Helicase Hmi1 stimulates the synthesis of concatemeric mitochondrial DNA molecules in yeast Saccharomyces cerevisiae

Hmi1p is a helicase in the yeast Saccharomyces cerevisiae required for maintenance of the wild-type mitochondrial genome. Disruption of the HMI1 ORF generates ^– and ⁰ cells. Here we demonstrate that, in ^– yeast strains, Hmi1p stimulates the synthesis of long concatemeric mitochondrial DNA molecules associated with a reduction in the number of nucleoids used for mitochondrial DNA packaging. Surprisingly, the ATPase negative mutants of Hmi1p can also stimulate the synthesis of long concatemeric ^– mitochondrial DNA molecules and support the maintenance of the wild-type mitochondrial genome, albeit with reduced efficiency. We show that, in the mutant hmi1–5 background, the wild-type mitochondrial DNA is fragmented; and we propose that, in hmi1 yeast cells, the loss of the wild-type mitochondrial genome is caused by this fragmentation of the mitochondrial DNA.     

A novel metalloprotease from Vipera lebetina venom induces human endothelial cell apoptosis.

A novel endothelial cell apoptosis inducing metalloprotease (VLAIP) was found in the snake venom of Vipera lebetina. This metalloprotease is a heterodimeric glycoprotein with molecular mass of about 106 kDa. The protease hydrolyzes azocasein, fibrinogen and oxidized insulin B-chain. The enzyme readily hydrolyzes the Aalpha-chain and more slowly Bbeta-chain of fibrinogen. VLAIP does not cleave fibrin. The complete amino acid sequences of the two different monomers of VLAIP are deduced from the nucleotide sequences of cDNAs encoding these proteins. The full-length cDNA sequences of the VLAIP-A and VLAIP-B encode open reading frames of 616 and 614 amino acids that include signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. VLAIP belongs to the metalloprotease/disintegrin family of reprolysins and has high identity with the proteins that induce apoptosis of endothelial cells. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes cell death. We demonstrated that VLAIP inhibits endothelial cell adhesion to extracellular matrix proteins: fibrinogen, fibronectin, vitronectin, collagen I, and collagen IV. The induction of apoptosis by VLAIP was shown by means of a typical DNA fragmentation pattern of apoptotic cells as well as by monitoring phosphatidylserine externalization using annexin V-FITC staining and flow cytometric analysis.     

Recombinant CD44-HABD is a novel and potent direct angiogenesis inhibitor enforcing endothelial cell-specific growth inhibition independently of hyaluronic acid binding

CD44 is the main cellular receptor for hyaluronic acid (HA). We previously found that overexpression of CD44 inhibited tumor growth of mouse fibrosarcoma cells in mice. Here, we show that soluble recombinant CD44 HA-binding domain (CD44-HABD) acts directly onto endothelial cells by inhibiting endothelial cell proliferation in a cell-specific manner. Consequently, soluble recombinant CD44-HABD also blocked angiogenesis in vivo in chick and mouse, and thereby inhibited tumor growth of various origins at very low doses (0.25 mg/kg x day). The antiangiogenic effect of CD44 is independent of its HA-binding capacity, since mutants deficient in HA binding still maintain their antiangiogenic and antiproliferative properties. Recombinant CD44-HABD represents a novel class of angiogenesis inhibitors based on a cell-surface receptor.     

The tumor suppressor CYLD interacts with TRIP and regulates negatively nuclear factor kappaB activation by tumor necrosis factor

Cylindromas are benign adnexal skin tumors caused by germline mutations in the CYLD gene. In most cases the second wild-type allele is lost in tumor tissue, suggesting that CYLD functions as tumor suppressor. CYLD is a protein of 956 amino acids harboring a functional deubiquitinating domain at the COOH-terminal end. To shed more light on the function of CYLD, we have performed a yeast two hybrid screen using an HaCaT cDNA library that identified the RING finger protein TRIP (TRAF-interacting protein) as interactor with full-length CYLD. Mapping of the interacting domains revealed that the central domain of CYLD binds to the COOH-terminal end of TRIP. Far Western analysis and coimmunoprecipitations in mammalian cells confirmed that full-length CYLD binds to the COOH-terminal domain of TRIP. Because TRIP is an inhibitor of nuclear factor (NF)-kappaB activation by tumor necrosis factor (TNF), the effect of CYLD on NF-kappaB activation was investigated in HeLa cells. The results established that CYLD down-regulates NF-kappaB activation by TNF-alpha. The inhibition by CYLD depends on the presence of the central domain interacting with TRIP and its deubiquitinating activity. These findings indicate that cylindromas arise through constitutive NF-kappaB activation leading to hyperproliferation and tumor growth.     

