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Biomedical subjects

M Frame

Publications and source records attributed to M Frame.

18 recordsLinked to original sources
PubMed2002

The role of the gut flora in health and disease, and its modification as therapy.

The gut flora is a vast interior ecosystem whose nature is only beginning to be unravelled, due to the emergence of sophisticated molecular tools. Techniques such as 16S ribosomal RNA analysis, polymerase chain reaction amplification and the use of DNA microarrays now facilitate rapid identification and characterization of species resistant to conventional culture and possibly unknown species. Life-long cross-talk between the host and the gut flora determines whether health is maintained or disease intervenes. An understanding of these bacteria-bacteria and bacteria-host immune and epithelial cell interactions is likely to lead to a greater insight into disease pathogenesis. Studies of single organism-epithelial interactions have revealed the large range of metabolic processes that gut bacteria may influence. In inflammatory bowel diseases, bacteria drive the inflammatory process, and genetic predisposition to disease identified to date, such as the recently described NOD2/CARD15 gene variants, may relate to altered bacterial recognition. Extra-intestinal disorders, such as atopy and arthritis, may also have an altered gut milieu as their basis. Clinical evidence is emerging that the modification of this internal environment, using either antibiotics or probiotic bacteria, is beneficial in preventing and treating disease. This natural and apparently safe approach holds great appeal.

Bacteria↗
PubMed2001

The inhibitory gamma subunit of the type 6 retinal cyclic guanosine monophosphate phosphodiesterase is a novel intermediate regulating p42/p44 mitogen-activated protein kinase signaling in human embryonic kidney 293 cells.

The inhibitory gamma subunits of the retinal rod and cone photoreceptor type 6 retinal cyclic guanosine monophosphate phosphodiesterase (PDEgamma) are expressed in non-retinal tissues. Here, we show that PDEgamma interacts with the G-protein-coupled receptor kinase 2 signaling system to regulate the epidermal growth factor- and thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase in human embryonic kidney 293 cells. This is based upon several lines of evidence. First, the transfection of cells with an antisense rod PDEgamma plasmid construct, which reduced endogenous rod PDEgamma expression, ablated the epidermal growth factor- and thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase. Second, the transfection of cells with recombinant rod or cone PDEgamma and/or G-protein-coupled receptor kinase 2 increased the stimulation of p42/p44 mitogen-activated protein kinase by epidermal growth factor or thrombin. In contrast, a G-protein-coupled receptor kinase 2 phosphorylation-resistant rod PDEgamma mutant failed to increase the epidermal growth factor- or thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase and, in fact, functioned as a dominant negative. Thrombin also stimulated the association of endogenous rod PDEgamma with dynamin II, which was increased in cells transfected with rod PDEgamma or G-protein-coupled receptor kinase 2. Dynamin II plays a critical role in regulating endocytosis of receptor signal complexes required for activation of p42/p44 mitogen-activated protein kinase. Therefore, PDEgamma may have an important role in promoting endocytosis of receptor signal complexes leading to the activation of p42/p44 mitogen-activated protein kinase. We conclude that PDEgamma is an entirely novel intermediate regulating mitogenic signaling from both receptor tyrosine kinase and G-protein-coupled receptors in human embryonic kidney 293 cells.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2001

The gamma subunit of the rod photoreceptor cGMP phosphodiesterase can modulate the proteolysis of two cGMP binding cGMP-specific phosphodiesterases (PDE6 and PDE5) by caspase-3.

