Nucleosome core protein: Asymmetric dissociation of the octamer

The octameric nucleosomal core-histone complex, (H2A)2-(H2B)2-(H3)2-(H4)2, isolated from rat liver, undergoes dissociation during gel exclusion chromatography as a result of dilution occurring in the columns. The elution pattern at pH 7.0 and 4°C showed a sharp leading peak containing all four histones but predominantly H3 and H4, and a trailing peak containing equal amounts of histones H2A and H2B. As column length was increased the area under the leading peak decreased and that under the trailing peak increased. In addition the relative positions of the two peaks varied with column length. From an analysis of the data on increase in elution volume of the leading peak in relation to column length an apparent molecular weight of 86 000 was calculated for the undissociated molecule. Its apparent molecular weight, histone composition and pattern of further dissociation in relation to column length suggest that this species is the hexamer, (H2A-H2B)-(H3)2-(H4)2. At pH 7.0 and 4°C the dissociation of the core complex appears to be as follows: (H2A)2-(H2B)2-(H3)2-(H4)2 → (H2A-H2B) + (H2A-H2B)-(H3)2-(H4)2 → 2(H2A-H2B) + (H3)2-(H4)2 This dissociation was accelerated by an increase in temperature or decrease in pH and was accompanied by marked conformational changes as judged by circular dichroism measurements.

Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver.

The homogeneous serine hydroxymethyltransferase purified from monkey liver, by the use of Blue Sepharose affinity chromatography, exhibited positive homotropic co-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactions with L-serine and nicotinamide nucleotides. The enzyme had an unusually high temperature optimum of 60 degrees C and was protected against thermal inactivation by L-serine. The allosteric effects were abolished when the monkey liver enzyme was purified by using a heat-denaturation step in the presence of L-serine, a procedure adopted by earlier workers for the purification of this enzyme from mammalian and bacterial sources. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterin and D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors. The insoluble monkey liver enzyme-antibody complex was catalytically active and failed to show positive homotropic co-operative interactions with tetrahydrofolate (h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. These results highlight the pitfalls in using a heat-denaturation step in the purification of allosteric enzymes.

Optimising between and within family selection for harvest weight in prawns with restricted harvest size.

Manual grading of prawns restricts the number that can be harvested. A restricted harvest size places a limit on the opposing within family and between family sources of selection pressure. A simulation study with inbreeding constrained at 0.5% per generation, a harvest size of 2000, heritability of 0.3, common family environmental effect of 0.1, indicates that maximum response to selection could be achieved with as few as 40 families. Increasing the number of families above 80 may reduce total selection response. It is important to be aware that increasing the number of families may not always yield a greater genetic response.

