Diaphragm and cardiac mitochondrial creatine kinases are impaired in sepsis.

Previous studies indicate that ATP formation by the electron transport chain is impaired in sepsis. However, it is not known whether sepsis affects the mitochondrial ATP transport system. We hypothesized that sepsis inactivates the mitochondrial creatine kinase (MtCK)-high energy phosphate transport system. To examine this issue, we assessed the effects of endotoxin administration on mitochondrial membrane-bound creatine kinase, an important trans-mitochondrial ATP transport system. Diaphragms and hearts were isolated from control (n = 12) and endotoxin-treated (8 mg.kg(-1).day(-1); n = 13) rats after pentobarbital anesthesia. We isolated mitochondria using techniques that allow evaluation of the functional coupling of mitochondrial creatine kinase MtCK activity to oxidative phosphorylation. MtCK functional activity was established by 1) determining ATP/creatine-stimulated oxygen consumption and 2) assessing total creatine kinase activity in mitochondria using an enzyme-linked assay. We examined MtCK protein content using Western blots. Endotoxin markedly reduced diaphragm and cardiac MtCK activity, as determined both by ATP/creatine-stimulated oxygen consumption and by the enzyme-linked assay (e.g., ATP/creatine-stimulated mitochondrial respiration was 173.8 +/- 7.3, 60.5 +/- 9.3, 210.7 +/- 18.9, was 67.9 +/- 7.3 natoms O.min(-1).mg(-1) in diaphragm control, diaphragm septic, cardiac control, and cardiac septic samples, respectively; P < 0.001 for each tissue comparison). Endotoxin also reduced diaphragm and cardiac MtCK protein levels (e.g., protein levels declined by 39.5% in diaphragm mitochondria and by 44.2% in cardiac mitochondria; P < 0.001 and P = 0.009, respectively, comparing sepsis to control conditions). Our data indicate that endotoxin markedly impairs the MtCK-ATP transporter system; this phenomenon may have significant effects on diaphragm and cardiac function.     

Mutations in both gp120 and gp41 Are Responsible for the Broad Neutralization Resistance of Variant Human Immunodeficiency Virus Type 1 MN to Antibodies Directed at V3 and Non-V3 Epitopes

The escape of human immunodeficiency virus type 1 from effects of neutralizing antibodies was studied by using neutralization-resistant (NR) variants generated by growing the neutralization-sensitive (NS) wild-type MN virus in the presence of human serum with neutralizing antibodies, more than 99% of which were directed at the V3 region of gp120. The variants obtained had broad neutralization resistance to human sera, without limitation with respect to the V3 specificity of the sera. The molecular basis for the resistance was evaluated with molecularly cloned viruses, as well as with pseudoviruses expressing envelope glycoproteins of the NS and NR phenotypes. Nucleotide sequence analyses comparing NS and NR clones revealed a number of polymorphisms, including six in the V1/V2 region, two in C4/V5 of gp120, three in the leucine zipper (LZ) domain of gp41, and two in the second external putative α-helix region of gp41. A series of chimeras from NS and NR env genes was constructed, and each was presented on pseudoviruses to locate the domain(s) which conferred the phenotypic changes. The neutralization phenotypes of the chimeric clones were found to be dependent on mutations in both the C4/V5 region of gp120 and the LZ region of gp41. Additionally, interaction between mutations in gp120 and gp41 was demonstrated in that a chimeric env gene consisting of a gp120 coding sequence from an NS clone and a gp41 sequence from an NR clone yielded a pseudovirus with minimal infectivity. The possible significance of predicted amino acid changes in these domains is discussed. The results indicate that polyvalent antibodies predominantly directed against V3 can induce NR through selection for mutations that alter interactions of other domains in the envelope complex.     

Host Cell-Virus Cross Talk: Phosphorylation of a Hepatitis B Virus Envelope Protein Mediates Intracellular Signaling

Phosphorylation of cytosolic pre-S domains of the duck hepatitis B virus (DHBV) large envelope protein (L) was identified as a regulatory modification involved in intracellular signaling. By using biochemical and mass spectrometric analyses of phosphopeptides obtained from metabolically radiolabeled L protein, a single phosphorylation site was identified at serine 118 as part of a PX(S/T)P motif, which is strongly preferred by ERK-type mitogen-activated protein kinases (MAP kinases). ERK2 specifically phosphorylated L at serine 118 in vitro, and L phosphorylation was inhibited by a coexpressed MAP kinase-specific phosphatase. Furthermore, L phosphorylation and ERK activation were shown to be induced in parallel by various stimuli. Functional analysis with transfected cells showed that DHBV L possesses the ability to activate gene expression in trans and, by using mutations eliminating (S→A) or mimicking (S→D) serine phosphorylation, that this function correlates with L phosphorylation. These mutations had, however, no major effects on virus production in cell culture and in vivo, indicating that L phosphorylation and transactivation are not essential for hepadnavirus replication and morphogenesis. Together, these data suggest a role of the L protein in intracellular host-virus cross talk by varying the levels of pre-S phosphorylation in response to the state of the cell.     

