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PubMed1991

Fluorescent tetradecanoylphorbol acetate: a novel probe of phorbol ester binding domains.

Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol-13-acetate-12-N-methyl-N-4-(N,N'-di(2-hydroxyethyl)amino)-7-n itr obenz-2-oxa-1,3-diazole-aminododecanoate (N-C12-Ac(13)) of 12-O-tetradecanoylphorbol-13-acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N-C12-Ac(13) is close to 488 nm, facilitating its use in argon-ion laser flow and imaging cytometry. When incubated with 100 nM N-C12-Ac(13) at 25 degrees C, P3HR-1 Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20-fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N-C12-Ac(13) stained cells in a dose-dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [3H]-phorbol-12,13-dibutyrate ([3H]-PBu2) from rat brain cytosol with N-C12-Ac(13) gave an apparent dissociation constant (Kd) of 11 nM. N-C12-Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N-C12-Ac(13), at a lower concentration (51 nM), induced expression of Epstein-Barr viral glycoprotein in P3HR-1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N-C12-Ac(13) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non-specific binding of N-C12-Ac(13), this probe is not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non-specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.

Animals↗

PubMed1995

Stability of nuclear segments in human neutrophils and evidence against a role for microfilaments or microtubules in their genesis during differentiation of HL60 myelocytes.

The nucleus of the mature human neutrophil is segmented into three to five interconnected lobes. The physiological purpose of this segmentation is unknown, as is the mechanism by which the lobes are formed during differentiation. Using video observation of migrating human neutrophils simultaneously illuminated for fluorescence and phase-contrast microscopy, we analyzed nuclear movements with respect to cell shape changes. The number of nuclear lobes and their relative size remained constant during observation (up to 1 h). The thin connecting segments between the lobes elongated and attenuated extensively but never separated. Electron microscopic analysis of neutrophil nuclei revealed no specialized nuclear or cytoplasmic structures in the vicinity of connecting segments. With fluorescence in situ hybridization of whole chromosome probes, we determined that chromosomes are randomly distributed among neutrophil nuclear lobes. HL60 cells are a human myelocytic line that, with retinoic acid treatment, segment their nuclei and differentiate into neutrophil-like cells over several days. Using a rapidly responding variant line termed HL60/S4 (Cancer Res. 52, 949-954), we found that segmentation could be induced within 24 h. We tested the role of cytoskeletal elements in the process of nuclear segmentation. Neither the microtubule inhibitor nocodazole nor the microfilament inhibitor cytochalasin D prevented nuclear segmentation. Together, our studies suggest that nuclear lobes in neutrophils are relatively stable structures that are not generated by microtubule- or microfilament-dependent forces.

Actin Cytoskeleton↗

PubMed1987

Lymph heart musculature in birds.

Development and innervation of the lymph heart musculature of chicken, emu, rhea, and duck was studied by electron microscopy at post-hatch ages from 3 days to adulthood. Development of innervation was monitored by acetylcholinesterase staining. Horseradish peroxidase was used to determine the extent of the transverse tubule network. Chickens were unusual among these birds in that lymph heart myocytes had already undergone a definitive differentiation and degeneration by 3 days. In ducks and ratite birds, lymph heart myocytes more slowly but progressively differentiate a cytomorphology that does not conform in all characteristics to cardiac or skeletal muscle and even resembles in some aspects, smooth muscle. Myofibrils become the dominant cytoplasmic structure, transverse tubules form "internal couplings" with agranular reticulum cisternae, and "external couplings" are formed between myocytes at myomyal junctions. The myomyal junctions also contain AChE-positive reaction product and some subplasmalemmal vesicles that lack a dense core. The lymph heart myocardium of ducks of 2 weeks demonstrated mitotic figures. In adult ducks the myosatellite cell numbers diminish and a characteristic pattern of myocyte degeneration appears. In juvenile ducks and ratites some myocytes differentiate to conductile cells, much as the conductile myocytes and myofibers of the blood heart. The lymph heart innervation is described, and the role of nerve in differentiation and maintenance of myocyte morphology in the lymph heart is discussed.

Animals↗

PubMed1994

Heat shock alters Alzheimer's beta amyloid precursor protein expression in human endothelial cells.

