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PubMed1990

Implication of mitochondria in the replication of Nodamura virus in larvae of the Lepidoptera, Galleria mellonella (L.) and in suckling mice.

The replication of Nodamura virus, type species of the family Nodaviridae, takes place principally into the interfibrillar spaces of the sarcoplasm in close association with mitochondria in the infected muscles of both G. mellonella and suckling mice. The most striking event in infection of G. mellonella muscle cells was the aggregation and shape modification of numerous mitochondria (elongation, interdigitation, and vesiculation) at an early stage of infection. These clusters of mitochondria were cemented by a thick electron-dense material at the periphery of which viral particles undergo maturation process. In a later stage, degenerated and dilated mitochondria showed a clear assembling of virus particles on their outer membrane and occasionally on some inner membranes. These facts and the localization of RNA by RNase-gold technique in the assembling sites of viruses around and inside the mitochondria allowed us to postulate a role for the mitochondria as supports and/or energy suppliers for viral RNA synthesis and translation.

Animals↗
PubMed1994

Local sprouting of neurites from cultured PC12D cells in response to a concentration gradient of nerve growth factor.

PC12D cells, a subline of PC12 cells, extend neurites very rapidly in response to NGF, even when RNA synthesis is blocked. Several minutes after the initiation of a concentration gradient of NGF from a micropipette in the vicinity of PC12D cells, clear projections emerged from cells on the side facing the micropipette while no significant changes in morphology were observed on the other side of cells. A control solution administered from a micropipette did not produce any changes in morphology. Longer exposure to the gradient of NGF of aggregates of PC12D cells increased the length of neurites extending toward the source of NGF. The observations indicate that the sprouting of neurites occurs locally in regions of PC12D cells that are exposed to an elevated concentration of NGF.

Animals↗
PubMed1976

Messenger ribonucleic acid metabolism in mammalian mitochondria. Isolation and characterization of polyribosomes from Ehrlich ascites mitochondria.

The Mg2+ precipitation procedure of R. D. Palmiter ((1974) Biochemistry 13, 3606) has been used for preparative scale isolation of polysomes from Ehrlich ascites mitochondria. Digitonin-washed metochondria used for isolating the polysomes contain no detectable reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and over 200-fold reduced hexokinase activity. The mitochondrial polysomes exhibit a heterogeneous sedimentation and appear to contain highly aggregated particlses ranging over hexamers. These polysomes are sensitive to RNase, (ethylenedinitrilo)tetraacetic acid and puromycin. Mitochondrial polysomes are active in portein synthesis when supplied with supernatant enzymes from the homologous mitochondrial source or from Escherichia coli. Cytoplasmic enzymes, however, appear to be completely inactive. Protein synthesis by mitochrondrial polysomes is sensitive to chloramphenicol and resistant to cycloheximide and emetine. The procedure yields particles containing intact rRNAs. The extent of cytoplasmic RNA contaminating the total mitochondrial RNA or mitochondrial polysomal RNA has been estimated to be negligible.

Animals↗
PubMed1983

On the evolution of orbiviruses.

The genomes of orbiviruses consist of 10 segments of double-stranded RNA. In cells simultaneously infected with two or more related viruses, recombinants are derived by independent reassortment of parental genes. The process is analogous to sexual reproduction in higher organisms and provides a mechanism for generating extensive diversity within this group of viruses. This genetic diversity can be explained by reference to modern concepts of the structure of natural populations of organisms. A species in the phylogenetic sense is the largest aggregate of individual organisms that evolves as a unit. The biological species concept stresses the community gene pool and reproductive isolation. The orbiviruses are now classified into 13 distinct serological groups, but confusion exists in defining species. Classification by reference to concepts of evolutionary species would define genetically interacting groups, estimate the extent of diversity within these groups, and establish phylogenetic relationships between species.

Biological Evolution↗
PubMed2002

Expression of osteopontin at sites of bone erosion in a murine experimental arthritis model of collagen-induced arthritis: possible involvement of osteopontin in bone destruction in arthritis.