Effects of dairy proteins and calcium on diet-induced obesity in mice

Diet high in dairy products is inversely associated with body mass index, risk of metabolic syndrome and prevalence of type 2 diabetes in several populations. Also a number of intervention studies support the role of increased dairy intake in the prevention and treatment of obesity. Dairy calcium has been suggested to account for the effect of dairy on body weight, but it has been repeatedly shown that the effect of dairy is superior to the effect of supplemental calcium. Dairy proteins are postulated to either enhance the effect of calcium or have an independent effect on body weight, but studies in the area are scarce. The aim of this study was to evaluate the potential of dairy proteins and calcium in the prevention and treatment of diet-induced obesity in C57Bl/6J mice. The effect of dairy proteins and calcium on the liver and adipose tissue was also investigated in order to characterise the potential mechanisms explaining the reduction of risk for metabolic syndrome and type 2 diabetes. A high-calcium diet (1.8%) in combination with dietary whey protein inhibited body weight and fat gain and accelerated body weight and fat loss in high-fat-fed C57Bl/6J mice during long-term studies of 14 to 21 weeks. α-lactalbumin, one of the major whey proteins, was the most effective whey protein fraction showing significantly accelerated weight and fat loss during energy restriction and reduced the amount of visceral fat gain during ad libitum feeding after weight loss. The microarray data suggest sensitisation of insulin signalling in the adipose tissue as a result of a calcium-rich whey protein diet. Lipidomic analysis revealed that weight loss on whey protein-based high-calcium diet was characterised by significant decreases in diabetogenic diacylglycerols and lipotoxic ceramide species. The calcium supplementation led to a small, but statistically significant decrease in fat absorption independent of the protein source of the diet. This augments, but does not fully explain the effects of the studied diets on body weight. A whey protein-containing high-calcium diet had a protective effect against a high-fat diet-induced decline of β3 adrenergic receptor expression in adipose tissue. In addition, a high-calcium diet with whey protein increased the adipose tissue leptin expression which is decreased in this obesity-prone mouse strain. These changes are likely to contribute to the inhibition of weight gain. The potential sensitisation of insulin signalling in adipose tissue together with the less lipotoxic and diabetogenic hepatic lipid profile suggest a novel mechanistic link to explain why increased dairy intake is associated with a lower prevalence of metabolic syndrome and type 2 diabetes in epidemiological studies. Taken together, the intake of a high-calcium diet with dairy proteins has a body weight lowering effect in high-fat-fed C57Bl/6J mice. High-calcium diets containing whey protein prevent weight gain and enhance weight loss, α-lactalbumin being the most effective whey protein fraction. Whey proteins and calcium have also beneficial effects on hepatic lipid profile and adipose tissue gene expression, which suggest a novel mechanistic link to explain the epidemiological findings on dairy intake and metabolic syndrome. The clinical relevance of these findings and the precise mechanisms of action remain an intriguing field of future research.

Inhibition of H2O2 generation in rat liver mitochondria by radical quenchers and phenolic compounds

Generation of H2O2 by rat liver mitochondria with choline, glycerol 1-phosphate and proline as substrates has been shown by using high-concentration phosphate buffer. Rates obtained under these conditions were higher and more consistent as compared with the earlier reports with high-concentration mannitol/sucrose/Tris buffer. Sulphate ions could replace phosphate indicating a requirement for a high concentration of oxygen-containing anions. H2O2 generation was dependent on the presence of native mitochondria and substrate. Maximal rates with various substrates were found to be the same as with succinate. Values of Km and Vmax for H2O2 generation were considerably less than those obtained for respective dehydrogenase activities, measured by dye reduction. Scavengers of O2-. and OH. inhibited generation of H2O2. ATP, ADP, thyronine derivatives and a number of phenolic compounds also showed very potent inhibitory effects of H2O2 generation, whereas phenyl compound had no effect. Phenolic compounds did not have any effect on mitochondrial superoxide dismutase and choline dehydrogenase activities as well as on O2-. generation by the xanthine-xanthine oxidase system. Inhibition by phenolic compounds may have potential for regulation of the intracellular concentration of H2O2, that is not considered to have a "second messenger' function.

Identification of amino acid residues essential for enzyme activity of sheep liver 5,10-methylenetetrahydrofolate reductase

Sheep liver 5,10-methylenetetrahydrofolate reductase was subjected to specific chemical modification with phenylglyoxal, diethyl pyrocarbonate and N-bromosuccinimide. The second-order rate constants for inactivation were calculated to be 54 M-1 X min-1, 103 M-1 X min-1 and 154 M-1 X min-1 respectively. This inactivation could be prevented by incubation with substrates or products, suggesting that the residues modified, namely arginine, histidine and tryptophan, are essential for enzyme activity.

