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B J Murphy

Publications and source records attributed to B J Murphy.

At least 37 records · Page 2Linked to original sources
PubMed1996

The ribosome as affinity matrix': efficient purification scheme for translation factors.

A convenient method to purify each of the non-ribosomal proteins required to translate a native mRNA in vitro is described. In this scheme, the ribosome is used as an 'affinity' matrix to selectively elute the non-ribosomal proteins required for translation that are bound to these particles. Different sets of these proteins can be eluted with solutions of Mg2+ and NH4+ of various concentrations from either 70S, or 30S and 50S particles. A scheme for the purification of each initiation, elongation and release factor and 20 aminoacyl-tRNA synthetases is described. Specific examples of the purification of the initiation (IF-1, IF-2, IF-3) and elongation (EF-Tu and EF-G) factors and for a protein called 'rescue', which affects the association of native ribosomal subunits, are given. A scheme for the purification of EF-P, which stimulates peptide-bond synthesis and one of the W proteins, which permit reconstitution of translation is also described. The procedure markedly simplifies the isolation, in homogeneous form, of all the non-ribosomal proteins required to reconstruct translation.

Amino Acyl-tRNA Synthetases↗
PubMed1996

Physical and functional sensitivity of zinc finger transcription factors to redox change.

Redox regulation of DNA-binding proteins through the reversible oxidation of key cysteine sulfhydryl groups has been demonstrated to occur in vitro for a range of transcription factors. The direct redox regulation of DNA binding has not been described in vivo, possibly because most protein thiol groups are strongly buffered against oxidation by the highly reduced intracellular environment mediated by glutathione, thioredoxin, and associated pathways. For this reason, only accessible protein thiol groups with high thiol-disulfide oxidation potentials are likely to be responsive to intracellular redox changes. In this article, we demonstrate that zinc finger DNA-binding proteins, in particular members of the Sp-1 family, appear to contain such redox-sensitive -SH groups. These proteins displayed a higher sensitivity to redox regulation than other redox-responsive factors both in vitro and in vivo. This effect was reflected in the hyperoxidative repression of transcription from promoters with essential Sp-1 binding sites, including the simian virus 40 early region, glycolytic enzyme, and dihydrofolate reductase genes. Promoter analyses implicated the Sp-1 sites in this repression. Non-Sp-1-dependent redox-regulated genes including metallothionein and heme oxygenase were induced by the same hyperoxic stress. The studies demonstrate that cellular redox changes can directly regulate gene expression in vivo by determining the level of occupancy of strategically positioned GC-binding sites.

Base Sequence↗
PubMed1996

MR imaging of the rotator cuff: peritendinous and bone abnormalities in an asymptomatic population.

OBJECTIVE: MR imaging of the shoulder was performed in an asymptomatic population to determine the prevalence of MR-evident peritendinous and bone abnormalities. Findings were correlated with subject age and rotator cuff abnormalities. SUBJECTS AND METHODS: Dual-echo T2-weighted oblique coronal MR images of the shoulder were evaluated for 100 asymptomatic volunteers who were 19-88 years old. Twenty symptomatic patients with arthroscopic correlation were included as controls. Images were assessed independently by two reviewers with no knowledge of subject history or symptomatology. Bursal, peribursal, and bone findings were correlated with subject age and the appearance of the rotator cuff tendon. RESULTS: Changes characteristic of acromioclavicular joint osteoarthrosis were present in three fourths of the shoulders. One third had subacromial spurs. Changes in the peribursal fat plane and the presence of fluid in the subacromial-subdeltoid bursa paralleled the degree of MR-evident rotator cuff abnormalities. Joint fluid was observed in nearly all subjects. CONCLUSION: Our findings reveal a high prevalence of MR-evident bone and peritendinous shoulder abnormalities among asymptomatic individuals. The prevalence of subacromial spurs and humeral head cysts correlated closely with the severity of MR-evident rotator cuff abnormalities, as did changes in the bursa and peribursal fat. Acromioclavicular joint osteoarthrosis is seen in many shoulders independently of rotator cuff disease; therefore, its presence alone does not appear to be a reliable indicator of pain or tendon disease.

Acromioclavicular Joint↗
PubMed1995

Sites of selective cAMP-dependent phosphorylation of the L-type calcium channel alpha 1 subunit from intact rabbit skeletal muscle myotubes.

