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PubMed1995

Effects of expressing human Hsp70 and its deletion derivatives on heat killing and on RNA and protein synthesis.

We have established rat cell lines stably and constitutively expressing intact human heat shock protein (hsp) 70 and its deletion mutant derivatives. Using these stable cell lines, the functions of the various domains of human hsp70 were studied. An elevated level of human hsp70 or fragments containing the C-terminal portion of the protein, but not fragments containing the amino-terminal portion of the protein, is found to protect cells against thermal killing. Only the expression of intact human hsp70, however, can reduce the degree of heat-induced inhibition of translation and most effectively accelerate the recovery from heat-induced inhibition of transcription and translation. The peptide-binding domain in the carboxyl portion of hsp70 is of primary importance in protecting cells from thermal stress, probably through its binding to unfolded or partially folded polypeptides; this interaction, in turn, retards detrimental thermal denaturation or aggregation of key cellular proteins and protects cells from thermal stress. ATP binding and hydrolysis, on the other hand, are presumably necessary in the refolding and reassembly of thermally damaged proteins through multiple cycles of interactions between hsp70 and its targets.

Animals↗
PubMed1999

Cell-cell interaction modulates myoD-induced skeletal myogenesis of pluripotent P19 cells in vitro.

P19 embryonal carcinoma cells can be induced to differentiate in culture to develop into a wide variety of cell types that include skeletal muscle. Skeletal myogenesis is controlled by transcription factors of the bHLH class, such as myoD. Expression of myoD from transfected genes did not induce significant amounts of myogenesis in P19 cells and it was possible to establish lines of undifferentiated P19[myoD] cells that express high levels of myoD mRNA. These P19[myoD] cells remained undifferentiated when cultured on solid surfaces but when allowed to aggregate, P19[myoD] cells differentiated efficiently into skeletal muscle. Aggregation did not increase the amount of myoD mRNA or the amount of myoD protein in P19[myoD] cells. The myoD protein was present in the nucleus in cells grown as attached or aggregated cultures and, in both culture conditions, the myoD protein was associated with transcription factors of the E2A family and was able to bind DNA at E-box sequences. Thus, the aggregation-induced myogenesis of P19[myoD] cells occurs in the absence of change in the myoD protein, suggesting that the cell-cell contact achieved in aggregates may result in the induction of an activity that increases accessibility of the myoD transcription factor to muscle-specific genes in chromatin.

Animals↗
PubMed1995

Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines.

Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed in Escherichia coli as periplasmic inclusion bodies. Their variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II of Drosophila melanogaster and rat MAb Yol1/34, specific for pig brain alpha-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv')2 was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental MAbs and fourfold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.

Amino Acid Sequence↗
PubMed1995

Improved method for polyamine determination in TMV, a rod-shaped virus.

Polyamines were measured in viruses using different techniques. An improved method of polyamine analysis is reported for tobacco mosaic virus (TMV), a rod-shaped virus (95% protein and 5% RNA), based on HPLC of sonicated PCA-treated highly purified virus suspensions. This method allowed higher and more reliable recovery of TMV polyamines (putrescine, spermidine and spermine) when compared to the HPLC of non-sonicated samples and to thin layer chromatography. It is suggested that sonication acts on PCA-precipitated protein aggregates causing the release of trapped polyamine molecules.

Chromatography, High Pressure Liquid↗
PubMed2006

Epimorphin acts extracellularly to promote cell sorting and aggregation during the condensation of vertebral cartilage.

Formation of vertebrae occurs via endochondral ossification, a process involving condensation of precartilaginous cells. Here, we provide the first molecular evidence of mechanism that underlies initiation of this process by showing that the extracellular factor, Epimorphin, plays a role during early steps in vertebral cartilage condensation. Epimorphin mRNA is predominantly localized in the vertebral primordium. When provided exogenously in ovo, it causes precocious differentiation of chondrocytes, resulting in the formation of supernumerary vertebral cartilage in chicken embryos. To further analyze its mode of action, we used an in vitro co-culture system in which labeled 10T1/2 or sclerotomal prechondrogenic cells were co-cultured with unlabeled Epimorphin-producing cells. In the presence of Epimorphin, the labeled cells formed tightly packed aggregates, and sclerotomal cells displayed augmented accumulation of NCAM and other early markers of chondrocyte differentiation. Finally, we found that the Epimorphin expression is initiated during vertebrogenesis by Sonic hedgehog from the notochord mediated by Sox 9. We present a model in which successive action of Epimorphin in recruiting and stacking sclerotomal cells leads to a sequential elongation of a vertebral primordium.

Amino Acid Sequence↗
PubMed2003

Photon correlation spectroscopy investigations of proteins.

