Correlation of hypoxia inducible factor-1α and vascular endothelium growth factor in rat myocardium during aerobic and anaerobic exercise.

Background: Exercise increases the need for oxygen to generate ATP through oxidative phosphorylation. If the high energy demand during exercise is not balanced by sufficient oxygen supply, hypoxia occurs in skeletal muscle tissue leading to upregulation of hypoxia inducible factor-1α (HIF-1α). The activity of HIF-1α increases the expression of various genes in order to reduce the metabolic dependence on oxygen and to increase oxygen supply to the tissue, e.g., VEGF which plays a role in angiogenesis. In myocardium, it is unclear whether exercise leads to hypoxia and whether HIF-1α and VEGF play a role in the mechanism of hypoxic adaptation. This study aimed to investigate the correlation of HIF-1α and VEGF in heart muscle tissue of rats during aerobic and anaerobic exercise. Methods: A rat treadmill was used with a specific exercise program for 1, 3, 7 and 10 days. The concentrations of HIF-1α and VEGF were measured the myocardium. Results: Both, HIF-1α protein and VEGF were increased (p < 0.05) in the groups with aerobic and anaerobic exercise. Concentrations of HIF-1α were highest on the first day of activity, being higher in the anaerobic than in the aerobic group (156.8 ± 33.1 vs. 116.03 ± 5.66). Likewise, the highest concentration of VEGF in the group with anaerobic exercise occurred on the first day (36.37 ± 2:35), while in the aerobic group, VEGF concentration was highest on day 3 (40.66 ± 1.73). The correlation between the myocardial tissue consentrations of HIF-1α and VEGF is moderate (r = 0.59) in the aerobic group and strong in the anaerobic group (r = 0.69). Conclusion: Aerobic and anaerobic exercise increase HIF-1α and VEGF concentrations in rat myocardium in specific patterns. The anaerobic condition triggers vascularization stronger and obviously earlier than aerobic exercise.

Correlation between hypoxia inducible factor -1α and renin expression in rats kidney induced by cobalt chloride.

Background: Cobalt chloride can be used as an agent to stabilize hypoxia inducible factor-1α (HIF-1α) and to imitate hypoxia without low levels of oxygen inside the body. We intended to investigate if there was any regulation of renin expression by HIF-1α. Therefore, we conducted several studies to clarify this possibility starting with the induction of hypoxic mimicry in rats by intra-peritoneal (IP) injection of cobalt chloride (CoCl2) to obtain the levels and pattern of HIF-1α and renin mRNA and protein expression. Methods: Twenty-four rats were randomly divided into four groups, control group and incubation groups 2, 8, and 24 hours after intra-peritoneal injection of 30 mg CoCl2 per kg BW. After the rats were sacrificed, kidneys were excised, weighed and kidney weight compared to BW. Tissue parameters were measured such as RNA concentration, HIF-1α protein by ELISA, and renin mRNA by RT-PCR. Results: Differences between the groups in the ratios of kidney weight to BW and in the concentrations of HIF-1α protein were statistically not significant (p > 0.05). Relative expression of renin mRNA increased markedly starting 8 hours after CoCl2 IP injection (30 times over controls) and further rising until 24 hours (2465 times over controls). Correlation between HIF-1α and renin mRNA by Pearson analysis was strongly positive, but not significant (R = 0.91; p = 0.09). Conclusion: Renin gene regulation in renal hypoxic mimicry strongly correlates with HIF-1α.

Correlation between oxidative stress and tumor grade in glioma cells from patients in Jakarta.

Background: The goal of this study was to analyze the correlation of oxidative stress in human glioma cells with tumor grade in order to explore the role of oxidative stress as a marker in determining the tumor progression. Methods: Samples were 21 brain tumors and 5 normal brain tissues from glioma patients. Oxidative stress was analyzed by measuring malondialdehyde (MDA), carbonyl and 8-hydroxy-2’-deoxyguanosine (8-OhdG). Additionaly, we analyzed MnSOD expression by measuring the MnSOD mRNA using real time RT-PCR and MnSOD enzyme activity using RanSOD kit. Tumor grade was determined by histopathologic examination. Data was statistically analyzed using t-test and Pearson correlation. Results: Levels of MDA, carbonyl and 8-OHdG reflecting oxidative stress in glioma cells were significantly higher than in normal brain tissue. The MDA and carbonyl levels were significantly correlated with tumor grade. Relative expression of MnSOD mRNA and specific enzyme activity in glioma cells were significantly higher than in normal brain cells. The relative expression of MnSOD mRNA increased significantly in accordance with the tumor grade. Surprisingly, MnSOD specific activity was significantly lower in high grade than in low grade glioma indicating a discrepancy between mRNA synthesis and its enzyme specific activity. Furthermore, there was a positive correlation between MnSOD mRNA and MDA levels. Conclusion: The high level of oxidative damage in human glioma cells was significantly correlated with tumor grade. The high level of MnSOD expression in human glioma cells was correlated with the high level of oxidative damage.

