Cell nuclear DNases: multiplicity and heterogeneity

In order to determine the ratio of activities of major endonucleases of rat liver chromatin, a stepwise fractionation of cell nuclear extracts by chromatography on phosphocellulose and gel filtration through Toyopearl HW60 was carried out. This procedure resulted in partially purified preparations of Ca2+,Mg2+-dependent endonuclease (55 +/- 10 kD), Ca2+,Mg2+-dependent endonuclease (30 +/- 10 kD), Mn2+-dependent endonuclease (30 +/- 5 kD) and acid cation-independent endonuclease. The Ca2+,Mg2+-dependent endonuclease with Mr of 55 +/- 10 kD made up to 57% of the nuclear extract activity in the presence of Ca2+ + Mg2+ and revealed a high calcium-magnesium synergism. Under the same experimental conditions, the 30 +/- 10 kD enzyme made up to 33% of the nuclear extract activity and revealed a low synergism. The activity of Mn2+-dependent endonuclease made up to 26% of the total nuclear extract activity in the presence of Mn2+, that of acid endonuclease--11% of the extract activity in 1 mM EDTA at pH 5.0. It was assumed that the low molecular weight Ca2+,Mg2+-dependent endonuclease represents a product of limited proteolysis of high molecular weight Ca2+,Mg2+-dependent endonuclease.     

The interaction between manganese and calcium fluxes in pancreatic beta-cells.

Electrothermal atomic-absorption spectroscopy was employed for measuring manganese in beta-cell-rich pancreatic islets isolated from ob/ob mice. The efflux from preloaded islets was estimated from the amounts remaining after 30 min of subsequent test incubations in the absence of Mn2+. An increase in the extracellular Mg2+ concentration promoted the Mn2+ efflux and removal of Na+ from a Ca2+-deficient medium had the opposite effect. Addition of 25 mM-K+ failed to affect Mn2+ outflow as did 3-isobutyl-1-methylxanthine and dibutyryl cyclic AMP. Whereas tolbutamide caused retention of manganese, the ionophore Br-X537A promoted an efflux. D-Glucose was equally potent in retaining the islet manganese when the external Ca2+ concentration ranged from 15 microM to 6.30 mM. Subcellular-fractionation experiments indicated a glucose-stimulated incorporation of manganese into all fractions except the microsomes. The effect was most pronounced in the mitochondrial fraction, being as high as 164%. The glucose-induced uptake of intracellular 45Ca was abolished in the presence of 0.25 mM-Mn2+. When added to medium containing 2.5 mM-Mn2+, glucose even tended to decrease 45Ca2+ uptake. The inhibitory effect of Mn2+ was apparent also from a diminished uptake of 45Ca into all subcellular fractions. The efflux of 45Ca2+ was markedly influenced by Mn2+ as manifested in a prominent stimulation followed by inhibition. In addition to demonstrating marked interactions between fluxes of Mn2+ and Ca2+, the present studies support the view that the glucose inhibition of the efflux of bivalent cations from pancreatic beta-cells is accounted for by their accumulation in the mitochondria.     

Effect of manganese poisoning on the levels of manganese and iron in rat viscera

Twenty rats were poisoned by manganese inhalation, and sacrificed six months after the first exposure. The tissue concentrations (in microgram/g dry weight) of manganese and iron were found in liver, pancreas, lung, kidney and suprarenal gland for atomic absorption spectrometry in control and experimental animals. The measurements show high tissue concentrations of manganese, especially in liver and pancreas of the experimental animals, as well as a little increase in the tissue concentrations of iron, fundamentally in liver, kidney and suprarenal gland.     