We have investigated whether the proteolysis of members of the cGMP binding phosphodiesterases (PDE6, PDE5A1, and PDE10A2) by caspase-3 is modulated by the gamma inhibitor subunit of PDE6. We show here that purified caspase-3 proteolyses PDE6, an enzyme composed of two nonidentical catalytic subunits (termed alpha and beta) with molecular mass of 88 and 84 kDa. The proteolysis of PDE6 produced a single fragment with a molecular mass of 78 kDa. This corresponds to the possible cleavage of the caspase-3 consensus DFVD site (amino acids: 164-168) in the alpha subunit and leads to a 50% decrease in the cGMP hydrolysing activity of the enzyme. The addition of rod PDEgamma to the incubation completely blocked the cleavage of PDE6 by caspase-3. In contrast, rod PDEgamma converted PDE5A1 (molecular mass of 98 kDa) to a better substrate for caspase-3. This resulted in the formation of four major fragments with molecular mass of 82-83, 67, 43, and 34 kDa. In addition, caspase-3 induced an approximately 80% reduction in the activity of a partially purified preparation of PDE5A1 in the presence of rod PDEgamma. Caspase-3 also cleaved PDE10A2 (molecular mass of 95 kDa) to a single 48-kDa fragment. This was consistent with cleavage of the DLFD site (amino acids: 312-315) in PDE10A2. In contrast with both PDE6 and PDE5A1, rod PDEgamma was without effect on this enzyme. These data show that rod PDEgamma interacts with at least two members of the cGMP binding PDE family (PDE5A1 and PDE6) and can exert differential effects on the cleavage of these enzymes by caspase-3.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2000

Regulation of both apoptosis and cell survival by the v-Src oncoprotein.

A number of oncogenes alter the regulation of the cell cycle and cell death, contributing to the altered growth of tumours. Expression of the v-Src oncoprotein in Rat-1 fibroblasts prevented cell cycle exit in response to growth factor withdrawal. Here we investigated whether survival of v-Src transformed cells in low serum is dependent on v-Src activity. We used a temperature sensitive v-Src to study the effect inactivating v-Src on transformed cells growing under low serum conditions. We found when we switched off v-Src the cells died by apoptosis characterised by activation of caspases and the stress-activated kinases, JNK (Jun N-terminal kinase) and p38 MAP (mitogen activated protein) kinase. We were able to prevent cell death by addition of serum or overexpression of Bcl-2. Thus v-Src transformed Rat-1 cells can be protected from apoptosis by serum, v-Src, or Bcl-2. We investigated how v-Src protects from apoptosis under these conditions. Amongst other effects, v-Src activates two kinases which have been shown to protect cells from apoptosis, phosphatidylinositol 3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK1/2). We found that switching off v-Src led to a decrease in the activity of both PI3-K and ERK1/2, however, we found that adding a specific inhibitor of PI3-K (LY294002) to v-Src transformed Rat-1 cells grown in low serum induced apoptosis while a specific ERK kinase (MEK1) inhibitor (PD98059) had no effect. This suggests that v-Src protects from apoptosis under low serum conditions by activating PI3-K.

Animals↗
PubMed1996

The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5).

The PDE4A (type IV) cAMP-specific, rolipram-inhibited phosphodiesterase RPDE-6 (RNPDE4A5), when transiently expressed in COS7 cells, could be complexed with the v-Src-SH3 domain expressed as a glutathione S-transferase (GST) fusion protein. RPDE-6 did not interact with GST itself. This complex was not disrupted by treatment with high NaCl concentration together with Triton X-100. Interaction was apparently determined by the N-terminal splice region of RPDE-6, as the PDE4A splice variant RPDE-39, which differs from RPDE-6 at the extreme N-terminus, failed to associate with v-Src-SH3; met26RD1 (where RD1 is rat 'dunc-like' PDE), which has the N-terminal splice region deleted, failed to associate with v-Src-SH3, and the association of RPDE-6 and v-Src-SH3 was blocked by a fusion protein formed from the N-terminal splice region. RDPE-6 showed binding to GST fusion proteins of both the intact Src kinase and an SH2-SH3 construct but did not bind to the Src-SH2 domain or to the adaptor protein Grb-2. RPDE-6 could be co-immunoprecipitated from cytosol extracts of transfected cells by using anti-Src antiserum. RPDE-6 exhibited selectivity in binding to the SH3 domains of c-Abl, Crk, Csk, Lck, Lyn, Fyn and v-Src, with binding to the SH3 regions of the Src-related tyrosyl kinases Lyn and Fyn being the most effective. The binding of RPDE-6 to the SH3 domains of Crk, Csk and Lck led to a marked reduction in PDE activity, but no change was apparent in complexes with other species. Endogenous RPDE-6 from brain, but not endogenous RPDE-39 from testis, bound to the Src-SH3 domain. We suggest that the PDE4A splice variant RPDE-6 has a propensity for interaction with selective SH3 domains, in particular those from Src and the Src-related tyrosyl kinases Lyn and Fyn. This interaction seems to be governed by alternative splicing of the PDE4A gene, because RPDE-39, a splice variant that lacks the proline-rich N-terminal splice region of RPDE-6, does not interact with these SH3 domains. It is proposed that the binding site on RPDE-6 for SH3 domains lies within the unique first 102 residues of its N-terminal splice domain, where two motifs representing Class I SH3 binding sites with selectivity for Src kinase SH3 domains can be identified and one motif for a putative Class II SH3 binding site.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

