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PubMed1958

The contribution of lower oxides of osmium to the density of biological specimens in electron microscopy.

Centrifugally stratified eggs of the sand-dollar, Dendraster excentricus, have cytoplasmic structures segregated into distinct layers. Fat droplets, yolk granules, and mitochondria are separated by "hyaline" layers of protoplasm. Aggregations of particles of 150 to 200 A diameter ("heavy bodies") are found near the mitochondrial layer and a concentration gradient of free 150 to 200 A particles corresponds to a similar gradient of basophilia in thick sections. Eggs fixed in buffered osmium-tetroxide at pH 7.4 and embedded in methacrylate were sectioned and floated on a "bleaching" solution of acidified hydrogen peroxide. "Bleached" sections showed a considerable loss in general density and especially a loss in the sharp images of cellular membranes. It was shown that such loss is not due to sublimation of structure in the electron beam. Assuming that the "bleach" acts principally to reoxidize lower oxides of osmium, it was concluded that reduced and bound lower oxides of osmium play a major role in creation of the electron micrograph image, especially in the delineation of phospholipide components of cellular membranes. Particles of 150 to 200 A diameter showed little or no loss in density, but rather a high intrinsic electron density. Refractometric data were presented to substantiate the tentative conclusions.

Animals↗
PubMed1981

Cell-substrate contacts illuminated by total internal reflection fluorescence.

A technique for exciting fluorescence exclusively from regions of contact between cultured cells and the substrate is presented. The technique utilizes the evanescent wave of a totally internally reflecting laser beam to excite only those fluorescent molecules within one light wavelength or less of the substrate surface. Demonstrations of this technique are given for two types of cell cultures: rat primary myotubes with acetylcholine receptors labeled by fluorescent alpha-bungarotoxin and human skin fibroblasts labeled by a fluorescent lipid probe. Total internal reflection fluorescence examination of cells appears to have promising applications, including visualization of the membrane and underlying cytoplasmic structures at cell-substrate contacts, dramatic reduction of autofluorescence from debris and thick cells, mapping of membranes topography, and visualization of reversible bound fluorescent ligands at membrane receptors.

Animals↗
PubMed1961

Electron microscopy of plasma-cell tumors of the mouse. I. MPC-1 and X5563 tumors.

An electron microscope study was made of a series of transplanted MPC-1 plasma-cell tumors carried by BALB/c mice. Large numbers of particles similar in morphology to virus particles were present inside the endoplasmic reticulum of tumor plasma cells. Very few particles were seen outside the cells or in ultracentrifuged preparations of the plasma or ascites fluid. In very early tumors particles were occasionally seen free in the cytoplasm adjacent to finely granular material. In general, the distribution of these particles inside endoplasmic reticulum is similar in early and late tumors. A few transplanted X5563 tumors of C3H mice were also examined. Large numbers of particles were found in the region of the Golgi apparatus in late X5663 tumors. A newly described cytoplasmic structure of plasma cells, here called a "granular body," appears to be associated with the formation of the particles. Particles present in MPC-1 tumors are exclusively of a doughnut form, whereas some of those in the inclusions of the late X5563 tumors show a dense center. Normal plasma cells, produced by inoculation of a modified Freund adjuvant into BALB/c mice. have been compared morphologically with tumor plasma cells of both tumor lines.

Animals↗
PubMed1983

Association between the mammalian 110,000-dalton heat-shock protein and nucleoli.

A rabbit antiserum has been prepared using as antigen the 110,000-dalton mammalian heat-shock protein. This protein was purified for injection by two-dimensional PAGE of heat-shocked Chinese hamster ovary cells. Characterization by immunoautoradiography and immunoprecipitation reveals that the antiserum is specific for the 110,000-dalton protein. Both techniques also reveal that the protein against which the antiserum is directed is induced by heat shock. Indirect immunofluorescence shows that the antigen is primarily localized at or near the nucleolus in cultured cells and numerous murine tissues. Treatment of cultured cells with deoxyribonuclease destroys the organization of staining within the nucleus while ribonuclease appears to completely release the antigen from the nucleus. A binding of the antiserum to cytoplasmic structures is also observed by immunofluorescence. This association with nucleoli may have implications in the regulatory aspects of the heat-shock response.