Low birth weight in MZ twins discordant for birth weight is associated with shorter telomere length and lower IQ, but not anxiety/depression in later life

Shorter telomere length (TL) has found to be associated with lower birth weight and with lower cognitive ability and psychiatric disorders. However, the direction of causation of these associations and the extent to which they are genetically or environmentally mediated are unclear. Within-pair comparisons of monozygotic (MZ) and dizygotic (DZ) twins can throw light on these questions. We investigated correlations of within pair differences in telomere length, IQ, and anxiety/depression in an initial sample from Brisbane (242 MZ pairs, 245 DZ same sex (DZSS) pairs) and in replication samples from Amsterdam (514 MZ pairs, 233 DZSS pairs) and Melbourne (19 pairs selected for extreme high or low birth weight difference). Intra-pair differences of birth weight and telomere length were significantly correlated in MZ twins, but not in DZSS twins. Greater intra-pair differences of telomere length were observed in the 10% of MZ twins with the greatest difference in birth weight compared to the bottom 90% in both samples and also in the Melbourne sample. Intra-pair differences of telomere length and IQ, but not of TL and anxiety/depression, were correlated in MZ twins, and to a smaller extent in DZSS twins. Our findings suggest that the same prenatal effects that reduce birth weight also influence telomere length in MZ twins. The association between telomere length and IQ is partly driven by the same prenatal effects that decrease birth weight.

Stimulation of liver tryptophan pyrrolase during heat exposure

Exposure of rats to heat (39 +/- 1 degree C) stimulated liver tryptophan pyrrolase 2-fold between 3 and 48 h. Plasma corticosterone increased 2-fold after 1 h of heat exposure and decreased to a low value of 50% by 16 h. The effect of heat exposure on the enzyme was obtained in adrenalectomized animals. Stimulation by cortisol and tryptophan of the enzyme was also obtained in heat exposure, and the effects seemed to be additive. The concentration of tryptophan in the liver remained unchanged, and that in the plasma decreased to about 50% at 8 h exposure to heat and reverted to normal by 46 h. Simultaneous administration of noradrenaline to heat-exposed rats had no effect, whereas that of thyroxine partly prevented the stimulation of the enzyme activity. Hypothyroid conditions obtained by thyroidectomy or treatment with propylthiouracil significantly stimulated the enzyme activity. Cycloheximide treatment of heat-exposed rats did not prevent the stimulation of the enzyme activity. The results indicate that the effect of heat exposure on liver tryptophan pyrrolase is obtained, due to the accompanying hypothyroid condition, by increasing the activity of the existing protein by a mechanism possibly different from those known at present.

Heat exposure and hypothyroid conditions decrease hydrogen peroxide generation in liver mitochondria

Exposure of rats to heat (39 +/- 1 degree C) decreased H2O2 generation in mitochondria of the liver, but not of the kidney or the heart. The effect was obtained with three substrates, succinate, glycerol 1-phosphate and choline, with a decrease to 50% in the first 2-3 days of exposure, and a further decrease on longer exposure. The dehydrogenase activity with only glycerol 1-phosphate decreased, which is indicative of the hypothyroid condition, whereas choline dehydrogenase activity remained unchanged and that of succinate dehydrogenase decreased on long exposure. The serum concentration of thyroxine decreased in heat-exposed rats. Thyroxine treatment of rats increased H2O2 generation. Hypothyroid conditions obtained by treatment with propylthiouracil or thyroidectomy caused a decrease in H2O2 generation and changes in dehydrogenase activities similar to those with heat exposure. Treatment of heat-exposed or thyroidectomized rats with thyroxine stimulated H2O2 generation by a mechanism apparently involving fresh protein synthesis. The results indicate that H2O2 generation in mitochondria of heat-exposed animals is determined by thyroid status.

Inflammation-induced atherogenesis, liver alterations, and cardiovascular outcome

