Effectiveness of chemical mutagenesis in multiple damage to one or both strands of plasmid DNA

Methods for site-directed multiple modification of DNA have been developed and used for modification of either one or two strands of plasmid DNA. Plasmid DNAs modified in the region of the tet gene were transformed into Escherichia coli cells and Tet colonies were screened. It was shown that multiple lesions in one DNA strand performed using either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium bisulfite were effectively repaired in the cell by error-free mechanism. In contrast, modification of two DNA strands led to induction of mutations. The efficiency of mutagenesis in the case of modification of a local region of one DNA strand with sodium bisulfite and modification of the other strand with MNNG was 1.1-7.9%. Mutations were analysed by restriction mapping and sequencing. All of them were G----A transitions.     

Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-gamma

By using a preparation of inactivated rabies virus, the blood mononuclear cells from five rabies vaccine recipients were stimulated in vitro in the presence of interleukin 2. T cell lines that displayed significant proliferative responses to whole rabies virus and to preparations of rabies glycoprotein and nucleocapsid were obtained from all the individuals. Other antigens, such as diphtheria and tetanus toxoids, influenza A virus, hepatitis B surface antigen, and serum albumin, failed to induce the proliferation of the T cell lines. One of these rabies-specific T cell lines was found to proliferate in response to rabies antigens only when the antigen-presenting cells expressed homologous HLA-DR antigens. The use of mouse monoclonal antibodies specific for human T cell surface markers revealed that most of the cells of these rabies-reactive lines were of the helper/inducer class of T lymphocytes. Stimulation of the T cell lines with the rabies antigens induced the production of interferon-gamma, a lymphokine with potent antiviral activity. Several T cell clones were isolated from two of these cell lines, and most of them appeared to be specific for the antigenic components of the viral nucleocapsid. Two T cell clones specific for the rabies glycoprotein were also isolated from one of these lymphocyte interleukin 2-dependent lines. Further in vitro studies with rabies-specific T cells could help us to understand in more depth the role of regulatory T cells in the human immune response to rabies virus.     

Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled (²H/¹⁵N/¹³C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1β were assessed by surface plasmon resonance yielding K_D values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.     

Loss of CpG dinucleotides from DNA. VI. Methylation of mitochondrial and chloroplast genes

From nucleotide sequences of mitochondrial and chloroplast genes the probable frequency of the CpG----TpG + CpA substitutions was determined. These substitutions may indicate the level of prior DNA methylation. It was found that the level of this methylation is significantly lower in mitochondrial DNA (mtDNA) and chloroplast DNA (chDNA) than in nuclear DNA (nDNA) of the same species. The species (taxon) specificity of mtDNA and chDNA methylation was revealed. A correlation was found between the level of CpG methylation in nDNA, and mtDNA and chDNA in different organisms. It is shown that cytosine residues in CpG were not subjected to significant methylation in the fungi and invertebrate mtDNA and also in the algae chDNA. In contrast, the vertebrate mtDNA bears the impress of CpG-supression, which is confirmed by direct data on methylation of these DNA. Here the first data on the possible enzymatic methylation of the plant mtDNA and chDNA were obtained. It was shown that the degree of CpG-suppression in the 5S rRNA nuclear genes of lower and higher plants is significantly higher in the chloroplast genes of 4,5S and 5S rRNA. From data on pea chDNA hydrolysis with MspI and HpaII it was established that in CCGG sequences this DNA is not methylated. The role of DNA methylation in increasing the mutation rate and in accelerating the evolutionary rates of vertebrate mtDNA is discussed.     

Characterization of cytoplasmic arginine-rich basic protein of Guerin epithelioma

Summary Arginine-rich basic protein from cytoplasma of Guerin epitheliomas has been isolated and characterized. It contains five amino acids: arginine, lysine, glycine, alanine and glutamic acid which make together 74 per cent of all amino acid residues. The protein has a cationic character with an isoelectric point of 8.2. No carbohydrate component was found in this protein. The significance of arginine-rich basic protein in the cytoplasma of Guerin epithelioma is discussed briefly.     

Influence of b2 adrenergic receptor polymorphism (rs1042713 and rs1042714) on anthropometric,hormonal and lipid profiles in polycystic ovarian syndrome

BackgroundPolycystic ovarian syndrome (PCOS) is a frequently encountered disorder. This study aimed to identify polymorphisms in ADRB2 in Saudi PCOS development and to study its influence on lipids, hormones, and anthropometric parameters.MethodsSaudi females (100) suffering from PCOS and healthy controls (100) were investigated. The estimation of cholesterol, triglycerides, high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), plasma glucose, leptin Insulin, and ghrelin were carried out. The DNA was extracted, and ADRB2 fragment carrying the exon 1 was amplified and sequenced.ResultsThe waist, W/H ratio, lipids, glucose, and insulin were significantly higher in the obese PCOS compared to the normal weight group. The leptin and ghrelin were not different. Two single nucleotide polymorphisms (SNPs): rs1042713 (Arg16Gly; A>G) and rs1042714 (Gln27Glu; C>G) were identified. The genotype and allele frequency of rs1042713 did not differ in the total PCOS and normal weight, and obese PCOS compare to the controls. However, rs1042714 was significantly associated with PCOS development, where the minor G allele was protective against PCOS development.ConclusionsThe rs1042714 polymorphism of the ADRB associates with PCOS development in Saudis, while rs1042713 does not. However, the GG genotype of rs1042713 associates significantly with elevated BMI, waist, hip, W/H, and leptin, and decreased ghrelin. On the other hand, rs1042714 genotypes do not associate with any abnormality except the homozygous GG have higher triglycerides and lower HDL-C. Interestingly, glucose showed different correlation patterns in individuals carrying different genotypes of the two studied SNP, indicating clearly that the metabolic responses to a normal nutrient are significantly influenced by the genotypes of the SNPs in ADRB2.     

Fast reactivation of photosynthesis in arctic phytoplankton during the polar night1

Arctic microalgae experience long periods of continuous darkness during the polar night, when they are unable to photosynthesize. Despite numerous studies on overwintering strategies, such as utilization of stored energy products, formation of resting stages, reduction of metabolic rates and heterotrophic lifestyles, there have been few attempts to assess the in situ physiological state and restoration of the photosynthetic apparatus upon re‐illumination. In this study, we found diverse and active marine phytoplankton communities during the polar night at 78°N. Furthermore, we observed rapid changes (≤20 min) in the efficiency of photosynthetic electron transport upon re‐illumination. High photosynthetic capacity and net primary production were established after 24 h of re‐illumination. Our results suggest that some Arctic autotrophs maintain fully functional photosystem II and downstream electron acceptors during the polar night even though the low in situ net primary production levels measured in January prove that light was not sufficient to support any measurable primary production. Due to low temperatures resulting in low respiratory rates as well as the absence of photodamage during the polar night, maintenance of basic photosynthetic machinery may actually pose relatively low metabolic costs for algal cells. This could allow Arctic microalgae to endure the polar night without the formation of dormant stages, enabling them to recover and take advantage of light immediately upon the suns return during the winter–spring transition.