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PubMed2001

Contents and uptake rates of Mn, Fe, Co, Zn, and Se in Se-deficient rat liver cell fractions.

The contents of manganese (Mn), iron (Fe), cobalt (Co), zinc (Zn), and selenium (Se) in nuclear (NU), mitochondrial (MT), microsomal (MC), and cytosolic (CS) fractions of liver homogenates of normal and selenium-deficient (SeD) rats were determined by instrumental neutron activation analysis (INAA). The uptake rates of these elements in the liver cell fractions of both groups of rats were determined by multitracer analysis (MTA). The results indicated that Se-deficiency caused a significant increase in the content of Fe in the MC fractions. The MTA showed that the uptake rate of Fe was highest in the MC fraction, and that the uptake rate in the fraction was similar between the SeD and normal rats.

Alanine Transaminase↗
PubMedDate not supplied

Intracellular localization of two betaine lipids by cell fractionation and immunomicroscopy.

The cellular localization of the betaine lipids diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and diacylglycerylhydroxymethyl-N,N,N-trimethyl-beta-alanine (DGTA) was investigated by a) chemical analysis of subcellular fractions and b) immunochemical methods using specific antisera and either fluorescence microscopy or electron microscopy for detection of the label. A homogenate of Lycopodium annotinum (Pteridophyta) was fractionated by differential and density gradient centrifugation. The particulate fractions obtained were analyzed for chlorophyll, cyt c oxidase, NADH-cyt c reductase and DGTS. Non-plastidial fractions were enriched in DGTS and only minor amounts of this lipid could be attributed to chloroplasts. Anti-DGTS and anti-DGTA sera were produced by immunization of rabbits. The monospecificity of the antisera was examined with cells of Chlamydomonas reinhardtii (Chlorophyceae) containing DGTS, Pavlova lutheri (Haptophyceae) containing DGTA and Ochromonas danica (Chrysophyceae) containing both DGTS and DGTA. Euglena gracilis which is free of betaine lipids, was used as a control. For the test, a FITC-coupled goat anti-rabbit antibody was used and detected by fluorescence microscopy. Thin sections of Ochromonas and Pavlova were incubated first with the anti-lipid sera and subsequently with a gold-coupled anti-rabbit serum and then examined in the electron microscope. With Ochromonas, anti-DGTS as well as anti-DGTA sera gave an accumulation of gold label in the cytoplasmic space but not in the chloroplasts. Similar results were obtained with Pavlova using anti-DGTA serum. These results describe for the first time the cytochemical localization of DGTS and DGTA strongly suggesting both these lipids to be associated mainly with non-plastidial structures.

Alanine↗
PubMed1975

Biological characterization of Fusobacterium necrophorum. Cell fractions in preparation for toxin and immunization studies.

Fusobacterium necrophorum isolated from bovine liver abscesses was grown in bulk at 37 C for 24 h under a strict anaerobic atmosphere. Harvested washed cells were disrupted ultrasonically and fractionated by differential centrifugation into the intracellular (cytoplasm) and cell wall fractions. Both intact cells and cell fractions induced generalized cytopathic effect on primary pig kidney cultures and caused a variety of signs of illness and/or death of intraperitoneally injected mice. The intact cells, disrupted cells, and cell walls produced necrotic lesions and erythema on intradermally injected guinea pigs and rabbits, whereas the cytoplasm mainly erythema. By contrast, the used culture medium (culture filtrate) of F. necrophorum did not show any detectable toxicity. The toxic component of the cytoplasm appears to be associated with nondialyzable, hemolytic, high-molecular-weight proteins and its toxicity is reduced by trypsin and pronase. Heating at 60 C for 10 min decreased markedly its erythemal and cytotoxic ability, wheras the toxicity of the cell walls appeared to be only slightly affected even when heated at 100 C for 1 h. These results suggest that at leasttwo distinct cell-bound toxic factors are present in F. necrophorum cells.

Amino Acids↗
PubMed1994

Productive HIV-1 infection of macrophages restricted to the cell fraction with proliferative capacity.

