Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients

DNA methylation is believed to regulate gene expression during adulthood in response to the constant changes in environment. The methylome is therefore proposed to be a biomarker of health through age. ANGPTL2 is a circulating pro-inflammatory protein that increases with age and prematurely in patients with coronary artery diseases; integrating the methylation pattern of the promoter may help differentiate age- vs. disease-related change in its expression. We believe that in a pro-inflammatory environment, ANGPTL2 is differentially methylated, regulating ANGPTL2 expression. To test this hypothesis we investigated the changes in promoter methylation of ANGPTL2 gene in leukocytes from patients suffering from post-acute coronary syndrome (ACS). DNA was extracted from circulating leukocytes of post-ACS patients with cardiovascular risk factors and from healthy young and age-matched controls. Methylation sites (CpGs) found in the ANGPTL2 gene were targeted for specific DNA methylation quantification. The functionality of ANGPTL2 methylation was assessed by an in vitro luciferase assay. In post-ACS patients, C-reactive protein and ANGPTL2 circulating levels increased significantly when compared to healthy controls. Decreased methylation of specific CpGs were found in the promoter of ANGPTL2 and allowed to discriminate age vs. disease associated methylation. In vitro DNA methylation of specific CpG lead to inhibition of ANGPTL2 promoter activity. Reduced leukocyte DNA methylation in the promoter region of ANGPTL2 is associated with the pro-inflammatory environment that characterizes patients with post-ACS differently from age-matched healthy controls. Methylation of different CpGs in ANGPTL2 gene may prove to be a reliable biomarker of coronary disease.     

Temperature dependence of the electronic spin-lattice relaxation time in a 2-iron-2-sulphur model complex

The complex [Fe2S2(S2-o-xylyl)2]2- in DMF (dimethylformamide), DMSO (dimethylsulphoxide) or a 1:1 DMF/DMSO mixture, a model for the chromophore in the 2Fe-2S proteins (ferredoxins), has been reduced and studied by conventional EPR over a temperature range. The low-field feature of the spectrum, Hz, has been computer simulated in order to analyse the lineshape in terms of a convolution product of Lorentzian and Gaussian distributions. The Gaussian contribution to the linewidth and a fixed part of the Lorentzian contribution, which is a function of the solvent and the way it freezes, were measured at a low temperature (less than or equal to 100 K) and subtracted from the linewidths in the higher-temperature range (130-200 K). The variable Lorentzian contribution thus obtained was related to spin-lattice relaxation times. The spin-lattice relaxation times of the sample having 1:1 DMSO/DMF solvent were measured in the range 6 to 11 K by the saturating pulse technique and in the range 10 to 65 K by continuous saturation methods. Up to 65 K the results follow the law 1/T1 alpha T4.5, a relationship which is not readily interpreted in terms of a simple Debye model. At higher temperatures the results may be interpreted in terms either of a dominant Orbach mechanism involving excited states at approx. 900 +/- 50 cm-1 (DMSO, DMF) or 770 +/- 50 cm-1 (1:1 DMSO/DMF), or of a Raman process in which 1/T1 alpha T7.5. The former is compatible with the two-phonon process already described in some ferredoxins, especially those with little anisotropy (gy - gx approximately 0.0) which have characteristically high [J] values.     

Nodule formation in soybeans by exopolysaccharide mutants of Rhizobium fredii USDA 191

Production of exopolysaccharides by Rhizobium has been linked with efficient invasion and nodulation of leguminous plant roots by the bacteria. Exopolysaccharide-deficient (exo) mutants of Rhizobium fredii USDA 191 were isolated following Tn5-insertion mutagenesis. Five phenotypically unique exo mutants were investigated for exopolysaccharide synthesis and their ability to nodulate soybeans. The exopolysaccharides produced by these mutants were analysed for polysaccharide composition by column chromatography and thin-layer chromatography. Two mutants designed exo-3 and exo-5 were deficient in both neutral glucan and exopolysaccharide synthesis, but each induced some functional nodules on Glycine max (Peking). The remaining three mutants (exo-1, exo-2 and exo-4) synthesized neutral glucans at levels higher or lower than those in wild-type and exhibited partial exopolysaccharide deficiencies. The data imply that neither exopolysaccharides nor neutral glucans are essential for the induction of determinate nodules by R. fredii.     

High-level expression of the FtsA protein inhibits cell septation in Escherichia coli K-12.

DNA fragments encoding the ftsA gene were subcloned into plasmids downstream of a lac promoter or a tac promoter. These plasmid constructs, when transformed into wild-type and mutant strains, inhibited normal cell septation, causing the formation of long nonseptate filaments. This phenotype is due to overproduction of the FtsA protein.     