Cardiovascular diseases, which presently are considered inflammatory diseases, affect millions of people worldwide. Chronic infections may contribute to the systemic inflammation suggested to increase the risk for cardiovascular diseases. Such chronic infections are periodontitis and Chlamydia pneumoniae infection. They are highly prevalent as approximately 10% of adult population and 30% of people over 50 years old are affected by severe periodontitis and 70-80% of elderly people are seropositive for C. pneumoniae. Our general aim was to investigate the role of infection and inflammation in atherosclerosis both in animal and human studies. We aimed to determine how the two pathogens alter the atherosclerosis-associated parameters, and how they affect the liver inflammation and lipid composition. Furthermore, we evaluated the association between matrix metalloproteinase-8 (MMP-8), a proteinase playing a major role in inflammation, and the future cardiovascular diseases (CVD) events in a population-based cohort. For the animal experiments, we used atherosclerosis-susceptible apolipoprotein E deficient (apoE-/-) mice. They were kept in germ free conditions and fed with a normal chow diet. The bacteria were administered either intravenously (A. actinomycetemcomitans) or intranasally (C. pneumoniae). Several factors were determined from serum as well as from aortic and hepatic tissues. We also determined how cholesterol efflux, a major event in the removal of excess cholesterol from the tissues, and endothelial function were affected by these pathogens. In the human study, serum MMP-8 and its tissue inhibitor (TIMP-1) concentrations were measured and their associations during the follow-up time of 10 years with CVD events were determined. An infection with A. actinomycetemcomitans increased concentrations of inflammatory mediators, MMP production, and cholesterol deposit in macrophages, decreased lipoprotein particle size, and induced liver inflammation. C. pneumoniae infection also elicited an inflammatory response and endothelial dysfunction, as well as induced liver inflammation, microvesicular appearance and altered fatty acid profile. In the population-based cohort, men with increased serum MMP-8 concentration together with subclinical atherosclerosis (carotid artery intima media thickness > 1mm) had a three-fold increased risk for CVD death during the follow-up. The results show that infections with A. actinomycetemcomitans and C. pneumoniae induce proatherogenic changes, as well as affect the liver. These data therefore support the concept that common infections have systemic effects and could be considered as cardiovascular risk factors. Furthermore, our data indicate that, as an independent predictor of fatal CVD event, serum MMP-8 could have a clinical significance in diagnosing cardiovascular diseases.

Residue Potential of Norsesquiterpene Glycosides in Tissues of Cattle Fed Austral Bracken (Pteridium esculentum).

Austral bracken, Pteridium esculentum, occurs widely in Australian grazing lands and contains both the known carcinogen ptaquiloside and its hydroxy analogue, ptesculentoside, with untested carcinogenic potential. Calves were fed a diet containing 19% P. esculentum that delivered 1.8 mg of ptaquiloside and 4.0 mg of ptesculentoside per kilogram of body weight (bw) per day to explore the carcass residue potential of these compounds. Concentrations of ptaquiloside and ptesculentoside in the liver, kidney, skeletal muscle, heart, and blood of these calves were determined as their respective elimination products, pterosin B and pterosin G, by HPLC-UV analysis. Plasma concentrations of up to 0.97 mu g/mL ptaquiloside and 1.30 mu g/mL ptesculentoside were found, but were shown to deplete to <10% of these values within 24 h of bracken consumption. Both glycosides were also detected in all tissues assayed, with ptesculentoside appearing to be more residual than ptaquiloside. Up to 0.42 and 0.32 mu g/g ptesculentoside was present in skeletal muscle and liver, respectively, 15 days after bracken consumption ended. This detection of residual glycosides in tissues of cattle feeding on Austral bracken raises health concerns for consumers and warrants further investigation.

Identification of active-site residues of sheep liver serine hydroxymethyltransferase

Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5'-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.

Functional changes in rat liver mitochondria on administration of 2-methyl-4-dimethylaminoazobenzene

Administration of 2-methyl-4-dimethylaminobenzene in the diet (0.1%, w/w) for 85-90 days doubled the content of mitochondria in the livers of rats. The azodye was covalently bound to liver proteins, and about 15% of the amount found in liver was associated with the mitochondrial fraction. Mitochondria isolated from the livers of azodye-fed animals showed drastically lowered ability to oxidize NAD+-linked substrates. The inhibited electron-transfer step was the reduction of ubiquinone. The organelles showed a large increase in succinate oxidase activity. The activity of cytochrome oxidase and the content of cytochrome aa3 were substantially higher in these organelles. Azodye-fed animals showed depressed serum cholesterol concentrations. The content of ubiquinone in liver also registered a small increase.