Retroviruses establish productive infection only in proliferating cells. Macrophages are often considered to be non-proliferating in vitro yet are susceptible to HIV-1 infection. This has led to the conclusion that HIV-1 can establish infection independent of host cell proliferation. We here report that a small proportion of macrophages does have proliferative capacity. A comparable small fraction of monocyte derived macrophages (MDM) supported productive HIV-1 infection as demonstrated in limiting dilution culture. Fluorescence activated cell sorting on the basis of incorporation of BrdUrd, a thymidine analog, and subsequent PCR analysis revealed the presence of proviral DNA only in the BrdUrd positive cell fraction with DNA synthesizing activity. To identify which phase of cell cycle is required for establishment of productive infection, growth arrest in G1 or G1/S phase prior to inoculation was performed. gamma-Irradiation, which arrests primary cells in G1, prevented both cell proliferation and establishment of productive infection in MDM. Treatment of MDM with aphidicolin, a specific inhibitor of DNA polymerase alpha and delta which arrests cells in G1/S phase of the cell cycle, also inhibited DNA synthesis but did not prevent establishment of productive infection which is completely analogous to observations in T cells. Our data thus indicate that not cell division itself but cellular conditions that coincide with cell proliferation are apparently indispensable for establishment of productive infection.

Aphidicolin↗
PubMed1975

Subpopulations of mouse spleen lymphocytes. II. Immunological reactivity of spleen cells fractionated on BSA density gradients.

We found in previous experiments that fractionation of non-immune mouse spleen cells on bovine serum albumin density gradients yields two subpopulations of T cells, one of high, the other of low density. Both subopopulations could be stimulated in corporate thymidine by the T cell-specific mitogen concanavalin A (con A). In the present investigation, spleen cells of mice immunized to sheep red cells (SRC) were similarly fractionated and the fractions recovered were assayed for: (a) reactivity to con A; (B) REACTIVITY TO SRC and (c) capacity to function as helper cells when stimulated with the homologous (SRC) or with a heterologous (donkey red cells) (DRC) antigen. Two subpopulations of cells reacting to con A were found in the spleens of the primed mice, corresponding to the subpopulations found in the non-immune mice. Both subpopulations contained cells responding to SRC (as measured by thymidine incorporation) and cells endowed with helper activity. The two subpopulations appeared to differ, however, in their specificity: while the denser cells could only exert their helper effect when stimulated by the specific antigen, the light cells could be effectively stimulated by both the specific (SRC) and the nonspecific (DRC) antigen.

Animals↗
PubMed1975

The ability of cell fractions of mouse spleen to repopulate the hemopoietic tissue and to mount graft-versus-host reaction.

Mouse spleen cells were separated into seven fractions on a discontinuous gradient of dextran. The lightest, uppermost fraction contained colony-forming units and was capable of repopulating the hemopoietic tissues damaged by irradiation. The fraction below it also contained the colony-forming units, but its repopulating ability was limited. Heavy fractions contained immunocompetent cells capable of mounting graft-versus-host reaction, but the performance of the cells depended on the type of assay, i.e. on whether the cells were assayed in irradiated or in non-irradiated recipients. It is concluded, firstly, that the technique used resolved the colony-forming units into two classes - those capable of self-renewal and those committed to differentiation - which were concentrated in different portions of the light part of the gradient; and secondly, that the cells implementing graft-versus-host reaction were probably also resolved into two classes - the inciting and the proliferating cells - which were concentrated in different portions of the heavy part of the gradient.

Animals↗
PubMed2000

Exposure of rainbow trout (Oncorhynchus mykiss) to nonylphenol is associated with an increased chloride cell fractional surface area.

