Disabling poxvirus pathogenesis by inhibition of Abl-family tyrosine kinases

The Poxviridae family members vaccinia and variola virus enter mammalian cells, replicate outside the nucleus and produce virions that travel to the cell surface along microtubules, fuse with the plasma membrane and egress from infected cells toward apposing cells on actin-filled membranous protrusions. We show that cell-associated enveloped virions (CEV) use Abl- and Src-family tyrosine kinases for actin motility, and that these kinases act in a redundant fashion, perhaps permitting motility in a greater range of cell types. Additionally, release of CEV from the cell requires Abl- but not Src-family tyrosine kinases, and is blocked by STI-571 (Gleevec), an Abl-family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Finally, we show that STI-571 reduces viral dissemination by five orders of magnitude and promotes survival in infected mice, suggesting possible use for this drug in treating smallpox or complications associated with vaccination. This therapeutic approach may prove generally efficacious in treating microbial infections that rely on host tyrosine kinases, and, because the drug targets host but not viral molecules, this strategy is much less likely to engender resistance compared to conventional antimicrobial therapies.     

Primary contact sites in intrinsically unstructured proteins: the case of calpastatin and microtubule-associated protein 2

Intrinsically unstructured proteins (IUPs) exist in a disordered conformational state, often considered to be equivalent with the random-coil structure. We challenge this simplifying view by limited proteolysis, circular dichroism (CD) spectroscopy, and solid-state (1)H NMR, to show short- and long-range structural organization in two IUPs, the first inhibitory domain of calpastatin (CSD1) and microtubule-associated protein 2c (MAP2c). Proteases of either narrow (trypsin, chymotrypsin, and plasmin) or broad (subtilisin and proteinase K) substrate specificity, applied at very low concentrations, preferentially cleaved both proteins in regions, i.e., subdomains A, B, and C in CSD1 and the proline-rich region (PRR) in MAP2c, that are destined to form contacts with their targets. For CSD1, nonadditivity of the CD spectra of its two halves and suboptimal hydration of the full-length protein measured by solid-state NMR demonstrate that long-range tertiary interactions provide the structural background of this structural feature. In MAP2c, such tertiary interactions are absent, which points to the importance of local structural constraints. In fact, urea and temperature dependence of the CD spectrum of its PRR reveals the presence of the extended and rather stiff polyproline II helix conformation that keeps the interaction site exposed. These data suggest that functionally significant residual structure exists in both of these IUPs. This structure, manifest as either transient local and/or global organization, ensures the spatial exposure of short contact segments on the surface. Pertinent data from other IUPs suggest that the presence of such recognition motifs may be a general feature of disordered proteins. To emphasize the possible importance of this structural trait, we propose that these motifs be called primary contact sites in IUPs.     

NMR relaxation studies on the hydrate layer of intrinsically unstructured proteins

Intrinsically unstructured/disordered proteins (IUPs) exist in a disordered and largely solvent-exposed, still functional, structural state under physiological conditions. As their function is often directly linked with structural disorder, understanding their structure-function relationship in detail is a great challenge to structural biology. In particular, their hydration and residual structure, both closely linked with their mechanism of action, require close attention. Here we demonstrate that the hydration of IUPs can be adequately approached by a technique so far unexplored with respect to IUPs, solid-state NMR relaxation measurements. This technique provides quantitative information on various features of hydrate water bound to these proteins. By freezing nonhydrate (bulk) water out, we have been able to measure free induction decays pertaining to protons of bound water from which the amount of hydrate water, its activation energy, and correlation times could be calculated. Thus, for three IUPs, the first inhibitory domain of calpastatin, microtubule-associated protein 2c, and plant dehydrin early responsive to dehydration 10, we demonstrate that they bind a significantly larger amount of water than globular proteins, whereas their suboptimal hydration and relaxation parameters are correlated with their differing modes of function. The theoretical treatment and experimental approach presented in this article may have general utility in characterizing proteins that belong to this novel structural class.     

