Differential Functions of C- and N-Terminal Hepatitis B x Protein in Liver Cells Treated with Doxorubicin in Normoxic or Hypoxic Condition

Hepatitis viral B x protein (HBx), a hepatocarcinogen, is frequently mutated. Hypoxia influences the growth of HCC and also the sensitivity of tumor cells to treatments. We aimed to test the role of HBx and acute hypoxia in the efficacy of chemotherapy. In this study, we established 4 Chang liver cell lines with the full-length HBx (HBx), the first 50 amino acids of N-terminal HBx (HBx/50), the last 104 amino acids of C-terminal HBx (HBx/51) and empty vector (CL), respectively. MTT and TNUEL assays were used to assess cell viability and apoptosis respectively. Western blot was used to determine the expression of relevant proteins. Results showed that among 4 cell lines, doxorubicin was most effective in decreasing the viability and enhancing apoptosis in HBx/51 cells, while HBx/50 cells were most resistant to the treatment. Cells in hypoxia were more susceptible to doxorubicin than cells in normoxia. Hypoxia facilitated the Bid cleavage especially in HBx/51 cells via phosphorylating p38 MAPK. p38 MAPK inhibitor significantly reduced the tBid level and increased cell viability. In conclusion, N-terminal HBx and C-terminal HBx function differentially in their ability to regulate cell growth, with the former being promotive but the latter being inhibitory. The acute hypoxia may overcome the HBx-induced resistance and facilitate the chemotherapy.     

Evaluation of DNA damage in white blood cells of healthy human volunteers using the alkaline comet assay and the chromosome aberration test

The present study was undertaken to contribute to the characterization of the degree of variability in baseline damage in white blood cells from control population, and to investigate how this variability is associated with external and internal factors. Altogether 170 healthy volunteers, randomly selected from the general population of the Republic of Croatia, participated in the study. Two sensitive tests: the alkaline comet assay and the chromosome aberration test were applied to study the background levels of DNA damage in their white blood cells. The results point to inter-individual differences, indicating different genome sensitivity. As revealed by both assays, the background levels of DNA damage were mostly influenced by smoking habit as well as medical exposure (especially to diagnostic X-rays). Sex and age of subjects did not significantly influence the values of DNA damage recorded in the white blood cells. Although higher levels of DNA damage were recorded in blood samples collected during winter and autumn, they were mostly influenced by medicinal exposure and smoking habit. Statistical evaluation of the data confirmed that a positive correlation exists between DNA migration and the number of long-tailed nuclei found with the comet assay and the total number of chromosome aberrations. The data obtained can serve as control values in forthcoming biomonitoring studies.     

Space asymmetry directs preferential sperm entry in the absence of polarity in the mouse oocyte

Knowledge about the mechanism that establishes embryonic polarity is fundamental in understanding mammalian development. In re-addressing several controversial claims, we recently proposed a model in which mouse embryonic polarity is not specified until the blastocyst stage. Before fertilization, the fully differentiated oocyte has been characterized as “polarized,” and we indeed observed that the sperm preferentially enters the polar body half. Here we show that preferential sperm entry is not due to an intrinsic polarity of the oocyte, since fertilization takes place uniformly when the zona pellucida is removed. We suggest that the term “asymmetry” denotes morphological differences, whereas “polarity” in addition implies developmental consequences. Thus, the mouse oocyte can be considered “asymmetric” but “non-polarized.” The penetration through the zona pellucida is also random, and a significant proportion of sperm binds to the oocyte membrane at a point distant from the zona penetration site. Time-lapse recordings confirmed that sperm swim around the perivitelline space before fertilization. Experimental enlargement of the perivitelline space in the non-polar body half increased the regional probability of fertilization. Based on these experiments, we propose a model in which the space asymmetry exerted by the first polar body and the zona pellucida directs sperm entry preferentially to the polar body half, with no need for oocyte polarity.     

Ribosomal protein S6 gene haploinsufficiency is associated with activation of a p53-dependent checkpoint during gastrulation

Nascent ribosome biogenesis is required during cell growth. To gain insight into the importance of this process during mouse oogenesis and embryonic development, we deleted one allele of the ribosomal protein S6 gene in growing oocytes and generated S6-heterozygous embryos. Oogenesis and embryonic development until embryonic day 5.5 (E5.5) were normal. However, inhibition of entry into M phase of the cell cycle and apoptosis became evident post-E5.5 and led to perigastrulation lethality. Genetic inactivation of p53 bypassed this checkpoint and prolonged development until E12.5, when the embryos died, showing decreased expression of D-type cyclins, diminished fetal liver erythropoiesis, and placental defects. Thus, a p53-dependent checkpoint is activated during gastrulation in response to ribosome insufficiency to prevent improper execution of the developmental program.     

