A guanyloribonuclease of mouse liver cytosol

The acid RNase activity of mouse liver cytosol has been resolved into two different enzymes named acid RNase I and acid RNase II respectively. Acid RNase I is a typical pancreatic-type enzyme hydrolyzing CpN and UpN bonds. Acid RNase II, however, hydrolyzes GpN bonds in non-hydrogen-bonded regions of the substrate.     

A cysteine metalloproteinase from mouse liver cytosol

A cysteine metalloproteinase that degrades 125I-insulin B chain at neutral pH values was isolated from C3H mouse liver. The enzyme was partially purified from the 100,000g supernatant fraction by ammonium sulfate precipitation, DEAE-cellulose chromatography, and fast protein liquid chromatography. The molecular weight of the proteinase was estimated to be 190,000 by gel filtration on Sephadex G-200. Degradation of 125I-insulin B chain by the proteinase was inhibited by p-hydroxymercuribenzoate (PHMB) and iodoacetate (cysteine proteinase inhibitors) and by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (metalloproteinase inhibitors). The proteinase also degraded 125I-glucagon but did not hydrolyze 125I-insulin, leucine-2-naphthylamide, or several large proteins. Equivalent levels of EDTA- and PHMB-inhibitable 125I-insulin B chain-degrading activity were observed in the 100,000g supernatant fractions of brain, liver, lung, kidney, heart, and spleen from four mouse strains (C3H/HeN, CBA/J, ICR, and C57BL/6). High levels of 125I-insulin B chain-degrading activity were found in the particulate fraction of kidneys and lungs from these four mouse strains; these activities were inhibited by EDTA but not by PHMB. The activity of the soluble liver cysteine metalloproteinase was not altered in C3H mice treated ip with metal chelators, bacterial endotoxin, phenobarbital, dexamethasone, or insulin. Starvation for 24 or 48 hr and alloxan-induced diabetes diminished total activity of this enzyme in liver by about 50 and 30%, respectively. This soluble polypeptide-degrading enzyme appears to be ubiquitous in mice and to be regulated by nutritional conditions.     

Apoptosis of mouse embryonic stem cells induced by single cell suspension

Embryonic stem cells (ES cells) are pluripotential, and are therefore used to construct gene knock-out mice. We found that the apoptosis of mouse ES cells was induced when the cells were dispersed as single cells, whereas this process was suppressed when they proliferated in aggregates. The apoptosis of ES cells was repressed when the cells were cultured on feeders prepared from STO cells, a cell line established from embryonic fibroblasts. Culture supernatants from STO cells did not block the apoptosis of ES cells, which suggests that a direct interaction between ES cells and STO cells is required for the suppression of apoptosis. The viability of ES cells examined by the trypan blue exclusion test or by the MTT ((3-4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay decreased dramatically when the cells were dispersed in phosphate-buffered saline PBS. Cellular activity was restored by the addition of culture medium for ES cells. Glucose in the medium was found to be a major factor responsible for the restoration. Amino acids also restored the decrease in reduction of MTT. Suspension of the ES cells in PBS(-) caused leakage of the nucleosome into cytoplasm. Results indicate that the single cell suspension of ES cells leads to leakage of substrates for oxidative phosphorylation from the mitochondria, and that these cells finally become committed to apoptosis.     

Purification of a fluoroacetate-specific defluorinase from mouse liver cytosol

Fluoraocetate-specific defluorinase, an enzyme which catalyzes the release of fluoride ion from the rodenticide fluoroacetate, has been purified 347-fold from mouse liver cytosol and shown to be distinct from multiple cationic and anionic glutathione S-transferase isozymes. Fluoroacetate-specific defluorinase was obtained at a final specific activity of 659 nmol of F-/min/mg of protein and was prepared in an overall yield of 12%. The isoelectric point of this hepatic enzyme was acidic, at pH 6.4, as determined by column chromatofocusing. The molecular weight of the active species was estimated at 41,000, and sodium dodecyl sulfate-polyacrylamide gels of the purified defluorinase demonstrated a predominant subunit, Mr = 27,000. Chromatofocusing completely partitioned the fluoroacetate-specific defluorinase from two separate peaks of murine anionic glutathione S-transferase activity. Rabbit antibodies prepared against the purified hepatic defluorinase quantitatively precipitated native defluorinase from mouse and rat liver, but were unable to immunoprecipitate cationic or anionic glutathione S-transferase enzymes from the same preparation. The evidence presented suggests that fluoroacetate-specific defluorinase and glutathione S-transferase activities are catalyzed by separate proteins present in the cytosol of mouse liver.     

