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At least 1,009 records · Page 56Linked to original sources
PubMed1978

Characteristics of a coupled cell-free transcription and translation system directed by vaccinia cores.

1. A coupled transcription and translation system is described in which protein synthesis is directed by mRNA synthesised in situ by vaccinia virus cores. The cell-free system is based on a micrococcal-nuclease-treated reticulocyte lysate. 2. The polypeptides made in vitro include many authentic early vaccinia proteins, but also other proteins which were not detected in infected cells. 3. Concentrations of cores which inhibit host cell protein synthesis in vivo caused a delayed inhibition of translation in vitro; this was partly, but not entirely, due to dsRNA associated with the cores. 4. The mRNA made was methylated by core enzymes. Inhibition of methylation reduced the rate of translation tenfold; unmethylated RNA bound ribosomes poorly, but was nevertheless translated faithfully.

Animals↗
PubMed1980

A high production rate of translatable IgG mRNA accounts for the amplified synthesis of IgG in myeloma cells.

The aim of the present work was to determine whether the accumulation of Ig mRNA in myeloma cells is due to a high rate of production or to a high stability of these molecules. Specific mRNAs for the light and heavy polypeptide chains of IgG were isolated from the murine MPC-11 myeloma tumor cells by immune precipitation of polysomes which synthesize these chains. It was found that the immune-precipitated polysomes were enriched 10--30-fold in the gamma and chi mRNA sequences respectively. In the wheat germ cell-free system the chi mRNA preparation was translated mainly into three polypeptides of Mr 25 000, 18 000, and 15 000. The method of immune precipitation of polysomes was also used to characterize three variant clones of MPC-11 myeloma. It was found that little if any gamma-chain polysomes are present in the L-chain producer and non-producer clones, while a substantial amount of chi-chain polysomes was present in the non-producer clone. This may be due to the presence in the non-producer cells of the constant region chi-chain fragment. In order to determine the relative synthesis rate of chi and gamma mRNAs, pulse-labelled polysomes were immune precipitated using antibodies to chi and gamma chains. It was found that chi and gamma mRNA molecules are produced at a very high relative rate each accounting for 10--15% of the total labeled mRNA after 1 h of labeling. These values are higher than the steady-state pool size of chi and gamma mRNA, which was 5--6%, and indicates that the half-life of these molecules is not unusually high. It is concluded that the amplified synthesis of immunoglobulin chains in myeloma cells is mainly due to a high rate of production of translatable chi and gamma mRNAs.

Animals↗
PubMed1981

Regulation of the synthesis of ribulose-1,5-bisphosphate carboxylase and its subunits in the flagellate Chlorogonium elongatum. Different levels of translatable messenger RNAs for the large and the small subunits in autotrophic and heterotrophic cells as determined by immunological techniques.

Poly(A)-rich and poly(A)-free RNAs were isolated from autotrophic and heterotrophic cells of the phytoflagellate Chlorogonium elongatum and translated in an mRNA-depleted reticulocyte lysate system. Immunoprecipitation methods were improved to detect large and small subunits of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase synthesized in vitro. Large-subunit polypeptides were shown to be the translation products of poly(A)-free RNA having the same molecular weight as large subunits made in vivo. Small-subunit polypeptides were synthesized when poly(A)-rich RNA was used as a template. They were made in vitro as a precursor, with an Mr about 6000 larger than mature small subunits. Cells growing heterotrophically in the dark with acetate are provided with lower levels of mRNA activities for the large and the small subunits is at least partially controlled by the amounts of translatable mRNAs.

Animals↗
PubMed1994

Heterotrimeric G proteins involved in the modulation of voltage-dependent calcium channels of neuroendocrine cells.

Various mechanisms have been identified by which hormones and neurotransmitters, interacting with heptahelical receptors, modulate the intracellular Ca2+ concentration in neuronal, endocrine, and neuroendocrine cells. All of them involve heterotrimeric G proteins. Best documented are hormonal stimulations and inhibitions of voltage-dependent Ca2+ channels. Stimulation is caused by agonists interacting with receptors known to induce phosphatidylinositol 4,5-bisphosphate hydrolysis, that is, a PI response. Although the PI response triggers a transient secretion by fast Ca2+ release, the stimulation of Ca2+ channels is assumed to be responsible for prolonged cell responses and for refilling of IP3-sensitive Ca2+ pools after repeated stimulations. Using antisense oligonucleotide microinjection in rat pituitary GH3 cells, Gi2 has been identified as the pertussis toxin-sensitive G protein stimulating Ca2+ channels, whereas Gq/G11 are involved in the concurrent PI response with subsequent protein kinase C activation, which is required for Ca2+ channel stimulation. Inhibitory modulations of Ca2+ channels are assumed to be the basis of inhibitions of transmitter or hormone secretion. Experiments in GH3 cells have revealed that Go subforms composed of alpha o1 x beta 3 x gamma 4 and alpha o2 x beta 1 x gamma 3 are the active G-protein heterotrimers transferring inhibitory signals from muscarinic M4 and somatostatin receptors to the Ca2+ channel, respectively.

