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PubMed1985

Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener's granulomatosis.

Immunoglobulin G (IgG) autoantibodies against extranuclear components of polymorphonuclear granulocytes were detected in 25 of 27 serum samples from patients with active Wegener's granulomatosis and in only 4 of 32 samples from patients without signs of disease activity. In a prospective study of 19 patients these antibodies proved to be better markers of disease activity than several other laboratory measurements used previously. The autoantibodies were disease specific and the titres were related to the results of an in-vitro granulocyte phagocytosis test, in which 7S IgG antibodies were internalised after specific binding to the cell, resulting in gradual formation of ring-like cytoplasmic structures. This autoantibody may have a pathogenetic role in Wegener's granulomatosis. The detection of this antibody is valuable for diagnosis and estimation of disease activity.

Adult↗

PubMed1998

A Plasmodium falciparum aminopeptidase gene belonging to the M1 family of zinc-metallopeptidases is expressed in erythrocytic stages.

A new single copy gene has been isolated from Plasmodium falciparum, by immunoscreening a genomic DNA expression library. The gene appears devoid of introns, displays the classical A + T richness and codon usage of P. falciparum genes, and is transcribed into a 4 kb mRNA in erythrocytic stages. The deduced amino acid sequence corresponds to a 1056 residue protein (122 kDa) containing the canonical HExxHx18E signature of zinc-metallopeptidase active sites of the M1 family at position 467-490, a downstream conserved tyrosine residue involved in catalysis in position 551, and the GAMEN conserved motif characteristic of aminopeptidases in the M1 family, at position 431-435. The greatest similarities were found with aminopeptidases N of Escherichia coli and Haemophilius influenza (more than 80% identical residues in the canonical signature of the active site) but significant similarities centred on the active site region exist with all other members of the M1 family such as other prokaryotic aminopeptidases, eukaryotic aminopeptidases A and N and leukotriene A4 hydrolases (40-50% identical residues in the canonical signature of the active site). A polyclonal serum raised to a synthetic peptide deduced from the gene labelled schizont proteins of 96 and 68 kDa purified to homogeneity and both displaying aminopeptidase activity, as well as cytoplasmic structures in schizont stages.

Amino Acid Sequence↗

PubMed2002

Whatever happened to the 'microtrabecular concept'?

Keith Porter culminated his stellar career as the founding father of biological electron microscopy by acquiring, in the late 1970s, a high-voltage electron microscope (HVEM). With this magnificent instrument he examined whole-mounts of cultured cells, and perceived within them a structured cytoplasmic matrix he named the "microtrabecular lattice". Over the next decade Porter published a series of studies, together with a team of outstanding young colleagues, which elaborated his broader "microtrabecular concept." This concept posited that microtrabeculae were real physical entities that represented the fundamental organization the cytoplasm, and that they were the physical basis of cytoplasmic motility and of cell-shape determination. The present review presents Porter's original images of microtrabeculae, after conversion to a more interpretable "digital-anaglyph" form, and discusses the rise and fall of the microtrabecular concept. Further, it explains how the HVEM images of microtrabeculae finally came to be considered as an artifact of the preparative methods Porter used to prepare whole cells for HVEM. Still, Keith's "microtrabecular concept" foretold of our current appreciation of the complexity and pervasiveness of the cytoskeleton, which has now been found by more modern methods of EM to actually be the fundamental organizing principle of the cytoplasmic matrix. During the impending eclipse of Porter's microtrabecular concept in the late 1980s, many of Keith's colleagues fondly described the cell as being filled, not with protoplasm, but with "Porterplasm." Despite the fact that Keith's view was clouded by the methods of his time, it would be fitting and apt to retain this name, still today, for the ordered matrix of cytoskeletal macromolecules that exists in the living cell. In the end, the story of what happened to Porter's microtrabecular concept should be an object lesson in scientific hubris that should humble and inform all of us in cell biology, even today--particularly when we begin to think that our most recent methods and observations are achieving "the last word".

