The cardiac extracellular matrix, composed predominantly of collagenous fibers, forms a stress-tolerant network that facilitates the distribution of forces generated in the heart and provides for proper alignment of cardiac myocytes. Although considerable information exists regarding the morphological organization of the heart extracellular matrix, little is known about the regulation of the synthesis and accumulation of extracellular matrix components. A potentially significant factor in the cardiovascular system is mechanical stimulation including changes in physical tension and pressure. We recently have developed an in vitro model system to elucidate the effects of mechanical stretch on isolated populations of heart cells. In the present study, we have used biochemical and molecular biological techniques to analyze changes in collagen synthesis by cardiac fibroblasts in response to mechanical stretch. These studies show that the ratio of collagen type III to collagen type I increases in mechanically stretched cells. They also show that type III collagen mRNA levels are increased in response to cyclic mechanical stretch for durations as short as 12 hours. Type I collagen mRNA levels were not found to change under the stretch conditions used in this study. Our results emphasize the potential regulatory role of mechanical stimulation in the expression of specific genes in the heart and support previous studies indicating this to be an intriguing in vitro model of cardiac hypertrophy.

A major mouse T-lymphoma surface glycoprotein (gp180) has been identified by labeling cells with 1251 and [3H]glucosamine. After ligand-induced receptor patching and/or capping, the amount of gp180 in the membrane-associated cytoskeleton fraction increases in direct proportion to the percentage of patched/capped cells. There is a parallel increase in the amount of fodrin in the membrane-associated cytoskeleton fraction. Evidence is presented that gp180 is the same as or very similar to the T-lymphocyte-specific glycoprotein T-200. An immunobinding assay of Nonidet P-40-solubilized plasma membrane selectively co-isolates gp180 and fodrin. After induction of receptor rearrangement, double-label immunofluorescence reveals that fodrin accumulated directly beneath gp180 patches and caps. Membrane extraction with Triton X-114 followed by sucrose gradient centrifugation permits isolation of a gp180-fodrin complex with a 1:1 molar ratio and sedimentation coefficient(s) of approximately 20. This complex remains stable during isoelectric focusing and exhibits a pl in the range of 5.2-5.7. On the basis of our results we conclude that gp180, an intergral membrane glycoprotein, and fodrin, a component of the membrane-associated cytoskeleton, are closely associated into a complex. Furthermore, we contend that, through fodrin's association with actin, this complex is of functional significance in ligand-induced patching and capping of gp180. We also propose that, through lateral interactions in the plane of the membrane, the gp180-fodrin complex might be responsible for linking other surface receptors to the intracellular microfilament network during lymphocyte patching and capping.The involvement of contractile microfilaments in the redistribution of cell surface receptors during patching and capping was first described by Taylor et al. in 1971 (1). Since then, numerous reports have confirmed this observation for a variety of different surface receptors of mouse and human lymphocytes (2, 3). Double-label immunofluorescence microscopy reveals that cytoplasmic actin and myosin accumulate directly beneath patches and caps; that is, beneath aggregated surface receptors (4-7). More conclusively, isolation of the plasma membrane (PM) t and associated cytoskeletal elements with non-ionic detergents demonstrates that, during capping, the surface receptors form a specific association with the actin-containing cytoskeleton (8, 9). However, the nature of the linkage between membrane surface receptors and the Abbreviations used in this paper." IEF, isoelectric focusing; NP-40, Nonidet P-40; PBES, phosphate-buffered Earle's balanced salt solution; PM, plasma membrane. cytoskeleton is not understood.Recently, analogs to proteins of the erythrocyte membranecytoskeleton complex (such as spectrin, ankyrin, and glycophorin) have been identified in nonerythroid cells (10)(11)(12)(13)(14). Two distinct, spectrin-like proteins have been identified: fodrin (15-18) isolated from brain ; and TW 260/240 (16,17) isolated from epithelial cells...

ESI FTICR mass spectrometry is the only technique currently used for accurate molecular weight analysis of PCR products above 100 bp in size. This is important in demonstrating the potential for MS in making major contributions in the molecular biology and genomics areas. In the near future, it is more likely that less expensive, more user friendly MS techniques will be used for high-throughput analyses (including MALDI TOF and ESI quadrupole). There have been numerous reports on the use of MALDI TOF. The current report is to the first to evaluate the use of ESI-quadrupole analysis of PCR products. Synthetic oligonucleotides (30 and 89 mers) and polymerase chain reaction products of varying molecular weight (62, 88, 89, and 114 bp) were analyzed by ESI using a quadrupole MS. The mass accuracy for nucleic acids in the 30-62 bp range was shown to allow determination of nucleotide substitutions and additions/deletions. For higher molecular weight PCR products (88-114 bp), the mass accuracy of ESI-MS distinguishes single or multiple nucleotide insertions/deletions. In addition, ESI quadrupole MS allows determination of molecular weight of both strands of higher molecular weight ds PCR products and can distinguish nucleotide modifications (e.g., with biotin). In conclusion, it is demonstrated that ESI-MS occupies an intermediate position (as compared to MALDI TOF and ESI FTICR) with regard to mass accuracy and resolution in analysis of nucleic acids.