Karyometry of nuclei in liver cells

Summary In untreated adult male albino rats nuclear volume and the percentage of binucleate cells were determined in the first layer of hepatocytes adjacent to hepatic venous branches of varying diameters (<40 m, 40 m–80 m, 80 m–120m, 120 m–160 m, >160 m), and in the third and fourth layer of hepatocytes in the remainder of the perivenous parenchyma. In the first layer of hepatocytes adjacent to the vascular structures means of nuclear volume are significantly lower and percentage of binucleate cells significantly higher than in the cells of the remainder of the perivenous parenchyma. Within each area measured distribution curves of nuclear volume classes were homogeneous but showed heterogeneity in comparison with each other. The morphometric data presented in this study strongly support the opinion of the heterogeneity of liver cells in the perivenous zone, as previously postulated on the basis of histochemical investigations.Supported by the Deutsche Forschungsgemeinschaft (Hi 318/2-1)     

Changes in chromosomal proteins from embryo induced carrot cells

The chromosomal histone and non-histone proteins from embryoinduced carrot cell cultures were analyzed by polyacrylamidegel electrophoresis and compared with those from non-inducedcultures. 16 protein bands were detected in the non-histoneproteins of non-induced control cultures and their patternsremained stable throughout the culture period. Generally, similar protein patterns were found in the non-histoneproteins of induced cultures. However, limited but distinctdifferences were observed. A group of proteins of low molecularweight was observed to be missing in samples shortly after theinduction of embryogenesis. In contrast, no changes were detectedin histones after the induction of embryogenesis. ¹Present address: Department of Agricultural Chemistry, KyotoUniversity, Kyoto, Japan. (Received May 2, 1974; )     

Ultrastructural demonstration of NAD-pyrophosphorylase activity in mouse liver nuclei

Summary Ultrastructural demonstration of NAD-pyrophosphorylase activity (E.C. 2.7.7.1) in isolated mouse liver nuclei was investigated with the use of an electronhistochemical procedure based on the precipitation of pyrophosphate ions with lead ions under conditions permitting simultaneous ATPase inhibition by formaldehyde/ethanol prefixation. In isolated mouse liver nuclei activity of NAD-pyrophosphorylase was found in nucleoli, in interchromatin granules, coiled bodies and strand-like structures in nucleoplasm.     

The enzymatic defluorination of fluoroacetate in mouse liver cytosol: the separation of defluorination activity from several glutathione S-transferases of mouse liver

The liberation of free fluoride ion from fluoroacetate (FAc) proceeds as an enzyme-catalyzed dehalogenation reaction in the soluble fractions of several organs of the CFW Swiss mouse. Liver contained the highest FAc defluorinating activity. The enzyme activity in other organs decreased in the order kidney greater than lung greater than heart greater than testes. No activity was detected in the brain. Experiments were designed to characterize and identify the enzyme species responsible for FAc metabolism in liver. Enzyme activity was dependent on the concentration of glutathione (GSH) in the assay mixture, with maximal activity occurring above 5 mM. The dehalogenation of FAc had an apparent Km of 7.0 mM when measured in the presence of a saturating concentration of GSH. An increase in the pH of the assay mixture enhanced fluoride release in both phosphate and borate buffer. The defluorination activity was reduced to negligible levels when stored for 24 h at 4 degrees C. The addition of either GSH, dithiothreitol, or 2-mercaptoethanol increased stability, with the latter providing protection for greater than 150 h at a concentration of 15 mM. DEAE anion-exchange chromatography separated the defluorinating activity from 90% of the soluble GSH S-transferase activity measured with 1-chloro-2,4-dinitrobenzene. FAc defluorination activity did not bind to a GSH affinity column which selectively separates it from a group of anionic GSH S-transferases. The GSH-dependent enzyme which dehalogenates FAc has unique properties and can be separated from the liver GSH S-transferases previously described in the literature.     

