UCP2 is a mitochondrial transporter with an unusual very short half-life

This study focused on the stability of UCP2 (uncoupling protein 2), a mitochondrial carrier located in the inner membrane of mitochondrion. UCP2 is very unstable, with a half-life close to 30min, compared to 30h for its homologue UCP1, a difference that may highlight different physiological functions. Heat production by UCP1 in brown adipocytes is generally a long and adaptive phenomenon, whereas control of mitochondrial ROS by UCP2 needs more subtle regulation. We show that a mutation in UCP2 shown to modify its activity, actually decreases its stability.     

Reverse genetic analysis of the yeast RSC chromatin remodeler reveals a role for RSC3 and SNF5 homolog 1 in ploidy maintenance

The yeast “remodels the structure of chromatin” (RSC) complex is a multi-subunit “switching deficient/sucrose non-fermenting” type ATP-dependent nucleosome remodeler, with human counterparts that are well-established tumor suppressors. Using temperature-inducible degron fusions of all the essential RSC subunits, we set out to map RSC requirement as a function of the mitotic cell cycle. We found that RSC executes essential functions during G1, G2, and mitosis. Remarkably, we observed a doubling of chromosome complements when degron alleles of the RSC subunit SFH1, the yeast hSNF5 tumor suppressor ortholog, and RSC3 were combined. The requirement for simultaneous deregulation of SFH1 and RSC3 to induce these ploidy shifts was eliminated by knockout of the S-phase cyclin CLB5 and by transient depletion of replication origin licensing factor Cdc6p. Further, combination of the degron alleles of SFH1 and RSC3, with deletion alleles of each of the nine Cdc28/Cdk1-associated cyclins, revealed a strong and specific genetic interaction between the S-phase cyclin genes CLB5 and RSC3, indicating a role for Rsc3p in proper S-phase regulation. Taken together, our results implicate RSC in regulation of the G1/S-phase transition and establish a hitherto unanticipated role for RSC-mediated chromatin remodeling in ploidy maintenance.     

Lost and found testes: the importance of the hCG stimulation test and other testicular markers to confirm a surgical declaration of anorchia

BACKGROUND: In patients with impalpable testes,laparoscopy or open surgery is considered conclusive in establishing the absence of testicular tissue. METHODS: Retrospective chart review. RESULTS: Over a 22-year period, 4 out of 82 patients with a diagnosis of bilateral anorchia by laparoscopy or laparotomy had persistent testicular tissue suggested by endocrine evaluations. The clue to the presence of testicular tissue was: (1) a pubertal rise in plasma testosterone (2 patients); (2) the presence of possible Müllerian structures and of a detectable plasma anti-Müllerian hormone (1 patient), and (3) the fact that one of the gonads had not been seen at surgery (1 patient who still had a testosterone response to hCG postoperatively). Testes were localized by venography (3 patients) and laparotomy (1 patient). CONCLUSION: A surgical diagnosis of bilateral anorchia needs to be confirmed by hCG stimulation, gonadotropin levels, or other markers of testicular function.     

Hormones, IgG and lactose changes around parturition in plasma, and colostrum or saliva of multiparous sows

Blood, colostrum and saliva samples were serially taken from 6 multiparous sows from day 109 of gestation until day 3 postpartum. Plasma was assayed for oestradiol-17beta (E2), progesterone (P4), prolactin (PRL), cortisol, immunoglobulin G (IgG) and lactose. Colostrum was assayed for E2, P4, IgG and lactose. Lactoserum, obtained after ultra centrifugation of colostrum, was assayed for PRL. Saliva was assayed for cortisol. Time-related variations in hormone, IgG and lactose concentrations measured in plasma were parallel to those measured in colostrum, lactoserum or saliva. However, the concentrations were higher in colostrum or lactoserum and lower in saliva than in plasma. Ratios of concentrations of cortisol in saliva and PRL in lactoserum over those in plasma did not vary with time and averaged 0.2 and 1.6, respectively. Conversely, the ratios of concentrations of E2 and P4 in colostrum over those in plasma varied with time (P < 0.05) but were quite constant before the end of parturition, averaging 2.7 and 3.6, respectively. The ratios of concentrations of IgG and lactose in colostrum over those in plasma also varied with time (P < 0.05). The concentrations of hormones in plasma on the one hand and in colostrum, lactoserum or saliva on the other hand were significantly correlated but correlations varied with time (PRL across periods: r = 0.31; cortisol across periods: r = 0.60; E2 during parturition: r = 0.83; P4 before parturition: r = 0.82; P4 during parturition: r = 0.67). The present results indicate that around parturition, assays of hormones in colostrum or saliva can be used to study the hormonal status of sows. Furthermore, variations in colostrum and plasma concentrations of IgG and lactose are good indicators of the transition from colostrum to milk synthesis.     

