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PubMed1978

Studies on secretory glycoproteins in the rat exocrine pancreas. III. Intracellular transport of fucose-labeled proteins as studied by cell fractionation.

The transcellular movement of fucosylated glycoproteins has been studied in vitro using rat pancreatic lobules and cell fractionation procedures, and has been compared with the well established pathway of secretory proteins. Using tritiated leucine as pulse label for the latter, their translocation from the rough endoplasmatic reticulum into the Golgi complex and finally into zymogen granules could be followed. In the case of glycoproteins, 14C-fucose was incorporated mainly into the smooth microsomal fraction (representative of the Golgi complex) and only one third of this specific activity was transported into the zymogen granule fraction. A detailed analysis of this fraction after separation of the content of zymogen granules from their membranes revealed a predominant labeling of membrane glycoproteins by 14C-fucose. In comparison, leucine-labeled bulk proteins were found almost exclusively in the zymogen granule content fraction, with little radioactivity in the membrane fraction. The data indicate a concomitant synthesis of fucosylated glycoproteins destined in part for the zymogen granule membrane and to a greater amount associated with the smooth microsomal fraction. The results are discussed in the light of recent findings indicating that about 40% of the proteins in the zymogen granule membrane are made up of one major glycoprotein which could be involved in the mechanism of exocytosis.

Amylases↗
PubMed1976

Some biological properties of mouse spleen cells fractionated by the adherence of Sephadex G 25 and glass bead columns.

The possibilities of separation of haemopoietic cells from lymphocytes capable of eliciting the graft-versus-host reaction through column chromatography were investigated. Strain-A mouse spleen cells were fractionated into the adherent and non-adherent fraction on Sephadex G-25, glass bead columns and glass beads coated with antibody against mouse globulin. Increased numbers of cells forming haemopoietic colonies were found in the cell fraction which did not adhere to the antibody-coated glass beads and in cells reversibly adhering to glass beads. No significant decrease in local graft-versus-host reaction was found in any fraction obtained, and the prolonged survival of irradiated semiallogeneic recipients was observed in both fractions obtained on Sephadex G-25 columns.

Animals↗
PubMed1975

Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions.

The recirculating perfusion of adult rat liver with a Ca-++-free Hanks' solution produces a release of the adhesiveness of cells and a cleaving of the desmosomes. The addition of collagenase and hyaluronidase to the perfusion medium leads to complete dissociation of the liver tissue into a mixture of isolated cells and cell cords in which the hepatocytes remain connected with specific junctional differentiations, namely the gap and tight junctions. Individual cells are released by submitting the suspension of cell trabeculae to a gentle rolling. The gap junctions are ruptured at least in one of the two adjacent cells and remain generally attached to the other cell taking with them a small portion of cytoplasm. This technique of isolation of hepatocytes yields about 60-65% of the parenchymal cells contained in a liver; endothelial cells and other cells of the connective tissue are not recovered. The ultrastructural preservation of the isolated hepatocytes is excellent and the glucose-6-phosphatase activity, confined to the endoplasmic reticulum, appears unaltered in most cells. Protein, DNA and RNA recovery in the preparations of isolated hepatocytes is satisfactory, amounting to 70% of that found in liver homogenate; glycogen, the most labile component examined, is partly lost or degraded during the manipulations. Cell diameters measured by different methods confirm the preservation of the original volume of the in situ hepatocytes and the presence of more than one type of parenchymal cell. By submitting this heterogeneous cell population to an isopycnic density gradient centrifugation, two types of hepatocytes can be distinguished: the light hepatocytes, with a mean diameter of 20.5 mum and a mean density of 1.10, are characterized by an extended smooth-walled endoplasmic reticulum entrapping dispersed alpha-glycogen particles; the heavy hepatocytes, with a mean diameter of 19.0 mum and a mean density of 1.14, present a relatively reduced compartment of smooth endoplasmic reticulum, but large accumulations of glycogen. It is suggested that the cell fraction of low density is enriched in centrolobular cells and the high density fraction in perilobular hepatocytes.