Animals↗
PubMed2001

Available space and extracellular transport of macromolecules: effects of pore size and connectedness.

Molecular exclusion in tumor tissues is one of the limiting factors for drug delivery to tumor cells. It can be quantified by the available volume fraction of solutes (K(AV)). We found in a previous study that K(AV) of dextran in tumor tissues decreased sharply when the molecular weight (MW) of dextran was increased from 40,000 to 70,000. Outside this range, K(AV) was less sensitive to the MW of dextran. To understand the mechanisms of the MW dependence of K(AV), we investigated K(AV) in tissue phantoms composed of tumor cells in 1% agarose gels, and performed numerical simulations of the available volume fraction in pore networks. We found that the MW dependence of K(AV) in tissue phantoms was similar to that in tumor tissues when the volume fraction of cells in the former was approximately 30%. Our numerical simulations revealed that the sharp decrease in K(AV) required two necessary conditions: (i) the existence of at least two populations of pores and (ii) the lack of connectedness of available pores in the interstitial space. Furthermore, results in this study suggest that it is important to consider not only the local structures of pores but also their connectedness in analyses of molecular transport in tissues.

Biological Transport, Active↗
PubMed1988

An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly.

Splicing of nuclear precursor messenger RNA (pre-mRNA) occurs on a large ribonucleoprotein complex, the spliceosome. Several small nuclear ribonucleoproteins (snRNP's) are subunits of this complex that assembles on the pre-mRNA. Although the U1 snRNP is known to recognize the 5' splice site, its roles in spliceosome formation and splice site alignment have been unclear. A new affinity purification method for the spliceosome is described which has provided insight into the very early stages of spliceosome formation in a yeast in vitro splicing system. Surprisingly, the U1 snRNP initially recognizes sequences at or near both splice junctions in the intron. This interaction must occur before the other snRNP's (U2, U4, U5, and U6) can join the complex. The results suggest that interaction of the two splice site regions occurs at an early stage of spliceosome formation and is probably mediated by U1 snRNP and perhaps other factors.

Actins↗
PubMed1995

Characterization of Pseudomonas aeruginosa-induced MDCK cell injury: glycosylation-defective host cells are resistant to bacterial killing.

As a model for bacterium-induced epithelial cell injury, we have studied the interaction of Pseudomonas aeruginosa with polarized Madin-Darby canine kidney (MDCK) cells grown on filters. Following an initial period of bacterial adhesion, foci of injured host cells, which consisted of a central region of cell debris, surrounded by cells that were permeable and apparently necrotic, were formed. Host cell death was quantified by measuring the increased permeability of the monolayer to the macromolecular tracer [14C]inulin. Using this MDCK model system, we have identified bacterial and host cell factors necessary for the host cell damage. The ability of P. aeruginosa to cause MDCK cell damage was independent of elastase or exotoxin A production. In contrast, bacteria with a mutation in the regulatory locus exsA (which are deficient in exoenzyme S production) neither bound to nor caused host cell injury. MDCK cells with defects in cell surface glycosylation were resistant to cell injury, indicating that bacteria may require host cell glycolipids and/or glycoproteins as points of adhesion to cause subsequent host cell injury.

Animals↗
PubMed1994

Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens.

Protein phosphatase 2A consists of three subunits, the catalytic subunit (C) and two regulatory subunits (A and B). The A subunit has a rod-like shape and consists of 15 nonidentical repeats. It binds the catalytic subunit through repeats 11 to 15 at the C terminus and the tumor antigens encoded by small DNA tumor viruses through overlapping but distinct regions at N-terminal repeats 2 to 8. A model of the A subunit was developed on the basis of the fact that uncharged or hydrophobic amino acids are conserved at eight defined positions within each repeat. Helical wheel projections suggested that each repeat can be arranged as two interacting amphipathic helixes connected by a short loop. Mutational analysis of the A subunit revealed that the proposed loops are important for binding of tumor antigens, the B subunit, and the C subunit. Native gel analysis of mutant A subunits synthesized in vitro demonstrated that the binding region for the B subunit, previously thought to include repeats 2 to 8, covers repeats 1 to 10 and that the B and C subunits cooperate in binding to the A subunit.