Formaldehyde dehydrogenase from the recombinant yeast Hansenula polymorpha: isolation and bioanalytic application

A recombinant yeast clone, a derivative of the recipient Hansenula polymorpha strain NCYC 495, was chosen as an NAD and glutathione-dependent formaldehyde dehydrogenase overproducer. Optimal cultivation conditions for the highest yield of enzyme were established. A simple scheme for the isolation of formaldehyde dehydrogenase from the recombinant strain was proposed, and some characteristics of the purified enzyme were studied. An enzymatic method for formaldehyde assay based on formaldehyde dehydrogenase was developed and used for testing real samples.     

Comparison of Verbal and Pictorial Measures of Hunger During Fasting in Normal Weight and Obese Subjects

Objective: Friedman, Ulrich, and Mattes described a new pictorial instrument for assessing hunger wherein respondents outline areas on a drawing of a human figure to depict the location of their hunger sensations. The present study compared normal weight and obese individuals on the pictorial measure and on more traditional verbal hunger measures during a 22‐hour fast. Research Methods and Procedures: The pictorial measure, along with 13 verbal items assessing hunger and hunger‐related symptoms, was administered to 29 normal weight college students and 46 overweight clinic patients four times during a 22‐hour fast. Factor analyses of verbal hunger items produced Hunger, Somatic Symptoms, and Stomach Symptoms factors. The pictorial measure was divided into peripheral (arms, legs, head) and central (trunk) body areas. Results: The increases in hunger during the fast were greater when measured using the pictorial as opposed to the verbal instrument. Correlations between and within the three verbal hunger measures and two pictorial measures were generally few in number and modest in size. The overall pattern of correlations suggested that the verbally based hunger measures more adequately reflected the experience of hunger in normal weight than in obese individuals. A significant interaction between weight status and assessment period was found for the pictorial measure, indicating that normal weight subjects experienced more bodily hunger than overweight subjects initially but experienced less hunger than obese subjects after a prolonged period of food deprivation. Discussion: Although more testing is needed, these results suggest that the pictorial hunger assessment provides information about the experience of hunger that could complement information provided by traditional verbally based hunger measures.     

C-shaped cells caused by expression of an ftsA mutation in Escherichia coli.

A plasmid, pDLL4, was isolated from a Tn5tac1 mutagenesis experiment with plasmid pZAQ. When pDLL4 was transformed into wild-type rod-shaped cells, it caused cells in the population to become curved (C-shaped or convoluted). The Tn5tac1 transposon was integrated within the carboxyl end of the ftsA gene in pDLL4. This mutation was designated ftsAc. Subcloning ftsAc DNA into another plasmid vector verified that the curved-cell phenotype was caused by the expression of this altered gene. DNA sequence analysis of the ftsAc mutation revealed that the transposition event changed the DNA so that the last 28 amino acids of the FtsA protein were lost and 5 new amino acids were added. A radioactive peptide band corresponding to this truncated FtsAc protein was identified by a T7 promoter-T7 polymerase protein labeling system. Observations of thin sections of these curved cells with an electron microscope revealed aggregates of striated cylindrical structures traversing the cytoplasm. The ends of these aggregates appear to be at or near the cell membrane. The linear periodicity of the cylinders was approximately 11 nm, and the diameter of a cylinder was about 15 nm. Aggregates of as many as five cylinders were arrayed diagonally to the long axis of the curved cells, a finding that suggests that some type of internal organization may be causing the curved cell shape.     

Spin lattice relaxation and exchange interaction in a 2-iron, 2-sulphur protein.

A two-iron-two-sulphur non-haem iron protein, the ferredoxin from Spirulina maxima, has been studied by means of electron paramagnetic resonance (EPR) in the range where the spectrum loses resolution with increasing temperature. The spin-lattice relaxation times were deduced from linewidths measured by spectral simulation and their variation as a function of temperature is interpreted in terms of an Orbach mechanism. On this basis, the exchange integral between the two iron atoms, assuming as antiferromagnetic interaction between them, is estimated to be - 83 cm-1.     

Activities of the enzymes of formaldehyde catabolism in recombinant strains of <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Activities of the enzymes of formaldehyde (FA) catabolism in recombinant strains of the methylotrophic yeast Hansenula polymorpha overproducing NAD⁺- and glutathione-dependent formaldehyde dehydrogenase (FADH) were studied under different cultivation conditions and at elevated FA content. Southern dot-blot analysis confirmed the presence of six to eight copies of the target FLD1 gene in stable recombinant clones of H. polymorpha. Under certain cultivation conditions, the transformants resistant to elevated FA concentrations were shown to produce FADH and other bioanalytically important enzymes: formate dehydrogenase, alcohol dehydrogenase, alcohol oxidase, and formaldehyde reductase. The optimal cultivation conditions for recombinants were determined, resulting in maximum synthesis of FADH: methanol as a carbon source, methylamine as a nitrogen source, FA as an inducer, temperature of 37°C, and cells in the early exponential phase of growth.