The principal (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive (L-type) calcium channels is present in full-length (212 kDa) and COOH-terminal truncated (190 kDa) forms, which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. Immunoprecipitation of the calcium channel from rabbit muscle myotubes in primary cell culture followed by phosphorylation with cA-PK, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed comparable phosphorylation of three COOH-terminal phosphopeptides found in the purified full-length alpha 1 subunit. Stimulation of muscle myotubes with a permeant cAMP analogue, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate, prior to immunoprecipitation of alpha 1 results in a 60-80% reduction of cA-PK catalyzed "back" phosphorylation of each of these sites in vitro in calcium channels purified from the cells, indicating that these sites are phosphorylated in vivo in response to increased intracellular cAMP. Serine 687, the most rapidly phosphorylated site in the truncated 190-kDa alpha 1 subunit, was observed as a minor phosphopeptide whose level of phosphorylation was not significantly affected by stimulation of endogenous cA-PK in the myotubes. The COOH-terminal sites, designated tryptic phosphopeptides 4, 5, and 6, were identified as serine 1757 (phosphopeptides 4 and 6) and 1854 (phosphopeptide 5) by a combination of protease cleavage, phosphorylation of synthetic peptides and fusion proteins, specific immunoprecipitation, and phosphopeptide mapping. Phosphorylation of serines 1757 and 1854 in the COOH-terminal region of the 212-kDa alpha 1 subunit in intact skeletal muscle cells may play a pivotal role in the regulation of calcium channel function by cA-PK.

Animals↗
PubMed1995

Functional co-expression of the beta 1 and type IIA alpha subunits of sodium channels in a mammalian cell line.

Brain sodium channels are a complex of alpha (260 kDa), beta 1 (36 kDa), and beta 2 (33 kDa) subunits, alpha subunits are functional as voltage-gated sodium channels by themselves. When expressed in Xenopus oocytes, beta 1 subunits accelerate the time course of sodium channel activation and inactivation by shifting them to a fast gating mode, but alpha subunits expressed alone in mammalian cells activate and inactivate rapidly without co-expression of beta 1 subunits. In these experiments, we show that the Chinese hamster cell lines CHO and 1610 do not express endogenous beta 1 subunits as determined by Northern blotting, immunoblotting, and assay for beta 1 subunit function by expression of cellular mRNA in Xenopus oocytes. alpha subunits expressed alone in stable lines of these cells activate and inactivate rapidly. Co-expression of beta 1 subunits increases the level of sodium channels 2- to 4-fold as determined from saxitoxin binding, but does not affect the Kd for saxitoxin. Co-expression of beta 1 subunits also shifts the voltage dependence of sodium channel inactivation to more negative membrane potentials by 10 to 12 mV and shifts the voltage dependence of channel activation to more negative membrane potentials by 2 to 11 mV. These effects of beta 1 subunits on sodium channel function in mammalian cells may be physiologically important determinants of sodium channel function in vivo.

Animals↗
PubMed1995

Abnormal findings on magnetic resonance images of asymptomatic shoulders.

Magnetic resonance images of the shoulders of ninety-six asymptomatic individuals were evaluated to determine the prevalence of findings consistent with a tear of the rotator cuff. The scans were reviewed independently by two diagnostic radiologists who are experienced in the interpretation of magnetic resonance images of the shoulder. The over-all prevalence of tears of the rotator cuff in all age-groups was 34 per cent (thirty-three). There were fourteen full-thickness tears (15 per cent) and nineteen partial-thickness tears (20 per cent). The frequency of full-thickness and partial-thickness tears increased significantly with age (p < 0.001 and 0.05, respectively). Twenty-five (54 per cent) of the forty-six individuals who were more than sixty years old had a tear of the rotator cuff: thirteen (28 per cent) had a full-thickness tear and twelve (26 per cent) had a partial-thickness tear. Of the twenty-five individuals who were forty to sixty years old, one (4 per cent) had a full-thickness tear and six (24 per cent) had a partial-thickness tear. Of the twenty-five individuals who were nineteen to thirty-nine years old, none had a full-thickness tear and one (4 per cent) had a partial-thickness tear. Magnetic resonance imaging identified a high prevalence of tears of the rotator cuff in asymptomatic individuals. These tears were increasingly frequent with advancing age and were compatible with normal, painless, functional activity. The results of the present study emphasize the potential hazards of the use of magnetic resonance imaging scans alone as a basis for the determination of operative intervention in the absence of associated clinical findings.

Adult↗
PubMed1994

Metallothionein IIA is up-regulated by hypoxia in human A431 squamous carcinoma cells.

