Development of a novel method to determine very low density lipoprotein kinetics

Isotopic tracer methods of determining triglyceride-rich lipoprotein (TRL) kinetics are costly, time-consuming, and labor-intensive. This study aimed to develop a simpler and cost-effective method of obtaining TRL kinetic data, based on the fact that chylomicrons compete with large VLDL (VLDL(1); S(f) = 60-400) for the same catalytic pathway. Ten healthy subjects [seven men; fasting triglyceride (TG), 44.3-407.6 mg/dl; body mass index, 21-35 kg/m(2)] were given an intravenous infusion of a chylomicron-like TG emulsion (Intralipid; 0.1 g/kg bolus followed by 0.1 g/kg/h infusion) for 75-120 min to prevent the clearance of VLDL(1) by lipoprotein lipase. Multiple blood samples were taken during and after infusion for separation of Intralipid, VLDL(1), and VLDL(2) by ultracentrifugation. VLDL(1)-apolipoprotein B (apoB) and TG production rates were calculated from their linear increases in the VLDL(1) fraction during the infusion. Intralipid-TG clearance rate was determined from its exponential decay after infusion. The production rates of VLDL(1)-apoB and VLDL(1)-TG were (mean +/- SEM) 25.4 +/- 3.9 and 1,076.7 +/- 224.7 mg/h, respectively, and the Intralipid-TG clearance rate was 66.9 +/- 11.7 pools/day. Kinetic data obtained from this method agree with values obtained from stable isotope methods and show the expected relationships with indices of body fatness and insulin resistance (all P < 0.05). The protocol is relatively quick, inexpensive, and transferable to nonspecialist laboratories.     

Characterization of insulin/IGF hybrid receptors: contributions of the insulin receptor L2 and Fn1 domains and the alternatively spliced exon 11 sequence to ligand binding and receptor activation

The IR (insulin receptor) and IGFR (type I insulin-like growth factor receptor) are found as homodimers, but the respective pro-receptors can also heterodimerize to form insulin-IGF hybrid receptors. There are conflicting data on the ligand affinity of hybrids, and especially on the influence of different IR isoforms. To investigate further the contribution of individual ligand binding epitopes to affinity and specificity in the IR/IGFR family, we generated hybrids incorporating both IR isoforms (A and B) and IR/IGFR domain-swap chimaeras, by ectopic co-expression of receptor constructs in Chinese hamster ovary cells, and studied ligand binding using both radioligand competition and bioluminescence resonance energy transfer assays. We found that IR-A-IGFR and IR-B-IGFR hybrids bound insulin with similar relatively low affinity, which was intermediate between that of homodimeric IR and homodimeric IGFR. However, both IR-A-IGFR and IR-B-IGFR hybrids bound IGF-I and IGF-II with high affinity, at a level comparable with homodimeric IGFR. Incorporation of a significant fraction of either IR-A or IR-B into hybrids resulted in abrogation of insulin- but not IGF-I-stimulated autophosphorylation. We conclude that the sequence of 12 amino acids encoded by exon 11 of the IR gene has little or no effect on ligand binding and activation of IR-IGFR hybrids, and that hybrid receptors bind IGFs but not insulin at physiological concentrations regardless of the IR isoform they contained. To reconstitute high affinity insulin binding within a hybrid receptor, chimaeras in which the IGFR L1 or L2 domains had been replaced by equivalent IR domains were co-expressed with full-length IR-A or IR-B. In the context of an IR-A-IGFR hybrid, replacement of IR residues 325-524 (containing the L2 domain and part of the first fibronectin domain) with the corresponding IGFR sequence increased the affinity for insulin by 20-fold. We conclude that the L2 and/or first fibronectin domains of IR contribute in trans with the L1 domain to create a high affinity insulin-binding site within a dimeric receptor.     

Regulation of DAPK1 by Natural Products: An Important Target in Treatment of Stroke

Neurochemical Research - Stroke is a sudden neurological disorder that occurs due to impaired blood flow to an area of the brain. Stroke can be caused by the blockage or rupture of a blood vessel...     

Salt-stress induced physiological and proteomic changes in tomato (Solanum lycopersicum) seedlings

Soil salinity is one of the major abiotic stress limiting crop productivity and the geographical distribution of many important crops worldwide. To gain a better understanding of the salinity stress responses at physiological and molecular level in cultivated tomato (Solanum lycopersicum. cv. Supermarmande), we carried out a comparative physiological and proteomic analysis. The tomato seedlings were cultivated using a hydroponic system in the controlled environment growth chamber. The salt stress (NaCl) was applied (0, 50, 100, 150 and 200?mM), and maintained for 14 days. Salt treatment induced a plant growth reduction estimated as fresh-dry weight. Photosynthetic pigments (chlorophyll a, b) content of NaCl-treated tomato plants was significantly decreased as the salinity level increased. Proline accumulation levels in leaf and root tissues increased significantly with increasing NaCl concentration. Relative electrolyte leakage known as an indicator of membrane damage caused by salt stress was increased proportionally according to the NaCl concentrations. Roots of control and salt-stressed plants were also sampled for phenol protein extraction. Proteins were separated by two-dimensional gel electrophoresis (2-DGE). Several proteins showed up- and downregulation during salt stress. MALDI-TOF/MS analysis and database searching of some of the identified proteins indicated that the proteins are known to be in a wide range of physiological processes, that is, energy metabolism, ROS (reactive oxygen species) scavenging and detoxification, protein translation, processing and degradation, signal transduction, hormone and amino acid metabolism, and cell wall modifications. All proteins might work cooperatively to reestablish cellular homeostasis under salt stress, water deficiency, and ionic toxicity.     

