Molecular Cloning, Expression Pattern, and Chromosomal Localization of the Human Na–Cl Thiazide-Sensitive Cotransporter (SLC12A3)

Electrolyte homeostasis is maintained by several ion transport systems. Na–(K)–Cl cotransporters promote the electrically silent movement of chloride across the membrane in absorptive and secretory epithelia. Two kidney-specific Na–(K)–Cl cotransporter isoforms are known, so far, according to their sensitivity to specific inhibitors. We have cloned the human cDNA coding for the renal Na–Cl cotransporter selectively inhibited by the thiazide class of diuretic agents. The predicted protein sequence of 1021 amino acids (112 kDa) shows a structure common to the other members of the Na–(K)–Cl cotransporter family: a central region harboring 12 transmembrane domains and the 2 intracellular hydrophilic amino and carboxyl termini. The ex- pression pattern of the human Na–Cl thiazide-sensitive cotransporter (hTSC, HGMW-approved symbol SLC12A3) confirms the kidney specificity. hTSC has been mapped to human chromosome 16q13 by fluorescencein situhybridization. The cloning and characterization of hTSC now render it possible to study the involvement of this cotransport system in the pathogenesis of tubulopathies such as Gitelman syndrome.     

Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications

Multiple system atrophy (MSA) is a fatal rapidly progressive α-synucleinopathy, characterized by α-synuclein accumulation in oligodendrocytes. It is accepted that the pathological α-synuclein accumulation in the brain of MSA patients plays a leading role in the disease process, but little is known about the events in the early stages of the disease. In this study we aimed to define potential roles of the miRNA-mRNA regulatory network in the early pre-motor stages of the disease, i.e., downstream of α-synuclein accumulation in oligodendroglia, as assessed in a transgenic mouse model of MSA. We investigated the expression patterns of miRNAs and their mRNA targets in substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage in the MSA model, by microarray and RNA-seq analysis, respectively. Analysis was performed at a time point when α-synuclein accumulation was already present in oligodendrocytes at neuropathological examination, but no neuronal loss nor deficits of motor function had yet occurred. Our data provide a first evidence for the leading role of gene dysregulation associated with deficits in immune and inflammatory responses in the very early, non-symptomatic disease stages of MSA. While dysfunctional homeostasis and oxidative stress were prominent in SN in the early stages of MSA, in striatum differential gene expression in the non-symptomatic phase was linked to oligodendroglial dysfunction, disturbed protein handling, lipid metabolism, transmembrane transport and altered cell death control, respectively. A large number of putative miRNA-mRNAs interaction partners were identified in relation to the control of these processes in the MSA model. Our results support the role of early changes in the miRNA-mRNA regulatory network in the pathogenesis of MSA preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of MSA.     

Migration of Naphthalene through Low Organic Content Sandy Soil Columns: Comparison of Unsaturated and Saturated Conditions

The transient transport of naphthalene through low organic matter content soil columns was investigated in different water-saturation and flow conditions. Some parameters were tested as flow rate, column height, and water saturation conditions. The soil was a clayed sandy soil from the Algerian coast near Boumerdes. The organic carbon content was 0.13% and the main mineral components were quartz (88%), clays minerals (7%) and calcite (3%). The height of the packing of the soil column (5.1 cm in diameter) varied from 15 to 40 cm. Simultaneous step injections of inert tracer (calcium chloride) and naphthalene at 10 mg L^?1 were performed. Tracer and naphthalene breakthrough curves (BTCs) were measured continuously by conductimetry and UV – 220 nm, respectively. The BTCs were simulated using the classical mixing cells in series with exchange model (MCE). In unsaturated conditions the comparison of the mean residence time of tracer BTCs with the geometrical pore volume gave us access to average water saturation along the column as a function of height. The higher the soil bed was, the higher the mean water saturation. The comparison of naphthalene distribution coefficients (K_d) in different flow conditions with the theoretical value from the Karickhoff law showed that in saturated conditions the obtained value was close to the theoretical one. In unsaturated conditions, the measured naphthalene K_d's were much lower than the theoretical value and correlated to the water saturation.     

