Reversible covalent binding of peptide nitriles to papain

The dissociation constants for reversible covalent binding of twelve peptide nitrile inhibitors to the active site of papain have been measured by means of fluorescence titration. The binding constants generally parallel the kinetic specificity constants (kcat/Km) for related papain substrates, supporting earlier suggestions that peptide nitriles behave as transition state analog inhibitors of papain. In ten cases the temperature dependence of binding was analyzed to determine the enthalpic and entropic contributions to the binding energy. A compensation plot of delta H vs. T delta S resulted in two parallel lines, one for 'specific' nitriles (i.e., N-Ac-L-aa-NHCH2CN; aa = Phe, Leu, Met) and the other for 'non-specific' nitriles (e.g., N-Ac-D-Phe-NHCH2CN, PhCH2CH2CONHCH2CN hippurylnitrile, etc.). For both specific and nonspecific nitriles representing an 1800-fold range of Kd values (0.27 microM-490 microM), the solvent deuterium isotope effect on binding (Kd(H2O)/Kd(D2O) = DKd) was very close to 2.0. This isotope effect could be accounted for entirely by the simple protonic change which occurs upon the reversible addition of the active site sulfhydryl of papain to the nitrile group of the peptide derivative to form a covalent thioimidate linkage. In contrast, six closely related non-nitrile ligands containing identical peptide side chains but having C-terminal groups incapable of binding covalently to papain had unmeasureably high dissociation constants. Collectively, these results indicate that strong binding of peptide nitrile substrate analogs to papain requires a combination of (1) hydrophobic interaction (especially at the P2 position), (2) specific intermolecular hydrogen bonding and (3) covalent interaction of the nitrile with the active site sulfhydryl group.     

Purification and properties of D-myo-inositol 1,4,5-trisphosphate 3-kinase from rat brain. Susceptibility to calpain

A new, rapid method for purification of inositol(1,4,5)P3 3-kinase in high yield from rat brain is described. Purified enzyme exhibited a polypeptide of Mr = 53,000 on sodium dodecyl sulfate-polyacrylamide gel and a specific activity of 29 mumol/min/mg at 37 degrees C in the absence of calmodulin. Inclusion of calpain inhibitors was critical for obtaining the 53-kDa protein as the major product and 0.1% of the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylamino]-2-propanesulfonate, was necessary to stabilize enzyme activity. In the absence of calpain inhibitors, the 53-kDa protein degraded progressively during purification and yielded a mixture containing polypeptides of various sizes. Relative intensity of these degradation products on sodium dodecyl sulfate-polyacrylamide gel varied from one preparation to another. However, broad band(s) at the 42-45 kDa region and a band at 35 kDa were always weak, while bands of 53, 51, 40 (sometimes doublets), 33, and 32 KDa were usually strong. The fact that all of these polypeptides including the weak bands of 42-45 and 35 kDa were derived from the 53 kDa form was confirmed by their immunocross-reactivity with monoclonal antibodies to the 53 kDa form. When the 51, 40, and a mixture of the 33 and 32 kDa forms were obtained separately and nearly free from other forms, each of them exhibited catalytic activity. Nevertheless, calmodulin binds to polypeptides larger than 35,000 but not to the 33 and 32 kDa forms. Incubation of the purified 53 kDa form with calpain generated a fragmentation pattern nearly identical to that generated during purification in the absence of calpain inhibitors. Incubation with five other endoproteases produced proteolytic fragments slightly different from those by calpain. However, the general fragmentation patterns generated by the proteases were similar, suggesting that inositol(1,4,5)P3 3-kinase contains several motifs susceptible to a variety of proteases.     

Review of the East Asian species of the genera Hybrizon Fallén and Ghilaromma Tobias (Hymenoptera: Ichneumonidae: Hybrizontinae)

East Asian species of the genera Hybrizon and Ghilaromma are reviewed. Four species of Hybrizon, H. buccatus (Brébisson 1825), H. ghilarovi Tobias, 1988, H. juncoi (Ceballos 1957) and H. flavofacialis Tobias, 1988 and two species of Ghilaromma, G. orientalis Tobias, 1988 and G. ussuriensis Tobias, 1988, were recognized. H. ghilarovi was recorded from Korea, Japan and China, while H. juncoi was recorded from Korea, for the first time. The specimens recorded from Japan as G. fuliginosi (Wilkinson, 1930) by Watanabe (1984) are referred to G. orientalis herein. This species is newly recorded from Korea and Japan. Keys to East Asian species of Hybrizon and the world species of Ghilaromma are also provided.     