The expression of metallothionein IIA (MT-IIA) was investigated in A431 human squamous carcinoma cells exposed to hypoxia (pO < or = 0.01% of atmospheric pO2) and subsequent reoxygenation. Northern analysis showed that MT-IIA mRNA levels were significantly increased during 14 h of hypoxia and during reoxygenation. Western blotting confirmed that total MT protein levels were also increased in response to these stresses. Evidence of the transcriptional control of MT-IIA expression in hypoxic and in reoxygenated A431 cells was found using a 0.2-kilobase sequence of the proximal 5'-regulatory region of the MT-IIA gene in a chloramphenicol acetyltransferase reporter gene construct. Thus the proximal promoter of the human MT-IIA gene appears to contain a hypoxic response element(s). These observations indicate that MT-IIA may have an important role in the stress responses of cells in solid tumors.

Carcinoma, Squamous Cell↗
PubMed1994

Selective down-regulation of integrin receptors in spheroids of squamous cell carcinoma.

Integrin expression in squamous cell carcinomas have been reported to be down-regulated in vivo. Because integrins are molecules whose functions include the reorganization of cytoskeleton in response to the surrounding extracellular matrix, we have examined the expression of integrins in the transformed cell line A431 when grown as multicellular spheroids or as monolayers. The spheroids were grown to sizes of approximately 100 microns and approximately 600 microns. Since larger A431 spheroids require epidermal growth factor for growth, we also investigated the effect of this growth factor on the expression of integrins in cells grown as monolayers or as small spheroids. Immunostaining studies using monoclonal antibodies specific for alpha 6, beta 1, and beta 4 subunits revealed a strong staining pattern in the periphery of the spheroids. The interior cells of the spheroids showed a moderate, positive reaction with the beta 1 antibody but significantly reduced from that at the periphery. Anti-alpha 2 antibody, on the other hand, revealed a uniform staining around the cells throughout the spheroids. Western blot analyses confirmed an overall diminution of alpha 6 and beta 1 protein levels in the spheroids compared with monolayers. Northern blot analyses showed that the low expression of integrin subunits alpha 6, beta 1, and beta 4 in spheroids was due to a reduction in mRNA transcripts. Northern blot analyses, however, showed no significant change in the expressions of alpha 2, alpha 5, or beta 5 mRNA. Conversely, the expression of alpha v was slightly reduced in spheroids. Epidermal growth factor increased the mRNA expression of alpha 2, alpha 6, beta 1, and beta 4 integrin subunits in cells grown either as monolayers or as spheroids whereas epidermal growth factor had no detectable effect on the expression of alpha v or beta 5. These results mimic the pattern of expression found in vivo and indicate that cell-cell contact and the microenvironments of cells within a spheroid regulate the expression and distribution of a subset of integrin molecules.

Animals↗
PubMed1994

Rupture of the distal biceps tendon: evaluation with MR imaging.

PURPOSE: To assess the usefulness of magnetic resonance (MR) imaging in the diagnosis of distal biceps tendon ruptures. MATERIALS AND METHODS: MR images of the elbow in 20 patients with suspected distal biceps injury were retrospectively reviewed along with those of eight asymptomatic volunteers. Surgical confirmation was obtained in nine cases. RESULTS: MR imaging helped diagnose 10 complete ruptures with depiction of the absence of the tendon distally (n = 10), a fluid-filled tendon sheath (n = 9), an antecubital fossa mass (n = 3), muscle edema (n = 3), and atrophy (n = 2). Six partial tears were depicted with high signal intensity within the tendon (n = 6), fluid in the biceps tendon sheath (n = 6), and thinning (n = 3) or thickening (n = 3) of the distal tendon. Of the remaining patients, one each had tendinosis, tenosynovitis, a biceps hematoma, and a brachialis contusion. CONCLUSION: MR imaging helped confirm distal biceps tendon ruptures because it distinguished complete from partial ruptures and other entities that may mimic ruptures.

Adult↗
PubMed1993

Identification of the sites of selective phosphorylation and dephosphorylation of the rat brain Na+ channel alpha subunit by cAMP-dependent protein kinase and phosphoprotein phosphatases.

