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PubMedDate not supplied

[Influence of dibutyryl cyclic AMP and theophylline on the frequency of nuclear microtubules and microfilaments of sympathetic neurons].

In vivo dibutyryl cyclic AMP and theophylline, as does electrical stimulation, increase the frequency of nuclear microtubules and microfilaments in neurons of cat stellate ganglia. These and previous results lead us to conclude that cyclic AMP mediates the process of assembly of these two structural components from preformed protein subunits and thus triggers the formation of highly organized nuclear inclusions corresponding to the so-called intranuclear rodlets of light microscopy.

Animals↗
PubMed1991

Stress proteins and cardiovascular disease.

Understanding the molecular basis by which cells of the heart and blood vessels adapt to physiological stress conditions is an important goal for cardiovascular investigators. The ubiquitous heat shock response provides a model for cellular adaptations to metabolic stresses that are encountered in cardiac disease. Stress-induced synthesis of a family of highly conserved proteins serves to protect cells from injury. In addition, members of this family have essential roles in protein processing and assembly of macromolecular complexes, and in regulation of gene expression, even in unstressed cells. Research concerning the regulation and function of stress proteins potentially is pertinent to the pathophysiology of myocardial hypertrophy, remodeling, and failure, to age-related changes in the cardiovascular system, as well as to ischemic heart disease.

Animals↗
PubMed1991

Evidence supporting a passive role for the insulin receptor transmembrane domain in insulin-dependent signal transduction.

We previously have demonstrated that intramolecular interactions between alpha beta-alpha beta subunits are necessary for insulin-dependent activation of the protein kinase domain within a single alpha 2 beta 2 heterotetrameric insulin-receptor complex (Wilden, P. A., Morrison, B. D., and Pessin, J. E. (1989) Biochemistry 28, 785-792). To evaluate the role of the beta subunit transmembrane domain in the insulin-dependent signalling mechanism, mutant human insulin receptors containing a series of nested transmembrane domain deletions (amino acids 941-945) were generated and stable Chinese hamster ovary-transfected cell lines were obtained. In addition, a substitution of Val-938 for Glu (E/V938) similar to the oncogenic mutation found in the neu transmembrane domain was also introduced into the insulin receptor. Scatchard analysis of insulin binding to the stable Chinese hamster ovary cell lines expressing either wild type or mutant insulin receptors indicated equivalent receptor number (2-4 x 10(6)/cell) and similar high affinity binding constants (Kd 0.1-0.3 nM). 125I-Insulin affinity cross-linking demonstrated that all of the expressed insulin receptors were assembled and processed into alpha 2 beta 2 heterotetrameric complexes. Surprisingly, all the mutant insulin receptors retained insulin-stimulated autophosphorylation both in vivo and in vitro. Furthermore, endogenous substrate phosphorylation in vivo as well as insulin-stimulated thymidine incorporation into DNA were unaffected by the transmembrane domain mutations. These data demonstrate that marked structural alterations in the insulin receptor transmembrane domain do not interfere with insulin-dependent signal transduction.

Amino Acid Sequence↗
PubMed1990

Colchicine binding to tubulin from brain homogenates in the presence of sugars, glycols and metal ions. Effect of nickel ions on the tubulin solubility.

The fact that glycerol preserves microtubules from depolymerizing in vitro, and that some ions such as Ca(II) and Mg(II), regulate the assembly-disassembly process of these structures, induced us to study the effect of several sugars, glycols and metal ions on solubility and colchicine affinity of tubulin in rat brain homogenates, and of purified microtubular protein. Inhibition of colchicine binding was significant with glycerol, polyethylene glycol 1000 (PEG-2) and the ions Al(III), Co(II), Ni(II), while compounds structurally related to glycerol (glucose and sucrose) did not inhibit it. Mannitol, instead, increased the activity a 47% over control. Apparently the presence of some compounds in brain homogenates [PEG-2 (1000) and Ni(II)] favored tubulin sedimentation when these latter were centrifuged at 100,000 x g for 150 min at 20 degrees C, but the form in which tubulin becomes aggregated in the pellet is unknown. Nickel ion made insoluble microtubular protein of homogenates and the purified one by more than 90% without causing significant inhibition of the colchicine binding. The sediment containing nickel-treated two cycles purified microtubular protein observed with the electron microscope did not present microtubules, but it revealed the presence of irregular, wavy and stretched structures bearing highly dense dotted material. The sediments became soluble in phosphate-glutamate buffer (pH 6.8) and, when incubated in polymerizing conditions, gave rise to microtubules undistinguishable from those prepared with untreated purified protein.

