Genetic factors in the pathogenesis of CPPD crystal deposition disease

Crystal deposition is a very complex process ruled by numerous factors. A small but important proportion of cases of chondrocalcinosis are monogenic, and many of the genes involved have been identified. These genetic findings strongly point to control of the level of extracellular inorganic pyrophosphate as the primary mechanism for their association with either calcium pyrophosphate dihydrate or hydroxyapatite deposition. However, effects on extracellular inorganic pyrophosphate levels do not explain the mechanism of association in all of these monogenic diseases. Further, there are likely to be several as yet unidentified genes that are important in this common condition. This review highlights what genetic studies have demonstrated about the processes involved in these diverse but related disorders.

Insecticide resistance and implications for future aphid management in Australian grains and pastures: A review

Aphids can cause substantial damage to cereals, oilseeds and legumes through direct feeding and through the transmission of plant pathogenic viruses. Aphid-resistant varieties are only available for a limited number of crops. In Australia, growers often use prophylactic sprays to control aphids, but this strategy can lead to non-target effects and the development of insecticide resistance. Insecticide resistance is a problem in one aphid pest of Australian grains in Australia, the green peach aphid (Myzus persicae). Molecular analyses of field-collected samples demonstrate that amplified E4 esterase resistance to organophosphate insecticides is widespread in Australian grains across Australia. Knockdown resistance to pyrethroids is less abundant, but has an increased frequency in areas with known frequent use of these insecticides. Modified acetylcholinesterase resistance to dimethyl carbamates, such as pirimicarb, has not been found in Australia, nor has resistance to imidacloprid. Australian grain growers should consider control options that are less likely to promote insecticide resistance, and have reduced impacts on natural enemies. Research is ongoing in Australia and overseas to provide new strategies for aphid management in the future.

Occurrence and properties of steryl ferulates and glycosides in wheat and rye

Cereal kernels are known to contain a number of minor components that possess beneficial health attributes. In this thesis rye and wheat were studied as sources of steryl ferulates and steryl glycosides and their behaviour in processing were evaluated. Further, enzymatic hydrolysis of these conjugates was studied, as well as the capacity of steryl ferulates to inhibit lipid oxidation at different temperatures. Steryl ferulates were shown to have a strong positive correlation with dietary fibre contents in milling fractions from the outer parts of the kernels obtained from a commercial scale mill. Highest contents of steryl ferulates were found in the bran in both cereals, with the content decreasing once moving towards the inner parts of the kernel. Variation in the contents of steryl ferulates was higher in wheat fractions than rye fractions. Steryl glycosides, on the other hand, had either negative or no correlation with dietary fibre, and the range of the steryl glycoside contents was much narrower than that of steryl ferulates in both cereals. There were significant differences in the sterol compositions of these steryl conjugates when compared with each other or with the total plant sterols in the corresponding fractions. Properties of steryl ferulates and steryl glycosides were evaluated after common processing methods and in enzymatic hydrolysis. Thermal and mechanical processing had only minor or no effects on the contents of steryl conjugates from rye and wheat bran. Enzymatic treatments on the other hand caused some changes, especially in the contents of glycosylated sterols. When steryl ferulates extracted from rye or wheat bran were subjected to enzymatic treatments by steryl esterase, significant differences in the rates of hydrolysis were observed between steryl ferulates from different sources with differing sterol compositions. Further, differences were also observed between enzymes from different sources. Steryl glycosides were shown to be hydrolysed by β-glucosidase (cellobiase) from A. niger, but less with β-glucosidases from other sources. Steryl ferulates showed good antioxidant activity at both moderate and high temperatures. In bulk and emulsion systems of methyl linoleate at 40°C steryl ferulates extracted from rye and wheat bran inhibited hydroperoxide formation much more effectively than synthetic steryl ferulates or those extracted from rice (γ-oryzanol), demonstrating that the sterol composition has an effect on the activity. At cooking (100°C) and frying temperatures (180°C) sitostanyl ferulate was shown to inhibit polymer formation significantly and, especially at 100°C, comparably to α-tocopherol. The rate of antioxidant degradation was slower for sitostanyl ferulate, showing higher heat stability than α-tocopherol. When evaluated as a mixture, no synergistic effect was observed between these two antioxidants. The data presented in this thesis provides information that may henceforth be applied when evaluating the intakes of steryl conjugates from cereal sources, as well as their possible influences as minor bioactive components. Wheat and rye both are good sources of steryl ferulates and steryl glycosides and, especially with steryl ferulates, what may be lost out to some other cereals on quantity is compensated with quality of the sterol composition.