Prevalence of moderate and severe depression among Croatian patients infected with human immunodeficiency virus

The aim of the study was to assess the prevalence of depression among Croatian patients infected with human immunodeficiency virus (HIV) and to make a comparison with patients with other acute and chronic infectious diseases. We assessed the depressive disorder using the Beck Depression Inventory questionnaire (BDI), without clinical confirmation. The BDI scores were examined in 80 HIV-infected persons and compared to 80 persons with chronic viral hepatitis and 78 with acute infectious diarrhea. All examinees were treated as outpatients at the University Hospital for Infectious Diseases in Zagreb in March and April of 2003. Prevalence of moderate and severe depression among HIV-infected was 16/80 (20%) with a 95% confidence interval 11% to 29%. Male patients with HIV or chronic viral hepatitis had a significantly higher BDI scores than males with acute infectious diarrhea (p = 0.017, Kruskall-Wallis, d.f. 2). Female patients with HIV infection tended to have a lower BDI score than females with chronic viral hepatitis or acute infectious diarrhea (p = 0.087, Kruskall-Wallis, d.f. 2). Prevalence of moderate and severe depression among Croatian HIV-positive patients is higher than the upper estimate for general population. Croatian males with chronic infectious disease have higher rate of depression than those with acute infectious disease.     

Dietary habits and folate status in women of childbearing age in Croatia

Folic acid and folates have an important role in prevention of neural tube defect that appears in the first weeks of pregnancy, when women are still not aware of their pregnancy, especially when pregnancy is not planned. Since ensuring sufficient quantities of folates and folic acid in this period is essential, dietary habits of childbearing age women are very important. In line with that the intake of folates and folic acid in nutrition of women age group 20-30 years is examined, as well as the frequency of consumption of foodstuffs rich in vitamins and folic acid supplements. Values of folates in serum are presented, dependent on their nutritional habits. Obtained results indicate that in spite of inadequate intake of folates and folic acid from foodstuffs, clinical deficit is not recorded, which is the result of frequent consumption of dietary supplements. In accordance with these dietary habits, differences in the folates status of examinees were observed.     

Frequency of ventricular premature beats and ventricular tachycardia in STEMI treated with fibrinolytics

To determine in acute myocardial infarction with an ST elevation (STEMI) treated with fibrinolytics frequency of ventricular premature beats (VPBs) and ventricular tachycardia (VT) according to the damaged area and residual cardiac function. With anterolateral infarction with ejection fraction (EF) < 45%, incidence of VPBs < 10/h was statistically significantly reduced (p < 0.001) while incidence of VPBs > or = 10/h as well as VPBs in a pair and VT was increased (p < 0.001). With anteroseptal infarction with EF < 45%, incidence of VPBs < 10/h was statistically reduced (p = 0.06) and incidence of VPBs > 10/h, VPBs in a pair and VT was increased (p = 0.06). With inferior and inferoposterior infarction with EF < 45%, incidence of VPBs < 10/h was reduced and incidence of VPBs > or = 10/h, VPBs in a pair and VT was increased. However, such difference was not statistically significant. Along with reduced residual cardiac function, one can also expect increase in frequency of VPBs and VT in all forms of STEMI regardless the area of damage. Such frequency is significant with all forms of anterior infarction, that is to say, slightly more with anterolateral infarction in relation to anteroseptal one. However, with inferior and inferoposterior infarction this frequency of VPBs i VT is not significant.     

Efficiency of free energy transfer and entropy production in photosynthetic systems

The diagram method for the calculation of entropy production and efficiency associated with very first steps of photosynthetic mechanism is developed. By using one simple model for the bacteriorhodopsin light activated proton pump action, it is shown that in the steady state of biological interest, both entropy production and efficiency of transfer of light energy are maximal for the same optimal values of kinetic constants.     

Cyanide-Resistant Respiration of a Neurospora crassa Membrane Mutant

Choline starvation of the Neurospora crassa chol-1 mutant leads to a decrease in respiration through the cytochrome chain and a concomitant induction of the alternate oxidase.     

Inhibition of K+ Transport through Na+, K+-ATPase by Capsazepine: Role of Membrane Span 10 of the α-Subunit in the Modulation of Ion Gating

Capsazepine (CPZ) inhibits Na⁺,K⁺-ATPase-mediated K⁺-dependent ATP hydrolysis with no effect on Na⁺-ATPase activity. In this study we have investigated the functional effects of CPZ on Na⁺,K⁺-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na⁺,K⁺-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na⁺,K⁺-ATPase current in the presence of extracellular K⁺. In contrast, CPZ stimulated pump current in the absence of extracellular K⁺. Similar conclusions were attained using HEK293 cells loaded with the Na⁺ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na⁺,K⁺-ATPase indicated that CPZ stabilizes ion interaction with the K⁺ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na⁺ congener at the Na⁺ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na⁺,K⁺-ATPase. CPZ increased oxonol VI fluorescence in the absence of K⁺, reflecting increased Na⁺ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K⁺, likely indicating opening of an intracellular pathway selective for K⁺. As revealed by the recent crystal structure of the E₁.AlF₄ ^-.ADP.3Na⁺ form of the pig kidney Na⁺,K⁺-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K⁺ sites in Na⁺,K⁺-ATPase.