Biocontrol agent of root-knot nematode Meloidogyne javanica and root-rot fungi, Fusarium solani in okra morphological, anatomical characteristics and productivity under greenhouse conditions.

This study was conducted to evaluate the ability of some fungal culture filtrate, as biocontrol agents against okra wilt caused by Fusarium solani. and Meloidogyne javanica. In the present study, fungal culture filtrates (FCFs) of Aspergillus terreus (1), Aspergillus terreus (2), Penicillium chrysogenum, and Trichoderma spp. were tested against M. javanica in vitro. The effects of P. chrysogenum and Trichoderma spp. (FCFs) in controlling root-rot fungi and root-knot nematode disease complex on okra plants were studied under greenhouse conditions (In vivo). In vitro experiment, the results revealed cumulative rate of J2s mortality of M. javanica reached to 97.67 and 95% by P. chrysogenum and Trichoderma spp., respectively, after 72 h. incubation. Additionally, Trichoderma spp exhibited the most effective inhibitory activity against the pathogen's radial growth, with a percentage of 68%. P. chrysogenum ranked second with 53.88%, while A. terreus (2) demonstrated the weakest inhibitory effect of 24.11%. T6 [Nematode infection (M. javanica) + Fungus infection (F. solani) + Overflowed with fungal culture filtrate (P. chrysogenum)] and T8 [Nematode infection (M. javanica) + Fungus infection (F. solani) + spray with fungal culture filtrate (P. chrysogenum)] had the greatest effects on nematode galling indices on okra roots and substantially reduced the reproductive factors in the greenhouse (In vivo experiment). T6 was the best treatment to decrease disease severity, as reached (28%) relatively. On the other hand, T12 [(Fungus infection (F. solani) + (Dovex 50% fungicide with irrigation water)] recorded the lowest disease severity reaching (8%) relatively. The results showed that nematode infection or fungus infection or both decreased all studied anatomical characteristics of okra root, stem, and leaves. We concluded from this study that root-knot nematode and root-rot fungi were reduced by using fungal culture filtrates and could improve plant growth.

Interferon-γ inducible protein-10 and interleukin 28B gene polymorphism as predictive markers for genotype 4 hepatitis C virus treatment response.

HCV genotype 4 dominates the HCV epidemic in Egypt. Drug resistance was the most serious side effect that reflects bad clinical outcome. Several studies had demonstrated that baseline serum interferon-γ-inducible-protein 10 (IP-10) levels and interleukin 28B polymorphisms were associated with the resistance to the standard of care pegylated interferon alpha and ribavirin (PEG-IFNα/RBV) therapy and development of post-treatment relapse. Our purpose was to assess the predictive value of combining IP-10 levels and IL28B genotypes to PEG-IFNα/RBV therapy response in Egyptian chronic HCV infection patients with genotype 4. Ninety Egyptian patients chronically infected by HCV genotype-4 treated with pegylated interferon alpha and ribavirin (PEG-IFNα/RBV) therapy were enrolled. Serum IP-10 levels were determined by enzyme linked immunosorbent assay pre- and post- treatment. IL-28B (rs12979860 and rs8099917) polymorphisms were performed by PCR-RFLP in all patients. Overall, 38 patients (42.2%) achieved sustained virologic response (SVR) and 52 (57.8%) patients have non-viral response (NVR). Pretreatment serum IP-10 mean levels were significantly lower in patients who achieved SVR than in NVR (P<0.05). CC genotype in IL28B polymorphism (rs12979860) was the favorable genotype as 65.8% achieved SVR, while TT genotype in IL-28B polymorphism (rs8099917) was the favorable genotype as 81.5% achieved SVR. Baseline IP-10 was significantly correlated to genotypes CC in rs12979860 and TT in rs8099917. Combined use of serum baseline IP-10 levels with IL-28B polymorphisms could improve the prediction of SVR to PEG-IFNα/RBV therapy in Egyptian chronic HCV infection patients with genotype 4.

Assessment of interleukin 17 and transforming growth factor-beta 1 in hepatitis C patients with disease progression.