Characterization of components of the mismatch repair machinery in Trypanosoma brucei

Mismatch repair is one of a number of DNA repair pathways that cells possess to deal with damage to their genome. Mismatch repair is concerned with the recognition and correction of incorrectly paired bases, which can be base-base mismatches or insertions or deletions of a few bases, and appears to have been conserved throughout evolution. Primarily, this is concerned with increasing the fidelity of DNA replication, but also has important roles in the regulation of homologous recombination and the correction of chemical damage. In this study, we describe five genes in the protistan parasite Trypanosoma brucei that are likely to be involved in nuclear mismatch repair. The predicted T. brucei mismatch repair genes are diverged compared with their likely counterparts in the other eukaryotes examined to date. To demonstrate that these do indeed encode a functional nuclear mismatch repair system, we made T. brucei null mutants in two of the genes, MSH2 and MLH1, that are likely to be central to the functioning of the mismatch repair machinery. These mutations resulted in increased rates of sequence variation at a number of microsatellite loci in the parasite genome, and led to increased tolerance to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, both phenotypes consistent with mismatch repair impairment.     

Characterization of a non-mitochondrial type I phosphatidylserine decarboxylase in Plasmodium falciparum

In search of key enzymes in Plasmodium phospholipid metabolism, we demonstrate the presence of a parasite-encoded phosphatidylserine decarboxylase (PSD) in the membrane fraction of Plasmodium falciparum-infected erythrocytes. PSD cDNA, encoding phosphatidylserine decarboxylase (PfPSD), was cloned by screening a directional cDNA library derived from the trophozoite erythrocytic stage. The corresponding PfPSD gene is located on chromosome 9 of P. falciparum, contains one intron of 938 nucleotides and is transcribed into a 3.7 kb mRNA. PfPSD cDNA encodes a putative protein of 362 amino acids, with a predicted molecular mass of 42.6 kDa, which clearly belongs to the type I PSD family. Only a 35 kDa polypeptide was detected in the parasite using a specific rabbit antiserum. PfPSD has a 314VGSS317 sequence near its carboxyl-terminus that is related to the Escherichia coli, yeast and human LGST motif, which is the site of proenzyme processing. PSD enzyme was expressed in E. coli with a KM of 63 +/- 19 microM and a VMAX of 680 +/- 49 nmol of phosphatidylethanolamine formed h-1 mg-1 protein. Site-directed mutagenesis of the VGSS active site demonstrated that the PfPSD proenzyme was processed into two non-identical subunits (alpha and beta) and revealed the crucial role played by each residue in enzyme processing and activity. Using indirect immunofluorescence, PfPSD labelling was co-localized with an endoplasmic reticulum marker, but not with a mitochondrial vital dye. This P. falciparum PSD is the first type I PSD identified in the endoplasmic reticulum compartment.     

Discrimination between lumenal and cytosolic sites of deglycosylation in endoplasmic reticulum-associated degradation of glycoproteins by using benzyl mannose in CHO cell lines