Effect of Immune Activation on the Dynamics of Human Immunodeficiency Virus Replication and on the Distribution of Viral Quasispecies

Virus replication in a human immunodeficiency virus (HIV)-infected individual, as determined by the steady-state level of plasma viremia, reflects a complex balance of viral and host factors. We have previously demonstrated that immunization of HIV-infected individuals with the common recall antigen, tetanus toxoid, disrupts this steady state, resulting in transient bursts of plasma viremia after immunization. The present study defines the viral genetic basis for the transient bursts in viremia after immune activation. Tetanus immunization was associated with dramatic and generally reversible shifts in the composition of plasma viral quasispecies. The viral bursts in most cases reflected a nonspecific increase in viral replication secondary to an expanded pool of susceptible CD4⁺ T cells. An exception to this was in a patient who harbored viruses of differing tropisms (syncytium inducing and non-syncytium inducing [NSI]). In this situation, immunization appeared to select for the replication of NSI viruses. In one of three patients, the data suggested that immune activation resulted in the appearance in plasma of virus induced from latently infected cells. These findings illustrate certain mechanisms whereby antigenic stimulation may influence the dynamics of HIV replication, including the relative expression of different viral variants.     

Leaf anatomical changes in Populus trichocarpa, Quercus rubra, Pseudotsuga menziesii and Pinus ponderosa exposed to enhanced ultraviolet‐B radiation

Leaf anatomical characteristics are important in determining the degree of injury sustained when plants are exposed to natural and enhanced levels of ultraviolet-B (UV-B) radiation (280–320 nm). The degree to which leaf anatomy can adapt to the increasing levels of UV-B radiation reaching the earth's surface is poorly understood in most tree species. We examined four tree species, representing a wide range of leaf anatomical characteristics, to determine responses of leaf area, specific leaf weight, and leaf tissue parameters after exposure to ambient and enhanced levels of UV-B radiation. Seedlings were grown in a greenhouse with photosynthetically active radiation of 39 mol m^?2 day^?1 and under one of three daily irradiances of biologically effective UV-B radiation (UV-B_BE) supplied for 10 h per day: (1) approximate ambient level received at Pullman, Washington on June 21 (1 x ); two times ambient (2 x ), or three times ambient (3 x ). We hypothesized the response of each species to UV-B radiation would be related to inherent anatomical differences. We found that the conifers responded anatomically to nearly an equal degree as the broad-leaved trees, but that different tissues were involved. Populus trichocarpa, an indeterminate broadleaf species, showed significantly thicker palisade parenchyma in recently mature leaves at the 3 x level and in older leaves under the 2 x level. In addition, individual leaf area was generally greater with increased UV-B irradiance. Quercus rubra, a semi-determinate broadleaf species, exhibited significantly thicker palisade parenchyma at the 2 x and 3 x levels as compared to controls. Psuedotsuga menziesii, an evergreen coniferous species with bifacially flattened needles, and Pinus ponderosa, an evergreen coniferous species with a complete hypodermis, showed no significant change in leaf area or specific leaf weight under enhanced UV-B radiation. Epidermal thickness was unchanged in P. menziesii. However, P. ponderosa increased the thickness and number of hypodermal layers produced, presumably decreasing penetration of UV-B radiation into the leaf. We concluded that differences in inherent leaf anatomy of the four species examined are important in the responses to enhanced levels of UV-B radiation.     

Effect of intense interval workouts on running economy using three recovery durations

The purposes of this study were to determine whether running economy (RE) is adversely affected following intense interval bouts of 10 × 400-m running, and whether there is an interaction effect between RE and recovery duration during the workouts. Twelve highly trained male endurance athletes [maximal oxygen consumption; V˙O_{2 max} =72.5 (4.3) ml·kg⁻¹·min⁻¹; mean (SD)] performed three interval running workouts of 10 × 400 m with a minimum of 4 days between runs. Recovery duration between the repetitions was randomly assigned at 60, 120 or 180 s. The velocity for each 400-m run was determined from a treadmill V˙O_{2 max} test. The average running velocity was 357.9 (9.0) m · min⁻¹. Following the workout, the rating of perceived exertion (RPE) increased significantly (P < 0.01) as recovery duration between the 400-m repetitions decreased (14.4, 16.1, and 17.7 at 180s, 120s, and 60 s recovery, respectively). Prior to and following each workout, RE was measured at speeds of 200 and 268 m · min⁻¹. Changes in RE from pre- to post-workout, as well as heart rate (HR) and respiratory exchange ratio (R) were similar for the three recovery conditions. When averaged across conditions, oxygen consumption (V˙O₂) increased significantly (P < 0.01) from pre- to post-test (from 38.5 to 40.5 ml · kg⁻¹ · min⁻¹ at 200 m · min⁻¹, and from 53.1 to 54.5 ml · kg⁻¹ · min⁻¹ at 268 m · min⁻¹, respectively). HR increased (from 124 to 138, and from 151 to 157 beats · min⁻¹ respectively) and R decreased (from 0.90 to 0.78, and from 0.93 to 0.89, respectively) at 200 and 268 m · min⁻¹, respectively (P < 0.01). This study showed that RE can be perturbed after a high-intensity interval workout and that the changes in V˙O₂, HR and R were independent of the recovery duration between the repetitions. Accepted: 23 June 1997     