Amino Acid Sequence↗
PubMed1994

Cell-free assembly of the herpes simplex virus capsid.

Herpes simplex virus type 1 (HSV-1) capsids were found to assemble spontaneously in a cell-free system consisting of extracts prepared from insect cells that had been infected with recombinant baculoviruses coding for HSV-1 capsid proteins. The capsids formed in this system resembled native HSV-1 capsids in morphology as judged by electron microscopy, in sedimentation rate on sucrose density gradients, in protein composition, and in their ability to react with antibodies specific for the HSV-1 major capsid protein, VP5. Optimal capsid assembly required the presence of extracts containing capsid proteins VP5, VP19, VP23, VP22a, and the maturational protease (product of the UL26 gene). Assembly was more efficient at 27 degrees C than at 4 degrees C. The availability of a cell-free assay for HSV-1 capsid formation will be of help in identifying the morphogenetic steps that occur during capsid assembly in vivo and in evaluating candidate antiherpes therapeutics directed at capsid assembly.

Capsid↗
PubMed2006

Polymersomes.

Polymersomes are self-assembled polymer shells composed of block copolymer amphiphiles. These synthetic amphiphiles have amphiphilicity similar to lipids, but they have much larger molecular weights, so for this reason--along with others reviewed here--comparisons of polymersomes with viral capsids composed of large polypeptide chains are highly appropriate. We summarize the wide range of polymers used to make polymersomes along with descriptions of physical properties such as stability and permeability. We also elaborate on emerging studies of in vivo stealthiness, programmed disassembly for controlled release, targeting in vitro, and tumor-shrinkage in vivo. Comparisons of polymersomes with viral capsids are shown to encompass and inspire many aspects of current designs.

Biocompatible Materials↗
PubMed2007

Drug targeting to the brain.

The central nervous system (CNS) is a sanctuary site and is protected by various barriers. These regulate brain homeostasis and the transport of endogenous and exogenous compounds by controlling their selective and specific uptake, efflux, and metabolism in the brain. Unfortunately, potential drugs for the treatment of most brain diseases are therefore often not able to cross these barriers. As a result, various drug delivery and targeting strategies are currently being developed to enhance the transport and distribution of drugs into the brain. Here we discuss briefly the biology and physiology of the blood-brain barrier (BBB) and the blood-cerebro-spinal-fluid barrier (BCSFB), and, in more detail, the possibilities for delivering large-molecular-weight drugs by local and global delivery and by viral and receptor-mediated nonviral drug delivery to the (human) brain.

Biological Transport↗
PubMed1980

Role of intracellular proteins in the regulation of calcium action and transmitter release during exocytosis.

A central problem in cellular neurobiology is how the process of exocytosis of transmitters and hormones is regulated at the molecular level. Calcium is important in the process and may act by initiating the formation of fusion complexes of secretory granules to each other and to plasma membranes. We have discovered and isolated a new 47,000 MW protein (synexin) from adrenal medulla tissue that fuses chromaffin granule membranes only in the presence of calcium. Synexin activity was detected in a number of secretory tissues including human platelets and bovine brain, and the synexin molecule was found by immunofluorescent cytochemistry to be localized to the cytoplasm of chromaffin cells. Purified synexin molecules were found to self-associate in the presence of Ca++ to form paracrystalline arrays of 50 X 150 A rods, and the association was dependent on [Ca++] in an identical fashion to the Ca++ dependence of granule membrane fusion. On the basis of these data we suggest that synexin may be the intracellular receptor for calcium during exocytosis. However, the actual release event of 'fission' of the secretory vesicle-plasma membrane complex did not appear to be related to synexin action, and we have considered the hypothesis that the chemiosmotic mechanism for ATP, Cl--dependent chromaffin granule lysis might provide the necessary localized force. We have now shown that the granule model successfully predicts the secretory properties of human platelets, bovine parathyroid cells, and bovine chromaffin cells. Like the granule lysis system, secretion from these cells required specific anions in the medium was inhibited by anion transport blocking drugs and proton ionophores, and was suppressed by elevated osmotic strength. We suggest that secretory granules fused to plasma membranes in secreting cells, perhaps by synexin, may experience net solute uptake and subsequently undergo local, outwardly directed osmotic lysis, or exocytosis.

