Muscle metabolic responses during 16 hours of intermittent heavy exercise

The alterations in muscle metabolism were investigated in response to repeated sessions of heavy intermittent exercise performed over 16 h. Tissue samples were extracted from the vastus lateralis muscle before (B) and after (A) 6 min of cycling at approximately 91% peak aerobic power at repetitions one (R1), two (R2), nine (R9), and sixteen (R16) in 13 untrained volunteers (peak aerobic power = 44.3 +/- 0.66 mL.kg-1.min-1, mean +/- SE). Metabolite content (mmol.(kg dry mass)-1) in homogenates at R1 indicated decreases (p < 0.05) in ATP (21.9 +/- 0.62 vs. 17.7 +/- 0.68) and phosphocreatine (80.3 +/- 2.0 vs. 8.56 +/- 1.5) and increases (p < 0.05) in inosine monophosphate (IMP, 0.077 +/- 0.12 vs. 3.63 +/- 0.85) and lactate (3.80 +/- 0.57 vs. 84.6 +/- 10.3). The content (micromol.(kg dry mass)-1) of calculated free ADP ([ADPf], 86.4 +/- 5.5 vs. 1014 +/- 237) and free AMP ([AMPf], 0.32 +/- 0.03 vs. 78.4 +/- 31) also increased (p < 0.05). No differences were observed between R1 and R2. By R9 and continuing to R16, pronounced reductions (p < 0.05) at A were observed in IMP (72.2%), [ADPf] (58.7%), [AMPf] (85.5%), and lactate (41.3%). The 16-hour protocol resulted in an 89.7% depletion (p < 0.05) of muscle glycogen. Repetition-dependent increases were also observed in oxygen consumption during exercise. It is concluded that repetitive heavy exercise results in less of a disturbance in phosphorylation potential, possibly as a result of increased mitochondrial respiration during the rest-to-work non-steady-state transition.     

Multiple pathways regulating the anti-apoptotic protein clusterin in breast cancer

Cancer chemotherapy inhibits tumor growth, in part, by triggering apoptosis, and anti-apoptotic proteins reduce the effectiveness of chemotherapy. Clusterin, a chaperone-like protein that binds to apoptotic and DNA repair proteins, is induced by chemotherapy and promotes tumor cell survival. Histone deacetylase inhibitors (HDIs) such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA) are pharmacological agents that induce differentiation and apoptosis in cancer cells by altering chromatin structure, and we have found that combinations of chemotherapeutic drugs such as doxorubicin and HDIs efficiently induce apoptosis, even though they paradoxically induce high levels of clusterin. The hyper-expressed form of clusterin localizes to mitochondria, inhibits cytochrome c release, and is inhibited by the proteasome. When HDIs are used as single agents, clusterin suppresses cytochrome c release and apoptosis. However, doxorubicin/HDI-induced apoptosis is not inhibited by clusterin, and clusterin-resistant apoptosis corresponds with markers of the extrinsic/receptor-mediated apoptotic pathway. Thus, chemotherapy-HDI combinations are capable of overcoming an innate anti-apoptotic pathway of tumor cells, suggesting that chemotherapy-HDI combinations have potential for treating advanced stage breast cancer.     

Structural origins of fibrin clot rheology

The origins of clot rheological behavior associated with network morphology and factor XIIIa-induced cross-linking were studied in fibrin clots. Network morphology was manipulated by varying the concentrations of fibrinogen, thrombin, and calcium ion, and cross-linking was controlled by a synthetic, active-center inhibitor of FXIIIa. Quantitative measurements of network features (fiber lengths, fiber diameters, and fiber and branching densities) were made by analyzing computerized three-dimensional models constructed from stereo pairs of scanning electron micrographs. Large fiber diameters and lengths were established only when branching was minimal, and increases in fiber length were generally associated with increases in fiber diameter. Junctions at which three fibers joined were the dominant branchpoint type. Viscoelastic properties of the clots were measured with a rheometer and were correlated with structural features of the networks. At constant fibrinogen but varying thrombin and calcium concentrations, maximal rigidities were established in samples (both cross-linked and noncross-linked) which displayed a balance between large fiber sizes and great branching. Clot rigidity was also enhanced by increasing fiber and branchpoint densities at greater fibrinogen concentrations. Network morphology is only minimally altered by the FXIIIa-catalyzed cross-linking reaction, which seems to augment clot rigidity most likely by the stiffening of existing fibers.     