Model-Based Simulation and Prediction of an Antiviral Strategy against Influenza A Infection

There is a strong need to develop novel strategies in using antiviral agents to efficiently treat influenza infections. Thus, we constructed a rule-based mathematical model that reflects the complicated interactions of the host immunity and viral life cycle and analyzed the key controlling steps of influenza infections. The main characteristics of the pandemic and seasonal influenza strains were estimated using parameter values derived from cells infected with Influenza A/California/04/2009 and Influenza A/NewCaledonia/20/99, respectively. The quantitative dynamics of the infected host cells revealed a more aggressive progression of the pandemic strain than the seasonal strain. The perturbation of each parameter in the model was then tested for its effects on viral production. In both the seasonal and pandemic strains, the inhibition of the viral release (k_C), the reinforcement of viral attachment (k_V), and an increased transition rate of infected cells into activated cells (k_I) exhibited significant suppression effects on the viral production; however, these inhibitory effects were only observed when the numerical perturbations were performed at the early stages of the infection. In contrast, combinatorial perturbations of both the inhibition of viral release and either the reinforcement of the activation of infected cells or the viral attachment exhibited a significant reduction in the viral production even at a later stage of infection. These results suggest that, in addition to blocking the viral release, a combination therapy that also enhances either the viral attachment or the transition of the infected cells might provide an alternative for effectively controlling progressed influenza infection.     

High-affinity phlorizin binding in Mytilus gill.

The gill of the marine mussel, Mytilus, contains a high affinity, Na-dependent D-glucose transporter capable of accumulating glucose directly from sea water. We examined the ability of the beta-glucoside, phlorizin, to act as a high-affinity ligand of this process in intact gills and isolated brush border membrane vesicles (BBMV). The time course of association of nanomolar [3H]phlorizin to gills and BBMV was slow, with t50 values between 10 and 30 min, and a half-time for dissociation of approx. 30 min. 1 mM D-glucose reduced equilibrium binding of 1 nM phlorizin by 90-95%, indicating that there was little non-specific binding of this ligand to the gill. In addition, there was little, if any, hydrolysis by the gill of phlorizin to its constituents, glucose and phloretin. Phlorizin binding to gills and BBMV was significantly inhibited by the addition of 50 microM concentrations of D-glucose and alpha-methyl-D-glucose, and unaffected by the addition of L-glucose and fructose. Binding to gills and BBMV was reduced by greater than 90% when Na+ was replaced by K+. Replacement of Na+ by Li+ effectively blocked binding to the intact gill, although Li+ did support a limited amount of glucose-specific phlorizin binding in BBMV. The Kd values for glucose-specific phlorizin binding in intact gills and BBMV were 0.5 nM and 6 nM, respectively. We conclude that phlorizin binds with extremely high affinity to the Na-dependent glucose transporter of Mytilus gill, which may be useful in future efforts to isolate and purify the protein(s) involved in integumental glucose transport.     

Novel transmembrane protein 126A (TMEM126A) couples with CD137L reverse signals in myeloid cells

Members of the TNF family can promote signals in myeloid cells and both positively and negatively regulate the production of pro-inflammatory cytokines depending on the target myeloid cell type. Using the yeast-two hybrid system, we identified transmembrane protein 126A (TMEM126A) as a binding partner for CD137L (4-1BB ligand). We found that TMEM126A associated and co-localized with CD137L in a mouse macrophage cell line and knockdown of TMEM126A with siRNA abolished the CD137L-induced tyrosine phosphorylation as well as the up-regulation of M-CSF, IL-1β and TN-C expressions. Knockdown of TMEM126A also blocked the down-regulation of IL-1β and IL-6 expressions induced by CD137L in thioglycollate-elicited primary peritoneal macrophages. Knockdown of TMEM126A by stable retroviral TMEM126A shRNA transduction also abolished CD137L-induced tyrosine phosphorylation and cell adherence. These findings identify a novel molecule that bridges TNF family cytokines and pro-inflammatory cytokine secretion in myeloid cells.     

Population-Based Analysis of Invasive Nontypeable Pneumococci Reveals That Most Have Defective Capsule Synthesis Genes

Since nasopharyngeal carriage of pneumococcus precedes invasive pneumococcal disease, characteristics of carriage isolates could be incorrectly assumed to reflect those of invasive isolates. While most pneumococci express a capsular polysaccharide, nontypeable pneumococci are sometimes isolated. Carriage nontypeables tend to encode novel surface proteins in place of a capsular polysaccharide synthetic locus, the cps locus. In contrast, capsular polysaccharide is believed to be indispensable for invasive pneumococcal disease, and nontypeables from population-based invasive pneumococcal disease surveillance have not been extensively characterized. We received 14,328 invasive pneumococcal isolates through the Active Bacterial Core surveillance program during 2006–2009. Isolates that were nontypeable by Quellung serotyping were characterized by PCR serotyping, sequence analyses of the cps locus, and multilocus sequence typing. Eighty-eight isolates were Quellung-nontypeable (0.61%). Of these, 79 (89.8%) contained cps loci. Twenty-two nontypeables exhibited serotype 8 cps loci with defects, primarily within wchA. Six of the remaining nine isolates contained previously-described aliB homologs in place of cps loci. Multilocus sequence typing revealed that most nontypeables that lacked capsular biosynthetic genes were related to established non-encapsulated lineages. Thus, invasive pneumococcal disease caused by nontypeable pneumococcus remains rare in the United States, and while carriage nontypeables lacking cps loci are frequently isolated, such nontypeable are extremely rare in invasive pneumococcal disease. Most invasive nontypeable pneumococci possess defective cps locus genes, with an over-representation of defective serotype 8 cps variants.