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At least 307 records · Page 17Linked to original sources
PubMed1992

Cellular localization and characterization of proteins that bind high density lipoprotein.

High density lipoprotein (HDL) stimulates excretion of excess intracellular cholesterol from cells, presumably by interacting with a cell-surface receptor. A 110 kDa membrane protein that is a candidate for the HDL receptor has been identified by ligand blot analysis. In this study we determined the cellular localization of this and other HDL-binding proteins and characterized their properties. The plasma membranes (PM) of cultured bovine aortic endothelial cells were labeled with trace amounts of [3H]cholesterol, and cell homogenates were fractionated on sucrose and Percoll gradients. Ligand blot analysis of homogenates of cultured bovine aortic endothelial cells demonstrated that cells contain multiple proteins that bind HDL3, including a major membrane protein with an apparent M(r) of 110 kDa and two minor ones with M(r) of 105 and 130 kDa. The gradient distribution of the 105, 110, and 130 kDa HDL-binding proteins mirrored that of labeled cholesterol and 5'-nucleotidase, both PM markers. Treatment of intact cells with the water-soluble cross-linker bis(sulfosuccinimidyl)suberate abolished the HDL binding activity of the 110 and 130 kDa proteins but not that of the 105 kDa protein. These findings suggest that the 105, 110, and 130 kDa HDL-binding proteins are localized to the PM and that at least two of these proteins are exposed to the extracellular fluid. Solubilized 110 and 130 kDa proteins were retained on wheat-germ agglutinin and abrin lectin columns, showing that they are glycoproteins. The cellular localization and physical properties of the 110 and 130 kDa proteins suggest that they may play a role in binding of HDL to the cell surface.

Abrin↗
PubMedDate not supplied

Assessment of retrovirus-expressed nucleoprotein as a vaccine against lethal influenza virus infections of chickens.

Hemagglutinin-based influenza vaccines stimulate protection in chickens that is limited to the serotype of the expressed hemagglutinin. To evaluate whether a more highly conserved influenza virus protein might stimulate a broader protective response, the influenza virus nucleoprotein (NP) was introduced into a retroviral vector (mRCAS/NP). NP is an internal influenza virus protein that has been shown to stimulate cytotoxic T-cell responses in influenza-virus-infected mice. Cells infected with mRCAS/NP expressed approximately 10% of the level of NP observed in influenza-virus-infected chicken embryo fibroblasts. Immunocompetent chicks were vaccinated intramuscularly with approximately 1 x 10(5) NP-expressing units of mRCAS/NP. Four weeks later, chicks were bled and challenged with a highly pathogenic avian influenza virus (A/Chicken/Victoria/1/85). The NP-expressing vector stimulated an influenza-virus-specific response, as indicated by the presence of antibody to NP, but failed to protect against the lethal challenge.

Animals↗
PubMed1992

The effects on lipid metabolism of 4-phenyl-5,5-dicarbethoxy-2-pyrrolidenone in Sprague Dawley rats.

4-Phenyl-5,5-dicarbethoxy-2-pyrrolidenone [XIV] treatment in Sprague Dawley rats caused significant reduction of serum cholesterol and triglyceride levels with reduction of VLDL and LDL cholesterol levels. The compound significantly reduced regulatory enzyme activities, e.g. ATP dependent citrate lyase, HMG CoA reductase, acyl CoA cholesterol acyl transferase, cholesterol-7-alpha-hydroxylase, sn-glycerol-3-phosphate acyl transferase and phosphatidylate phosphohydrolase. In tissue cultured cells, the compound suppressed LDL receptor activity and degradation, and elevated HDL receptor activity and HDL degradation. Rat bile cholesterol and phospholipids were elevated; however, overall bile acids were reduced. In situ loop studies suggest that the agent interfered with interhepatic reabsorption of cholesterol and cholic acids. At the therapeutic dose of compound XIV, no deleterious effects were demonstrated in mice.

Animals↗
PubMed1992

The MRS1 gene of S. douglasii: co-evolution of mitochondrial introns and specific splicing proteins encoded by nuclear genes.