One of the pathological lesions in Alzheimer's disease (AD) is the amyloid or senile plaque. The plaque core is predominantly made up of amyloid beta peptide (A beta), a 42-43 amino acid peptide derived from amyloid precursor protein (APP). APP is a membrane bound glycoprotein which is expressed ubiquitously in many cells. Although normal or pathological functions for APP are not well understood, several observations suggest that APP may play a role in cellular stress and inflammation at the endothelial cell/vascular barrier. APP is found in platelets and endothelial cells, it can inhibit a blood coagulation factor, and secreted APP can be neuroprotective. Changes in expression of APP during cellular stress or inflammation may contribute to pathological deposition of A beta. In the present studies, expression of APP in human endothelial cells was examined following heat shock. In human umbilical vein endothelial cells (HUVECs) exposed to 42 degrees C for 30 min, there was a five- to eight-fold increase in APP mRNA levels which peaked at 4 hr. The increase in APP mRNA was followed by an increase in APP protein immunoreactivity in the cytoplasm in a perinuclear Golgi-like region, and in discrete granular cytoplasmic structures. Immunoblot analysis of APP in the cell media found a transient increase in APP which peaked at 1 hr after heat shock. These results suggest that cellular stress induces the secretion of APP from endothelial cells followed by a subsequent increase in APP mRNA and protein synthesis. The upregulation of APP mRNA and protein supports a cellular stress role for APP.

Alzheimer Disease↗

PubMed1987

Morphology and fertilizability of frozen human oocytes.

Human oocytes were frozen and thawed by four methods previously used for cryopreservation of human embryos. Most of these oocytes were inseminated after thawing to assess their capacity to fertilize and form pronuclear ova. Their morphology was assessed by phase-contrast microscopy used in routine IVF. Twenty-three oocytes were examined by electron microscopy to critically evaluate the effects of cooling and cryopreservation and to confirm fertilization. Morphological survival was observed in more than 60% of the oocytes examined after freeze-thawing. The main features of cryoinjury were cracks in the zona pellucida, disruption of the plasma membrane and extensive disorganization of the ooplasm. Subtle changes in the cytosol of cumulus cells was also observed. Cooling to 0 degrees C or -6 degrees C had little effect on cytoplasmic structure. Spindles were damaged in two frozen oocytes. Cumulus cell activity, sperm binding to the zona, sperm penetration of the zona seem to be largely unaffected by freeze-thawing. Fertilization was observed in eight oocytes after postthaw insemination and three embryos (8-cell to morula stages) were developed from pronuclear ova on further culture. Both monospermic and polyspermic fertilization were confirmed by electron microscopy and micronuclei were detected in three pronuclear ova. The genetic implications of these nuclear aberrations are discussed. These preliminary studies indicate that oocyte freezing needs to be integrated cautiously with clinical IVF by further assessment of embryos developed from frozen oocytes.

Female↗

PubMed1992

Differentiation profiles of normal blast cell colonies derived from mononucleated cells, T cell depleted nonadherent cells and CD33-negative, CD34-positive normal human bone marrow cells.

Blast cell colonies can be grown reproducibly in methylcellulose from normal human mononuclear bone marrow cells (MNC) in the presence of 30% human plasma, 1 U human recombinant erythropoietin and 10% of medium conditioned by phytohemagglutinin stimulated leukocytes as a source of growth factors. The colonies can be recognized by inverted microscopy by their display of highly refractile cytoplasmic structures that resemble small vacuoles. A proportion of cells within these colonies remains CD34-positive (CD34+). The blast-like morphology of these cells was sustained for at least 14 to 21 days. The frequency of blast cell colony forming units (CFU-BL) can be increased by a series of separation procedures to produce E-rosette depleted, nonadherent cells (E-NAC), CD34+ cells and CD33-CD34+ cells. The mean number of CFU-BL per 10(5) cells was 1.9 +/- 1.6 in MNC, 5.7 +/- 2.7 in E-NAC, 108 +/- 51 in CD34+ cells and 112 +/- 109 in CD33-CD34+ cells. The enrichment procedures were not specific for CFU-BL but also resulted in a similar increase of multilineage and single lineage progenitors. The relative proportions of CFU-BL and other progenitors remained unchanged among these subpopulations. The size of individual blast cell colonies on day 16 varied from 20 to 1,600 cells. After 14 to 21 days, cells within blast cell colonies acquired mature morphological features. The majority of blast cell colonies developed into multilineage colonies (62%); some (38%) were restricted to a single hemopoietic lineage. Larger blast cell colonies had a higher probability of developing into multilineage colonies than smaller blast cell colonies.

