Degradation of Cdt1 during S phase is Skp2-independent and is required for efficient progression of mammalian cells through S phase

Previous reports have shown that the N terminus of Cdt1 is required for its degradation during S phase (Li, X., Zhao, Q., Liao, R., Sun, P., and Wu, X. (2003) J. Biol. Chem. 278, 30854-30858; Nishitani, H., Lygerou, Z., and Nishimoto, T. (2004) J. Biol. Chem. 279, 30807-30816). The stabilization was attributed to deletion of the cyclin binding motif (Cy motif), which is required for its phosphorylation by cyclin-dependent kinases. Phosphorylated Cdt1 is subsequently recognized by the F-box protein Skp2 and targeted for proteasomal mediated degradation. Using phosphopeptide mapping and mutagenesis studies, we found that threonine 29 within the N terminus of Cdt1 is phosphorylated by Cdk2 and required for interaction with Skp2. However, threonine 29 and the Cy motif are not necessary for proteolysis of Cdt1 during S phase. Mutants of Cdt1 that do not stably associate with Skp2 or cyclins are still degraded in S phase to the same extent as wild type Cdt1, indicating that other determinants within the N terminus of Cdt1 are required for degrading Cdt1. We localized the region necessary for Cdt1 degradation to the first 32 residues. Overexpression of stable forms of Cdt1 significantly delayed entry into and completion of S phase, suggesting that failure to degrade Cdt1 prevents normal progression through S phase. In contrast, Cdt1 mutants that fail to interact with Skp2 and cyclins progress through S phase with similar kinetics as wild type Cdt1 but stimulate the re-replication caused by overexpressing Cdt1. Therefore, a Skp2-independent pathway that requires the N-terminal 32 residues of Cdt1 is critical for the degradation of Cdt1 in S phase, and this degradation is necessary for the optimum progression of cells through S phase.     

Analysis of the molecular variation between and within cultivated and wild Pistacia species using AFLPs

Knowledge of pistachio genetic diversity is necessary for the formulation of appropriate management strategies for the conservation of these species. We analysed amplified fragment length polymorphisms in a total of 216 pistachio accessions, which included seven populations from three wild species (Pistacia vera, Pistacia khinjuk and Pistacia atlantica subsp. kurdica) and most of the important cultivars from Iran, together with some foreign cultivars. High levels of genetic diversity were detected within the Iranian cultivars, and they showed a clear separation from foreign cultivars, as revealed by unweighted pair group method with arithmetic averaging and supported by analysis of molecular variance. The lowest amount of polymorphism was observed in P. atlantica subsp. kurdica, which showed the lowest number of total bands as compared to the other species. This revealed strong genetic erosion of P. atlantica subsp. kurdica, which reflected a severe decline in habitat and over-exploitation. Based on these findings, strategies are proposed for the genetic conservation and management of pistachio species and cultivars.     

Variation of DAT1 VNTR alleles and genotypes among old ethnic groups in Mesopotamia to the Oxus region

Variation of a VNTR in the DAT1 gene in seven ethnic groups of the Middle East was used to infer the history and affinities of these groups. The populations consisted of Assyrian, Jewish, Zoroastrian, Armenian, Turkmen, and Arab peoples of Iran, Iraq, and Kuwait. Three hundred forty subjects from these seven ethnic groups were screened for DAT1. DAT1 VNTR genotyping showed 3, 6, 7, 8, 9, 10, 11, and 12 alleles in the samples. Analysis of these data revealed differentiation and relationship among the populations. In this region, which covers an area of 2-2.5 million km2, the influence of geography and especially of linguistic characteristics has had potentially major effects on differentiation. Religion also has played a major role in imposing restrictions on some ethnic groups, who as a consequence have maintained their community. Overall, these ethnic groups showed greater heterogeneity compared to other populations.     

Ablation of Promyelocytic Leukemia Protein (PML) Re-patterns Energy Balance and Protects Mice from Obesity Induced by a Western Diet

The promyelocytic leukemia protein is a well known tumor suppressor, but its role in metabolism is largely unknown. Mice with a deletion in the gene for PML (KO mice) exhibit altered gene expression in liver, adipose tissue, and skeletal muscle, an accelerated rate of fatty acid metabolism, abnormal glucose metabolism, constitutive AMP-activating kinase (AMPK) activation, and insulin resistance in skeletal muscle. Last, an increased rate of energy expenditure protects PML KO mice from the effects of obesity induced by a Western diet. Collectively, our study uncovers a previously unappreciated role of PML in the regulation of metabolism and energy balance in mice.     

KIAA0101 interacts with BRCA1 and regulates centrosome number

To find genes and proteins that collaborate with BRCA1 or BRCA2 in the pathogenesis of breast cancer, we used an informatics approach and found a candidate BRCA interactor, KIAA0101, to function like BRCA1 in exerting a powerful control over centrosome number. The effect of KIAA0101 on centrosomes is likely direct, as its depletion does not affect the cell cycle, KIAA0101 localizes to regions coincident with the centrosomes, and KIAA0101 binds to BRCA1. We analyzed whether KIAA0101 protein is overexpressed in breast cancer tumor samples in tissue microarrays, and we found that overexpression of KIAA0101 correlated with positive Ki67 staining, a biomarker associated with increased disease severity. Furthermore, overexpression of the KIAA0101 gene in breast tumors was found to be associated with significantly decreased survival time. This study identifies KIAA0101 as a protein important for breast tumorigenesis, and as this factor has been reported as a UV repair factor, it may link the UV damage response to centrosome control.     