We have developed a rapid and simple methodology to locate yeast genes within cloned inserts, obtain partial sequence information, and construct chromosomal disruptions of these genes. This methodology has been used to study a nuclear gene from the yeast S. douglasii (a close relative of S. cerevisiae), which is essential for the excision of the mitochondrial intron aI1 of S. douglasii (the first intron in the gene encoding subunit I of cytochrome oxidase), an intron which is not present in the mitochondrial genome of S. cerevisiae. We have shown that this gene is the homologue of the S. cerevisiae MRS1 gene, which is essential for the excision of the mitochondrial introns bI3 and aI5 beta of S. cerevisiae, but is unable to assure the excision of the intron aI1 from the coxI gene of S. douglasii. The two genes are very similar, with only 13% nucleotide substitutions in the coding region, transitions being 2.5 times more frequent than transvertions. At the protein level there are 86% identical residues and 7% conservative substitutions. The divergence of the MRS1 genes of S. cerevisiae and S. douglasii, and the concomitant changes in the structure of their mitochondrial genomes is an interesting example of the co-evolution of nuclear and mitochondrial genomes.

Amino Acid Sequence↗
PubMed1992

Effects of isoxazolidine or triazolidine on rat serum lipids in vivo and LDL and HDL binding and degradation in human and rodent cultured cells in vitro.

2-(3,4,5-Trimethoxybenzoyl)-4,4-diethyl-3,5-isoxazolidione (TDI) and 1-acetyl-4-phenyl-1,2,4-triazolidine-3,5-dione (APTD) are two chemically related derivatives which have demonstrated potent hypolipidemic activity in mice at 20 mg/kg/day I.P. for 16 days. The purpose of this study is to correlate in vivo effects of TDI and APTD on rat serum lipoprotein lipids and apoprotein levels as well as plasma clearance and tissue uptake with effects of the agents on tissue cultured cells' LDL and HDL receptor binding, internalization and degradation. This study also correlates in vivo effects of TDI and APTD with endogenous enzyme activities regulated by these high affinity receptors. In rats at 20 mg/kg/day orally serum cholesterol, triglyceride, and VLDL-cholesterol levels were effectively reduced while HDL cholesterol levels were significantly elevated with both agents. These compounds in human hepatocytes lowered LDL receptor binding and degradation, whereas HDL receptor binding and degradation were elevated in human hepatocytes, rat small intestinal epithelium cells, human BG fibroblasts, rat aorta cells and mouse macrophages. These drugs inhibited HMG CoA reductase and sn-glycerol-3-phosphate acyl transferase activities, findings consistent with the observed in vivo reductions in serum cholesterol and triglyceride levels. Both drugs reduced activity of acyl CoA:cholesterol acyl transferase and accelerated activity of neutral cholesterol hydrolase in liver and aorta cells. This modulation by the drugs should reduce disposition of cholesterol esters in these tissues especially aorta wall; this effect was indeed observed in vivo. In the presence of TDI and APTD, HDL uptake of intracellular cholesterol from fibroblasts was accelerated. This was consistent with results from in vivo rat studies showing that HDL clearance was faster after treatment while clearance of LDL slowed. Tissue uptake of HDL and LDL after drug treatment was reduced for the major organs; however the liver accumulation was elevated. The accelerated uptake in the liver was probably due to the observed higher levels of Apo-E and Apo-AI in HDL after drug treatment. Increased excretion of cholesterol from the liver to the bile after drug treatment indicated that the reserve cholesterol transport system by HDL was accelerated by the agents in vivo.

Animals↗
PubMed1992

A human polyadenylation factor is a G protein beta-subunit homologue.

Cleavage stimulation factor (CstF) is one of the multiple factors required for polyadenylation of mammalian pre-mRNAs in vitro. We have shown previously that this factor is composed of three distinct subunits of 77, 64, and 50 kDa, and that the 64-kDa subunit can be UV-cross-linked to RNA in a polyadenylation signal (AAUAAA)-dependent manner. By molecular cloning, the 64-kDa subunit was shown to contain a ribonucleoprotein-type RNA binding domain and a novel repeat structure. To study the functions of the other subunits, we have now isolated cDNAs encoding the 50-kDa subunit of human CstF. This subunit shares extensive homology with mammalian G protein beta-subunits and has a characteristic repeat structure (transducin repeat), in which an approximately 44-amino acid-long sequence is repeated seven times. To our knowledge, the 50-kDa subunit is the first example of a functional beta-subunit-like protein in vertebrates. Possible roles of the transducin repeat, both in CstF function specifically and in other beta-subunit homologues more generally, are discussed.

Amino Acid Sequence↗
PubMed1992

The RNP motif protein family.