OBJECTIVE: To investigate the involvement of osteopontin (OPN) in bone destruction in a murine experimental arthritis model of collagen-induced arthritis (CIA). METHODS: The expression of OPN was examined at both the messenger RNA (mRNA) and protein levels in various arthritic lesions in mice with CIA by in situ hybridization and immunohistochemistry, respectively. In addition, the expression of alpha(v)beta3 integrin, a receptor for OPN, the ligation of which is thought to be essential for bone resorption by osteoclasts, was examined by immunohistochemistry. Plasma concentrations of OPN were measured at different time points in the course of CIA by enzyme-linked immunosorbent assay. RESULTS: OPN mRNA was detected mainly at sites of bone erosion in arthritic lesions, where activated osteoclasts were present; OPN protein was also detected at sites of bone erosion. In the arthritic synovium, OPN was predominantly expressed in the synovial lining layer, but not in lymphoid aggregates. In addition, alpha(v)beta3 integrin was detected coincident with OPN at sites of bone erosion (bone-pannus junction). Plasma OPN levels were markedly elevated at the time points that corresponded to arthritis flares, and higher levels were maintained during the progression of arthritis. CONCLUSION: OPN may mediate bone resorption by osteoclasts in arthritis through ligation with its receptor, alpha(v)beta3 integrin. OPN may be a useful therapeutic target molecule in the prevention of bone destruction in arthritis.

Animals↗
PubMed1997

Accumulation of misfolded protein aggregates leads to the formation of russell body-like dilated endoplasmic reticulum in yeast.

RNAP-1, an aspartic proteinase from a filamentous fungus Rhizopus niveus, is secreted very efficiently in Saccharomyces cerevisiae. It is synthesized first as a precursor form with signal sequence and prosequence in its amino-terminus. Our previous study indicated that the prosequence of RNAP-I had important roles in its correct folding and secretion in yeast, and that a prosequence-deleted derivative of RNAP-I, delta pro, was not secreted but was retained and degraded in the yeast endoplasmic reticulum (ER). In the present study, we show that the accumulation of delta pro in the yeast ER caused elevated synthesis of ER resident chaperones, indicating that delta pro is recognized as an unfolded protein species in the ER. Our biochemical data demonstrated that delta pro formed aggregates which contained BiP, but not protein disulfide isomerase (PDI), in the ER. Immunoelectron microscopical analysis revealed that the delta pro aggregates were indeed visible as electron-dense regions in the ER and nuclear envelope. Such 'chaperone-associated misfolded protein bodies' were observed for the first time in yeast. Morphologies of the ER and nucleus were drastically altered by the accumulation of the delta pro aggregates. The ER lost its flat cisternal shape; the ER lumen extended aberrantly and the ER membrane irregularly proliferated. The misfolded delta pro proteins are probably sorted from the ordinary ER lumen to form the aggregates so that the ER function would not be grossly impaired, and the dilated ER may represent an ER subcompartment where the delta pro aggregates are degraded.

Amino Acid Sequence↗
PubMed1993

An amber mutation of prion protein in Gerstmann-Sträussler syndrome with mutant PrP plaques.

We found an amber mutation in the open reading frame of the prion protein (PrP) gene. The codon 145 mutation (tyrosine to stop) was recognized on a PrP allele of a patient with Alzheimer-type clinical course. Pathologic examination revealed many amyloid plaques and neurofibrillary changes. However, the amyloid plaques in this patient were not composed of beta/A4 protein, but of PrP. Both wild and mutant PrP alleles were detected in the cerebral mRNA; however, only C-terminal truncated PrP was detected in the kuru plaques. We herein present evidence that only mutant PrP aggregates to make kuru plaques in the central nervous system.

Adult↗
PubMed1993

Association between ICAM-1 expression and metastatic capacity of murine B-cell hybridomas.

We previously reported that a derivative of the interleukin-6 (IL-6)-dependent B9 B-cell hybridoma (B9/LPNU1L) constitutively expressing an interleukin-1 alpha (IL-1 alpha) gene introduced by retrovirus-mediated gene transfer preferentially metastasized to bone marrow following intravenous injection into unirradiated syngeneic BALB/c mice. B9/LPNU1L cells recovered from the femoral marrow of a recipient with hind limb paralysis (denoted B9/BM1) retained their IL-6-dependency yet displayed enhanced metastatic capacity during serial transplantation in vivo. In contrast, autonomously-growing B9 variants spontaneously arising in vitro or IL-6-independent B9 derivatives created by infection with recombinant IL-6 retroviruses rarely gave rise to experimental metastases in syngeneic BALB/c or nude mice. Examination of cell adhesion molecule profiles by immunofluorescence flow cytometry has revealed high levels of CD44, moderate levels of VLA-4 and low levels of LFA-1 on all B9-series cells. By comparison, ICAM-1 expression was significantly elevated on B9/BM1 cells, with independent isolates stably expressing about 4-fold higher levels which were paralleled by corresponding increases in the steady-state levels of ICAM-1 mRNA. L-Selectin was not expressed by any of the cell lines. Despite higher ICAM-1 levels, cell aggregation assays revealed that LFA-1-ICAM-1 adhesive interactions were not involved in the homotypic adhesion of B9/BM1 cells but rather that binding of CD44 to endogenously-synthesized hyaluronan was responsible. Furthermore, B9/BM1 cells expressing high levels of ICAM-1 were found to be less susceptible to cytolysis by natural killer (NK) cells than their weakly metastatic or nonmetastatic counterparts.

