Concatemerization of tRNA molecules in the presence of trivaline derivative.
(57/34186)
The interaction of tRNA with trivaline dansyl hydrazide trifluoroacetate (DHTV) has been studied. The shape of curves of fluorimetric titration of tRNA with DHTV and vice versa can be explained only by formation of DHTV dimers on tRNA molecules, and subsequent association of DHTV-saturated tRNA molecules with each other. The ability of tRNA molecules to form concatemers in solution in the presence of DHTV has been demonstrated by electron microscopy. Electron microscopy of the tRNA-DHTV complexes stained with uranyl acetate revealed flexible rods 6-7 nm thick and up to several micrometers long. (+info)
Chromatin nu bodies: isolation, subfractionation and physical characterization.
(58/34186)
Monomer chromatin subunit particles (nu1) have been isolated in gram quantities by large-scale zonal centrifugation of micrococcal nuclease digests of chicken erythrocyte nuclei. nu1 can be stored, apparently indefinitely, frozen in 0.2 mM EDTA (pH 7.0) at less than or equal to 25 degrees C. Aliquots of the stored monomers have been subfractionated by dialysis against 0.1 M KCl buffers into a soluble fraction containing equimolar amounts of H4, H3, H2A, H2B associated with a DNA fragment of approximately 130-140 nucleotide pairs, and a precipitated fraction containing all of the histones including H5 and H1 associated with DNA fragments. The total nu1 and the KCl-soluble fraction of nu1 have been examined by sedimentation, diffusion, sedimentation equilibrium ultracentrifugation, low-angle X-ray diffraction, and electron microscopy. Physical parameters from all of these techniques are presented and correlated in this study. (+info)
An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3.
(59/34186)
Steroid hormones may enter cells by diffusion through the plasma membrane. However, we demonstrate here that some steroid hormones are taken up by receptor-mediated endocytosis of steroid-carrier complexes. We show that 25-(OH) vitamin D3 in complex with its plasma carrier, the vitamin D-binding protein, is filtered through the glomerulus and reabsorbed in the proximal tubules by the endocytic receptor megalin. Endocytosis is required to preserve 25-(OH) vitamin D3 and to deliver to the cells the precursor for generation of 1,25-(OH)2 vitamin D3, a regulator of the calcium metabolism. Megalin-/- mice are unable to retrieve the steroid from the glomerular filtrate and develop vitamin D deficiency and bone disease. (+info)
Early ontogeny of monocytes and macrophages in the pig.
(60/34186)
Prenatal development of cord blood monocytes and tissue macrophages was studied in pig foetuses by immunophenotyping and functional assays. The function of peripheral blood monocytes was compared in germ-free and conventional piglets. First macrophages were identified by electron microscopy in foetal liver on the 25th day of gestation. Monoclonal antibodies against porcine CD45 and SWC3 antigens were used for flow cytometric identification of myelomonocytic cells in cell suspensions prepared from the yolk sac, foetal liver, spleen and cord blood. Leukocytes expressing the common myelomonocytic antigen SWC3 were found in all organs studied since the earliest stages of development. Opsonized zymosan ingestion assay was used to determine the phagocytic capacity of foetal mononuclear phagocytes isolated from cord blood, liver and spleen. In the foetal liver, avid phagocytosis of apoptic cells had been found to occur before cells were able to ingest zymosan in vitro. The first cells capable of ingesting zymosan particles were found on the 40th day of gestation in umbilical blood and 17 days later in foetal spleen and liver. Their relative proportion increased with age. Cord blood monocytes and peripheral blood monocytes in germ-free piglets had low oxidatory burst activity as shown by iodonitrophenyl tetrazolium reduction assay. A remarkable increase of oxidatory burst activity was observed in conventional piglets, probably due to activation of immune mechanisms by the microflora colonizing gastrointestinal tract. (+info)
Lysosomal alterations induced in cultured rat fibroblasts by long-term exposure to low concentrations of azithromycin.
(61/34186)
Computer-aided simulations suggest that the doses and schedules of administration of azithromycin proposed in treatment and prophylaxis of Mycobacterium avium complex (MAC) in AIDS patients will result in drug concentrations in serum and extracellular fluids remaining for sustained periods of time in the 0.03-0.1 mg/L range. We exposed cultured rat embryo fibroblasts to these concentrations (and multiples up to 20 mg/L) for up to 16 days. Electron microscopy showed that after 7 days' incubation in 0.03 mg/L azithromycin, there was conspicuous accumulation of osmiophilic, lamellar structures (myeloid bodies) in lysosomes, suggesting the onset of a phospholipidosis. Assay of total cell phospholipids and cholesterol showed significant increases in cells exposed to > or = 1 to 5 mg/L of azithromycin in association with hyperactivity of the lysosomal enzyme cathepsin B. The data suggest that azithromycin, at extracellular concentrations pertinent to its use for MAC treatment, and perhaps also prophylaxis, causes limited morphological alterations of the lysosomes in cultured cells which are of the same nature as those developing rapidly and extensively at higher concentrations. (+info)
Ultramicroscopic structures of the leptomeninx of mice with communicating hydrocephalus induced by human recombinant transforming growth factor-beta 1.
