Human DNA replication-related element binding factor (hDREF) self-association via hATC domain is necessary for its nuclear accumulation and DNA binding

We previously demonstrated that hDREF, a human homologue of Drosophila DNA replication-related element binding factor (dDREF), is a DNA-binding protein predominantly distributed with granular structures in the nucleus. Here, glutathione S-transferase pulldown and chemical cross-linking assays showed that the carboxyl-terminal hATC domain of hDREF, highly conserved among hAT transposase family members, possesses self-association activity. Immunoprecipitation analyses demonstrated that hDREF self-associates in vivo, dependent on hATC domain. Moreover, analyses using a series of hDREF mutants carrying amino acid substitutions in the hATC domain revealed that conserved hydrophobic amino acids are essential for self-association. Immunofluorescence studies further showed that all hDREF mutants lacking self-association activity failed to accumulate in the nucleus. Self-association-defective hDREF mutants also lost association with endogenous importin beta1. Moreover, electrophoretic gel-mobility shift assays revealed that the mutations completely abolished the DNA binding activity of hDREF. These results suggest that self-association of hDREF via the hATC domain is necessary for its nuclear accumulation and DNA binding. We also found that ZBED4/KIAA0637, another member of the human hAT family, also self-associates, again dependent on the hATC domain, with deletion resulting in loss of efficient nuclear accumulation. Thus, hATC domains of human hAT family members appear to have conserved functions in self-association that are required for nuclear accumulation.     

Current knowledge of vector-borne zoonotic pathogens in Zambia: A clarion call to scaling-up “One Health” research in the wake of emerging and re-emerging infectious diseases

BackgroundAlthough vector-borne zoonotic diseases are a major public health threat globally, they are usually neglected, especially among resource-constrained countries, including those in sub-Saharan Africa. This scoping review examined the current knowledge and identified research gaps of vector-borne zoonotic pathogens in Zambia.Methods and findingsMajor scientific databases (Web of Science, PubMed, Scopus, Google Scholar, CABI, Scientific Information Database (SID)) were searched for articles describing vector-borne (mosquitoes, ticks, fleas and tsetse flies) zoonotic pathogens in Zambia. Several mosquito-borne arboviruses have been reported including Yellow fever, Ntaya, Mayaro, Dengue, Zika, West Nile, Chikungunya, Sindbis, and Rift Valley fever viruses. Flea-borne zoonotic pathogens reported include Yersinia pestis and Rickettsia felis. Trypanosoma sp. was the only tsetse fly-borne pathogen identified. Further, tick-borne zoonotic pathogens reported included Crimean-Congo Haemorrhagic fever virus, Rickettsia sp., Anaplasma sp., Ehrlichia sp., Borrelia sp., and Coxiella burnetii.ConclusionsThis study revealed the presence of many vector-borne zoonotic pathogens circulating in vectors and animals in Zambia. Though reports of human clinical cases were limited, several serological studies provided considerable evidence of zoonotic transmission of vector-borne pathogens in humans. However, the disease burden in humans attributable to vector-borne zoonotic infections could not be ascertained from the available reports and this precludes the formulation of national policies that could help in the control and mitigation of the impact of these diseases in Zambia. Therefore, there is an urgent need to scale-up “One Health” research in emerging and re-emerging infectious diseases to enable the country to prepare for future epidemics, including pandemics.     

Participation of cAMP in the facilitatory action of beta,gamma-methylene ATP on the noradrenaline release from rabbit ear artery

beta, gamma-Methylene ATP (betagamma-mATP) significantly facilitated the electrically (4 Hz) evoked release of noradrenaline (NA) from the rabbit ear artery by activation of prejunctional purinoceptors on the sympathetic nerve terminals. In the present study, we investigated whether intracellular cAMP is involved in the purinoceptor mediated facilitatory mechanisms. Forskolin, an adenylate cyclase activator, and 8-bromo cAMP, a cAMP analogue, significantly enhanced the NA-release. The enhancement of NA-release by betagamma-mATP was significantly potentiated by Ro20-1724, a phosphodiesterase inhibitor, but abolished by SQ22536, an adenylate cyclase inhibitor. Both drugs alone had no effect on the NA-release. N-ethylmaleimide and pertussis toxin, inhibitors of Gi-proteins, did not affect the NA-release, or the enhancement of NA-release by betagamma-mATP. Alone Cholera toxin (CTX), an activator of Gs-proteins, significantly increased the NA-release, but in the presence of CTX, betagamma-mATP could not produce further enhancement of the NA-release. These results suggest that cAMP is closely associated with the facilitatory action of betagamma-mATP on NA-release in the rabbit ear artery.     