Antigens, CD↗

PubMed1997

Analysis of expressed sequence tags (ESTs) of the parasitic protozoa Entamoeba histolytica.

A directional cDNA library constructed from mRNA of the trophozoite of Entamoeba histolytica HM-1:IMSS strain was used for the generation of expressed sequence tags (ESTs). From 5' ends of the distinct cDNA clones, 105 ESTs were obtained. Of these, 30 clones (29%) were previously known E. histolytica genes. Forty-five clones (42%) had matches with entries for other organisms in the databases. These new E. histolytica genes constituted a broad range of transcripts distributed among cytoplasmic structural and regulatory proteins, enzymes, nuclear and other proteins, and proteins of unknown function. Thirty clones (29%) had no significant database matches and thus potentially represent E. histolytica-specific genes. These data of E. histolytica genes identified by nucleotide sequencing indicate the value of the adoption of genome sequencing strategies for the rapid increase in knowledge of organisms causing dysentery and liver abscess.

Animals↗

PubMed1999

Expression of vasa(vas)-related genes in germline cells and totipotent somatic stem cells of planarians.

Planarians are known for their strong regenerative ability. This ability has been considered to reside in the totipotent somatic stem cell called the "neoblast." Neoblasts contain a unique cytoplasmic structure called the "chromatoid body," which has similar characteristics to the germline granules of germline cells of other animals. The chromatoid bodies decrease in number and size during cytodifferentiation and disappear in completely differentiated cells during regeneration. However, germ cells maintain the chromatoid body during their differentiation from neoblasts. These observations suggest that the chromatoid body is concerned with the totipotency of cells. To understand the molecular nature of the chromatoid body in the neoblast, we focused on vasa (vas)-related genes, since VAS and VAS-related proteins are known to be components of the germline granules in Drosophila and Caenorhabditis elegans. By PCR, two vas-related genes (Dugesia japonica vasa-like gene, DjvlgA and DjvlgB) were isolated, and they were shown to be expressed in germ cells. Interestingly, DjvlgA was also expressed in a number of somatic cells in the mesenchymal space. In regenerating planarians, accumulation of DjvlgA-expressing cells was observed in both the blastema and the blastema-proximal region. In X-ray-irradiated planarians, which had lost regenerative capacity, the number of DjvlgA-expressing cells decreased drastically. These results suggest that the product of DjvlgA may be a component of the chromatoid body and may be involved in the totipotency of the neoblast.

Amino Acid Sequence↗

PubMed1996

Localization of heat shock proteins in mouse male germ cells: an immunoelectron microscopical study.

We have examined the intracellular distribution of heat shock proteins HSP27 and HSP90 by means of specific antibodies and ultrastructural immunocytochemistry in the normal mouse testis as well as after heat shock. In the unstressed testis, these heat shock proteins are present in the cytoplasm and to a lesser extent in the nucleus throughout spermatogenesis. They do not show preferential association with any specific cytoplasmic structures and are absent from mitochondria. They disappear from the cell nucleus at the stage of elongating spermatids. After heat shock (42 degrees C), both HSP90 and HSP27 increase and partly relocate to the nucleus. Similarly to the localization in unstressed cells, they are mainly associated with perichromatin fibrils and the nucleolus. Moreover, a remarkable increase in the frequency of perichromatin fibrils in Sertoli cells, spermatogonia, and primary spermatocytes is also observed upon heat shock. Finally, a sharp increase in the labeling of HSPs in chromatoid bodies of round spermatids occurs following hyperthermic treatment. Interestingly, these two HSPs are localized on nuclear structures which are actively involved in RNA synthesis and processing, suggesting that they may have protective functions in these processes in a tissue which is particularly sensitive to heat stresses.

Animals↗

PubMed2001

Association of HSPB2, a member of the small heat shock protein family, with mitochondria.