Aluminum↗
PubMed1990

The use of the orthographic lexicon in reading kana words.

A lexical decision task was used to investigate the dual-route hypothesis that, in reading words conventionally written in katakana, a Japanese syllabic script, lexical access may be achieved by both a process of assembled segmental phonology and the use of a visual orthographic lexicon. The variables examined were lexicality (words and nonwords), kana type (katakana and hiragana), string length (long and short), and vocal interference (silence and concurrent vocalization). The main effects of the first three variables were significant, but more important, we found that subjects could be divided into two groups--phonological assemblers and orthographic lexicon users. Words that assume sighting were also identified. Collectively, the results were interpreted to uphold the dual-route hypothesis and to reject the common view that kana words are accessed on the basis of phonological mediation alone.

Adult↗
PubMed1988

Synthesis and assembly of a functionally active recombinant platelet-derived growth factor AB heterodimer.

A Chinese hamster ovary cell line that stably expresses transfected human platelet-derived growth factor (PDGF) A and B chain precursors was established. All three dimeric combinations of PDGF chains were produced by this cell line; their biosynthesis, assembly, and processing were followed by pulse-chase analyses. PDGF-AA, PDGF-AB, and PDGF-BB were processed to Mr values of about 30,000 and were accumulated in these forms in the medium. In addition, PDGF-BB was further processed to a 24-kDa component, which remained cell-associated. The major secreted component was PDGF-AB, which was purified and shown to have structural and functional characteristics indistinguishable from PDGF-AB purified from human platelets.

Animals↗
PubMed1988

Biosynthesis of the plasma membrane H+-ATPase of Neurospora crassa.

The plasma membrane H+-ATPase of Neurospora is a 100-kDa integral membrane protein which appears, on the basis of hydropathy analysis of its amino acid sequence, to span the lipid bilayer at least eight times. To investigate the assembly and processing of the ATPase, a full-length cDNA has been constructed for use in in vitro transcription and translation experiments. Comparison of three different forms of the ATPase (nascent protein, nascent protein cotranslationally inserted into membranes, and mature protein) revealed no difference in electrophoretic mobility. Furthermore, the nascent and mature forms gave identical peptide patterns after partial proteolysis with Staphylococcus aureus V8 protease, suggesting that the ATPase does not contain an NH2-terminal signal peptide which is cleaved upon membrane insertion. Consistent with this interpretation, the NH2-terminal peptide has been purified from a tryptic digest of the ATPase and found to lack only the initiator methionine residue; the penultimate alanine is acetylated based on analysis by fast atom bombardment mass spectroscopy. Although the ATPase contains one potential site of N-linked glycosylation, its electrophoretic mobility was unchanged following digestion with endoglycosidase H and it did not incorporate [3H]mannose or bind concanavalin A. Thus, the Neurospora plasma membrane-ATPase appears to undergo minimal post-translational processing, and its membrane insertion is probably mediated by internal sequences.

Amino Acid Sequence↗
PubMedDate not supplied

[The role of structural changes in T4 bacteriophage tail proteins].

Conformational changes in bacteriophage tail proteins after heating and ionic strength alteration leading to dissociation of tail sheath have been studied using protein fluorescence, differential scanning microcalorimetry and electron microscopy methods. Autonomous structural changes in tube-baseplate proteins have been revealed. They take place under the same conditions as those which release the bonds holding the sheath protein subunits to those of the tube in isolated sheathed tails. The conformational changes in the tube-baseplates are reversible similarly to the process of assembly and disassembly of the extended sheath. Morphological changes in the tube have been found at the temperature above the transition registered by protein fluorescence but not by calorimetry. This suggests that revealed spectral alterations reflect changes in quaternary structure of tail tube in particular.

Calorimetry, Differential Scanning↗
PubMed1983

Intranuclear membranes (vesicles, lamellae, annulate lamellae) in oocytes of the ascidian, Styela partita.