Physical principles of photon correlation spectroscopy (PCS), mathematical treatment of the PCS data (converting autocorrelation functions to distribution functions or average characteristics), and PCS applications to study proteins and other biomacromolecules in aqueous media are described and analysed. The PCS investigations of conformational changes in protein molecules, their aggregation itself or in consequence of interaction with other molecules or organic (polymers) and inorganic (e.g. fumed silica) fine particles as well as the influence of low molecular compounds (surfactants, drugs, salts, metal ions, etc.) reveal unique capability of the PCS techniques for elucidation of important native functions of proteins and other biomacromolecules (DNA, RNA, etc.) or microorganisms (Escherichia coli, Pseudomonas putida, Dunaliella viridis, etc.). Special attention is paid to the interaction of proteins with fumed oxides and the impact of polymers and fine oxide particles on the motion of living flagellar microorganisms analysed by means of PCS.

Bacterial Proteins↗
PubMed2004

Sox6 overexpression causes cellular aggregation and the neuronal differentiation of P19 embryonic carcinoma cells in the absence of retinoic acid.

The Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia and fibroblast-like cells. In this report, we have shown that Sox6 mRNA increased rapidly in P19 cells during RA induction and then decreased during the differentiation of P19 into neuronal cells. To explore the possible roles of Sox6 during this process, stably Sox6-overexpressing P19 cell lines (P19[Sox6]) were established. These P19[Sox6] had acquired both characteristics of the wild-type P19 induced by RA. First, P19[Sox6] cells showed a marked cellular aggregation in the absence of RA. Second, P19[Sox6] could differentiate into microtubule-associated protein 2 (MAP2)-expressing neuronal cells in the absence of RA. Sox6 expression could cause the activation of endogenous genes including the neuronal transcription factor Mash-1, the neuronal development-related gene Wnt-1, the neuron-specific cell adhesion molecule N-cadherin, and the neuron-specific protein MAP2, resulting in neurogenesis. Moreover, E-cadherin, a major cell adhesion molecule of wild-type P19, was strongly induced by Sox6, resulting in cellular aggregation without RA. Thus Sox6 may play a critical role in cellular aggregation and neuronal differentiation of P19 cells.

Animals↗
PubMed1971

Cytochemistry, cytogenetics and ultrastructure of hamster tumour cells carrying mouse sarcoma viral genome (HT-1 cells).

The pleomorphic cytology of the HT-1 cell line is described. Cytochemical studies indicated the presence of glycogen and lysosomes in these cells. Cytogenetic studies demonstrated the presence of a large telocentric chromosome (M1) and two minute chromosomes (M2) not found in normal hamster cells. A cloned line was isolated which did not contain these marker chromosomes. All cells were hyperdiploid with chromosome number around triploidy, although none was a true triploid. Ultrastructural studies revealed the presence of "nuclear bodies", "dense bodies" and "interchromatinic granules" which have been previously described in viral induced malignancies. A few of the cells contained in their cytoplasm structures of approximately 700 [unk] in diameter which bore some resemblance to immature virus particles. Both light and electron microscopy demonstrated some nuclei lacking chromatin aggregates. This is interpreted to mean that the total genetic material of these nuclei was dispersed as free DNA not linked with histone to form chromatin aggregates.

Animals↗
PubMed1993

Molecular cloning and adhesive properties of murine platelet/endothelial cell adhesion molecule 1.

We describe the isolation and characterization of a functional murine platelet/endothelial cell adhesion molecule (PECAM) 1 cDNA clone from a mouse lung library. At the nucleotide level, the coding sequence of murine PECAM-1 is 73% identical to human PECAM-1, and at the amino acid level, the sequence is 79% homologous to its human counterpart. Southern hybridization reveals that one copy of the gene exists in the mouse genome; Northern hybridization reveals a single mRNA species in mouse lung tissue. COS-7 and mouse L cells transfected with murine PECAM-1 expressed a 130-kDa glycoprotein on their surfaces that reacted with anti-murine PECAM-1 monoclonal antibody and comigrated on SDS/PAGE with human PECAM-1. Stable L-cell transfectants aggregate with each other in a PECAM-dependent, homophilic manner.

Amino Acid Sequence↗
PubMed1997

Mechanism of homophilic binding mediated by ninjurin, a novel widely expressed adhesion molecule.

