A mutation in the STN1 gene triggers an alternative lengthening of telomere-like runaway recombinational telomere elongation and rapid deletion in yeast

Some human cancer cells achieve immortalization by using a recombinational mechanism termed ALT (alternative lengthening of telomeres). A characteristic feature of ALT cells is the presence of extremely long and heterogeneous telomeres. The molecular mechanism triggering and maintaining this pathway is currently unknown. In Kluyveromyces lactis, we have identified a novel allele of the STN1 gene that produces a runaway ALT-like telomeric phenotype by recombination despite the presence of an active telomerase pathway. Additionally, stn1-M1 cells are synthetically lethal in combination with rad52 and display chronic growth and telomere capping defects including extensive 3' single-stranded telomere DNA and highly elevated subtelomere gene conversion. Strikingly, stn1-M1 cells undergo a very high rate of telomere rapid deletion (TRD) upon reintroduction of STN1. Our results suggest that the protein encoded by STN1, which protects the terminal 3' telomere DNA, can regulate both ALT and TRD.     

Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha

The activation function 2/ligand-dependent interaction between nuclear receptors and their coregulators is mediated by a short consensus motif, the so-called nuclear receptor (NR) box. Nuclear receptors exhibit distinct preferences for such motifs depending both on the bound ligand and on the NR box sequence. To better understand the structural basis of motif recognition, we characterized the interaction between estrogen receptor alpha and the NR box regions of the p160 coactivator TIF2. We have determined the crystal structures of complexes between the ligand-binding domain of estrogen receptor alpha and 12-mer peptides from the Box B2 and Box B3 regions of TIF2. Surprisingly, the Box B3 module displays an unexpected binding mode that is distinct from the canonical LXXLL interaction observed in other ligand-binding domain/NR box crystal structures. The peptide is shifted along the coactivator binding site in such a way that the interaction motif becomes LXXYL rather than the classical LXXLL. However, analysis of the binding properties of wild type NR box peptides, as well as mutant peptides designed to probe the Box B3 orientation, suggests that the Box B3 peptide primarily adopts the "classical" LXXLL orientation in solution. These results highlight the potential difficulties in interpretation of protein-protein interactions based on co-crystal structures using short peptide motifs.     

Antibody to herpes simplex virus type 2 as serological marker of sexual lifestyle in populations.

OBJECTIVES--To examine the epidemiology of antibody to herpes simplex virus type 2 and to assess its suitability as a serological marker of sexual behaviour in populations with high and low prevalences. DESIGN--Cross sectional survey. SETTING--Department of genitourinary medicine and blood donation centre in central London. SUBJECTS--Representative sample of 869 patients attending department between November 1990 and December 1991, and 1494 consecutive blood donors attending for donation between February and April 1992. METHOD--Participants had a blood sample taken for antibody testing with a novel type specific assay and completed a questionnaire. RESULTS--Prevalence of antibody differed significantly between the two groups (188/833 (22.7%) clinic attenders; 102/1347 (7.6%) blood donors). In both populations antibody was strongly associated with sex, sexual orientation, years of sexual activity, number of lifetime sexual partners, and past infection with sexually transmitted diseases after other factors were controlled for. Only 130 (45%) of all those with antibody had symptoms suggestive of genital herpes, and 79 (27.4%) had had genital herpes diagnosed. Of those without antibody to herpes simplex viruses type 1 and 2, 8.0% reported genital blisters or sores and 1.1% had had genital herpes diagnosed by a doctor. CONCLUSIONS--The strong relation between herpes simplex virus type 2 and sexual lifestyle suggests that the presence of antibody to the virus may be suitable for use as an objective, serological marker of patterns of sexual behaviour in different populations. These data show that only a minority of those infected with herpes simplex virus type 2 have a diagnosis of genital herpes or express clinical symptoms, making serological determinants of infection essential for epidemiological studies.     

