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OBJECTIVES: Tobacco smoke exposure reduces CFTR functional expression in vitro and contributes to acquired CFTR dysfunction. We investigated whether it also inhibits the clinical benefit of CFTR modulators, focusing on tezacaftor/ivacaftor, approved in February 2018 for individuals with CF age ≥12 years. METHODS: A retrospective longitudinal analysis of encounter-based data from the CF Foundation Patient Registry (2016-2018) compared the slope of change in lung function (GLI FEV(1)% predicted) before and after tezacaftor/ivacaftor initiation in smoke-exposed vs unexposed age-eligible pediatric patients. Tobacco smoke exposure (Ever/Never) was determined from caregiver self-report. Statistical analyses used hierarchical linear mixed modeling and fixed effects regression modeling. RESULTS: The sample included 6,653 individuals with a total of 105,539 person-period observations. Tezacaftor/ivacaftor was prescribed to 19% (1,251) of individuals, mean age 17 years, mean baseline ppFEV(1) 83%, 28% smoke-exposed. Tezacaftor/ivacaftor users who were smoke-exposed had a lower baseline ppFEV(1) and experienced a greater lung function decline. Over two years, the difference in ppFEV(1) by smoke exposure among tezacaftor/ivacaftor users increased by 1.2% (7.6% to 8.8%, p

Polyketide natural products are valuable sources of bioactive molecules such as nutraceuticals and pharmaceuticals. The tremendous development of the genome sequence database revealed that the majority of the biosynthetic gene clusters (BGCs) are cryptic. Activation of these cryptic BGCs and identification of the related products is essential for finding more lead compounds for pharmaceuticals. On the other hand, 99% of microbes in nature cannot be cultured in regular conditions, which greatly hinders the efforts to explore their biosynthetic potentials. Expression of polyketide BGCs in heterologous hosts with better growth, good genetic characteristics, and amenable molecular tools is a robust approach to identify new polyketides and characterize their biosynthesis. This review outlines the challenges in the heterologous production of polyketide BGCs of bacterial origins.

The RPMI 2650 cell line has been a subject of evaluation as a physiological and pharmacological model of the nasal epithelial barrier. However, its suitability for drug permeability assays has not yet been established on a sufficiently large set of model drugs. We investigated two RPMI 2650 cell models (air-liquid and liquid-liquid) for nasal drug permeability determination by adopting the most recent regulatory guidelines on showing suitability of in vitro permeability methods for drug permeability classification. The permeability of 23 model drugs and several zero permeability markers across the cell models was assessed. The functional expression of two efflux transporters P-glycoprotein (P-gp) and Breast Cancer Resistant Protein (BCRP) was shown to be negligible by bidirectional transport studies using appropriate transporter substrates and inhibitors. The model drug permeability determined in the two RPMI 2650 cell models was correlated with the fully differentiated nasal epithelial model (MucilAir™). Additionally, correlations between the drug permeability in the investigated cell models and the ones determined in the Caco-2 cells and isolated rat jejunum were established. In conclusion, the air-liquid RPMI 2650 cell model is a promising pharmacological model of the nasal epithelial barrier and is much more suitable than the liquid-liquid model for nasal drug permeability prediction.

The majority of tissues within human body are constituted of designated structures to enable specific functions. Much effort has been done to engineer artificial fabric cell-laden scaffolds which are widely used for a great diversity of linear tissue constructs. For this purpose, collagen microfibers are of great concern among diverse materials while the control of cell-laden fiber formation and orientated structure is still unsolvable. Here, we developed a novel microfluidic-based strategy for continuous fabrication and assembly of three-dimensional (3D) cell-laden oriented collagen hydrogel microfibers. Inspired by the flow-introduced shear force in a microfluidic chip, collagen hydrogel microfibers obtained the oriented fabric structure which could guide rat pheochromocytoma cells (PC12) oriented spreading and enhance relative cellular functional expression. Rat aortic endothelial cells (RAOECs) were introduced to construct a co-cultured microfiber model, which further facilitated the functional expression of neural cells due to the synergistic effect of both vascularized-like cells and neural-like cells. Moreover, the ability of assembling collagen microfibers into larger constructs will benefit a variety of applications in tissue engineering and biomedical research.