We previously identified HSPB2, a new member of the small heat shock protein family, expressed in heart and skeletal muscles. In this study, we used a polyclonal anti-HSPB2 antibody and examined the subcellular localization of HSPB2 in differentiated C2C12 cells, KNS-81 cells, and NIH3T3 transfectants expressing human HSPB2. Double staining with anti-HSPB2 and various markers for cytoplasmic structures showed that HSPB2 was present in the cytosol as granules, some of which colocalized with mitochondria. This colocalization was not altered by a colchicine treatment, indicating that it is independent of microtubules. The subcellular fractionation of differentiated C2C12 cells revealed that HSPB2 was mainly detected in the postmitochondrial supernatant, but mild heat treatment enriched the amount of HSPB2 in the mitochondrial fraction. The expression of HSPB2 protected the cells from heat-induced cell death. In addition, Northern blot analysis revealed that expression of HSPB2 mRNA is higher in slow-twitch muscle than in fast-twitch muscle, which correlates with the amounts of mitochondria present in these two types of tissue. Taken together, these results suggest that HSPB2 may not localize in the matrix, but rather associates with the outer membrane components of the mitochondria and thus plays a role in the stress response.

Animals↗

PubMed2002

DN-cadherin is required for spatial arrangement of nerve terminals and ultrastructural organization of synapses.

We studied roles of DN-cadherin, the Drosophila major neuronal cadherin, in neuronal connections in the visual system. In DN-cadherin mutants, axon terminals of a large subset of photoreceptor cells reached and associated with their target interneurons, but their characteristic spatial arrangement was disrupted as synaptogenesis proceeded. Although synapses were formed at contact sites between the axon terminals and target neurons, underlying cytoplasmic structures were not fully specialized at both pre- and postsynaptic terminals and synaptic vesicles appeared to accumulate at the presynapses. These results suggest that the cadherin adhesion system is required for interaction between pre- and postsynaptic terminals and for generation of the mature synaptic structures.

Animals↗

PubMed1995

Identification of the rubella virus nonstructural proteins.

Five segments of the rubella virus (RUB) nonstructural protein open reading frame (NSP-ORF) were cloned into pATH (trpE) bacterial fusion protein expression plasmid vectors. Antisera raised in rabbits against these fusion proteins were used to identify RUB nonstructural polypeptides in lysates from RUB-infected Vero cells and from BHK cells transfected with pTM3/nsRUB, a vector from which the RUB NSP-ORF is expressed. In both systems, three polypeptides were immunoprecipitated. A 200-kDa polypeptide (P200) was immunoprecipitated by all of the antisera and therefore is the primary translation product of the ORF. A 150-kDa polypeptide (P150) was immunoprecipitated by antisera against fusion proteins containing N-terminal regions of the ORF, and a 90-kDa polypeptide (P90) was immunoprecipitated by sera against fusion proteins containing C-terminal regions of the ORF. The order of these polypeptides within the NSP-ORF is thus NH2-P150-P90-COOH. It was shown in a previous study that a protease within P200 catalyzes cleavage of P200 (L. D. Marr et al., Virology 198, 586-592, 1994). When hypertonic block was used to synchronize initiation of translation in RUB-infected cells, P200 was detected within 10 min following release of the block, while P150 and P90 were not detected until 20 min, indicating that translation of the precursor is completed before proteolytic cleavage occurs. Pulse-chase experiments showed that cleavage of the P200 precursor was complete within 90 min of synthesis. P150 was stable for 24 hr following processing, while turnover of P90 was detected within 90 min. In immunofluorescence experiments on RUB-infected cells, antisera that recognized P150 stained a perinuclear focus, a thread-like cytoplasmic structure, and the nuclear membrane.

Animals↗

PubMed1995

Biosynthesis and localization of the Autographa californica nuclear polyhedrosis virus 25K gene product.

Mutations of the AcMNPV 25K gene are associated with the "few polyhedra" phenotype (M. J. Fraser et al., 1983, J. Virol. 47, 287-300; B. Beames and M. D. Summers, 1989, Virology 168, 344-353). Polyclonal antisera was produced and used to investigate the time course of expression and localization of the 25K protein in infected cells. Western blot analysis detected 25K protein in both cytosolic and nuclear extracts from 18-24 hr p.i. through 96 hr p.i. and also in purified viral occlusions, but not in purified virions. Immunogold electron microscopy revealed that 25K protein was predominantly associated with amorphous cytoplasmic structures and to a lesser extent with a more electron-dense structure in the nucleus. Viral occlusions in cell sections were not specifically labeled by 25K antibody. Observations of purified viral occlusions and nuclei prepared for immunogold EM revealed the presence of contaminating amorphous material that was labeled with 25K antibody.