Identification of mitogen-activated protein/extracellular signal-responsive kinase kinase 2 as a novel partner of the scaffolding protein human homolog of disc-large

Human disc-large homolog (hDlg), also known as synapse-associated protein 97, is a scaffold protein, a member of the membrane-associated guanylate kinase family, implicated in neuronal synapses and epithelial-epithelial cell junctions whose expression and function remains poorly characterized in most tissues, particularly in the vasculature. In human vascular tissues, hDlg is highly expressed in smooth muscle cells (VSMCs). Using the yeast two-hybrid system to screen a human aorta cDNA library, we identified mitogen-activated protein/extracellular signal-responsive kinase (ERK) kinase (MEK)2, a member of the ERK cascade, as an hDlg binding partner. Site-directed mutagenesis showed a major involvement of the PSD-95, disc-large, ZO-1 domain-2 of hDlg and the C-terminal sequence RTAV of MEK2 in this interaction. Coimmunoprecipitation assays in both human VSMCs and human embryonic kidney 293 cells, demonstrated that endogenous hDlg physically interacts with MEK2 but not with MEK1. Confocal microscopy suggested a colocalization of the two proteins at the inner layer of the plasma membrane of confluent human embryonic kidney 293 cells, and in a perinuclear area in human VSMCs. Additionally, hDlg also associates with the endoplasmic reticulum and microtubules in these latter cells. Taken together, these findings allow us to hypothesize that hDlg acts as a MEK2-specific scaffold protein for the ERK signaling pathway, and may improve our understanding of how scaffold proteins, such as hDlg, differentially tune MEK1/MEK2 signaling and cell responses.     

Musical experience and the aging auditory system: implications for cognitive abilities and hearing speech in noise

Much of our daily communication occurs in the presence of background noise, compromising our ability to hear. While understanding speech in noise is a challenge for everyone, it becomes increasingly difficult as we age. Although aging is generally accompanied by hearing loss, this perceptual decline cannot fully account for the difficulties experienced by older adults for hearing in noise. Decreased cognitive skills concurrent with reduced perceptual acuity are thought to contribute to the difficulty older adults experience understanding speech in noise. Given that musical experience positively impacts speech perception in noise in young adults (ages 18-30), we asked whether musical experience benefits an older cohort of musicians (ages 45-65), potentially offsetting the age-related decline in speech-in-noise perceptual abilities and associated cognitive function (i.e., working memory). Consistent with performance in young adults, older musicians demonstrated enhanced speech-in-noise perception relative to nonmusicians along with greater auditory, but not visual, working memory capacity. By demonstrating that speech-in-noise perception and related cognitive function are enhanced in older musicians, our results imply that musical training may reduce the impact of age-related auditory decline.     

MRI-based finite-element analysis of left ventricular aneurysm

Tagged MRI and finite-element (FE) analysis are valuable tools in analyzing cardiac mechanics. To determine systolic material parameters in three-dimensional stress-strain relationships, we used tagged MRI to validate FE models of left ventricular (LV) aneurysm. Five sheep underwent anteroapical myocardial infarction (25% of LV mass) and 22 wk later underwent tagged MRI. Asymmetric FE models of the LV were formed to in vivo geometry from MRI and included aneurysm material properties measured with biaxial stretching, LV pressure measurements, and myofiber helix angles measured with diffusion tensor MRI. Systolic material parameters were determined that enabled FE models to reproduce midwall, systolic myocardial strains from tagged MRI (630 +/- 187 strain comparisons/animal). When contractile stress equal to 40% of the myofiber stress was added transverse to the muscle fiber, myocardial strain agreement improved by 27% between FE model predictions and experimental measurements (RMS error decreased from 0.074 +/- 0.016 to 0.054 +/- 0.011, P < 0.05). In infarct border zone (BZ), end-systolic midwall stress was elevated in both fiber (24.2 +/- 2.7 to 29.9 +/- 2.4 kPa, P < 0.01) and cross-fiber (5.5 +/- 0.7 to 11.7 +/- 1.3 kPa, P = 0.02) directions relative to noninfarct regions. Contrary to previous hypotheses but consistent with biaxial stretching experiments, active cross-fiber stress development is an integral part of LV systole; FE analysis with only uniaxial contracting stress is insufficient. Stress calculations from these validated models show 24% increase in fiber stress and 115% increase in cross-fiber stress at the BZ relative to remote regions, which may contribute to LV remodeling.     

Antimicrobial activity and main chemical composition of two smoke condensates from Peganum harmala seeds

The smoke of Peganum harmala seeds is traditionally used in Iran as a disinfectant agent. The aim of this study was to determine the antimicrobial activity of two smoke condensates from Peganum harmala seeds. Furthermore the composition of smoke preparations was studied using gas chromatography and mass spectroscopy analysis. The most prevalent compound detected in a dichloromethane extract was harmine. Standard harmine as well as the dichloromethane extract showed antimicrobial activity against all test strains. Harmine was not detected in an n-hexane extract and we did not observe antimicrobial activity from this smoke preparation at the tested concentrations.