Heat stress reduces cerebral blood velocity and markedly impairs orthostatic tolerance in humans

Orthostatic tolerance is reduced in the heat-stressed human. This study tested the following hypotheses: 1) whole body heat stress reduces cerebral blood velocity (CBV) and increases cerebral vascular resistance (CVR); and 2) reductions in CBV and increases in CVR in response to an orthostatic challenge will be greater while subjects are heat stressed. Fifteen subjects were instrumented for measurements of CBV (transcranial ultrasonography), mean arterial blood pressure (MAP), heart rate, and internal temperature. Whole body heating increased both internal temperature (36.4+/-0.1 to 37.3+/-0.1 degrees C) and heart rate (59+/-3 to 90+/-3 beats/min); P<0.001. Whole body heating also reduced CBV (62+/-3 to 53+/-2 cm/s) primarily via an elevation in CVR (1.35+/-0.06 to 1.63+/-0.07 mmHg.cm-1.s; P<0.001. A subset of subjects (n=8) were exposed to lower-body negative pressure (LBNP 10, 20, 30, 40 mmHg) in both normothermic and heat-stressed conditions. During normothermia, LBNP of 30 mmHg (highest level of LBNP achieved by the majority of subjects in both thermal conditions) did not significantly alter CBV, CVR, or MAP. During whole body heating, this LBNP decreased MAP (81+/-2 to 75+/-3 mmHg), decreased CBV (50+/-4 to 39+/-1 cm/s), and increased CVR (1.67+/-0.17 to 1.92+/-0.12 mmHg.cm-1.s); P<0.05. These data indicate that heat stress decreases CBV, and the reduction in CBV for a given orthostatic challenge is greater during heat stress. These outcomes reduce the reserve to buffer further decreases in cerebral perfusion before presyncope. Increases in CVR during whole body heating, coupled with even greater increases in CVR during orthostasis and heat stress, likely contribute to orthostatic intolerance.     

Potential of chlorpyrifos and cypermethrin forming DNA adducts

DNA adducts consist of DNA monoadducts, DNA intrastrand crosslinks, DNA interstrand crosslinks, and DNA-protein crosslinks. If not repaired or mistakenly repaired, DNA adducts may lead to gene mutations and initiate carcinogenesis. Two insecticides, chlorpyrifos and cypermethrin, were studied for their potential of forming DNA monoadducts, DNA interstrand crosslinks, and DNA-protein crosslinks in primary mouse hepatocytes via the assays of bioluminescence, ethidium bromide fluorescence, and K+-SDS precipitation. DNA interstrand crosslinks were also measured on calf thymus DNA. It was shown that chlorpyrifos could not form DNA adducts. Cypermethrin formed DNA monoadducts and DNA interstrand crosslinks in hepatocytes. However, cypermethrin didn't form DNA interstrand crosslinks on calf thymus DNA and in hepatocytes treated with SKF-525A, a cytochrome P450 inhibitor, which suggests that active metabolites of cypermethrin instead of cypermethrin itself caused DNA interstrand crosslinks and that cytochrome P450 may be involved in the activation of cypermethrin.     

Non-sense mutations in the dihydropyridine receptor beta1 gene, CACNB1, paralyze zebrafish relaxed mutants

Contractions by skeletal muscle require proper excitation-contraction (EC) coupling, whereby depolarization of the muscle membrane leads to an increase in cytosolic Ca(2+) and contraction. Changes in membrane voltage are detected by dihydropyridine receptors (DHPR) that directly interact with and activate ryanodine receptors to release Ca(2+) from the sarcoplasmic reticulum into the cytosol. A genetic screen for motility mutations isolated a new allele of the immotile zebrafish mutant relaxed. Muscles in relaxed embryos do not contract in response to potassium chloride (KCl) thus appear unresponsive to membrane depolarization, but do contract when stimulated by caffeine, an agonist of ryanodine receptors. This suggests that relaxed mutant muscles are defective in EC coupling. Indeed, immunohistochemical analysis demonstrated that mutants lack DHPRs in skeletal muscles. The mutant phenotype results from non-sense mutations in the zebrafish CACNB1 gene that encodes the DHPR beta1 subunit. The zebrafish CACNB1 gene is expressed in skeletal muscles, PNS and CNS. Electrophysiological recordings showed no obvious abnormalities in the motor output of relaxed mutants, presumably due to redundancy provided by other beta subunits. The structural and functional homology of CACNB1 in zebrafish and mammals, suggests that zebrafish can be useful for studying EC coupling and potential neuronal function of CACNB1.     

