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PubMed1986

A subset of human cord blood mononuclear cells is similar to Langerhans cells of the skin: a study with peanut agglutinin and monoclonal antibodies.

Mononuclear cells were fractionated from human cord blood by affinity chromatography on immobilized peanut agglutinin, as previously described (Rosenberg et al., Hum Immunol 7:67, 1983). The PNA+ subset was found to be composed mainly of a population of cells phenotyped as Ia+, T6+, M01+, and MY4+. The presence of mononuclear cells coexpressing these antigens was demonstrated by three techniques: double labeling immunofluorescence using FITC and rhodamine conjugated goat antimouse IgG; fluorescence activated cell sorter (FACS); and by direct counting (under the microscope) of cells stained by either individual or a combination of a variety of monoclonal antibodies. The PNA+ cells expressed cytoplasmic structures similar to Birbeck granules. In view of the fact that Langerhans cells of the skin share a similar phenotype and express Birbeck granules, we suggest that this subset may be the precursor of the Langerhans cells of the skin. In addition, these cells may also be the precursors of the dendritic cells found in the spleen, lymph nodes, thymus, and liver.

Antibodies, Monoclonal↗
PubMed1980

Chemical characterization, synthesis and distribution of proteinase inhibitor in newborn rat epidermis.

A protein solubilized in Tris-HCl/saline buffer from keratinized cells of newborn rat epidermis exhibited inhibitor activity to papain and ficin, but not to trypsin, cathepsin D and pepsin. This protein was purified from keratinized cells as well as nonkeratinized and germinative cells by means of IgG affinity chromatography. The inhibitors extracted from all cell layers were immunologically identical and had a molecular weight of approximately 12,500 +/- 500. Since amino acid analysis showed that the inhibitor contains about 35 residues of glycine per mol, [3H]glycine was used to investigate synthesis of the protein. The inhibitor from nonkeratinized and germinative cells was radioactively labeled by 2 h after injection and appeared in keratinized cells by 48 h after injection. Indirect immunofluorescence microscopy demonstrated in situ distribution of the protein in the entire epidermis, and the protein localized by the plasma membrane in granular cells and diffusely in keratinized cells was shown to be insoluble in Tris-HCl saline buffer. The results indicate that a thiol-proteinase inhibitor is synthesized in epidermal cells during keratinization and is retained as part of the cytoplasmic structure

Amino Acids↗
PubMed1981

Ca2+-dependent cross-linking processes in human platelets.

When platelet cytoplasmic Ca2+ is increased by the ionophore A23187 in the presence of the protease inhibitor leupeptin, there is the coincident appearance of a cross-linked polymer and the partial disappearance of monomeric protein and glycoprotein units. In the absence of leupeptin only 30% of the polymer was formed. The disappearance of monomeric protein bands, as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is prevented by histamine, which as a pseudodonor amine is a known inhibitor of transglutaminase-catalyzed cross-linking [14C]Histamine, at a tracer concentration, is incorporated into the polymer as well as into myosin, glycoproteins IIb and III, actin and tropomyosin. The loss of monomeric protein bands is mostly due to their conversion into polymers. Control measurements show that leupeptin effectively inhibited platelet Ca2+-dependent proteases. The cross-linking processes bringing about the observed increase in polymer formation are thus the result of a Ca2+-dependent platelet transglutaminase activity. The latter is located in the platelet cytosol and has been identified as platelet factor XIII on the basis of its specific cross-linking of fibrin. Platelet factor XIII, upon activation, may function physiologically to couple membrane proteins to cytoplasmic structural proteins. Thus, a new concept is proposed for the stabilization of platelet membranes and platelets as they form the hemostatic plug.

Blood Platelets↗
PubMed1995

Preovulatory overripeness of the oocyte as a cause of ovarian dysfunction in the human female.