Animals↗

PubMed1997

Slowly triggered excitotoxicity occurs by necrosis in cortical cultures.

This study examined the possibility that the excitotoxin-induced death of cultured cortical neurons might occur by apoptosis, specifically focusing on the slowly triggered death induced by low concentrations of excitotoxin. Cultured murine cortical neurons (days in vitro 10-12) were exposed continuously to N-methyl-D-aspartate (10-15 microM), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (3-100 microM) or kainate (30-60 microM) over 24 h. Within 2 h of exposure onset, neuronal cell body swelling was visible under phase-contrast optics. At this point, transmission electron microscopy revealed disruption of cell membranes and organelles, mitochondrial swelling and scattered chromatin condensation at the periphery of nuclei. By 8 h after exposure onset, many neurons were devoid of cytoplasmic structures, but nuclear membranes remained relatively intact. This excitotoxic degeneration was not blocked by the protein synthesis inhibitor, cycloheximide, or the growth factors, brain-derived neurotrophic factor or insulin-like growth factor-1, agents that did block serum deprivation-induced apoptosis death in other cultures. DNA agarose gel electrophoresis, however, revealed the transient occurrence of internucleosomal DNA fragmentation, appearing 4-8 h after exposure onset, but absent 24 h after exposure onset. The present results suggest that even slowly triggered excitotoxicity occurs by necrosis, and raise a cautionary note in interpreting internucleosomal DNA fragmentation in isolation as evidence for apoptosis.

Animals↗

PubMed1998

Intermediate filaments and their associated proteins: multiple dynamic personalities.

A fusion of mouse and human genetics has now proven that intermediate filaments form a flexible scaffold essential for structuring cytoplasm in a variety of cell contexts. In some cases, the formation of this scaffold is achieved through a newly identified family of intermediate-filament-associated proteins that form cross-bridges between intermediate filaments and other cytoskeletal elements, including actin and microtubules.

Animals↗

PubMed2002

Tea3p is a cell end marker activating polarized growth in Schizosaccharomyces pombe.

Eukaryotic cells are often polarized in their cytoplasmic structures, and this can be important for their function. The fission yeast Schizosaccharomyces pombe is a highly polarized cell that extends bipolarly along a single axis to generate a rod-shaped cell. It divides by medial fission to generate two equal-sized daughter cells that resume growth only at the old end. Once these cells have reached a particular length, they undergo NETO, new end take-off, whereby growth is activated at the other end to generate bipolarly extending cells. The activation and positioning of these growth zones are essential for maintaining growth in a straight line. Genetic analyses have identified many proteins involved in this process, like the cell end markers Tea1p and Pom1p and the kinases Orb2p/Shk1p/Pak1, Ssp1p, and Wee1p. Here, we describe tea3, a gene encoding a tea1-like protein with some similarities to ERM proteins. Tea3p is required for efficient NETO and for the proper placement of the septum. Like Pom1p, Tea3p localizes to cell ends, and its localization depends on microtubules and Tea1p. We propose that Tea3p is a novel cell end marker required specifically to activate polarized cell growth at the second end during NETO.

Cell Division↗

PubMed2003

Chaperonin-mediated protein folding: fate of substrate polypeptide.