The non-assumptive projection pursuit regression (NA-PPR) method, which is an exploratory data analysis method for analyzing high-dimensional data, was applied to predict the hydration heat of Portland-cement-based cementitious systems with mortar blends. The hydration mechanisms of cementitious systems were investigated by the Krstulovic–Dabic model, which was based on the proposed NA-PPR model of hydration heat. The new ridge functions and three-layer-grouping iterative optimization method were used to develop the NA-PPR model to improve the performance and address the non-uniqueness problem of parameter selection and subjective assumptions. It was shown that the proposed NA-PPR model is a promising non-assumptive method, with favorable prediction accuracy and a visible functional expression. Moreover, the Krstulovic–Dabic model combined with the NA-PPR model was able to well simulate the hydration process of the Portland-cement-based cementitious materials systems with mortar blends.

The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate “revertants” viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria.

Salicin is a bitter compound with important pharmaceutical values. The detection of salicin in pharmaceutical industry usually employs standard methods of analytical chemistry such as high performance liquid chromatography, which is not suitable for primary detection due to the complicated operation process and expensive systems. In this study, we explore the feasibility of using a label-free bioengineered cell-based biosensor to detect salicin. A human bitter taste receptor, T2R16, which could specifically respond to salicin, was expressed in HEK-293 cells as sensing elements of the biosensor. The structural and functional expression of T2R16 was validated by immunofluorescent staining and calcium imaging. The cell-impedance sensor was utilized as secondary transducer to detected cellular responses to salicin. The cell-impedance response of the bioengineered cells to salicin was investigated by analyzing the response intensity under various concentrations. The cell-impedance results revealed that this bioengineered cells could respond to salicin in a dose-dependent relationship. Finally, the performance of this bioengineered cell-based biosensor was evaluated. In cell-impedance measurement, non-transfected HEK-293 cells were not responsive to salicin, which proved the specificity of the biosensor. The detection of salicin at two unmeasured concentrations in standard response curve also proved this biosensor was sensitive in salicin detection. With the advantage of label-free measurement, this bioengineered may provide a promising and valuable approach for primary and quick detection of salicin.

The C-terminal domain (CTD) of MMP-2, which includes a hemopexin-like domain, has been increasingly studied as an alternative target in developing selective intervention strategies towards MMP-2. Moreover, The CTD itself has been implicated in a growing number of biological events, either MMP-dependent or –independent. The production of CTD, however, has been mostly based on the uncontrolled lysis of the latent ProMMP-2 or fusion protein expression that leaves a fusion tag. In this work we present a facile production of the untagged CTD in E. coli. The target protein was expressed as inclusion bodies, and we established an efficient wash and refolding strategy that allows us to obtain the target protein in extremely high purity. The yield was established at ~6 mg/L of the culture medium, which would greatly facilitate the production and hence the biological study of CTD. The method described herein might also prove useful for related (domain) proteins in MMP family and beyond.

