Sequence and expression analysis of cardiac ryanodine receptor 2 in broilers that died from sudden death syndrome.

Sudden death syndrome (SDS) is a stress-related disease in broilers with no diagnostic clinical or necropsy findings. SDS is associated with ventricular tachycardia (VT) and ventricular fibrillation (VF); however, its pathogenesis is not precisely described at the molecular level. Dysfunction of ryanodine receptor 2 (RYR2), that controls rapid release of Ca2+ from the sarcoplasmic reticulum (SR) into the cytosol during muscle contraction, has been associated with VT and sudden cardiac death (SCD) in human patients with structurally normal heart, but there is no report describing abnormalities in RYR2 in diseased broilers. In order to advance our knowledge on the molecular mechanisms predisposing broilers to fatal arrhythmia, the present study was conducted to determine the occurrence of possible mutations and changes in the expression level of the chicken RYR2 gene (chRYR2) in broilers that died from SDS. An increase in mRNA expression level and nine novel point mutations in chRYR2 were found in relation to SDS. In conclusion, susceptibility to lethal cardiac arrhythmia in SDS may be associated with specific changes in intracellular Ca2+ cycling components such as RYR2 due to mutation and dysregulation. Finding the probable association of SDS with gene defects can be applied to select for chickens with lower susceptibility to SDS and decrease the poultry industry losses due to SDS mortality. RESEARCH HIGHLIGHTS Investigation of the occurrence of possible mutations and changes in the expression level of chicken RYR2 gene (chRYR2) in broilers that died from SDS. Increase in the mRNA expression level of chRYR2 in relation to SDS. Nine novel point mutations in chRYR2 of broilers that died from SDS. Possible connection between susceptibility to lethal cardiac arrhythmia in SDS and changes in intracellular Ca2+ cycling machinery, such as RYR2, due to mutation and dysregulation.

Polymorphism identification and cardiac gene expression analysis of the calsequestrin 2 gene in broiler chickens with sudden death syndrome.

Sudden death syndrome (SDS) in broilers is a cardiac disease associated with ventricular tachycardia (VT) and ventricular fibrillation (VF); however, its pathogenesis at the molecular level is not precisely determined. Downregulation and mutations of calsequestrin 2 (CASQ2), a major intracellular Ca(2+) buffer, have been associated with VT and sudden cardiac death (SCD) in humans but in chickens there is no report describing CASQ2 abnormalities in cardiac diseases. In order to better understand the molecular mechanisms predisposing the myocardium to fatal arrhythmia in broilers, the mRNA expression level of chicken CASQ2 gene (chCASQ2) in the left ventricle of dead broilers with SDS was determined and compared to healthy broilers using quantitative real-time PCR (qPCR). To determine the probable mutations in chCASQ2, PCR and direct sequencing were also done. Results showed a reduction in chCASQ2 expression in broilers dead by SDS. Three novel mutations (K289R, P308S, D310H) which are absent in healthy broilers were observed in chCASQ2. It is concluded that susceptibility to fatal cardiac arrhythmia in SDS may be associated with changes in intracellular Ca(2+) balance due to mutation and downregulation of chCASQ2.

Rhodanese activity in different tissues of the ostrich.

1. The purpose of this investigation was to determine the activity, and compare the pattern of distribution, of rhodanese (thiosulphate: cyanide sulphurtransferase, EC. 2.8.1.1) in different tissues of male and female ostriches. 2. Tissue samples from male and female Blue Neck ostriches were assayed for rhodanese activity by the determination of thiocyanate formed by the action of the enzyme on thiosulphate and KCN. 3. Rhodanese was present in all tissues, and the highest activity was observed in the kidney and liver. Other tissues which contained significant activities of rhodanese were the duodenum, pancreas, heart, caecum and rectum. 4. Unlike other birds, the proventiculus does not appear to have an important role in cyanide detoxification in ostrich and, like mammals, the kidney and liver perform this function. 5. The results suggest that the main organs harbouring high rhodanese activity in the ostrich are associated with sites likely to be required in rhodanese mediated cyanide detoxification.