Hepatitis C virus (HCV) infection in Egypt is the most serious health problem. Identifying HCV-positive persons at high risk of early complications can help prioritize treatment decisions. Recently, attention has been directed to non-invasive, accurate alternatives using serum biochemical markers. The transforming growth factor ß 1/interleukins pathway plays an important role in the process of cell injury and inflammation. Thus, TGF-ß1 and IL-17 were assessed in serum of chronic HCV patients with correlation to hepatic inflammatory and fibrotic status. The quantitative serum levels of TGF-ß1 and IL-17 were analyzed among chronic hepatitis C (CHC) patients (n=75) and normal control (NC) subjects (n=15). Disease severity in patients was assessed using the Child-Pugh scores and METAVIR. Serum levels of TGF-ß1 and IL-17 were significantly increased in HCV patients compared to control group. Furthermore, the levels of TGF-ß1 and Il-17 were positively correlated to serum transaminases and alpha-fetoprotein and they were negatively correlated with serum albumin and platelets. Additionally, the serum levels of TGF-ß1 and Il-17 were associated with inflammation grades and stages of liver fibrosis. TGF-ß1 and IL-17 may be hopeful serum biomarkers concerned in the progression of liver inflammation and fibrosis accompanying chronic HCV infection. Therefore, they could be used in the future as targets for anti-fibrotic therapy of chronic HCV to ameliorate the disease progress.

Assessment of interleukin 17 and transforming growth factor-beta 1 in hepatitis C patients with disease progression

@#Hepatitis C virus (HCV) infection in Egypt is the most serious health problem. Identifying HCV-positive persons at high risk of early complications can help prioritize treatment decisions. Recently, attention has been directed to non-invasive, accurate alternatives using serum biochemical markers. The transforming growth factor β 1/interleukins pathway plays an important role in the process of cell injury and inflammation. Thus, TGF-β1 and IL-17 were assessed in serum of chronic HCV patients with correlation to hepatic inflammatory and fibrotic status. The quantitative serum levels of TGF-β1 and IL-17 were analyzed among chronic hepatitis C (CHC) patients (n=75) and normal control (NC) subjects (n=15). Disease severity in patients was assessed using the Child-Pugh scores and METAVIR. Serum levels of TGF-β1 and IL-17 were significantly increased in HCV patients compared to control group. Furthermore, the levels of TGF-β1 and Il-17 were positively correlated to serum transaminases and alpha-fetoprotein and they were negatively correlated with serum albumin and platelets. Additionally, the serum levels of TGF-β1 and Il-17 were associated with inflammation grades and stages of liver fibrosis. TGF-β1 and IL-17 may be hopeful serum biomarkers concerned in the progression of liver inflammation and fibrosis accompanying chronic HCV infection. Therefore, they could be used in the future as targets for anti-fibrotic therapy of chronic HCV to ameliorate the disease progress.

Interferon-γ inducible protein-10 and interleukin 28B gene polymorphism as predictive markers for genotype 4 hepatitis C virus treatment response

@#HCV genotype 4 dominates the HCV epidemic in Egypt. Drug resistance was the most serious side effect that reflects bad clinical outcome. Several studies had demonstrated that baseline serum interferon-γ-inducible-protein 10 (IP-10) levels and interleukin 28B polymorphisms were associated with the resistance to the standard of care pegylated interferon alpha and ribavirin (PEG-IFNα/RBV) therapy and development of post-treatment relapse. Our purpose was to assess the predictive value of combining IP-10 levels and IL28B genotypes to PEG-IFNα/RBV therapy response in Egyptian chronic HCV infection patients with genotype 4. Ninety Egyptian patients chronically infected by HCV genotype-4 treated with pegylated interferon alpha and ribavirin (PEG-IFNα/RBV) therapy were enrolled. Serum IP-10 levels were determined by enzyme linked immunosorbent assay pre- and post- treatment. IL-28B (rs12979860 and rs8099917) polymorphisms were performed by PCR-RFLP in all patients. Overall, 38 patients (42.2%) achieved sustained virologic response (SVR) and 52 (57.8%) patients have non-viral response (NVR). Pretreatment serum IP-10 mean levels were significantly lower in patients who achieved SVR than in NVR (P<0.05). CC genotype in IL28B polymorphism (rs12979860) was the favorable genotype as 65.8% achieved SVR, while TT genotype in IL-28B polymorphism (rs8099917) was the favorable genotype as 81.5% achieved SVR. Baseline IP-10 was significantly correlated to genotypes CC in rs12979860 and TT in rs8099917. Combined use of serum baseline IP-10 levels with IL-28B polymorphisms could improve the prediction of SVR to PEG-IFNα/RBV therapy in Egyptian chronic HCV infection patients with genotype 4.

