Expression and secretion of the atrial natriuretic peptide in human adipose tissue and preadipocytes.

OBJECTIVE: Atrial natriuretic peptide (ANP) is a secretory hormone displaying diuretic, natriuretic, and vasorelaxant activities. Recently, its lipolytic activity has been reported. Since the expression of ANP in adipose tissue has not been documented, we used real-time reverse transcriptase polymerase chain reaction (RT-PCR) to investigate the expression of ANP in human adipose tissue and preadipocytes. RESEARCH METHODS AND PROCEDURES: RNA was extracted from the human adipose tissue of severely obese premenopausal women as well as from human preadipocytes. For human preadipocytes, two cell systems were investigated: the human preadipose immortalized (Chub-S7) cells, a well-characterized human preadipose cell line, and primary preadipocytes derived from the stromal vascular fraction of the human adipose tissue. We measured the mRNA of ANP, of corin (a transmembrane serine protease involved in the conversion of pro-ANP to ANP) and of uncoupling protein 2 (UCP2; a control gene known to be ubiquitously expressed). The expression of ANP was also investigated using immunofluorescence and radioimmunoassay in Chub-S7 cells and human primary preadipocytes in culture. RESULTS: Our results indicate that ANP and corin are expressed at the mRNA level in human adipose tissue and preadipocytes. Immunofluorescence experiments demonstrated that pro-ANP was expressed in Chub-S7 cells. In addition, ANP secretion could be measured in Chub-S7 cells and human primary preadipocytes in culture. Rosiglitazone, a selective peroxisome proliferator-activated receptor type gamma (PPAR-gamma) agonist promoting adipocyte differentiation, was found to modulate both ANP expression and secretion in preadipocytes. DISCUSSION: Our findings suggest the existence of an autocrine/paracrine system for ANP in the human adipose tissue whose implications in lipolysis and cardiovascular function need to be further explored.

Use of constant denaturant capillary electrophoresis of pooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1a and Scnn1b) encoding the alpha and beta subunits of the epithelial sodium channel.

BACKGROUND: The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta and the gamma subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the alpha subunit, has not been investigated. METHODS: We screened for mutations in the COOH termini of the alpha and beta subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of alpha- and beta-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples. RESULTS: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of alpha-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G-->T and 1987A-->G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of beta-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C-->T (T594M). CONCLUSION: CDCE rapidly detects point mutations in these candidate disease genes.

La biologie moléculaire est-elle utile au praticien [Is molecular biology useful to the practitioner?]

The relative importance of molecular biology in clinical practice is often underestimated. However, numerous procedures in clinical diagnosis and new therapeutic drugs have resulted from basic molecular research. Furthermore, understanding of the physiological and physiopathological mechanisms underlying several human diseases has been improved by the results of basic molecular research. For example, cloning of the gene encoding leptin has provided spectacular insights into the understanding of the mechanisms involved in the control of food intake and body weight maintenance in man. In cystic fibrosis, the cloning and identification of several mutations in the gene encoding the chloride channel transmembrane regulator (CFTR) have resolved several important issues in clinical practice: cystic fibrosis constitutes a molecular defect of a single gene. There is a strong correlation between the clinical manifestations or the severity of the disease (phenotype) with the type of mutations present in the CFTR gene (genotype). More recently, identification of mutations in the gene encoding a subunit of the renal sodium channel in the Liddle syndrome has provided important insight into the physiopathological understanding of mechanisms involved in this form of hereditary hypertension. Salt retention and secondary high blood pressure are the result of constitutive activation of the renal sodium channel by mutations in the gene encoding the renal sodium channel. It is speculated that less severe mutations in this channel could result in a less severe form of hypertension which may correspond to patients suffering from high blood pressure with low plasma renin activity. Several tools, most notably PCR, are derived from molecular research and are used in everyday practice, i.e. in prenatal diagnosis and in the diagnosis of several infectious diseases including tuberculosis and hepatitis. Finally, the production of recombinant proteins at lower cost and with fewer side effects is used in everyday clinical practice. Gene therapy remains an extraordinary challenge in correcting severe hereditary or acquired diseases. The use of genetically modified animal cell lines producing growth factors, insulin or erythropoetin, which are subsequently encapsulated and transferred to man, represents an attractive approach for gene therapy.