A 14-kDa immediate-early phosphoprotein is specifically expressed in cells infected with oncogenic Marek's disease virus strains and their attenuated derivatives.

Previously, we reported two cDNAs derived from the Marek's disease virus (MDV) long internal repeat region. A 14-kDa polypeptide (p14) encoded by two small open reading frames (ORFs) from at least two distinct cDNAs is expressed in cells lytically infected with both oncogenic and attenuated MDV as well as in cells latently infected and transformed by MDV. In this study, we demonstrate that p14 is serotype 1 specific and highly phosphorylated. Given the degree of phosphorylation and lack of homology to known proteins, we propose the name pp14 for the polypeptide encoded by ORF1a and ORF1b. Further analysis reveals that pp14 is predominantly found in cytoplasmic fractions of MDV-infected cells and can be detected in the cytoplasm of MDV-infected cells by immunofluorescence with polyclonal antisera prepared against pp14-glutathione S-transferase fusion protein.

Animals↗
PubMed1994

Functions of the v-Src protein tyrosine kinase.

The peripheral non-receptor tyrosine kinase oncoprotein, v-Src, has pleiotropic effects. It is a mitogen for quiescent cells, substituting for both competence and progression factor-mediated signals but it also induces cellular morphological transformation. We are dissecting the activities of v-Src by studying mutant proteins, including those with temperature sensitive (ts) effects, in different cellular backgrounds. Activation of a ts v-Src kinase rapidly increases activity of both the transcription factor, AP-1, and MAP kinase, an enzyme that enhances AP-1 activity by both phosphorylation of c-Jun and increased c-fos transcription; the relative contribution of these two events depends on the cells in which v-Src is expressed. Transient early AP-1 activation requires proper location of v-Src at the cell periphery and it is essential for mitogenesis. It is not, however, sufficient for entry into S-phase, there being a second need for v-Src later in G1. Transformation by v-Src does not require AP-1 activation but seems linked to events at the cell periphery, notably phosphorylation of proteins that bind to the v-Src SH3 domain such as the p85 subunit of PI-3 kinase.

Animals↗
PubMed1994

ATP-sensitive K+ channel opener acts as a potent Cl- channel inhibitor in vascular smooth muscle cells.

We describe the activation of a K+ current and inhibition of a Cl- current by a cyanoguanidine activator of ATP-sensitive K+ channels (KATP) in the smooth muscle cell line A10. The efficacy of U83757, an analogue of pinacidil, as an activator of KATP was confirmed in single channel experiments on isolated ventricular myocytes. The effects of U83757 were examined in the clonal smooth muscle cell line A10 using voltage-sensitive dyes and digital fluorescent imaging techniques. Exposure of A10 cells to U83757 (10 nM to 1 microM) produced a rapid membrane hyperpolarization as monitored by the membrane potential-sensitive dye bis-oxonol ([diBAC4(3)], 5 microM). The U83757-induced hyperpolarization was antagonized by glyburide and tetrapropylammonium (TPrA) but not by tetraethlyl-ammonium (TEA) or charybdotoxin (ChTX). The molecular basis of the observed hyperpolarization was studied in whole-cell, voltage-clamp experiments. Exposure of voltage-clamped cells to U83757 (300 nM to 300 microM) produced a hyperpolarizing shift in the zero current potential; however, the hyperpolarizing shift in reversal potential was associated with either an increase or decrease in membrane conductance. In solutions where EK = -82 mV and ECl = 0 mV, the reversal potential of the U83757-sensitive current was approximately -70 mV in those experiments where an increase in membrane conductance was observed. In experiments in which a decrease in conductance was observed, the reversal potential of the U83757-sensitive current was approximately 0 mV, suggesting that U83757 might be acting as a Cl- channel blocker as well as a K+ channel opener. In experiments in which Cl- current activation was specifically brought about by cellular swelling and performed in solutions where Cl- was the major permeant ion, U83757 (300 nM to 300 microM) produced a dose-dependent current inhibition. Taken together these results (i) demonstrate the presence of a K(+)-selective current which is sensitive to KATP channel openers in A10 cells and (ii) indicate that the hyperpolarizing effects of K+ channel openers in vascular smooth muscle may be due to both the inhibition of Cl- currents as well as the activation of a K(+)-selective current.