Rapid cytochemical identification of phagosomes in various tissues of the rat and their differentiation from mitochondria by the peroxidase method

1. Granules characterized by their ability to segregate foreign proteins (phagosomes) were identified in the cells of many rat organs after intravenous administration of horseradish peroxidase, by using the conventional test with benzidine for the histochemical detection of peroxidase. The largest numbers of phagosomes were identified in kidney and liver. Considerable numbers were observed cytochemically in pancreas, prostate, epididymis, thymus, spleen, bone marrow, small intestine, heart, pituitary, and mouse mammary carcinoma. 2. The variation in size of the phagosomes ranging from the limit of microscopic visibility up to 5 micro diameter, previously described for kidney, was also observed to occur in many of the other organs. The average size of the phagosomes in different organs was also different, the phagosomes of the liver, for example being on the average smaller than those of the kidney, pancreas, and prostate. 3. In squash preparations of kidney and liver, the phagosomes appeared often in curved rows following the course of the cell membranes of epithelial cells. In several other organs, they appeared aggregated in cells located in the vicinity of blood or lymphatic vessels or capillaries. 4. After injection of peroxidase directly into the brain of a rabbit, a striking concentration of peroxidase was observed in phagosomes of endothelial cells of capillaries and vessels, surrounding the site of injection. It was suggested that this localization may offer an explanation for the so called blood-brain barrier. 5. The cytochemical peroxidase method was applied to smears of isolated fractions of kidney and liver. Only the isolated phagosomes, but not the isolated nuclei, mitochondria, and microsomes, reacted with benzidine after administration of peroxidase. The contamination of conventionally prepared nuclear, mitochondrial, and microsomal fractions of kidney and liver with phagosomes of different sizes was observed. By correlating the cytochemical peroxidase test of smears of isolated fractions with the colorimetric determination of peroxidase, acid phosphatase, and cytochrome oxidase in the same fractions, the differentiation of the phagosomes from mitochondria and other cell granules was facilitated. 6. The marked difference in the osmotic properties of phagosomes and mitochondria, detectable after treatment with 70 per cent alcohol, and the difference in their affinities towards basic fuchsin, made it possible to differentiate the phagosomes from the mitochondria. It was found by this simple procedure that kidney cells of normal rats contain a large number of phagosomes ranging in size from 0.5 to 3 micro, whereas liver cells of normal rats contain relatively few phagosomes of this size but many smaller ones (0.2 to 0.5 micro diameter). These increased in size after treatment of the rats with horseradish peroxidase.     

Stimulation of rat kidney, spleen and brain DNA-(cytosine-5)-methyltransferases by divalent cobalt ions

Rat kidney, spleen, brain, and liver DNA-methylases were partially purified by chromatography on DEAE-Trisacryl columns and their catalytic properties were studied. Crude extracts contain one or several inhibitors which are thermostable and resistant to acidic or alkaline treatments and which can be eliminated by dialysis, or by chromatography on DEAE-Trisacryl. These are most probably divalent ions, such as, Pb2+, Zn2+, Cu2+, Fe2+, Mg2+, Mn2+ or Ca2+, which inhibit the DNA-methylase activity. However, Co2+, at concentrations ranging from 0.05 mM to 1 mM, has an efficient stimulatory action on spleen, kidney or brain DNA-methylase activity. The spleen DNA-methylase activity on chicken erythrocyte DNA could be increased 10-fold, by a 0.2 mM concentration of Co2+, but no stimulation was found with liver DNA-methylase. The fact that significant differences exist between the DNA-methylases from the different organs in their behavior towards Co2+ could indicate that these enzymes are different.     

Metal ion interactions in the control of haem oxygenase induction in liver and kidney.