Effect of actinidin on the protein solubility, water holding capacity, texture, electrophoretic pattern of beef, and on the quality attributes of a sausage product.

The objective of this study was to study the effect of actinidin, a sulfhydryl protease from kiwi fruit, on the protein solubility (nitrogen solubility index [NSI]), water holding capacity (WHC), texture, and SDS-PAGE pattern of beef and to evaluate the effect of pretreatment of beef with actinidin on the quality attributes of a sausage product. Actinidin was partially purified by precipitation with ammonium sulfate, followed by DEAE-Sephadex column chromatography. Actinidin significantly (P < 0.05) increased NSI and WHC of beef; the highest NSI and WHC (approximately 20% and 8% increase, respectively) was observed when beef was incubated with 0.9 unit enzyme/g beef. Texture analysis indicated increased tenderization (10% decrease in shear force) when slices of cattle beef were treated with actinidin at 37 degrees C for 2 h. SDS-PAGE results indicated appearance of several low molecular weight bands (<10 kDa) after treating beef with different levels of actinidin for 30 or 60 min. Slight changes in protein band in the range of 100 to 120 kDa and 13 to 25 kDa were also observed. Use of actinidin-tenderized beef significantly improved emulsion stability, texture, and organoleptic properties of the sausage product.

Antibacterial activity of dextran-conjugated lysozyme against Escherichia coli and Staphylococcus aureus in cheese curd.

The purposes of this research were to glycosylate lysozyme with dextran under Maillard reaction conditions and assess the antimicrobial characteristics of the lysozyme-dextran conjugate in a culture medium and cheese curd. Solutions containing lysozyme and dextran were incubated at 60 degrees C and at 79% relative humidity. Gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to follow the glycosylation process. Under optimum conditions 3.7 mol of dextran were coupled to 1 mol of lysozyme. Lytic activity of the conjugate against the cell wall of Micrococcus luteus was about 62% of that of native lysozyme. Evaluation of the lysozyme-dextran conjugate against test microorganisms (Staphylococcus aureus and Escherichia coli) in culture media indicated a progressive increase in antimicrobial activity, with an increase in enzyme-conjugate concentration. The lysozyme-dextran conjugate was also effective against E. coli in a natural food system, as it reduced the bacterial count by 3 log in cheese curd after 40 days of storage. Unlike E. coli, the antimicrobial action of lysozyme against S. aureus was not improved by conjugation with dextran in both in vitro and in vivo tests. Antimicrobial activity of the lysozyme-dextran conjugate against gram-negative bacteria is probably related to the remaining lytic activity as well as the excellent surfactant properties of the lysozyme-dextran conjugate. These results might increase the applicability of lysozyme as a natural antimicrobial ingredient in different food products.

Distribution of rhodanese in different parts of the urogenital systems of sheep at pre- and post-natal stages.

The enzyme rhodanese (thiosulfate:cyanide sulfurtransferase) is a ubiquitous enzyme present in all living organisms, from bacteria to humans and plays a central role in cyanide detoxification. The purpose of this investigation is to determine and compare rhodanese activity in different parts of urogenital systems of male and female sheep fetuses at 2.5, 3, 3.5, 4, 4.5, and 5 months of age. The highest activity of rhodanese in male fetus was in kidney cortex, followed by medulla of the kidney. No significant difference was observed in other organs. In female fetus, the highest activity was in kidney cortex followed by oviduct and medulla of kidney. The enzyme activity of tissues increased with age. There was no significant difference (P > 0.05) between male and female fetuses in levels of rhodanese activity of different tissues except in urinary bladder at 2.5 and 3 months and in urethra at 4.5 months of age. The results of this study might indicate the involvement of rhodanese in cyanide detoxification in tissues which are more exposed to cyanide. On the other hand, rhodanese might perform other functions which are specific in these tissues.

Rhodanese (thiosulfate: cyanide sulfurtransferase) in the digestive tract of chicken at different stages of development.