Transmission electron microscope studies on developing oocytes of Styela partita that were preserved with a primary fixative of buffered glutaraldehyde and a secondary fixative of buffered osmium tetroxide have dealt with the origin, distribution, and associations of intranuclear annulate lamellae in both previtellogenic and vitellogenic oocytes. These studies (1) substantiate that, developmentally, intranuclear vesicles appear to precede differentiated intranuclear annulate lamellae although intranuclear vesicles and intranuclear annulate lamellae may coexist in the same nucleus (germinal vesicle), (2) substantiate an earlier demonstration that the inner membrane of the nuclear envelope forms blebs which could, after detachment, become intranuclear vesicles, (3) confirm that the pore-associated material of the pores in intranuclear annulate lamellae appears to be structurally identical to that of the pores in the nuclear envelope, (4) demonstrate that at certain times during oogenesis electron-dense material inside the nucleus, thought to represent nucleolar-derived material, is continuous with the pore-associated material of pores in both intranuclear annulate lamellae and the nuclear envelope, (5) propose a function for intranuclear annulate lamellae that considers the pores to play a role in processing or assembling various RNA's and proteins to become cytoplasmic polyribosomes as has recently been suggested also for the pores in the nuclear envelope as well as pores in cytoplasmic or extranuclear annulate lamellae, and (6) provide additional evidence that intranuclear annulate lamellae are not remnants of the nuclear envelope persisting after mitosis as has been described to be the case in certain other cells.

Animals↗
PubMed1984

Dissociation of heme metabolic activities from the microsomal cytochrome P-450 turnover in testis of hypophysectomized rats.

In the rat testis, 7 days after hypophysectomy, the microsomal content of cytochrome P-450 decreased to a negligible level. The sodium dodecyl sulfate gel electrophoresis of the microsomal preparation did not reveal a decrease in apocytochrome P-450; however, in this preparation, heme was undetectable. The latter did not reflect decreases in the activities of the heme biosynthesis enzymes. Also, an increase in heme oxygenase activity did not appear responsible for the suppression of the cytochrome levels. The cellular basis for the depression of the cytochrome was explored by measuring the incorporation of [14C]delta-aminolevulinate into the testicular microsomal and mitochondrial hemoproteins, and determining the relative affinity of microsomal heme for the endoplasmic reticulum membranes. In comparison with the sham-operated animals, in hypophysectomized rats, the specific 14C activity of heme in mitochondrial fraction was not decreased; however, that of the microsomal fraction was markedly reduced. The latter appeared to reflect a lowered binding affinity of the apoprotein moiety of cytochrome P-450 for heme. The treatment of hypophysectomized rats with human chorionic gonadotropin partially restored the normal level of the cytochrome. It is suggested that the anterior pituitary hormones control the level of cytochrome P-450 in the testis through factors which do not involve the production of heme; rather, the control appears to involve the processes of assembly of the hemoprotein and the association of the heme molecule with the apoprotein.

5-Aminolevulinate Synthetase↗
PubMed1980

The incorporation of radioactive lysine or tyrosine into cardiac and skeletal myofibrillar and non-myofibrillar contractile proteins.

The labelled amino acids incorporation into cardiac and skeletal contractile proteins has been compared after a single injection of 3H lysine, repeated injections of 3H lysine during 1 or 6 hours or after a continuous infusion of both 3H-lysine and 14C-tyrosine. The myofibrillar incorporation was higher in the heart than in the skeletal muscle. Myosin heavy chains and actin have been prepared using gel filtration. The incorporation was again higher in the heart for both these proteins but the labelling of actin in both the muscles reaches rapidly a plateau in contrast with myosin, suggesting that these two proteins possess a different precursor pool. Myosin heavy chains prepared from the supernatant obtained after a relaxing treatment were more labelled than those extracted from myofibrils. These heavy chains from the supernatant were presumably newly synthetized and not yet incorporated into myofibrils. They also were more labelled in the heart than in the skeletal muscle which means that the myosin synthesis itself was different and not the process of assembly of the myofibrils.

Actins↗
PubMed1980

Proteolysis of the C5b-7 complex: cleavage of the C5b and C6 subunits and its effect on the interaction of the complex with phospholipid bilayers.

The present study was conducted to gain insight into the process of assembly of the C5b-7 complex on the phospholipid bilayer. The C5b-6 complex, C7, and 2 different forms of the C5b-7 complex (the C5b-7 complex either bound to artificial phospholipid bilayers or in fluid phase) were digested with trypsin, and the resulting products were analyzed by SDS-polyacrylamide gel electrophoresis. The alpha'-chains of C5b and C6 in the C5b-6 complex and C7 were susceptible to trypsin. The formation of the C5b-7 complex from the C5b-6 complex and C7 was accompanied by alteration in susceptibility to trypsin to C7: the C7 subunit of the fluid phase complex as well as of the complex bound to phospholipid vesicles is not susceptible to trypsin. On the other hand, the alpha'-chains of C5b and C6 in the C5b-7 complex bound to phospholipid vesicles as well as in fluid phase remained accessible and susceptible to trypsin, and cleaved as in the C5b-6 complex. Upon ultracentrifugation in a sucrose density gradient, the proteolyzed C5b-7 complex in fluid phase dissociated into subunits. However, none of the subunits in the proteolyzed C5b-7 complex bound to phospholipid vesicles dissociated from vesicles upon sucrose density gradient ultracentrifugation. Thus, the molecule of the C5b-7 complex became stable and resistant to proteolytic dissociation upon binding to the phospholipid bilayer. It is proposed that each subunit of the C5b-7 complex participates in the stable interaction of the complex with the phospholipid bilayer. In addition, the possible significance of the alteration in susceptibility to trypsin of C7 associated with the formation of the C5b-7 complex is discussed.

