Effect of avian influenza virus subtype H9N2 on the expression of complement-associated genes in chicken erythrocytes.

1. The H9N2 subtype avian influenza virus can infect both chickens and humans. Previous studies have reported a role for erythrocytes in immunity. However, the role of H9N2 against chicken erythrocytes and the presence of complement-related genes in erythrocytes has not been studied. This research investigated the effect of H9N2 on complement-associated gene expression in chicken erythrocytes.2. The expression of complement-associated genes (C1s, C1q, C2, C3, C3ar1, C4, C4a, C5, C5ar1, C7, CD93 and CFD) was detected by reverse transcription-polymerase chain reaction (RT-PCR). Quantitative Real-Time PCR (qRT-PCR) was used to analyse the differential expression of complement-associated genes in chicken erythrocytes at 0 h, 2 h, 6 h and 10 h after the interaction between H9N2 virus and chicken erythrocytes in vitro and 3, 7 and 14 d after H9N2 virus nasal infection of chicks.3. Expression levels of C1q, C4, C1s, C2, C3, C5, C7 and CD93 were significantly up-regulated at 2 h and significantly down-regulated at 10 h. Gene expression levels of C1q, C3ar1, C4a, CFD and C5ar1 were seen to be different at each time point. The expression levels of C1q, C4, C1s, C2, C3, C5, C7, CFD, C3ar1, C4a and C5ar1 were significantly up-regulated at 7 d and the gene expression of levels of C3, CD93 and C5ar1 were seen to be different at each time point.4. The results confirmed that all the complement-associated genes were expressed in chicken erythrocytes and showed the H9N2 virus interaction with chicken erythrocytes and subsequent regulation of chicken erythrocyte complement-associated genes expression. This study reported, for the first time, the relationship between H9N2 and complement system of chicken erythrocytes, which will provide a foundation for further research into the prevention and control of H9N2 infection.

NF-кB pathway genes expression in chicken erythrocytes infected with avian influenza virus subtype H9N2.

1. Chicken erythrocytes in blood vessels are the most abundant circulating cells, which participate in the host's immune responses. The transcription factor nuclear factor-kappa B (NF-κB) plays a vital role in the inflammatory response following viral infections. However, the expression of the NF-κB pathway, and other immune-related genes in chicken erythrocytes infected with low pathogenic avian influenza virus (LPAIV H9N2), has not been extensively studied.2. The following study determined the interaction of LPAIV H9N2 with chicken erythrocytes using indirect immunofluorescence microscopy. This was followed by investigating myeloid differentiation primary response 88 (MyD88), C-C motif chemokine ligand 5 (CCL5), melanoma differentiation-associated protein 5 (MDA5), the inhibitor of nuclear factor-kappa B kinase subunit epsilon (IKBKE), NF-κB inhibitor alpha (NFKBIA), NF-κB inhibitor epsilon (NFKBIE), interferon-alpha (IFN-α), colony-stimulating factor 3 (CSF3) and tumour necrosis factor receptor-associated factor 6 (TRAF6) by mRNA expression using quantitative real-time PCR (qRT-PCR) at four different time intervals (0, 2, 6 and 10 h).3. There was a significant interaction between erythrocytes and LPAIV H9N2 virus. Furthermore, the mRNA expression of the NF-κB pathway and other immune-related genes were significantly up-regulated at 2 h post-infection in infected chicken erythrocytes, except for TRAF6, which were significantly downregulated. While at 0 h post-infection, IFN-α and CSF3 were significantly upregulated, whereas NFKBIA was significantly downregulated. Further expression of MDA5, CCL5 and NFKBIA was upregulated, while TRAF6 was downregulated at 6 h post-infection. In infected erythrocytes, expression of MyD88, CCL5 and IKBKE was upregulated. However, IFN-α and TRAF6 were downregulated at 10 h post-infection.4. These results give initial evidence that the NF-κB pathway, and other genes related to immunity, in chicken erythrocytes may contribute to LPAIV subtype H9N2 and induce host immune responses.

Glutathione-S-transferase A3 protein suppresses thiram-induced tibial dyschondroplasia by regulating prostaglandin-related genes expression.

