Kaposi's sarcoma-associated herpesvirus induces the phosphatidylinositol 3-kinase-PKC-zeta-MEK-ERK signaling pathway in target cells early during infection: implications for infectivity

Human herpesvirus 8 (HHV-8) is implicated in the pathogenesis of Kaposi's sarcoma. HHV-8 envelope glycoprotein B (gB) possesses the RGD motif known to interact with integrin molecules, and HHV-8 infectivity was inhibited by RGD peptides, by antibodies against alpha3 and beta1 integrins, and by soluble alpha3beta1 integrin (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Anti-gB antibodies immunoprecipitated the virus alpha3 and beta1 complexes, and virus-binding studies suggest a role for alpha3beta1 in HHV-8 entry. HHV-8 infection induced the integrin-mediated activation of focal adhesion kinase (FAK), implicating a role for integrin and the associated signaling pathways in HHV-8 entry into the target cells. Immediately after infection, target cells exhibited morphological changes and cytoskeletal rearrangements, suggesting the induction of signal pathways. As early as 5 min postinfection, HHV-8 activated the MEK-ERK1/2 pathway. The focal adhesion components phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C-zeta (PKC-zeta) were recruited as upstream mediators of the HHV-8-induced ERK pathway. Anti-HHV-8 gB-neutralizing antibodies and soluble alpha3beta1 integrin inhibited the virus-induced signaling pathways. Early kinetics of the cellular signaling pathway and its activation by UV-inactivated HHV-8 suggest a role for virus binding and/or entry but not viral gene expression in this induction. Studies with human alpha3 integrin-transfected Chinese hamster ovary cells and FAK-negative mouse DU3 cells suggest that the alpha3beta1 integrin and FAK play roles in the HHV-8 mediated signal induction. Inhibitors specific for PI 3-kinase, PKC-zeta, MEK, and ERK significantly reduced the virus infectivity without affecting virus binding to the target cells. Examination of viral DNA entry suggests a role for PI 3-kinase in HHV-8 entry into the target cells and a role for PKC-zeta, MEK, and ERK at a post-viral entry stage of infection. These findings implicate a critical role for integrin-associated mitogenic signaling in HHV-8's infection of target cells and suggest that, by orchestrating the signal cascade, HHV-8 may create an appropriate intracellular environment to facilitate the infection.     

Analysis of cardiac signals using spatial filling index and time-frequency domain

Background  

Analysis of heart rate variation (HRV) has become a popular noninvasive tool for assessing the activities of the autonomic nervous system (ANS). HRV analysis is based on the concept that fast fluctuations may specifically reflect changes of sympathetic and vagal activity. It shows that the structure generating the signal is not simply linear, but also involves nonlinear contributions. These signals are essentially non-stationary; may contain indicators of current disease, or even warnings about impending diseases. The indicators may be present at all times or may occur at random in the time scale. However, to study and pinpoint abnormalities in voluminous data collected over several hours is strenuous and time consuming.     

Equipoise,design bias,and randomized controlled trials: the elusive ethics of new drug development

The concept of 'equipoise', or the 'uncertainty principle', has been represented as a central ethical principle, and holds that a subject may be enrolled in a randomized controlled trial (RCT) only if there is true uncertainty about which of the trial arms is most likely to benefit the patient. We sought to estimate the frequency with which equipoise conditions were met in industry-sponsored RCTs in rheumatology, to explore the reasons for any deviations from equipoise, to examine the concept of 'design bias', and to consider alternative ethical formulations that might improve subject safety and autonomy. We studied abstracts accepted for the 2001 American College of Rheumatology meetings that reported RCTs, acknowledged industry sponsorship, and had clinical end-points (n = 45), and examined the proportion of studies that favored the registration or marketing of the sponsor's drug. In every trial (45/45) results were favorable to the sponsor, indicating that results could have been predicted in advance solely by knowledge of sponsorship (P < 0.0001). Equipoise clearly was being systematically violated. Publication bias appeared to be an incomplete explanation for this dramatic result; this bias occurs after a study is completed. Rather, we hypothesize that 'design bias', in which extensive preliminary data are used to design studies with a high likelihood of being positive, is the major cause of the asymmetric results. Design 'bias' occurs before the trial is begun and is inconsistent with the equipoise principle. However, design bias increases scientific efficiency, decreases drug development costs, and limits the number of subjects required, probably reducing aggregate risks to participants. Conceptual and ethical issues were found with the equipoise principle, which encourages performance of negative studies; ignores patient values, patient autonomy, and social benefits; is applied at a conceptually inappropriate decision point (after randomization rather than before); and is in conflict with the Belmont, Nuremberg, and other sets of ethical principles, as well as with US Food and Drug Administration procedures. We propose a principle of 'positive expected outcomes', which informs the assessment that a trial is ethical, together with a restatement of the priority of personal autonomy.     