Expression of manganese superoxide dismutase in rat blood, heart and brain during induced systemic hypoxia.

Background: Hypoxia results in an increased generation of ROS. Until now, little is known about the role of MnSOD - a major endogenous antioxidant enzyme - on the cell adaptation response against hypoxia. The aim of this study was to determine the MnSOD mRNA expression and levels of specific activity in blood, heart and brain of rats during induced systemic hypoxia. Methods: Twenty-five male Sprague Dawley rats were subjected to systemic hypoxia in an hypoxic chamber (at 8-10% O2) for 0, 1, 7, 14 and 21 days, respectively. The mRNA relative expression of MnSOD was analyzed using Real Time RT-PCR. MnSOD specific activity was determined using xanthine oxidase inhibition assay. Results: The MnSOD mRNA relative expression in rat blood and heart was decreased during early induced systemic hypoxia (day 1) and increased as hypoxia continued, whereas the mRNA expression in brain was increased since day 1 and reached its maximum level at day 7. The result of MnSOD specific activity during early systemic hypoxia was similar to the mRNA expression. Under very late hypoxic condition (day 21), MnSOD specific activity in blood, heart and brain was significantly decreased. We demonstrate a positive correlation between MnSOD mRNA expression and specific activity in these 3 tissues during day 0-14 of induced systemic hypoxia. Furthermore, mRNA expression and specific activity levels in heart strongly correlate with those in blood. Conclusion: The MnSOD expression at early and late phases of induced systemic hypoxia is distinctly regulated. The MnSOD expression in brain differs from that in blood and heart revealing that brain tissue can possibly survive better from induced systemic hypoxia than heart and blood. The determination of MnSOD expression in blood can be used to describe its expression in heart under systemic hypoxic condition.

Polyclonal VDAC3 antibody decreases human sperm motility: a novel approach to male contraception.

Background: Voltage dependent anion channels (VDAC) mediate transport of anions, cations and ATP which play an important role in sperm motility. This study was aimed to examine the effect of polyclonal VDAC3 antiserum to human sperm motility. Methods: Polyclonal VDAC3 antiserum used in this study was produced in rabbits by immunization of VDAC3-specific synthetic peptides. Preimmunserum was collected before immunization and used for control experiment. Recognition of VDAC3 antiserum to antigen in human sperm was performed by western blot. Thirty sperm samples obtained from fertile men which had high quality of sperm motility were washed and collected by Percoll gradient. Sperm motility was assessed by means of evaluation of sperm velocity (seconds per 0.1 mm distance) and the number of unmoved sperm (million per ml) which were observed 0 minute, 30 minutes and 60 minutes after addition of VDAC3 antiserum and preimmunserum as a control. Both data were analyzed by SPSS 13.0 software. Results: VDAC3 antiserum recognized VDAC3 protein in human sperm. Statistical analysis demonstrated that there were increasing numbers of unmoved spermatozoa after addition of anti-VDAC3 antiserum in vitro for 60 minutes observation compared with preimmunserum (control). We found also that sperm velocity decreased signifi cantly after giving anti-VDAC3 antiserum in vitro for 0 minute, 30 minutes, and 60 minutes compared with pre-immunee serum (control). Conclusion: VDAC3 antiserum can decrease motility of human sperm. and may provide a novel principle of male contraception in the future.

CYP2A6 gene polymorphisms impact to nicotine metabolism.

Nicotine is a major addictive compound in tobacco cigarette smoke. After being absorbed by the lung nicotine is rapidly metabolized and mainly inactivated to cotinine by hepatic cytochrome P450 2A6 (CYP2A6) enzyme. Genetic polymorphisms in CYP2A6 may play a role in smoking behavior and nicotine dependence. CYP2A6*1A is the wild type of the CYP2A6 gene which is associated with normal or extensive nicotine metabolism. In the CYP2A6 gene, several polymorphic alleles have been reported such as CYP2A6*4, CYP2A6*7, CYP2A6*9, and CYP2A6*10 which are related to decreasing nicotine metabolism activity. The variation of nicotine metabolism activity could alter nicotine plasma levels. Smokers need a certain level of nicotine in their brain and must smoke regularly because of nicotine’s short half-life; this increases the number of smoked cigarettes in extensive metabolizers. Meanwhile, in slow metabolizers, nicotine plasma level may increase and results in nicotine toxicity. This will eventually lower the risk of dependence.

Gene expression of manganese superoxide dismutase in human glioma cells.