Animals↗
PubMed1978

Cell fractionation and cytological analysis of human lymphatic cells from tonsil and blood.

No differences could be detected in the discontinuous density gradient when comparing the distribution pattern of tonsillar cells and peripheral lymphocytes. When the Kiel nomenclature is applied, the presence of centrocytes, centroblasts, lymphocytes and plasma cells in the tonsil is shown by a cytological analysis. The biological importance of these results is discussed from the point of cellular proliferation and tonsillar function.

Adult↗
PubMed1979

Distribution of newly formed ribosomal proteins in HeLa cell fractions.

The distribution of newly formed ribosomal proteins between cytoplasmic, nucleoplasmic, and nucleolar fractions of HeLa cells was determined. All but a few of the newly formed ribosomal proteins were concentrated 10- to 50-fold in the nucleolus and two- to fivefold in the nucleoplasm. Nevertheless, substantial amounts were found in the cytoplasm. Pretreatment of cells with actinomycin D to deplete the nucleolar pool of ribosomal precursor RNA had no effect on the concentration of newly formed ribosomal proteins in the nucleus, but did lead to an increased amount in the nucleoplasm at the expense of the nucleolus.

Cell Nucleolus↗
PubMed1978

Stimulation of human B lymphocytes by Listeria cell wall fraction.

Cell wall fraction of Listeria monocytogenes (LCWF), a B cell mitogen for mouse spleen cells, is also mitogenic for human adult and cord peripheral blood lymphocytes. Purified B-cell suspensions responded to LCWF in vitro proliferation, to a similar extent as the unfractionated suspensions. Furthermore, LCWF-induced B cell differentiation into IgM-containing cells and their percentage correlated significantly with the extent of lymphocyte proliferation.

Adult↗
PubMed1978

DNA synthesis and thymidine kinase activity of rat colon epithelial cells fractionated by discontinuous Ficoll gradient.

Epithelial cells from colons of adult Sprague-Dawley rats were fractionated on a discontinuous Ficoll gradient at low centrifugal forces (170 x g) for approximately 60 minutes. Epithelial cells were separated into three distinct zones, whereas cell debris, yeast, and bacteria remained at the top of the gradient. The percentage of cells in each zone was inversely related to the density of the gradient. More than 95% of the cells were morphologically intact and viable (excluded trypan blue). Cells sedimenting at higher densities of Ficoll exhibited higher thymidine kinase activity and DNA synthesis, suggestive of active cell division. The cells sedimenting at lower densities of Ficoll showed the least thymidine kinase activity and DNA synthesis, properties that are compatible with those of mature absorptive cells. Tall columnar cells with vesicular nuclei were predominant in the fraction sedimenting at the lowest density (top fraction). At higher densities (middle and lower fractions), most of the cells were short and columnar with basally located condensed dark-staining nuclei.

Animals↗
PubMed1975

Biosynthesis of liver catalase in rats treated with allylisopropylacetylcarbamide. I. Immunochemical assay of catalase in liver cell fractions.

Rats were injected twice intraperitoneally with 20 mg of allylisopropylacetylcarbamide (Sedormid) per 100 g of body weight at an interval of 12 hr. The level of catalase [EC 1.11.1.6] in various liver cell fractions was determined both enzymatically and immunochemically 12 hr after the second injection. 1. The decrease in catalase protein assayed by the immunochemical method directly confirmed the inhibition of biosynthesis of the enzyme by this porphyrinogenic drug. 2. The occurrence of a considerable amount of catalase protein with no enzymatic activity was demonstrated both in the peroxisomes and in the supernatant fraction. 3. The amount of catalase-synthesizing polysomes in hepatic cell was reduced in Sedormid-treated rats by the extent comparable to the decrease in the concentration of liver catalase.

Animals↗
PubMed1975

Migration inhibition with various cell fractions in human colorectal cancer.