Nonylphenol is a biodegradation product of a widely used group of non-ionic detergents. Because of its ubiquitous distribution and persistence, nonylphenol is present in surface waters as a pollutant. Little is known about its biological effects at environmentally relevant concentrations other than its action as a xenoestrogen. The goal of the present paper was to study the trout gill surface epithelium as the major interface between fish and water in view of possible morphological alterations due to exposure to nonylphenol. Rainbow trout were intermittently exposed to 10 micrograms/l nonylphenol and gill samples from experimental and control animals were investigated by scanning electron microscopy. Gill surface epithelium was scrutinised for changes in chloride cell density and their status regarding cell surface modifications. In addition, chloride cell fractional surface area (CCFA) was determined by morphometrical methods. Statistical analysis revealed a highly significant increase of CCFA in animals exposed to nonylphenol as compared to control animals (P = 0.0001). Semi-quantitative assessment of the other parameters suggested a higher chloride cell density and a larger proportion of chloride cells bearing microvilli. Taken together, these results provide evidence that exposure of trout to nonylphenol is associated with a substantial increase in the active interface of chloride cells with water. We interpret these findings as being a means to further the fish's capacity for calcium exchange.

Animals↗
PubMed1986

A sensitive diffusion plate assay for screening inhibitors of protease activity in plant cell fractions.

Proteolytic activity was detected, using a sensitive radial diffusion plate assay, in the plasma membrane fractions of corn (Zea mays L.) roots and from roots of several other plant species. The proteases could be effectively inhibited in corn with phenylmethane sulfonyl fluoride or chymostatin. Protease activity of oat roots, however, was not significantly reduced by these inhibitors. The results of diffusion plate assay were confirmed with the less sensitive azocasein assay using crude cell homogenates. Chymostatin and phenylmethane sulfonyl fluoride were effective in preventing protease degradation of polypeptides as revealed by electrophoresis. The diffusion plate assay uses a permanent support for a 0.75 millimeter thick agarose slab containing 200 micrograms per milliliter casein. By staining the fixed and dried gel with Coomassie blue R-250, proteolytic activity was visualized as a cleared area around the sample well with a detection limit of about 0.3 nanograms trypsin. The diffusion plate assay should prove useful for screening inhibitors of proteases where limited amounts of material are available, such as with plant cell fractions or highly purified proteins.

Journal Article↗
PubMed1979

[Sulfhydryl and amino groups in the cell fractions of white rat tissues: ontogenetic characteristics].

The author investigated total protein, percentage of protein sulfhydril and amino-groups of those in the cytoplasm and total sulfhydril groups in mitochondria, lysosomes and cytoplasm in liver, kidney and spleen of rats. There was a tendency to an increase of total protein in single tissue components with the advancement of age. The amount of sulfhydril groups was altered with advancement of age as well. Their maximal value was in animals, aged 5 days. In all examined cell fractions there was a statisticaly significant lowering in the subsequent age group, which tendency continued till the end of the examined period (5, 10, 15, 20, 25, and 30 -- day rats). The changes, observed in amino-groups were statisticaly significant and reflected the specificity in the metabolism of the three examined organs in accordance with their functions. Statistificaly significant lowering of the present sulfhydril groups, determined by DYNB, and nonsignificant changes of amino-groups showed that proteins in the examined tissues exposed various amount of sulfhydril groups probably due to the various water content of the tissues at different age periods. Perhaps there could be an effect of other factors as well, having connection with oxidation and reduction of sulfhydril groups.

Aging↗
PubMed1993

Antimutagenicity of cell fractions of microorganisms on potent mutagenic pyrolysates.

The inactivation of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (Glu-P-1) by binding of mutagenic pyrolysate to fractions of microorganisms (their desmutagenic and bio-antimutagenic activity) was investigated. All strains bound Trp-P-1 effectively, but Glu-P-1 to a lesser extent. The Gram-negative bacteria (GNB) could bind about 10-20 micrograms/mg of Trp-P-1 more than the Gram-positive bacteria (GPB), and about 50-60 micrograms/mg more than the yeasts. The cell wall skeletons of all strains tested had great binding ability, but in the cytoplasm of all strains tested it was lower. The peptidoglycan, outer membrane, and glucan isolate from the cell wall skeletons showed the highest binding ability to Trp-P-1. The cell wall skeletons of the tested strains greatly inhibited the mutagenicity induced by Trp-P-1, and to a lower extent that of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Although the cultured broth and solution of cells extracted by phosphate buffer (pH 7.0) showed antimutagenicity against Trp-P-1, this activity was lower than the binding of Trp-P-1 to the cells. The cultured broth and freeze-dried cytoplasms of yeast cells showed bio-antimutagenicity towards Trp-P-1, but those of all bacteria tested did not.