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.

A novel TMPRSS3 missense mutation in a DFNB8/10 family prevents proteolytic activation of the protein.

Pathogenic mutations in TMPRSS3, which encodes a transmembrane serine protease, cause non-syndromic deafness DFNB8/10. Missense mutations map in the low density-lipoprotein receptor A (LDLRA), scavenger-receptor cysteine-rich (SRCR), and protease domains of the protein, indicating that all domains are important for its function. TMPRSS3 undergoes proteolytic cleavage and activates the ENaC sodium channel in a Xenopus oocyte model system. To assess the importance of this gene in non-syndromic childhood or congenital deafness in Turkey, we screened for mutations affected members of 25 unrelated Turkish families. The three families with the highest LOD score for linkage to chromosome 21q22.3 were shown to harbor P404L, R216L, or Q398X mutations, suggesting that mutations in TMPRSS3 are a considerable contributor to non-syndromic deafness in the Turkish population. The mutant TMPRSS3 harboring the novel R216L missense mutation within the predicted cleavage site of the protein fails to undergo proteolytic cleavage and is unable to activate ENaC, thus providing evidence that pre-cleavage of TMPRSS3 is mandatory for normal function.

Critical flux and fouling in ultrafiltration of proteins

In this thesis different parameters influencing critical flux in protein ultrafiltration and membrane foul-ing were studied. Short reviews of proteins, cross-flow ultrafiltration, flux decline and criticalflux and the basic theory of Partial Least Square analysis (PLS) are given at the beginning. The experiments were mainly performed using dilute solutions of globular proteins, commercial polymeric membranes and laboratory scale apparatuses. Fouling was studied by flux, streaming potential and FTIR-ATR measurements. Critical flux was evaluated by different kinds of stepwise procedures and by both con-stant pressure and constant flux methods. The critical flux was affected by transmembrane pressure, flow velocity, protein concentration, mem-brane hydrophobicity and protein and membrane charges. Generally, the lowest critical fluxes were obtained at the isoelectric points of the protein and the highest in the presence of electrostatic repulsion between the membrane surface and the protein molecules. In the laminar flow regime the critical flux increased with flow velocity, but not any more above this region. An increase in concentration de-creased the critical flux. Hydrophobic membranes showed fouling in all charge conditionsand, furthermore, especially at the beginning of the experiment even at very low transmembrane pressures. Fouling of these membranes was thought to be due to protein adsorption by hydrophobic interactions. The hydrophilic membranes used suffered more from reversible fouling and concentration polarisation than from irreversible foul-ing. They became fouled at higher transmembrane pressures becauseof pore blocking. In this thesis some new aspects on critical flux are presented that are important for ultrafiltration and fractionation of proteins.

ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins

Background: Annotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms. Results: While characterizing genes from the retinitis pigmentosa locus RP26 at 2q31-q33, we have identified a new gene, ORMDL1, that belongs to a novel gene family comprising three genes in humans (ORMDL1, ORMDL2 and ORMDL3), and homologs in yeast, microsporidia, plants, Drosophila, urochordates and vertebrates. The human genes are expressed ubiquitously in adult and fetal tissues. The Drosophila ORMDL homolog is also expressed throughout embryonic and larval stages, particularly in ectodermally derived tissues. The ORMDL genes encode transmembrane proteins anchored in the endoplasmic reticulum (ER). Double knockout of the two Saccharomyces cerevisiae homologs leads to decreased growth rate and greater sensitivity to tunicamycin and dithiothreitol. Yeast mutants can be rescued by human ORMDL homologs. Conclusions: From protein sequence comparisons we have defined a novel gene family, not previously recognized because of the absence of a characterized functional signature. The sequence conservation of this family from yeast to vertebrates, the maintenance of duplicate copies in different lineages, the ubiquitous pattern of expression in human and Drosophila, the partial functional redundancy of the yeast homologs and phenotypic rescue by the human homologs, strongly support functional conservation. Subcellular localization and the response of yeast mutants to specific agents point to the involvement of ORMDL in protein folding in the ER.