Role of alpha-tocopherol in the regulation of mitochondrial permeability transition

We previously showed that Ca2+-induced cyclosporin A-sensitive membrane permeability transition (MPT) of mitochondria occurred with concomitant generation of reactive oxygen species (ROS) and release of cytochrome c (Free Rad. Res.38, 29-35, 2004). To elucidate the role of alpha-tocopherol in MPT, we investigated the effect of alpha-tocopherol on mitochondrial ROS generation, swelling and cytochrome c release induced by Ca2+ or hydroxyl radicals. Biochemical analysis revealed that alpha-tocopherol suppressed Ca2+-induced ROS generation and oxidation of critical thiol groups of mitochondrial adenine nucleotide translocase (ANT) but not swelling and cytochrome c release. Hydroxyl radicals also induced cyclosporin A-sensitive MPT of mitochondria. alpha-Tocopherol suppressed the hydroxyl radical-induced lipid peroxidation, swelling and cytochrome c release from mitochondria. These results indicate that alpha-tocopherol inhibits ROS generation, ANT oxidation, lipid peroxidation and the opening of MPT, thereby playing important roles in the prevention of oxidative cell death.     

Mutually antagonistic effects of androgen and activin in the regulation of prostate cancer cell growth

Activin, a member of the TGFbeta superfamily, is expressed in the prostate and inhibits growth. We demonstrate that the effects of activin and androgen on regulation of prostate cancer cell growth are mutually antagonistic. In the absence of androgen, activin induced apoptosis in the androgen-dependent human prostate cancer cell line LNCaP, an effect suppressed by androgen administration. Although activin by itself did not alter the cell cycle distribution, it potently suppressed androgen- induced progression of cells into S-phase of the cell cycle and thus inhibited androgen-stimulated growth of LNCaP cells. Expression changes in cell cycle regulatory proteins such as Rb, E2F-1, and p27 demonstrated a strong correlation with the mutually antagonistic growth regulatory effects of activin and androgen. The inhibitory effect of activin on growth was independent of serine, serine, valine, serine motif phosphorylation of Smad3. Despite their antagonistic effect on growth, activin and androgen costimulated the expression of prostate-specific antigen through a Smad3-mediated mechanism. These observations indicate the existence of a complex cross talk between activin and androgen signaling in regulation of gene expression and growth of the prostate.     

Simultaneous determination of isoflavones and bisphenol A in rat serum by high-performance liquid chromatography coupled with coulometric array detection

A method for simultaneous detection and quantification is presented to determine the presence of isoflavones and bisphenol A in a biological sample. A coulometric array detector was used with reversed-phase high-performance liquid chromatography (HPLC). Daidzein (1), glycitein (2), genistein (3) and their glucoside conjugates, daidzin (4), glycitin (5) and genistin (6), were measured as phytochemicals. Also assayed here was equol (7), a metabolite from compound 1, and bisphenol A (8), an industrial chemical that acts as an endocrine disrupter. All chemicals were simultaneously detected by using a 600-mV single detection voltage with high efficacy. A mixture of 1, 3 and 8 was orally administered to rats, and the levels of these three chemicals in the serum were clearly increased after a 4 kU beta-glucuronidase treatment. The levels of compounds 1 and 3 in the serum were detected at 1665 and 2040 ng/ml, while 8 was at a low level of 417 ng/ml. Compound 7 in the serum was not detected until after enzymatic hydrolysis (72 ng/ml). These results suggest that this analytical method would be useful for metabolic and pharmacokinetic studies on isoflavones and bisphenol A.     

Nitrous oxide-forming codenitrification catalyzed by cytochrome P450nor

Intact cells of the denitrifying fungus Fusarium oxysporum were previously shown to catalyze codenitrification to form a hybrid nitrous oxide (N2O) species from nitrite and other nitrogen compounds such as azide and ammonia. Here we show that cytochrome P450nor can catalyze the codenitrification reaction to form N2O from nitric oxide (NO) but not nitrite, and azide or ammonia. The results show that the direct substrate of the codenitrification by intact cells should not be nitrite but NO, which is formed from nitrite by the reaction of a dissimilatory nitrite reductase.