Voltage-sensitive brain Na+ channels are regulated by cAMP-dependent protein kinase (cA-PK) and protein kinase C. Using synthetic peptides and protein microsequencing, we have determined that the alpha subunit of rat brain Na+ channel is selectively phosphorylated by cA-PK in vitro and in intact cells on 4 serine residues in the intracellular loop connecting homologous domains I and II. Ser-623 was most rapidly and extensively phosphorylated in vitro, whereas Ser-573, Ser-610, and Ser-687 were phosphorylated to lesser extents. In contrast, serine 687 was most extensively phosphorylated in mammalian cells transfected with the alpha subunit of type IIA Na+ channel in response to an increase in intracellular cAMP. Purified protein phosphatases dephosphorylated these sites selectively. Calcineurin rapidly and extensively dephosphorylated Ser-623 and also dephosphorylated Ser-573, Ser-610, and Ser-687 to lesser extents. Phosphatase 2A selectively dephosphorylated Ser-610. Together these results indicate that modulation of neuronal Na+ channel activity and therefore neuronal excitability by cAMP-dependent phosphorylation results from selective phosphorylation and dephosphorylation of four sites in the intracellular loop connecting homologous domains I and II of the alpha subunit.

Amino Acid Sequence↗
PubMed1993

A 60 kDa polypeptide of skeletal-muscle sarcoplasmic reticulum is a calmodulin-dependent protein kinase that associates with and phosphorylates several membrane proteins.

Activation of a calmodulin (CaM)-dependent protein kinase associated with rabbit skeletal-muscle sarcoplasmic reticulum (SR) results in the phosphorylation of polypeptides of 450, 360, 165, 105, 89, 60, 34 and 20 kDa. Radioligand-binding studies indicated that a membrane-bound 60 kDa polypeptide contained both CaM- and ATP-binding domains. Under renaturing conditions on nitrocellulose blots, the 60 kDa polypeptide of the membrane exhibited CaM-dependent autophosphorylation activity, suggesting that it was the CaM-dependent protein kinase of SR. Ca2+/CaM-independent autophosphorylation of polypeptides of 62 and 45 kDa was found to occur in the light SR, whereas the Ca2+/CaM-dependent autophosphorylation activity was enriched in the heavy SR. Both these kinase activities were absent from transverse tubules, although these membranes were enriched in CaM-binding polypeptides of 160, 100 and 80 kDa. In the absence of Ca2+, CaM bound to a 33 kDa polypeptide of the membrane. The purified ryanodine receptor was not phosphorylated by the purified CaM kinase, although it was a substrate for protein kinase C. Affinity-purified antibodies to brain CaM kinase II cross-reacted with the 60 kDa polypeptide in Western blots and immunoprecipitated the 60 kDa polypeptide, along with the 360, 105, 89, 34 and 20 kDa phosphoproteins, from Nonidet-P-40-solubilized SR membranes. Antibodies raised against the 60 kDa kinase polypeptide did not cross-react with the other phosphoproteins, suggesting that these polypeptides were distinct and unrelated. Subcellular distribution of the 60 kDa kinase indicated the specific association of the polypeptide with the junctional-face membrane of SR. The CaM-dependent incorporation of 32P into various membrane proteins was inhibited by the CaM kinase II fragment (290-309), with an IC50 value of 2 nM for the inhibition of incorporation into the 60 kDa kinase polypeptide. Recent studies [Wang and Best (1992) Nature (London) 359, 739-741] have shown that a CaM kinase activity intrinsic to the membrane can inactivate the Ca(2+)-release channel of skeletal muscle SR. Since our results demonstrate that the 60 kDa polypeptide of SR is a CaM-dependent protein kinase, we suggest that this kinase, through its associations, may be responsible for gating the Ca(2+)-release channel.

Adenosine Triphosphate↗
PubMed1993

Convergent regulation of sodium channels by protein kinase C and cAMP-dependent protein kinase.

The function of voltage-gated sodium channels that are responsible for action potential generation in mammalian brain neurons is modulated by phosphorylation by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (cA-PK) and by protein kinase C (PKC). Reduction of peak sodium currents by cA-PK in intact cells required concurrent activation of PKC and was prevented by blocking phosphorylation of serine 1506, a site in the inactivation gate of the channel that is phosphorylated by PKC but not by cA-PK. Replacement of serine 1506 with negatively charged amino acids mimicked the effect of phosphorylation. Conversion of the consensus sequence surrounding serine 1506 to one more favorable for cA-PK enhanced modulation of sodium currents by cA-PK. Convergent modulation of sodium channels required phosphorylation of serine 1506 by PKC accompanied by phosphorylation of additional sites by cA-PK. This regulatory mechanism may serve to integrate neuronal signals mediated through these parallel signaling pathways.