The unseparated leucocytes, separated mononuclear cells and granulocytes of six control subjects and nine patients with colorectal cancer have been studied by a direct cell migration inhibition technique. A migratory index was calculated from the migration in the presence and absence of a perchloric acid extract of large bowel tumours. In 10% homologous AB serum, no significant migration inhibition occurred with any of the cells from control subjects. Five of the nine cancer patients showed significant inhibition with their unseparated leucocytes, seven of seven with their mononuclear cells, and none of nine with their granulocytes. In 10% autologous serum, some controls exhibited migration inhibition with their unseparated leucocytes and their granulocyte fraction, but not with the mononuclear cell fraction. Migration inhibition was also now apparent in the granulocyte fraction of the cancer patients. It is concluded that, with a soluble tumour antigen preparation, a mononuclear cell population increases the sensitivity of the direct migration inhibition test and that autologous serum may interfere directly with the migration of granulocytes, by an action not dependent upon the release of inhibitory factors from sensitized lymphocytes. This could explain some of the inconsistencies of the assay when using an unseparated leucocyte population.

ABO Blood-Group System↗
PubMed1979

Uptake and metabolism of female sex steroids by isolated small neurons and other cell fractions from the rat medial basal hypothalamus.

Rat medial basal hypothalami (MBH) and sections of cerebral cortex (CC) were dissociated with trypsin to prepare single cells and subcellular fractions. They were then separated into four fractions on a discontinuous sucrose gradient. The small neurons in Fraction D were highly purified. Fraction A had synaptosomes, myelin and other cell particulates. Fraction B had glial cells, neurons and a few synaptosomes. Fraction C had large neurons and red blood cells. All four fractions contained LHRH, but most (62.5%) of this hormone was present in Fraction A. Dissociated cell suspensions were incubated with [3H]-steroids, with and without a 100-fold excess of unlabeled steroids, then separated on sucrose gradients. In most fractions the total uptake and specific uptake of [3H]-progesterone, [3H]-5 alpha-pregnane-3,20-dione (5 alpha-dihydroprogesterone) and [3H]-17 beta-estradiol were greater for the dissociated cells from the MBH than the CC. The dissociated cells and cell particulates in all four fractions from the MBH and CC metabolized progesterone, 5 alpha-dihydroprogesterone and 17 beta-estradiol. These results indicate that hypothalamic neurons contain small amounts of LHRH and retain the ability to take up and metabolize progesterone, 5 alpha-dihydroprogesterone and 17 beta-estradiol.

20-alpha-Dihydroprogesterone↗
PubMed1975

Influence of subtotal hepatectomy on peroxisomes and peroxisomal enzymes of rat liver and isolated liver cell fractions.

The activities of peroxisomal enzymes of rat liver were followed 1 to 10 days after subtotal (60-70%) hepatectomy in homogenates prepared from regenerating livers and in cell fractions isolated from them. Catalase activity was found to be depressed in the total liver homogenate (H) as well as in the mitochondrial (M) and soluble (S) fractions, while it did not change appreciably in the microsomal (Mc) and lysosomal (L) fractions. Alpha-hydroxyacid oxidase behaved in a similar fashion. In contrast to these enzymes, urate oxidase activity remained unchanged in H, whereas it was decreased in M and increased in L and Mc during the first 5 days after operation. These results agree well with the assumption that microbody proliferation is initiated by the fragmentation of large peroxisomes. The different relations of peroxisomal enzyme activities during regeneration time are discussed with respect to the possible existence of various kinds of peroxisomes with different enzyme equipments and with different turnover rates. Biochemical examinations ions were paralleled to morphological and histochemical studies. An early increase in number of peroxisomes was found to occur during the first day after partial hepatectomy, which is accompanied by decrease in particle size. During the first mitotic wave (24-36 hrs post op.) the number of peroxisomes per cell was reduced to about the half. After this time number and size of the particles began to increase. Positive staining of ribosomes was frequently observed in the vicinity of peroxisomes after the application of the cytochemical catalase reaction (alkaline diaminobenzidine medium). This phenomenon is interpreted to represent rather a diffusion artifact than the cytochemical identification of newly synthesized catalase.

Alcohol Oxidoreductases↗
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↗
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↗