Recent studies demonstrated that deglycosylation step is a prerequisite for endoplasmic reticulum (ER)-associated degradation of misfolded glycoproteins. Here, we report the advantages of using benzyl mannose during pulse-chase experiments to study the subcellular location of the deglycosylation step in Chinese hamster ovary (CHO) cell lines. Benzyl mannose inhibited both the ER-to-cytosol transport of oligomannosides and the trimming of cytosolic-labeled oligomannosides by the cytosolic mannosidase in vivo. We pointed out the occurrence of two subcellular sites of deglycosylation. The first one is located in the ER lumen, and led to the formation of Man8GlcNAc2 (isomer B) in wild-type CHO cell line and Man4GlcNAc2 in Man-P-Dol-deficient cell line. The second one was revealed in CHO mutant cell lines for which a high rate of glycoprotein degradation was required. It occurred in the cytosol and led to the liberation of oligosaccharides species with one GlcNAc residue and with a pattern similar to the one bound onto glycoproteins. The cytosolic deglycosylation site was not specific for CHO mutant cell lines, since we demonstrated the occurrence of cytosolic pathway when the formation of truncated glycans was induced in wild-type cells.     

The tissue-specific distribution of 3H-seocalcitol (EB 1089) and 3H-calcitriol in rats

Seocalcitol (EB 1089) is under development for the treatment of hepato-cellular carcinoma (HCC). The tissue distribution of 3H-seocalcitol was investigated in comparison to 3H-calcitriol in rats. Quantitative whole-body autoradiography was used to quantify the tissue distribution. The greatest difference in distribution between the two compounds was observed in the bloodstream. For most tissues the ratio seocalcitol/calcitriol varied between 0.2 and 3.1. The concentration of radioactivity in the liver was almost the same for the two compounds. For seocalcitol the concentration in the liver was 10 times higher than in serum. Assuming that the liver/serum concentration ratio is the same in rats and humans, the concentration of seocalcitol in the human liver is expected to be higher than the concentration resulting in more than 50% inhibition of cancer cell proliferation, and thus pharmacologically effective in HCC. It is questionable whether calcitriol would be present in the human liver in sufficient concentrations to be effective for the treatment of HCC, as the antiproliferative activity of calcitriol is generally more than 10-fold lower compared to that of seocalcitol and as calcitriol can only be administered at a dose that is ca. three-fold lower than the dose of seocalcitol.     

CO2- and anaerobiosis-induced changes in physiology and gene expression of different Listeria monocytogenes strains

Although carbon dioxide (CO(2)) is known to inhibit growth of most bacteria, very little is known about the cellular response. The food-borne pathogen Listeria monocytogenes is characterized by its ability to grow in high CO(2) concentrations at refrigeration temperatures. We examined the listerial responses of different strains to growth in air, 100% N(2), and 100% CO(2). The CO(2)-induced changes in membrane lipid fatty acid composition and expression of selected genes were strain dependent. The acid-tolerant L. monocytogenes LO28 responded in the same manner to CO(2) as to other anaerobic, slightly acidic environments (100% N(2), pH 5.7). An increase in the expression of the genes encoding glutamate decarboxylase (essential for survival in strong acid) as well as an increased amount of branched-chain fatty acids in the membrane was observed in both atmospheres. In contrast, the acid-sensitive L. monocytogenes strain EGD responded differently to CO(2) and N(2) at the same pH. In a separate experiment with L. monocytogenes 412, an increased isocitrate dehydrogenase activity level was observed for cells grown in CO(2)-containing atmospheres. Together, our findings demonstrate that the CO(2)-response is a partly strain-dependent complex mechanism. The possible links between the CO(2)-dependent changes in isocitrate dehydrogenase activity, glutamate metabolism and branched fatty acid biosynthesis are discussed.     

New bicyclam-GalCer analogue conjugates: synthesis and in vitro anti-HIV activity

The synthesis of bipharmacophore anti-HIV compounds which, in a single molecule, combine two ligands, that is, the bicyclam AMD3100 and a GalCer analogue, that might inhibit several steps of the complex virus/cell cascade interactions has been performed. The 'double-drug' Gal-AMD3100 conjugates elicited inhibitory effects on T (or X4)-tropic HIV-1 replication in all CXCR4 expressing cell lines with EC(50) values ranging from 0.25 to 6.0 microM which were however approximately 40- to 125-fold lower than that of AMD3100. Concerning the mechanism of inhibition of the Gal-AMD3100 conjugates, experiments performed with X4 or R5HIV-1 strains and GHOST cells genetically modified to express CD4 and CXCR4 or CCR5 indicated clearly that the conjugates interact with CXCR4 and not with CCR5.