This study sought to examine the co-expression of the following purinergic receptor subunits: P2X1, P2X1del, P2X4, and P2X7 and characterize the P2X response in human monocyte-derived macrophages (MDMs). Single-cell RT-PCR shows the presence of P2X1, P2X1del, P2X4, and P2X7 mRNA in 40%, 5%, 20%, and 90% of human MDMs, respectively. Of the studied human MDMs, 25% co-expressed P2X1 and P2X7 mRNA; 5% co-expressed P2X4 and P2X7; and 15% co-expressed P2X1, P2X4, and P2X7 mRNA. In whole-cell patch clamp recordings of human MDMs, rapid application of ATP (0.01 mM) evoked fast current activation and two different desensitization kinetics: 1. a rapid desensitizing current antagonized by PPADS (1 μM), reminiscent of the P2X1 receptor's current; 2. a slow desensitizing current, insensitive to PPADS but potentiated by ivermectin (3 μM), similar to the P2X4 receptor's current. Application of 5 mM ATP induced three current modalities: 1. slow current activation with no desensitization, similar to the P2X7 receptor current, present in 69% of human macrophages and antagonized by A-804598 (0.1 μM); 2. fast current activation and fast desensitization, present in 15% of human MDMs; 3. fast activation current followed by biphasic desensitization, observed in 15% of human MDMs. Both rapid and biphasic desensitization kinetics resemble those observed for the recombinant human P2X1 receptor expressed in oocytes. These data demonstrate, for the first time, the co-expression of P2X1, P2X4, and P2X7 transcripts and confirm the presence of functional P2X1, P2X4, and P2X7 receptors in human macrophages.

Appendix A. Supplementary data: Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015.03.066.; International audience; We report a dose-dependent detection of androstenone in solution, as one of the boar taint compounds, based on related OR7D4 olfactory receptors immobilized on a gold electrode through their 6-His tag and NTA-copper complex, as visualized through fluorescence microscopy. Square wave voltammetry (SWV) is for the first time, the method used to monitor the olfactory receptor/odorant recognition process. The relative variation of the Cu(I)-Cu(II) current peak increases linearly versus log (concentration of androstenone) from 10(-14) M to 10(-4) M, in buffer solution. Negative tests were performed, using an unrelated odorant, helional, itself a ligand of OR 1740. Cross-selectivity was also tested after immobilization of OR 1740.

[EN] Laccases are multicopper oxidoreductases considered by many in the biotechonology field as the ultimate >green catalysts>. This is mainly due to their broad substrate specificity and relative autonomy (they use molecular oxygen from air as an electron acceptor and they only produce water as by-product), making them suitable for a wide array of applications: biofuel production, bioremediation, organic synthesis, pulp biobleaching, textiles, the beverage and food industries, biosensor and biofuel cell development. Since the beginning of the 21st century, specific features of bacterial and fungal laccases have been exhaustively adapted in order to reach the industrial demands for high catalytic activity and stability in conjunction with reduced production cost. Among the goals established for laccase engineering, heterologous functional expression, improved activity and thermostability, tolerance to non-natural media (organic solvents, ionic liquids, physiological fluids) and resistance to different types of inhibitors are all challenges that have been met, while obtaining a more comprehensive understanding of laccase structure-function relationships. In this review we examine the most significant advances in this exciting research area in which rational, semi-rational and directed evolution approaches have been employed to ultimately convert laccases into high value-added biocatalysts., EU (FP7-KBBE-2013-7-613549-INDOX, FP7-People2013-ITN-607793 and COST-Action CM1303 Systems Biocatalysis) and the Spanish Government (BIO2010-19697-EVOFACEL, BIO2013-43407-RDEWRY and CAMBIOS-RTC-2014-1777-3)

High-temperature heat-harm of deep mine seriously affects the safety mining of coal mine, so at present the prediction of deep mine's underground temperature of and underground climate conditions and the treatment of heat- harm have already become the significant safety technology issues of coal mine production. Mine climate was actually measured along with mainly intake and return air routes of Zhangshuanglou coal mine, using drilling hole to measurement temperature and app lying inversion theory of mathematical physics and MATLAB to simulate. The conclusion that the temperature of wall rock of roadways was higher with increasing of depth then to a fixed value while the geothermal gradient was lower with increasing of depth then approached to zero was obtained. Finally the functional expression of the regulating ring of wall rock of laneway was drawn by using the third-order polynomial approximated method, and the deep underground temperature of mining workface 9409, 9109, 7109 and 7505 were predicted, the results of which are accurate, reliable and acceptable.

