Biomedical subjects
Publications and source records attributed to S J Morris.
Three children presented with soft tissue masses which were clinically suspected to be soft tissue sarcomas. The identification of phleboliths on initial radiological studies should have suggested the correct diagnosis of benign intramuscular haemangiomata. Subsequent referral to a paediatric oncological unit, and the unnecessary parental anxiety so generated, could have been avoided.
The complexity of signal transduction is becoming increasingly apparent following the cloning of multiple families of receptors, G proteins, and effectors. Therefore, new tools are needed to assess the importance of particular subtypes in receptor-mediated signal transduction. One such tool is the use of antisense approaches to specifically 'knockout' particular G protein subtypes and then assess the functional consequences for receptor-signalling pathways. In this article by Paul Albert and Stephen Morris, various antisense approaches (including transfection of full-length cDNA) are discussed and compared for their specificity and efficiency. The antisense approach is argued to be applicable to a wide variety of signal-transduction systems, including G-protein-coupled receptor signalling, for analysis of the downstream events that dictate biological responsiveness.
In this study we demonstrated that individual melanotropes expressed differing levels of POMC mRNA. Interspersed among the melanotropes was a small population of process-bearing cells expressing GFAP. We compared cellular resolution and feasibility for quantitation of hybrids formed by oligonucleotide probes labeled with two different markers using in situ hybridization histochemistry. In hybridizations using [35S]-labeled probes the signal could not easily be assigned to individual melanotropes, although digoxigenin-labeled probes provided good cellular resolution. Heterogeneous staining intensities of individual melanotropes for POMC mRNA were measured, and relative quantitation of changes in POMC mRNA levels following interactions with the D2 dopamine receptor was performed. We combined in situ hybridization histochemistry with immunohistochemistry to demonstrate that cells within the lobe parenchyma not expressing POMC mRNA were immunoreactive for GFAP.
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The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve".
A selective age-related decrease in both the protein and mRNA levels of the most abundant GABAA receptor subunits has been revealed in the rat inferior colliculus. The number (not affinity) of the native and fully assembled GABAA receptors assayed by 3H-muscimol binding was also decreased (35-49%). The decrease in GABA receptors was accompanied by a decrease in the protein and mRNA of the GABA-synthesizing enzyme glutamic acid decarboxylase. No other region of the rat brain showed such large age-related changes in these GABAergic synaptic molecules. Specific antibodies and riboprobes in conjunction with a computerized image analysis system were used to quantify immunocytochemistry and in situ hybridization. In old Sprague-Dawley rats, the combination of beta 2 and beta 3 peptide subunits was reduced 55%, while the beta 2 and beta 3 mRNAs were decreased 31% and 22%, respectively. The gamma 2S and gamma 2L subunit proteins decreased 43% and 21%, respectively, while the gamma 2 mRNA, including both short and long forms, was reduced 61%. The alpha 1 subunit protein was decreased 28%, whereas the alpha 1 mRNA decreased 40%. The glutamic acid decarboxylase protein was reduced 62% while GAD65 mRNA decreased 42%. Similar age-related changes were also observed in the inferior colliculus of Fischer-344 rats. In contrast, no changes were observed in the level of expression of some glial and/or neuronal proteins such as S-100, glial fibrillary acidic protein, and 160 KDa neurofilament protein in the inferior colliculus.(ABSTRACT TRUNCATED AT 250 WORDS)
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The heat-stable enterotoxin B (STB) of Escherichia coli is a 48-amino acid extracellular peptide that induces rapid fluid accumulation in animal intestinal models. Unlike other E. coli enterotoxins that elicit cAMP or cGMP responses in the gut [heat-labile toxin (LT) and heat-stable toxin A (STA), respectively], STB induces fluid loss by an undefined mechanism that is independent of cyclic nucleotide elevation. Here we studied the effects of STB on intracellular calcium concentration ([Ca2+]i), another known mediator of intestinal ion and fluid movement. Ca2+ and pH measurements were performed on different cell types including Madin-Darby canine kidney (MDCK), HT-29/C1 intestinal epithelial cells, and primary rat pituitary cells. Ca2+ and pH determinations were performed by simultaneous real-time fluorescence imaging at four emission wavelengths. This allowed dual imaging of the Ca(2+)- and pH-specific ratio dyes (indo-1 and SNARF-1, respectively). STB treatment induced a dose-dependent increase in [Ca2+]i with virtually no effect on internal pH in all of the cell types tested. STB-mediated [Ca2+]i elevation was not inhibited by drugs that block voltage-gated Ca2+ channels including nitrendipine, verapamil (L-type), omega-conotoxin (N-type), and Ni2+ (T-type). The increase in [Ca2+]i was dependent on a source of extracellular Ca2+ and was not affected by prior treatment of MDCK cells with thapsigargin or cyclopiazonic acid, agents that deplete and block internal Ca2+ stores. In contrast to these results, somatostatin and pertussis toxin pretreatment of MDCK cells completely blocked the STB-induced increase in [Ca2+]i. Taken together, these data suggest that STB opens a GTP-binding regulatory protein-linked receptor-operated Ca2+ channel in the plasma membrane. The nature of the STB-sensitive Ca2+ channel is presently under investigation.