Microbial activities in boreal soils: Biodegradation of organic contaminants at low temperature and ammonia oxidation

This thesis deals with the response of biodegradation of selected anthropogenic organic contaminants and natural autochthonous organic matter to low temperature in boreal surface soils. Furthermore, the thesis describes activity, diversity and population size of autotrophic ammonia-oxidizing bacteria (AOB) in a boreal soil used for landfarming of oil-refinery wastes, and presents a new approach, in which the particular AOB were enriched and cultivated in situ from the landfarming soil onto cation exchange membranes. This thesis demonstrates that rhizosphere fraction of natural forest humus soil and agricultural clay loam soil from Helsinki Metropolitan area were capable of degrading of low to moderate concentrations (0.2 50 µg cm-3) of PCP, phenanthrene and 2,4,5-TCP at temperatures realistic to boreal climate (-2.5 to +15 °C). At the low temperatures, the biodegradation of PCP, phenanthrene and 2,4,5-TCP was more effective (Q10-values from 1.6 to 7.6) in the rhizosphere fraction of the forest soil than in the agricultural soil. Q10-values of endogenous soil respiration (carbon dioxide evolution) and selected hydrolytic enzyme activities (acetate-esterase, butyrate-esterase and β-glucosidase) in acid coniferous forest soil were 1.6 to 2.8 at temperatures from -3 to +30 °C. The results indicated that the temperature dependence of decomposition of natural autochthonous soil organic matter in the studied coniferous forest was only moderate. The numbers of AOB in the landfarming (sandy clay loam) soil were determined with quantitative polymerase chain reaction (real-time PCR) and with Most Probable Number (MPN) methods, and potential ammonium oxidation activity was measured with the chlorate inhibition technique. The results indicated presence of large and active AOB populations in the heavily oil-contaminated and urea-fertilised landfarming soil. Assessment of the populations of AOB with denaturing gradient gel electrophoresis (DGGE) profiling and sequence analysis of PCR-amplified 16S rRNA genes showed that Nitrosospira-like AOB in clusters 2 and 3 were predominant in the oily landfarming soil. This observation was supported by fluorescence in situ hybridization (FISH) analysis of the AOB grown on the soil-incubated cation-exchange membranes. The results of this thesis expand the suggested importance of Nitrosospira-like AOB in terrestrial environments to include chronically oil-contaminated soils.

Fatty acid component of vitamin A ester in sheep liver

Vitamin A, when extracted along with other lipids from sheep liver, had an E1cm.1% value of 14.4, which was raised to 45.57 on removal of the phospholipids by cold acetone. Selective hydrolysis of triglycerides by an extract of acetone-dried sheep pancreas in the presence of HgCl2 as inhibitor of vitamin A esterase, followed by chromatography through alumina gave a product with E1cm.1% value of 276. This on chromatography through magnesium oxide raised the E1cm.1, value to 601.5, representing 64% pure vitamin A ester calculated as palmitate, and the total recovery was 23% of the starting oil. The purified ester preparation, when subjected to reverse-phase chromatography on silicone-impregnated paper, gave a single ultraviolet fluorescent band. The fluorescent band on hydrolysis gave only one fatty acid. This was conclusively identified to be palmitic acid.