Animals↗
PubMed2002

Aggregation of influenza virus ribonucleocapsids at low pH.

Uncoating of influenza occurs in endosomes where the acid environment is instrumental in membrane fusion and the dissociation of the ribonucleoprotein (RNP) from matrix protein by the action of the hemagglutinin and M2 protein ion channels, respectively. Earlier studies have shown that low pH treatment results in the release of M1 protein from RNP. To obtain RNP free of M1 protein, we attempted to isolate RNP by velocity sedimentation on pH 5 glycerol gradients; however, the RNP sedimented as pellets under centrifugation conditions that had previously resolved RNP on neutral gradients. The increase in sedimentation rate occurred between pH 5.6 and 6.0 and was reversible for a portion of the RNP on raising the pH to neutrality. RNP isolated from infected cells or virions sedimented on acidification and was seen to form clumps visible by electron microscopy. If acidification preceded NP40 detergent lysis, virion RNP appeared to be released as genomic complexes. The pH threshold for viral membrane fusion was 5.8 indicating that the same pH condition also resulted in aggregation of RNP. Because exposure of virions to pH 5 occurs during uncoating in endosomes and is essential for infectivity, it is possible that low pH-induced RNP aggregation may facilitate aspects of viral uncoating such as dissociation of RNP from M1 or transport of genomes to the nucleus.

Cell Line↗
PubMed2005

Folding a nonbiological polymer into a compact multihelical structure.

The only molecules that are currently known to fold into unique three-dimensional conformations and perform sophisticated functions are biological polymers - proteins and some RNA molecules. Our aim is to create a nonbiological sequence-specific polymer that folds in aqueous solution. Toward that end, we synthesized sequence-specific 30mer, 45mer, and 60mer peptoid oligomers (N-substituted glycine polymers) consisting of 15mer units we chained together by disulfide and oxime linkages to mimic the helical bundle structures commonly found in proteins. Because these 15mer sequences were previously shown to form defined helical structures that aggregate together at submillimolar concentrations, we expected that by covalently linking multiple 15mers together, they might fold as helical bundles. To probe whether they folded, we used fluorescence resonance energy transfer (FRET) reporter groups. We found that certain constructs fold up with a hydrophobic core and have cooperative folding transitions. Such molecules may ultimately provide a platform for designing specific functions resembling those of proteins.

Chromatography, High Pressure Liquid↗
PubMed2001

Interaction of morin-cetyltrimethylammonium bromide with nucleic acids and determination of nucleic acids at nanograms per milliliter levels based on the enhancement of preresonance light scattering.

A new preresonance light scattering (PRLS) assay of nucleic acids is presented. At pH 7.30, the weak PRLS of morin-cetyltrimethylammonium bromide (CTMAB) can be greatly enhanced by the addition of nucleic acids, owing to the interaction between the nucleic acid and morin-CTMAB. After the addition of morin and CTMAB to DNA, the zeta potential of DNA decreases and changes from negative to positive, which is due to the formation of an associate, the aggregation of morin on nucleic acids and the electric neutralization between DNA and the cationic surfactant CTMAB. Mechanism studies showed that the enhanced PRLS comes from the aggregation of morin in the presence of nucleic acids and CTMAB. The enhanced intensity of PRLS is in proportion to the concentration of nucleic acids in the range 7.5 x 10(-9)-1.0 x 10(-5) g ml(-1) for calf thymus DNA, 7.5 x 10(-9)-1.0 x 10(-6) g ml(-1) for salmon sperm DNA and 1.0 x 10(-8)-1.0 x 10(-6) g ml(-1) for yeast RNA. The detection limits are 3.4, 6.2 and 4.1 ng ml(-1) for calf thymus DNA, salmon sperm DNA and yeast RNA, respectively. Synthetic samples were analyzed satisfactorily.