Animals↗
PubMed1984

Detection of single microtubules in living cells: particle transport can occur in both directions along the same microtubule.

Video-enhanced contrast/differential interference-contrast microscopy was used in conjunction with whole mount electron microscopy to study particle transport along linear elements in fibroblasts. Keratocytes from the corneal stroma of Rana pipiens were grown on gold indicator grids and examined with video microscopy. Video records were taken of the linear elements and associated particle transport until lysis and/or fixation of the cells was completed. The preparations were then processed for whole mount electron microscopy. By combining these two methods, we demonstrated that linear elements detected in the living cell could be identified as single microtubules, and that filaments as small as 10 nm could be detected in lysed and fixed cells. The visibility of different cytoplasmic structures changed after lysis with many more cellular components becoming visible. Microtubules became more difficult to detect after lysis while bundles of microfilaments became more prominent. All particle translocations were observed to take place along linear elements composed of one or more microtubules. Furthermore, particles were observed to translocate in one or both directions on the same microtubule.

Animals↗
PubMed1993

Flow cytometric identification of proliferative subpopulations within normal human epidermis and the localization of the primary hyperproliferative population in psoriasis.

In this study we define the proliferative compartments of in vivo human epidermis, using specific antibodies related to cell differentiation (beta 1 and beta 4 integrins and K1/K10 differentiation keratins) and cell cycle (proliferating cell nuclear antigen [PCNA]) in combination with flow cytometric quantitation of the DNA content and optical characteristics of the cells. The beta 1 integrin (CD29) marked both of the potentially proliferative subsets in normal epidermis. One subset of normal epidermis is CD29+ K1/K10-, which was predominantly basal, and found to be comprised of slow cycling, small cells with primitive cytoplasmic organization. The vast majority (95.5%) of these cells were in a quiescent state (G0/early G1) as indicated by their lack of the cyclin, PCNA. The other proliferative subset of normal epidermis was CD29+ K1/K10+, which was suprabasal and occasional basal, highly proliferative, larger in size, and which exhibited a more complex cytoplasmic structure. Because early differentiation (K1/K10 expression) has begun in the CD29+ K1/K10+ subset, it is highly likely that they represent the proliferative population which is capable of transiently amplifying itself before terminal differentiation. Within lesional psoriatic epidermis, similar proliferative cell populations were present as in normal epidermis, and the hyperproliferative defect was localized to the beta 1 and beta 4 integrin+, K1/K10- populations, which in normal epidermis is basally located and quiescent with regard to cell cycle. In psoriatic epidermis, a six- to sevenfold increase in the number of cells in the S/G2+M phase of cell cycle was found among CD29+ K1/K10- cells (p < 0.05). Furthermore, all lesional K1/K10- cells showed high PCNA positivity, indicating that all these cells had been recently induced into cell cycle. By contrast, the proportion of cycling cells among lesional psoriatic CD29+ K1/K10+ keratinocytes was similar to normals. Anti-HLA-DR, CD45, and vimentin antibodies were used to concomitantly track the proliferative states of Langerhans cell, melanocyte, and infiltrating leukocyte populations. In normal epidermis, the cycling fractions (cells in S/G2/M phase) of these cells were similar to the CD29+K1/K10- keratinocytes, whereas in lesional epidermis their cycling pools were increased relative to normal, but not so much as the proliferative fractions of psoriatic CD29+ K1/K10- keratinocytes. These data demonstrate the use of simultaneous analysis of integrin expression, differentiation keratins, cyclin, cell cycle status, and optical characteristics of freshly isolated human epidermal cells. Such analysis allowed the physical identification and quantification of cy cling populations in normal human skin, and has enabled the precise location of the primary epidermal proliferative defect in psoriasis.