Mn2+ and Zn2+ exhibit a striking ability to block the induction by Sn2+ and Ni2+ of haem oxygenase (EC 1.14.99.3) in kidney. The blocking effects of Mn2+ and Zn2+ were found to be greatest on simultaneous administration, time-dependent when administered up to 8 h before the inducing metal ions, and ineffective when administered as little as 10 min after the inducing metal ions. The decreases in cytochrome P-450 and haem contents and the sequential changes in delta-aminolaevulinate synthase (EC 2.3.1.37) activity that occur concomitant with haem oxygenase induction were largely eliminated with simultaneous or prior treatment with Mn2+ or Zn2+, but not when Mn2+ or Zn2+ was administered after Sn2+ or Ni2+. Mn2+ and Zn2+ did not increase the catabolism of the enzyme in vivo. Zn2+ on simultaneous administration was also able substantially to block the induction of haem oxygenase by Co2+, Cd2+ and Ni2+ in liver. The Zn2+ blockade of Cd2+ induction was examined in detail, and prior or simultaneous administration of Zn2+ was found to be effective in blocking the induction of haem oxygenase and the concomitant decreases in cytochrome P-450 and haem contents, ethylmorphine demethylase activity and the sequential changes in delta-aminolaevulinate synthase activity. Zn2+ administration 10 min or more after Cd2+ was ineffective in preventing the occurrence of these perturbations in haem metabolism. These findings describe a new and striking biological property of Mn2+ and Zn2+, and indicate the existence of significant metal ion interactions in the control of haem metabolism.     

Differential response of testicular and ovarian heme oxygenase activity to metal ions

The response of the microsomal heme oxygenase in the testis to metal ions distinctly differed from that of the ovarian source. The activity of the ovarian enzyme in rats treated with Co2+ (250 mumol/kg, 24 h) responded in consonance with that of the liver and the kidney, i.e., heme oxygenase activity was elevated. In contrast, similar treatments did not increase the activity of testicular heme oxygenase. In addition, other metal ions, such as Cu2+, Sn2+, Pb2+, and Hg2+, known for their potency to increase heme oxygenase activity, were ineffective in increasing the enzyme activity in the testis. The unprecedented response of heme oxygenase in the testis to metal ions did not reflect an unusual nature of the enzyme protein insofar as it displayed a similar cofactor requirement and inhibition by known inhibitors of the enzyme activity, such as KCN and NaN3. Moreover, the apparent Km's for oxidation of hematoheme by the testicular and ovarian microsomal fractions were comparable and measured 2.3 and 1.4 microM, respectively. In the testis of Co2+-treated rats, the concentration of cytochrome P-450 in the rough and smooth endoplasmic reticular fractions was significantly decreased. The decrease in the hemoprotein level, however, did not reciprocate the activity of heme oxygenase in the fractions. The inability of metal ions to induce heme oxygenase activity in the testis did not represent the general refractory nature of the enzymes of heme metabolism to metal ions in this organ, since in rats treated with Co2+ the activity of delta-aminolevulinate synthetase was significantly decreased 24 h after treatment. However, the activities of uroporphyrinogen-I synthetase, delta-aminolevulinate dehydratase, and ferrochelatase and the content of porphyrins were not altered in the testis of rats treated with Co2+. The response of delta-aminolevulinate synthetase in the ovarian tissue to Co2+ treatment contrasted that of the testis. In the ovary, the enzyme activity significantly decreased 6 h after treatment. This decrease was followed by a rebound increase at 24 h after administration of Co2+. The presently described inability of metal ions to induce testicular heme oxygenase activity suggests that the activity of the enzyme in the testis is controlled by factor(s) which differ from those regulating the enzyme activity in other organs, including another steroidogenic organ, the ovary.     

Rapid Cytochemical Identification of Phagosomes in Various Tissues of the Rat and their Differentiation from Mitochondria by the Peroxidase Method