Ninjurin is a novel protein that is up-regulated after nerve injury both in dorsal root ganglion (DRG) neurons and in Schwann cells. We previously reported that ninjurin demonstrates properties of a homophilic adhesion molecule and promotes neurite outgrowth from primary cultured DRG neurons. We have now found that ninjurin is widely expressed in both adult and embryonic tissues, primarily in those of epithelial origin. Aggregation assays were used to demonstrate that ninjurin-mediated adhesion requires divalent cations and is an energy-dependent process. The critical domain for ninjurin-mediated homophilic adhesion was localized to an 11-residue region (between Pro26 and Asn37) by mutagenesis and by employing synthetic oligopeptides as competitive inhibitors of ninjurin-mediated adhesion. Of particular importance are the Trp residue at position 29 and the 3 arginines in the region. Furthermore, we show that the peptide which inhibits aggregation of Jurkat cells expressing ninjurin is also capable of blocking the ability of ninjurin to promote neurite extension from DRG neurons. Using FISH analysis, the ninjurin gene was localized to human chromosome 9q22. Several genetic diseases of unknown etiology have been mapped to this region, including hereditary sensory neuropathy type 1, self-healing squamous epithelioma, split-hand/foot deformity type 1, and familial dilated cardiomyopathy.

Amino Acid Sequence↗
PubMed1990

Gene expression in visceral endoderm: a comparison of mutant and wild-type F9 embryonal carcinoma cell differentiation.

We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.

Animals↗
PubMed1993

Patterns of cytokine production by mycobacterium-reactive human T-cell clones.

To gain insight into the functional capacity of human T cells in the immune response against Mycobacterium tuberculosis, we evaluated the spectrum of cytokines produced by mycobacterium-reactive human T-cell clones. Nine of 11 T-cell clones bearing alpha beta or gamma delta T-cell receptors produced both Th1 and Th2 cytokines, a pattern resembling that of murine Th0 clones. The most frequent pattern was secretion of gamma interferon, tumor necrosis factor alpha (TNF), and interleukin-10 (IL-10), in combination with IL-2, IL-5, or both. Two clones produced only Th1 cytokines, and none produced exclusively Th2 cytokines. Although IL-4 was not detected in cell culture supernatants, IL-4 mRNA was detected by polymerase chain reaction amplification in two of six clones. There were no differences between the cytokine profiles of alpha beta and gamma delta T cells. A striking finding was the markedly elevated concentrations of TNF in clone supernatants, independent of the other cytokines produced. Supernatants from mycobacterium-stimulated T-cell clones, in combination with granulocyte-macrophage colony-stimulating factor, induced aggregation of bone-marrow-derived macrophages, and this effect was abrogated by antibodies to TNF. The addition of recombinant TNF to granulocyte-macrophage colony-stimulating factor markedly enhanced macrophage aggregation, indicating that TNF produced by T cells may be an important costimulus for the granulomatous host response to mycobacteria. The cytokines produced by T cells may exert immunoregulatory and immunopathologic effects and thus mediate some of the clinical manifestations of tuberculosis.

Antigens, Bacterial↗
PubMed2006

Overexpression of the RADICAL-INDUCED CELL DEATH1 (RCD1) gene of Arabidopsis causes weak rcd1 phenotype with compromised oxidative-stress responses.

rcd1 is a mutant of Arabidopsis thaliana that is more resistant to methyl viologen, but more sensitive to ozone than the wild type. rcd1-2 is caused by a single nucleotide substitution that results in a premature stop codon at Trp-332. The rcd1-2 mRNA level does not change significantly with the mutation. Since overexpression of rcd1-1 cDNA has been shown to bring about an rcd1-like phenotype, we created and examined the overexpression lines of RCD1 by the use of the cauliflower mosaic virus 35S promoter. The transgenic lines exhibited a weak rcd1-like phenotype, although no resistance to methyl viologen was observed. Further, they fully complemented the aberrant rcd1-2 phenotype. Subcellular localization of RCD1 was examined by transiently expressing green fluorescent protein (GFP) fused with RCD1 in onion epidermal cells. GFP signals are observed as aggregated foci in the inner nuclear matrix-like region.

Arabidopsis↗
PubMed2001

The mechanism of the growth-inhibitory effect of coxsackie and adenovirus receptor (CAR) on human bladder cancer: a functional analysis of car protein structure.

The coxsackie and adenovirus receptor (CAR) is identified as a high-affinity receptor for adenovirus type 5. We observed that invasive bladder cancer specimens had significantly reduced CAR mRNA levels compared with superficial bladder cancer specimens, which suggests that CAR may play a role in the progression of bladder cancer. Elevated CAR expression in the T24 cell line (CAR-negative cells) increased its sensitivity to adenovirus infection and significantly inhibited its in vitro growth, accompanied by p21 and hypophosphorylated retinoblastoma accumulation. Conversely, decreased CAR levels in both RT4 and 253J cell lines (CAR-positive cells) promoted their in vitro growth. To unveil the mechanism of action of CAR, we showed that the extracellular domain of CAR facilitated intercellular adhesion. Furthermore, interrupting intercellular adhesion of CAR by a specific antibody alleviates the growth-inhibitory effect of CAR. We also demonstrated that both the transmembrane and intracellular domains of CAR were critical for its growth-inhibitory activity. These data indicate that the cell-cell contact initiated by membrane-bound CAR can elicit a negative signal cascade to modulate cell cycle regulators inside the nucleus of bladder cancer cells. Therefore, the presence of CAR cannot only facilitate viral uptake of adenovirus but also inhibit cell growth. These results can be integrated to formulate a new strategy for bladder cancer therapy.