Effects of habitat fragmentation on provisioning rates, diet and breeding success in two species of tit (great tit and blue tit)

The aim of this study was to examine the effects of forest fragmentation on the ability of parent birds to provide their young with an adequate food supply. To examine whether prey population densities of the great tit (Parus major L.) and the blue tit (P. caeruleus L.) vary between study areas in different forest size classes we compared provisioning rates and chick diet and related these parameters to breeding success. We filmed 217 nests over two breeding seasons and collected data on frass fall as a general estimate of caterpillar availability. Nests which were attended by none or one parent only during filming (n = 46) were excluded from the analyses. In both years and for both species feeding rates were highest in the smallest fragments and lowest in the large forest. There was also a suggestion that differences in feeding rates between areas vary between years. We found no consistent tendency for prey size to change with forest size, although both species brought slightly smaller prey items to the nest in the smallest forest fragments and feeding rates correlated negatively with prey size. Caterpillars were the main item fed to nestlings, in both species. We found no evidence to suggest that either frass fall or the proportion of caterpillars in the diet varied with forest size. There was also no correlation between mean frass fall and the total number of caterpillars brought to the nests, in either species. Breeding success, as measured by clutch size, brood size, fledging weight and fledging success, did not differ between the small fragments and the large forest, in either species. There was also no relationship between provisioning rate (as concerns volume of prey fed to nestlings and the quality of chick diet) and breeding success parameters. In conclusion, this study does not suggest suboptimal foraging or breeding conditions in small fragments compared to a nearby large forest, for either species. Received: 23 June 1997 / Accepted: 29 December 1997     

Mental retardation and abnormal skeletal development (Dyggve-Melchior-Clausen dysplasia) due to mutations in a novel,evolutionarily conserved gene

Dyggve-Melchior-Clausen dysplasia (DMC) and Smith-McCort dysplasia (SMC) are similar, rare autosomal recessive osteochondrodysplasias. The radiographic features and cartilage histology in DMC and SMC are identical. However, patients with DMC exhibit significant developmental delay and mental retardation, the major features that distinguish the two conditions. Linkage studies localized the SMC and DMC disease genes to chromosome 18q12-21.1, providing evidence suggesting that they are allelic disorders. Sequence analysis of the coding exons of the FLJ90130 gene, a highly evolutionarily conserved gene within the recombination interval defined in the linkage study, identified mutations in SMC and DMC patients. The affected individuals in two consanguinous DMC families were homozygous for a stop codon mutation and a frameshift mutation, respectively, demonstrating that DMC represents the FLJ90130-null phenotype. The data confirm the hypothesis that SMC and DMC are allelic disorders and identify a gene necessary for normal skeletal development and brain function.     

Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice

The Rho GTPase and Fyn tyrosine kinase have been implicated previously in positive control of keratinocyte cell-cell adhesion. Here, we show that Rho and Fyn operate along the same signaling pathway. Endogenous Rho activity increases in differentiating keratinocytes and is required for both Fyn kinase activation and increased tyrosine phosphorylation of beta- and gamma-catenin, which is associated with the establishment of keratinocyte cell-cell adhesion. Conversely, expression of constitutive active Rho is sufficient to promote cell-cell adhesion through a tyrosine kinase- and Fyn-dependent mechanism, trigger Fyn kinase activation, and induce tyrosine phosphorylation of beta- and gamma-catenin and p120ctn. The positive effects of activated Rho on cell-cell adhesion are not induced by an activated Rho mutant with defective binding to the serine/threonine PRK2/PKN kinases. Endogenous PRK2 kinase activity increases with keratinocyte differentiation, and, like activated Rho, increased PRK2 activity promotes keratinocyte cell-cell adhesion and induces tyrosine phosphorylation of beta- and gamma-catenin and Fyn kinase activation. Thus, these findings reveal a novel role of Fyn as a downstream mediator of Rho in control of keratinocyte cell-cell adhesion and implicate the PRK2 kinase, a direct Rho effector, as a link between Rho and Fyn activation.     