A variety of gynaecological phenomena such as menstrual disorders, infertility and premature menopause are associated with ovarian dysfunction. In this paper, a factor is considered that may play a role in the aetiology of ovarian dysfunction. Animal research has shown that preovulatory overripeness of the oocyte may cause various developmental and chromosomal anomalies. Among the developmental anomalies observed in amphibia and fish are a number of gonadal aberrations, often occurring in otherwise normal specimens. This may be the consequence of a degeneration of the 'germinal cytoplasm', cytoplasmic structures located in the vegetative pole region of the oocyte, destined to become part of future primordial germ cells. Indirect evidence suggests that preovulatory overripeness of the oocyte also plays a significant role in human reproductive failure. An increased risk of preovulatory overripeness is expected during periods of endocrine irregularities, such as the first postmenarcheal years, the last premenopausal years, the first months after a pregnancy and, possibly, during certain seasons. If the overripeness-induced gonadal maldevelopment observed in animals also occurs in humans, this may explain variations in reproductive function according to month of birth, as observed in women. It is hypothesized that females conceived during periods of maternal endocrine irregularities face an increased risk of ovarian dysfunction through overripeness-induced gonadal maldevelopment.

Amphibians↗
PubMed1996

Testosterone effects on the neuronal ultrastructure in the medial preoptic nucleus of male Japanese quail.

Dorsolateral neurons of the medial preoptic nucleus (POM) of male Japanese quail are sensitive to the plasma levels of testosterone: their volume and optical density in Nissl-stained sections increase in castrated birds treated with testosterone. The present study was performed on castrated male quail treated or not with Silastic implants filled with testosterone to describe the ultrastructural variations induced by testosterone in these neurons. Gonadally intact male birds were included as controls. The ultrastructure of neurons, taken from the dorsolateral portion of the POM, was dramatically affected by the endocrine manipulations. Quantitative evaluations demonstrated a significant decrease in castrated birds of the rough endoplasmic reticulum (RER), of free polyribosomes, of Golgi complexes, and of dense bodies; these changes paralleled the decrease in cell size. The cell size and the percentage of volume occupied by the intracellular organelles in castrated birds treated with testosterone were comparable to values observed in controls. These ultrastructural changes are similar to those observed in neuronal targets for other gonadal hormones, supporting the idea that testosterone stimulates the development of cytoplasmic structures involved in protein synthesis and secretion. In addition, exposure to testosterone affects the synaptic inputs to POM. These ultrastructural changes are presumably related to the physiological effects (e.g., activation of male sexual behavior) exerted by testosterone on this preoptic region.

Analysis of Variance↗
PubMed1995

Identification of new Schistosoma mansoni genes by the EST strategy using a directional cDNA library.

A directional size-selected cDNA library constructed from Schistosoma mansoni (Sm) adult worm RNA was used for the generation of expressed sequence tags (EST). From one or both ends of 429 distinct cDNA clones 607 EST were obtained. Of these, only 16% were previously known Sm genes. More than 22% of the clones had matches with entries for other organisms in the databases. These new Sm genes constituted a broad range of transcripts distributed among cytoplasmic structural and regulatory proteins, enzymes, membrane, nuclear and secretory proteins, and proteins with other functions. Almost 33% of the clones had no significant database matches and thus potentially represent Sm-specific genes. Among the latter, several clones, as judged by their redundancy in the library, appear to represent abundant transcripts. The data, taken as a whole, more than double the number of Sm genes identified by nucleotide sequencing and indicate the potential value of the adoption of genome sequencing strategies for the rapid increase in knowledge of complex disease-causing organisms.

Animals↗
PubMed1992

Positional information in cells and organisms.

The processes of pattern formation are usually considered to be quite different in unicellular and multicellular organisms. The only unifying ideas have been very general, such as those concerning regional differences and organization along a polar axis. Concepts like induction, fields and gradients have generally been applied only to the development of multicellular organisms. Here, Joseph Frankel suggests that pattern formation by multicellular organisms evolved in their progenitors in response to multiplication of cytoplasmic structural units rather than of nuclei. Ciliates provide a living example of complex patterning in a compound uninucleate organism.

Journal Article↗
PubMed2005

Centrosome inheritance in the male germ line of Drosophila requires hu-li tai-shao function.