The discovery of odorant receptors led to endeavors in matching them with their cognate ligands. Although it has been challenging to functionally express odorant receptors in heterologous cells, previous studies have linked efficient odorant receptor expression with N-terminal modifications and accessory proteins, including the receptor-transporting proteins (RTPs) and Ric8b. Here we have shown that a shorter form of RTP1, RTP1S, supports robust cell-surface and functional expression of representative odorant receptors. Using a combination of accessory proteins, including RTP1S, Ric8b, and G(alphaolf), a diverse set of untagged odorant receptors were successfully expressed heterologously due to the synergistic effects among the various accessory proteins. Furthermore, the addition of an N-terminal rhodopsin tag to the odorant receptors, along with the same set of accessory proteins, exhibits an additional level of synergism, inducing enhanced odorant receptor responses to odorants and thus defining a more efficient heterologous expression system. We then showed that the presence or absence of different N-terminal tags has little effect on the ligand specificity of odorant receptors, although the amount of receptor expressed can play a role in the ligand response profile. The accuracy of the odorant receptor heterologous expression system involving tagged odorant receptors and various accessory proteins promises success in high throughput de-orphaning of mammalian odorant receptors.

Cathepsin L (CL)-like proteases are important candidate vaccine antigens for protection against helminth infections. We previously identified an immunogenic 32 kDa protein specifically present in newly excysted juveniles (NEJs) of Fasciola hepatica. Here we show by N-terminal protein sequencing that this protein represents a CL-like protease still containing the propeptide. Two cDNAs encoding this CL were subsequently isolated from NEJs by RT-PCR. The predicted amino acid sequences of these cDNAs showed approximately 70% sequence homology to both CL1 and CL2 sequences isolated from adult stage F. hepatica and are, therefore, referred to as CL3. The CL3 clones encoded asparagine at position PI of the propeptide cleavage site, suggesting a dependence on asparaginyl endopeptidases for maturation. Recombinant expression of a CL3 cDNA in Saccharomyces cerevisiae resulted in secretion of the proenzyme form. The propeptide of CL-like proteins was predicted to contain important B-cell epitopes. To determine the contribution of the propeptide to protective immunity, rats were vaccinated with Keyhole Limpet Haemocyanin-conjugated synthetic peptides encoding these putative B-cell epitopes derived from the CL1 or CL3 sequence. A subsequent challenge infection resulted in a significant (P

The gastrin releasing peptide (GRP) receptor is becoming an increasingly attractive target for development of new radiolabeled peptides with diagnostic and therapeutic potential. The attractiveness of the GRP receptor as a target is based upon the functional expression of GRP receptors in several tumors of neuroendocrine origin including prostate, breast, and small cell lung cancer. This concise review outlines some of the efforts currently underway to develop new GRP receptor specific radiopharmaceuticals by employing a variety of radiometal chelation systems.

The production of sufficient amounts of chemically and conformationally homogenous protein is a major requirement for successful crystallization and structure determination. With membrane proteins, this constitutes a particular problem because the membrane volume is limited and the organisms are usually very sensitive to changes in membrane properties brought about by massive protein insertion. Moreover, the extraction of membrane proteins from the membrane with detergents is generally a harsh treatment, which gives rise to conformational aberrations. A number of successful procedures for functional expression followed by purification are reviewed here together with nonfunctional expression into inclusion bodies and subsequent (re)folding to produce functional proteins. Most of the data are for prokaryotic outer membrane proteins, but the outer membrane proteins of eukaryotic organelles are also considered as they do show similar features.