Tibial dyschondroplasia (TD) is an intractable avian cartilage disease in which proximal growth plates of tibia lack blood vessels and contain nonviable cells, and it leads to the inflammatory response. Prostaglandins (PGs) genes have not been studied yet in TD chicken, and they might play role in skeletal metabolism, therefore we planned to explore the role of recombinant glutathione-S-transferase A3 (rGSTA3) protein and PG-related genes. In this study, qRT-PCR, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) analysis were used to identify the expression patterns of eight PG-related genes in the tibial growth plate of broiler chicken. The results showed that the expression of PG-related genes glutathione-S-transferase A3 (GSTA3), cyclooxygenase 2 (COX-2), prostaglandin D2 synthase (PTGDS), prostaglandin E synthase (PTGES), prostaglandin E2 receptor (PTGER) 3, PTGER4, prostaglandin reductase 1 (PTGR1) and hematopoietic prostaglandin D synthases (HPGDS) expression were identified and could significantly respond to thiram-induced TD chicken. Interestingly, the expression of rate-limiting enzyme COX-2 and PGE2 were induced after the treatment of rGSTA3 protein. These findings demonstrated that the occurrence of TD is closely related to the inhibition of PGs. Moreover, rGSTA3 protein participated in the recovery of TD by strengthening the expression of PG-related genes.

The expression of prostaglandins-related genes in erythrocytes of broiler chicken responds to thiram-induced tibial dyschondroplasia and recombinant glutathione-S-transferase A3 protein.

Tibial dyschondroplasia (TD) is a type of bone deformity found in fast-growing chickens, which induce inflammatory responses. Prostaglandins (PGs) implicate in bone formation and bone resorption, associated with inflammation in an autocrine/paracrine manner. This study used qRT-PCR and immunohistochemistry analysis to identify the expression patterns of PG-related genes in the erythrocytes of broiler chickens and explore the effects of thiram-induced TD and the recombinant glutathione-S-transferase A3 (rGSTA3) protein on the expression of PG-related genes: GSTA3, cyclooxygenase 2 (COX-2), prostaglandin D2 synthase (PTGDS), prostaglandin E synthase (PTGES), prostaglandin E2 receptor (PTGER) 3, PTGER4 and prostaglandin reductase 1 (PTGR1). Interestingly, the results showed that these seven PG-related genes expression was identified in the erythrocytes of broiler chicken, and thiram-induced TD suppressed the expression of these PG-related genes in the initial stage of TD and promoted their expression in TD recovery. These findings demonstrated that the immunoregulatory function of erythrocytes can be inhibited in the early stage of TD and promoted in the recovery stage by modulating the expression of PG-related genes. Further, the rGSTA3 protein can modulate the expression of PG-related genes in erythrocytes and participate in the recovery of TD.

Identification of apoptosis-related genes in erythrocytes of broiler chickens and their response to thiram-induced tibial dyschondroplasia and recombinant glutathione-S-transferase A3 protein.

Thiram, a carbamate pesticide, is known to induce tibial dyschondroplasia (TD) in broiler chickens. This study used a thiram-induced TD model to explore whether apoptosis-related genes were expressed in erythrocytes of broiler chickens and the impacts of thiram-induced TD and the recombinant GSTA3 protein in regulating these genes expression. In this study, mRNA and protein expression of six types of apoptosis-related genes (Bcl-2, Bax, Murine double minute MDM2, Bcl-2-associated athanogene BAG-1, BAG-3, STAT3) were identified in erythrocytes of broiler chickens by real-time PCR and immunohistochemistry, and we also found that thiram-induced TD induced the decreased expression of these antiapoptotic genes in the initial stage of TD and promoted their expression in TD recovery, which suggested that the expression of these apoptosis-related genes in erythrocytes is highly related to the development of TD. Further, the recombinant GSTA3 protein promoted the expression of all apoptosis-related genes in the initial stage of TD and recovered the normal expression of these genes in the recovery stage of TD, which indicated that the recombinant GSTA3 protein may participate in the recovery of TD. Further studies are needed to elucidate the mechanism of the response of erythrocytes to thiram-induced TD and the recombinant protein GSTA3 in broiler chickens.

Specific and Sensitive Detection of Enterobacter mori Using Reliable RT-PCR.

Enterobacter mori, the causal agent of bacterial wilt in mulberry, is becoming a serious disease in mulberry orchards in China. Because no effective control strategy has been devised for this disease, the reliable screening of mulberry material for latent infection became necessary. Hence, a fast polymerase chain reaction (PCR) assay for the detection of E. mori was developed in this study. The primers were designed within regions of the RNA polymerase ß-subunit (rpoB) gene. The method is fast and simple and showed 100% sensitivity (no false negatives) and 100% specificity (no false positives), which was tested with 4 representative E. mori strains, 9 Enterobacter type strains, 2 strains of the other major mulberry bacterial pathogens (Ralstonia solanacearum and Pseudomonas syringae pv. mori) in China, 7 strains of other plant-associated pathogens, and 50 unidentified epiphytic bacterial isolates from mulberry plants. The real-time PCR assays reliably detected the DNA at at least 10 fg/µl and the bacterial cells at 102 CFU/ml from mulberry shoots and roots suspension. The strong positive reaction in testing of all symptomatic plants (with 100% positive) and parts of asymptomatic latent infected plant samples (with 36.4% positive) provided proof that this method is reliable and sensitive and suitable for screening plant material with latent infections of E. mori.