Calorimetric studies of ligand binding in R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) is a novel bacterial protein that possesses 222 symmetry and a single active site pore. Although the 222 symmetry implies that four symmetry-related binding sites must exist for each substrate as well as for each cofactor, various studies indicate only two molecules bind. Three possible combinations include two dihydrofolate molecules, two NADPH molecules, or one substrate plus one cofactor. The latter is the productive ternary complex. To explore the role of various ligand substituents during binding, numerous analogues, inhibitors, and fragments of NADPH and/or folate were used in both isothermal titration calorimetry (ITC) and K(i) studies. Not surprisingly, as the length of the molecule is shortened, affinity is lost, indicating that ligand connectivity is important in binding. The observed enthalpy change in ITC measurements arises from all components involved in the binding process, including proton uptake. As a buffer dependence for binding of folate was observed, this likely correlates with perturbation of the bound N3 pK(a), such that a neutral pteridine ring is preferred for pairwise interaction with the protein. Of interest, there is no enthalpic signal for binding of folate fragments such as dihydrobiopterin where the p-aminobenzoylglutamate tail has been removed, pointing to the tail as providing most of the enthalpic signal. For binding of NADPH and its analogues, the nicotinamide carboxamide is quite important. Differences between binary (binding of two identical ligands) and ternary complex formation are observed, indicating interligand pairing preferences. For example, while aminopterin and methotrexate both form binary complexes, albeit weakly, neither readily forms ternary complexes with the cofactor. These observations suggest a role for the O4 atom of folate in a pairing preference with NADPH, which ultimately facilitates catalysis.     

Molecular basis of pH and Ca2+ regulation of aquaporin water permeability

Aquaporins facilitate the diffusion of water across cell membranes. We previously showed that acid pH or low Ca(2+) increase the water permeability of bovine AQP0 expressed in Xenopus oocytes. We now show that external histidines in loops A and C mediate the pH dependence. Furthermore, the position of histidines in different members of the aquaporin family can "tune" the pH sensitivity toward alkaline or acid pH ranges. In bovine AQP0, replacement of His40 in loop A by Cys, while keeping His122 in loop C, shifted the pH sensitivity from acid to alkaline. In the killifish AQP0 homologue, MIPfun, with His at position 39 in loop A, alkaline rather than acid pH increased water permeability. Moving His39 to His40 in MIPfun, to mimic bovine AQP0 loop A, shifted the pH sensitivity back to the acid range. pH regulation was also found in two other members of the aquaporin family. Alkaline pH increased the water permeability of AQP4 that contains His at position 129 in loop C. Acid and alkaline pH sensitivity was induced in AQP1 by adding histidines 48 (in loop A) and 130 (in loop C). We conclude that external histidines in loops A and C that span the outer vestibule contribute to pH sensitivity. In addition, we show that when AQP0 (bovine or killifish) and a crippled calmodulin mutant were coexpressed, Ca(2+) sensitivity was lost but pH sensitivity was maintained. These results demonstrate that Ca(2+) and pH modulation are separable and arise from processes on opposite sides of the membrane.     

Biomarker indicators of chemoprevention among subjects occupationally exposed to metals

Chemoprevention with chelating agent Humetta for three months was performed, due to anaemia and other haematologic disorders, immunotoxicological alterations and/or increased chromosome aberration rate among galvanisers and goldsmiths occupationally exposed to precious and heavy metals. Twenty-two of altogether 47 subjects took part voluntarily in the chemoprevention, and the rest of the subjects served as untreated controls. Complex clinical laboratory testing including detailed anamneses; genotoxicological and immunotoxicological monitoring were performed before and after administration of chemopreventive agent. After chemoprevention a significant improvement was observed in anaemia and serum glucose levels, while a less marked improvement was found in serum cholesterol levels and liver functions. Altered chromosome aberration and apoptotic cell fraction also tended to normalise after treatment. Immunological parameters were not affected by the treatment. The obtained results may suggest that chemoprevention with chelating agents as Humetta can help in the prevention of harmful effects of occupational exposures to metals.     