The dissociation of NO from nitrosylhemoglobin

The reaction between nitrosylhemoglobin and an excess of deoxymyoglobin has been used to study the kinetics of ligand dissociation from Hb4(NO)4 and Hb4(no)1 species. The kinetics of the dissociation of the first NO molecule from Hb4(no)4 was studied by the ligand replacement method. The results indicate that: (a) the ligand dissociation reaction in Hb4(NO)4 is a cooperative process. This is consistent with the results of Moore and Gibson (Moore, E.G., and Gibson, Q.H. (1977) J. Biol. Chem. 251, 2788-2794). (b) alpha and beta chains in the T state formed by adding IHP to Hb4(NO)4 show kinetic heterogeneity. (c) A similar kinetic heterogeneity is shown by alpha and beta chains in the species Hb4NO in the absence of IHP.(d) The value for the NO dissociation rate constant calculated from the slow phases observed in (b) and (c) is similar to that estimated for the R state. These results suggest that the R to T transition brought about with or without inositol hexaphosphate changes the ligand affinity of one type of the chains much more than of the other. On the basis of IR and EPR studies, it is suggested that alpha chains undergo larger functional changes in R to T transition (or vice versa) in nitrosylhemoglobin. The kinetic parameters for HbNO are compared with those of HbO2 and HbCO and the implications of the results for the reaction mechanism are discussed.     

Suppression of stimulating cell activity by microtubule-disrupting alkaloids

Microtubule-disrupting alkaloids and protein fixatives were used to investigate the nature of an active process that must occur within stimulator cells in order for them to intiate a unidirectional mixed lymphocyte response (MLR). Brief treatment of the stimulator cells (SC) with glutaraldehyde (0.15%), formalin (0.6%), or lanthanum chloride (10^?3 M) abolished their capacity to activate responder cells (RC). Pre-treatment of SC with the microtubule-disrupting alkaloids, colchicine (c) (10^?4 to 10^?6) or clochicine + vincristine (c+v) (10^?4 to 10^?6 M) also abrogated their stimulating capacity. This capacity was not restored by the addition of supernates from untreated cultures, thereby excluding the possibility that the alkaloids acted by decreasing the release of soluble stimulatory factors from SC. The introduction of alkaloid-inactivated, mitomycin-treated RC as drug carriers did not affect the mitogenic response of untreated RC to concanavalin A. This excluded a significant leakage of alkaloids from the treated SC and uptake by RC during culture. Lumicolchicine produced no decrease in the stimulating capacity of SC. This suggested that the suppression induced by low concentrations of colchicine resulted from its specific disruption of microtubules. None of the above treatements quantitatively reduced the antigenicity of SC, as evaluated by humoral and cell-mediated lysis of the treated cells. Also, these treatements produced no significant changes in the specific binding of concanavalin A by SC. These indicate there is a functional interaction of microtubular structures with cell surface antigens that appears to regulate either the capacity of SC to associate with RC, or the ability of SC to form and stabilize stimulatory antigenic configurations on the cell surface.     

"May I Buy a Pack of Marlboros,Please?" A Systematic Review of Evidence to Improve the Validity and Impact of Youth Undercover Buy Inspections

Most smokers become addicted to tobacco products before they are legally able to purchase these products. We systematically reviewed the literature on protocols to assess underage purchase and their ecological validity. We conducted a systematic search in May 2015 in PubMed and PsycINFO. We independently screened records for inclusion. We conducted a narrative review and examined implications of two types of legal authority for protocols that govern underage buy enforcement in the United States: criminal (state-level laws prohibiting sales to youth) and administrative (federal regulations prohibiting sales to youth). Ten studies experimentally assessed underage buy protocols and 44 studies assessed the association between youth characteristics and tobacco sales. Protocols that mimicked real-world youth behaviors were consistently associated with substantially greater likelihood of a sale to a youth. Many of the tested protocols appear to be designed for compliance with criminal law rather than administrative enforcement in ways that limited ecological validity. This may be due to concerns about entrapment. For administrative enforcement in particular, entrapment may be less of an issue than commonly thought. Commonly used underage buy protocols poorly represent the reality of youths'' access to tobacco from retailers. Compliance check programs should allow youth to present themselves naturally and attempt to match the community’s demographic makeup.     

Genome-wide identification of specific oligonucleotides using artificial neural network and computational genomic analysis

Background  

Genome-wide identification of specific oligonucleotides (oligos) is a computationally-intensive task and is a requirement for designing microarray probes, primers, and siRNAs. An artificial neural network (ANN) is a machine learning technique that can effectively process complex and high noise data. Here, ANNs are applied to process the unique subsequence distribution for prediction of specific oligos.     

Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium

K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area.