Adenosine Triphosphate↗
PubMed1990

Photoinduced electron transfer within complexes between plastocyanin and ruthenium bisbipyridine dicarboxybipyridine cytochrome c derivatives.

A new technique has been developed to measure intracomplex electron transfer between cytochrome c and its redox partners. Cytochrome c derivatives labeled at single lysine amino groups with ruthenium bisbipyridine dicarboxybipyridine were prepared as previously described [Pan, L.P., Durham, B., Wolinska, J., & Millett, F. (1988) Biochemistry 27, 7180-7184]. Excitation of RuII with a short light pulse resulted in the formation of the excited-state RuII*, which rapidly transferred an electron to the ferric heme group to form FeII and RuIII. Aniline was included in the buffer to reduce RuIII to RuII, leaving the heme group in the ferrous state. This process was complete within the lifetime of the light pulse. When plastocyanin was present in the solution, electron transfer from the ferrous heme of cytochrome c to CuII in plastocyanin was observed. All of the ruthenium cytochrome c derivatives formed electrostatic complexes with plastocyanin at low ionic strength, allowing intracomplex electron-transfer rate constants to be measured. The rate constants for derivatives modified at the indicated lysines were as follows: Lys 13, 1920 s-1; Lys 8, 1480 s-1; Lys 7, 1340 s-1; Lys 86, 1020 s-1; Lys 25, 820 s-1; Lys 72, 800 s-1; Lys 27, 530 s-1. It is interesting that the derivative modified at lysine 13 at the top of the heme crevice had the largest rate constant, while lysine 27 at the right side of the heme crevice had the smallest.(ABSTRACT TRUNCATED AT 250 WORDS)

2,2'-Dipyridyl↗
PubMed1982

Comparison of twice daily administration of a new corticosteroid budesonide with beclomethasone dipropionate four times daily in the treatment of chronic asthma.

The efficacy and side-effects of a new corticosteroid aerosol, budesonide and beclomethasone dipropionate were assessed in 30 patients with chronic asthma in a double-blind cross-over study. Budesonide was administered, 200 micrograms twice daily, from a conventional pressurized aerosol with a tube spacer extension attached and beclomethasone was given via a conventional inhaler in the recommended four times daily dose of 100 micrograms, each treatment being administered for one month. No clinically significant differences were found between the two treatments and no significant side-effects were observed.

Adolescent↗
PubMedDate not supplied

Expression of the normal H-ras1 gene can suppress the transformed and tumorigenic phenotypes induced by mutant ras genes.

The transformed phenotype of rat 208F cells transfected with the T24 H-ras1 oncogene is suppressed by simultaneous or subsequent transfection with the normal H-ras1 gene. The suppressed cells express both the normal and mutant forms of ras p21 but the normal form predominates. Rare transformed cells obtained after simultaneous transfection express mainly the T24 p21. Some suppressed cells induce tumours in nude mice after a long lag period and these tumour cell lines have much reduced expression of normal p21. The normal H-ras1 gene also suppresses the transformed phenotype induced by mutant N-ras, albeit less effectively. The tumorigenicity of the EJ bladder carcinoma cell line, which contains only the T24 mutant allele of H-ras1, is also suppressed following transfection with the normal H-ras1 gene. The results suggest that transforming alleles of ras genes do not behave in a fully dominant manner and that expression of the normal allele at elevated levels can lead to suppression of the transformed and tumorigenic phenotypes.

Animals↗