Carboxilesterase como biomarcador de hipoxia em peixes

Quando as esterases acetilcolinesterase (AChE), butirilcolinesterase (BChE) e carboxilesterase (CarbE) hidrolisam ésteres de fosfato seus sítios ativos sofrem fosfatação inibitória. Por isto, tal fosfatação pode proteger seres vivos contra o espalhamento de xenobióticos organofosforados dentro de seus corpos, já que estas enzimas têm a capacidade de captar moléculas de pesticidas organofosforados estequiometricamente. Os organismos terrestres vivem em um ambiente com mais oxigênio do que os organismos aquáticos. Na água, quando o nível de oxigênio atinge aproximadamente 2,6 mg/L o ambiente está em hipoxia. Este fenômeno afeta ecossistemas aquáticos, uma vez que muitos organismos não conseguem se adaptar à baixa do oxigênio. Estudamos peixes em hipoxia e hiperoxia para entender melhor a bioquímica do funcionamento de suas enzimas captadoras de organofosforados quando eles estão expostos às variações físico-químicas de seus habitats. Dois grupos de no mínimo seis pacus (Piaractus mesopotamicus), seis peixes dourados (Carassius auratus auratus), seis tilápias (Oreochromis niloticus niloticus), seis piavussus (Leporinus macrocephalus), seis apaiaris (Astronotus ocellatus), ou seis carpas (Cyprinus carpio carpio) foram aclimatados à temperatura ambiente em dois aquários de 250 L. No primeiro aquário, pelo menos três animais ensaio de cada espécie sofreram hipoxia por diminuição da concentração de oxigênio até 0,5 mg/L através de borbulhamento de nitrogênio na água. Quando estes animais atingiram a hipoxia foram mantidos a 0,5 mg/L de oxigênio por 6, 8, 24 ou, no máximo, por 42 horas. Três peixes controle de cada espécie foram mantidos em normoxia (4,5 até 7,0 mg/L de oxigênio). Após estes tempos houve a retirada de cerca de 3,5 mL de sangue e dos fígados. Depois de coagular, o sangue foi centrifugado para retirada do soro sobrenadante, que foi usado como amostra para ensaios das esterases. Os fígados foram armazenados em freezer a -70 C e, no momento do ensaio, homogeneizados e centrifugados para obter as frações citosólica e microssomal. As atividades das esterases foram ensaiadas em espectrofotômetro com os substratos acetiltiocolina, butiriltiocolina ou p-nitrofenilacetato. As atividades sobre p-nitrofenilacetato (CarbE) do soro e do fígado sofreram queda em todos os exemplares das espécies submetidos à hipoxia. Tipicamente, esta atividade caiu cerca de 50% nos soros de pacus mantidos por 42 h sob concentrações de oxigênio abaixo de 1,0 mg/L. O tempo para que ocorresse a queda desta atividade enzimática variou de espécie para espécie.

Biological activities of skin secretions of the salamander Tylototriton verrucosus

Water-soluble skin secretions of salamander Tylototriton venucosus, first described by Anderson in 1871, were studied for their biological and enzymatic activities. They were found to be toxic to mice with an intraperitoneal LD50 of 11.5 mg/kg. Using Sephadex G-75 gel filtration, it was proven that the toxic components of the secretions are proteins with molecular weights ranging from 30,000 to 50,000 Da. The secretions of T. venucosus display a wide spectrum of antimicrobial activities and also contain both proteolytic activity and trypsin inhibitory activity. In contrast, neither hemolytic nor hemorrhagic activities were found. The secretions were determined to have phospholipase A(2) activity; however, no acetylcholine esterase activity was detectable under the assay conditions.

ISOLATION AND PROPERTIES OF A BLOOD-COAGULATION FACTOR-X ACTIVATOR FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

Biochemical and biological properties of Trimeresurus jerdonii venom and characterization of a platelet aggregation-inhibiting acidic phospholipase A(2)

Several biochemical and biological activities such as phospholipase A(2), arginine esterase, proteolytic, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase, thrombin-like, anticoagulant, and hemorrhagic activities were determined for whole desiccated venom of Trimeresurus jerdonii. An acidic phospholipase (named TJ-PLA(2)) was purified by anionic exchange chromatography, gel filtration, and reverse phase HPLC. TJ-PLA(2) had a molecular weight of 16,000 and a pI of 4.8. TJ-PLA(2) was non-lethal to mice up to an i.p. dose of 15 mg/kg body weight and lacked neurotoxicity and myotoxicity. It induced edema in the footpads of mice. The purified enzyme inhibited ADP- and collagen-induced human platelet aggregation in a manner which was both dose- and time-dependent.

Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms

Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5' nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes. (C) 2003 Elsevier Ltd. All rights reserved.