This study was undertaken to investigate the relationships between stage of embryonic development and early posthatch growth and the level of rhodanese (thiosulfate:cyanide sulfurtransferase) activity in different regions of the digestive tract and liver of chickens. The embryos were studied at 14, 17, and 20 d and chickens were 1, 2, and 3 wk old. All tissues studied contained rhodanese. The highest specific activity of rhodanese was present in the liver followed by the proventriculus (P < 0.05). The lowest level was in the esophagus. The level of rhodanese was found to increase with age in the proventriculus and duodenum. The highest rhodanese activity in 3-wk-old chickens was in the proventriculus followed by the liver. These results are discussed in terms of the role of different sections of the digestive tract of the chicken in cyanide metabolism.

Isoprenaline induces biosynthesis of proline-rich proteins in the salivary glands of rat but not in sheep.

Long-term treatment of rats with isoprenaline resulted in induction of proline-rich proteins (PRPs) in the salivary glands, which were subsequently purified by TCA solubility and column chromatography. When rats were removed from beta-agonist regimen, then these proteins were no longer observed. Treatment of sheep with isoprenaline did not reveal the induction of PRPs.

Rhodanese (thiosulfate:cyanide sulfurtransferase) distribution in the digestive tract of chicken.

In this study the level of rhodanese (thiosulfate:cyanide sulfurtransferase) activity in different regions of the digestive tract of chicken was determined and compared with that in the liver, heart, kidney, and lung. All tissues studied contained rhodanese. The highest specific activity of rhodanese was in the submucosal layer of proventriculus, followed by liver, heart, the mucosal layer of cecum, rectum, and kidney. The lowest level was present in lung. These results suggest that in the chicken part of the ingested cyanide is detoxified in the digestive tract, mainly by the proventriculus, and part of the absorbed cyanide is metabolized by hepatic rhodanese.

Changes in arginase, aminotransferases and rhodanese in sera of domestic animals with experimentally induced liver necrosis.

Changes in the serum concentrations of aspartic aminotransferase (AST), alanine aminotransferase (ALT), rhodanese and arginase were measured in dogs, sheep and cattle with hepatic necrosis induced by the oral administration of carbon tetrachloride. A new method for arginase assay was based on the determination of remaining arginine (after its conversion to urea and ornithine) by its reaction with p-nitrophenyl glyoxal (PNPG). In all species studied the serum arginase increased 6-12 h after liver damage, reached a peak value in 48 h and returned to normal thereafter. Rhodanese activity did not change in dogs but rose significantly in sheep and, to a lesser extent, in cattle. AST increased strikingly in sheep as compared with dogs and cattle and remained high for > 5 days. In dogs ALT rose sharply and remained elevated for > 10 days. No change in ALT was seen in sheep or cattle. The determination of arginase by a simple procedure such as the PNPG method, in conjunction with AST or ALT assay, may be of value in assessing the stage of liver necrosis.

The cyanide-metabolizing enzyme rhodanese in different parts of the respiratory systems of sheep and dog.

Studies on the rhodanese activity of respiratory systems of sheep and dog showed that a significant difference exists in the pattern of distribution of this enzyme in different parts of the respiratory system of both species. In sheep, larynx, trachea, bronchiole, and lung contain higher activity than nasal cavity and pharynx. In dog, significantly greater rhodanese activity was found in nasal cavity than in other parts of the respiratory system. In regions with high rhodanese activity the enzyme was more concentrated in the mucosa than in the underneath tissues. These results are discussed in terms of the possible role of rhodanese in cyanide metabolism in respiratory tract and the efficacy of this organ in inhaled cyanide detoxification in these species of animals.

A novel colorimetric method for assaying arginase activity.

Arginase catalyzes the conversion of arginine to urea and ornithine in the liver of ureotelic animals. Higher activity of this enzyme is found in tumors as well as in the sera of patients with hepatic diseases. We have developed a simple colorimetric method for its determination. This is based on the determination of residual arginine, after its conversion with p-nitrophenyl glyoxal (PNPG) at pH 9.0 in the presence of sodium ascorbate. The reaction product obeys Beer's law in the range of 0.01-0.20 mmol/L arginine with an arginine-equivalent molar extinction coefficient of 0.65 x 10(4) M-1 cm-1. The decrease in absorbance in the presence of arginase correlates with the enzyme activity. Color development as well as termination of enzyme activity is achieved by addition of a single reagent, thereby obviating the use of many chemicals necessary in other methods. The sensitivity of this method is equivalent to those of currently available procedures but has the added advantages of greater convenience.

