The Implication of Early Chromatin Changes in X Chromosome Inactivation

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ICOS Gene Polymorphisms (IVS1 + 173 T/C and c. 1624 C/T) in Primary Sjögren’s Syndrome Patients: Analysis of ICOS Expression

Background: Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease, which affects exocrine glands. T cell activation is a trigger mechanism in the immune response. Hyperreactivity of T cells and antibody production are features in pSS. ICOS can be critical in the pathogenesis of pSS. Methods: A total of 134 pSS patients and 134 control subjects (CS) were included. Genotyping was performed by PCR-RFLP. ICOS mRNA expression was quantified by real-time PCR, and CD4+ ICOS+ T cells were determined by flow cytometry. Results: The ICOS IVS1 + 173 T>C polymorphisms were not associated with susceptibility to pSS (p = 0.393, CI = 0.503–1.311). However, the c.1624 C>T polymorphism was associated with a reduction in the risk of development of pSS (p = 0.015, CI = 0.294–0.884). An increase in ICOS mRNA expression in patients was observed (3.7-fold). Furthermore, pSS patients showed an increase in membranal-ICOS expression (mICOS). High expression of mICOS (MFI) was associated with lymphocytic infiltration. Conclusions: The IVS1 + 173 polymorphism is not a genetic marker for the development of pSS, while c.1624 T allele was associated with a low risk. However, elevated mICOS expression in pSS patients with high lymphocytic infiltration was found. ICOS may have an important role in the immunopathogenesis of pSS and should be analyzed in T cell subsets in pSS patients as a possible disease marker.     

The mitochondrial dicarboxylate carrier is essential for the growth of Saccharomyces cerevisiae on ethanol or acetate as the sole carbon source

The dicarboxylate carrier (DIC) is an integral membrane protein that catalyses a dicarboxylate-phosphate exchange across the inner mitochondrial membrane. We generated a yeast mutant lacking the gene for the DIC. The deletion mutant failed to grow on acetate or ethanol as sole carbon source but was viable on glucose, galactose, pyruvate, lactate and glycerol. The growth on ethanol or acetate was largely restored by the addition of low concentrations of aspartate, glutamate, fumarate, citrate, oxoglutarate, oxaloacetate and glucose, but not of succinate, leucine and lysine. The expression of the DIC gene in wild-type yeast was repressed in media containing ethanol or acetate with or without glycerol. These results indicate that the primary function of DIC is to transport cytoplasmic dicarboxylates into the mitochondrial matrix rather than to direct carbon flux to gluconeogenesis by exporting malate from the mitochondria. The delta DIC mutant may serve as a convenient host for overexpression of DIC and for the demonstration of its correct targeting and assembly.     

Role for the Rac1 exchange factor Vav in the signaling pathways leading to NK cell cytotoxicity

Here we investigate the activation of and a possible role for the hematopoietic Rac1 exchange factor, Vav, in the signaling mechanisms leading to NK cell-mediated cytotoxicity. Our data show that direct contact of NK cells with a panel of sensitive tumor targets leads to a rapid and transient tyrosine phosphorylation of Vav and to its association with tyrosine-phosphorylated Syk. Vav tyrosine phosphorylation is also observed following the activation of NK cells through the low-affinity Fc receptor for IgG (Fc gamma RIII). In addition, we demonstrate that both direct and Ab-mediated NK cell binding to target cells result in the activation of nucleotide exchange on endogenous Rac1. Furthermore, Vav antisense oligodeoxynucleotide treatment leads to an impairment of NK cytotoxicity, with Fc gamma RIII-mediated killing being more sensitive to the abrogation of Vav expression. These results provide new insight into the signaling pathways leading to cytotoxic effector function and define a role for Vav in the activation of NK cell-mediated killing.     

Inhibition of Na+-K+-2Cl- cotransport by mercury

Mercury alters thefunction of proteins by reacting with cysteinyl sulfhydryl(SH) groups. Theinorganic form (Hg²⁺) is toxicto epithelial tissues and interacts with various transport proteinsincluding the Na⁺ pump andCl channels. In this study,we determined whether theNa⁺-K⁺-Clcotransporter type 1 (NKCC1), a major ion pathway in secretory tissues,is also affected by mercurial substrates. To characterize theinteraction, we measured the effect ofHg²⁺ on ion transport by thesecretory shark and human cotransporters expressed in HEK-293 cells.Our studies show that Hg²⁺inhibitsNa⁺-K⁺-Clcotransport, with inhibitor constant(K_i) values of25 µM for the shark carrier (sNKCC1) and 43 µM for thehuman carrier. In further studies, we took advantage of speciesdifferences in Hg²⁺ affinity toidentify residues involved in the interaction. An analysis ofhuman-shark chimeras and of an sNKCC1 mutant(Cys-697Leu) reveals that transmembrane domain 11 plays an essential role in Hg²⁺binding. We also show that modification of additionalSH groups by thiol-reactingcompounds brings about inhibition and that the binding sites are notexposed on the extracellular face of the membrane.

     

Amphibians as a model system for the investigation of respiratory control development

Recent medical advances have made it possible for babies to survive premature birth at increasingly earlier developmental stages. This population requires costly and sophisticated medical care to address the problems associated with immaturity of the respiratory system. In addition to pulmonary complications, respiratory instability and apnea reflecting immaturity of the respiratory control system are major causes of hospitalization and morbidity in this highly vulnerable population. These medical concerns, combined with the curiosity of physiologists, have contributed to the expansion of research in respiratory neurobiology. While most researchers working in this field commonly use rodents as an animal model, recent research using in vitro brainstem preparation from bullfrogs (Rana catesbeiana) have revealed the technical advantages of this animal model, and shown that the basic principles underlying respiratory control and its ontogeny are very similar between these two groups of vertebrates. The present review highlights the recent advances in the area of research with a focus on intermittent (episodic) breathing and the role of serotonergic and GABAergic modulation of respiratory activity during development.     

Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.