Aim This study analyze the MnSOD gene expression as endogenous antioxidant in human glioma cells compared with leucocyte cells as control. Methods MnSOD gene expression of 20 glioma patients was analyzed by measuring the relative expression of mRNA and enzyme activity of MnSOD in brain and leucocyte cells. The relative expression of mRNA MnSOD was determined by using quantitative Real Time RT-PCR and the enzyme activity of MnSOD using biochemical kit assay (xantine oxidase inhibition). Statistic analysis for mRNA and enzyme activity of MnSOD was performed using Kruskal Wallis test. Results mRNA of MnSOD in glioma cells of 70 % sample was 0.015–0.627 lower, 10 % was 1.002-1.059 and 20 % was 1.409-6.915 higher than in leucocyte cells. Also the specific activity of MnSOD enzyme in glioma cells of 80 % sample showed 0,064-0,506 lower and 20 % sample was 1.249-2.718 higher than in leucocyte cells. Conclusion MnSOD gene expression in human glioma cells are significantly lower than its expression in leucocytes cells.

Detection of Cryptosporidium sp infection by PCR and modifi ed acid fast staining from potassium dichromate preserved stool.

Aim To identify the frequency of Cryptosporidium infection in children below 3 years old by examining concentrated long term preserved stool using PCR detection of 18S rRNA gene and compared with modifi ed acid fast staining technique. Methods Hundred eighty eight stools from children ≤ 3 years old were stored for 13 months in 2.5% K2Cr2O7 solution at 40C. Cryptosporidium oocysts were isolated by water-ether concentration technique. The concentrates were smeared onto object glass and stained with modifi ed acid fast staining, and the rest of the concentrates were DNA extracted by freezing and thawing cycles and proteinase K digestion, then direct PCR was done to detect 18S rRNA gene. Result The proportion of positive stools for Cryptosporidium sp by acid fast staining from concentrated stools and 18S rRNA PCR were 4.8% and 34.6% respectively, which showed statistically signifi cant difference. Conclusion The frequency of Cryptosporidium infection among children ≤ 3 years old was very high and stool storage in K2Cr2O7 for 13 months did not affect the PCR result. High prevalence of Cryptosporidium infection indicated high transmission in that area and the potential to be transmitted to other individuals such as the immunocompromised.

Expression of hypoxia inducible factor-1a (HIF-1a) gene and apoptosis in the heart induced by systemic hypoxia.

Aim This study explored the expression of HIF-1α in hypoxic cardiac muscle in mice, and observed the evidence of apoptosis in hypoxia induced cardiomyocyte. Methods Male Sprague-Dawley rats, were randomized into 7 groups (n= 4 per group): control normoxia group that was exposed to atmospheric oxygen and hypoxia groups that were housed in hypoxic chambers (O2 level 8%) for 1, 3, 7, 14, 21, and 28 days respectively. Animals were sacrificed, hearts were rapidly excised, total RNA was extracted with an mRNA isolation kit and the expression of HIF-1α mRNA was then detected by real-time RT-PCR. Apoptosis was assessed by TUNEL method. Results For rat in hypoxia group, the expression of HIF-1α mRNA in cardiac myocytes was clearly up-regulated compared to the control normoxia group. Further, HIF-1α expression level elevated gradually and reached a peak at 21 days of hypoxia. No cell labeled by the TUNEL method was detected in the control group. Compared with the control group, the apoptotic index was significantly increased in the hypoxia group (P < 0.05). There was no significant correlation between the elevation of HIF-1α mRNA and the elevation of apoptotic index. Conclusion Systemic chronic hypoxia caused the elevation of HIF-1α mRNA and apoptosis in cardiac myocytes.

Malaria in Jayapura District, Papua Province, Indonesia and resistance to sulfadoxine-pyrimethamine.

Malaria poses a major public health problem in Papua. The rapid spread of drug-resistant parasites and deterioration of the malaria control program following the monetary crisis and civil strife in the late 90’es have worsened the malaria situation in many areas of Papua. To re-assess the current magnitude of malaria problem, a malariometric survey was conducted in three sub-districts of Jayapura District, as well as antimalarial drug sensitivity test using sulfadoxine-pyrimethamine (SP) at the outpatient clinics of Abepura General Hospital (RSU), Hamadi Primary Health Center and Panti Asuhan Katolik Clinics, Sentani, Jayapura District, Papua Province. Screening of 543 subjects in three sub-districts revealed 185 malaria positive subjects, either with P. falciparum, P. vivax or mixed of the two species. All of the malaria cases were asymptomatic. In the SP drug sensitivity test, of the 56 subjects involved, 32 were classified as early treatment failure and two subjects were classified as late treatment failure. The remaining 22 subjects were classified as adequate clinical and parasitological response (ACPR). These results indicated that the surveyed area has a high malaria prevalence and treatment failure to SP and therefore alert to the need of deploying new antimalarial regiment that suit the remote setting.