Action Potentials↗
PubMed1993

Enhancement of heme oxygenase expression and activity in A431 squamous carcinoma multicellular tumor spheroids.

We have investigated the effects of the growth of A431 human squamous carcinoma cells as three-dimensional aggregates (multicellular tumor spheroids) on the expression and enzyme activity of heme oxygenase (HO). We demonstrate that A431 squamous carcinoma cells grown as day 4 spheroids selectively increase the expression of heme oxygenase 1 (HO-1), caused, directly or indirectly, by three-dimensional cell-cell contact effects. Steady-state levels of both mRNA and protein are significantly enhanced in spheroids compared with day 4 monolayers (approximately 13-fold). Because of the similarity of apparent half-lives between monolayers (2.7 h) and spheroids (2.1 h), it appears that the increases are caused at least partly by altered transcriptional rates. Total HO enzyme activity, measured by carbon monoxide production, is also up-regulated (2.6-fold) in spheroids, compared to that in monolayers. This increase indicates that the up-regulation in HO-1 protein expression corresponds to an increase in functional enzyme levels. We propose that HO may play a more complex role in cellular metabolism than would be evident from studies using two-dimensional monolayer cultures.

Blotting, Northern↗
PubMed1992

Enhanced epidermal growth factor receptor synthesis in human squamous carcinoma cells exposed to low levels of oxygen.

Exposure of human A431 squamous carcinoma cells to levels of hypoxia found in some solid tumors causes 2-fold increases in epidermal growth-factor receptor (EGF-R) mRNA levels and rate of receptor protein synthesis compared with aerobic cells. Similar results are shown for receptor message from other squamous carcinoma cells, human keratinocytes, and human W138 fibroblasts. Less basal tyrosine phosphorylation of the receptor occurs in hypoxic compared with aerobic A431 cells. Scatchard analysis also shows that reoxygenated A431 cells display enhanced surface expression of the EGF-R compared with aerobic control cells. Possible mechanisms and implications for tumor therapy are discussed.

Aerobiosis↗
PubMed1992

Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C.

Phosphorylation of voltage-sensitive Na+ channels in neurons by protein kinase C slows Na+ channel inactivation and reduces peak Na+ currents. Na+ channels purified from rat brain and reconstituted into phospholipid vesicles under conditions that restore Na+ channel function were rapidly phosphorylated by protein kinase C on their 260-kDa alpha subunit. The phosphorylation reaction required Ca2+, diolein, and phosphatidylserine for activation of protein kinase C, and the rate of phosphorylation of reconstituted Na+ channels was 3- to 4-fold faster than for Na+ channels in detergent solution. Phosphorylation was on serine residues in three distinct tryptic phosphopeptides designated A, B, and C. Up to 2.5 mol of phosphate were incorporated per mol of Na+ channel. Following maximum phosphorylation by protein kinase C, cAMP-dependent protein kinase was able to incorporate more than 2.25 mol of phosphate per mol of Na+ channel indicating that these two kinases phosphorylate distinct sites. However, prior phosphorylation by cAMP-dependent protein kinase prevented phosphorylation of phosphopeptide B indicating that both kinases phosphorylate the site in this peptide. Phosphopeptide B shown here to be phosphorylated by protein kinase C and phosphopeptide 7 previously shown to be phosphorylated by cAMP-dependent protein kinase co-migrate on two-dimensional phosphopeptide maps and evidently are identical. The reduction in peak Na+ currents caused by both protein kinase C and cAMP-dependent protein kinase may result from phosphorylation of this single common site.

Animals↗
PubMed1992

Posterior tibial tendon dysfunction.

Posterior tibial tendon dysfunction, a common entity, frequently is unrecognized and inappropriately managed. Acutely, pain and swelling are present over the medial ankle and longitudinal arch. Long-standing inflammation can lead to tendon rupture, resulting in a progressive planovalgus or "flat foot" deformity. Plain radiographs illustrate the changes in bony anatomy associated with chronic posterior tibial deficiency, while magnetic resonance imaging scans can identify the three stages of posterior tibial tendon pathology. Most cases are amenable to conservative therapy, including rest and administration of nonsteroidal antiflammatory agents. Often a short period of immobilization in a cast or the use of an orthosis is beneficial. In cases with persistent tenosynovitis, complete tendon rupture, or progressive deformity, surgical intervention is indicated.

Biomechanical Phenomena↗