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Biomedical subjects

B J Murphy

Publications and source records attributed to B J Murphy.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Enhancement of transforming growth factor-alpha synthesis in multicellular tumour spheroids of A431 squamous carcinoma cells.

Multicellular tumour spheroids are cellular aggregates that can be prepared from many types of tumour cells. These three-dimensional structures provide a model for analysing the effects of cell-cell contact and intercellular microenvironments on phenomena such as autocrine regulation of growth factor synthesis. Autoregulation of the synthesis of transforming growth factor-alpha (TGF-alpha) was investigated at the message and protein levels in spheroid and monolayer cultures prepared from the A431 human squamous carcinoma cell line. The epidermal growth factor receptor (EGF-R) of these monolayer A431 cells had an average surface density of 2.2 x 10(6)/cell. Constitutive expression of TGF-alpha mRNA was an average of 3-fold greater in A431 spheroids than in monolayers, even for densely packed, confluent monolayers. This effect did not depend on hypoxic stress within the spheroids. TGF-alpha protein synthesis was enhanced in comparison with that in monolayer culture, reaching a value of up to 2-fold greater on a per cell basis. These results are discussed in the context of a TGF-alpha/EGF-R autocrine loop operating within cells that produce high local concentrations of TGF-alpha in the three-dimensional architecture of a spheroid.

Blotting, Northern↗
PubMed1992

Bone signal abnormalities in the posterolateral tibia and lateral femoral condyle in complete tears of the anterior cruciate ligament: a specific sign?

Thirty-two patients with acute, complete tears of the anterior cruciate ligament (ACL) proved at surgery underwent examination with magnetic resonance (MR) imaging. Bone impaction sites were present in the posterolateral tibial plateau in 30 patients (94%) and in the lateral femoral condyle (LFC) in 29 patients (91%). The bone abnormalities had low signal intensity on T1-weighted images and high signal intensity on T2-weighted images when compared with the signal intensity of normal marrow. It is assumed that the bone changes occur during injury when the LFC impacts into the posterior tibia, either during the initial rotary subluxation or as the LFC recoils to return to anatomic alignment. Only one of six partial ACL tears had a bone signal change. In patients with acute knee injury, bone impaction sites in the posterolateral tibia and the LFC suggest that a complete ACL tear is present.

Anterior Cruciate Ligament Injuries↗
PubMed1992

MR imaging of the elbow.

Of 27 patients who underwent magnetic resonance (MR) imaging of the elbow, 11 underwent elbow arthroscopy and/or an open surgical procedure. Surgical findings were compared with those from MR imaging. Five healthy volunteers also underwent MR imaging to demonstrate anatomic relationships. Transchondral fracture (osteochondritis dissecans) was identified in three of the 11 patients and was proved at surgery. Loose bodies were suspected at MR imaging in the three patients but were found in only two. One complete avulsion of the ulnar collateral ligament (UCL) and four cases of intact, thickened UCLs were identified at MR imaging and surgery. Loose bodies from the olecranon tip were found in three patients at surgery but were seen on MR images in only two. MR imaging depicted olecranal osteophytes in three cases, which were confirmed at surgery. Two complete avulsions of the biceps tendon and one partial triceps tendon tear were identified with MR imaging and proved at surgery. A postoperative soft-tissue infection and a synovial cyst were also seen at MR imaging and surgery. These results suggest that MR imaging is useful in the evaluation of the elbow.

Adolescent↗
PubMed1992

Iliotibial band friction syndrome: MR imaging findings.

Six patients with clinical histories and physical examination results consistent with iliotibial band friction syndrome (ITBFS) were examined with magnetic resonance (MR) imaging. Ill-defined decreased signal intensity on T1-weighted images and increased signal intensity on T2-weighted images was present deep to the iliotibial band, adjacent to the lateral femoral epicondyle. Axial fast imaging with steady-state precession (FISP) gradient-echo sequences were essential in differentiating the ill-defined signal intensity abnormality associated with ITBFS from fluid in the lateral knee joint. None of these patients were found to have lateral meniscal tears, and all responded to conservative measures directed at treating ITBFS. The authors conclude that MR imaging may be useful in confirming or establishing the diagnosis of ITBFS in patients with the appropriate clinical history and distal lateral thigh or lateral knee pain.