Antigens, CD↗
PubMed1996

Mitochondrial control of nuclear apoptosis.

Anucleate cells can be induced to undergo programmed cell death (PCD), indicating the existence of a cytoplasmic PCD pathway that functions independently from the nucleus. Cytoplasmic structures including mitochondria have been shown to participate in the control of apoptotic nuclear disintegration. Before cells exhibit common signs of nuclear apoptosis (chromatin condensation and endonuclease-mediated DNA fragmentation), they undergo a reduction of the mitochondrial transmembrane potential (delta psi m) that may be due to the opening of mitochondrial permeability transition (PT) pores. Here, we present direct evidence indicating that mitochondrial PT constitutes a critical early event of the apoptotic process. In a cell-free system combining purified mitochondria and nuclei, mitochondria undergoing PT suffice to induce chromatin condensation and DNA fragmentation. Induction of PT by pharmacological agents augments the apoptosis-inducing potential of mitochondria. In contrast, prevention of PT by pharmacological agents impedes nuclear apoptosis, both in vitro and in vivo. Mitochondria from hepatocytes or lymphoid cells undergoing apoptosis, but not those from normal cells, induce disintegration of isolated Hela nuclei. A specific ligand of the mitochondrial adenine nucleotide translocator (ANT), bongkreik acid, inhibits PT and reduces apoptosis induction by mitochondria in a cell-free system. Moreover, it inhibits the induction of apoptosis in intact cells. Several pieces of evidence suggest that the proto-oncogene product Bcl-2 inhibits apoptosis by preventing mitochondrial PT. First, to inhibit nuclear apoptosis, Bcl-2 must be localized in mitochondrial but not nuclear membranes. Second, transfection-enforced hyperexpression of Bcl-2 directly abolishes the induction of mitochondrial PT in response to a protonophore, a pro-oxidant, as well as to the ANT ligand atractyloside, correlating with its apoptosis-inhibitory effect. In conclusion, mitochondrial PT appears to be a critical step of the apoptotic cascade.

Animals↗
PubMed1951

The nature and significance of changes in adrenal cytology, weight, and cortical/medullary ratio in experimental renal hypertension and clinical hypertension.

There is a direct proportionality between the degree of adrenal and that of cardiac enlargement in experimental renal hypertension. This is evidence for the participation of the adrenal to an extent dependent on the severity of the hypertension as reflected in the degree of cardiac hypertrophy. The greater part of the weight increase of the adrenal is due to growth of the cortex. However, the relative degree of medullary growth exceeds that of cortical growth. There is evidence of medullary hyperplasia, but the cortical growth may be due to cell hypertrophy alone. In view of the known relationship between prolonged skeletal muscle work and the size of the adrenals, in which there is likewise a relatively greater growth of the medulla, the possibility that adrenal growth in experimental renal hypertension is a consequence of the metabolic demand of increased heart muscle work must be considered. The hyperplastic, hypertrophic medulla of the renal hypertensive rat is presumed to be producing increased amounts of a hormone whose action is not known. The adrenal medulla is not necessary for the development of moderate renal hypertension though this does not exclude the possibility that it may be involved somehow in the maintainance of the hypertensive state. Large, periodic-Schiff-positive, hyaline droplets are found in the cytoplasm of adrenal cortical cells with nuclear evidence of active function. There is some evidence that these indicate hypophyseal stimulation. Similar particles are found in the medulla, suggesting an interrelationship between the two parts of the adrenal. The close resemblance of changes in adrenal weight, nuclear/cytoplasmic ratio, and nuclear and cytoplasmic structure, indicates that similar physiological and morphological controlling mechanisms are involved in the pathogenesis and maintainance of experimental renal hypertension and clinical essential hypertension.