Identifying the membrane proteome of HIV-1 latently infected cells

Profiling integral plasma membrane proteins is of particular importance for the identification of new biomarkers for diagnosis and for drug development. We report in this study the identification of surface markers by performing comparative proteomics of established human immunodeficiency virus-1 (HIV-1) latent cell models and parental cell lines. To this end we isolated integral membrane proteins using a biotin-directed affinity purification method. Isolated proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) after in gel digestion. Seventeen different proteins were found to vary on the surface of T-cells due to HIV-1 infection. Of these proteins, 47% were integral membrane proteins, and 18% were membrane-associated. Through the use of complementary techniques such as Western blotting and fluorescent staining, we confirmed the differential expression of some of the proteins identified by MALDI-TOF including Bruton's tyrosine kinase and X-linked inhibitor of apoptosis. Finally, using phosphatidylinositol 3-kinase inhibitors and flavopiridol to inhibit Bruton's tyrosine kinase localization at the membrane and X-linked inhibitor of apoptosis protein expression, respectively, we showed that HIV-1 latently infected cells are more sensitive to these drugs than uninfected cells. This suggests that HIV-1 latently infected cells may be targeted with drugs that alter several pathways that are essential for the establishment and maintenance of latency.     

Involvement of vesicle-cytoskeleton interaction in AQP5 trafficking in AQP5-gene-transfected HSG cells

We examined the regulatory role of a reduction/oxidation (redox) control protein, thioredoxin (TRX), in tumor necrosis factor-alpha (TNF-alpha)-induced p38 MAP kinase activation and p38 MAP kinase-mediated cytokine expression utilizing TRX-transfected murine L929 cells (TRX14). The results showed that TNF-alpha-induced p38 MAP kinase activation and interleukin-6 (IL-6) production by TRX 14 were less than those by the parental L cells and the control transfected L cells (Neo-1). SB 203580 as the specific inhibitor for p38 MAP kinase activity inhibited TNF-alpha-induced IL-6 production by the parental L cells, indicating that TNF-alpha-activated p38 MAP kinase regulates IL-6 production by the cell lines used in this study. These results showed that overexpression of TRX negatively regulates p38 MAP kinase activation and p38 MAP kinase-mediated IL-6 production by TNF-alpha-stimulated cells, indicating that TRX is critical for p38 MAP kinase activation which regulates cytokine expression.     

Vibrio cholerae O139 persists in Dhaka,Bangladesh since 1993

BackgroundAfter a multi-country Asian outbreak of cholera due to Vibrio cholerae serogroup O139 which started in 1992, it is rarely detected from any country in Asia and has not been detected from patients in Africa.Methodology/Principal findingsWe extracted surveillance data from the Dhaka and Matlab Hospitals of International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) to review trends in isolation of Vibrio cholerae O139 in Bangladesh. Data from the Dhaka Hospital is a 2% sample of > 100,000 diarrhoeal patients treated annually. Data from the Matlab Hospital includes all diarrhoeal patients who hail from the villages included in the Matlab Health and Demographic Surveillance System. Vibrio cholerae O139 was first isolated in Dhaka in 1993 and had been isolated every year since then except for a gap between 2005 and 2008. An average of thirteen isolates was detected annually from the Dhaka Hospital during the last ten years, yielding an estimated 650 cases annually at this hospital. During the last ten years, cases due to serogroup O139 represented 0.47% of all cholera cases; the others being due to serogroup O1. No cases with serogroup O139 were identified at Matlab since 2006. Clinical signs and symptoms of cholera due to serogroup O139 were similar to cases due to serogroup O1 though more of the O139 cases were not dehydrated. Most isolates of O139 remained sensitive to tetracycline, ciprofloxacin, and azithromycin, but they became resistant to erythromycin starting in 2009.Conclusions/SignificanceCholera due to Vibrio cholerae serogroup O139 continues to cause typical cholera in Dhaka, Bangladesh.     

Investigation of tRNA<Superscript>Leu/Lys</Superscript> and ATPase 6 Genes Mutations in Huntington’s Disease

Huntington disease (HD) is a genetically dominant condition caused by expanded CAG repeats which code for glutamine in the HD gene product, huntingtin. Huntingtin is expressed in almost all tissues, so abnormalities outside the brain can also be expected. Involvement of nuclei and mitochondria in HD pathophysiology has been suggested. In fact mitochondrial dysfunction is reported in brains of patients suffering from HD. The tRNA gene mutations are one of hot spots that can cause mitochondrial disorders. In this study, possible mitochondrial DNA (mtDNA) damage was evaluated by screening for mutations in the tRNA^leu/lys and ATPase 6 genes of 20 patients with HD, using PCR and automated DNA sequencing. Mutations including an A8656G mutation in one patient were observed, which may be causal to the disease. Understanding the role of mitochondria in the pathogenesis of neurodegenerative diseases could potentially be important for the development of therapeutic strategies in HD.