Purification of a reconstitutively active K+/H+ antiporter from rat liver mitochondria.

We describe purification of three different states of the 82-kDa K+/H+ antiporter from rat liver mitochondria. The denatured 82-kDa protein, identified by its selective labeling with [14C]dicyclohexylcarbodiimide (DCCD), was purified by preparative two-dimensional gel electrophoresis. This purified product was used to raise and immunopurify monospecific polyclonal antibodies. Western blot analysis showed that the [14C] DCCD-labeled 82-kDa protein is not a DCCD-crosslinked product. The native, [14C]DCCD-labeled, 82-kDa protein was purified by (NH4)2SO4 fractionation and column chromatography, using 14C labeling and gel electrophoresis to track the protein. The native, non-DCCD-labeled 82-kDa protein was purified by similar procedures, using immunopurified antibodies to track the protein. DCCD binding had no effect on chromatographic behavior of the antiporter protein. This protocol resulted in purification of the 82-kDa protein to apparent homogeneity. The purified, native 82-kDa protein was reconstituted into proteoliposomes and assayed for K+ transport with the new fluorescent probe, PBFI. K+ transport was electroneutral and was inhibited by DCCD, Mg2+, and timolol. The turnover number for K+ transport was about 1000 s-1, very similar to the value previously estimated in intact mitochondria.

The grid-blot: a procedure for screening large numbers of monoclonal antibodies for specificity to native and denatured proteins.

This report describes a procedure referred to as a grid-blot for simultaneously testing up to 30 monoclonal antibodies for specificity with an equivalent number of different proteins on a single sheet of nitrocellulose paper. Only 150 microliters of hybridoma culture supernatant is required for the screening and the entire procedure can be completed in less than five hours. This assay was developed to quickly identify those hybridoma cultures producing antibodies that preferentially recognize the native form of a protein and those that also recognize the SDS denatured form and were optimal for use in Western blots. Monoclonal antibodies raised against two distinct proteins, myofibril C-protein (120 antibodies) and the catalytic subunit of cyclic-AMP dependent protein kinase (240 antibodies) were tested. The grid-blot results indicated that 85 of the C-protein antibodies and 55 of the catalytic subunit antibodies were monospecific. Only 4 of the C-protein and 9 catalytic subunit antibodies showed a preferential staining for the appropriate native protein. The antibodies that stained the denatured protein most intensely in the grid-blot corresponded with those that produced the best immunostain in the Western blot. Finally, a version of the grid-blot was found to be an efficient means of determining antibody isotypes.

Inhibitory effect of polycations on phosphorylation of glycogen synthase by glycogen synthase kinase 3.

Several polycations were tested for their abilities to inhibit the activity of glycogen synthase kinase 3 (GSK-3). L-Polylysine was the most powerful inhibitor of GSK-3 with half-maximal inhibition of glycogen synthase phosphorylation occurring at approx. 100 nM. D-Polylysine and histone H1 were also inhibitory, but the concentration dependence was complex, and DL-polylysine was the least effective inhibitor. Spermine caused about 50% inhibition of GSK-3 at 0.7 mM and 70% inhibition at 4 mM. Inhibition of GSK-3 by L-polylysine could be blocked or reversed by heparin. A heat-stable polycation antagonist isolated from swine kidney cortex also blocked the inhibitory effect of L-polylysine on GSK-3 and blocked histone H1 stimulation of protein phosphatase 2A activity. Under the conditions tested, L-polylysine also inhibited GSK-3 catalyzed phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase and a 63 kDa brain protein, but only slightly inhibited phosphorylation of inhibitor 2 or proteolytic fragments of glycogen synthase that contain site 3 (a + b + c). L-Polylysine at a concentration (200 nM) that caused nearly complete inhibition of GSK-3 stimulated casein kinase I and casein kinase II, but had virtually no effect on the catalytic subunit of cAMP-dependent protein kinase. These results suggest that polycations can be useful in controlling GSK-3 activity. Polycations have the potential to decrease the phosphorylation state of glycogen synthase at site 3, both by inhibiting GKS-3 as shown in this study and by stimulating the phosphatase reaction as shown previously (Pelech, S. and Cohen, P. (1985) Eur. J. Biochem. 148, 245-251).