Adolescent↗
PubMed1991

Dihydropyridine receptor of L-type Ca2+ channels: identification of binding domains for [3H](+)-PN200-110 and [3H]azidopine within the alpha 1 subunit.

To identify the binding domain for dihydropyridine Ca2+ antagonists, skeletal muscle Ca2+ channels were photolabeled with [3H](+)-PN200-110 and [3H]azidopine. Regions of alpha 1 photolabeled by these ligands were then identified by antibody mapping of proteolytic fragments. Approximately 50% of the specific labeling by both ligands was incorporated in domain III. [3H]Azidopine labeled peptide Gln-989-Arg-1022, which contains a portion of the connecting loop between transmembrane segments IIIS5 and IIIS6 (IIIS5/S6), and peptide Ala-1023-Lys-1077, which contains IIIS6 itself and some adjacent amino acid residues. In contrast, [3H](+)-PN200-110 labeling occurred almost exclusively in the fragment containing IIIS6. A second site labeled by both ligands was identified in transmembrane segment S6 of domain IV and adjacent residues. In contrast to azidopine, the photoreactive benzofurazane group of (+)-PN200-110 is located in close proximity to the essential dihydropyridine ring. Therefore, the regions photolabeled by [3H](+)-PN200-110 within or adjacent to transmembrane segments IIIS6 and IVS6 must participate in the formation of the dihydropyridine binding site. As IIIS5/S6 is preferentially labeled by [3H]azidopine, it may contribute to drug binding by interaction with the long side chain of some dihydropyridines like azidopine. It is proposed, based on physiological studies, that these three peptide segments interact to form a receptor site accessible from the extracellular surface of the Ca2+ channel.

Amino Acid Sequence↗
PubMed1991

A phosphorylation site in the Na+ channel required for modulation by protein kinase C.

Voltage-gated sodium channels are responsible for generation of action potentials in excitable cells. Activation of protein kinase C slows inactivation of sodium channels and reduces peak sodium currents. Phosphorylation of a single residue, serine 1506, that is located in the conserved intracellular loop between domains III and IV and is involved in inactivation of the sodium channel, is required for both modulatory effects. Mutant sodium channels lacking this phosphorylation site have normal functional properties in unstimulated cells but do not respond to activation of protein kinase C. Phosphorylation of this conserved site in sodium channel alpha subunits may regulate electrical activity in a wide range of excitable cells.

Amino Acid Sequence↗
PubMed1991

Oxygen regulated 80 kDa protein and glucose regulated 78kDa protein are identical.

Ischemic stress of cells within solid tumors arises from inadequate perfusion of regions of the tumor and results in microenvironments which are hypoxic and deficient in nutrient delivery and waste product removal. Stressed cells within these microenvironments show growth inhibition and synthesize unique sets of proteins referred to as glucose and oxygen regulated proteins (GRPs and ORPs respectively). The commonality of proteins induced by glucose-starvation and hypoxia has not been proven. To this end, ORPs were induced in Chinese hamster ovary cells in the presence of high glucose concentration in the media and ORP 80 isolated from two dimension gels. Eleven tryptic peptides of the 80 kDa ORP were sequenced and found to be identical to GRP 78 sequences. The data demonstrate that GRP 78 and ORP 80 have the same primary amino acid sequence and suggest that glucose-starvation and hypoxia can induce the same cellular responses.

Amino Acid Sequence↗
PubMed1991

Two major antigens of heart sarcolemma are Ca2(+)-binding glycoproteins that copurify with the dihydropyridine receptor.