In this study, we investigated the role of Ras and the mitogen-activated protein kinase (MAPK) pathway in the modulation of the inward rectifier potassium channel IRK1. We show that although expression of IRK1 in HEK 293 cells leads to the appearance of a potassium current with strong inward rectifying properties, coexpression of the constitutively active form of Ras (Ras-L61) results in a significant reduction of the mean current density without altering the biophysical properties of the channel. The inhibitory effect of Ras-L61 is not due to a decreased expression of IRK1 since Northern analysis indicates that IRK1 mRNA level is not affected by Ras-L61 co-expression. Moreover, the inhibition can be relieved by treatment with the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD98059. Confocal microscopy analysis of cells transfected with the fusion construct green fluorescent protein-IRK1 shows that the channel is mainly localized at the plasma membrane. Coexpression of Ras-L61 delocalizes fluorescence to the cytoplasm, whereas treatment with PD98059 partially restores the membrane localization. In conclusion, our data indicate that the Ras-MAPK pathway modulates IRK1 current by affecting the subcellular localization of the channel. This suggests a role for Ras signaling in regulating the intracellular trafficking of this channel.

Transcription factor GATA-2 contains two copies of a highly conserved zinc finger domain and plays unique roles at an early stage of hematopoietic differentiation. In the mouse pituitary gland, Pit-1-GATA-2 protein-protein interaction has been shown to lead to gene-specific actions to obtain cell-specific roles. In this study, we investigated the expression of GATA-2 and Pit-1 in human pituitary adenomas using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical techniques. By immunohistochemical analysis, GATA-2 was detected in all of the gonadotropin-subunit (Gn-su)-positive adenomas (n = 8) and in four of five thyroid-stimulating hormone (TSH)-secreting adenomas, but its incidence was low in the other types of adenomas. Pit-1 protein was detected in 4 of 5 TSH-secreting adenomas and in 10 of 10 growth hormone (GH)-secreting adenomas. By RT-PCR analysis, GATA-2 was detected in all Gn-su-positive adenomas and TSH-secreting adenomas, and Pit-1 was detected in all TSH-secreting adenomas and GH-secreting adenomas. These results suggested that GATA-2 contributes to the functional expression and the differentiation of Gn-su-positive adenomas and the TSH-secreting adenomas and that the interaction between GATA-2 and Pit-1 can lead to gene-specific action and differentiation of TSH-secreting adenomas. It is further speculated that GATA-2 and transcriptional interaction with Pit-1 play roles in the functional differentiation of specific pituitary adenomas.

Non-caloric sweeteners are widely used for the formulation of calorie-reduced beverages for health-conscious consumers. However, disadvantages such as undesired off-tastes limit the use of non-nutritive sweeteners. Therefore, the food industry is constantly searching for novel sweeteners and frequently resorts to using blends combining non-caloric sweeteners in a single formulation. The earliest blend allowing higher sweetness levels with reduced bitter off-taste combined saccharin with cyclamate. However, the mechanism by which sweetener blends become superior to single compounds remained obscure. By functional expression of human bitter taste receptors, we found the explanation for the phenomenon observed ∼60 years ago. We demonstrate that cyclamate potently blocks the receptors responsible for saccharin's bitter off-taste. This effect occurs at concentrations where cyclamate itself does not elicit a side taste. Intriguingly, also saccharin inhibits cyclamate-activated bitter receptors. Our experiments demonstrate that heterologous assays are useful for understanding perceptual phenomena and the development of novel tastant formulations.

We have isolated a cDNA from a rabbit intestinal cDNA library which, when co-expressed with the heavy chain of the human 4F2 antigen (4F2hc) in mammalian cells, induces system L-like amino acid transport activity. This protein, called LAT2, consists of 535 amino acids and is distinct from LAT1 which also interacts with 4F2hc to induce system L-like amino acid transport activity. LAT2 does not interact with rBAT, a protein with a significant structural similarity to 4F2hc. The 4F2hc/LAT2-mediated transport process differs from the 4F2hc/LAT1-mediated transport in substrate specificity, substrate affinity, tissue distribution, interaction with D-amino acids, and pH-dependence. The 4F2hc/LAT2-associated transport process has a broad specificity towards neutral amino acids with K(t) values in the range of 100-1000 microM, does not interact with D-amino acids to any significant extent, and is stimulated by acidic pH. In contrast, the 4F2hc/LAT1-associated transport process has a narrower specificity towards neutral amino acids, but with comparatively higher affinity (K(t) values in the range of 10-20 microM), interacts with some D-amino acids with high affinity, and is not influenced by pH. LAT2 is expressed primarily in the small intestine and kidney, whereas LAT1 exhibits a much broader tissue distribution.

