Analysis of IL-12 p40 subunit gene and IFN-γ G5644A polymorphisms in Idiopathic Pulmonary Fibrosis

Background

Genes encoding cytokine mediators are prime candidates for genetic analysis in conditions with T-helper (Th) cell disease driven imbalance. Idiopathic Pulmonary Fibrosis (IPF) is a predominantly Th2 mediated disease associated with a paucity of interferon-gamma (IFN-γ). The paucity of IFN-γ may favor the development of progressive fibrosis in IPF. Interleukin-12 (IL-12) plays a key role in inducing IFN-γ production. The aim of the current study was to assess whether the 1188 (A/C) 3'UTR single nucleotide polymorphism (SNP) in the IL-12 p40 subunit gene which was recently found to be functional and the 5644 (G/A) 3' UTR SNP of the IFN-γ gene were associated with susceptibility to IPF.

Methods

We investigated the allelic distribution in these loci in UK white Caucasoid subjects comprising 73 patients with IPF and 157 healthy controls. The SNPs were determined using the polymerase chain reaction in association with sequence-specific primers incorporating mismatches at the 3'-end.

Results

Our results showed that these polymorphisms were distributed similarly in the IPF and control groups

Conclusion

We conclude that these two potentially important candidate gene single nucleotide polymorphisms are not associated with susceptibility to IPF.     

Efficient gene delivery into murine ovarian cells by intraovarian injection of plasmid DNA and subsequent in vivo electroporation

We describe the use of direct injection of circular plasmid DNA and subsequent in vivo electroporation (EP) for efficient gene delivery to the ovarian cells, including follicular cells and oocytes of mice. When Trypan blue (TB) was injected into the central portion of an ovary by a glass micropipette, rapid dispersion of TB to each preantral and antral follicle was observed. Injections of lacZ-expressing plasmid DNA and subsequent in vivo EP resulted in transfection of follicles with efficiencies ranging from 8-60%, together with cells in the thecal portion of the ovary. Of the lacZ-positive follicles, some oocytes were also positive for lacZ activity. These findings suggest that a solution introduced inside the ovary is rapidly dispersed to each follicle. With this technique, we expect great progress in genetic engineering in murine ovary.     

Nested PCR for detection of mutans streptococci in dental plaque

AIMS: Mutans streptococci such as Streptococcus mutans and Streptococcus sobrinus have been implicated in human dental caries. In an attempt to develop a rapid and sensitive method for detecting Strep. mutans and Strep. sobrinus in dental plaque, a nested PCR amplification based on the 16S rRNA gene was employed. METHODS AND RESULTS: A universal set of PCR primers for bacterial 16S rRNA gene was introduced for the first PCR, and then two sets of primers specific for the 16S rRNA gene sequences of either Strep. mutans or Strep. sobrinus were used for the second PCR. Eighteen plaque samples were analyzed, and a nested PCR was shown to be more sensitive for detecting Strep. mutans and Strep. sobrinus than direct PCR. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The 16S rRNA gene-based nested PCR method is a rapid and sensitive method for the detection of mutans streptococci, and may also be suitable for carrying out large-scale studies on the cariogenicity of mutans streptococci.     

Erythrina poeppigiana-derived phytochemical exhibiting antimicrobial activity against Candida albicans and methicillin-resistant Staphylococcus aureus

AIMS: To screen five phytochemicals isolated from Erythrina poeppigiana (Leguminosae) for antimicrobial activity against both Candida albicans and methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: Roots of E. poeppigiana were macerated with acetone and the chloroform-soluble fraction of the residue was subjected to repeated silica gel column chromatography using various eluting solvents. Structures of the isolated compounds were determined by extensive spectroscopic studies. Each compound was dissolved in dimethyl sulphoxide and added to agar plates (final concentration: 1.56-100 microg ml(-1)) and minimum inhibitory concentrations (MICs) against C. albicans and MRSA were determined. Spectral data indicated the presence of three different types of phytochemicals; isoflavonoids (erypoegin A, demethylmedicarpin and sandwicensin), alpha-methyldeoxybenzoin (angolensin) and cinnamylphenol (erypostyrene). While all compounds showed anti-MRSA activity in this concentration range, isoflavonoids and alpha-methyldeoxybenzoin failed to inhibit the growth of C. albicans. Erypostyrene (E-1-[2-hydroxy-4-methoxy-5-(gamma,gamma-dimethylallyl)benzyl]-2-(4-hydroxyphenyl)ethylene) exhibited not only the highest anti-MRSA activity (MIC value of 6.25 microg ml(-1)) but also anti-candidal potency (MIC value of 50 microg ml(-1)). The compound reduced viable cell numbers of C. albicans and MRSA by approximately 1 of 2000 and 1 of 1000 after 1 h incubation at each MIC, respectively. CONCLUSIONS: A new cinnamylphenol, erypostyrene, possessed anti-candidal and anti-MRSA activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Erypostyrene could be a leading candidate for development of antimicrobial agents with anti-candidal and anti-MRSA activity.     