Subcellular localization of glycogen synthase with monoclonal antibodies.

Two monoclonal antibodies, designated 7H5 and 8E11, were produced against glycogen synthase purified from rabbit skeletal muscle. Both antibodies were of the IgG1 (k) isotype. Western blot analysis of extracts of rat and rabbit tissues showed that antibody 7H5 recognized glycogen synthase from skeletal and cardiac muscles, but not from liver. Antibody 8E11 gave similar results but the responses were weaker. Antibody 7H5 also recognized a 69,000 dalton tryptic fragment of glycogen synthase whereas antibody 8E11 did not bind this fragment. Immunocytochemical staining of rabbit skeletal muscle with antibody 7H5 indicated two major sites of glycogen synthase localization. A granular localization present in the cytoplasm and a band-like staining associated with the Z-disk region of the myofibrils. Rabbit cardiac muscle presented a similar pattern though less cytoplasmic staining was apparent. An assay of subcellular fractions for glycogen synthase indicated that the enzyme in cardiac and skeletal muscles is distributed between the soluble (80-90%) and myofibrilar (10-20%) fractions of the tissues. These results provide direct evidence for the presence of glycogen synthase in subcellular fractions other than the soluble fraction of skeletal and cardiac muscles.

Modulation of glycogen synthase kinase activity of skeletal and smooth muscle casein kinase I by spermine.

Casein kinase I (CK-I) from skeletal muscle was stimulated 2-3 fold by 0.25-1 mM spermine. The polyamine also stimulated the phosphorylation of glycogen synthase by another casein kinase purified from aortic smooth muscle [DiSalvo et al. (1986) Biochem. Biophys. Res. Commun. 136, 789-796]. Phosphopeptide maps and phosphoamino acid analysis of [32P]glycogen synthase revealed that smooth muscle casein kinase phosphorylated glycogen synthase in the same sites that undergo phosphorylation by CK-I. The stimulatory effect of spermine on glycogen synthase kinase activity of CK-I was accompanied by increased phosphorylation of all peptide sites of glycogen synthase. Increased phosphorylation was observed in both seryl and threonyl residues. Higher concentrations (4 mM) of spermine inhibited CK-I activity by about 50%. These results indicate that aortic smooth muscle casein kinase is a CK-I enzyme and that skeletal and smooth muscle CK-I can be modulated by spermine.

Phosphorylation of protein B-50 (GAP-43) from adult rat brain cortex by casein kinase II.

The phosphoprotein B-50 (GAP-43) was purified from adult rat brain cortex and phosphorylated by casein kinase II. Phosphorylation of B-50 by casein kinase II approached 1.2 mol phosphate/mol B-50. The apparent Km of casein kinase II for B-50 was 4 microM with an apparent Vmax of 13 nmol.min-1.mg-1. A tryptic phosphopeptide map on reversed phase HPLC and phosphoamino acid analysis of [32P]B-50 showed that casein kinase II phosphorylated in serine residue(s) which were located in a single tryptic peptide. Phosphorylation of B-50 by casein kinase II was inhibited more than 90% by 5 micrograms heparin/ml or 2.4 mM peptide substrate specific for casein kinase II (RRREEETEEE). The initial phosphorylation rate was increased about 2-fold by 1 mM spermine.

Calcium-dependent phosphorylation of bovine cardiac C-protein by phosphorylase kinase.

Phosphorylase kinase catalyzed the calcium-dependent phosphorylation of bovine cardiac C-protein. Phosphorylation of C-protein by phosphorylase kinase reached nearly 2 mol [32P]/mol C-protein. Tryptic phosphopeptide mapping and phosphoamino acid analysis indicated that phosphorylase kinase maybe phosphorylating some of the same seryl residues that undergo phosphorylation by cAMP-dependent protein kinase and that C-protein from bovine and chicken heart are structurally different. Bovine cardiac C-protein was not a substrate for a number of calcium and cyclic nucleotide-independent protein kinases, suggesting that phosphorylation of cardiac C-protein is restricted to protein kinases which are modulated by calcium and cAMP.

