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PubMed1996

Nicotinic receptor subunits alpha 3, alpha 4, and beta 2 and high affinity nicotine binding sites are expressed by P19 embryonal cells.

Controlled exposure to retinoic acid (RA) induces the murine embryonal carcinoma cell line P19S18O1A1 (P19) to differentiate into a variety of cell types. One of the cell types exhibits neuronal-like morphology and expresses neuronal markers including neurofilament proteins, glutamate receptors, and the cholinergic enzymes choline acetyl-transferase and acetylcholinesterase. In this study we use Northern blot analysis, double-label immunocytochemistry, and single cell RNA analysis using polymerase chain reaction to show that RA-treated P19 cells with neuronal-like morphology also express neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha 3, alpha 4, and beta 2. Greater than 80% of RA-treated P19 cells with a neuronal-like phenotype express nAChR alpha 4 subunit transcripts and both alpha 4 and beta 2 protein. The RA-induced expression of alpha 3 transcripts accounts for a comparably small number of nAChR-containing cells (< 20%) of which half coexpress alpha 4 transcripts. Expression of high-levels of alpha 4 RNA is dependent upon both cell-cell contact and RA exposure. The appearance of nAChR subunits also coincides with RA-induced expression of high affinity [3H]-nicotine binding receptors. The P19 cell line offers an inducible neuronal cell system to study mammalian neuronal nicotinic receptor expression and the development of high affinity nicotinic binding sites similar to those expressed in the mammalian central nervous system.

Animals↗
PubMed1995

Identification of constitutive and inducible forms of nitric oxide synthase in human platelets.

A number of cell types possess an L-arginine-nitric oxide (NO) pathway. We studied the presence of constitutive and inducible forms of NO synthase in human platelets. N omega-nitro-L-arginine, an inhibitor of NO synthase, potentiated thrombin-induced aggregation of washed human platelets, whereas L-arginine inhibited it. The direct evidence for the presence of constitutive form of NO synthase came from the observation of conversion of tritium-labeled L-arginine to tritium-labeled L-citrulline by washed platelets suspended in Ca(++)-rich but not in Ca(++)-free buffer. Incubation of washed platelets in Ca(++)-free buffer with cytokines (tumor necrosis factor-alpha and interferon-gamma) or cytokines plus lipopolysaccharide caused a marked increase in the conversion of [3H]L-arginine to [3H]L-citrulline, suggesting the presence of inducible form of NO synthase. Gel electrophoresis identified an approximately 130 kd protein band with NO synthase in the platelet cytosol, which on isolation converted [3H]L-arginine to [3H]L-citrulline. This 130 kd protein required the presence of Ca++, reduced nicotinamide adenine dinucleotide phosphate tetrahydro-L-biopterin, and flavin adenine dinucleotide for expression of NO synthase activity. Platelet sonicates demonstrated presence of nitrite, and its concentrations were lowered by preincubation of platelets with NG-nitro-L-arginine methyl ester and enhanced in cytokine-treated platelets. Reverse-transcription polymerase chain reaction demonstrated messenger RNA expression of the constitutive endothelial (but not brain) and inducible isoforms of NO synthase in platelets. These observations indicate that human platelet cytosol possesses both constitutive and inducible forms of NO synthase.

Amino Acid Oxidoreductases↗
PubMed1989

Assembly of hybrid RNAs with tobacco mosaic virus coat protein. Evidence for incorporation of disks in 5'-elongation along the major RNA tail.