The functional expression of Ca2+-activated K+ channels (KCa) in developing chick ciliary ganglion (CG) neurons requires interactions with target tissues and preganglionic innervation. Here, we show that the stimulatory effects of target tissues are mediated by an isoform of TGFbeta. Exposure of cultured CG neurons to TGFbeta1, but not TGFbeta2 or TGFbeta3, caused robust stimulation of KCa. The KCa stimulatory effects of target tissue extracts were blocked by a neutralizing pan-TGFbeta antiserum but not by specific TGFbeta2 or TGFbeta3 antisera. Intraocular injection of TGFbeta1 caused robust stimulation of KCa, whereas intraocular injection of pan-TGFbeta antiserum inhibited expression of KCa in CG neurons developing in vivo. The effects of TGFbeta1 were potentiated by beta-neuregulin-1, a differentiation factor expressed in preganglionic neurons.

Directed enzyme evolution has emerged in the past few years as a powerful alternative to rational approaches for engineering biocatalysts. Prerequisites for successful directed evolution are functional expression in a suitable microbial host, a rapid screen for the desired feature(s) and a well-thought-out working strategy for navigating protein landscapes. The rapidly growing body of literature on enzyme evolution in vitro includes techniques for creating and searching combinatorial enzyme libraries, as well as several successful examples of different evolutionary strategies being used.

Growth of Bacteria and Archaea has been observed at temperatures up to 95 and 110 degrees C, respectively. These thermophiles are adapted to environments of high temperature by changes in the membrane lipid composition, higher thermostabilities of the (membrane) proteins, higher turnover rates of the energy transducing enzymes, and/or the (exclusive) use of sodium-ions rather than protons as coupling ion in energy transduction. The proton permeability of the cytoplasmic membrane of bacteria and archaea was observed to increase with the temperature. This increased proton permeability limits the maximum temperature of growth of bacteria. Higher growth temperatures can be reached by an increased proton pumping activity by using the less permeable sodium ions as coupling ions or by changing the lipid composition of the cytoplasmic membrane. The Na+/H+/glutamate transport proteins of the thermophiles Bacillus stearothermophilus (GltT(Bc)) and Bacillus caldotenax (GltT(Bc)) were studied extensively. These transportproteins have unique features. Transport of L-glutamate occurs in symport with 1 Na+ and 1 H+ when the transport proteins are expressed in their natural environment. The sodium ion dependency of the GltT transporters of these Bacillus strains was found to increase with temperature. However, when the GltT proteins are expressed in the mesophile Escherichia coli, electrogenic symport of L-glutamate occurs with greater than or equal to 2 H+. These observations suggest that the conformation of the transport proteins in the E. coli and the Bacillus membranes differs, and that the conformation influences the coupling ion selectivity. The Na+/H+/glutamate transport proteins of B. stearothermophilus (GltT(Bc)) and B. caldotenax (GltT(Bc)) are homologous to transport systems of glutamate and structurally related compounds from mesophilic organisms. Both sodium, as well as proton coupled transporters, belong to this family of carboxylate transporters (FCT). (C) 1997 Elsevier Science Inc.