Screening of 20 patients with X-linked mental retardation using chromosome X-specific array-MAPH

The rapid advancement of high-resolution DNA copy number assessment methods revealed the significant contribution of submicroscopic genetic imbalances to abnormal phenotypes, including mental retardation. In order to detect submicroscopic genetic imbalances, we have screened 20 families with X-linked mental retardation (XLMR) using a chromosome X-specific array-MAPH platform with median resolution of 238 kb. Among the 20 families, 18 were experimental, as they were not previously screened with any microarray method, and two were blind controls with known aberrations, as they were previously screened by array-CGH. This study presents the first clinical application of chromosome X-specific array-MAPH methodology. The screening of 20 affected males from 20 unrelated XLMR families resulted in the detection of an unknown deletion, spanning a region of 7–23 kb. Family studies and population screening demonstrated that the detected deletion is an unknown rare copy number variant. One of the control samples, carrying approximately 6-Mb duplication was correctly identified, moreover it was found to be interrupted by a previously unknown 19 kb region of normal copy number. The second control 50 kb deletion was not identified, as this particular region was not covered by array-MAPH probes. This study demonstrates that the chromosome X-specific array-MAPH platform is a valuable tool for screening patients with XLMR, or other X-linked disorders, and emerges the need for introducing new high-resolution screening methods for the detection of genetic imbalances.     

Aerobic–anaerobic transition intensity measured via EMG signals in athletes with different physical activity patterns

The purpose of the present study was to investigate the use of electromyographic signals (EMG), to determine the EMG threshold (EMGT) in four lower extremity muscles and to compare these thresholds with the second ventilatory threshold (VT2) in subjects participating in different sports and at different performance levels. Forty-nine subjects (23.8 ± 5.7 years, 182.7 ± 5.3 cm, 79.1 ± 8.6 kg) including eleven cyclists, ten team-handball players, nine kayakers, eight power lifters and eleven controls were investigated utilizing a cycle ergometer. Respiratory gas exchange measures were collected and EMG activity was continuously recorded from four muscles (vastus lateralis, vastus medialis, biceps femoris and gastrocnemius lateralis). The VO₂max averaged 56.1 ± 11.1 ml kg⁻¹ min⁻¹, the average aerobic power was 348.5 ± 61.0 W and the corresponding VT2 occurred at 271.4 ± 64.0 W. The EMGT ranged from 80 to 98% of power output for the different muscles. The VT2 and EMG thresholds from four different muscles were not different. When thresholds were analyzed among different groups of subjects, no significant difference was observed between VT2 and EMGT despite threshold differences between the groups. All four EMGT were significantly related to maximal aerobic power (r = 0.73–0.83) and were highly correlated to each other (r = 0.57–0.88). In conclusion, EMGT can be used to determine the VT2 for individuals independent of sport specificity or performance level.     

Overexpressed human CD44s promotes lung colonization during micrometastasis of murine fibrosarcoma cells: Facilitated retention in the lung vasculature

Normally nonmetastatic murine sis-transformed BALB/c 3T3 cells, transfected with human CD44s gene (hCD44s), acquire spontaneous metastatic capacity to the lung. The mechanism(s) of this facilitated micrometastasis was analyzed in an experimental metastasis model. Human CD44s overexpression promoted the earliest stages severalfold (initial implantation and subsequent stabilization of tumor cells) but was irrelevant for later stages (subsequent outgrowth) of lung experimental micrometastasis. By injecting mixed populations of parental (nonmetastatic) and CD44s-transfected cells, it was shown that cell–cell adhesion between tumor and parental cells was not promoted by hCD44s but that promotion of cell–cell adhesion to lung endothelium or specifically between transfected cells (via hyaluronan) are likely mechanisms. Results obtained with hCD44s-negative primary tumor cells and hCD44s-positive or -negative variants of lung micrometastatic cells (after s.c. injection of transfectants) confirmed the importance of CD44s overexpression for early but not late stages of experimental lung metastasis. Therefore, CD44s represents a metastasis-facilitating molecule that is irrelevant for primary tumor outgrowth but that promotes micrometastasis to the lungs at the very earliest stages.     

Effects of amlodipine and candesartan on oxidized LDL level in patients with mild to moderate essential hypertension

Objective. To compare the effects of amlodipine and candesartan on oxidized low-density lipoprotein (OxLDL), conjugated dienes (CD) and baseline diene conjugation in circulating low-density lipoproteins (LDL-BDC) level during antihypertensive treatment. Methods. Forty-nine patients with untreated mild to moderate essential hypertension were recruited in a randomized double-blind study to receive a daily dose either of 8 mg candesartan or 5 mg amlodipine for 16 weeks. Blood pressure, OxLDL, CD, LDL-BDC, triglycerides (TG), total cholesterol and lipoprotein cholesterol were measured at baseline, at week 2 and at week 16. Results. During treatment, in addition to a significant decrease in systolic and diastolic blood pressure, high level of OxLDL decreased significantly reaching practically upper kit reference values. Both treatment groups were similar with regard to the studied parameters at all time points. At the same time serum TG, lipoprotein and total cholesterol levels as well as LDL-BDC did not change and CD levels did not exceed endemic normal. Decrease in both systolic and diastolic blood pressure was associated with decrease in LDL-BDC/LDL. Conclusions. Besides their antihypertensive effects, both candesartan and amlodipine are efficient drugs for reducing OxLDL level, being neutral with regard to serum lipids.