Characterization of GSK-M, a glycogen synthase kinase from rat skeletal muscle.

A form of glycogen synthase kinase designated GSK-M3 was purified 4000-fold from rat skeletal muscle by phosphocellulose, Affi-Gel blue, Sephacryl S-300 and carboxymethyl-Sephadex column chromatography. Separation of GSK-M from the catalytic subunit of the cAMP-dependent protein kinase was facilitated by converting the catalytic subunit to the holoenzyme form by addition of the regulatory subunit prior to the gel filtration step. GSK-M had an apparent Mr 62,000 (based on gel filtration), an apparent Km of 11 microM for ATP, and an apparent Km of 4 microM for rat skeletal muscle glycogen synthase. The kinase had very little activity with 0.2 mM GTP as the phosphate donor. Kinase activity was not affected by the addition of cyclic nucleotides, EGTA, heparin, glucose 6-P, glycogen, or the heat-stable inhibitor of cAMP-dependent protein kinase. Phosphorylation of glycogen synthase from rat skeletal muscle by GSK-M reduced the activity ratio (activity in the absence of Glc-6-P/activity in the presence of Glc-6-P X 100) from 90 to 25% when approximately 1.2 mol of phosphate was incorporated per mole of glycogen synthase subunit. Phosphopeptide maps of glycogen synthase obtained after digestion with CNBr or trypsin showed that this kinase phosphorylated glycogen synthase in serine residues found in the peptides containing the sites known as site 2, which is located in the N-terminal CNBr peptide, and site 3, which is located in the C-terminal CNBr peptide of glycogen synthase. In addition to phosphorylating glycogen synthase, GSK-M phosphorylated inhibitor 2 and activated ATP-Mg-dependent protein phosphatase. Activation of the protein phosphatase by GSK-M was dependent on ATP and was virtually absent when ATP was replaced with GTP. GSK-M had minimal activity toward phosphorylase b, casein, phosvitin, and mixed histones. These data indicate that GSK-M, a major form of glycogen synthase kinase from rat skeletal muscle, differs from the known glycogen synthase kinases isolated from rabbit skeletal muscle.

Dephosphorylation of cardiac myofibril C-protein by protein phosphatase 1 and protein phosphatase 2A.

C-protein purified from chicken cardiac myofibrils was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase to nearly 3 mol [32P]phosphate/mol C protein. Digestion of 32P-labeled C-protein with trypsin revealed that the radioactivity was nearly equally distributed in three tryptic peptides which were separated by reversed-phase HPLC. Fragmentation of 32P-labeled C-protein with CNBr showed that the isotope was incorporated at different ratios in three CNBr fragments which were separated on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphorylation was present in both serine and threonine residues. Incubation of 32P-labeled C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of the [32P]phosphate. The major site(s) dephosphorylated by either one of the phosphatases was a phosphothreonine residue(s) apparently located on the same tryptic peptide and on the same CNBr fragment. CNBr fragmentation also revealed a minor phosphorylation site which was dephosphorylated by either of the phosphatases. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A at high concentrations could completely dephosphorylate C-protein. These results demonstrate that C-protein phosphorylated with cAMP-dependent protein kinase can be dephosphorylated by protein phosphatases 1 and 2A. It is suggested that the enzyme responsible for dephosphorylation of C-protein in vivo is phosphatase 2A.

Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps.

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.

Separation of the catalytic and stimulatory regulatory subunits of rat brain adenylate cyclase.

The catalytic subunit of rat brain adenylate cyclase was separated from its stimulatory subunit with loss of activation by 5'-guanylylimidodiphosphate. With respect to other properties, the resolved catalytic subunit was similar to the native enzyme. Nucleotide activation was restored after reconstitution with the isolated regulatory subunit. A key step in resolution was preliminary exposure of isolated membranes to increased ionic strength. After solubilization with nonionic detergent solution, the subunits were separated by gel filtration. The apparent molecular size of the catalytic subunit was unchanged by salt treatment, whereas the Stokes' radius of the regulatory subunit decreased from 72 A to 48 A.