Extrahepatic cells contribute to the progenitor/stem cell response following reduced-size liver transplantation in mice

The extent to which extrahepatic cells participate in liver regeneration following transplantation is not known. Either full-size or reduced-size livers from wild-type mice were implanted into green fluorescent protein-positive (GFP(+)) transgenic recipient mice to determine whether regenerated liver contained host-derived GFP(+) hepatic cells. After reduced-size liver transplantation, GFP(+) cells were localized to the portal zone of the liver lobule. Interestingly, GFP(+) cells stained for CD117, a marker for progenitor cells, beginning 2 days after transplantation. A significant number of GFP(+) CD117(+) cells were identified in donor livers after 28 days. GFP(+) cells comprised nearly 9% of the donor liver 28 days after reduced-size liver transplant. Moreover, GFP(+) cells also expressed the hepatic progenitor cell marker A6 and novel marker hepatic-specific antigen (HSA), as well as stem cell antigen-1 (Sca-1). Interestingly, some GFP(-) cells also were stained for CD117 and A6, suggesting that both extrahepatic and intrahepatic stem cells were present and may have contributed to the regenerative response under these conditions. Reduced-size liver transplantation using GFP(+) transgenic mice supports the hypothesis that recipient-derived progenitor cells are present and may contribute to liver regeneration following transplantation.     

MS-275, a potent orally available inhibitor of histone deacetylases--the development of an anticancer agent

In the last few years it was found that beside genetic aberrations, epigenetic changes also play an important role in tumorigenesis. Acetylation and deacetylation of histones have been found to contribute to a significant extent to epigenetic regulation of gene expression. Analyses of various tumor models and patient samples revealed that the enzyme class of histone deacetylases is associated with many types of cancer and that, for example, over-expression of these enzymes leads to a disturbed balance between acetylation and deacetylation of histones, resulting in differences in the gene expression patterns between normal and cancer cells. Consequently, this class of enzymes has been considered as a potential target for cancer therapy. Numerous inhibitors have been identified and several are in clinical development. Although, with SAHA, one inhibitor has been approved by the FDA for a tumor indication, many open questions remain regarding the mode of action of these inhibitors. In this review, various aspects of preclinical and clinical research of the HDAC inhibitor MS-275 are described, to provide insight into the development of such a compound.     

Mycoplasma infection and hypoxia initiate succinate accumulation and release in the VM-M3 cancer cells

Succinate is known to act as an inflammatory signal in classically activated macrophages through stabilization of HIF-1α leading to IL-1β production. Relevant to this, hypoxia is known to drive succinate accumulation and release into the extracellular milieu. The metabolic alterations associated with succinate release during inflammation and under hypoxia are poorly understood. Data are presented showing that Mycoplasma arginini infection of VM-M3 cancer cells enhances the Warburg effect associated with succinate production in mitochondria and eventual release into the extracellular milieu. We investigated how succinate production and release was related to the changes of other soluble metabolites, including itaconate and 2-HG. Furthermore, we found that hypoxia alone could induce succinate release from the VM-M3 cells and that this could occur in the absence of glucose-driven lactate production. Our results elucidate metabolic pathways responsible for succinate accumulation and release in cancer cells, thus identifying potential targets involved in both inflammation and hypoxia. This article is part of a Special Issue entitled 20th European Bioenergetics Conference, edited by László Zimányi and László Tretter.     

Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K(m) values of approximately 20-30 microM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1'-(13)C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly alpha-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.     

Tenotomy decreases reporter protein synthesis via the 3'-untranslated region of the beta-myosin heavy chain mRNA

We tested thehypothesis that the -myosin heavy chain (-MHC) 3'-untranslatedregion (UTR) mediates decreased protein expression after tenotomy ofthe rat soleus. We also tested the hypothesis that decreased proteinexpression is the result of RNA-protein interactions within the 3'-UTR.-MHC was chosen for study because of its critical role in thefunction of postural muscles such as soleus. Adult rat soleus muscleswere directly injected with luciferase (LUC) reporter constructscontaining either the -MHC or SV40 3'-UTR. After 48 h oftenotomy, there was no significant effect on LUC expression in the SV403'-UTR group. In the -MHC 3'-UTR group, LUC expression was 37.3 ± 4% (n = 5, P = 0.03) of that in shamcontrols. Gel mobility shift assays showed that a protein factorspecifically interacts with the -MHC 3'-UTR and that tenotomysignificantly increases the level of this interaction (25 ± 7%,n = 5, P = 0.02). Thus the -MHC3'-UTR is directly involved in decreased protein expression that isprobably due to increased RNA-protein binding within the UTR.