1. Granules characterized by their ability to segregate foreign proteins (phagosomes) were identified in the cells of many rat organs after intravenous administration of horseradish peroxidase, by using the conventional test with benzidine for the histochemical detection of peroxidase. The largest numbers of phagosomes were identified in kidney and liver. Considerable numbers were observed cytochemically in pancreas, prostate, epididymis, thymus, spleen, bone marrow, small intestine, heart, pituitary, and mouse mammary carcinoma. 2. The variation in size of the phagosomes ranging from the limit of microscopic visibility up to 5 µ diameter, previously described for kidney, was also observed to occur in many of the other organs. The average size of the phagosomes in different organs was also different, the phagosomes of the liver, for example being on the average smaller than those of the kidney, pancreas, and prostate. 3. In squash preparations of kidney and liver, the phagosomes appeared often in curved rows following the course of the cell membranes of epithelial cells. In several other organs, they appeared aggregated in cells located in the vicinity of blood or lymphatic vessels or capillaries. 4. After injection of peroxidase directly into the brain of a rabbit, a striking concentration of peroxidase was observed in phagosomes of endothelial cells of capillaries and vessels, surrounding the site of injection. It was suggested that this localization may offer an explanation for the so called blood-brain barrier. 5. The cytochemical peroxidase method was applied to smears of isolated fractions of kidney and liver. Only the isolated phagosomes, but not the isolated nuclei, mitochondria, and microsomes, reacted with benzidine after administration of peroxidase. The contamination of conventionally prepared nuclear, mitochondrial, and microsomal fractions of kidney and liver with phagosomes of different sizes was observed. By correlating the cytochemical peroxidase test of smears of isolated fractions with the colorimetric determination of peroxidase, acid phosphatase, and cytochrome oxidase in the same fractions, the differentiation of the phagosomes from mitochondria and other cell granules was facilitated. 6. The marked difference in the osmotic properties of phagosomes and mitochondria, detectable after treatment with 70 per cent alcohol, and the difference in their affinities towards basic fuchsin, made it possible to differentiate the phagosomes from the mitochondria. It was found by this simple procedure that kidney cells of normal rats contain a large number of phagosomes ranging in size from 0.5 to 3 µ, whereas liver cells of normal rats contain relatively few phagosomes of this size but many smaller ones (0.2 to 0.5 µ diameter). These increased in size after treatment of the rats with horseradish peroxidase.     

Metabolism of tritiated water in the cell nucleus and intracellular substances of the rat

Tritiated water was given to rats in single oral doses, and the cell fractions for each organ were prepared by ultracentrifugation for measurement of the concentration of tissue-bound tritium. The concentration of tissue-bound tritium reached a peak relatively soon after intubation, 1-4 days after administration. The initial concentration of tissue-bound tritium in liver and kidney was high in the mitochondrial and microsomal fractions but low in the nuclear and cytosol fractions. The initial tissue-bound tritium concentration in the brain was high in the mitochondrial and microsomal fractions but low in the nuclear and cytosol fractions. The initial concentration of tissue-bound tritium in the testes was high in the mitochondrial, microsomal, and cytosol fractions but low in the nuclear fraction. The half-life for the long component was larger in the nuclear, mitochondrial, and microsomal fractions of the brain than in the other organs according to an interorgan comparison of each fraction. As for the testes, the values for the mitochondrial and microsomal fractions were larger than those for the other organs.     

Acquisition of manganous ions by mutans group streptococci.

The cariogenic bacteria Streptococcus sobrinus and S. cricetus were shown to have an absolute requirement for manganous ion in order to bind glucans or to adhere to glass in the presence of sucrose. The bacteria possessed a reasonably high affinity transport system for 54Mn2+, yielding a Km of about 12 microM. The Vmax for uptake of 54Mn2+ in S. sobrinus was increased when the bacteria were grown in Mn-depleted medium, but the Km remained the same. There was no evidence for two Mn2+ uptake systems, commonly observed for many bacteria. Ions such as Ca2+, Co2+, Co3+, Cu2+, Fe2+, Fe3+, Hg2+, Mg2+, Ni2+, and Zn2+ did not inhibit the uptake of 54Mn2+ by the bacteria, although Cd2+ was a potent inhibitor. Fractionation experiments showed that manganese was distributed in protoplasts (67%) and in the cell wall (33%). Approximately 80% of the 54Mn2+ in S. sobrinus was rapidly exchangeable with nonradioactive Mn2+. Electron spin resonance experiments showed that all of the manganese was bound or restricted in mobility. Proton motive force-dissipating agents increased the acquisition of 54Mn2+ by the streptococci, probably because the wall became more negatively charged when the cell could no longer produce protons.     

