Suppression of Viral RNA Binding and the Assembly of Infectious Hepatitis C Virus Particles In Vitro by Cyclophilin Inhibitors

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) is an indispensable component of the HCV replication and assembly machineries. Although its precise mechanism of action is not yet clear, current evidence indicates that its structure and function are regulated by the cellular peptidylprolyl isomerase cyclophilin A (CyPA). CyPA binds to proline residues in the C-terminal half of NS5A, in a distributed fashion, and modulates the structure of the disordered domains II and III. Cyclophilin inhibitors (CPIs), including cyclosporine (CsA) and its nonimmunosuppressive derivatives, inhibit HCV infection of diverse genotypes, both in vitro and in vivo. Here we report a mechanism by which CPIs inhibit HCV infection and demonstrate that CPIs can suppress HCV assembly in addition to their well-documented inhibitory effect on RNA replication. Although the interaction between NS5A and other viral proteins is not affected by CPIs, RNA binding by NS5A in cell culture-based HCV (HCVcc)-infected cells is significantly inhibited by CPI treatment, and sensitivity of RNA binding is correlated with previously characterized CyPA dependence or CsA sensitivity of HCV mutants. Furthermore, the difference in CyPA dependence between a subgenomic and a full-length replicon of JFH-1 was due, at least in part, to an additional role that CyPA plays in HCV assembly, a conclusion that is supported by experiments with the clinical CPI alisporivir. The host-directed nature and the ability to interfere with more than one step in the HCV life cycle may result in a higher genetic barrier to resistance for this class of HCV inhibitors.     

The application of a potential-sensitive cyanine dye to rat small intestinal brush border membrane vesicles

The sensitivity of the fluorescent dye, 3,3′-diethylthiadicarbocyanine (DiS-C₂(5)), was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl₃ or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na⁺-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.     

Nitrogenase activity,hydrogen evolution and biomass production in different Casuarina symbioses

Nitrogenase activity, hydrogen evolution, biomass production and nodulation were studied in threeCasuarina species,C. equisetifolia Forst.,C. glauca Sieber ex Spreng andC. obesa Miq., either inoculated with a crushed nodule inoculum prepared fromC. glauca nodules or inoculated with the pure cultureHFP CcI3. Nodulation was also studied inC. cristata Miq. inoculated with the above mentionedFrankia sources. C. equisetifolia, C. glauca andC. obesa were nodulated when inoculated with both of theFrankia inoculum, whileC. cristata was very poorly nodulated. Nitrogenase activity per plant and on a nodule dry weight basis was significantly highest inC. glauca inoculated withC. glauca inoculum after 150 days from planting. This difference decreased and at 217 days from planting there was no significant difference between the symbioses, except forC. obesa inoculated withC. glauca inoculum which showed the significantly lowest nitrogenase activity. After 150 days from planting relative efficiency of nitrogenase was lowest inC. equisetifolia inoculated withHFP CcI3 and inC. equisetifolia inoculated withC. glauca inoculum. Biomass production was similar inC. glauca inoculated withC. glauca inoculum, inC. equisetifolia inoculated withHFP CcI3 and inC. obesa inoculated withHFP CcI3 at the final harvest. The data presented here show that there is a strong interrelationship between host plant and endobiont. This interrelationship is of considerable importance when introducing Casuarina symbioses for production of fuel wood.     

Angiotensin III and IV activation of the brain AT1 receptor subtype in cardiovascular function

The present investigation determined that native angiotensins II and III (ANG II and III) were equipotent as pressor agents when ICV infused in alert rats, whereas native angiotensin IV (ANG IV) was less potent. An analogue of each of these angiotensins was prepared with a hydroxyethylamine (HEA) amide bond replacement at the N-terminus, yielding additional resistance to degradation. These three angiotensin analogues, HEA-ANG II, HEA-ANG III, and HEA-ANG IV, were equivalent with respect to maximum elevation in pressor responses when ICV infused; and each evidenced significantly extended durations of effect compared with their respective native angiotensin. Comparing analogues, HEA-ANG II had a significantly longer effect compared with HEA-ANG III, and HEA-ANG IV, whereas the latter were equivalent. Pretreatment with the AT₁ receptor subtype antagonist, Losartan (DuP753), blocked subsequent pressor responses to each of these analogues, suggesting that these responses were mediated by the AT₁ receptor subtype. Pretreatment with the specific AT₄ receptor subtype antagonist, Divalinal (HED 1291), failed to influence pressor responses induced by the subsequent infusion of these analogues. These results suggest an important role for Ang III, and perhaps ANG IV, in brain angiotensin pressor responses mediated by the AT₁ receptor subtype.     