Cardiac G protein-activated Kir (GIRK) channels may assemble as heterotetrameric polypeptides from two subunits, Kir3.1 and Kir3.4. For a functional comparison with native channels in the CNS we investigated all possible combinations of heteromeric channel formation from brain Kir3.1, Kir3.2, Kir3.3, and Kir3.4 subunits in mRNA-injected Xenopus oocytes. Analysis of macroscopic current amplitudes and channel gating kinetics indicated that individual subunits or combinations of Kir3.2, Kir3.3, and Kir3.4 formed functional channels ineffectively. Each of these subunits gave rise to prominent currents with distinct characteristics only in the presence of Kir3.1 subunits. Functional expression of concatemeric constructs between Kir3.1 and Kir3.2/3.4 subunits as well as coimmunoprecipitations with subunit-specific antibodies confirmed heteromeric channel formation. Mutational swapping between subunits of a single pore loop residue (Kir3.1F137S; Kir3.3S114F; a phenylalanine confers slow channel gating in Kir3.1 subunits) revealed that Kir3.1 subunits are an important constituent for native heteromeric channels and dominate their functional properties. However, homomeric channels from Kir3.1 subunits in vivo may not exist due to the spatial conflict of bulky phenylalanines in the pore structure.

Functional expression of gap junction proteins can be obtained conveniently with the paired oocyte cell–cell channel assay. So far all gap junction proteins (connexins), with the exception of one, have been found to make functional channels either by themselves or as hybrid channels (two hemichannels of different connexin composition). Connexin33 (cx33) appears not to follow this rule. Expression of cx33 in oocytes does not yield functional channels, and attempts to identify another connexin with which cx33 can form hybrid channels have failed so far. The observation was made that cx33 inhibits functional expression of other connexins in a connexin-specific way. While expression of cx32 remains unaffected by coinjection into oocytes of cx33 mRNA together with cx32 mRNA, junctional conductance obtained with cx43 is marginally reduced whereas coinjection of cx33 mRNA at equimolar concentrations almost completely abolishes functional cx37 expression. The fact that testis is the only tissue found to express significant levels of cx33 mRNA together with cx37 and cx43 suggests a possible functional role for an inhibitory connexin. A model is proposed where cx33 limits a cell's capability to make functional channels by allowing formation of heterotypic channels with other cells while formation of homotypic channels is disallowed. Such a mechanism would permit asynchronous maturation of germ cells while at the same time allowing communication between germ cells and Sertoli cells.

Mammalian kidney is known to express a transport system specific for small peptides and pharmacologically active aminocephalosporins. This system is energized by a transmembrane electrochemical H + gradient. Recently, a H + -coupled peptide transporter has been cloned from rabbit and human intestine (Fei et al. (1994) Nature 368, 563-566; Liang et al., J. Biol. Chem., in press). Functional studies have established that the renal peptide transport system is similar but not identical to its intestinal counterpart. Therefore, in an attempt to isolate the renal H + /peptide cotransporter cDNA, we screened a human kidney cDNA library with a probe derived from the rabbit intestinal H + /peptide cotransporter cDNA. This has resulted in the isolation of a positive clone with a 2190 bp long open reading frame. The predicted protein consists of 729 amino acids. Hydropathy analysis of the amino acid sequence indicates the presence of twelve putative transmembrane domains. The primary structure of this protein exhibits 50% identity and 70% similarity to the human intestinal H + /peptide cotransporter. Functional expression of the kidney cDNA in HeLa cells results in the induction of a H + -coupled transport system specific for small peptides and aminocephalosporins. Reverse transcription-coupled polymerase chain reaction demonstrates that the cloned transporter is expressed in human kidney but not in human intestine. This transporter, henceforth called PEPT 2, represents a new member in the growing family of H + -coupled transport systems in the mammalian plasma membrane.

The short QT syndrome (SQTS) is a rare channelopathy associated with a high risk of life-threatening arrhythmias, sudden cardiac death (SCD) or sudden death (SD). This study sought to evaluate the phenotypic and functional expression of an apparent hotspot mutation associated with SQTS. Probands

The current state of knowledge concerning the structure, organization, and functional expression of immunoglobulin genes in chondrichthyan and osteichthyan fish is presented.