Ca2+ binding has been studied in isolated heart sarcolemmal membranes using the 45Ca overlay technique. 45Ca bound to two sarcolemmal polypeptides of 125 kDa and 97 kDa in preparations from dog, rabbit, cow and pig. During fractionation on DEAE ion-exchange and wheat-germ lectin affinity columns, the two Ca2(+)-binding polypeptides copurified with the dihydropyridine receptor associated with the voltage gated Ca2+ channel. These polypeptides were the major proteins in the isolated fraction as judged by silver staining in SDS-PAGE. Antisera raised against purified dog heart, sarcolemma indicated that the 125 and 97 kDa polypeptides were highly antigenic components of this membrane. The antisera cross-reacted with similar polypeptides in cardiac sarcolemmal preparations from rabbit, cow and pig, but not sarcoplasmic reticulum membranes. Purified antibodies against the 125 kDa polypeptide did not cross-react with the 97 kDa polypeptide, while antibodies against the 97 kDa polypeptide did not cross-react with the 125 kDa polypeptide. Both the 125 kDa and 97 kDa polypeptides bound wheat-germ lectin, suggesting both were glycoproteins. It is unlikely that these Ca2+ binding glycoproteins represent subunits of the dihydropyridine receptor-Ca2+ channel in this membrane.

Animals↗
PubMed1991

Gastric manifestations of AIDS: radiographic findings on upper gastrointestinal examination.

The radiographic findings in the stomach in the upper gastrointestinal examination in 11 patients with the Acquired Immune Deficiency Syndrome (AIDS) were reviewed. Four cases of Kaposi's sarcoma (KS) showed multiple submucosal nodular filling defects; in one of these cases, ulceration of some of the nodules was present. Gastric lymphoma occurred in two cases and appeared as one or several large nodular masses; one of these involved the esophagogastric junction. Five cases of infectious gastritis, caused by cytomegalovirus (CMV), Cryptosporidium, and Toxoplasma, were manifested as thickened gastric folds and/or antral narrowing. Thus, in patients with AIDS, gastric neoplastic lesions can be suggested when nodular filling defects or masses are identified, whereas inflammatory processes tend to show thickened gastric folds or antral narrowing. However, endoscopic biopsy is required to enable proper treatment.

Acquired Immunodeficiency Syndrome↗
PubMed1991

The identification of heme oxygenase as a major hypoxic stress protein in Chinese hamster ovary cells.

Chronic hypoxia increases the expression of a set of stress proteins (oxygen regulated proteins or ORPs) which is implicated in the development of drug resistance and radiation sensitivity in tumour cells. Five major ORPs have been documented, and two, ORP 80 and ORP 100, are considered to be identical to the glucose regulated stress proteins GRP78 and GRP94, respectively. We report here that ORP 33 is a form of the heme catabolic enzyme, heme oxygenase, using evidence obtained from northern blotting, two-dimensional polyacrylamide gel electrophoresis and western analysis. Heme oxygenase is believed to be an important component of the cellular response to oxidative stress. The significance of heme oxygenase as a hypoxia-induced stress protein is discussed.

Aerobiosis↗
PubMed1991

Hypoxia-induced drug resistance: comparison to P-glycoprotein-associated drug resistance.

In this report, we investigate several examples of hypoxia-induced drug resistance and compare them with P-glycoprotein associated multidrug resistance (MDR). EMT6/Ro cells exposed to drugs in air immediately after hypoxic treatment developed resistance to adriamycin, 5-fluorouracil, and actinomycin D. However, these cells did not develop resistance to colchicine, vincristine or cisplatin. When the cells were returned to a normal oxygen environment, they lost resistance. There was no correlation between the content of adriamycin and the development of adriamycin resistance induced by hypoxia. There was no difference between the efflux of adriamycin from aerobic cells and that from hypoxia-treated cells. The mRNA for P-glycoprotein was not detected in the hypoxia-treated cells. These results suggest that hypoxia-induced drug resistance is different from P-glycoprotein associated multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Modification of a discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide gel system for minigel electrophoresis.