Animals↗
PubMed1995

A human-mouse chimeric Lym-1 monoclonal antibody with specificity for human lymphomas expressed in a baculovirus system.

A murine anti-human B-cell monoclonal antibody, Lym-1, has shown considerable promise for the treatment of human malignant lymphomas and has been utilized as a new radioimmunotherapy for refractory lymphoma. In order to enhance its clinical potential, a genetically engineered chimeric Lym-1 (chLym-1) with murine variable regions and human gamma 1 and kappa constant regions was constructed and expressed. The goal of this study was to generate a Lym-1 reagent with decreased immunogenicity and improved effector functions. Murine Lym-1 variable region cDNAs were isolated from the murine Lym-1 hybridoma cell line, fused to gamma 1 and kappa constant region cDNAs, and expressed in an insect cell expression system with the baculovirus transfer vector pAcUW31. The chLym-1 antibody expressed in this system was correctly processed and assembled into the expected immunoglobulin monomer. Chimeric Lym-1 bound to both target antigen-bearing Raji cells and a Lym-1 anti-idiotype antibody and had a similar binding affinity as murine Lym-1. The chimeric and murine versions of Lym-1 were assayed for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and to induce complement-mediated cytotoxicity (CMC) against Raji targets. Chimeric Lym-1 mediated a two-fold higher level of ADCC than murine Lym-1 and slightly lower levels of CMC than murine Lym-1. In addition, in Raji lymphoma-bearing nude mice, chLym-1 localized to the tumor with approximately equal uptake at 24 and 72 hours. Chimeric Lym-1, however, cleared from the blood of nontumor-bearing mice approximately 5 times faster than murine Lym-1 (20 h vs. 5 days), as expected for a xenogeneic protein. The improved in vitro and in vivo activities of this genetically engineered monoclonal antibody render it a new potential immunotherapeutic reagent for the treatment of human malignant lymphomas.

Animals↗
PubMed1995

The intracellular transport of MHC class II molecules in the absence of HLA-DM.

The HLA-DM alpha and HLA-DM beta genes encode a nonpolymorphic, class II-like molecule that functions by an as yet undefined mechanism in the assembly of processed antigen-HLA class II complexes. Mutant cells that fail to express HLA-DM are deficient in Ag processing. We previously isolated a subcellular compartment in mouse B cells in which functional processed Ag-class II complexes are first formed, referred to as the peptide-loading compartment. Here, evidence is provided that HLA-DM resides in a subcellular compartment with the characteristics of a peptide-loading compartment in a human B lymphoblastoid cell line, but is not required for the intracellular transport of HLA-DR3 molecules to a corresponding compartment in HLA-DM-deficient cells. Thus, the primary defect in HLA-DM-deficient cells does not appear to be a failure in the intracellular trafficking of class II molecules.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1993

A developmentally regulated glycoprotein complex from Dictyostelium discoideum.

The monoclonal antibody MUD50 recognizes an epitope on a family of developmentally regulated proteins in Dictyostelium discoideum (Alexander, S., Smith, E., Davis, L., Gooley, A., Por, S. B., Browne, L., and Williams, K. L. (1988) Differentiation 38, 82-90). This epitope is an O-linked oligosaccharide that requires the wild-type modB allele for expression. These glycoproteins are structurally diverse. Some are integral membrane proteins, while others are soluble and do not require detergent for solubilization. One of the soluble glycoproteins, PsB, is also recognized by the monoclonal antibody MUD102, which binds to the polypeptide backbone. The antibody recognizes a single 100-kDa band in Western analysis. In this report we demonstrate that the PsB molecule is part of a specific developmentally regulated multiprotein complex containing six different proteins. The complex is held together by both covalent and noncovalent bonds. Interestingly, only the PsB glycoprotein has the MUD50 O-linked oligosaccharide determinant. None of the proteins in the complex is N-glycosylated as assessed by sensitivity to peptide N-glycosidase F. Only one of the complex proteins is heavily phosphorylated. This work establishes the foundation for additional studies on the biosynthesis, processing, and assembly of the O-glycosylated PsB protein.