Animals↗

PubMed1975

Human and experimental reticulum cell sarcoma (microglioma) of the nervous system.

The histology and ultrastructure of primary central nervous system and metastatic (epidural) reticulum cell sarcoma is described. The tumors showed varying numbers of argyrophilic microglial cells which in all cases constituted a minority of all tumor cells. A primitive, undifferentiated cell, common to all tumors, was a small cell with a large dense nucleus and a narrow rim of poorly structured cytoplasm. These cells frequently showed small fatty inclusions and were tentatively identified as primitive multipotential cells from which all other tumor cells develop. It is concluded that reticulum cell sarcoma of the nervous system (primary or metastatic) and microglioma or microgliomatosis are one and the same tumor.

Adult↗

PubMed1983

Ultrastructural aspects of rapid-frozen, deep-etched and rotary-shadowed synaptosomes.

Isolated synaptic terminals were rapid-frozen with a liquid-propane jet, deep-etched, and rotary-shadowed replicas produced. The replicas show a three-dimensional view of the cytoplasmic components. Intraterminal mitochondria display a rough inner membrane with numerous stalked globular particles. Microfilaments connect the diverse organelles of the nerve terminal. The main cytoplasmic structure is a tubular membrane system, probably formed by the smooth endoplasmic reticulum (SER). Rapid-frozen synaptosomes reveal sparse profiles of synaptic vesicles in contrast to the current structural features shown by the techniques employing cryoprotectants and/or fixatives. The structural lability of these SER-profiles seems to be very high, to the extent that varying morphological features are observed in the same replica. It is proposed that the synaptic vesicles originate from cisternae of the SER, and that the synaptic vesicles and the SER are in close structural and functional relation. The morphological relationship of both structures to the cell membrane still remains unclear.

Animals↗

PubMed1978

The growth of follicles in the rat ovary under the influence of busulphan and endoxan.

Busulphan and Endoxan inhibit the normal course of ovarian follicular growth. Older secondary as well as an antral follicles perish within a very short time. Growing follicles, which at the beginning of the experiment exhibited only one layer, remained intact and single-layered during the entire duration of the experiment. The oocytes, however, continue growing, and the cytoplasmic structures, which are characteristic of older growing follicles, develop in them as well as in the follicle cells. Even a theca formation develops. In some of the "growing" follicles which have remained single-layered, after 10 days of Busulphan administration, some liquor folliculi is produced and accumulates in a fissure-shaped "antrum" between the zona pellucida and the follicular epithelium.

Animals↗

PubMed1979

A dense-cored filamentous body in Leydig cells of the golden hamster.

A unique cytoplasmic structure has been observed in Leydig cells of the golden hamster. It consists of a laminar core made up of electron dense material surrounded by a filamentous matrix of lower density, and is tentatively called a dense-cored filamentous body (DCFB). DCFBs vary in overall size and in configuration of the centrally disposed dense lamina. They are typically located in the vicinity of the centrosome and the Golgi complex. The body has no limiting membrane, and may be in contact with virtually every type of organelle. The DCFB is well developed in active Leydig cells, whereas it is small in the quiescent stage of the secretory cell. It is likely that the DCFB is a constant organelle in the hamster Leydig cell and may be involved in the physiological function of the Leydig cell, which remains to be specified.

Animals↗

PubMed1991

Immunohistochemical localization of basic fibroblast growth factor in A431 human epidermoid carcinoma cells.

The intracellular location of basic fibroblast growth factor (bFGF) was determined in A431 human epidermoid carcinoma cells both on immunofluorescence and on immunoelectron microscopy using a monoclonal anti-bFGF antibody. The immunofluorescence was located in the cytoplasm in quiescent cells. Following the addition of FCS to the culture medium of quiescent sparse cells the growth factor was translocated to and accumulated in the nucleolus. Immunogold particles were dense near the ribosomes, but were not recognized in the cytoplasmic structures concerned with the usual secretory pathway such as the rough endoplasmic reticulum, the Golgi apparatus, and secretory granules. These results suggest that endogenous bFGF undergoes intracellular sorting and enters the nucleoli in A431 cells according to an extracellular growth signal.

Carcinoma, Squamous Cell↗