STUDIES ON HAEM BIOSYNTHESIS IN RAT BRAIN

Abstract— Abnormalities involving haem biosynthesis have been postulated as underlying mechanisms in the aetiology of the neural manifestations of acute porphyria and of lead poisoning. This paper reports a study of the enzymes of the haem biosynthetic pathway and their control in mammalian brain. The activity of rat brain 6-aminolaevulinate synthetase (ALA synthetase), 6-aminolaevulinate dehydratase (ALA dehydratase), uroporphyrinogen I synthetase, uroporphyrinogen decarboxylase and ferrochelatase were found to be between 12.5 and 0.002% of the corresponding values for liver. This accords with the lower concentrations of total haem and cytochrome P₄₅₀ found in brain and with the slower rate of incorporation of [4-¹⁴C]ALA into brain haem in vivo . The subcellular distribution of radioactivity following intraventricular injection of [4-¹⁴C]ALA confirmed that the bulk of brain haemoproteins are intramitochondrial in contrast to liver where the major portion is microsomal. Brain haem biosynthesis was apparently unaffected by factors known to influence this pathway in liver, including starvation and treatment with allylisopropylacetamide or phenobarbitone. These findings suggest that brain haem requirements are considerably less than those of liver and are not subject to significant fluctuations under normal circumstances. Apparent non-inducibility of ALA synthetase suggests that deficient haem and consequently haemoprotein production could result where other enzymes in the pathway become rate-limiting due to genetic defects or inhibition by exogenous agents such as lead.     

Effect of ethanol on cholesterol and phospholipid composition of HeLa cells

Chronic exposure of animals to ethanol leads to changes in membrane lipid composition which may be related to the development of tolerance and physical dependence. The object of the present study was to investigate this phenomenon at a cellular level. HeLa cells were grown in the presence of ethanol (86 mM) for periods of up to 9 days. Both the cholesterol and phospholipid concentration of these cells increased during this period but the cholesterol:phospholipid ratio remained unchanged. Among the phospholipid classes phosphatidic acid decreased while phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine increased rapidly, returning toward control values by 9 days. Significant decreases were observed in saturated (14:0, 16:0) and monoenoic (16:1, 18:1) fatty acids while the major polyenoic fatty acid (20:4) increased. It is concluded that cultured mammalian cells represent a useful model for investigation of the direct effects of ethanol on membrane lipid metabolism.     

Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells

Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcgammaRIIA or both FcgammaRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcgammaRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcgammaRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcgammaRIIA under identical conditions displayed little periphagosomal Ca2+ signaling. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca2+ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca2+ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.     

Improved colorimetric procedure for quantitating N-carbamoyl-β-alanine with minimum dihydrouracil interference

After modifying the Prescott-Jones colorimetric method for carbamoyl compound determination, it was possible to measure the concentration of N-carbamoyl-β-alanine (β-ureidopropionate) with little interference from its metabolic precursor, dihydrouracil. Color formation at 70°C was linear with respect to N-carbamoyl-β-alanine up to a concentration of 0.07 mm.     

Effects of chronic ethanol treatment and aging on brain phosphoinositide turnover and adenylate cyclase activity

Inositol phosphate accumulation and adenylate cyclase activity were investigated in the cortex of young and aged ethanol-treated rats. Three months of ethanol treatment of young rats decreased maximal stimulation of inositol phosphate accumulation by carbachol by 26%, from 494 ± 76% of basal turnover in control animals to 396 ± 54% in ethanol-treated animals (mean ± SD). In aged rats ethanol-related changes were no longer observed but age-related changes were evident. EC₅₀ was significantly higher than in young animals and maximal stimulation was significantly lower. Basal adenylate cyclase activity in cortical membranes of all groups of animals was not different. Forskolin-stimulated adenylate cyclase activity was not affected by ethanol treatment, but was higher in aged animals. The activity of forskolin-stimulated adenylate cyclase in the presence of carbachol was higher in both young and aged ethanol-treated animals, when compared to young controls. These results suggest that both ethanol and aging impair the efficiency of receptor/effector coupling.     

Out of the Loop: Why Research Rarely Reaches Policy Makers and the Public and What Can be Done

Most of the world's population that derives their livelihoods or part of their livelihoods from forests are out of the information loop. Exclusion of public users of natural resources from access to scientific research results is not an oversight; it is a systemic problem that has costly ramifications for conservation and development. Results of a survey of 268 researchers from 29 countries indicate that institutional incentives support the linear, top-down communication of results through peer-reviewed journal articles, which often guarantees positive performance measurement. While the largest percentage of respondents (34%) ranked scientists as the most important audience for their work, only 15 percent of respondents considered peer-reviewed journals effective in promoting conservation and/or development. Respondents perceived that local initiatives (27%) and training (16%) were likely to lead to success in conservation and development; but few scientists invest in these activities. Engagement with the media (5%), production of training and educational materials (4%) and popular publications (5%) as outlets for scientific findings was perceived as inconsequential (<14%) in measuring scientific performance. Less than 3 percent of respondents ranked corporate actors as an important audience for their work. To ensure science is shared with those who need it, a shift in incentive structures is needed that rewards actual impact rather than only 'high-impact' journals. Widely used approaches and theoretical underpinnings from the social sciences, which underlie popular education and communication for social change, could enhance communication by linking knowledge and action in conservation biology.     

Heterogeneity of lipoprotein B

Combined very low and low density lipoproteins were derived from human plasma by polyanion precipitation and the low density lipoprotein fraction (density 1.027–1.050 g/ml) was isolated by sequential ultracentrifugation. When this fraction was applied to Sepharose column chromatography, three lipoproteins were eluted. The first and third peaks were minor components while the second peak represented the bulk of LDL. Further chromatographic and electrophoretic studies indicated that the component representing the second peak was heterogeneous. This component was subsequently delipidated at pH 4 in a quaternary biphasic solvent system. The apoproteins remained soluble after delipidation and were treated with various deaggregating agents. On column isoelectric focusing in the presence of 4 M urea the apoproteins banded as broad overlapping peaks between pH 3 and 7. When hexanol was added to the system, distinct apoprotein subfractions were resolved.     

Purification and some properties of cytosine deaminase from Salmonella typhimurium

Cytosine deaminase (EC 3.5.4.1) from Salmonella typhimurium has been purified 419-fold to apparent homogeneity. SDS polyacrylamide gel electrophoresis indicated that the final cytosine deaminase preparation was homogenous. The molecular weight of cytosine deaminase was determined to be approx. 230 000 containing four identical subunits with each subunit having a molecular weight of 54 000. Cytosine deaminase has a pH optimum of 7.30 to 7.50 and a temperature optimum of 45 to 50°C. Cytosine was deaminated specifically; 5-fluorocytosine was deaminated to a lesser extent. The K_m and V values for cytosine were 0.74 mM and 47.16 μmole/min, respectively. As effectors of enzyme activity, PP_i stimulated the deamination while metal ions and orotidine monophosphate inhibited it. The physical characteristics of cytosine deaminase lend credence to its proposed salvage role in pyrimidine metabolism as indicated previously by physiological studies (West, T.P. and O'Donovan, G.A., J. Bacteriol. (1982) 149, 1171–1174).