Identification of differentially expressed genes in the growth plate of broiler chickens with thiram-induced tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is characterized by expansion of the proximal growth plates of the tibiotarsus that fail to form bone, lack blood vessels, and contain non-viable cells. Thiram (a carbamate pesticide), when fed to young broiler chicks, induces TD with high regularity and precision. We used this experimental model to understand the cause of the defects associated with TD by selecting and identifying the genes differentially expressed in the TD growth plate of broiler chickens. Broiler chicks at 7 days of age were randomly divided into two groups. After fasting overnight, they were fed with regular diet (control) or the same diet containing 100 mg/kg thiram for 96 h to induce TD (thiram-fed). mRNA was purified from the growth plates of control and thiram-fed broilers. Forward and reverse-subtracted cDNA libraries were generated by suppression subtractive hybridization technology. Ten selected genes from cDNA libraries were identified by real-time quantitative polymerase chain reaction. All were differentially expressed in TD growth plates (P<0.05 or P<0.01). The levels of collagen type X (Col X), pro-alpha-1 collagen type I (Col I alpha1), collagen type IX (Col IX), NADH dehydrogenase (NADH DH), cytochrome C oxidase subunit III (COX III), enolase 1, alpha (ENO1), carbonic anhydrase II (CA2) and heat shock protein 90 (Hsp90) mRNA transcripts were up-regulated, while the expression levels of Matrilin 3 (MATN3) and chondromodulin-I (ChM-I) were down-regulated. Col I and Hsp90 were detected by immunohistochemistry at different stages. Given that these genes are involved in matrix formation, endochondral ossification, developmental regulation, electron transport in the mitochondrial respiratory chain and vascularization, our findings may provide new insights into understanding the pathogenesis of TD.

Effects of osmolytes on unfolding of chicken liver fatty acid synthase.

Urea-induced aggregation of chicken liver fatty acid synthase [acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85] was studied. The aggregation was facilitated at increased ionic strength. Methyl-beta-cyclodextrin and some osmolytes, such as glycerol, sucrose, proline, glycine, and heparin, could effectively prevent the aggregation, implying an artificial chaperone role of those substances during fatty acid synthase unfolding. The osmolytes also protected the enzyme from inactivation.

Unfolding and inactivation of fatty acid synthase from chicken liver during urea denaturation.

The inactivation and conformational changes of the multifunctional fatty acid synthase (acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85) from chicken liver have been studied in urea solution. The results show that complete inactivation of the fatty acid synthase occurs before obvious conformational changes with regard to the overall, beta-ketoacyl reduction and acetoacetyl-CoA reduction reactions. Significant conformational changes indicated by the changes of the intrinsic fluorescence emission and the circular dichroism spectra occurred at higher urea concentrations. The kinetic rate constants for the two phase inactivation and unfolding reactions were measured and semilogarithmic plots of the activity versus time gave curves which could be resolved into two straight lines, indicating that both the inactivation and unfolding processes consisted of fast and slow phases as a first-order reaction. The results from Lineweaver-Burk plots indicated that urea is a competitive inhibitor for acetyl-CoA and malonyl-CoA, with K(m) increasing with increasing urea concentrations. However, urea is a noncompetitive inhibitor for NADPH, the substrate of the overall reaction and beta-ketoacyl reduction reaction, and acetylacetate, the substrate of the beta-ketoacyl reduction reaction. Activation by low concentrations of urea was observed although this activation was only temporarily induced in an early stage of inactivation. The aggregation phenomenon of the fatty acid synthase in a certain concentration range of urea (3-4 M) was also observed during unfolding. This result shows that this multifunctional enzyme unfolds with competition with misfolding in the folding pathway. Comparison of inactivation and conformational changes of the enzyme as well as aggregation imply that unfolding intermediates may exist during urea denaturation. The possible unfolding pathway of fatty acid synthase is also discussed in this paper.

Inactivation and conformational changes of fatty acid synthase from chicken liver during unfolding by sodium dodecyl sulfate.