We have shown that during the reassembly of tobacco mosaic virus (TMV) RNA, with the coat protein supplied as a "disk preparation", the lengths of RNA protected from nuclease are "quantized" with steps which correspond to incorporation of the subunits from either a single or, more commonly, both rings of a disk. This interpretation has been challenged and it was suggested that the pattern was due to special, though unspecified features of the sequence of TMV RNA. To test whether the specific sequence of TMV RNA is important during the elongation, rather than just during nucleation, we have now followed growth of particles containing hybrid RNAs, with the TMV RNA origin of assembly but otherwise non-TMV sequences. We have prepared in vitro RNA transcripts containing heterologous RNA 5' to the origin of assembly sequence from TMV RNA, i.e. with a heterologous RNA tail in place of the natural major 5'-tail and no minor tail, and used these for assembly experiments. In each case we observe a banding pattern very similar to that which we had found with native TMV RNA and with a dominant quantum step of just over 100 bases, and sometimes also a step of 50 bases, strongly suggesting that this is not due to any feature of the TMV RNA. This same repeat is also visible even with a heterologous RNA chosen because it had a sequence repeat of 135 or 136 bases, confirming that the quantization is due to a feature of the elongation reaction and in no way to the RNA sequence being encapsidated. We have also followed elongation with the origin of assembly located 5' to the heterologous RNA. This leads to a slower elongation along this 3'-tail, after the initial rapid encapsidation of the origin RNA, which lacks any quantization of length protected. These results are fully compatible with the hypothesis we had advanced earlier, that the major growth along the 5'-tail is from performed aggregates ("disks") while the minor growth along the 3'-tail is from subunits in the "A-protein" adding singly or a few at a time.

Base Sequence↗
PubMed1993

Solution oligomerization of the rev protein of HIV-1: implications for function.

rev is an RNA-binding protein of human immunodeficiency virus-1 and is required for the expression of incompletely spliced viral transcripts. Oligomerization of rev is thought to be associated with RNA binding and rev function. Here, we have characterized the oligomerization of rev using equilibrium analytical centrifugation. rev is predominantly monomeric at low concentrations, but reversibly polymerizes to produce large aggregates at higher concentrations. The data fit well to an unlimited isodesmic self-association model in which the association constants for the addition of a monomer to each aggregate are equal [K = 1.08 x 10(6) M-1 at 4 degrees C]. The association constant is essentially independent of monovalent salt concentration from 0.15 to 2 M at pH 6-9. Thermodynamic parameters derived from the temperature dependence of the association constant over the limited range of 0-30 degrees C reveal that the primary contribution to the free energy of oligomerization is a large negative enthalpy. Binding of rev to the rev-responsive element of RNA was characterized under the same conditions as the centrifugation experiments using a nitrocellulose filter assay. rev binds to the RRE at a protein concentration where rev is predominantly monomeric, suggesting that solution multimerization of rev is not required for rev function.

Cloning, Molecular↗
PubMed1994

Use of aryl azide cross-linkers to investigate protein-protein interactions: an optimization of important conditions as applied to Escherichia coli RNA polymerase and localization of a sigma 70-alpha cross-link to the C-terminal region of alpha.

In an effort to better understand protein-protein photoaffinity cross-linking using aryl azides, we have tested a number of factors influencing the cross-linking of the sigma 70 subunit of Escherichia coli RNA polymerase to core RNA polymerase. These factors include the effect of the incubations necessary for the derivatization of the protein on enzyme activity, the effect of overhead lighting on azide stability, the effect of reducing agents on azide stability, aggregation of the derivatized protein, and a comparison of two types of aryl azide cross-linkers, N-[(5-azido-2-nitrobenzoyl)oxy]succinimide (ANB-NOS) and (N-hydroxysuccinimidyl)-4-azidosalicylic acid (NHS-ASA). We found that derivatization proceeds effectively in a buffer similar to the buffer used during protein purification, that overderivatization can cause protein aggregation, that room lighting does not appreciably destroy aryl azides, and that 0.1 mM DTT is a better choice of reducing agent than 5 mM 2-mercaptoethanol. The cross-link products were separated by SDS gel electrophoresis and identified on Western blots by cross-reactivity with monoclonal antibodies to the individual subunits of RNA polymerase. In agreement with previous work (Coggins et al., 1977; Hillel & Wu, 1977), it was possible to cross-link sigma 70 to all three of the subunits of RNA polymerase. With a combination of gel analysis, chemical cleavage, and immunodetection, it is possible to demonstrate that sigma 70 cross-links to the alpha subunit between residues 209 and 329.

Azides↗
PubMed2003

Detection of oncofetal h19 RNA in rheumatoid arthritis synovial tissue.