(62/34186)
An experimental model of communicating hydrocephalus was developed based on intrathecal injection of human recombinant transforming growth factor-beta 1 (hrTGF-beta 1) in the mouse. To clarify the mechanism of this hydrocephalus model, the ultrastructure of the leptomeninx in the process of ventricular dilation was examined in C57/BL6 mice injected intrathecally with 60 ng of hrTGF-beta 1. The leptomeninx was examined at various periods after injection by light and electron microscopy. Immunostaining for fibroblasts and macrophages was also performed. Leptomeninx within a week after injection showed that the thin cytoplasmic processes of leptomeningeal cells formed a laminated structure with a meshwork, which was almost the same as the controls. In the second week, many cells with a round nucleus appeared in the leptomeninx. Immunohistochemically, these cells were positive for anti-fibroblast antibody and negative for anti-Mac-1 and anti-macrophage BM-8 antibodies. Three weeks later, the laminated structure was disrupted and abundant deposition of collagen fibers was found in the inter-cellular space of the leptomeninx. Such inter-meningeal fibrosis would disturb cerebrospinal fluid flow in the mouse leptomeninx and cause slowly progressive ventricular dilation. (+info)
Altered gene expression and functions of mitochondria in human nephrotic syndrome.
(63/34186)
The molecular basis of glomerular permselectivity remains largely unknown. The congenital nephrotic syndrome of the Finnish type (CNF) characterized by massive proteinuria already present but without extrarenal symptoms is a unique human disease model of pure proteinuria. In search of genes and pathophysiologic mechanisms associated with proteinuria, we used differential display-PCR to identify differences in gene expression between glomeruli from CNF and control kidneys. A distinctly underexpressed PCR product of the CNF kidneys showed over 98% identity with a mitochondrially encoded cytochrome c oxidase (COX I). Using a full-length COX I cDNA probe, we verified down-regulation of COX I mRNA to 1/4 of normal kidney values on Northern blots. In addition, transcripts of other mitochondrially encoded respiratory chain complexes showed a similar down-regulation whereas the respective nuclearly encoded complexes were expressed at comparable levels. Additional studies using histochemical, immunohistochemical, in situ hybridization, RT-PCR, and biochemical and electron microscopic methods all showed a mitochondrial involvement in the diseased kidneys but not in extrarenal blood vessels. As a secondary sign of mitochondrial dysfunction, excess lipid peroxidation products were found in glomerular structures in CNF samples. Our data suggest that mitochondrial dysfunction occurs in the kidneys of patients with CNF, with subsequent lipid peroxidation at the glomerular basement membrane. Our additional studies have revealed similar down-regulation of mitochondrial functions in experimental models of proteinuria. Thus, mitochondrial dysfunction may be a crucial pathophysiologic factor in this symptom. (+info)
Cholesteryl ester hydrolysis in J774 macrophages occurs in the cytoplasm and lysosomes.
(64/34186)
The relationship of cholesteryl ester hydrolysis to the physical state of the cholesteryl ester in J774 murine macrophages was explored in cells induced to store cholesteryl esters either in anisotropic (ordered) inclusions or isotropic (liquid) inclusions. In contrast to other cell systems, the rate of cholesteryl ester hydrolysis was faster in cells containing anisotropic inclusions than in cells containing isotropic inclusions. Two contributing factors were identified. Kinetic analyses of the rates of hydrolysis are consistent with a substrate competition by co-deposited triglyceride in cells with isotropic inclusions. In addition, hydrolysis of cholesteryl esters in cells with anisotropic droplets is mediated by both cytoplasmic and lysosomal lipolytic enzymes, as shown by using the lysosomotropic agent, chloroquine, and an inhibitor of neutral cholesteryl ester hydrolase, umbelliferyl diethylphosphate. In cells containing anisotropic inclusions, hydrolysis was partially inhibited by incubation in media containing either chloroquine or umbelliferyl diethylphosphate. Together, chloroquine and umbelliferyl diethylphosphate completely inhibited hydrolysis. However, when cells containing isotropic inclusions were incubated with umbelliferyl diethylphosphate, cholesteryl ester hydrolysis was completely inhibited, but chloroquine had no effect. Transmission electron microscopy demonstrated a primarily lysosomal location for lipid droplets in cells with anisotropic droplets and both non-lysosomal and lysosomal populations of lipid droplets in cells with isotropic droplets. These results support the conclusion that there is a lysosomal component to the hydrolysis of stored cholesteryl esters in foam cells. (+info)