Cytokinesis partitions a centrosome to each daughter cell at cell division that will duplicate and assemble a bipolar spindle in the subsequent M phase. Cytokinesis is incomplete in proliferating germ cells in Drosophila and cytoplasmic channels connect sibling germ cells. Although centrosomes are essential to male fertility, the molecular mechanism that retains centrosomes in parental germ cells is not known. Cortical cytoplasmic structures known as fusomes extend through ring canals and connect cells within the cyst. Fusome assembly in males requires function of hu-li tai-shao (hts), an adducin like protein found in fusomes and in the cortical membrane cytoskeleton of somatic cells. This work used immunological and cytological methods to place hts mutants in an allelic series. Male fertile hts mutants express hts protein and generate apparently normal or fragmented fusomes. A male sterile allele does not express hts protein or show fusome structures. Gonial cells in all hts mutants showed 2 centrosomes and mitotic spindles were bipolar. Yet, primary spermatocytes, with and without fusome structures, frequently contained too many or too few centrosomes. Although spindle structures were not found in spermatocytes without centrosomes, meiotic spermatocytes with centrosomes generated bipolar, monopolar, and multipolar spindles. Collectively, these results indicate that hts function is necessary for centrosome inheritance in spermatocytes as well as for male fertility.

Animals↗
PubMed2004

Multiple cell death programs: Charon's lifts to Hades.

Cells use different pathways for active self-destruction as reflected by different morphology: while in apoptosis (or "type I") nuclear fragmentation associated with cytoplasmic condensation but preservation of organelles is predominant, autophagic degradation of cytoplasmic structures preceding nuclear collapse is a characteristic of a second type of programmed cell death (PCD). The diverse morphologies can be attributed--at least to some extent--to distinct biochemical and molecular events (e.g. caspase-dependent and -independent death programs; DAP-kinase activity, Ras-expression). However, apoptosis and autophagic PCD are not mutually exclusive phenomena. Rather, diverse PCD programs emerged during evolution, the conservation of which apparently allows cells a flexible response to environmental changes, either physiological or pathological.

Apoptosis↗
PubMed2006

Association of PAT proteins with lipid storage droplets in term fetal membranes.

As depots for neutral lipids, lipid storage droplets (LDs) accumulate with advancing gestation within the fetal membranes. Little is currently known about the proteins associated with the LDs of these cells. The PAT family [perilipin, adipose differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47)] represents a unique group of proteins thought to contribute to LD formation and function. We examined the association of each of the PAT proteins with LDs of term fetal membranes. We found that large LDs of amnion epithelial cells were reactive for neutral lipid stains and simultaneously encoated with ADRP and TIP47, but not perilipin. Within the remaining cell types, LDs were frequently co-labeled with antibodies recognizing ADRP and TIP47; however, in cells harboring only small LDs, the majority of TIP47 labeling was cytoplasmic. Structures labeled with perilipin antibodies were present only in chorion laeve trophoblasts. Gene and protein expression analyses suggested this to be a small molecular weight perilipin isoform, such as that seen in steroidogenic cells. We conclude that LDs are heterogeneous among differing cell types of the fetal membranes. Subclassification of LDs based on associated proteins suggests that these organelles may serve specialized functions within individual cells.

3T3 Cells↗
PubMed2006

Tudor-related proteins TDRD1/MTR-1, TDRD6 and TDRD7/TRAP: domain composition, intracellular localization, and function in male germ cells in mice.

The germ-line cells of many animals possess a characteristic cytoplasmic structure termed nuage or germinal granules. In mice, nuage that is prominent in postnatal male germ cells is also called intermitochondrial cement or chromatoid bodies. TDRD1/MTR-1, which contains Tudor domain repeats, is a specific component of the mouse nuage, analogously to Drosophila Tudor, a constituent of polar granules/nuage in oocytes and embryos. We show that TDRD6 and TDRD7/TRAP, which also contain multiple Tudor domains, specifically localize to nuage and form a ribonucleoprotein complex together with TDRD1/MTR-1. The characteristic co-localization of TDRD1, 6 and 7 was disrupted in a mutant of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), which encodes another evolutionarily conserved component of nuage. In vivo over-expression experiments of the TDRD proteins and truncated forms during male germ cell differentiation showed that a single Tudor domain is a structural unit that localizes or accumulates to nuage, but the expression of the truncated, putative dominant negative forms is detrimental to meiotic spermatocytes. These results indicate that the Tudor-related proteins, which contain multiple repeats of the Tudor domain, constitute an evolutionarily conserved class of nuage components in the germ-line, and their localization or accumulation to nuage is likely conferred by a Tudor domain structure and downstream of Mvh, while the characteristic repeated architecture of the domain is functionally essential for the differentiation of germ cells.