The expression of oncofetal H19 RNA and its localization/cellular source was analyzed in synovial tissue (ST) and isolated synovial macrophages (Mphi) or synovial fibroblasts (SFBs) by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR showed significantly higher H19 expression in ST from patients with rheumatoid arthritis (RA) (P = 0.000) and osteoarthritis (OA) (P = 0.009) than in normal/joint trauma controls (N/JT), but comparable levels in reactive arthritis. In situ hybridization demonstrated strong signals in all RA-ST samples (n = 8), with > or =85% positive cells in the lining layer, diffuse infiltrates, and stroma regions. In lymphoid aggregates and endothelial cells only 20% were positive. RA-ST contained a significantly higher percentage of strongly positive lining cells than OA-ST and N/JT-ST. H19 RNA was expressed in both Mphi and SFBs, as confirmed by RT-PCR in isolated RA Mphi and SFBs (n = 3). In RA-SFBs, low constitutive H19 RNA expression in culture (10% fetal calf serum) was strongly increased on starvation (3.5-fold, 1% fetal calf serum), with or without the addition of interleukin-1beta (10 to 100 U/ml), tumor necrosis factor-alpha (1 to 25 ng/ml), or platelet-derived growth factor-BB (2.5 to 10 U/ml). In OA-SFBs, this starvation-induced increase was lower (twofold), reaching significant differences compared with RA-SFBs after stimulation with interleukin-1beta and platelet-derived growth factor-BB. In both RA- and OA-SFBs, the MAP-kinase ERK-1/2 pathway and the phosphatidylinositol-3 kinase pathway influenced H19 RNA expression, as shown by inhibitor studies. Significant overexpression of H19 RNA and its increased sensitivity to starvation/cytokine regulation in RA suggests a pathogenetic role of this oncofetal gene, possibly reflecting embryonal dedifferentiation of the adult ST and/or ongoing inflammatory/oxidative stress.

Adult↗
PubMed2006

Role of neurofilament aggregation in motor neuron disease.

A major question in the pathogenesis of motor neuron disease is why motor neurons are selectively susceptible to mutations in widely expressed gene products. Reexamination of motor neuron degeneration due to alterations of neurofilament (NF) expression suggests that disruption of assembly with aggregation of the light neurofilament (NFL) protein may be an upstream event and contributing factor leading to the preferential degeneration of motor neurons. The implications of these findings are that aggregation of NFL is not only a triggering mechanism to account for the hallmark aggregates of NF protein in sporadic and familial forms of amyotrophic lateral sclerosis, but that aggregates of NFL may also promote aggregation of wildly expressed proteins that are destabilized by missense mutations, such as by mutations in superoxide dismutase-1 protein. This review examines the potential role of NFs in determining and promoting the preferential degeneration of motor neurons in motor neuron disease. The underlying premise is that motor neurons are selectively susceptible to alterations in NF expression, that alterations in NF expression lead to NF aggregates in motor neurons, and that elevated levels of NF aggregates provide a favorable microenvironment for the formation of neurotoxic aggregation and degeneration of motor neurons.

Animals↗
PubMed2006

Transforming omics data into context: bioinformatics on genomics and proteomics raw data.

Differential gene expression analysis and proteomics have exerted significant impact on the elucidation of concerted cellular processes, as simultaneous measurement of hundreds to thousands of individual objects on the level of RNA and protein ensembles became technically feasible. The availability of such data sets has promised a profound understanding of phenomena on an aggregate level, expressed as the phenotypic response (observables) of cells, e.g., in the presence of drugs, or characterization of cells and tissue displaying distinct patho-physiological states. However, the step of transforming these data into context, i.e., linking distinct expression or abundance patterns with phenotypic observables - and furthermore enabling a sound biological interpretation on the level of reaction networks and concerted pathways, is still a major shortcoming. This finding is certainly based on the enormous complexity embedded in cellular reaction networks, but a variety of computational approaches have been developed over the last few years to overcome these issues. This review provides an overview on computational procedures for analysis of genomic and proteomic data introducing a sequential analysis workflow: Explorative statistics for deriving a first, from the purely statistical viewpoint, relevant candidate gene/protein list, followed by co-regulation and network analysis to biologically expand this core list toward functional networks and pathways. The review on these procedures is complemented by example applications tailored at identification of disease-associated proteins. Optimization of computational procedures involved, in conjunction with the continuous increase in additional biological data, clearly has the potential of boosting our understanding of processes on a cell-wide level.