Detection of lysozyme-like enzymatic activity secreted by an immune-responsive mosquito cell line

Using molecular approaches, we have recently shown that the C7-10 mosquito cell line from Aedes albopictus, and the Aag-2 line from Aedes aegypti, secrete a variety of immune peptides into the culture medium, including cecropins, defensins, transferrin, and lysozyme. The diversity of these peptides makes it difficult to quantify the relative activities of each molecule, because possible synergistic interactions may occur. Using a microtiter plate assay with live bacteria, we now show that C7-10 cells secrete an activity that is more potent against the Gram-positive bacterium, Micrococcus luteus, than against Gram-negative Escherichia coli. This lysozyme-like activity is accompanied by production of a lytic zone in an agarose plate assay containing commercially available, lyophilized M. luteus. Properties of the lysozyme-like activity from C7-10 cells included a broad pH optimum from 5.5 to 6.5, and heat-sensitivity above 42 degrees C. Amounts of secreted activity increased during the initial 24h of incubation with heat-killed bacteria. During this induction, lysozyme-like activity was found primarily in the cell culture supernatant.     

Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo

The innate immune response, and in particular the alpha/beta interferon (IFN-alpha/beta) system, plays a critical role in the control of viral infections. Interferons alpha and beta exert their antiviral effects through the induction of hundreds of interferon-induced (or -stimulated) genes (ISGs). While several of these ISGs have characterized antiviral functions, their actions alone do not explain all of the effects mediated by IFN-alpha/beta. To identify additional IFN-induced antiviral molecules, we utilized a recombinant chimeric Sindbis virus to express selected ISGs in IFN-alpha/beta receptor (IFN-alpha/betaR)(-/-) mice and looked for attenuation of Sindbis virus infection. Using this approach, we identified a ubiquitin homolog, interferon-stimulated gene 15 (ISG15), as having antiviral activity. ISG15 expression protected against Sindbis virus-induced lethality and decreased Sindbis virus replication in multiple organs without inhibiting the spread of virus throughout the host. We establish that, much like ubiquitin, ISG15 requires its C-terminal LRLRGG motif to form intracellular conjugates. Finally, we demonstrate that ISG15's LRLRGG motif is also required for its antiviral activity. We conclude that ISG15 can be directly antiviral.     

Integrins Influence the Size and Dynamics of Signaling Microclusters in a Pyk2-dependent Manner

Integrin engagement on lymphocytes initiates “outside-in” signaling that is required for cytoskeleton remodeling and the formation of the synaptic interface. However, the mechanism by which the “outside-in” signal contributes to receptor-mediated intracellular signaling that regulates the kinetics of granule delivery and efficiency of cytolytic activity is not well understood. We have found that variations in ICAM-1 expression on tumor cells influence killing kinetics of these cells by CD16.NK-92 cytolytic effectors suggesting that changes in integrin ligation on the effector cells regulate the kinetics of cytolytic activity by the effector cells. To understand how variations of the integrin receptor ligation may alter cytolytic activity of CD16.NK-92 cells, we analyzed molecular events at the contact area of these cells exposed to planar lipid bilayers that display integrin ligands at different densities and activating CD16-specific antibodies. Changes in the extent of integrin ligation on CD16.NK-92 cells at the cell/bilayer interface revealed that the integrin signal influences the size and the dynamics of activating receptor microclusters in a Pyk2-dependent manner. Integrin-mediated changes of the intracellular signaling significantly affected the kinetics of degranulation of CD16.NK-92 cells providing evidence that integrins regulate the rate of target cell destruction in antibody-dependent cell cytotoxicity (ADCC).