Saccharomyces cerevisiae Grx6 and Grx7 are monothiol glutaredoxins associated with the early secretory pathway

Saccharomyces cerevisiae Grx6 and Grx7 are two monothiol glutaredoxins whose active-site sequences (CSYS and CPYS, respectively) are reminiscent of the CPYC active-site sequence of classical dithiol glutaredoxins. Both proteins contain an N-terminal transmembrane domain which is responsible for their association to membranes of the early secretory pathway vesicles, facing the luminal side. Thus, Grx6 localizes at the endoplasmic reticulum and Golgi compartments, while Grx7 is mostly at the Golgi. Expression of GRX6 is modestly upregulated by several stresses (calcium, sodium, and peroxides) in a manner dependent on the Crz1-calcineurin pathway. Some of these stresses also upregulate GRX7 expression under the control of the Msn2/4 transcription factor. The N glycosylation inhibitor tunicamycin induces the expression of both genes along with protein accumulation. Mutants lacking both glutaredoxins display reduced sensitivity to tunicamycin, although the drug is still able to manifest its inhibitory effect on a reporter glycoprotein. Grx6 and Grx7 have measurable oxidoreductase activity in vivo, which is increased in the presence of tunicamycin. Both glutaredoxins could be responsible for the regulation of the sulfhydryl oxidative state at the oxidant conditions of the early secretory pathway vesicles. However, the differences in location and expression responses against stresses suggest that their functions are not totally overlapping.

ErbB Ligands in Angiogenesis

The formation of new blood vessels, i.e. angiogenesis, is an important phenomenon during normal development and wound repair, as well as during various pathological processes, such as tumor growth and metastasis. Specific growth factors regulate angiogenesis by modulating the different cellular functions of endothelial cells (EC), and periendothelial cells, such as pericytes (PC) and smooth muscle cells (SMC), which interact with ECs in a paracrine manner. ErbB receptors form a subgroup of transmembrane receptor tyrosine kinases that interact with growth factors of the epidermal growth factor (EGF) family. ErbB receptors regulate behaviour of a variety of normal as well as tumor cell types. Cancer drugs that target epidermal growth factor receptor (EGFR, ErbB1) or ErbB2 receptor have been approved for clinical use. It has been speculated that part of the antitumor activity of ErbB inhibitor compounds result from an antiangiogenic mechanism. The results presented here indicate a role for endothelial-derived EGF-like growth factors heparin binding EGF-like growth factor (HB-EGF) and neuregulin-1 (NRG-1) in the paracrine regulation of angiogenesis. HB-EGF, EGFR and ErbB2 are shown to mediate interaction between ECs and SMCs in vitro, and gefitinib, an inhibitor of EGFR kinase activity, suppresses recruitment of PCs/SMCs in vivo. NRG-1 is shown to regulate EC functions in vitro and angiogenesis in vivo by indirect mechanisms involving vascular endothelial growth factor-A (VEGF-A) and VEGF receptor-2 (VEGFR-2). Furthermore, EGFR activity is demonstrated to regulate recruitment of bone marrow-derived perivascular cells during tumor neovascularization in vivo. These results indicate that ErbB signaling is involved in the cellular processes of new blood vessel formation. This study gives new information about the role of ErbB ligands and receptors in angiogenesis and vasculogenesis and about the mechanisms by which ErbB inhibitor drugs such as gefitinib affect tumor growth.

Spontaneous Cdc42 polarization independent of GDI-mediated extraction and actin-based trafficking.

The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules.

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties.

The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.

Combined loss of the BH3-only proteins Bim and Bmf restores B-cell development and function in TACI-Ig transgenic mice.

