Catalytic properties of cytochrome c oxidase purified from Nitrosomonas europaea

Cytochrome c oxidase of Nitrosomonas europaea reacts with not only the native cytochrome c (N. europaea cytochrome c-552) but also horse and yeast cytochromes c. The effects on its reactivity of various reagents were very different between the reactions with the native and eukaryotic cytochromes c as the electron donors. The oxidation of eukaryotic ferrocytochrome c by the oxidase was activated by addition of anionic detergents such as sodium dodecyl sulfate and sodium cholate, and anionic phospholipids such as cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine, while the reaction was not activated by Triton X-100, Tween 20, or phosphatidylcholine. However, the reaction with the native cytochrome c of the enzyme was hardly affected by any of the detergents and phospholipids mentioned above, while it was activated by the presence of poly-L-lysine.     

A new method for the isolation of fresh hepatocytes from periportal and pericentral regions of the liver lobule

A simple method which avoids the use of perfusion with calcium free buffer, hydrolytic enzymes and detergents has been developed to obtain fresh hepatocytes from periportal and pericentral regions of the liver lobule. Cylindrical plugs (200 x 500 microns) of periportal and pericentral areas of the rat liver lobule weighing about 1 mg were collected with a micropunch from fresh or perfused liver. Ninety percent of cells were intact as assessed from trypan blue staining. Glutamine synthetase activity was detected predominantly (ca. 85%) in plugs isolated from pericentral regions indicating that this method allows selective harvesting of pure sublobular zones of the liver lobule. Rates of oxygen uptake measured at 25 degrees C by plugs from livers perfused in the anterograde direction were 56 +/- 5 and 33 +/- 7 mumol/g/h by periportal and pericentral plugs, respectively, values similar to data obtained from the intact organ. This method provides new opportunities to study the regulation of basic metabolic processes in cells from sublobular areas under nearly physiological conditions.     

Modulation of diphthamide synthesis by 5'-deoxy-5'-methylthioadenosine in murine lymphoma cells

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5'-deoxy-5'-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.     

Simulation analysis of excitation conduction in the heart: Propagation of excitation in different tissues

The normal excitation and conduction in the heart are maintained by the coordination between the dynamics of ionic conductance of each cell and the electrical coupling between cells. To examine functional roles of these two factors, we proposed a spatially-discrete model of conduction of excitation in which the individual cells were assumed isopotential. This approximation was reasoned by comparing the apparent space constant with the measured junctional resistance between myocardial cells. We used the four reconstruction models previously reported for five kinds of myocardial cells. Coupling coefficients between adjacent cells were determined quantitatively from the apparent space constants. We first investigated to what extent the pacemaker activity of the sinoatrial node depends on the number and the coupling coefficient of its cells, by using a one-dimensional model system composed of the sinoatrial node cells and the atrial cells. Extensive computer simulation revealed the following two conditions for the pacemaker activity of the sinoatrial node. The number of the sinoatrial node cells and their coupling coefficients must be large enough to provide the atrium with the sufficient electric current flow. The number of the sinoatrial node cells must be large so that the period of the compound system is close to the intrinsic period of the sinoatrial node cell. In this simulation the same sinoatrial node cells produced action potentials of different shapes depending on where they were located in the sinoatrial node. Therefore it seems premature to classify the myocardial cells only from their waveforms obtained by electrical recordings in the compound tissue. Second, we investigated the very slow conduction in the atrioventricular node compared to, for example, the ventricle. This was assumed to be due to the inherent property of the membrane dynamics of the atrioventricular node cell, or to the small value of the coupling coefficient (weak intercellular coupling), or to the electrical load imposed on the atrioventricular node by the Purkinje fibers, because the relatively small atrioventricular node must provide the Purkinje fibers with sufficient electric current flow. Relative contributions of these three factors to the slow conduction were evaluated using the model system composed of only the atrioventricular cells or that composed of the atrioventricular and Purkinje cells. We found that the weak coupling has the strongest effect. In the model system composed of the atrioventricular cells, the propagation failure was not observed even for very small values of the coupling coefficient.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ammonia in the intestinal contents from germfree rats

The values of pH and the concentrations of nitrogen (N) and ammonia in the middle part of the small intestinal and cecal contents of germfree (GF) and conventionalized (CVZ) seven-week-old rats were compared. The pH of the small intestinal and cecal contents of GF rats was higher than that of CVZ rats. There was no difference in total N per fresh weight in contents from the middle part of the small intestine between GF and CVZ, whereas total N per fresh weight of the cecal contents was higher in CVZ than in GF rats. The ammonia concentrations per fresh weight or per total N in the intestinal and cecal contents of CVZ rats were higher than those of GF rats.     