Animals↗
PubMed1994

[Comorbidity and the physician's decision: the case of undertreatment in care of breast tumors].

When a physician has to come to a decision while caring for a given patient, he is supposed to take into account several kind of information related to the disease, illness or patients' personal characteristics. Although it is wellknown that the framework in which such decision has to be taken is complex, most of the scientific knowledge is based on pieces of evidence that derive from studies where complexity is avoided by applying restriction rules. The goal of this approach is to enhance the internal validity while preserving the generalizability of the findings. But in some cases this approach raises more doubts than certainty. It is the case of the process to assemble practice guidelines to reduce the gap between the best care and what is observed into practice using the available knowledge (i.e., available literature or experts' recommendations). As a variety of relevant clinical and patients' personal characteristics are not available at all or little is known on their impact on patients global health status, variables that actually do drive the practice are not included in guidelines that are intended to change it. This paper introduces the conceptual model of such debate and assesses the impact of co-morbidity--a variable seldom taken into account in effectiveness, quality and appropriateness studies- on the kind of medical care given to a sample of 1019 patients with early stage breast cancer. Empirical results and future implications are eventually discussed.

Adult↗
PubMed1993

Complementation of the radiosensitive phenotype in severe combined immunodeficient mice by human chromosome 8.

Severe combined immunodeficient (scid) C.B-17 mice are deficient in variable (diversity) joining region recombination, the process of assembling the immunoglobulin and T-cell receptor genes from gene segments, thereby creating much of the enormous diversity of antigen-binding capacity, scid mice are also sensitive to ionizing radiation, as a result of their deficiency in double-strand break repair. Here we report the complementation of the radiation-sensitive scid phenotype by transferring human chromosome 8 into scid cells. Somatic cell hybrids were generated by fusing scid cells with human HT-1080 cells, resulting in radioresistant hybrids with several human chromosomes. One of the identified human chromosomes in the radioresistant scid cell line 4.61, which retains only two human chromosomes, is a rearranged 8/21 translocation. Proof that chromosome 8 confers the complementation was achieved by transferring only human chromosome 8 into scid cells by microcell-mediated chromosome transfer (scid/hu8 cell line). The presence of chromosome 8 in our scid/hu8 cell line was monitored by fluorescence in situ hybridization and polymerase chain reaction. We demonstrated the radioresistance of this hybrid not only to high dose rate but also to low dose rate radiation. We also showed that transference of human chromosome 8 to scid cells fully complements the DNA double-strand break repair deficiency and the high sensitivity of scid cells to radiation-induced chromosome aberrations. Mapping the scid gene to human chromosome 8 is an important first step in cloning the scid gene, which will enhance our understanding of double-strand break repair pathways in humans.

Animals↗
PubMed1996

A chimeric Lym-1/interleukin 2 fusion protein for increasing tumor vascular permeability and enhancing antibody uptake.

A murine antihuman B-cell monoclonal antibody, Lym-1, has shown considerable promise for the treatment of human malignant lymphomas. To enhance its clinical potential, a genetically engineered fusion protein consisting of a chimeric Lym-1 (chLym-1) and interleukin 2 (IL-2) was tested for mediating cytotoxicity, increasing vasopermeability, and enhancing antibody uptake in human malignant lymphomas. The chLym-1/IL-2 fusion protein, which was expressed initially in a baculovirus system and more recently in the glutamine synthetase gene amplification system, was shown to be processed and assembled into a normal immunoglobulin monomer with two IL-2 molecules per antibody. It was found to be equivalent to the chLym-1 antibody in antigen-binding specificity and relative affinity. In addition, it maintains IL-2 cytokine activity as demonstrated by support of T-cell proliferation. Moreover, in antibody-dependent cellular cytotoxicity assays against Raji target cells, chLym-1/IL-2 had approximately 2-fold and 4-fold higher cytotoxicity than chLym-1 and murine Lym-1, respectively. Used as a pretreatment, chLym-1/IL-2 enhances the uptake of chLym-1 at the tumor site by altering the permeability of tumor vessels producing tumor:normal organ ratios of 420:1 for blood and 1708:1 for muscle at 3 days. The in vitro and in vivo activities of chLym-1/IL-2, therefore, suggest that this genetically engineered antibody fusion protein may represent a new immunotherapeutic reagent for the treatment of human malignant lymphomas.

Animals↗