Animals↗
PubMed1978

Decreased activity of peptide-elongation factors after treatment with cholesterol esterase.

1. Peptide-elongation factors were purified from rat liver and treated with cholesterol esterase and phospholipase A2 immobilized on Sepharose 4B. 2. Binding of L-[3H]-phenylalanyl-tRNA to 40S ribosomal subunits was decreased by approx. 70% and to polyribosomes by 30% in the presence of the binding factor incubated with cholesterol esterase. Treatment of this factor with immobilized phospholipase A2 decreased the binding to smaller ribosomal subunits by only about 15%. 3. Poly(U)-dependent phenylalanine polymerization by ribosomal subunits was decreased to approx. 30% of its original value by treatment of both elongation factors with cholesterol esterase. 4. The normal activity of esterase-treated elongation factor in both the binding reaction and peptide-elongation assay was fully recovered by the addition of cholesteryl 14-methyl-hexadecanoate. 5. Different classes of lipids present in peptide-elongation factor 1 have apparently different functions. Whereas phospholipids are required to maintain the strcture of heavy aggregates of this factor, the presence of cholesteryl 14-methylhexadecanoate is obviously necessary for the normal function of peptide-elongation factors.

Animals↗
PubMed2003

Identification of protein kinase C (PKC) isoforms in teleostean, amphibian and avian pigment cells.

The beta isoform of protein kinase C (PKC) has been described as the main isoform involved in the stimulation of melanogenesis in mammalian skin melanocytes. Little is known about PKC isoforms in non-mammalian pigment cells. In neopterigian fish (holostei and teleostei), PKC is associated with pigment granule aggregation within the pigment cells (skin lightening), whereas in elasmobranchs and tetrapods, the activation of PKC leads to pigment granule dispersion (skin darkening). In an attempt to a better understanding of this distinct functional behavior upon PKC activation, we decided to investigate the PKC isoforms expressed in pigment cell lines of teleost fish, amphibians and birds, using RT-PCR followed by cloning and sequencing. Our results demonstrate the presence of messenger RNA (mRNA) for the following PKC isoforms: beta 1, lambda and iota in GEM-81 cells (Carassius auratus erythrophoroma), beta 1, beta 2 and zeta in Xenopus laevis (amphibian) melanophores; beta 1 and lambda in Gallus gallus (chicken) primary melanocytes. Beta 1 PKC seems to be conserved throughout phylogeny, but the diversity of the other isoforms in the different groups may account for the functional differences after PKC activation, which are observed between teleost and tetrapod pigment cells.

Amino Acid Sequence↗
PubMed2000

Localization of E-cadherin in villous, extravillous and vascular trophoblasts during intrauterine, ectopic and molar pregnancy.

Previous reports have described down-regulation of E-cadherin in trophoblasts differentiating to an invasive phenotype. This study shows the localization of E-cadherin in a prospective design with stereological sampling of fetal and maternal first, second and third trimester tissue. E-cadherin was observed in villous cytotrophoblasts, and in non-proliferating, intermediate trophoblasts (IT) within cell columns and islands in intrauterine, ectopic and partial molar placentas. Highly proliferating IT with cytological atypia in complete molar placentas were also E-cadherin-positive. E-cadherin was present in trophoblasts throughout the anchoring cell columns. Trophoblasts undergoing epithelial mesenchymal transformation (EMT) detaching from the distal cell columns and deeper located single extravillous interstitial trophoblasts (EVT) showed E-cadherin-negative breaches in the cell membrane. Prior to the late second trimester, the relative number of E-cadherin-positive single EMT and EVT differed from the total number of cytokeratin-positive trophoblasts. Intraluminal, endovascular and perivascular trophoblasts adjacent to the maternal vessels were also E-cadherin-positive, but a highly varying pattern was observed at different ages of gestation. Our results indicate a temporary shift in E-cadherin expression in extravillous trophoblasts possessing a migrating and invasive potential. Functional E-cadherin may be restored as trophoblasts aggregate in the decidua and the vessel wall after completion of migration.

Antibodies, Monoclonal↗
PubMed1995

Use of glycerol, polyols and other protein structure stabilizing agents in protein crystallization.