Demonstration and partial characterization of cytosol receptors for testosterone.

Androgen uptake was investigated in several peripheral organs after administration of (1,2,6,7 minus -3H)testosterone to castrated male rats. The animals were killed after 30 min, the organs were taken out, and the radioactivity was determined after tissue combustion. A relatively high accumulation of androgen was found in pancreas, adrenals, spleen, thigh muscle, kidneys, and liver in addition to the classical androgen target organs coagulation glands, seminal vesicles, prostate, preputial glands, and harderian glands. In a second serier of experiments, nuclear and cytosol fractions were prepared from prostate, seminal vesicles, coagulation glands, preputial glands, spleen, submaxillary glands, kidneys, and pancreas from castrated male rats give (1,2,6,7 minus -3H)testosterone, and these fractions were then characterized by thin-layer and radio-gas chromatography with respect to their patterns of labeled steroids. Only prostate and seminal vesicles were found to contain significant amounts of nuclear 5alpha-(-3H)dihydrotestosterone. The major nuclear androgen was (-3H)testosterone that was the only detectable labeled steroid in coagulation glands, preputial glands, and spleen and that constituted 70% or more of the nuclear radioactivity in seminal vesicles, submaxillary glands, kidneys, and pancreas. These results indicate that testosterone itself may be the predominant active androgen principle in vivo in most androgen target organs and that conversion to 5alpha-dihydrotestosterone is generally not a prerequisite for androgen activity. Using an ultrasensitive micromodification of isoelectric focusing (cf. M. Katsumata and A. S. Goldman (1974), Biochem. Biophys. Acta 359, 112. It was possible to show that cytosol from kidney; submaxillary gland, thigh muscle, and levator ani muscle and nuclei from kidney and submaxillary gland contained androgen-binding proteins with pI's in the region 4.6-5.1 ("4.6 minus 5.1 Complex"). This complex also formed in vitro after incubation of (1,2,6,7 minus -3H)testosterone with cytosol from kidney and submaxillary gland. (1,2,6,7 minus -3H)Testosterone was bound with high affinity to receptor proteins in cytosol from both kidney, submaxillary gland, and thigh muscle with dissociation constants of 5.0 x 10 minus -12 M (kidney), 3.3 x 10 mi;nus -11 M and 4.1 x 10 minus -10 M (two types of binding sites, submaxillary gland), 2.4 x 10 minus -12 M (thigh muscle) and 1.9 x 10 minus -12 M (levator ani muscle). The number of binding sites was in all cases between 1 and 20 fmol/mg of protein. On the basis of these results the hypothesis is presented that a common class of testosterone receptors is present in most organs and that these receptors can be detected both in vivo and in vitro provided methods sensitive enough are utilized.     

Effect of manganese on some bioantioxidants in various organs of protein-deficient rats

The effect of manganese exposure (Mn2+ 4 mg Mn/kg intraperitoneally) on certain bioantioxidants in brain, liver, kidney and testes in growing rats maintained on 21% and 8% casein diet were investigated. Manganese administration for 30 days caused significant reduction in the level of GSH (reduced glutathione) in liver and testes and GR (glutathione reductase) and G-6-PDH (glucose-6-phosphate dehydrogenase) in brain, liver and testes. The magnitude of alteration was greater in 8% casein diet fed animals compared to rats maintained on 21% casein diet. These results indicate that protein deficient animals are more susceptible to the manganese induced biochemical changes in various tissues. The mechanism of such changes is discussed.     

Appearance of magnesium guanylate cyclase activity in rat liver with sodium azide activation.