Mammalian Fbh1 is important to restore normal mitotic progression following decatenation stress

We have addressed the role of the F-box helicase 1 (Fbh1) protein during genome maintenance in mammalian cells. For this, we generated two mouse embryonic stem cell lines deficient for Fbh1: one with a homozygous deletion of the N-terminal F-box domain (Fbh1^f/f), and the other with a homozygous disruption (Fbh1^?/?). Consistent with previous reports of Fbh1-deficiency in vertebrate cells, we found that Fbh1^?/? cells show a moderate increase in Rad51 localization to DNA damage, but no clear defect in chromosome break repair. In contrast, we found that Fbh1^f/f cells show a decrease in Rad51 localization to DNA damage and increased cytoplasmic localization of Rad51. However, these Fbh1^f/f cells show no clear defects in chromosome break repair. Since some Rad51 partners and F-box-associated proteins (Skp1-Cul1) have been implicated in progression through mitosis, we considered whether Fbh1 might play a role in this process. To test this hypothesis, we disrupted mitosis using catalytic topoisomerase II inhibitors (bisdioxopiperazines), which inhibit chromosome decatenation. We found that both Fbh1^f/f and Fbh1^?/? cells show hypersensitivity to topoisomerase II catalytic inhibitors, even though the degree of decatenation stress was not affected. Furthermore, following topoisomerase II catalytic inhibition, both Fbh1-deficient cell lines show substantial defects in anaphase separation of chromosomes. These results indicate that Fbh1 is important for restoration of normal mitotic progression following decatenation stress.     

Identification of proximal tubular transport functions in the established kidney cell line, OK

OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, L-glutamate, L-proline, L-alanine, and alpha-methyl-glucopyranoside (alpha-methyl D-glucoside) were accumulated through Na+-dependent and Na+-independent transport pathways. D-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na+. Influx of inorganic phosphate was only observed in the presence of Na+. Na+/alpha-methyl D-glucoside uptake was preferentially inhibited by phlorizin and D-glucose uptake by cytochalasin B. An amiloride-sensitive Na+-transport was also identified. In isolated apical vesicles (enriched 8-fold in gamma-glutamyltransferase), L-glutamate, L-proline, L-alanine, alpha-methyl D-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na+-gradient as compared to an inwardly directed K+-gradient. L-Glutamate transport required additionally intravesicular K+. D-Glucose transport was similar in the presence of a Na+- and a K+-gradient. Na+/alpha-methyl D-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na+/D-glucose transport. An amiloride-sensitive Na+/H+ exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na+-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. D-Glucose appears a poor substrate for the Na+/hexose transport system.     

Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells

Summary For patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na⁺ and K⁺ as well as to Cl^–. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.     

Role of tumour necrosis factor in the tumour-necrotizing activity of agents with diverging toxicity

Summary We investigated the ability of various tumournecrotizing agents with diverging toxicity to induce tumour necrosis factor (TNF) and cytostatic activity inPropionibacterium-acnes-primed Swiss and tumour-bearing BALB/c mice, and the capacity of anti-TNF antibodies to inhibit induction of tumour necrosis by the agents. Lipid A and especially its combination with muramyl dipeptide induced high TNF levels in Swiss mice, as measured in the serum. Lower levels were induced by detoxified lipid A and the nontoxic dsRNA, polyadenylic polyuridylic acid, either alone or combined with muramyl dipeptide. The toxic agents also appeared the strongest inducers of mediators with cytostatic activity against cultured endothelial cells and MethA tumour cells. Anti-TNF antibodies partially reduced the cytostatic activity of the sera against MethA cells. Tumour-bearing BALB/c mice produced only low levels of TNF and cytostatic factors in response to all agents. Recombinant mouse TNF hardly reduced the DNA synthesis of MethA cells, unless normal mouse serum was added. Serum fromP.-acnes-treated Swiss mice and tumour-bearing BALB/c mice, that were inhibitory on their own, failed to potentiate the action of TNF. Serum from Swiss mice treated with toxic, but not detoxified, lipid A caused extensive tumour necrosis upon injection into MethA-bearing BALB/c mice. This activity was completely abolished by pre-incubation of the serum with anti-TNF. The tumour-necrotizing activity of the agents could be partially reduced by prior injection of these antibodies. Results show that the capacity of the agents to induce TNF and cytostatic activity is not related to their antitumour potential. Although TNF is likely to be a crucial mediator of the tumour-necrotizing action of the toxic as well as the nontoxic agents, it is probably not the sole mediator. Data also indicate that induction of tumour necrosis does not require induction of high and, thus toxic, TNF levels in the serum.