Chaperonins are megadalton ring assemblies that mediate essential ATP-dependent assistance of protein folding to the native state in a variety of cellular compartments, including the mitochondrial matrix, the eukaryotic cytosol, and the bacterial cytoplasm. Structural studies of the bacterial chaperonin, GroEL, both alone and in complex with its co-chaperonin, GroES, have resolved the states of chaperonin that bind and fold non-native polypeptides. Functional studies have resolved the action of ATP binding and hydrolysis in driving the GroEL-GroES machine through its folding-active and binding-active states, respectively. Yet the exact fate of substrate polypeptide during these steps is only poorly understood. For example, while binding involves multivalent interactions between hydrophobic side-chains facing the central cavity of GroEL and exposed hydrophobic surfaces of the non-native protein, the structure of any polypeptide substrate while bound to GroEL remains unknown. It is also unclear whether binding to an open GroEL ring is accompanied by structural changes in the non-native substrate, in particular whether there is an unfolding action. As a polypeptide-bound ring becomes associated with GroES, do the large rigid-body movements of the GroEL apical domains serve as another source of a potential unfolding action? Regarding the encapsulated folding-active state, how does the central cavity itself influence the folding trajectory of a substrate? Finally, how do GroEL and GroES serve, as recently recognized, to assist the folding of substrates too large to be encapsulated inside the machine? Here, such questions are addressed with the findings available to date, and means of further resolving the states of chaperonin-associated polypeptide are discussed.

Adenosine Triphosphate↗

PubMed2004

Characteristics of invasive cells found in between zona pellucida and oocyte during follicular atresia in mice.

During the process of follicular atresia, cells are observed to invade the zona pellucida (invasive cells) where they presumably play an important role in eliminating degraded oocytes. Although our preliminary studies have suggested that these cells may originate from granulosa cells and not from macrophages, a detailed morphological analysis of the cells has not been conducted. The objective of this study was to characterize the cells more precisely by electron microscopy and immunohistochemistry, using sexually immature mice. The results show that the invasive cells were first observed within advanced primary (non-antral) atretic follicles. The cells frequently contained cytoplasmic lysosome-like granules after passing through the zona pellucida. F4/80 and Mac-1, reported as macrophage-specific antibodies, were reactive with the cells in most cases, but some immunonegative invasive cells were also observed. The ultrastructural features of the invasive cells were quite similar to those of granulosa cells, not macrophages. Gap junctions, which are typical cytoplasmic structures of epithelial cells, were frequently identified between neighbouring cells. Although direct evidence indicating a contribution by the cells to the elimination of degenerated oocytes was not obtained, our results strongly suggest that the invasive cells originated from granulosa cells surrounding the zona pellucida, and that they may have a macrophage-like cell function for the elimination of oocytes from atretic follicles in mice.

Animals↗

PubMed2001

Effects of ethanol administration on hepatocellular ultrastructure of regenerating liver induced by partial hepatectomy.

Acute ethanol administration partially inhibits DNA and protein syntheses during liver regeneration (LR) induced by partial hepatectomy (PH) in rats. Previous findings that the magnitude of ethanol's deleterious effects on LR are related to the route and timing of its administration led us to perform studies at the ultrastructural level, comparing ethanol effects on PH-induced LR, as a consequence of its administration route. PH promoted alterations on the endoplasmic reticulum and mitochondria, accompanied by decreased glycogen and increased lipid content in cytoplasm. Structural nuclear and nucleolar activities were also evident. Intragastric ethanol administration practically abolished the adaptative changes found in PH-promoted regenerating hepatocytes, whereas its administration through the intraperitoneal route induced later ultrastructural modifications, indicating cellular proliferation. These results suggest that ethanol, under certain conditions, could stimulate liver proliferation triggered by PH. The mechanism underlying this surprising effect of ethanol on LR remains to be elucidated. However, it is suggested that an altered ethanol metabolism by rats subjected to PH could be involved.

Alcoholism↗

PubMedDate not supplied

Axonal protein synthesis and transport.

Recent evidence has challenged our ideas about the nature of axonal protein synthesis and transport. Previous metabolic labeling evidence supported the idea that all axonal proteins were synthesized in the cell body and then transported as formed cytoplasmic structures into the axon. Recent evidence suggests that neither the synthesis nor the transport of axonal proteins is that simple. Though most axonal proteins do appear to be synthesized in the neuronal cell body, a small amount of protein appears to be synthesized intra-axonally in some axons. Though small in amount, intra-axonal protein synthesis may be important functionally in some axons. Recent experiments have also begun to identify the presence of a rich array of transport motors in axons, including many members of the kinesin, dynein and myosin families. Progress is being made in identifying which cargoes are being transported by which of these motors. Finally, recent experiments have addressed an old question about whether axoplasmic proteins are transported as filamentous polymers or as soluble components in axons. The answer is that both mechanism can be used in axons. For example, neurofilament protein can move in its particulate or polymeric state, while tubulin can move in its soluble or unpolymerized state.