Native soluble and particulate guanylate cyclase from several rat tissues preferred Mn2+ to Mg2+ as the sole cation cofactor. Wtih 4mM cation, activities with Mg2+ were less than 25% of the activities with Mn2+. The 1 mM NaN3 markedly increased the activity of soluble and particulate preparations from rat liver. Wtih NaN3 activation guanylate cyclase activities wite similar with Mn2+ and Mg2+. Co2+ was partially effective as a cofactor in the presence of NaN3, while Ca2+ was a poor cation with or without NaN3. Activities with Ba, Cu2+, or Zn2+ were not detectable without or with 1 mM NaN3. With soluble liver enzyme both manganese and magnesium activities were dependent upon excess Mn2+ or Mg2+ at a fixed MnGTP or MgGTP concentration of 0.4 mm; apparent Km values for excess Mn2+ and Mg2+ were 0.3 and 0.24 mM, respectively. After NaN3 activation, the activity was less dependent upon free Mn2+ and retained its dependence for free Mg2+, at 0.4 mM MgGTP the apparent Km for excess Mg2+ was 0.3 mM. The activity of soluble liver guanylate cyclase assayed with Mn2+ or Mg2+ was increased with Ca2+. After NaN3 activiation, Ca2+ had no effect or was somewhat inhibitory with either Mn2+. After NaN activation, Ca2+ had no effect or was somewhat inhibitory with either Mn2+ or Mg2+. The stimulatory effect of NaN2 on Mn2+-and Mg2+-dependent guanylate cyclase activity from liver or cerebral cortex supernatant fractions required the presence of the sodium azide-activator factor. With partially purified soluble liver guanylate cyclase and azide-activator factor, the concentration (1 mjM) of NaN3 that gave half-maximal activation with Mn2+ or Mg2+ was imilar. Thus, under some conditions guanylate cyclase can effectively use Mg2+ as a sole cation cofactor.     

Manganese accumulation in pancreatic beta-cells and its stimulation by glucose.

Electrothermal atomic-absorption spectroscopy was employed for measuring manganese in beta-cell-rich pancreatic islets microdissected from ob/ob mice. The islet content of endogenous manganese was 80 mumol/kg dry wt., which is about half as much as found in the exocrine pancreas. The initial uptake was characterized by two components, with approximate Km values of 35 microM and 3.7 microM respectively. After 60 min of incubation with 0.25 mM-Mn2+, the intracellular concentration of manganese corresponded to an almost 25-fold accumulation compared with that of the extracellular medium. When exposed to 20 mM-D-glucose, the islets retained more manganese, owing to suppression of its mobilization. The glucose inhibition of efflux was prompt and reversible, as indicated from direct recordings of manganese in a perifusion medium. D-Glucose was an equally potent inhibitor of efflux in the presence of 15 microM- and 1.28 mM-Ca2+. The inhibitory action disappeared when metabolism was suppressed by adding 0.1 mM-N-ethylmaleimide or by lowering the temperature from 37 degrees C to 2 degrees C. At a concentration of 0.25 mM, Mn2+ abolished the insulin-releasing action of D-glucose, exerting only moderate suppression of its metabolism. The addition of Mn2+ resulted in inhibition of basal insulin release in the presence of 1.28 mM-Ca2+, but not in a Ca2+-deficient medium. The studies indicate that the previously observed phenomenon of glucose inhibition of 45Ca efflux has a counterpart in the suppression of manganese mobilization from the pancreatic islets. With the demonstration of a pronounced glucose inhibition of manganese efflux, it is evident that Mn2+ may represent a useful tool for exploring the mechanism of glucose-induced retention of calcium in the pancreatic beta-cells.     

