Membrane cholesterol content modulates ClC-2 gating and sensitivity to oxidative stress

ClC-2 is a broadly expressed member of the voltage-gated ClC chloride channel family. In this study, we aimed to evaluate the role of the membrane lipid environment in ClC-2 function, and in particular the effect of cholesterol and ClC-2 distribution in membrane microdomains. Detergent-resistant and detergent-soluble microdomains (DSM) were isolated from stably transfected HEK293 cells by a discontinuous OptiPrep gradient. ClC-2 was found concentrated in detergent-insoluble membranes in basal conditions and relocalized to DSM upon cholesterol depletion by methyl-beta-cyclodextrin. As assessed by patch clamp recordings, relocalization was accompanied by acceleration of the activation kinetics of the channel. A similar distribution and activation pattern were obtained when cells were treated with the oxidant tert-butyl hydroperoxide and after ATP depletion. In both cases activation was prevented by cholesterol enrichment of cells. We conclude that the cholesterol environment regulates ClC-2 activity, and we provide evidence that the increase in ClC-2 activity in response to acute oxidative or metabolic stress involves relocalization of this channel to DSM.     

Lipase-catalyzed synthesis of a pure macrocyclic polyester from dimethyl terephthalate and diethylene glycol

The polymerization of dimethyl terephthalate and diethylene glycol by enzymic catalysis in toluene is described. The potential pi-pi interactions of the aromatic rings together with the relative flexibility of the diol segment have been regarded as synergy factors responsible for the formation of a pure cyclic compound. The so-called C2 macrocycle was characterized by (1)H and (13)C NMR, size-exclusion chromatography, differential scanning calorimetry, mass spectrometry, and X-ray diffraction on powder. Structurally speaking, it is made of two repeating units. The substitution of the central oxygen atom of the diol by -CH(2)- or -S- as well as the presence of ortho-substituents (-NH(2) or -NO(2)) on the phthalate were expected to disrupt the stacking of the phenyl groups more or less.     

Conditional Mutants of Meiosis in Yeast

Three temperature-sensitive mutants, spo1-1, spo2-1, and spo3-1, were characterized with respect to their behavior in sporulation medium at a restrictive temperature. The time of expression of the functions defective in the mutants was determined by temperature-shift experiments during the sporulation process. In addition, each mutant was examined for the following: (i) its ability to undergo the nuclear divisions of meiosis; (ii) deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis; (iii) protein turnover; and (iv) colony-forming ability after exposure to sporulation medium. Mutant spo1-1 is defective in a function which confers a temperature-sensitive period which extends over 32% of the sporulation cycle. The temperature-sensitive period of mutant spo2-1 occupies 34% of the cycle, whereas the temperature-sensitive period of mutant spo3-1 extends over 2% of the sporulation cycle. Cytological evidence indicates that all three mutants initiate but do not complete the meiotic nuclear divisions. The DNA content of sporulation cultures of mutants spo1-1 and spo3-1 did not increase to the wild-type level; DNA synthesis in spo2-1 was normal. All three strains exhibit a loss of colony-forming ability during incubation in sporulation medium at the restrictive temperature. RNA and protein synthesis and protein turnover occur in the mutants.     

A minority of 46,XX true hermaphrodites are positive for the Y-DNA sequence including SRY

Summary A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca²⁺-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca²⁺-influx. With purified lumisomes it has been shown that Ca²⁺-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with ²²Na and ⁴²K indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.     

NOV/CCN3 impairs muscle cell commitment and differentiation

NOV (nephroblastoma overexpressed) is a member of a family of proteins which encodes secreted matrix-associated proteins. NOV is expressed during development in dermomyotome and limb buds, but its functions are still poorly defined. In order to understand the role of NOV in myogenic differentiation, C2C12 cells overexpressing NOV (C2-NOV) were generated. These cells failed to engage into myogenic differentiation, whereas they retained the ability to differentiate into osteoblasts. In differentiating conditions, C2-NOV cells remained proliferative, failed to express differentiation markers and lost their ability to form myotubes. Inhibition of differentiation by NOV was also observed with human primary muscle cells. Further examination of C2-NOV cells revealed a strong downregulation of the myogenic determination genes MyoD and Myf5 and of IGF-II expression. MyoD forced expression in C2-NOV was sufficient to restore differentiation and IGF-II induction whereas 10(-6) M insulin treatment had no effects. NOV therefore acts upstream of MyoD and does not affect IGF-II induction and signaling. HES1, a target of Notch, previously proposed to mediate NOV action, was not implicated in the inhibition of differentiation. We propose that NOV is a specific cell fate regulator in the myogenic lineage, acting negatively on key myogenic genes thus controlling the transition from progenitor cells to myoblasts.