Fatty acid synthase is an important enzyme participating in energy metabolism in vivo. The inactivation and conformational changes of the multifunctional fatty acid synthase from chicken liver in SDS solutions have been studied. The results show that the denaturation of this multifunctional enzyme by SDS occurred in three stages. At low concentrations of SDS (less than 0.15 mM) the enzyme was completely inactivated with regard to the overall reaction. For each component of the enzyme, the loss of activity occurred at higher concentrations of SDS. Significant conformational changes (as indicated by the changes of the intrinsic fluorescence emission and the ultraviolet difference spectra) occurred at higher concentrations of SDS. Increasing the SDS concentration caused only slight changes of the CD spectra, indicating that SDS had no significant effect on the secondary structure of the enzyme. The results suggest that the active sites of the multifunctional fatty acid synthase display more conformational flexibility than the enzyme molecule as a whole.

Affinity labeling of chicken liver fatty acid synthase with chloroacetyl-CoA and bromopyruvate.

Fatty acid synthase of chicken liver is inactivated rapidly and irreversibly by incubation with chloroacetyl-CoA or with bromopyruvate. Inactivation by both reagents follows saturation kinetics, indicating the formation of an E ... I complex (dissociation constants of 0.36 microM for chloroacetyl-CoA and 31 microM for bromopyruvate) prior to alkylation. The limiting rate constants are 0.15 s-1 for bromopyruvate and 0.041 s-1 for chloroacetyl-CoA. Inactivation by both reagents is protected by NADPH and 200 mM KCl, and by saturating amounts of thioester substrates which reduced the limiting rate constants 6.5-30-fold. Active-site-directed reaction of chloroacetyl-CoA is supported by the ability of this compound to form a kinetically viable complex with the enzyme as competitive inhibitor of acetyl-CoA. Chloroacetyl-CoA interacts initially at the CoA binding pocket, since the nucleotide afforded competitive protection of inactivation and caused a large decrease in its affinity. Subsequently, the phosphopantetheine prosthetic group is alkylated. Evidence is presented to show that bromopyruvate competes with chloroacetyl-CoA for the same target site.

Studies on the reactivity of the essential sulfhydryl groups as a conformational probe for the fatty acid synthetase of chicken liver. Inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid) and intersubunit cross-linking of the inactivated enzyme.

Fatty acid synthetase of chicken liver is rapidly and reversibly inactivated by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) at a rate (k2 = 132 mM-1 S-1 in 3 mM EDTA, 1% (v/v) glycerol, pH 7.0, at 25 degrees C) up to 2200 times higher than the reaction of this reagent with simple thiol compounds. The inactivation is caused by the reaction of the phosphopantetheine SH group, since it is protected competitively by either acetyl- or malonyl-CoA, and since the inactivated enzyme is unreactive with the phosphopantetheine label chloroacetyl-CoA but reactive with the cysteine reagent 1,3-dibromopropanone. Moreover, chloroacetyl-CoA prevents the modification of the rapidly reacting essential SH group by DTNB. The number of SH groups involved in inactivation was determined by correlating activity loss with the extent of reaction and by stopped-flow analysis of substrate (or chloroacetyl-CoA) protection. Values between 0.91 and 1.15 SH groups/dimer were obtained, indicating the presence of substoichiometric amounts of the prosthetic group in the fatty acid synthetase preparations used in this study. Inactivation of the synthetase by DTNB is strongly inhibited by increasing salt concentration and protected noncompetitively by NADP+ and NADPH. Treatment of the enzyme inactivated at low salt by salt, NADP+, or NADPH also effectively reduced cross-linking between enzyme subunits. The parallel effects of these treatments on the reaction with DTNB and subsequent dimerization are consistent with a minimum model of two discreet conformation states for fatty acid synthetase. In the low salt conformer, the phosphopantetheine and cysteine SH groups are juxtaposed, and the DTNB reaction (k2 approximately 132 mM-1 S-1) and dimerization are both facilitated. Transition to the high salt conformer by the above treatments is accompanied by an approximately 20-fold reduction of reactivity with DTNB (k2 = 6.8 mM-1 S-1) and reduced dimerization, due to spatial separation of the SH groups. During palmitate synthesis, the enzyme may oscillate between these conformation states to permit the reaction of intermediates at different active sites. Results obtained by studies on the effect of pH on DTNB inactivation implicate a pK of 5.9-6.1 for the essential SH group independent of salt concentration. This value is 1.5-1.8 pH units lower than the pK of 7.6-7.7 for CoA and may explain the 23-fold increase of the rate constant from a value of 0.3 mM-1 S-1 for CoA to that of the high salt conformer.

Hybridization studies of chicken liver fatty acid synthetase. Evidence for the participation in palmitate synthesis of cysteine and phosphopantetheine sulfhydryl groups on adjacent subunits.