Amino Acid Sequence↗
PubMed2000

The proapoptotic BH3-only protein bim is expressed in hematopoietic, epithelial, neuronal, and germ cells.

Proapoptotic Bcl-2 family members activate cell death by neutralizing their anti-apoptotic relatives, which in turn maintain cell viability by regulating the activation of the cell death effectors, the caspases. Bim belongs to a distinct subgroup of proapoptotic proteins that only resemble other Bcl-2 family members within the short BH3 domain. Gene targeting experiments in mice have shown that Bim is essential for the execution of some but not all apoptotic stimuli, for hematopoietic cell homeostasis, and as a barrier against autoimmunity. There are three Bim isoforms, Bim(S), Bim(L), and Bim(EL), which have different proapoptotic potencies due at least in part to differences in interaction with the dynein motor complex. The expression pattern of Bim was investigated by immunohistochemical staining, immunoprecipitation followed by Western blotting, and in situ hybridization. Bim was found in hematopoietic, epithelial, neuronal, and germ cells. Bim(L) and Bim(EL) were coexpressed at similar levels in many cell types, but Bim(S) was not detected. Microscopic examination revealed a punctate pattern of Bim(L) and Bim(EL) immunostaining, indicating association with cytoplasmic structures. These results are discussed in the context of the phenotype of Bim-deficient mice and the post-translational regulation of Bim's pro-apoptotic activity.

3T3 Cells↗
PubMed2001

Effects of intermediate filaments on actin-based motility of Listeria monocytogenes.

How does subcellular architecture influence the intracellular movements of large organelles and macromolecular assemblies? To investigate the effects of mechanical changes in cytoplasmic structure on intracellular motility, we have characterized the actin-based motility of the intracellular bacterial pathogen Listeria monocytogenes in normal mouse fibroblasts and in fibroblasts lacking intermediate filaments. The apparent diffusion coefficient of L. monocytogenes was two-fold greater in vimentin-null fibroblasts than in wild-type fibroblasts, indicating that intermediate filaments significantly restrict the Brownian motion of bacteria. However, the mean speed of L. monocytogenes actin-based motility was statistically identical in vimentin-null and wild-type cells. Thus, environmental drag is not rate limiting for bacterial motility. Analysis of the temporal variations in speed measurements indicated that bacteria in vimentin-null cells displayed larger fluctuations in speed than did trajectories in wild-type cells. Similarly, the presence of the vimentin meshwork influenced the turning behavior of the bacteria; in the vimentin-null cells, bacteria made sharper turns than they did in wild-type cells. Taken together, these results suggest that a network of intermediate filaments constrains bacterial movement and operates over distances of several microns to reduce fluctuations in motile behavior.

Actins↗
PubMed1992

Identification and characterization of a new set of nucleolar ribonucleoproteins which line the chromosomes during mitosis.

We investigated the perichromosomal architecture established during mitosis. Entry into mitosis brings about a dramatic reorganization of both nuclear and cytoplasmic structures in preparation for cell division. While the nuclear envelope breaks down, nuclear proteins are redistributed during chromosome condensation. Some of these proteins are found around the chromosomes, but little is known concerning their nature and function. Ten autoimmune sera were used to study the microenvironment of chromosomes and, in particular, the chromosome periphery. They were selected for their anti-nucleolar specificity and were found to recognize three nucleolar proteins that coat the chromosomes during mitosis. The distribution of these antigens was followed through the cell cycle by confocal laser scanning microscopy. The antigens dispersed very early during prophase and simultaneously with the chromosome condensation suggesting a correlation between these two processes. The antigens have apparent molecular weights of 53, 66, and 103 kDa on SDS-PAGE migration. Elution of the antibodies and immunopurification showed that they are RNA-associated proteins. The coimmunoprecipitating RNA moiety involved in these RNPs appeared to be U3, but the antigens are not related to the fibrillarin family. Therefore, small nucleolar RNPs follow the same distribution during mitosis as that described for small nuclear RNPs. Possible functions for these antigens are discussed.