Pre-clinical methods for the determination of insulin sensitivity

We compared the hyperinsulinaemic euglycaemic glucose clamping (HEGC) procedure and the rapid insulin sensitivity test (RIST) to characterize insulin sensitivity in anaesthetized rats. The changes in insulin sensitivity were then supplemented with the direct measurement of insulin-stimulated glucose uptake using tissue accumulation of radioactive 2-deoxyglucose in skeletal muscle samples obtained from animals undergone either procedure. Studies of the recently described endogenous insulin sensitizer mechanism termed hepatic insulin sensitizing (HISS) mechanism, by the two methods yielded data for evaluation. The HISS mechanism is defined as an increase in tissue insulin sensitivity in response to post-prandial hepatic release of an undefined substance through a nitrergic pathway. For the HEGC method, insulin was infused to attain a stable plasma insulin immunoreactivity of 100 microU/ml determined by radioimmunoassay, whereas with the RIST method the HISS mechanism was activated by a 50 mg/kg i.v. insulin bolus. Euglycaemia was kept constant by means of glucose infusion. With the HEGC and the RIST methods, insulin sensitivity was defined as the average rate of glucose infusion and the amount of glucose/kg body weight/40 min (RIST index) infused to maintain euglycaemia and preinvestigation blood glucose level, respectively. During HEGC 16+/-4.2 mg/kg/min glucose was able to maintain euglycaemia, which decreased to 8+/-2.9 (p<0.05) after administration of 10 mg/kg NG-nitro-L-arginine methyl ester (L-NAME) (i.p.), a NO synthase inhibitor. Conversely, the RIST index decreased by 55+/-6.9% (p<0.05) after L-NAME. Similarly, 2-deoxyglucose uptake by the gastrocnemius muscle was decreased by 49.9+/-5.8 (p<0.05) and 52.3+/-7.4% (p<0.05) with the HEGC and the RIST methods, respectively. The results show that both the HEGC and the RIST methods supplemented with tissue radioactive 2-deoxyglucose uptake determinations are appropriate methods to characterize the alteration of insulin sensitivity in context of the HISS mechanism.     

New caligiform Copepod (Siphonostomatoida: Dissonidae) parasitic on Seriola hippos from western Australian waters, with new records and orphological variation for Dissonus nudiventris and Dissonus similis, and an updated key to the species of Dissonus

Dissonus hoi n. sp. is described from female and male specimens collected from the nasal cavities of a Samson Fish (Seriola hippos Günther) captured off Rottnest Island, Western Australia. Adult female D. hoi are distinguished from their congeners by possessing the following combination of characters: (1) a quadrangular genital complex, (2) ventral spines on the genital complex, (3) pair of postantennal processes, (4) a sternal furca, (5) 1-segmented abdomen, and (6) convoluted, uniseriate egg strings. New records and intraspecific morphological variation are also given for Dissonus nudiventris collected from Australia, and Dissonus similis collected from the tropical western Pacific. A key to the valid species of Dissonus is provided.     

Radiation Treatment of Foods: I. Radurization of Fresh Eviscerated Poultry1

Radurization processing of fresh eviscerated poultry has been microbiologically studied. The recommended dose of 0.5 Mrads extended the shelf life at 5 C by approximately 14 days. This treatment also effected a 10 and 11 log reduction in the number of viable Salmonella species and Staphylococcus aureus strains, respectively. Recycling these organisms at sublethal doses of irradiation resulted in strains possessing increased irradiation resistance. Shifts in the microbial ecology after irradiation and storage resulted, in some instances, in the isolation of organisms tentatively identified as Moraxella sp. and Herellea vaginicola.     

The Rate of Oxygen Uptake of Quiescent Cardiac Muscle

The rate of oxygen uptake of quiescent papillary muscle of the cat heart has been determined in a flow respirometer with the use of the oxygen electrode. The apparent rate of oxygen uptake as a function of the diameter of the muscle was also determined. It was found that papillary muscles from cat hearts use oxygen at a rate of 2.84 (microliters/mg. wet weight)/hour at a temperature of 35°C. Such muscles can be adequately supplied by diffusion when their surface is uniformly exposed to an atmosphere containing 95 per cent oxygen only if their diameter is 0.64 mm. or less. Papillary muscles from kitten hearts use oxygen at a rate of 4.05 (microliters/mg. wet weight)/hour at a temperature of 35°C. Such muscles can be adequately supplied by diffusion when their surface is uniformly exposed to an atmosphere containing 95 per cent oxygen only if their diameter is 0.53 mm. or less. If the muscles are small enough to be adequately supplied with oxygen by diffusion, the rate of oxygen uptake does not increase when the muscle is stretched.