Terminal differentiation of B cells depends on two interconnected survival pathways, elicited by the B-cell receptor (BCR) and the BAFF receptor (BAFF-R), respectively. Loss of either signaling pathway arrests B-cell development. Although BCR-dependent survival depends mainly on the activation of the v-AKT murine thymoma viral oncogene homolog 1 (AKT)/PI3-kinase network, BAFF/BAFF-R-mediated survival engages non-canonical NF-κB signaling as well as MAPK/extracellular-signal regulated kinase and AKT/PI3-kinase modules to allow proper B-cell development. Plasma cell survival, however, is independent of BAFF-R and regulated by APRIL that signals NF-κB activation via alternative receptors, that is, transmembrane activator and CAML interactor (TACI) or B-cell maturation (BCMA). All these complex signaling events are believed to secure survival by increased expression of anti-apoptotic B-cell lymphoma 2 (Bcl2) family proteins in developing and mature B cells. Curiously, how lack of BAFF- or APRIL-mediated signaling triggers B-cell apoptosis remains largely unexplored. Here, we show that two pro-apoptotic members of the 'Bcl2 homology domain 3-only' subgroup of the Bcl2 family, Bcl2 interacting mediator of cell death (Bim) and Bcl2 modifying factor (Bmf), mediate apoptosis in the context of TACI-Ig overexpression that effectively neutralizes BAFF as well as APRIL. Surprisingly, although Bcl2 overexpression triggers B-cell hyperplasia exceeding the one observed in Bim(-/-)Bmf(-/-) mice, Bcl2 transgenic B cells remain susceptible to the effects of TACI-Ig expression in vivo, leading to ameliorated pathology in Vav-Bcl2 transgenic mice. Together, our findings shed new light on the molecular machinery restricting B-cell survival during development, normal homeostasis and under pathological conditions. Our data further suggest that Bcl2 antagonists might improve the potency of BAFF/APRIL-depletion strategies in B-cell-driven pathologies.

The Human Acid-Sensing Ion Channel ASIC1a: Evidence for a Homotetrameric Assembly State at the Cell Surface.

The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here the oligomeric state of the functional ASIC1 in situ at the cell surface. The oligomeric states of functional ASIC1a and mutants with additional cysteines introduced in the extracellular pore vestibule were resolved on SDS-PAGE. The functional ASIC1 complexes were stabilized at the cell surface of Xenopus laevis oocytes or CHO cells either using the sulfhydryl crosslinker BMOE, or sodium tetrathionate (NaTT). Under these different crosslinking conditions ASIC1a migrates as four distinct oligomeric states that correspond by mass to multiples of a single ASIC1a subunit. The relative importance of each of the four ASIC1a oligomers was critically dependent on the availability of cysteines in the transmembrane domain for crosslinking, consistent with the presence of ASIC1a homo-oligomers. The expression of ASIC1a monomers, trimeric or tetrameric concatemeric cDNA constructs resulted in functional channels. The resulting ASIC1a complexes are resolved as a predominant tetramer over the other oligomeric forms, after stabilization with BMOE or NaTT and SDS-PAGE/western blot analysis. Our data identify a major ASIC1a homotetramer at the surface membrane of the cell expressing functional ASIC1a channel.

Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens

Background: Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely understood. Although patients with cystic fibrosis have mutations in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene. Methods: To investigate CBAVD at the molecular level, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clinical manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls. Results: Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 percent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the frequency of this allele in the general population was about 5 percent. Conclusions: Most patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mutation in the other copy is the most common cause of CBAVD. The 5T allele mutation has a wide range of clinical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men.

Vitamin D: a review on its effects on muscle strength, the risk of fall, and frailty.

Vitamin D is the main hormone of bone metabolism. However, the ubiquitary nature of vitamin D receptor (VDR) suggests potential for widespread effects, which has led to new research exploring the effects of vitamin D on a variety of tissues, especially in the skeletal muscle. In vitro studies have shown that the active form of vitamin D, calcitriol, acts in myocytes through genomic effects involving VDR activation in the cell nucleus to drive cellular differentiation and proliferation. A putative transmembrane receptor may be responsible for nongenomic effects leading to rapid influx of calcium within muscle cells. Hypovitaminosis D is consistently associated with decrease in muscle function and performance and increase in disability. On the contrary, vitamin D supplementation has been shown to improve muscle strength and gait in different settings, especially in elderly patients. Despite some controversies in the interpretation of meta-analysis, a reduced risk of falls has been attributed to vitamin D supplementation due to direct effects on muscle cells. Finally, a low vitamin D status is consistently associated with the frail phenotype. This is why many authorities recommend vitamin D supplementation in the frail patient.