RNA-protein interactions play critical roles in a variety of important processes in the cell, such as mRNA splicing and translation. Many RNA binding proteins contain an approximately 80-aminoacid conserved sequence, the RNP motif, that has been shown in several cases to constitute the binding domain. As a class, RNP motif proteins bind many different RNAs with varying degrees of sequence specificity. A major area of current interest is the elucidation of the structure-function relationships in these proteins: the structure of the motif and the residues that are important for sequence-specific binding, the roles of the individual motifs in proteins with multiple RNP motifs, and the functions of other domains found in RNP motif proteins. Recent experiments have provided some insight into these issues and have paved the way for a more detailed understanding of this protein family.

Amino Acid Sequence↗
PubMed1992

Identification of residues in GTPase-activating protein Src homology 2 domains that control binding to tyrosine phosphorylated growth factor receptors and p62.

Ras GTPase-activating protein (GAP) contains two Src homology 2 (SH2) domains which are implicated in binding to tyrosine-phosphorylated sites in specific activated growth factor receptors and to a cytoplasmic tyrosine-phosphorylated protein, p62. We have used site-directed mutagenesis of the two GAP SH2 domains (SH2-N and SH2-C) to identify residues involved in receptor and p62 binding. A bacterial fusion protein containing the precise SH2-N domain, as defined by sequence homology, associated with both the activated beta platelet-derived growth factor receptor and epidermal growth factor receptor, and p62 in vitro. However, short deletions at either the N or C termini of the SH2-N domain abolished binding, suggesting that the entire SH2 sequence is required for formation of an active domain. Conservative substitutions of 2 highly conserved basic residues in the SH2-N domain, an arginine and a histidine, resulted in complete loss of receptor and p62 binding, whereas other basic residues, and residues at variable SH2 sites, were more tolerant of substitution. The conserved arginine and histidine therefore appear critical for association with phosphotyrosine-containing proteins, possibly through an interaction with phosphotyrosine. The GAP SH2-C domain, unlike SH2-N, does not bind efficiently to activated receptors or p62 in vitro. The SH2-C domain lacks 3 residues which are otherwise well conserved, and contribute to high affinity SH2-N binding. Replacement of 1 of these residues, a cysteine, with the consensus glycine, conferred SH2-C binding activity toward tyrosine-phosphorylated p62 and epidermal growth factor receptor. Loss-of-function and gain-of-function mutations in the GAP SH2 domains can therefore be used to identify residues that are critical for receptor and p62 binding.

Amino Acid Sequence↗
PubMed1992

Endothelial monocyte-activating polypeptide II. A novel tumor-derived polypeptide that activates host-response mechanisms.

An important means by which tumor cells influence the vasculature is through the production of soluble mediators altering vascular properties. A approximately 22-kDa polypeptide was purified to homogeneity from conditioned medium of murine methylcholanthrene A (meth A) fibrosarcoma cells by ion-exchange chromatography and preparative sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE), based on its ability to induce tissue factor procoagulant activity in endothelial cells (ECs). The final product migrated as a broad band on reduced and nonreduced SDS-PAGE and had an unique amino-terminal sequence. This meth A-derived polypeptide modulated EC coagulant properties through the induction of tissue factor, induced monocyte migration and tissue factor expression, and was also chemotactic for granulocytes. Injection of the polypeptide into mouse footpads resulted in an inflammatory response with tissue swelling and polymorphonuclear leukocyte infiltration. The ability of this mediator to activate ECs and monocytes has led us to name it EMAP II (endothelial monocyte-activating polypeptide). EMAP II is distinct from a previously described approximately 40-kDa meth A-derived polypeptide termed EMAP I. Through its potential to activate host effector mechanisms, EMAP II could contribute to the biology of immunogenic tumors, such as the meth A fibrosarcoma.

Amino Acid Sequence↗
PubMed1992

The expression of the Hu (paraneoplastic encephalomyelitis/sensory neuronopathy) antigen in human normal and tumor tissues.

Using immunohistochemistry or Western blot analysis, the authors have studied the expression of the Hu antigen (a neuronal protein identified by the serum of patients with small cell lung cancer and paraneoplastic encephalomyelitis/sensory neuronopathy) in normal human tissues and 115 tumors of different histologic types. In normal tissue, the Hu antigen is highly restricted to the nervous system. In lung tumors, the Hu antigen is restricted in its expression to all small cell carcinomas. A few other neuroendocrine-related tumors, especially neuroblastomas (50%), also express the antigen.

Carcinoma, Small Cell↗
PubMedDate not supplied

Molecular analysis of the fragile X syndrome.