Citrate synthase mutants of Sinorhizobium fredii USDA257 form ineffective nodules with aberrant ultrastructure

The tricarboxylic acid (TCA) cycle plays an important role in generating the energy required by bacteroids to fix atmospheric nitrogen. Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle. We cloned and determined the nucleotide sequence of the gltA gene that encodes citrate synthase in Sinorhizobium fredii USDA257, a symbiont of soybeans (Glycine max [L.] Merr.) and several other legumes. The deduced citrate synthase protein has a molecular weight of 48,198 and exhibits sequence similarity to citrate synthases from several bacterial species, including Sinorhizobium meliloti and Rhizobium tropici. Southern blot analysis revealed that the fast-growing S. fredii strains and Rhizobium sp. strain NGR234 contained a single copy of the gene located in the bacterial chromosome. S. fredii USDA257 gltA mutant HBK-CS1, which had no detectable citrate synthase activity, had diminished nodulation capacity and produced ineffective nodules on soybean. Light and electron microscopy observations revealed that the nodules initiated by HBK-CS1 contained very few bacteroids. The infected cells contained large vacuoles and prominent starch grains. Within the vacuoles, membrane structures that appeared to be reminiscent of disintegrating bacteroids were detected. The citrate synthase mutant had altered cell surface characteristics and produced three times more exopolysaccarides than the wild type produced. A plasmid carrying the USDA257 gltA gene, when introduced into HBK-CS1, was able to restore all of the defects mentioned above. Our results demonstrate that a functional citrate synthase gene of S. fredii USDA257 is essential for efficient soybean nodulation and nitrogen fixation.     

Solution structure of delta-Am2766: a highly hydrophobic delta-conotoxin from Conus amadis that inhibits inactivation of neuronal voltage-gated sodium channels

The three-dimensional (3D) NMR solution structure (MeOH) of the highly hydrophobic delta-conotoxin delta-Am2766 from the molluscivorous snail Conus amadis has been determined. Fifteen converged structures were obtained on the basis of 262 distance constraints, 25 torsion-angle constraints, and ten constraints based on disulfide linkages and H-bonds. The root-mean-square deviations (rmsd) about the averaged coordinates of the backbone (N, C(alpha), C) and (all) heavy atoms were 0.62+/-0.20 and 1.12+/-0.23 A, respectively. The structures determined are of good stereochemical quality, as evidenced by the high percentage (100%) of backbone dihedral angles that occupy favorable and additionally allowed regions of the Ramachandran map. The structure of delta-Am2766 consists of a triple-stranded antiparallel beta-sheet, and of four turns. The three disulfides form the classical 'inhibitory cysteine knot' motif. So far, only one tertiary structure of a delta-conotoxin has been reported; thus, the tertiary structure of delta-Am2766 is the second such example. Another Conus peptide, Am2735 from C. amadis, has also been purified and sequenced. Am2735 shares 96% sequence identity with delta-Am2766. Unlike delta-Am2766, Am2735 does not inhibit the fast inactivation of Na+ currents in rat brain Na(v)1.2 Na+ channels at concentrations up to 200 nM.     

