Conformational dynamics of abasic DNA upon interactions with AP endonuclease 1 revealed by stopped-flow fluorescence analysis

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from exposure to UV light, ionizing radiation, alkylating agents, and oxygen radicals. In human cells, AP endonuclease 1 (APE1) recognizes this mutagenic lesion and initiates its repair via a specific incision of the phosphodiester backbone 5' to the AP site. We have investigated a detailed mechanism of APE1 functioning using fluorescently labeled DNA substrates. A fluorescent adenine analogue, 2-aminopurine, was introduced into DNA substrates adjacent to the abasic site to serve as an on-site reporter of conformational transitions in DNA during the catalytic cycle. Application of a pre-steady-state stopped-flow technique allows us to observe changes in the fluorescence intensity corresponding to different stages of the process in real time. We also detected an intrinsic Trp fluorescence of the enzyme during interactions with 2-aPu-containing substrates. Our data have revealed a conformational flexibility of the abasic DNA being processed by APE1. Quantitative analysis of fluorescent traces has yielded a minimal kinetic scheme and appropriate rate constants consisting of four steps. The results obtained from stopped-flow data have shown a substantial influence of the 2-aPu base location on completion of certain reaction steps. Using detailed molecular dynamics simulations of the DNA substrates, we have attributed structural distortions of AP-DNA to realization of specific binding, effective locking, and incision of the damaged DNA. The findings allowed us to accurately discern the step that corresponds to insertion of specific APE1 amino acid residues into the abasic DNA void in the course of stabilization of the precatalytic complex.     

A combined approach of VNTR and MLST analysis: improving molecular typing of Argentinean isolates of Leptospira interrogans

Leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. Regular outbreaks associated with specific risk factors have been reported in Argentina. However, there are no available data concerning the genetic population level for this pathogen. Therefore, the aim of this work was to describe the genetic diversity of Leptospira interrogans through the application of two molecular typing strategies: variable number of tandem repeats (VNTR) and multilocus sequence typing (MLST). For this purpose, seven reference strains and 18 non-epidemiologically related isolates from diverse hosts and Argentinean regions were analysed. Among them, nine genotypes and seven sequence types (STs), including three unreported STs, were described using VNTR and MLST, respectively. eBURST analysis demonstrated that ST37 was the most frequent and founder genotype of a clonal complex (CCs) containing STN1 and STN3, suggesting the importance of studying the serovars belonging to this CC in Argentina. The data from maximum parsimony analysis, which combined both techniques, achieved intra-serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. This study is the first to combine both strategies for L. interrogans typing to generate a more comprehensive molecular genotyping of isolates from Argentina in a global context.     

Regulation of Connexin43 Oligomerization is Saturable

We have used connexin constructs containing a C-terminal di-lysine-based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, we found that Cx43-HKKSL stably expressed at moderate levels by HeLa cells was retained in the ER and prevented from oligomerization. However, Cx43-HKKSL stably overexpressed by HeLa cells escaped from the ER and localized to a perinuclear region of the cell that included the Golgi apparatus. Overexpressed Cx43-HKKSL oligomerized into hexamers and also formed Triton X-100 insoluble, intracellular complexes that resembled gap junctions. Thus, the ability of HeLa cells to inhibit Cx43 oligomerization was saturable. HeLa cells stably overexpressing Cx43-HKKSL may provide a useful model system to evaluate pharmacologic agents and/or cDNAs encoding chaperones with the potential to regulate initial steps in Cx43 oligomerization.     

Identification of rab20 as a potential regulator of connexin 43 trafficking

Connexin oligomerization and trafficking are regulated processes. To identify proteins that control connexin 43 (Cx43), a screen was designed using HeLa cells expressing a Cx43 construct with di-lysine endoplasmic reticulum (ER)-retention/retrieval motif, Cx43-HKKSL. At moderate levels of expression, Cx43-HKKSL is retained in the ER as monomers; however, Cx43-HKKSL stably overexpressed by HeLa cells localizes to the perinuclear region and oligomerizes. HeLa/Cx43-HKKSL overexpressors were transiently transfected with pooled clones from a human kidney cDNA library and used immunofluorescence microscopy to identify cDNAs that enabled overexpressed Cx43-HKKSL to convert from a perinuclear to ER localization pattern. Using this approach, a small molecular weight GTPase, rab20, was identified as a candidate protein with the ability to regulate Cx43 trafficking. Enhanced green fluorescent protein (EGFP)-tagged rab20 showed a predominantly perinuclear and ER localization pattern and caused wild-type Cx43 to be retained inside the cell. By contrast, mutant EGFP-rab20T19N, which lacks the ability to bind GTP, had no effect on Cx43. These results suggest Cx43 is transported through an intracellular compartment regulated by rab20 along the secretory pathway.     