Axonal Transport↗

PubMed1979

Immunoreactive forms of human erythrocyte ankyrin are present in diverse cells and tissues.

Ankyrin is a polypeptide of molecular weight (MW) 200,000 which is tightly bound to the cytoplasmic surface of the human erythrocyte membrane and has been identified as the high-affinity membrane attachment protein for spectrin. This protein has also been shown to be associated with band 3 (ref. 4), the major transmembrane protein which links a cytoplasmic structural protein to an integral membrane protein. A water-soluble, 72,000-MW, proteolytic fragment of ankyrin has been purified which retains the ability to bind to spectrin, and competitively inhibits reassociation of spectrin with membranes. Monospecific antibodies directed against this fragment have been prepared and demonstrated to cross-react only with ankyrin among the erythrocyte membrane proteins. The present study reports the use of these antibodies to develop a radioimmunoassay capable of detecting femtomolar quantities of ankyrin, and demonstrates the presence of small but significant amounts of immunoreactivity in a variety of types of cells and tissues.

Blood Platelets↗

PubMed1987

Age-related retinal vasculopathy.

Second and third order retinal arterioles and venules were studied by immunohistochemistry and electron microscopy in the uncomplicated ageing process and in association with central retinal vascular occlusive disease (CRVOD). Tissue was obtained from eight enucleated human eyes (4 control, 4 with manifestations of CRVOD) and the investigation was directed towards abnormalities in the myocytes and the nature of collagenous materials which are deposited during the process of hyalinization. In normal ageing and in two cases of CRVOD, the endothelial cell monolayer and the underlying cells (subendothelial myocytes) were preserved in arterioles and venules: there was no evidence of fibrin leakage. Degenerative changes were found in the medial myocytes in control tissue from the fifth decade and these included myocyte shrinkage, accumulation of intracytoplasmic membranous structures, cytoplasmic vacuolation and fragmentation. The 'hyalinized' acellular vessel wall seen in CRVOD contains scattered activated fibroblast-like myocytes and macrophages lying within a matrix of fibronectin, 65 nm collagen and multilayered basement membrane material. Endothelial cells and cohort subendothelial myocytes are involved in the formation of capillaries which bud into the hyalinized vessel wall in CRVOD. In two cases of CRVOD there was extensive cellular degeneration and cell debris accumulated within the degenerate stroma: this was attributed to superadded total ischaemia. The pathogenesis of hyalinization remains obscure but a subtle age-related dysfunction of the morphologically intact lining endothelium could be responsible for metabolic damage to myocytes in the media.

Aged↗

PubMed1991

Participation of cytoplasmic organelles in E-rosette formation.

To elucidate the mechanism of E-rosette formation between T cells and sheep red blood cells (SRBC), the effect of various agents affecting the function of cytoplasmic structures (microtubules and microfilaments) and organelles (mitochondria and rough endoplasmic reticulum) was investigated. E-rosette formation was greatly inhibited by agents that block either the energy producing system (KCN and NaN3) or the integration of microfilaments (cytochalasin B, dihydrocytochalasin B and cytochalasin D). On the other hand, there was little or no suppression by either inhibitors of protein synthesis (cycloheximide and puromycin), agents that block the polymerization of microtubules (colchicine, podophyllotoxin and vinblastine), or chemicals disconnecting surface membrane proteins from the intracellular structural proteins (chlorpromazine, dibucaine, hydrocortisone, and propranolol). The calcium ionophore A23187, which transports Ca2+ into the cytosol, inhibited the E-rosette formation in the presence of Ca2+, but not in its absence. From these results, we concluded that new synthesis of ATP and the structural integration of microfilaments are indispensable for the E-rosette formation, which is triggered by an interaction between the ligand (T11TS) and its corresponding receptor (CD2). A certain level of intracellular Ca2+ is also involved in the E-rosette formation.