Animals↗
PubMed2006

Trypsin and thrombin accelerate aggregation of human endocrine pancreas precursor cells.

Human islet-derived precursor cells (hIPCs) and human pancreatic ductal carcinoma (PANC-1) cells can be induced to form aggregates that subsequently differentiate into hormone-expressing islet-like cell aggregates (ICAs). We show that challenge of hIPCs or PANC-1 cells with thrombin or trypsin resulted in stimulation of signaling via the inositol-tris-phosphate second messenger pathway leading to rapid, transient increases in cytosolic calcium ion concentration in the majority of the cells. Because we found that hIPCs, PANC-1 cells, human fetal pancreas, and human adult islets express two protease-activated receptors (PARs), PAR-1 and PAR-2, we tested whether the effects of thrombin and trypsin were mediated, at least in part, by these receptors. Peptide agonists that are relatively specific for PAR-1 (SFLLRN-amide) or PAR-2 (SLIGRL-amide) stimulated increases in inositol phosphates and cytosolic calcium ion concentration, and increased the phosphorylation of Rho, a small G-protein associated with cytoskeletal changes affecting cellular morphology and migration. Most importantly, we show that these agonists increased the rate of hIPC aggregation leading to the formation of more viable, smaller ICAs. Our data show that thrombin and trypsin accelerate aggregation, an early stage of hIPC differentiation in vitro, and imply that pancreatic trypsin and thrombin may be involved in islet development in vivo.

Calcium↗
PubMed2006

Recurrent hepatitis C after living donor liver transplantation detected by Tc-99m GSA liver scintigraphy.

Recurrence of hepatitis C virus (HCV) after living donor liver transplantation was investigated using technetium-99m- diethylenetriaminepentaacetic acid-galactosyl human serum albumin (Tc-99m-GSA) liver scintigraphy. Four patients with decompensated cirrhosis due to HCV infection were retrospectively reviewed in this study. Scintigraphy was performed to determine the hepatic uptake ratio of the tracer corrected for disappearance from the blood, as well as the maximal removal rate of the tracer by hepatocytes, as parameters of hepatic functional reserve. In all patients, serum HCV ribonucleic acid (RNA) was detected 3 months after transplantation. The corrected hepatic uptake ratio and removal rate showed little change after transplantation in two patients without the recurrence of HCV infection. In another two patients, these levels were decreased at 3 months after transplantation. In one patient, recurrent HCV infection was diagnosed by confirmatory histologic examination at 12 months after transplantation. In the other patient, both levels declined further at 8 months. Although treatment was initiated with a combination of interferon plus ribavirin, this patient died of progressive hepatic failure. In conclusion, a decrease in scintigraphic parameters at 3 months after transplantation suggests recurrent HCV infection affecting the graft. Tc-99m-GSA liver scintigraphy is a useful noninvasive method for evaluating graft functional reserve.

Female↗
PubMed1981

Regulation of terminal differentiation of cultured human keratinocytes by vitamin A.

Vitamin A is known to exert an important influence on epithelial differentiation. The fetal calf serum supplement of cell-culture medium contains enough of the vitamin to affect the differentiation of cultured keratinocytes derived from epidermis and from other stratified squamous epithelia. The cellular and molecular properties of the cultures are altered when the medium is supplemented with serum from which the vitamin A has been removed by solvent extraction (delipidized serum). Cell motility is reduced, the adhesiveness of cells increases and pattern formation is prevented. In both epidermal and conjunctival keratinocytes, removal of vitamin A leads to the synthesis of a 67 kd keratin characteristic of terminally differentiating epidermis and to much reduced synthesis of the 52 kd and 40 kd keratins typical of conjunctiva. These changes, both cellular and molecular, are reversed by the addition of retinyl acetate to the medium containing delipidized serum. Cell motility and pattern formation are restored, and detachment of the most mature cells from the surface of the stratified epithelium is promoted. Synthesis of the 67 kd keratin is prevented and the synthesis of the 40 and 52 kd keratins is stimulated. The nature of the keratins synthesized is regulated by the concentration of vitamin A, and each cell type adjusts its synthesis differently at a given vitamin concentration.

Cell Aggregation↗
PubMed2006

SALM synaptic cell adhesion-like molecules regulate the differentiation of excitatory synapses.