Influence of sex,strain, and species on trace metal status of insulin-deficient diabetic rodents

Previous studies have demonstrated marked alterations in trace metal metabolism in male Sprague-Dawley rats following chemical induction of the diabetic state. To determine whether such changes represented a general response to the insulin-deficient condition the levels of zinc, copper, and maganese in liver, kidney, and intestine of normal and streptozotocin (STZ)-diabetic male rats of the Sprague-Dawley, Wistar, and Long-Evans strains, female Sprague-Dawley rats, and male mice were measured. Significantly increased concentrations of zinc, copper, and maganese in liver, and zinc and copper in kidney were found in STZ-diabetic rats, regardless of sex and strain. In contrast, the zinc and copper contents in liver and kidney of control and STZ-diabetic mice were similar, but hepatic manganese levels were significantly elevated in both organs of the diabetic mouse. The concentrations of all three metals were similar in the intestine of control and diabetic rodents. Higher amounts of zinc and copper were bound to metallothionein in the liver and kidney of the diabetic rats. Nicotinamide injection prior to STZ administration protected rats against the development of diabetes and alterations in trace metal status. These data indicate that specific alterations in the metabolism of zinc, copper and manganese during episodes of pancreatic hormonal imbalance represent a general phenomenon in the rat. A possible explanation for the differential response of the STZ-diabetic mouse is discussed.     

Tissue antioxidant status in streptozotocin-induced diabetes in rats

Interactions between manganese (Mn) deficiency and streptozotocin (STZ)-diabetes with respect to tissue antioxidant status were investigated in male, Sprague-Dawley rats. All rats were fed either a Mn-deficient (1 ppm) or a Mn-sufficient (45 ppm) diet for 8 wk. Diabetes was then induced by tail-vein injection of STZ (60 mg/kg body weight), after which the rats were kept for an additional 4 or 8 wk. The control groups comprised rats not injected with STZ and fed either Mn-deficient or Mn-sufficient diets for a total of 12 wk. The Mn-deficient diet decreased the activities of manganese superoxide dismutase (MnSOD) in kidney and heart, and of copperzinc superoxide dismutase (CuZnSOD) in kidney, in the non-diabetic animals. In the diabetic rats, the Mn-deficient diet induced more pronounced decreases in activities of these same enzymes, and also increased liver MnSOD activity. Plasma and hepatic vitamin E levels increased progressively with the duration of diabetes, independent of dietary Mn intake. Lipid peroxidation, as measured by H₂O₂-induced production of thiobarbituric acid reactive substances in erythrocytes, also increased, concomitant with decreased liver and kidney glutathione (GSH) levels. These findings demonstrate for the first time an interactive effective between Mn deficiency and STZ-diabetes, resulting in amplification of tissue antioxidant changes seen with either Mn deficiency or STZ-diabetes alone. This effect of Mn deprivation in experimental diabetes suggests a physiological role for Mn as an antioxidant nutrient.     

Benzoate modulates renal and extrarenal nitrogen flow: metabolic mechanisms.

The mechanism by which benzoate enhances total nitrogen excretion was investigated in-situ and in separated rat renal proximal tubules. Orally administered benzoate augmented NH4+, urea and hippurate excretion 2, 1.9 and 76 fold respectively, as compared to baseline for control. Hippurate had similar effects. Benzoate augmented renal blood flow, glutamine extraction and total NH4+ production. Arterio-venous concentration differences of glutamine, glutamate, and NH4+ across the kidney, liver and gut demonstrated an increase in glutamine uptake by the kidney despite reduced release and uptake by the liver and gut, respectively; glutamate release by the kidney and gut was increased; NH4+ handling was unchanged at these three organs. Studies in separated rat renal proximal tubules demonstrated that benzoate stimulated glutamine dependent ammonia-genesis by activation of gamma-glutamyltransferase, via the synthesis of hippurate. The results demonstrate that benzoate can modulate the interorgan partitioning of nitrogen metabolites across several organs, the net effect of which is physiologically expressed as enhanced NH4+ , urea and hippurate excretion.     