Actin Cytoskeleton↗

PubMed2000

Inhibition of cell proliferation in HCC-9204 hepatoma cells by a c-myc specific ribozyme.

A ribozyme (RZ) gene targeting c-myc mRNA was synthesized and cloned. Cleavage reaction showed that cleavage of the RZ was efficient and specific. The RZ gene-containing retrovirus vector pDOR-RZ was transfected into HCC-9204 hepatoma cells, which constitutively express high levels of c-myc using Lipofectamine. Positively transfected cells were selected using G418. In situ hybridization showed that both pDOR-RZ and pDOR vectors had been integrated into the chromosome of HCC-9204 cells. Dot blot hybridization indicated that expression of the RZ was only evident in pDOR-RZ-transfected HCC-9204 cells. Avidin-biotin complex enzyme-linked immunosorbent assay showed that c-myc expression was down-regulated. Chromatin aggregation into compact masses, cytoplasmic vacuole degeneration, and blurring of cytoplasm structure were observed by transmission electron microscopy in HCC-9204-RZ cells. These results suggest that the use of a c-myc mRNA cleaving enzyme could be most effective in tumor cells that are highly proliferative and constitutively express high levels of c-myc.

Carcinoma, Hepatocellular↗

PubMed2003

Mammalian NADH diphosphatases of the Nudix family: cloning and characterization of the human peroxisomal NUDT12 protein.

The human NUDT12 Nudix hydrolase has been expressed in insect cells from a baculovirus vector as a His-tagged recombinant protein. In vitro, it efficiently hydrolyses NAD(P)H to NMNH and AMP (2',5'-ADP), and diadenosine diphosphate to AMP. It also has activity towards NAD(P)(+), ADP-ribose and diadenosine triphosphate. K (m) values for NADH, NADPH and NAD(+) are 11, 16 and 190 microM and k (cat) values are 11, 16 and 10.5 s(-1) respectively. Thus, like other NADH diphosphatases of the Nudix family, NUDT12 has a marked substrate preference for the reduced nicotinamide nucleotides. Optimal activity was supported by 50 microM Mn(2+) ions in vitro, with 3-fold lower activity at 0.4 mM Mg(2+). Expression of NUDT12 as a C-terminal fusion to green fluorescent protein revealed that it was targeted to peroxisomes by the C-terminal tripeptide PNL acting as a novel type 1 peroxisomal targeting signal. Deletion of PNL resulted in diffuse cellular fluorescence. In addition, C-terminal, but not N-terminal, fusions with or without the PNL signal accumulated in large, unidentified cytoplasmic structures. NUDT12 may act to regulate the concentration of peroxisomal nicotinamide nucleotide cofactors required for oxidative metabolism in this organelle.

Amino Acid Sequence↗

PubMed2001

The utility of cytokeratin subsets in distinguishing Barrett's-related oesophageal adenocarcinoma from gastric adenocarcinoma.