Arginase distribution in tissues of domestic animals.

1. A new colorimetric method was used for determination of arginase in different tissues of some domestic animals. 2. In all species studied liver was the richest source of arginase. 3. Significant differences were observed in the specific activity of arginase in livers from different species. 4. In all species, besides liver, kidney and brain also contained significant levels of arginase. 5. In the dog, in addition to the three organs mentioned above, lung, heart, spleen and skeletal muscle showed some arginase activity. 6. In sheep and cattle significant arginase activity was observed in the rumen. No differences were observed between epithelial and muscular layers of different parts of digestive system in all species studied. 7. These results are discussed in terms of the possible role of arginase in different tissues of animals.

Comparative studies on the distribution of rhodanese in different tissues of domestic animals.

1. The activity of rhodanese in different tissues of some domestic animals was measured. 2. Rhodanese was present in all tissues studied. 3. The activity of rhodanese in most tissues of sheep was higher than other animals studied. 4. In sheep and cattle the epithelium of rumen, omasum and reticulum were the richest sources of rhodanese. Significant activity of rhodanese was also present in liver and kidney. 5. In camel the liver contained the highest level of rhodanese followed by lung and rumen epithelium. Camel liver contained a third of the activity of sheep liver. 6. Equine liver had a third of the activity of sheep liver. Other tissues showed low levels of rhodanese activity. 7. Dog liver contained only 4% of the activity of sheep liver. In this animal, brain was the richest source of rhodanese. 8. The results are discussed in terms of efficacy of different tissues of animals in cyanide detoxification.

Determination of creatine kinase in the sera of patients with myocardial infarction by a rho-nitrophenylglyoxal method.

We have developed a new colorimetric method for the determination of creatine kinase (CK). This method is based on the reaction of creatine, formed enzymatically from creatine phosphate and ADP, with rho-nitrophenylglyoxal (PNPG) under mild alkaline conditions to produce a coloured complex which absorbs maximally at 480 nm. This method was applied to the sera of patients with myocardial infarction. The results obtained by the PNPG method agreed well with the results obtained by other available methods for CK. However, the PNPG method was more convenient and less expensive than other methods and required a single chromogenic reagent. The PNPG method might be easily adapted to routine clinical application.

Comparative studies on the distribution of rhodanese and beta-mercaptopyruvate sulfurtransferase in different organs of sheep (Ovis aries) and cattle (Bos taurus).

1. The activities of rhodanese and beta-mercaptopyruvate sulfurtransferase (MST) in different organs of sheep and cattle were measured. 2. Liver, kidney, omasum, and rumen were the richest sources of both enzymes. The activities of both enzymes in other organs of the sheep and the cattle decreased in the order of lung, brain, heart, abomasum, lymph node, urinary bladder, spleen, and the skeletal muscle. 3. The activities of both enzymes in most organs of the sheep were higher than the cattle. 4. Both enzymes showed higher activities in the epithelial layers than the muscular layers of rumen, omasum and reticulum. 5. In most of the tissues of both species the level of rhodanese activity was greater than MST.

A new colorimetric method for determination of creatine phosphokinase.

A new colorimetric method has been developed for the determination of creatine phosphokinase (CPK). This method is based on the reaction of creatine, formed enzymatically from creatine phosphate and ADP, with p-nitrophenylglyoxal (PNPG) under alkaline conditions to produce a colored product which absorbs maximally at 480 nm. At 25 degrees C the reaction was complete after 10 min in 0.1 M sodium pyrophosphate, pH 12, containing 0.15 M sodium ascorbate. The colored product was stable for at least 24 h and obeyed Beer's law in the range 0.005-0.05 mM creatine. The color reaction was used to determine the activity of CPK in serum and tissue extracts. The results obtained by this method agreed well with the results obtained by other available methods for CPK determination. However, the PNPG method was more rapid and more sensitive than other colorimetric methods and required a single chromogenic reagent.