Compensated hypertrophy of cardiac ventricles in aged transgenic FVB/N mice overexpressing calsequestrin

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed contractile parameters and hypertrophy in transgenic mice. To determine the long-term consequences of calsequestrin overexpression, the cardiac phenotype of young (2–3-months old) and aged (17 months old) transgenic FVB/N mice was characterized. Ventricular/body weight ratios, which were increased in young transgenics compared with wild-types, were unaltered with age. Left atria of aged transgenics exhibited enlargement and mineralization, but their ventricles did not display fibrosis, mineralization and other injuries. Although echocardiography suggested a time-dependent change in ventricular geometry and loading conditions in vivo, as well as an age-dependent reduction of left ventricular fractional shortening in transgenic mice, Langendorff-perfused hearts of young and aged transgenics indicated that there were no age-related reductions of contractile parameters (±dP/dt). Furthermore, neither genotype nor age altered lung/body weight ratios. Thus, our findings suggest that left ventricular performance in calsequestrin overexpressing mice becomes apparently depressed with age, but this depression is not associated with progressive reduction of left ventricular contractility and heart failure.     

Ser-130 of Natronobacterium pharaonis halorhodopsin is important for the chloride binding

Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump from Natronobacterium pharaonis. In order to clarify the role of Ser-130(phR) residue which corresponds to Ser-115(shR) for salinarum hR on the anion-binding affinity, the wild-type and Ser-130 mutants substituted with Thr, Cys and Ala were expressed in E. coli cells and solubilized with 0.1% n-dodecyl beta-D-maltopyranoside The absorption maximum (lambda(max)) of the S130T mutant indicated a blue shift from that of the wild type in the absence and presence of chloride. For S130A, a large red shift (12 nm) in the absence of chloride was observed. The wild-type and all mutants showed the blue-shift of lambda(max) upon Cl(-) addition, from which the dissociation constants of Cl(-) were determined. The dissociation constants were 5, 89, 153 and 159 mM for the wild-type, S130A, S130T and S130C, respectively, at pH 7.0 and 25 degrees C. Circular dichroic spectra of the wild-type and the Ser-130 mutants exhibited an oligomerization. The present study revealed that the Ser-130 of N. pharaonis halorhodopsin is important for the chloride binding.     

Superoxide and hydrogen peroxide production by Drosophila mitochondria

Drosophila melanogaster is a key model organism for genetic investigation of the role of free radicals in aging, but biochemical understanding is lacking. Superoxide production by Drosophila mitochondria was measured fluorometrically as hydrogen peroxide, using its dependence on substrates, inhibitors, and added superoxide dismutase to determine sites of production and their topology. Glycerol 3-phosphate dehydrogenase and center o of complex III in the presence of antimycin had the greatest maximum capacities to generate superoxide on the cytosolic side of the inner membrane. Complex I had significant capacity on the matrix side. Center i of complex III, cytochrome c, and complex IV produced no superoxide. Native superoxide generation by isolated mitochondria was also measured without added inhibitors. There was a high rate of superoxide production with sn-glycerol 3-phosphate as substrate; two-thirds mostly from glycerol 3-phosphate dehydrogenase on the cytosolic side and one-third on the matrix side from complex I following reverse electron transport. There was little superoxide production from any site with NADH-linked substrate. Superoxide production by complex I following reverse electron flow from glycerol 3-phosphate was particularly sensitive to membrane potential, decreasing 70% when potential decreased 10 mV, showing that mild uncoupling lowers superoxide production in the matrix very effectively.     

Natural resistance to Clover yellow vein virus in beans controlled by a single recessive locus

We characterized the resistance of the common bean cv. Jolanda to Clover yellow vein virus no. 30 (ClYVV). After inoculation, the virus was detected in neither inoculated nor upper leaves, suggesting that the resistance operates at either the viral replication or cell-to-cell movement level. To analyze the mechanism of resistance, we developed a green fluorescent protein (GFP)-tagged ClYVV, and monitored GFP fluorescence at sites of infection on ClYVV-inoculated leaves. No GFP fluorescence was detected in Jolanda, whereas its expression in single cells and spread on inoculated leaves were observed clearly in susceptible cultivars. ClYVV-introduced Jolanda cells were found to be still viable; therefore, it is unlikely that the restriction of multiplication was due to rapid cell death. Genetic analysis indicated that a single recessive locus controlled the resistant phenotype of Jolanda. We designated this locus desc (determinant of susceptibility to ClYVV). Meanwhile, a spontaneous mutant virus that overcomes the resistance (ClYVV-Br) was isolated. Inoculation assays using chimeric viruses suggested that a viral genome-linked protein (VPg) might be the avirulence determinant. The resistance mechanism may be associated with the role of VPg in the viral infection cycle.     

Presence of free D-amino acids in microalgae

Several species of microalgae (phytoplankton), 4 species of freshwater algae and 4 species of marine diatoms, were cultured germ-free in the laboratory. The presence of free D-amino acids was verified in these species by a reversed-phase HPLC analysis. D-Aspartate was detected in all the microalgae examined, but D-alanine was only present in the marine diatoms. The D-amino acid content in Asterionella sp. of the marine diatoms increased from the exponential phase to the stationary phase and then decreased to the phase of decline.     

A root-specific O-methyltransferase gene expressed in salt-tolerant barley

A cDNA encoding an O-methyltransferase (OMT) was isolated from salt-tolerant barley roots by subtraction hybridization with cDNAs of salt-tolerant barley roots as a tester cDNA and cDNAs of the salt-sensitive barley roots as a driver cDNA. The deduced amino acid sequence showed significant identity with plant caffeic acid/5-hydroxyferulic acid OMTs. Southern blot analysis showed that the OMT gene was a single copy in both salt-tolerant and -sensitive barley. The cloned gene was expressed in a wheat germ cell-free system to produce the OMT, which had methylating activity for caffeic acid. Northern blot analysis showed that the OMT gene was expressed constitutively in the salt-tolerant barley roots and the expression level was increased 1.5 times by salt stress, but the salt-sensitive barley showed no expression of the gene in roots and leaves.