Antibodies, Antinuclear↗
PubMed1978

Ultrastructure of perineurial cells during peripheral nerve regeneration. Electron microscopical investigations on the so-called amputation neuroma.

The ultrastructural morphology of perineurial cells was examined after transsection of the sciatic nerve of rats. The material prepared for investigation was taken from the proximal nerve stump and the so-called amputation neuroma at different times after operation. We found that perineurial cells begin to activate in the first week, and in the further course of regeneration they modulate their cytoplasmic structure corresponding to the function of proliferation or synthesis. Besides the reconstitution of a diffusion barrier the perineurial cells may exert a stabilizing function in regenerating nerve tissue comparable to myofibroblasts in granulation tissue, and may be engaged in remodeling of connective tissue fibers. The relations of perineurial cells to other cells of peripheral nerves under the condition of regeneration are discussed.

Animals↗
PubMed1999

Cytokeratin subsets can reliably distinguish Barrett's esophagus from intestinal metaplasia of the stomach.

The histological distinction between intestinal metaplasia involving the distal esophagus (Barrett's esophagus [BE]) and intestinal metaplasia of the stomach has important clinical implications and can be difficult even with the use of histochemical mucin stains. Cytokeratin (CK) 7 and 20 are cytoplasmic structural proteins that show restricted expression in normal and malignant epithelia of the gastrointestinal tract. The aim of this study was to determine the use of CK7 and 20 expression in the histological distinction of BE from gastric intestinal metaplasia. CK7 and 20 immunostaining was performed on randomly selected surgical resection (n = 31) and biopsy specimens (n = 34) from patients with long-segment BE and gastric resection specimens (n = 11) and gastric cardia biopsy specimens (n = 13) in patients with histological evidence of intestinal metaplasia. A unique pattern of immunoreactivity designated the Barrett's CK7/20 pattern showed superficial CK20 staining and strong CK7 staining of both superficial and deep glands in 29 of 31 (94%) esophageal resection specimens and 34 of 34 (100%) esophageal biopsy specimens form patients with long-segment BE. A Barrett's CK7/20 pattern was not observed in gastric cardia biopsy specimens (n = 13) or gastric resection specimens (n = 11) in patients with histological evidence of intestinal metaplasia. The sensitivity, specificity, and positive predictive value of a Barrett's CK7/20 pattern for a diagnosis of long-segment BE was 97%, 100%, and 100%, respectively. CK7 and 20 reactivity patterns can reliably identify the location of intestinal metaplasia in the esophagus and stomach using histological material from both routine endoscopic biopsy and surgical resection specimens.

Barrett Esophagus↗
PubMed1997

Intracellular localization of inducible nitric oxide synthase in neonatal rat cardiomyocytes in culture.

Recognition of the role of nitric oxide (NO) in cardiovascular regulations raised an acute interest in NO-generating enzymes-nitric oxide synthases (NOS). Nevertheless, the subcellular localization of inducible isoform of NOS (NOS II) and regulation of its expression in the cardiomyocyte still remains to be elucidated. Therefore, we focused this study on the subcellular localization of NOS II in cultured neonatal rat cardiomyocytes using immunocytochemical techniques at the light and electron microscopic level as well as the demonstration of NADPH-diaphorase activity and the Griess assay for NO measurement. Cultivation of neonatal cardiomyocytes during 2 and more days induced a moderate increase in the NOS II immunolabeling in defined cytoplasmic structures and a nuclear NOS II staining in some cells. Exposure of the cell cultures to exogenous cAMP markedly stimulated NO production with a concomitant enhancement of NOS II immunolabeling of cardiomyocytes. cAMP-induced changes were significantly attenuated by dexamethasone. This report provides evidence for the localization of NOS II in the perinuclear space, Golgi complex, mitochondria, plasma membrane and along contractile fibers of cardiomyocytes, as well as for the appearance of NOS II staining of the cell nuclei in the course of cultivation. In non-cardiomyocytes contaminating the cell culture, positive immunoreaction was detected in the Golgi complex and endoplasmic reticulum. Our data point to a notable constitutive expression of NOS II in rat cardiomyocytes apparently dependent on the developmental stage.

Animals↗