Studies on the metabolism of unsaturated fatty acids. XVI. Purification and general properties of 2,4-dienoyl-CoA reductase from Candida lipolytica

2,4-Dienoyl-CoA reductase has been purified to homogeneity from Candida lipolytica cultivated in the presence of linoleic acid. The native enzyme had a molecular weight close to 360,000 as estimated by gel filtration on Sepharose CL-4B, whereas the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 33,000. The purified 2,4-dienoyl-CoA reductase from C. lipolytica gave a single precipitin line with antibodies raised against the purified enzyme from C. lipolytica. The general properties of the 2,4-dienyl-CoA reductase from C. lipolytica were examined. The enzyme had optimal pH at 6.5 and was inactivated by heat treatment at 50 degrees C for 10 min. trans-2,trans-4-Octadienoyl-CoA was the most active substrate of the dienoyl-CoA esters examined.     

Common characteristics of mutant adenine phosphoribosyltransferases from four separate Japanese families with 2,8-dihydroxyadenine urolithiasis associated with partial enzyme deficiencies

Summary 2,8-Dihydroxyadenine urolithiasis associated with partial deficiencies of adenine phosphoribosyltransferase (APRT) has been found only among Japanese families. All Caucasian patients with the same lithiasis are completely deficient in this enzyme. Partially purified APRT from one of the Japanese families with the lithiasis associated with a partial deficiency of APRT had a reduced affinity for 5-phosphoribosyl-1-pyrophosphate (PRPP). In the present investigations, we have shown that this characteristic is common in mutant enzymes from all the four separate Japanese urolithiasis families associated with partial APRT deficiencies so far tested. The mutant enzymes also had several other characteristics in common including increased resistance to heat in the absence of PRPP and reduced sensitivity to the stabilizing effect of PRPP. These data suggest that these families have a common mutant allele (APRT ^* J) at the APRT gene locus.     

Effect of Streptococcus faecalis BIO-4R on intestinal flora of weanling piglets and calves.

The effect of oral administration of Streptococcus faecalis BIO-4R, an antibiotic-resistant lactic acid bacterium, on the intestinal flora of weanling piglets and cows reared on antibiotic-containing diet was investigated. Fourteen days after administration of the bacteria, the intestinal flora of the piglets was examined. Animals of the administered group had stabilized lactic flora such as bifidobacteria, streptococci, and lactobacilli, whereas most animals of control group had reduced lactic flora. On the other hand, abundant yeasts were detected from the cecum, colon, and feces of the control animals, but the levels were significantly lower in the animals given strain BIO-4R. The density of Salmonella in the intestine appeared to be reduced after the administration of strain BIO-4R. The number of BIO-4R cells was shown to be 10 times lower in the duodenum and jejunum than in the ileum, suggesting that strain BIO-4R might have grown transiently in the ileum. The similar trend toward stabilization of the lactic flora was also observed in cows after administration of BIO-4R. In addition, an antagonistic effect of the strain against yeasts and Salmonella was suggested. These findings indicate that the oral administration of strain BIO-4R is one of the useful methods whereby the potentially deleterious effect of antibiotics on the intestinal flora of farm animals may be minimized.     

Lipid composition of the vascular system during infancy, childhood, and young adulthood

The object of this study was to determine the changes in lipid composition that occur in blood vessels from infancy to young adulthood. Analyses included levels of total cholesterol, total triglyceride, phospholipid, and cholesteryl ester fatty acids, and the distribution of saturated and unsaturated fatty acids. Triglyceride, total and monoenoic fatty acids, and linoleic acid were lower in the ascending, thoracic, and abdominal aorta than in the pulmonary artery and inferior vena cava. Phospholipids and arachidonic acid were higher in aortic segments than in the other two vessels. Aortic lipids showed significant changes with increasing age: total cholesterol and total fatty acids decreased from <1 wk to 5 yr, then increased to 22 yr of age. Triglycerides decreased whereas cholesteryl esters increased from 10 to 22 yr of age. Saturated fatty acids decreased from 1 wk to 10 yr, then remained relatively constant. Linoleic acid (3.7-9.8% of total fatty acids) and arachidonic acid (15.8-21.7%) both increased with age; the increase in cholesteryl linoleate was highly significant. After 10 yr of age, total cholesterol and total fatty acids were significantly higher in abdominal than in ascending and thoracic segments of aorta.