Characterization of the mechanism of cytochrome P450 reductase-cytochrome P450-mediated nitric oxide and nitrosothiol generation from organic nitrates

Mammalian cytochrome P450 reductase (CPR) and cytochrome P450 (CP) play important roles in organic nitrate bioactivation; however, the mechanism by which they convert organic nitrate to NO remains unknown. Questions remain regarding the initial precursor of NO that serves to link organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of CPR-CP-mediated organic nitrate bioactivation, EPR, chemiluminescence NO analyzer, NO electrode, and immunoassay studies were performed. With rat hepatic microsomes or purified CPR, the presence of NADPH triggered organic nitrate reduction to NO2(-). The CPR flavin site inhibitor diphenyleneiodonium inhibited this NO2(-) generation, whereas the CP inhibitor clotrimazole did not. However, clotrimazole greatly inhibited NO2(-)-dependent NO generation. Therefore, CPR catalyzes organic nitrate reduction, producing nitrite, whereas CP can mediate further nitrite reduction to NO. Nitrite-dependent NO generation contributed <10% of the CPR-CP-mediated NO generation from organic nitrates; thus, NO2(-) is not the main precursor of NO. CPR-CP-mediated NO generation was largely thiol-dependent. Studies suggested that organic nitrite (R-O-NO) was produced from organic nitrate reduction by CPR. Further reaction of organic nitrite with free or microsome-associated thiols led to NO or nitrosothiol generation and thus stimulated the activation of sGC. Thus, organic nitrite is the initial product in the process of CRP-CP-mediated organic nitrate activation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.     

Cholesterol-induced apoptotic macrophages elicit an inflammatory response in phagocytes, which is partially attenuated by the Mer receptor

Macrophage apoptosis and the ability of phagocytes to clear these apoptotic cells are important processes in advanced atherosclerosis. Phagocytic clearance not only disposes of dead cells but usually elicits an anti-inflammatory response. To study this process in a model of advanced lesional macrophage death, macrophages rendered apoptotic by free cholesterol loading (FC-AMs) were incubated briefly with fresh macrophages ("phagocytes"). FC-AMs were promptly ingested by the phagocytes, which was dependent upon actin polymerization and the phagocyte Mer receptor. Surprisingly, this brief exposure to FC-AMs triggered a modest proinflammatory response in the phagocytes: tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta were induced, whereas the levels of transforming growth factor-beta and IL-10 were not increased. This response required cell contact between the FC-AMs and phagocytes but not FC-AM ingestion. TNF-alpha and IL-1beta induction required one or more proteins on the FC-AM surface and was dependent on signaling through extracellular signal-regulated kinase-1/2 mitogen-activated protein kinase and nuclear factor-kappaB in the phagocytes. TNF-alpha production was markedly greater when Mer-defective phagocytes were used, indicating that Mer attenuated the inflammatory response. Interestingly, a more typical anti-inflammatory response was elicited when phagocytes were exposed to macrophages rendered apoptotic by oxidized low density lipoprotein or UV radiation. Thus, the proinflammatory milieu of advanced atherosclerotic lesions may be promoted, or at least not dampened, by contact between FC-induced apoptotic macrophages and neighboring phagocytes prior to apoptotic cell ingestion.     

A finite element framework for studying the mechanical response of macromolecules: application to the gating of the mechanosensitive channel MscL