Adenoviridae↗
PubMed2026

Redox cycling of viral RNA polymerase controls picornavirus replication.

Picornaviruses, including foot-and-mouth disease virus (FMDV), enterovirus 71 (EV71) and encephalomyocarditis virus (EMCV), are important pathogens that cause fever, herpes, and myocarditis in humans and animals. The interplay between picornaviruses and their hosts remains enigmatic. Here we perform porcine genome-wide CRISPR/Cas9 screens and identify methionine sulfoxide reductase B3 (MSRB3) as an essential factor for FMDV. MSRB3 deficiency inhibits FMDV replication. Mechanistically, MSRB3 eliminates methionine oxidation of FMDV 3D polymerase and stabilizes its expression. Further studies show that radical SAM domain-containing protein 1 (RSAD1) catalyzes methionine oxidation of FMDV 3D polymerase and promotes its aggregation and subsequent degradation through the autophagy-lysosome pathway. Importantly, RSAD1-MSRB3-mediated redox modification also affects the stability of 3D polymerases of EV71 and EMCV, and regulates their infectivity and pathogenesis both in vitro and in vivo. Collectively, this study corroborates that RSAD1-MSRB3-mediated redox cycling of 3D polymerase plays a conserved function in modulating picornavirus infection, providing insights into viral pathogenesis and broad-spectrum antiviral development.

Animals↗
PubMed1980

On the mechanism of inactivation of Chikungunya virus by tannic acid.

The mechanism of the action of tannic acid (TA) Chikungunya virus (CHIKV) was investigated. Both infectivity and hemagglutination (HA) activity of CHIKV were reduced by treatment with TA in vitro. Aggregation of the TA-treated virus particles was observed by electron microscopy. The reaction was reversible, depending on the pH of the mixture. However, mere dilution of the TA-virus mixture or addition of other protein, such as bovine serum albumin, did not restore the lost infectivity. TA also suppressed the infectivity of RNA extracted from the virus and the HA activity of the viral membrane. The affinity between TA and CHIKV structural proteins (E1, E2, and C) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a discontinuous buffer system and the order of affinity was found to be C greater than E1 greater than E2. Specific conditions for binding of TA to each of the virus proteins were investigated.

Cell Membrane↗
PubMed2004

Hypothermia induces anti-inflammatory cytokines and inhibits nitric oxide and myeloperoxidase-mediated damage in the hearts of endotoxemic rats.

STUDY OBJECTIVE: s: The impairment of cardiac contractility during endotoxemia involves induction of nitric oxide formation through a cascade of events initiated by overexpression of proinflammatory cytokines. We previously showed that hypothermia attenuates endotoxin-induced overexpression of nitric oxide in rat lungs. In the present study, we tested the hypothesis that hypothermia protects against endotoxin-induced myocardial inflammation by changing the balance of pro- and anti-inflammatory cytokines, inhibiting myeloperoxidase, an indicator of neutrophil activity, and inhibiting nitric oxide-mediated protein damage. DESIGN: Rats were randomized to treatment with either hypothermia (n = 6; 18 to 24 degrees C) or normothermia (n = 6; 36 to 38 degrees C). Endotoxin (15 mg/kg) was administered intravascularly to anesthetized animals, and heart tissue was harvested 150 min later. MEASUREMENTS AND RESULTS: Using enzyme-linked immunosorbent assays (ELISAs), we found that hypothermia induced myocardial expression of the anti-inflammatory cytokines interleukin (IL)-4 and IL-10, while decreasing concentrations of the pro-inflammatory cytokines IL-1beta and growth-related oncogene/cytokine-induced neutrophil chemoattractant (rat homolog of IL-8). Electromobility shift assay revealed that hypothermia inhibited the nuclear translocation of nuclear factor-kappaB. Reverse transcriptase-polymerase chain reaction and Western blot assays revealed that hypothermia attenuated the endotoxin-induced overexpression of both inducible nitric oxide synthase (iNOS) messenger RNA and iNOS protein, respectively. Hypothermia also attenuated nitric oxide-mediated myocardial protein damage, as determined by a nitrotyrosine ELISA. Myocardial myeloperoxidase content, an indicator of neutrophil accumulation and oxidative activity, was also inhibited by hypothermia in endotoxemic rats. CONCLUSION: These data demonstrate that hypothermia induces an anti-inflammatory cytokine profile, inhibits neutrophil aggregation, and inhibits the formation of nitric oxide during endotoxemia in the rat.

Animals↗