Eosinophil major basic protein increases membrane permeability in mammalian urinary bladder epithelium

The eosinophilgranule protein major basic protein (MBP) is toxic to a wide variety ofcell types, by a poorly understood mechanism. To determine whether theaction of MBP involves an alteration in membrane permeability, wetested purified MBP on rabbit urinary bladder epithelium usingtransepithelial voltage-clamp techniques. Addition of nanomolarconcentrations of MBP to the mucosal solution caused an increase inapical membrane conductance only when the voltage across the apicalmembrane was cell interior negative. The magnitude of the MBP-inducedconductance was a function of MBP concentration, and the rate of theinitial increase in conductance was a function of the transepithelialvoltage. The MBP-induced conductance was nonselective forK⁺ andCl. MucosalCa²⁺ reversed the inducedconductance, whereas mucosal Mg²⁺partially blocked the induced conductance and slowed the rate of theincrease in conductance. The induced conductance was partially reversedby changing the voltage gradient across the apical membrane to cellinterior positive. Prolonged exposure resulted in an irreversible lossof the barrier function of the urinary bladder epithelium. Theseresults suggest that an increase in cell membrane ion permeability isan initial step in MBP-induced loss of barrier function.

     

Potassium-Stimulated Taurine Release and Nitric Oxide Synthase Activity During Quinolinic Acid Lesion of the Rat Striatum

The microdialysis technique was used to study the effect of nitric oxide synthase (NOS) activity on taurine release. Taurine release was characterized in rat striatum that was excitotoxically lesioned compared to normal conditions. The basal taurine level of the dialysate decreased during quinolinate (QUIN) lesion in parallel to the cell degeneration process. The K⁺-stimulated taurine concentration also decreased during QUIN-lesion, but to an extent that was different from that of basal values. K⁺-stimulated taurine levels were further markedly lowered by coapplication of the NOS inhibitor L-NAME in control and in lesioned animals up to 30 days after QUIN-injection. Postdegenerative tissue did not show any NOS-dependency in K⁺-induced taurine release. We conclude that a substantial part of K⁺-induced taurine release depends on NOS-activity both in normal brain tissue and in excitotoxically induced neurodegeneration. The main source of K⁺-induced taurine release in control rats are neurons but in lesioned animals are activated astroglial cells.     

On the application of polyelectrolyte “limiting laws” to the helix-coil transition of DNA. I. Excess univalent cations

A general theory of polyelectrolyte solutions is here used to calculate the differences in Gibbs free energy, enthalpy, and entropy between the coil and helix forms of DNA at any temperature and salt concentration. The salt has univalent cations and is assumed present in excess over the base concentration. The results are restricted to sufficiently dilute solutions. It is shown that the salt concentrations effect is entirely entropic in origin. When applied to the melting temperature, the calculations yield a relation between the enthalpy difference at the melting temperature and the slope of the plot of melting temperature vs. the logarithm of the salt concentration. In accord with observation, both the Gibbs free energy difference at any fixed temperature and the melting temperature are predicted to be linear functions of the log of the salt concentration. However, the theory is not in quantitative agreement with enthalpy data. Data on various colligative and transport properties of both helix and coil forms are reviewed in the text and in Appendix B, and good agreement is found with theory for both forms. No attempt is made to explain why the theory is quantitative for these properties but not for heat measurements. Finally, in Appendix A, an approximate calculation is made of the free energy contributions due to ionic effects not associated with the salt concentration.     

THE MYOFILAMENT LATTICE: STUDIES ON ISOLATED FIBERS : II. The Effects of Osmotic Strength, Ionic Concentration, and pH upon the Unit-Cell Volume

The effects of osmotic concentration, ionic strength, and pH on the myofilament lattice spacing of intact and skinned single fibers from the walking leg of crayfish (Orconectes) were determined by electron microscopy and low-angle X-ray diffraction. Sarcomere lengths were determined by light diffraction. It is demonstrated that the interfilament spacing in the intact fiber is a function of the volume of the fiber. It is also shown that the interfilament spacing of the skinned (but not of the intact) fiber is affected in a predictable manner by ionic strength and pH insofar as these parameters affect the electrostatic repulsive forces between the filaments. From these combined observations it is demonstrated that the unit-cell volume of the in vivo myofilament lattice behaves in a manner similar to that described for liquid-crystalline solutions.