), DIMM misexpression conferred accumulation of endogenous SYT-α and SYT-β proteins. Furthermore transgenic SYT-α does not appreciably accumulate in nonpeptidergic neurons in vivo but does so if DIMM is comisexpressed. Among Drosophila syt genes, only syt-α and syt-β RNA levels are upregulated by DIMM overexpression. Together, these data suggest that SYT-α and SYT-β are important for NE cell physiology, that one or both are integral membrane components of the large dense-core vesicles, and that they are closely regulated by DIMM at a post-transcriptional level.