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The interrelationships between changes in intracellular calcium concentration ([Ca2+]i) and intracellular pH in Madin-Darby canine kidney cells and kidney glomerular epithelial cells exposed to various stimuli were analyzed simultaneously using a new design of a fluorescence video microscope. Cells were double labeled with indo 1 and SNARF 1 dyes and were excited simultaneously at 350 and 540 nm. Images at four emission wavelengths were captured simultaneously at 405, 475, 575, and 640 nm at 30 frames/s for the two ratio dyes. SNARF sensitivity to pH between 6.5 and 8.0 was unchanged by [Ca2+]i. The SNARF ratio maps were used to correct the pH-dependent changes in the calculation of local cell calcium. NH4Cl loading produced the expected alkalinization and a concurrent rise in [Ca2+]i. When the NH4Cl was removed and the cells became acidic, a second rise in [Ca2+]i was recorded. Both changes in [Ca2+]i were from intracellular stores since they persisted in the absence of extracellular calcium. The findings demonstrate the need for pH correction of indo 1 recordings.
The regulatory activities of both intracellular calcium ([Ca2+]i) and intracellular pH (pHi) have greatly increased interest in the study of their interdependence. We have designed an epifluorescence video microscope that will image the fluorescence from two ratio dyes, indo-1 (for [Ca2+]i) and SNARF-1 (for pHi) at video rates. We examined primary cultures of pituitary intermediate lobe melanotropes loaded with both dyes. After experimentation, cells were positively identified by fluorescence immunohistochemistry. K(+)-induced depolarization of melanotropes produced increases in [Ca2+]i due to activation of L-type Ca channels. A secondary Ca2+ peak or oscillations were often seen. After treatment with carbonyl cyanide m-chlorophenylhydrozone, depolarization produced a rise in intracellular [Ca2+]i as well as oscillations. After thapsigargin or cyclopiazonic acid treatment, depolarization produced a primary Ca2+ elevation, but the secondary Ca2+ changes disappeared. This suggests that the oscillations were due to Ca2+ release from an endoplasmic reticulum type of intracellular store. All of these increases in [Ca2+]i were also directly coupled to a rise in intracellular H+. The close association between intracellular Ca2+ and H+ suggests that the observed pHi changes were due to the release of H+ upon binding of Ca2+ to intracellular buffers. This direct obligate coupling of intracellular Ca2+ and H+ suggests the possibility that pH-dependent cellular processes are directly activated by sudden increases in intracellular Ca2+ levels. This second messenger type of signaling system would be activated whether the Ca2+ was released from intracellular stores or entered the cell via plasma membrane Ca2+ channels.
Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.
An educational program to promote screening through primary health care for the squamous cell cancers of the buccal cavity, pharynx, and larynx as developed and implemented, and its impact on screening was documented. Providers of care for high-risk patients at seven inner-city health care sites in Boston were identified and targeted for training. Of the 327 providers who were targeted for training from December 1986 through June 1989, 261 (80%) attended educational sessions. Screening exams were documented on an average of 14.7 patients per targeted provider through December 1989. The educational program was associated with a large increase in documented screening for these cancers, compared with baseline rates. Several adaptations in the program were required, including a demonstration of efficient screening to address the concerns of these providers about time constraints. Variations in the quantity and quality of documented screening among health care sites were noted.