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system was recently described by J. P. Doucet and J. M. Trifaró [1988) Anal. Biochem. 168, 265-271). The system was developed to separate with high and broad resolution the components from large volume samples after an overnight electrophoresis. This system was found to have many advantages. However, when used directly as a minigel system, this method cannot sustain the high voltage inherent to minigel electrophoresis and produces artefacts, namely a double front and a loss of resolution in the low molecular weight range. These problems were eliminated using the buffer system of M. A. Porzio and A.M. Pearson [1977) Biochem. Biophys. Acta 490, 27-34) in the separating gel and in the electrode chambers. The resulting modified discontinuous minigel system has the same advantages as the technique described for large slab gel electrophoresis, including the effective and rapid transfer of high molecular weight proteins to nitrocellulose membranes, as well as the advantages of the minigel format.

Animals↗
PubMed1990

Dihydropyridine binding to the L-type Ca2+ channel in rabbit heart sarcolemma and skeletal muscle transverse-tubules: role of disulfide, sulfhydryl and phosphate groups.

The dihydropyridine receptor is associated with the L-type Ca2+ channel in the cell membrane. In this study we have examined the effects of group-specific modification on dihydropyridine binding in heart sarcolemmal membranes isolated from the rabbit. Specifically, dithiothreitol and glutathione were employed to assess the possible role of disulfide (-SS-) bonds in the binding of [3H]dihydropyridines. NEM, PCMS and iodoacetamide were employed to examine the effect of blocking free sulfhydryl groups (-SH) on the binding of [3H]dihydropyridines to their receptor in heart sarcolemma. Glutathione inhibited [3H]PN200-110 binding to sarcolemmal membranes 100%, with an IC50 value of 50 microM, while DTT inhibited maximally by 75% with an IC50 value in the millimolar range. Alkylation of free sulfhydryl groups by NEM or iodoacetamide inhibited binding of [3H]PN200-110 binding in cardiac sarcolemma approx. 40-60%. Blocking of free sulfhydryl groups by PCMS completely inhibited [3H]PN200-110 binding to their receptor in sarcolemmal membranes in a dose-dependent manner with an IC50 value of 20 microM. These results suggest the involvement of disulfide bonds and free sulfhydryl groups in DHP binding to the L-type Ca2+ channel in heart muscle. We also examined the effect of membrane phosphorylation on the specific binding of the dihydropyridine [3H]nitrendipine to its receptor. Phosphorylation was studied in cardiac sarcolemmal as well as skeletal muscle transverse-tubule membranes. Phosphorylation due to endogenous protein kinase and cAMP-dependent protein kinase was without effect on [3H]nitrendipine binding in both cardiac sarcolemmal and skeletal muscle membranes. Addition of exogenous calmodulin under conditions known to promote Ca2+/calmodulin-dependent phosphorylation increased [3H]nitrendipine binding 20% with no alteration in KD in both types of membrane preparation. These results suggest a role for calmodylin in dihydropyridine binding to L-type Ca2+ channels.

4-Chloromercuribenzenesulfonate↗
PubMed1990

A sensitive and rapid method for identification and characterization of low abundance receptors.

An improved method for detection of low intensity radioligand-receptor complexes resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is described. [3H]Azidopine-labeled 1,4-dihydropyridine (DHP) receptor from skeletal muscle resolved by SDS-PAGE was transferred to nitrocellulose and cut into strips and individual slices were analyzed for radioincorporation by liquid scintillation counting. [3H]Azidopine-labeled DHP binding subunit migrated as a single entity with a mass of 170 kDa and was confirmed using conventional methods. Results were obtained within 4 h after resolution by SDS-PAGE compared to 3-40 days using conventional methods. In addition, detection of extremely low signals (less than 50 cpm/lane), otherwise overwhelmed by background noise using conventional methods, was possible due to removal of free ligand during electro-transfer to nitrocellulose. This technique offers a rapid sensitive, cost effective alternative to fluorography or other conventional gel slice analysis methods for detecting low intensity radiolabeled complexes resolved by SDS-PAGE.

Affinity Labels↗