Synaptic cell adhesion molecules (CAMs) are known to play key roles in various aspects of synaptic structures and functions, including early differentiation, maintenance, and plasticity. We herein report the identification of a family of cell adhesion-like molecules termed SALM that interacts with the abundant postsynaptic density (PSD) protein PSD-95. SALM2, a SALM isoform, distributes to excitatory, but not inhibitory, synaptic sites. Overexpression of SALM2 increases the number of excitatory synapses and dendritic spines. Mislocalized expression of SALM2 disrupts excitatory synapses and dendritic spines. Bead-induced direct aggregation of SALM2 results in coclustering of PSD-95 and other postsynaptic proteins, including GKAP and AMPA receptors. Knockdown of SALM2 by RNA interference reduces the number of excitatory synapses and dendritic spines and the frequency, but not amplitude, of miniature excitatory postsynaptic currents. These results suggest that SALM2 is an important regulator of the differentiation of excitatory synapses.

Animals↗
PubMed1992

Cis-acting, orientation-dependent, positive control system activates pheromone-inducible conjugation functions at distances greater than 10 kilobases upstream from its target in Enterococcus faecalis.

The prgB gene encodes the surface protein, Asc10, which mediates cell aggregation, resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline-resistance plasmid pCF10 in Enterococcus faecalis. Messenger RNA analysis by Northern blot hybridization and primer extension indicates that prgB transcription is pheromone-inducible and monocistronic. Previous transposon mutagenesis and sequencing analysis of a 12-kilobase (kb) region of pCF10 indicated that several genes including prgR and prgS are required to activate expression of prgB. The distance (3-4 kb) between these regulatory genes and prgB suggested that the activation might function in trans. To test this, a promoterless lacZ gene fusion to prgB was constructed and cloned without some or all of the regulatory genes. Several restriction fragments of the regulatory region were cloned in a higher copy-number plasmid, and numerous complementation studies were carried out in E. faecalis. Complementation in trans was not observed in any of these experiments. However, when the regulatory region and target genes were cloned in different sites of the same plasmid, separated by as much as 12 kb, activation of prgB was observed. Interestingly, this activation occurred only when the regions were cloned in the same relative orientation in which they exist on wild-type pCF10. These results suggest that one or more regulatory molecules may bind to an upstream cis-acting site and track along the DNA to reach a target site to activate prgB transcription.

Bacterial Proteins↗
PubMed2001

Integrins regulate the intracellular distribution of eukaryotic initiation factor 4E in platelets. A checkpoint for translational control.

Recent evidence from our laboratory demonstrates that platelets synthesize numerous proteins in a signal-dependent fashion (Pabla, R., Weyrich, A. S., Dixon, D. A., Bray, P. F., McIntyre, T. M., Prescott, S. M., and Zimmerman, G. A. (1999) J. Cell Biol. 144, 175-184; Weyrich, A. S., Dixon, D. A., Pabla, R., Elstad, M. R., McIntyre, T. M., Prescott, S. M., and Zimmerman, G. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5556-5561). Protein synthesis in platelets is controlled at the translational level; however, the mechanisms of regulation are not known. Here we demonstrate that translation initiation factors are redistributed to mRNA-rich areas in aggregated platelets, an event that induces protein synthesis. Interrogation of cDNA arrays revealed that platelet-derived mRNAs are primarily associated with the cytoskeletal core. In contrast, eukaryotic initiation factor 4E (eIF4E), the essential mRNA cap-binding protein that controls global translation rates, is localized in the membrane skeleton and soluble fraction of platelets, physically separated from most mRNAs. Platelet activation redistributes eIF4E to the cytoskeleton and increases interactions of eIF4E with mRNA cap structures. Redistribution of eIF4E to the mRNA-rich cytoskeleton coincides with a marked increase in protein synthesis, a process that is blocked when intracellular actin is disrupted. Additional studies demonstrated that beta(3) integrins are the primary membrane receptor that distributes eIF4E within the cell. These results imply that integrins link receptor-mediated pathways with mRNA-rich cytoskeletal domains and thereby modulate the organization of intracellular translational complexes. They also indicate that the functional status of eIF4E is regulated by its intracellular distribution.

Arachidonic Acid↗