At the authors' institution, as part of the on-going Dornier National Biliary Lithotripsy Study, 174 patients with gallstones were randomly assigned to receive either ursodeoxycholic acid (UDCA) or placebo for 6 months after undergoing extracorporeal shock wave lithotripsy (ESWL). Six weeks after ESWL, 40 (23%) patients were re-treated for gallstone fragments larger than 5 mm in diameter. No significant difference in the UDCA- and placebo-treated patients was noted. The overall 6- and 12-month stone-free rates for all patients with initially noncalcified stones were as follows: UDCA patients, 31% and 36%, respectively, and placebo patients, 21% and 23%, respectively (P less than .05). In patients with a single, noncalcified gallstone that was at most 20 mm in diameter, the 12-month stone-free rates were as follows: UDCA patients, 60%, and placebo patients, 33% (P less than .01). Patients with noncalcified stones that were 11-20 mm in diameter who were treated with UDCA had a higher rate of stone clearance than placebo patients. At 6 months, the authors' previous study demonstrated no significant difference in the two groups of patients. However, the stone-free rate at 12 months was noted to be significantly higher for patients with a single, noncalcified stone that was at most 20 mm in diameter begun on UDCA than for all other groups of patients.
To identify the specific component(s) in the target membrane involved in fusion of vesicular stomatitis virus (VSV), we examined the interaction of the virus with human erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically by the octadecylrhodamine dequenching assay. Fusion of VSV with lipid-symmetric erythrocyte ghosts was rapid at 37 degrees C and low pH, whereas little or no fusion was observed with lipid-asymmetric ghosts. Conversion of phosphatidylserine in the lipid-symmetric ghost membrane to phosphatidylethanolamine by means of the enzyme phosphatidylserine decarboxylase did not alter the target membrane's susceptibility to VSV fusion. Spin-labeled phospholipid analogues with phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine headgroups incorporated into the outer leaflet of lipid-asymmetric erythrocytes did not render those membranes fusogenic. Electron spin resonance spectra showed an increased mobility of a phosphatidylcholine spin-label incorporated into the outer leaflet of lipid-symmetric erythrocyte ghosts as compared to that of lipid-asymmetric ghosts. These results indicate that the susceptibility to VSV fusion is not dependent on any particular phospholipid but rather is related to packing characteristics of the target membrane.
The hypothesis that the small portion of cellular phosphoinositide participating in signal transduction might be preferentially recycled within the plasma membrane was tested in rat glioma (C6) and murine neuroblastoma (N1E-115) cells. Percoll density gradient centrifugation was used to isolate a purified plasma membrane fraction and the subcellular distribution of all enzymes mediating phosphoinositide turnover was assessed. A small but significant proportion of PtdInsP2-specific phosphodiesterase was located in the plasma membrane but only two of the five enzymes required to replace PtdInsP2 (diacylglycerol kinase and PtdInsP kinase) also were present. CTP:phosphatidate cytidylyltransferase and CMP-phosphatidate:inositol phosphatidyltransferase were located exclusively in a microsomal fraction containing enriched levels of endoplasmic reticulum markers. Thus, diacylglycerol from agonist-stimulated cleavage of PtdInsP2, or phosphatidic acid formed from it, must be transferred to the endoplasmic reticulum for conversion to PtdIns. Plasma membrane also lacked PtdIns kinase. If the soluble PtdIns kinase has access to membrane-bound substrate, PtdIns may be phosphorylated to PtdInsP before or during transport to the plasma membrane. Phosphorylation by the predominantly plasma membrane PtdInsP kinase to form PtdInsP2 completes the cycle. PtdInsP phosphatase was present in all membrane fractions suggesting that PtdInsP can be returned to the PtdIns pool in plasma membrane and elsewhere. PtdInsP2 phosphatase was almost exclusively in the cytosol suggesting that reversible interchange between PtdInsP and PtdInsP2 in the plasma membrane may be modulated by the ability of this phosphatase to act on PtdInsP2 in the membrane. Thus, PtdIns resynthesis in the plasma membrane of these cells does not occur and is not required for phosphoinositide-mediated signal transduction.
An HPLC assay is described that can be used to study the covalent bonding interaction of carbinolamine-containing pyrrolo[2,1-c][1,4]benzodiazepines with the model nucleophile thiophenol, in order to evaluate electrophilicity at the C-11-position. Preliminary experiments with anthramycin, tomaymycin and neothramycin show that their reaction with thiophenol follows second-order kinetics, but the ranking order of reactivity (neothramycin greater than tomaymycin greater than anthramycin), does not correlate with either in vitro cytotoxicity or in vivo antitumour activity. This suggests that other factors such as non-covalent DNA-interaction or drug transport play a more crucial role in biological activity than simple alkylating ability. This assay should, however, prove a useful tool in the study of structure-activity relationships for this series of compounds and provide "C-11-electrophilicity" parameters for use in Hansch analysis and related studies.