Proton relaxation enhancement in tissue due to ingested manganese chloride: time course and dose response in the rat

MnCl2, a potential NMR contrast agent, was force fed by cannula to 27 fasted rats in an attempt to establish the efficacy of this route of administration. The animals were sacrificed and the relaxation times (T1 and T2) of liver, spleen, kidney, heart, and skeletal muscle were determined in vitro. One group of rats was subject to a range of doses (8.3-333.3 mg/kg) and sacrificed at 90 minutes. Another group received a single large dose (500 mg/kg) with animals sacrificed at intervals from 15-225 minutes. The single large dose caused a rapid and prolonged reduction in liver T1 (-93% of normal) and to a lesser degree affected the other organs. The dose response data shows that liver T1 is also affected at significantly lower dosages than the other tissues tested. These results suggest the oral route as a possible alternative to IV Mn+2. Further studies with inorganic and organic manganese are indicated.     

Properties of a bicarbonate-stimulated ATPase from rat uterus.

Crude subcellular fractions from rat uterus contain a HCO-3 -stimulated Mg2+ -ATPase with properties analogous to those previously reported for the enzyme in gastric mucosa, pancreas, salivary gland and liver lyosome. Estradiol-17 beta treatment of ovariectomized rats resulted in an increase in uterine mitochondrial (HCO-3 +Mg2+)-ATPase and Mg2+ -ATPase activity. In an early response (105 min) to estradiol-17 beta treatment of ovariectomized rats, the lysosomal enzyme, beta-N-acetylglucosaminidase increased in the nuclear and mitochondrial fractions and decreased in the microsomal and supernatant fractions.     

Calmodulin-stimulated protein methylation in rat liver cytosol

The in vitro methylation of three liver cytosolic proteins was found to be selectively stimulated by calmodulin. This effect was also seen, although to a much smaller degree, in kidney and lung, but not in testes, brain, or spleen. The three methylated proteins affected by calmodulin have apparent Mr = 29,000, 32,000, and 45,000. The stimulation of methylation by calmodulin was greatest for the Mr 29,000 protein; there was an equal degree of methylation of the other two proteins. Dialysis of liver cytosolic fractions also stimulated the methylation of these proteins; the methylation of the Mr 32,000 and 45,000 proteins was stimulated to a greater extent by dialysis than by calmodulin. The degree of stimulation of methylation of the Mr 29,000 protein by calmodulin and dialysis was equivalent, but the addition of calmodulin to dialyzed liver cytosolic fractions gave additive effects on the stimulation of methylation of the Mr 29,000 protein, but not of either the Mr 32,000 or 45,000 proteins. Troponin C stimulated the methylation of the Mr 29,000 protein, but not the Mr 32,000 or 45,000 proteins, whereas parvalbumin stimulated methylation of the Mr 32,000 protein, but not the Mr 29,000 or 45,000 proteins. The effects of calmodulin and dialysis on protein methylation are cation-dependent and substrate-specific; methylation of the Mr 29,000 was supported by Mn2+, Ca2+, and Co2+, and to a lesser degree by Mg2+, Ni2+, and Zn2+. Methylation of the Mr 32,000 protein was supported only by Mn2+ and Mg2+ and methylation of the Mr 45,000 protein by Mn2+, Mg2+, Ca2+, Ni2+, and Zn2+, and to a much smaller extent by Fe2+. In extracts of fetal liver, stimulation of protein methylation by calmodulin or dialysis was restricted to the Mr 45,000 protein. In regenerating liver, stimulation of the methylation of all three proteins was observed, but the stimulation provided by dialysis plus calmodulin was much less than that observed in preparations from intact adult liver, suggesting a possible negative correlation between the rate of cell division and calmodulin-dependent methylation of these hepatic proteins. These results are consistent with the presence in liver of a minimum of three distinct N-methyltransferases and a dialyzable inhibitor which antagonizes calmodulin-dependent protein methylation.