Participation of cytochrome b5 in CMP-N-acetylneuraminic acid hydroxylation in mouse liver cytosol

The activity of CMP-N-acetylneuraminic acid hydroxylase, that converts CMP-N-acetylneuraminic acid (CMP-NeuAc) to CPM-N-glycolylneuraminic acid (CMP-NeuGc), in mouse liver was determined by a newly developed HPLC method using non-radioactive CMP-NeuAc as a substrate. The activity was detected in the cytosol fraction but not in the microsomal fraction. Either NADH or NADPH was used as an electron donor by the cytosol enzyme, but NADH was much more efficiently used than NADPH. An antibody against cytochrome b5 markedly reduced the CMP-NeuAc hydroxylase activity when added to incubation mixture containing either NADH or NADPH as an electron donor. These data led us to postulate the following electron transport system, which is involved in the CMP-NeuAc hydroxylation in mouse liver cytosol: (formula; see text) where X, Y, and Z are components supposedly involved.     

Ultrastructural demonstration of NAD-pyrophosphorylase activity in mouse liver nuclei.

Ultrastructural demonstration of NAD-pyrophosphorylase activity (E.C.2.7.7.1) in isolated mouse liver nuclei was investigated with the use of an electronhistochemical procedure based on the precipitation of pyrophosphate ions with lead ions under conditions permitting simultaneous ATPase inhibition by formaldehyde/ethanol prefixation. In isolated mouse liver nuclei activity of NAD-pyrophosphorylase was found in nucleoli, in interchromatin granules, coiled bodies and strand-like structures in nucleoplasm.     

Chromocenters of interphase nuclei in the mouse liver contain satellite DNA]

In isolated interphase mouse liver nuclei after hypotonic treatment only the chromocenters belonging to the pericentromeric heterochromatin remain in dense form while the main mass of a chromatin is completely decondensed. The centromeric nature of these chromocenters is demonstrated by their capability for C-banding and for hybridization with a satellite mouse DNA.     

Apoptosis in granulose cells induced by intrafollicular peptide

Ovarian follicular fluid peptide (OFFP) purified from sheep ovaries has been earlier shown to induce degeneration of ovarian follicles in mice. In the present study, whether the effect of OFFP on granulosa cells was similar to apoptosis was studied using three parameters. Immature mice injected with pregnant mare serum gonadotropin on day 0 were administered with 10 or 20 μg of OFFP on day 1 and autopsied on day 2. The granulosa cells were collected from the ovarian follicles. The presence of apoptotic bodies were observed by staining the cells with acridine orange. DNA profiles of DAPI-stained cells analysed by flow cytometry also revealed apoptotic response to OFFP. Furthermore, agarose gel electrophoresis of low molecular weight DNA fraction extracted from the cells of OFFP-treated animals confirmed ladder formation and induction of apoptosis and not necrosis in granulosa cells. In conclusion, all the three parameters indicated apoptotic changes in granulosa cells of ovarian follicles in .mice treated with OFFP. The effect of OFFP seems to be exerted directly on the granulosa cells showing its autocrine role in the process of follicular atresia. This is discussed in the light of other intra/extra ovarian factors.     

Isolation of intact nuclei of high purity from mouse liver

Current methods of nuclear isolation from liver disrupt the plasmalemmae via homogenization and separation of the nuclei by high centrifugal force (HCF) through gradients of sucrose or other substances for up to 80 min. The use of HCF for such a long time increases the potential for nuclear damage and degradation by endogenous proteases. We compared four combinations of alterations to classical nuclear isolation methods as follows. Mouse liver was gently crushed through a fine mesh with and without in vivo perfusion with collagenase. The cell suspension was centrifuged at 600g to remove gross debris and then at moderate centrifugal force (MCF, 16,000g) or high centrifugal force (HCF, 70,000g) through sucrose gradients for 30 min. The purity of the isolated nuclei was assessed biologically and morphologically, including analyses of representative marker proteins for nuclei and cytoplasm. The results indicate that MCF and no collagenase provided the highest nuclear integrity and purity, whereas MCF with collagenase is a viable option if priority is given to yield. The method is especially suited for small samples and so should facilitate studies with human liver biopsies and livers from mice, the most widely used species for gene targeting.