The gating pathways of mechanosensitive channels of large conductance (MscL) in two bacteria (Mycobacterium tuberculosis and Escherichia coli) are studied using the finite element method. The phenomenological model treats transmembrane helices as elastic rods and the lipid membrane as an elastic sheet of finite thickness; the model is inspired by the crystal structure of MscL. The interactions between various continuum components are derived from molecular-mechanics energy calculations using the CHARMM all-atom force field. Both bacterial MscLs open fully upon in-plane tension in the membrane and the variation of pore diameter with membrane tension is found to be essentially linear. The estimated gating tension is close to the experimental value. The structural variations along the gating pathway are consistent with previous analyses based on structural models with experimental constraints and biased atomistic molecular-dynamics simulations. Upon membrane bending, neither MscL opens substantially, although there is notable and nonmonotonic variation in the pore radius. This emphasizes that the gating behavior of MscL depends critically on the form of the mechanical perturbation and reinforces the idea that the crucial gating parameter is lateral tension in the membrane rather than the curvature of the membrane. Compared to popular all-atom-based techniques such as targeted or steered molecular-dynamics simulations, the finite element method-based continuum-mechanics framework offers a unique alternative to bridge detailed intermolecular interactions and biological processes occurring at large spatial scales and long timescales. It is envisioned that such a hierarchical multiscale framework will find great value in the study of a variety of biological processes involving complex mechanical deformations such as muscle contraction and mechanotransduction.     

Low concentrations mono-butyl phthalate stimulates steroidogenesis by facilitating steroidogenic acute regulatory protein expression in mouse Leydig tumor cells (MLTC-1)

Di-n-butyl phthalate (DBP) is one of the most dominant phthalate esters and is widely distributed environmental contaminant. Although previous studies have demonstrated that DBP led to a variety of male reproductive abnormalities similar to those caused by androgen receptor antagonists, DBP and its active metabolite, mono-butyl phthalate (MBP), have been demonstrated no affinity for the androgen receptor, but rather exert anti-androgenic effect by altering testosterone biosynthesis. Furthermore, all these results were obtained from very high administrations of DBP or MBP. The purpose of this study was to determine the onset and the site of action of relatively low concentration of MBP on steroidogenesis in vitro. The mouse Leydig tumor cells (MLTC-1) was employed as a cellular model to investigate the effect of MBP on steroidogenesis. Various concentrations of MBP (1, 10, 100 and 1000nmol/l) and its solvent dimethyl sulfoxide (DMSO) were added to the medium for 24h followed by stimulation of some compounds such as human chorionic gonadotrophin (hCG), cholera toxin (CT), forskolin, cAMP analog 8-Br-cAMP, 22(R)-hydroxycholesterol (22R-HC) and pregnenolone. Progesterone in the medium and amounts of intracellular cAMP were measured by RIA. Expression of steroidogenic acute regulatory protein (StAR) was monitored by real-time PCR and Western blotting. The results revealed that the increases of progesterone production in the presence of hCG, CT, forskolin and 8-Br-cAMP were augmented by MBP. In contrast, the levels of intracellular cAMP exhibited no statistical significance when MLTC-1 cells were treated as above. These results implied that the site in the steroid biosynthesis pathway affected by MBP occurs after PKA activation in MLTC-1 cells. Moreover, supplementing the medium with 22R-HC and pregnenolone as progesterone precursors for P450 side chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD), respectively, resulted in no rise in progesterone production, making clear that MBP did not influence the P450scc and 3beta-HSD but on the rate-limiting step, cholesterol transportation into mitochondria. In fact, the above results were confirmed by the upgraded StAR expression in MBP-treated cells. These data support that MBP promotes steroid hormone production by facilitating StAR expression in MLTC-1 cells.     

Exploring the role of the active site cysteine in human muscle creatine kinase

All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pKa about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa of 5.4 observed in the WT V/K(creatine) pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.     

The temperature dependence of salt-protein association is sequence specific

Molecular dynamics (MD) simulations are used to probe the origin of the unexpected temperature dependence of salt accumulation in the C-terminal region of the protein human lymphotactin. As in previous MD simulations, sodium ions accumulate in an enhanced manner near the C-terminal helix at the lower temperature, while the temperature dependence of chloride accumulation is much weaker and slightly positive. In a designed mutant in which all positively charged residues in the C-terminal helix are replaced with neutral polar groups (Ser), the unexpected temperature dependence of the sodium ions is no longer observed. Therefore, these simulations convincingly verified the previous hypothesis that the temperature dependence of ion-protein association is sensitive to the local sequence. This is explained qualitatively in terms of the entropy of association between charged species in solution. These findings have general implications for the interpretation of thermodynamic quantities associated with binding events where ion release is important, such as protein-DNA interactions.