Adrenal Glands↗
PubMed2005

The role of microglial cellular senescence in the aging and Alzheimer diseased brain.

With each cell division, telomeres progressively shorten until they reach a critical length, at which point the cells enter cellular senescence. Microglia, a non-neuronal cell type residing within the central nervous system (CNS), play vital roles in maintaining neuronal function, health, and survival in both the normal and pathological CNS. A recent article described an increased incidence of microglial cytoplasmic structural abnormalities (i.e., swelling, twisted and shortened processes, and fragmentation) and dystrophy occurring in the cerebral cortex of human brains with age. These results suggest that microglial dystrophy may be a result of, or contribute to, their senescence, which in turn may impair their neuron-sustaining functions and ultimately lead to neuronal cell death.

Aging↗
PubMed2001

An intact dilysine-like motif in the carboxyl terminus of MAL is required for normal apical transport of the influenza virus hemagglutinin cargo protein in epithelial Madin-Darby canine kidney cells.

The MAL proteolipid, a component of the integral protein sorting machinery, has been demonstrated as being necessary for normal apical transport of the influenza virus hemagglutinin (HA) and the overall apical membrane proteins in Madin-Darby canine kidney (MDCK) cells. The MAL carboxy terminus ends with the sequence Arg-Trp-Lys-Ser-Ser (RWKSS), which resembles dilysine-based motifs involved in protein sorting. To investigate whether the RWKSS pentapeptide plays a role in modulating the distribution of MAL and/or its function in apical transport, we have expressed MAL proteins with distinct carboxy terminus in MDCK cells whose apical transport was impaired by depletion of endogenous MAL. Apical transport of HA was restored to normal levels by expression of MAL with an intact but not with modified carboxyl terminal sequences bearing mutations that impair the functioning of dilysine-based sorting signals, although all the MAL proteins analyzed incorporated efficiently into lipid rafts. Ultrastructural analysis indicated that compared with MAL bearing an intact RWKSS sequence, a mutant with lysine -3 substituted by serine showed a twofold increased presence in clathrin-coated cytoplasmic structures and a reduced expression on the plasma membrane. These results indicate that the carboxyl-terminal RWKSS sequence modulates the distribution of MAL in clathrin-coated elements and is necessary for HA transport to the apical surface.

Adaptor Protein Complex gamma Subunits↗
PubMed1995

Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms.

The mechanochemical ATPase kinesin is thought to move membrane-bounded organelles along microtubules in fast axonal transport. However, fast transport includes several classes of organelles moving at rates that differ by an order of magnitude. Further, the fact that cytoplasmic forms of kinesin exist suggests that kinesins might move cytoplasmic structures such as the cytoskeleton. To define cellular roles for kinesin, the axonal transport of kinesin was characterized. Retinal proteins were pulse-labeled, and movement of radiolabeled kinesin through optic nerve and tract into the terminals was monitored by immunoprecipitation. Heavy and light chains of kinesin appeared in nerve and tract at times consistent with fast transport. Little or no kinesin moved with slow axonal transport indicating that effectively all axonal kinesin is associated with membranous organelles. Both kinesin heavy chain molecular weight variants of 130,000 and 124,000 M(r) (KHC-A and KHC-B) moved in fast anterograde transport, but KHC-A moved at 5-6 times the rate of KHC-B. KHC-A cotransported with the synaptic vesicle marker synaptophysin, while a portion of KHC-B cotransported with the mitochondrial marker hexokinase. These results suggest that KHC-A is enriched on small tubulovesicular structures like synaptic vesicles and that at least one form of KHC-B is predominantly on mitochondria. Biochemical specialization may target kinesins to appropriate organelles and facilitate differential regulation of transport.

Animals↗
PubMed1998

A mutation in a novel yeast proteasomal gene, RPN11/MPR1, produces a cell cycle arrest, overreplication of nuclear and mitochondrial DNA, and an altered mitochondrial morphology.