Objective:Chronic pain is an emotionally and physically debilitating form of pain that activates the body's stress response and over time can result in lowered heart rate variability (HRV) power, which is associated with reduced resiliency and lower self-regulatory capacity. This pilot project was intended to determine the effectiveness of HRV coherence biofeedback (HRVCB) as a pain and stress management intervention for veterans with chronic pain and to estimate the effect sizes. It was hypothesized that HRVCB will increase parasympathetic activity resulting in higher HRV coherence measured as power and decrease self-reported pain symptoms in chronic pain patients.Study design:Fourteen veterans receiving treatment for chronic pain were enrolled in the pre-post intervention study. They were randomly assigned, with 8 subjects enrolled in the treatment group and 6 in the control group. The treatment group received biofeedback intervention plus standard care, and the other group received standard care only. The treatment group received four HRVCB training sessions as the intervention.Measures:Pre-post measurements of HRV amplitude, HRV power spectrum variables, cardiac coherence, and self-ratings of perceived pain, stress, negative emotions, and physical activity limitation were made for both treatment and control groups.Results:The mean pain severity for all subjects at baseline, using the self-scored Brief Pain Inventory (BPI), was 26.71 (SD=4.46; range=21–35) indicating a moderate to severe perceived pain level across the study subjects. There was no significant difference between the treatment and control groups at baseline on any of the measures. Post-HRVCB, the treatment group was significantly higher on coherence (P=.01) and lower (P=.02) on pain ratings than the control group. The treatment group showed marked and statistically significant (1-tailed) increases over the baseline in coherence ratio (191%, P=.04) and marked, significant (1-tailed) reduction in pain ratings (36%, P<.001), stress perception (16%, P=.02), negative emotions (49%, P<.001), and physical activity limitation (42%, P<.001). Significant between-group effects on all measures were found when pre-training values were used as covariates.Conclusions:HRVCB intervention was effective in increasing HRV coherence measured as power in the upper range of the LF band and reduced perceived pain, stress, negative emotions, and physical activity limitation in veterans suffering from chronic pain. HRVCB shows promise as an effective non-pharmacological intervention to support standard treatments for chronic pain.

This study was performed to compare the effects of three well-known phytoestrogens such as genistein, resveratrol, and quercetin on steroidogenesis in MA-10 mouse tumor Leydig cells. Addition of genistein or resveratrol to MA-10 cells resulted in decreases in the cAMP-stimulated progesterone secretion, but quercetin had an opposite response. Steroidogenic acute regulatory (StAR) mRNA expression and StAR promoter activity in transiently transfected MA-10 cells were significantly reduced by genistein or resveratrol, but increased by quercetin. Genistein was found to inhibit MA-10 cell proliferation, while resveratrol and quercetin had no effect. Quercetin-induced increase in cAMP-stimulated progesterone secretion was reversed by ICI 182,780, an estrogen receptor (ER) antagonist. However, ICI 182,780 had no effect on cAMP plus quercetin-stimulated StAR promoter activity. To examine whether non-ER factors are associated with quercetin-stimulated progesterone production, we treated MA-10 cells with EGTA to deprive them of extracellular Ca 2C. We found that EGTA inhibited quercetin-plus cAMP-stimulated progesterone secretion and StAR promoter activity. Blocking of Ca 2C influx through L-or Ttype voltage-gated Ca 2C channels with verapamil or mibefradil respectively, attenuated quercetin-stimulated progesterone secretion, while they had no effect on quercetinplus cAMP-stimulated StAR promoter activity. Blocking of intracellular Ca 2C efflux by sodium orthovanadate, a Ca 2C -pump inhibitor, blocked quercetin-plus cAMP-stimulated progesterone secretion and StAR promoter activity in MA-10 cells. Finally, EGTA or vanadate reduced quercetin and cAMP-increased in StAR mRNA expression in MA-10 cells, while ICI 182,780 had no effect. Taken together, these results indicate that phytoestrogens have differential effects on steroidogenesis in MA-10 cells.