Polyamines may regulate S-phase progression but not the dynamic changes of chromatin during the cell cycle

Several studies suggest that polyamines may stabilize chromatin and play a role in its structural alterations. In line with this idea, we found here by chromatin precipitation and micrococcal nuclease (MNase) digestion analyses, that spermidine and spermine stabilize or condense the nucleosomal organization of chromatin in vitro. We then investigated the possible physiological role of polyamines in the nucleosomal organization of chromatin during the cell cycle in Chinese hamster ovary (CHO) cells deficient in ornithine decarboxylase (ODC) activity. An extended polyamine deprivation (for 4 days) was found to arrest 70% of the odc⁻ cells in S phase. MNase digestion analyses revealed that these cells have a highly loosened and destabilized nucleosomal organization. However, no marked difference in the chromatin structure was detected between the control and polyamine-depleted cells following the synchronization of the cells at the S-phase. We also show in synchronized cells that polyamine deprivation retards the traverse of the cells through the S phase already in the first cell cycle. Depletion of polyamines had no significant effect on the nucleosomal organization of chromatin in G₁–early S. The polyamine-deprived cells were also capable of condensing the nucleosomal organization of chromatin in the S/G₂ phase of the cell cycle. These data indicate that polyamines do not regulate the chromatin condensation state during the cell cycle, although they might have some stabilizing effect on the chromatin structure. Polyamines may, however, play an important role in the control of S-phase progression. J. Cell Biochem. 68:200–212, 1998. © 1998 Wiley-Liss, Inc.     

Biodistribution of adenoviral vector to nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods.

Expression of transgene other than in the target tissue may cause side effects and safety problems in gene therapy. We analyzed biodistribution of transgene expression after intravascular and periadventitial gene delivery methods using the first generation nuclear-targeted lacZ adenovirus. RT-PCR and X-Gal stainings were used to study transgene expression 14 days after the gene transfer. After intravascular catheter-mediated gene transfer to rabbit aorta mimicking angioplasty procedure, the target vessel showed 1.1% +/- 0. 5 gene transfer efficiency. Other tissues showed varying lacZ gene expression indicating a systemic leakage of the vector with the highest transfection efficiency in hepatocytes (0.7% +/- 0.5). X-Gal staining of blood cells 24 h after the intravascular gene transfer indicated that a significant portion (1.8% +/- 0.8) of circulating monocytes was transfected. X-Gal-positive cells were also found in testis. After periadventitial gene transfer using a closed silicon capsule placed around the artery, 0.1% +/- 0.1 lacZ-positive cells were detected in the artery wall. Positive cells were also found in the liver and testis (<0.01%), indicating that the virus escapes even from the periadventitial space, although less extensively than during the intravascular application. We conclude that catheter-mediated intravascular and, to a lesser extent, periadventitial gene transfer lead to leakage of adenovirus to systemic circulation, followed by expression of the transgene in several tissues. Possible consequences of the ectopic expression of the transgene should be evaluated in gene therapy trials even if local gene delivery methods are used.     

An improved colloidal iron stain for glomerular sialoproteins

The thick sialoglycoprotein coat of the glomerular epithelial cells is a part of the glomerular filtration barrier. It can be demonstrated in paraffin histoslogy by means of the colloidal iron procedure. We have studied the special requirements of tissue processing to obtain reproducible results by this staining method. The requirements include proper fixation, avoiding heat during sectioning and ample rehydration. We have further tested a colloidal iron-PAS procedure for simultaneous demonstration of the glomerular epithelial coat and the basement membrane.     

Comparative molecular field analysis and molecular dynamics studies of α/β hydrolase domain containing 6 (ABHD6) inhibitors

The endocannabinoid system remains an attractive molecular target for pharmacological intervention due to its roles in the central nervous system in learning, thinking, emotional function, regulation of food intake or pain sensation, as well as in the peripheral nervous system, where it modulates the action of cardiovascular, immune, metabolic or reproductive function. α/β hydrolase domain containing 6 (ABHD6)—an enzyme forming part of the endocannabinoid system—is a newly discovered post-genomic protein acting as a 2-AG (2-arachidonoylglycerol) serine hydrolase. We have recently reported a series of 1,2,5-thiadiazole carbamates as potent and selective ABHD6 inhibitors. Here, we present comparative molecular field analysis (CoMFA) and molecular dynamics studies of these compounds. First, we performed a homology modeling study of ABHD6 based on the assumption that the catalytic triad of ABHD6 comprises Ser148–His306–Asp 278 and the oxyanion hole is formed by Met149 and Phe80. A total of 42 compounds was docked to the homology model using the Glide module from the Schrödinger suite of software and the selected docking poses were used for CoMFA alignment. A model with the following statistics was obtained: R ² = 0.98, Q ² = 0.55. In order to study the molecular interactions of the inhibitors with ABHD6 in detail, molecular dynamics was performed with the Desmond program. It was found that, during the simulations, the hydrogen bond between the inhibitor carbonyl group and the main chain of Phe80 is weakened, whereas a new hydrogen bond with the side chain of Ser148 is formed, facilitating the possible formation of a covalent bond.

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Graphical Abstract Left–right: Docking pose of 1 in the binding pocket of α/β hydrolase domain containing 6 (ABHD6) selected for molecular alignment; CoMFA steric and electrostatic contour fields; changes in potential energy of the complex during simulations for the complex of 6 and ABHD6

     

Determinants of accelerated metabolomic and epigenetic aging in a UK cohort

Markers of biological aging have potential utility in primary care and public health. We developed a model of age based on untargeted metabolic profiling across multiple platforms, including nuclear magnetic resonance spectroscopy and liquid chromatography–mass spectrometry in urine and serum, within a large sample (N = 2,239) from the UK Airwave cohort. We validated a subset of model predictors in a Finnish cohort including repeat measurements from 2,144 individuals. We investigated the determinants of accelerated aging, including lifestyle and psychological risk factors for premature mortality. The metabolomic age model was well correlated with chronological age (mean r = .86 across independent test sets). Increased metabolomic age acceleration (mAA) was associated after false discovery rate (FDR) correction with overweight/obesity, diabetes, heavy alcohol use and depression. DNA methylation age acceleration measures were uncorrelated with mAA. Increased DNA methylation phenotypic age acceleration (N = 1,110) was associated after FDR correction with heavy alcohol use, hypertension and low income. In conclusion, metabolomics is a promising approach for the assessment of biological age and appears complementary to established epigenetic clocks.