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24 degreesC growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36 degreesC on either carbon source. Microscopic observation of cells growing on glucose at 24 degreesC shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho degrees] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

Amino Acid Sequence↗
PubMed2006

POLKADOTS are foci of functional interactions in T-Cell receptor-mediated signaling to NF-kappaB.

Stimulation of the T-cell receptor (TCR) results in the activation of several transcription factors, including NF-kappaB, that are crucial for T-cell proliferation and gain of effector functions. On TCR engagement, several proteins within the TCR-directed NF-kappaB signaling pathway undergo dynamic spatial redistribution, but the significance of these redistribution events is largely unknown. We have previously described TCR-induced cytoplasmic structures called POLKADOTS (punctate and oligomeric killing or activating domains transducing signals) that are enriched in the NF-kappaB signaling intermediate, Bcl10. We now show that these structures are formed only under conditions that promote efficient NF-kappaB activation. Furthermore, POLKADOTS formation is dependent on functional domains of specific NF-kappaB signal transducers. Through use of a photoactivatable GFP, we demonstrate that POLKADOTS contain both a highly stable and a rapidly equilibrating protein component. FRET analyses show that POLKADOTS are sites of enriched interactions between Bcl10 and partner signaling proteins. These observations strongly suggest that POLKADOTS are focal sites of dynamic information exchange between cytosolic intermediates in the process of TCR activation of NF-kappaB.

Adaptor Proteins, Signal Transducing↗
PubMed1994

forked proteins are components of fiber bundles present in developing bristles of Drosophila melanogaster.

The forked (f) gene of Drosophila melanogaster encodes six different transcripts 6.4, 5.6, 5.4, 2.5, 1.9, and 1.1 kb long. These transcripts arise by the use of alternative promoters. A polyclonal antibody raised against a domain common to all of the forked-encoded products has been used to identify forked proteins on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and in Drosophila pupal tissues. The antibody stains fiber bundles present in bristle cells for about 15 hr during normal pupal development. Electron microscopy shows that these fibers are present from 40 to 53 hr in bristles of wild-type flies but are absent in the null f36a mutant. The forked protein(s) thus appear to be an essential part of the bristle fibers. The phenotype of the f36a mutation can be rescued by a 13-kb fragment of the forked locus containing the coding regions for the 2.5, 1.9, and 1.1-kb transcripts, suggesting that the proteins encoded by the three large forked RNAs are dispensable during bristle development. Increasing the copy number of a P[w+,f+] construct containing the 13-kb fragment induces a hypermorphic bristle phenotype whose severity correlates with the number of copies of P[w+,f+] present. These results indicate that alterations in the ratios among the forked proteins, or between forked products and other components of the fiber, result in abnormal assembly of the fibrillar cytoplasmic structures necessary for bristle morphogenesis.

Animals↗
PubMed1994

Overexpression of yeast homologs of the mammalian checkpoint gene RCC1 suppresses the class of alpha-tubulin mutations that arrest with excess microtubules.

Microtubules in eukaryotic cells participate in a variety of nuclear and cytoplasmic structures, reflecting functional requirements and cell cycle position. We are studying the cellular regulation of microtubule assembly and organization in the yeast Saccharomyces cerevisiae. We screened for genes that when overexpressed suppress the growth phenotype of conditional mutants in alpha-tubulin that arrest with excess microtubules at the nonpermissive temperature (class 2 mutations). Here we describe one such suppressing element, called ATS1 (for Alpha Tubulin Suppressor). Overexpression of this gene rescues both the growth and microtubule phenotypes of all class 2 mutations, but not the cold-sensitive mutations that arrest with no microtubules (class 1 mutations). Deletion of ATS1 confers a modest slow growth phenotype which is slightly enhanced in strains containing both a deletion of ATS1 and a class 2 tub 1 mutation. The predicted ATS1 protein contains 333 amino acids and has considerable structural homology to the products of both the mammalian mitotic control gene RCC1 and the S. cerevisiae gene SRM1/PRP20. Overexpression of SRM1/PRP20 also suppresses class 2 mutants. The results suggest that this family of genes may participate in regulatory interactions between microtubules and the cell cycle.