Prenatal alcohol exposure and early postnatal changes in the developing nerve-muscle system

BACKGROUND: Extensive research on prenatal alcohol exposure has proven the potent teratogenicity of this substance of abuse. Children born to alcoholic mothers are often diagnosed with fetal alcohol syndrome (FAS). Those afflicted with FAS often have muscle weakness, muscle wasting, and atrophy. This study assessed the effects of prenatal alcohol exposure on the developing rat neuromuscular system. METHODS: Pregnant Sprague-Dawley rats were injected intraperitoneally with 1.0 ml of 20% ethyl alcohol/100 gm body weight. Unexposed rats served as controls. The offspring were killed 2, 3, 4, and 5 weeks after birth, and their body weights were recorded. The tibialis anterior (TA) and extensor digitorum longus (EDL) muscles were recovered and weighed. The TA muscles were histochemically stained by silver cholinesterase in order to study the pattern of innervation. The EDL muscles were processed and stained by hematoxylin-eosin. The number and size of the EDL muscle fibers was quantified. The sciatic nerve was also removed and stained by Swank and Davenport's method to demonstrate the myelin pattern. RESULTS: Assessment at the neuromuscular junction showed a higher proportion of endplates polyneuronally innervated in the alcohol-exposed rats. The muscle weights, as well as the number and size of the muscle fibers, were significantly reduced in these animals. A light-microscopy examination of the nerve sections revealed alterations in the connectivity of myelin. CONCLUSIONS: The finding that a higher proportion of endplates were polyneuronally innervated in the alcohol-exposed rats indicates that the maturation process of the neuromuscular system was delayed, thus confirming the deleterious effects of alcohol on growth and maturation of the nerve-muscle system.     

Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae

The Niemann-Pick C1 (NPC1) protein regulates cholesterol transport from late endosomes-lysosomes to other intracellular compartments. In this article, cholesterol transport to caveolin-1 and caveolin-2 containing compartments, such as the trans-Golgi network (TGN) and plasma membrane caveolae, was examined in normal (NPC+/+), NPC heterozygous (NPC+/-), and NPC homozygous (NPC-/-) human fibroblasts. The expression and distribution of NPC1 in each cell type were similar, and characterized by a finely dispersed, granular staining pattern. The expression of caveolin-1 and caveolin-2 was increased in NPC+/- and NPC-/- fibroblasts, although the distribution in each cell type was similar and characterized by predominant staining of the TGN and plasma membrane. The TGN in NPC+/+ fibroblasts was relatively cholesterol-enriched, whereas the TGN in NPC+/- and NPC-/- fibroblasts was partially or completely cholesterol-deficient, respectively. Consistent with studies demonstrating the transport of cholesterol from the TGN to plasma membrane caveolae, the concentration of cholesterol in plasma membrane caveolae isolated from NPC+/- and NPC-/- fibroblasts was significantly decreased, even though the total concentration of plasma membrane cholesterol in each cell type was similar.These studies demonstrate that NPC1 regulates cholesterol transport to caveolin-1 and caveolin-2 containing compartments such as the TGN and plasma membrane caveolae.     

Phosphorylation of pyrimidine L-deoxynucleoside analog diphosphates. Kinetics of phosphorylation and dephosphorylation of nucleoside analog diphosphates and triphosphates by 3-phosphoglycerate kinase

Anticancer and antiviral D- and L-nucleoside analogs are phosphorylated stepwise in the cells to the pharmacologically active triphosphate metabolites. We recently reported that in the last step, L-deoxynucleoside analog diphosphates are phosphorylated by 3-phosphoglycerate kinase (PGK). To explain the preference of PGK for L- over D-deoxynucleoside analog diphosphates, the kinetics of their phosphorylation were compared with the dephosphorylation of the respective triphosphates using recombinant human PGK. The results attributed favorable phosphorylation of L-deoxynucleoside analog diphosphates by PGK to differences in k(cat), which were consequences of varied orientations of the sugar and diphosphates in the catalytic site of PGK. The amino acids involved in the catalytic reaction of PGK (including Glu(344), Lys(220), and Asn(337)) were therefore mutated. The impact of mutations on the phosphorylation of L- and D-deoxynucleoside analog diphosphates was different from those on dephosphorylation of the respective triphosphates. This suggested that the interactions of the nucleoside analogs with amino acids during the transition state are different in the phosphorylation and dephosphorylation reactions. Thus, reversible action of the enzyme may not involve the same configuration of the active site. Furthermore, the amino acid determinants of the action of PGK for L-deoxynucleotides were not the same as for the D-deoxynucleotides. This study also suggests the potential impact of nucleoside analog diphosphates and triphosphates on the multiple cellular functions of PGK, which may contribute to the action of the analogs.