AIMS: Accurate tumour classification is critical for meaningful epidemiological studies in the assessment of cancer incidence rates and trends. Differentiating primary gastric carcinoma from oesophageal carcinoma can be difficult, especially when tumours are large and involve both the oesophagus and stomach. Furthermore, adenocarcinomas of both organs typically are of intestinal histological type and arise in a background of intestinal metaplasia. Consequently, histological markers that reliably distinguish Barrett's-related oesophageal adenocarcinoma from gastric adenocarcinoma would be useful. Cytokeratins (CK)7 and 20 are cytoplasmic structural proteins with restricted expression that help to determine the origin of many epithelial tumours including those of the gastrointestinal tract. The aim of this study was to determine the utility of co-ordinate CK7 and 20 expression in the distinction of Barrett's-related oesophageal adenocarcinoma from gastric adenocarcinoma arising in a background of intestinal metaplasia. METHODS AND RESULTS: CK7 and 20 immunostaining was performed on randomly selected surgical resection specimens from patients with Barrett's-related oesophageal adenocarcinoma (n = 30) and intestinal type gastric adenocarcinoma (n = 14) arising in a background of intestinal metaplasia. A CK7+ CK20- immunophenotype was demonstrated in 27 of 30 (90%) patients with Barrett's-related oesophageal adenocarcinoma and only three of 14 (21%) gastric adenocarcinomas. The sensitivity, specificity and positive predictive value of a CK7+/20- immunophenotype for a diagnosis of Barrett's-related oesophageal adenocarcinoma was 90%, 79%, and 90%, respectively. CONCLUSIONS: A CK7+/20- tumour immunophenotype is associated with Barrett's-related oesophageal adenocarcinoma and may be useful in accurate tumour classification, thus facilitating improving epidemiological evaluation of tumours at the oesophagogastric junction.

Adenocarcinoma↗

PubMed2001

Localization of mitochondrial ribosomal RNA on the chromatoid bodies of marine planarian polyclad embryos.

Electron-dense cytoplasmic structures, referred to as chromatoid bodies, are observed in the somatic stem cells, called neoblasts, and germline cells in adult planarians. Although it has been revealed that the chromatoid bodies morphologically resemble germline granules in Drosophila and Xenopus embryos, what essential role it plays in the planarian has remained unclear. In the present study, to examine whether chromatoid bodies in planarian embryos are responsible for germline formation, the presence and behavior of chromatoid bodies during embryogenesis were examined. Mitochondrial large ribosomal RNA and mitochondrial small ribosomal RNA were used as candidate markers for components of the chromatoid body. Starting from the fertilized egg, extramitochondrial signals of both RNA (mtrRNA) were observed. At the ultrastructural level, mtrRNA were localized on the surface of the chromatoid bodies. At subsequent stages, the signals of mtrRNA were observed in certain restricted blastomeres that contribute to the formation of larval structures. The signals gradually decreased from the gastrula stage. These results suggest that the chromatoid bodies associated with mtrRNA in embryogenesis are not germline granules. The chromatoid bodies of blastomeres may be concerned with the toti- or pluripotency and cell differentiation as proposed in adult planarian neoblasts.

Animals↗

PubMedDate not supplied

Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.

Recent studies suggest that purified villous cytotrophoblasts are largely contaminated by mononucleated syncytial fragments and therefore unsuitable for studies of trophoblast differentiation. We assessed highly purified (>99.99 per cent) populations of villous trophoblasts for fragment contamination using the syncytial markers placental alkaline phosphatase (PLAP, by immunohistochemistry) and exteriorized phosphatidyl serine (ePS, by flow cytometric analysis). The preparations contained from 4-46 per cent syncytial fragments. However, we find that PLAP negative cells preferentially adhere to tissue culture surfaces and that all preparations were <2 per cent PLAP positive after routine plating and washing procedures. A second purification procedure eliminated dead (propidium iodide permeable) cells and separated viable syncytial fragments (ePS-positive) from viable cytotrophoblasts (ePS-negative) by two colour fluorescence activated cell sorting (FACS). Viable ePS-positive cells were ultrastructurally apoptotic, adhered poorly in culture and those that adhered rapidly underwent apoptosis. Viable ePS-negative cells contained large heterochromic nuclei and cytoplasmic structures, adhered strongly in culture and remained viable. The latter population (putative true villous CT) differentiated into syncytialized cells when cultured with EGF. We conclude that villous CT can be routinely purified, are viable in culture and can undergo syncytial fusion without extensive preformed syncytium.

Adult↗