Amino Acid Sequence↗
PubMed1989

Neuron-binding antibodies in Alzheimer's disease and Down's syndrome.

We used an indirect immunoperoxidase technique (Avidin-Biotin system) to study the sera of patients with "clinically probable" Alzheimer's disease (AD) and with Down's Syndrome (DS), compared with age-matched controls. Diluted sera were incubated with paraffin sections of hippocampus, frontal, temporal, and parieto-occipital lobes from normal human brains. Biotinylated anti-human goat gamma-globulins were used as secondary antisera. A significantly greater percentage of neurons were immunostained in all the brain regions (frontal, temporal, and parieto-occipital lobes and hippocampus) incubated with sera of AD patients than with sera of DS patients or of controls. This indicates that AD patients have an excess of circulating neuron-binding antibodies (NBAs), mainly reacting with cytoplasmic structures. NBAs could be either the cause or the result of the cerebral lesion found in AD. This study is not able to answer this question, but some previous data from our own and other laboratories suggest that NBAs have a role in the pathogenesis of AD lesions. Since we found no increase of NBAs in DS patients, the brain lesions in DS appear to have a different pathogenesis.

Aged↗
PubMed2003

NSDHL, an enzyme involved in cholesterol biosynthesis, traffics through the Golgi and accumulates on ER membranes and on the surface of lipid droplets.

NSDHL, for NAD(P)H steroid dehydrogenase-like, encodes a sterol dehydrogenase or decarboxylase involved in the sequential removal of two C-4 methyl groups in post-squalene cholesterol biosynthesis. Mutations in this gene are associated with human CHILD syndrome (congenital hemidysplasia with ichthyosiform nevus and limb defects), an X-linked, male lethal disorder, as well as the mouse mutations bare patches and striated. In the present study, we have investigated the subcellular localization of tagged proteins encoded by wild-type and selected mutant murine Nsdhl alleles using confocal microscopy. In addition to an ER localization commonly found for enzymes of post-squalene cholesterol biosynthesis, we have identified a novel association of NSDHL with lipid droplets, which are endoplasmic reticulum (ER)-derived cytoplasmic structures that contain a neutral lipid core. We further demonstrate that trafficking through the Golgi is necessary for ER membrane localization of the protein and propose a model for the association of NSDHL with lipid droplets. The dual localization of NSDHL within ER membranes and on the surface of lipid droplets may provide another mechanism for regulation of the levels and sites of accumulation of intracellular cholesterol.

3-Hydroxysteroid Dehydrogenases↗
PubMed1981

Persistent non-A, non-B hepatitis in experimentally infected chimpanzees.

Non-A, non-B (NANB) hepatitis was transmitted to six chimpanzees by intravenous inoculation of antihemophilic (factor VIII) materials, acute-phase chimpanzee liver, and chronic-phase plasma obtained from two NANB hepatitis-infected chimpanzees 10 and 16 months, respectively, after their inoculation. Five of six experimentally infected chimpanzees observed for more than one year demonstrated persistent or intermittent elevations in levels of serum alanine aminotransferase (ALT) indicative of continuing liver dysfunction. Liver biopsy specimens obtained from three chimpanzees with persistent elevations in levels of ALT were positive for hepatocyte cytoplasmic structures associated with NANB hepatitis for as long as 27 months after inoculation. Liver biopsy specimens obtained from four infected animals 13-30 months after inoculation also showed mild but persistent histopathologic lesions of undefined character. The detection of circulating immune complexes in one chimpanzee with persistent elevations in levels of ALT suggests that these complexes may be involved in the pathogenesis of NANB hepatitis.

Animals↗