Carriers of the fragile X mutation possess more than the normal number of copies of a trinucleotide repeat (CGG) within the coding region of a gene designated as FMR-1 in Xq27. The clinical phenotype is determined by the number of copies of the CGG repeat. DNA-based methods for the detection of the fragile X mutation are now available which greatly assist in the genetic diagnosis of this disorder. Direct detection of the mutation enables the identification of fragile X negative normal transmitting males and fragile X negative carrier females.

Fragile X Messenger Ribonucleoprotein 1↗
PubMed1992

Iron regulates the activity of the iron-responsive element binding protein without changing its rate of synthesis or degradation.

The iron-responsive element binding protein (IRE-BP) interacts with specific sequence/structure motifs (iron-responsive elements) within the mRNAs encoding ferritin and the transferrin receptor and thereby post-transcriptionally regulates the expression of these two proteins involved in cellular iron homeostasis. The activity of the IRE-BP is itself regulated by iron such that when cells are treated with an iron source, the RNA binding activity is decreased. The expression of recombinant human IRE-BP in murine cells has been examined as have the expressions of the endogenous IRE-BP of both human and rabbit cells. In all cases, iron down-modulated the RNA binding activity of the IRE-BP, but in no instance was this decrease in activity accompanied by a decrease in the level of the protein as judged by quantitative Western blots. Moreover, the rate of synthesis of the IRE-BP and its rate of degradation have been found to be unaltered by iron manipulation of cells in culture. Consistent with IRE-BP regulation occurring post-translationally, the iron regulation of its activity was found to be unaffected by cycloheximide. These data are discussed in terms of a model of IRE-BP regulation involving the modification of the protein's iron-sulfur center.

Animals↗
PubMed1992

Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of beta-tropomyosin pre-mRNA.

Previous studies of alternative splicing of the rat beta-tropomyosin gene have shown that nonmuscle cells contain factors that block the use of the skeletal muscle exon 7 (Guo, W., Mulligan, G. J., Wormsley, S., and Helfman, D. M. (1991) Genes & Dev. 5, 2095-2106). Using an RNA mobility-shift assay we have identified factors in HeLa cell nuclear extracts that specifically interact with sequences responsible for exon blockage. Here we present the purification to apparent homogeneity of a protein that exhibits these sequence specific RNA binding properties. This protein is identical to the polypyrimidine tract binding protein (PTB) which other studies have suggested is involved in the recognition and efficient use of 3'-splice sites. PTB binds to two distinct functional elements within intron 6 of the beta-tropomyosin pre-mRNA: 1) the polypyrimidine tract sequences required for the use of branch points associated with the splicing of exon 7, and 2) the intron regulatory element that is involved in the repression of exon 7. Our results demonstrate that the sequence requirements for PTB binding are different than previously reported and shows that PTB binding cannot be predicted solely on the basis of pyrimidine content. In addition, PTB fails to bind stably to sequences within intron 5 and intron 7 of beta-TM pre-mRNA, yet forms a stable complex with sequences in intron 6, which is not normally spliced in HeLa cells in vitro and in vivo. The nature of the interactions of PTB within this regulated intron reveals several new details about the binding specificity of PTB and suggests that PTB does not function exclusively in a positive manner in the recognition and use of 3'-splice sites.

Alternative Splicing↗
PubMedDate not supplied

Nephrotropic properties demonstrated by A/chicken/Alabama/75 (H4N8) following intravenous challenge of chickens.

Tissue tropism properties of A/chicken/Alabama/75 (H4N8) were examined after intravenous inoculation of 5-week-old specific-pathogen-free chickens. From 14 clinically normal chickens euthanatized on days 1-20 postinoculation, the frequencies of virus recovery were highest for cloacal swabs (86%), bursal swabs (64%), and kidney tissues (64%) and lowest for tracheal swabs (14%), thymus tissues (14%), bone-marrow swabs (7%), and brain tissues (0%). Evidence that the high frequency of virus recovery from kidney tissues was associated with virus replication in the kidney tissues was provided by high virus titers, ranging up to 10(9.5) mean embryo infectious dose per gram of kidney tissue, and by identification of intranuclear and intracytoplasmic type A influenza nucleoprotein in kidney cells using immunohistochemistry. Virus-recovery and virus titer results from three chickens that died on days 4 and 5 postinoculation paralleled the results from the clinically normal chickens. These findings indicate that A/chicken/Alabama/75 has nephrotropic properties similar to nephrotropic properties previously reported for waterfowl-origin type A influenza viruses and provide evidence that kidney lesions could be manifestations of systemic influenza infections in commercial laying chickens.

Animals↗