Amines↗
PubMed1970

Drosophila polyribosomes. The characterization of two populations by cell fractionation and isotopic labeling with nucleic acid and protein precursors.

Two populations of polyribosomes have been isolated from third instar larvae of D. melanogaster. One population appeared to be soluble while the second seemed membrane-bound. Short-term labeling of the two RNP fractions with radioactive nucleic acid and protein precursors was achieved by using a feeding stimulant. RNA was extracted from both polyribosomal fractions following 25, 40, and 60 min of in vivo uridine-(3)H incorporation. Soluble polyribosomes exhibited more rapid uptake of uridine into ribosomal and heterogeneous RNA fractions than did membrane-bound polyribosomes at comparable time periods. In vivo amino acid incorporation into the two polyribosomal populations was examined after 10, 20, 40, 60, and 80 min of incubation in leucine-(3)H. In this case, the membrane-bound polyribosomes reached a higher specific activity than did the soluble ones. These functional differences confirmed the observation, based on cellular fractionation studies, that the two classes of polyribosomes represented functionally distinct populations. These data have been compared with those from studies on other metazoan systems. In addition, dithiothreitol has been demonstrated to be a powerful ribonuclease inhibitor.

Amino Acids↗
PubMed1973

Localization of polysome-bound albumin and serine dehydratase in rat liver cell fractions.

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [(125)I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [(125)I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [(125)I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5-6 times those released from free polysomes. Anti-rat serum albumin-[(125)I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [(125)I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[(125)I]Fab to free polysomes was also obtained. The [(125)I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[(125)I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be demonstrated. These results indicate that the reaction of the Fab fragments are specific for polysomes that synthesize rat serum albumin or rat liver serine dehydratase. Furthermore, they demonstrate that even with this high degree of specificity, some polysomes in the fraction labeled "free" are in the process of synthesizing rat serum albumin while bound polysomes to a significant, if not major, degree are the site of the synthesis of rat liver serine dehydratase.

Animals↗
PubMed1990

Relationship of changes in pH and energy status to hypoxic cell fraction and hyperthermia sensitivity.

The relative concentrations of nucleotide triphosphates, creatine phosphate, inorganic phosphate, and pH have been evaluated as a function of tumor volume in a murine fibrosarcoma (FSaII) by 31P NMR spectroscopy. As the tumor volume increased from 60-1250 mm3, the ratio of phosphocreatine to inorganic phosphate systemically decreased. This decrease paralleled a decrease in the ratio of nucleotide triphosphate to inorganic phosphate in the same tumor volume range. The tumor pH as measured by 31P NMR decreased slightly with tumor growth. A pH of 7.17 +/- 0.07 (n = 17) was found for tumors between 60 and 150 mm3, whereas for tumors greater than 900 mm3, a pH of 7.05 +/- .03 (n = 6) was noted. Intermediate size tumors (151-900) had a pH of 7.12 +/- 0.09 (n = 18). The change in tumor energy status with tumor volume inversely paralleled the change in tumor radiobiologic hypoxic cell fraction and suggested a causal relationship between tumor nutrient status and energy status. Tumor thermal sensitivity also increased with tumor volume, suggesting a relationship between pH, energy status, and thermal sensitivity, as has been demonstrated under in vitro conditions. Each NMR parameter was found to correlate significantly with tumor volume independent of the other NMR parameters.

Adenosine Triphosphate↗
PubMed1975

[Studies on bradykinin-binding cell fractions. 1. Characterization of kininase activity of the microsomal and membrane fraction from the rat uterus].

In the membrane fraction of rat uterus, kinin-destroying activity was found and characterized as kininase II by means of several kininase inhibitors. It could be shown that sudies of bradykinin-receptor interactions requires the use of kininase inhibitors like phenanthroline or the use of a stable bradykinin analogue like [8-erythro-alpha-amino-beta-phenylbutyric acid]-bradykinin.

Angiotensin-Converting Enzyme Inhibitors↗