Adrenal Medulla↗
PubMed1990

Interactions of biospecific functional polymers with blood proteins and cells.

Biospecific functional polymers, i.e. synthetic or artificial polymers substituted with specific chemical functional groups carried by the macromolecular chain are designed to interact with living systems. These polymers are either insoluble or soluble, derived from polystyrene and dextran. Polymers substituted with aryl sulfonate and carboxyl groups specifically interact with antithrombin III and serine-proteases involved in the coagulation of blood. As a consequence, these polymers possess heparin-like activity and are therefore of low thrombogenicity when exposed to flowing blood. Other functional polymers have been prepared in order to interact with various components of the immune system. Soluble and insoluble functional polymers in contact with cells can affect both cell proliferation and metabolism. Some functional polymers have the ability to inhibit or to stimulate cell growth while others can alter cell function without a change in growth characteristics. The functional polymers described have possible applications as plasma expanders, non-thrombogenic catheters, non-complement activating surfaces and other applications in oncology, biotechnology and immunochemistry.

Adsorption↗
PubMed2003

Amphiphilic star-like macromolecules as novel carriers for topical delivery of nonsteroidal anti-inflammatory drugs.

The objective of this study was to evaluate amphiphilic star-like macromolecules (ASMs) as a topical drug delivery system. Indomethacin, piroxicam, and ketoprofen were individually encapsulated into the ASMs using coprecipitation. The effects of the ASMs on percutaneous permeation of nonsteroidal anti-inflammatory drugs (NSAIDs) across full thickness, hairless mouse skin were evaluated in vitro using modified Franz diffusion cells. In addition, solubility and in vitro release experiments were performed to characterize ASMs behavior in aqueous media. Poly(ethylene glycol) (PEG) and Pluronic P-85 were used as polymer controls to compare the role of PEG and amphiphilic behavior in the ASMs. In vitro release experiments indicated that ASMs can delay drug release (P <.05), whereas solubility measurements showed that ASMs can increase NSAIDs aqueous solubility (P <.05). Percutaneous permeation studies revealed that ASMs decreased both flux and Q24 of drugs compared with the control (P <.10). Skin pretreatment studies with ASM-containing solution before drug application demonstrated that pretreatment similarly influenced NSAID percutaneous permeation. In conclusion, ASMs likely slow drug permeation through 2 mechanisms, delayed drug diffusion from its core and skin dehydration by its shell. Thus, ASMs may be useful for delayed dermal delivery or prevention of compound permeation through the skin (eg, sunscreens, N,N-diethyl-m-toluamide [DEET]) from aqueous formulations.

Administration, Topical↗
PubMed1995

Structure-activity relationships of phenyl- and benzoylpyrroles.

Antitumor, antimicrobial, and phytotoxic activities of the marine antibiotic pentabromopseudilin (1a) and related phenyl-, benzyl- and benzoyl pyrroles were compared. All activities depended strongly on the substituent pattern, with the natural compound 1a being the most active one. As judged from model reactions, a covalent bond of nucleophiles to the pyrrole system may be involved in the inhibition of macromolecular syntheses.

Animals↗
PubMed1993

Establishment and characterization of a feline large granular lymphoma cell line expressing interleukin 2 receptor alpha-chain.

A feline large granular lymphocyte (LGL) cell line was established from a cat with an alimentary-form lymphoma. This cell line, designated as FGL, had many large azurophilic granules in the cytoplasm, which were typical to LGL cells. Proviral genome of feline leukemia virus was detected in the chromosomal DNA of FGL cells, and reverse transcriptase activity was also demonstrated in the culture supernatant. Furthermore, we found expression of interleukin 2 (IL-2) receptor alpha-chain on the cell surface of FGL and its natural killer activity against human erythroblastic leukemia cell line, K562.

Animals↗
PubMed2004

Using NMRView to visualize and analyze the NMR spectra of macromolecules.

Nuclear magnetic resonance (NMR) experiments on macromolecules can generate a tremendous amount of data that must be analyzed and correlated to generate conclusions about the structure and dynamics of the molecular system. NMRView is a computer program that is designed to be useful in visualizing and analyzing these data. NMRView works with various types of NMR datasets and can have multiple datasets and display windows opened simultaneously. Virtually all actions of the program can be controlled through the Tcl scripting language, and new graphical user interface components can be added with the Tk toolkit. NMR spectral peaks can be analyzed and assigned. A suite of tools exists within NMRView for assigning the data from triple-resonance experiments.

Algorithms↗