A protein preparation to be used for crystallization should be homogeneous and should remain so throughout the course of a prolonged crystallization experiment. General methods for preparation of pure proteins and for prevention of their covalent modification (through proteolysis, sulfhydryl oxidation, etc.) during prolonged incubation are well known. Crystallographers are less aware of general methods for stabilization of proteins against non-covalent modifications (partial denaturation, heterogeneous aggregation) which can also introduce structural heterogeneity into a protein preparation. Related to this issue are methods to suppress protein conformational flexibility which can be a source of dynamic structural heterogeneity and which presents an entropic barrier to crystallization. However, for many years agents which stabilize protein structure have been described in the biochemical literature. Recently the most widely used of these structure-stabilizing agents, glycerol, was used to crystallize T7 RNA polymerase. The observation that this compound has general structure-stabilizing effects and that it was essential for crystallization of at least this one protein led to the suggestion that it might be generally useful in crystallizing flexible proteins and in inducing order in disordered segments of crystalline proteins. Subsequently, glycerol was used with good effect in the crystallization of a number of proteins. Other recent results suggest that soaking crystals in solutions containing glycerol can have 'structure-ordering' effects on the crystalline protein. These observations support the utility of glycerol in protein crystallization and suggest that the information in the biochemical literature on protein structure-stabilizing agents will find useful application in the field of protein crystal growth.

Journal Article↗
PubMed2000

The evolution of reproductive isolation in the ectomycorrhizal Hebeloma crustuliniforme aggregate (Basidiomycetes) in northwestern Europe: a phylogenetic approach.

To reconstruct the evolution of reproductive isolation in the ectomycorrhizal Hebeloma crustuliniforme aggregate (Basidiomycetes), phylogenetic relationships were determined between strains that belong to a clade consisting of nine intercompatibility groups (ICGs, biological species). Four of these nine ICGs are partially compatible and belong to the H. crustuliniforme aggregate. Different levels of partial compatibility have been found between these four ICGs. Between ICGs 3 and 4, 15% of the combinations were compatible. One strain was compatible with all isolates of both ICGs 3 and 4 and also with one isolate of ICG 2. Both a nuclear phylogeny, based on ribosomal IGS sequence data, and a mitochondrial phylogeny, based on a group-I intron located in the large subunit ribosomal RNA gene (LrRNA), were reconstructed. The level of incompatibility was compared with the phylogenetic history of individuals belonging to this clade. Different relationships were found between the level of compatibility and the relative age of the most recent common ancestor (MRCA) for different ICGs. On the one hand, the evolution of incompatibility between ICGs 2 and 3/4 is most consistent with the class of "divergence- first" models because a positive correlation was found between the relative age of the MRCA and the level of incompatibility for ICG 2 versus 3/4. On the other hand, the lack of such a correlation for ICGs 3 and 4 shows that (partial) incompatibility between these ICGs has arisen without strong divergence. The ecological (and to a lesser extent geographical) differences found between ICGs 3 and 4 suggest that selection for incompatibility, associated with host tree preference, has been important in the evolution of incompatibility between these two ICGs. The incongruence between the nuclear and mitochondrial trees for ICG 1 could be explained by a hybrid origin of this ICG, with different donors of the mitochondrial and nuclear sequences.

Basidiomycota↗
PubMedDate not supplied

Radionuclide angiography and scintigraphy in hepatocellular carcinoma.

Among 1 257 patients subjected to liver-spleen (RES) scintigraphy and radionuclide angiography (RNA), there were 13 cases of histologically confirmed hepatocellular carcinoma (1%). All 13 patients had scintigraphic findings indicating cirrhosis. Histologically, cirrhosis was present in only 9 out of 11 cases in which liver parenchyma was available for examination. One patient had hemochromatosis without evidence of cirrhosis. In 11 cases, the tumor was clearly demonstrated as a defect in the static scintigram. However, in 2 cases with cirrhosis and poorly differentiated hepatocellular carcinoma, the tumor nodules were hardly discernible. In RNA, the tumor displayed high activity in the arterial phase and decreasing activity during the portal phase, ending up as a defect. At RNA, the lesion was clearly outlined in 12 cases, including the 2 patients in whom the scintigraphic findings were inconclusive; in one case with severe cirrhosis and a well differentiated hepatocellular carcinoma, the tumor was barely detectable. We conclude that a combination of RNA and scintigraphy is a valuable screening which usually provides a correct diagnosis in hepatocellular carcinoma.

Adult↗