Nicotinic acetylcholine receptors alpha4beta2 and alpha7 regulate myelo- and erythropoiesis within the bone marrow

Nicotine consumed upon smoking affects numerous physiological processes through nicotinic acetylcholine receptors, which mediate cholinergic regulation by the neuronal and endogenous acetylcholine. Consequently, nicotinic receptors are expressed in many non-excitable tissues including the blood. In spite of the documented effect of nicotine on hematopoiesis, little is known about the expression and role of nicotinic receptors in the course of blood cell differentiation. The aim of the present study was to investigate whether and how nicotinic receptors are involved in the development of myeloid and erythroid cells within the bone marrow. The presence of nicotinic receptors containing alpha4(beta2) and alpha7 subunits in the bone marrow cells of C57Bl/6 mice was shown by the binding of [125I]-alpha-bungarotoxin or [3H]-Epibatidine and by flow cytometry with subunit-specific antibodies or fluorescein-labeled alpha-cobratoxin. Both TER119+ (erythroid) and CD16+CD43med (myeloid) progenitor cells bound more alpha4-specific antibodies than their mature forms, while the binding of alpha-cobratoxin and alpha7-specific antibodies was also high in mature cells. According to morphological analysis, either the absence of alpha7-containing nicotinic receptors in knockout mice or their desensitization in mice chronically treated with nicotine decreased the number of myeloid and erythroid progenitors and junior cells. In contrast, the absence of beta2-containing receptors favored myelocyte generation and erythroid cell maturation. It is concluded that the development of both myeloid and erythroid cell lineages is regulated by endogenous cholinergic ligands and can be affected by nicotine through alpha7- and alpha4beta2-containing nicotinic receptors, which play different roles in the course of the cell maturation.     

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Generation of uniform low-temperature plasma in a pulsed non-self-sustained glow discharge with a large-area hollow cathode

Generation of plasma in a pulsed non-self-sustained glow discharge with a hollow cathode with an area of ≥2 m² at gas pressures of 0.4–1 Pa was studied experimentally. At an auxiliary arc-discharge current of 100 A and a main discharge voltage of 240 V, a pulse-periodic glow discharge with a current amplitude of 370 A, pulse duration of 340 μs, and repetition rate of 1 kHz was obtained. The possibility of creating a uniform gas-discharge plasma with a density of up to 10¹² cm^?3 and an electron temperature of 1 eV in a volume of >0.2 m³ was demonstrated. Such plasma can be efficiently used to treat material surfaces and generate pulsed ion beams with a current density of up to 15 mA/cm².     

Gamma-ray- and UV-sensitive strains of a radioresistant cell line: isolation and cross-sensitivity to other agents

Two gamma-ray-sensitive and two ultraviolet (UV)-sensitive variants were isolated from the gamma-ray- and UV-resistant TN-368 lepidopteran insect cell line. The isolation was performed by inducing mutations in the TN-368 cells using ethyl methanesulfonate, growing them for an expression period, irradiating with 137Cs gamma rays or 254-nm UV radiation, allowing cells to incorporate 5-bromodeoxyuridine (BrdU) in the presence of hydroxyurea (DNA repair synthesis), and finally irradiating with 365-nm UV radiation to cause DNA strand breakage at sites of BrdU incorporation with the intent of killing those cells that have undergone DNA repair synthesis and sparing those cells which, for a variety of reasons, did not. The survival of the Cs2 and Cs7 variants exposed to X rays is significantly different from the parent TN-368 line at the P less than 0.0001 level. The survival of the UV10 and UV19 variants exposed to UV radiation is different from the parent at the P less than 0.0001 and P less than 0.003 levels, respectively. In cross-sensitivity testing of the gamma-ray-sensitive variants, only Cs2 is more sensitive to 254-nm UV and only Cs7 is more sensitive to 44 degrees C heating; both are sensitive to PUVA. The UV-sensitive mutants are both sensitive to X irradiation, PUVA, and mitomycin C. However, UV10 is not sensitive to 44 degrees C heating while UV19 is, making UV19 the only variant strain sensitive to all agents examined. Despite the isolation procedure which was intended to select for DNA repair-deficient cells, the results suggest that a more general mechanism is responsible for the sensitivity of the variant cells to the agents tested.     

Estimation of cold stress effect on dairy cows

Twelve crossbred heifers (Slovak Spotted x Holstein-Friesian) were housed in an open, uninsulated barn with straw bedding and a concrete-floored yard. Minimum temperatures inside the barn were as low as –19°C. The average milk yield decreased as the temperatures approached these minima. Compared with the temperate conditions, the feed intake and blood levels of glucose and free fatty acids increased. The level of sodium declined significantly during the second cold period. Correlations and regressions between milk yield and biochemical parameters were calculated, and the results indicate that the concentrations of free fatty acids, cholesterol, and triiodothyronine and the haematocrit values may serve to predict milk production during periods of cold stress, or in lactations of 305 days.