Molecular mechanisms of numerous cellular processes in eukaryotes are conserved between yeast and mammalian cells. Because of the ease of microbiological manipulation and genetic characterization, Saccharomyces cerevisiae is commonly used as a surrogate host for studying the function of proteins from other eukaryotic cell types. Numerous vector systems developed for high-level production of heterologous proteins in yeast are available for use in this research, but the successful expression of a biologically active mammalian protein in yeast requires additional considerations. In this paper, we describe the available expression vectors and protein sorting systems, and discuss appropriate strategies for expression of functional mammalian proteins in S. cerevisiae.

The upstroke of the action potential in heart cells, as in nerve and skeletal muscle cells, is due to the movement of sodium ions through ion-selective channels in the surface membrane. Voltage-dependent sodium channels in the heart, while sharing many common electrophysiologic characteristics with sodium channels in nerve and skeletal muscle, are distinctive in that they also contribute current to the plateau phase of the action potential and have unique sensitivities to channel-active toxins and drugs, most notably the local anesthetic-type antiarrhythmic agents. With the recent cloning and functional expression of the rat and human cardiac sodium channel genes, we are now in a position to understand better the molecular mechanisms responsible for the unique characteristics of sodium channels in the heart.

Myotonic dystrophy (DM) is the most prevalent human dystrophy; it results from a spliceopathy in which muscleblind-like (MBNL) regulatory proteins are sequestered by expanded CUG triplet repeats. In order to investigate the specific role that MBNL proteins play on the functional expression of chloride (ClC-1) channels, we studied the chloride currents (ICl) in fibers isolated from FDB muscles of adult knockout (KO) mice lacking MBNL1, MBNL3, or both (MBNL1/3 DKO). ICl were recorded in fibers voltage clamped with 2 microelectrodes, internally equilibrated with 70 mM intracellular chloride, and bathed in TEA-Cl solution. We found that ICl records in fibers from the three knockout strains display kinetic and voltage-dependent properties comparable to those in control fibers (129SV mice). However, the maximal peak ICl (peak-IClmax), and the maximal conductance calculated from them (gClmax), varied markedly among strains. Both peak-IClmax and gClmax are significantly smaller (∼34%, p

γ6 subunit of voltage-gated calcium channels is expressed in the heart and in the brain. It modulates gating of T-type channels (Hansen et al., 2004; Lin et al., 2008). It also associates with CaV1.2 channels and enhances their inactivation in the presence of the β1b, but not β2b, subunit (Yang et al., 2010).We found that γ6 subunit dramatically reduces the number of functional α1/β2a channels expressed in tsA-201 cells. Channels with the γ6 are trapped in the Golgi complex. Deletions of N-termini of α1 and γ6 subunits restore functional targeting to the plasma membrane.We propose that the γ6 regulates functional expression of CaV1.2 channels by interacting with the pore-forming α1 subunit or with another protein in the complex.

QPatch HTX has the highest throughput in the QPatch series. With signature features such as unattended operations, eight pipettes and the integrated microfluidic glass flow channels of the QPlates, the QPatch HTXis well suited for testing ligand gated ion channels. The recent advent of a QPlate with 10 patch holes per measurement site, instead of the classical single patch holehas minimized problems with low expression or problems with stability of the recodings over time. The success rate of recording on QPatch HTX is therefore approaching 100% for a number of different applications. We have previously shown that compound profiling on a wide variety of ligand-gated ion channels is certainly possible on the QPatch. With the increased stability and success rates of QPatch HTX, we demonstrate that the QPatch also can be used as an efficient screening tool for ligand gated ion channels. In this study, we have tested 3 different ion channels in a screening assay on QPatch HTX: GABA α1β1γ2 (γ-amino butyric acid receptor), ASIC a1 (acid sensing ion channel) and nAchR α1 (nicotinic acetylcholine receptor). These assays each have their own unique set of challenges: Low functional expression, extremely high elicited current amplitudes, and fast desensitization respectively. In this poster we present data to compare throughput and assay quality between the QPatch HTX in multi-hole mode and the classic single-hole mode in a screening scenario.