Enzymatically inactive variants of chicken liver fatty acid synthetase have been prepared by specific chemical modification of the active cysteine SH group with iodoacetamide, and the phosphopantetheine SH group with chloroacetyl-CoA. Hybridization of each of these variants with the unmodified enzyme yielded (modified)-(unmodified) hybrid dimers which possessed 50% synthetase activity. A 50% active (iodoacetamide-modified)-(chloroacetyl-CoA-modified) hybrid dimer was also demonstrated by recombination of these variants with each other. These results indicate that the two functional sites on the synthetase are independently active, and that each is comprised of a cysteine SH group from one subunit and a complementary phosphopantetheine SH group from the other subunit as depicted by the head-to-tail arrangement proposed by Wakil and co-workers (Wakil, S. J., Stoops, J. K., and Joshi, V.C.

Transient kinetic studies of fatty acid synthetase. A kinetic self-editing mechanism for the loading of acetyl and malonyl residues and the role of coenzyme A.

A kinetic self-editing mechanism for correcting errors in the loading of thioester substrates is described for the animal fatty acid synthetase reaction. In the catalyzed reaction, these substrates load competitively on a common phosphopantetheine site, and during each of the eight loading steps the enzyme sites are partitioned between competent and incompetent substrate molecules. The incompetently bound substrate is removed by CoA through reversal of the loading reaction and partitioning again occurs. The loading-unloading cycle is repeated until competent enzyme complex is formed and the reaction proceeds. Furthermore, at each step the loading of a malonyl residue is competitively favored as is the unloading of enzyme-bound acetyl groups. This mechanism is entirely consistent with the recently postulated role (Stern, A., Sedgwick, B., and Smith, S. J. Biol. Chem. (1982) 257, 799-803) of CoA as a co-substrate. Supporting evidence is obtained by monitoring the progress curves of NADPH oxidation by chicken liver fatty acid synthetase in the stopped flow apparatus. At noninhibiting acetyl-CoA, the reaction shows an initial lag period as the result of preferential formation of malonyl-enzyme and time-dependent recycling of the loading step to obtain competent acetyl-enzyme. At a malonyl-CoA/acetyl-CoA ratio of 2:1, the induction time of the reaction is 1.02 +/- 0.05 s at 6 degrees C. It decreases with increasing acetyl-CoA concentration or preincubation of the enzyme with acetyl-CoA which promotes acetyl-enzyme formation but is slightly increased upon preincubation with malonyl-CoA. Increasing acetyl-CoA causes a parallel decrease in steady state cycle time (i.e. the average time required to complete a single malonyl-CoA condensation cycle), suggesting that the latter is limited by the lag period. At inhibitory acetyl-CoA, the steady state cycle time is lengthened due to acetyl-enzyme formation at malonyl-CoA loading steps and to the recycling necessary to obtain competent malonyl-enzyme. A requirement of CoA for the first condensation cycle is unequivocally demonstrated in conventional spectrophometric assays and stopped flow experiments by using phosphotransacetylase and acetyl phosphate as a CoA trap. This requirement at each loading step is normally met by CoA generated through initial loading. At noninhibitory acetyl-CoA, added CoA inhibits the reaction and slightly increases the lag.(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of the rate constant of enzyme modification by measuring the substrate reaction in the presence of the modifier.

On the basis of the equations derived previously [Tsou, C. L. (1965) Sheng Wu Hua Hsueh Yu Sheng Wu Wu Li Hsueh Pao 5, 398-408, 409-417] for the substrate reaction during the course of enzyme modification, the kinetic behavior of the system chymotrypsin-substrate-modifier has been studied. The kinetics of benzoyltyrosine ester hydrolysis during the course of irreversible inhibition of the enzyme has been found to be in satisfactory agreement with equations obtained previously. The apparent rate constant between the enzyme and an irreversible inhibitor can be easily obtained in one single experiment by following the course of substrate hydrolysis in the presence of the inhibitor. The results are also in accord with the assumption that diisopropyl fluorophosphate can be classified as an irreversible competitive inhibitor. For both phenylmethanesulfonyl fluoride and L-1-[(p-toluene-sulfonyl)amino]-2-phenylethyl chloromethyl ketone, the inhibition has been found to be in agreement with the kinetics of the complexing type; i.e., a noncovalent enzyme-inhibitor complex is formed before irreversible enzyme modification. Both the equilibrium constants for the complex formation and the first-order rate constants for the irreversible modification step have been determined also by following the course of substrate hydrolysis in the presence of the irreversible inhibitor.