Biomedical subjects
Publications and source records attributed to K Rose.
Pain is a common complaint in patients with secondary upper limb lymphoedema related to breast cancer. This study shows how standard conservative treatment of lymphoedema can result in a significant reduction in pain and decreased limb volume.
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The aim of the present in vitro study was to provide information on the pharmacologic properties of the muscularis mucosae in three regions of the rabbit colon. Proximal muscularis mucosae exhibited spontaneous contractions whose frequency was independent of endogenous acetylcholine. In the mid and distal colon, spontaneous contractile frequencies were depressed by atropine and enhanced by eserine. Muscularis mucosae from all regions responded to acetylcholine, ADP, AMP, ATP, bradykinin, histamine, methoxamine, substance P, vasoactive intestinal polypeptide but not cholecystokinin octapeptide or gamma-aminobutyric acid. Low concentrations of norepinephrine caused propranolol-sensitive relaxations of proximal colonic muscularis mucosae whereas high concentrations evoked phentolamine-sensitive contractions. In the mid and distal colon, norepinephrine caused relaxations which were poorly antagonized by propranolol. Proximal colonic muscularis mucosae responded to electrical stimulation with an atropine- and tetrodotoxin-sensitive "on contraction." Responses from the mid and distal colon were tetrodotoxin-sensitive and consisted of an atropine-sensitive "duration contraction" followed by a propranolol-insensitive "off relaxation" which was not mediated by prostaglandin synthesis, a purine, or vasoactive intestinal polypeptide. These data suggest that the rabbit colonic muscularis mucosae possesses alpha-1 adrenoceptors, histamine H1, muscarinic, P2 purinoceptors and beta adrenoceptors. However, their relative importance and the nature of the intrinsic innervation suggests considerable specialization of this muscle layer in different regions of the rabbit colon.
The production of semisynthetic human insulin for therapeutic purposes is of considerable importance. During trypsin-catalysed transformation of pig insulin into an ester of insulin of human sequence, the alanyl residue at position B30 is removed and replaced with an esterified residue of threonine. We have carried out this transformation in a medium enriched in 18OH2 and studied the product by MS. In contrast to a previous report, we find that incorporation of label into the B29 - B30 peptide bond occurs during the transformation with threonine methyl ester in aqueous N,N-dimethylacetamide. Quantitative data are presented and the implications of these findings are discussed.
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Removal of extracellular Mg2+ triggered the onset of repetitive excitatory discharges in cultured murine cortical neurons, detected by recording with patch electrodes in the whole cell configuration. The discharges were suppressed by 100 microM D-2-amino-5-phosphonovalerate. Over the next 24-72 h substantial numbers of neurons, but not glia, degenerated, releasing lactate dehydrogenase to the bathing medium. The neuronal death induced by removal of extracellular Mg2+ could be attenuated by either 3 microM tetrodotoxin or 50 microM dextrorphan, and thus likely reflects excessive activation of N-methyl-D-aspartate receptors triggered by excitatory discharges. This Mg2+ removal model may be a useful model in which to study certain aspects of epileptic neocortical injury.
Repeated pregnancy without lactation in rats has been reported to produce permanently elevated carcass fat mass and hyperplasia in subcutaneous fat depot. In the present study, the pregnancy outcome, intravenous glucose tolerance ability (IVGTT), and carcass composition were further examined in Osborne-Mendel rats. Female rats were divided into pregnancy-lactation (PL), pregnancy-no lactation (PNL), and control (CON) groups. Half of the rats were killed after three pregnancy/lactation cycles, whereas the rest were given a 12-week rest period before killing. It was found that rats in PNL group had fasting hyperglycemia and insulin insensitivity during the third pregnancy (P less than or equal to .05), elevated spontaneous abortion rate (P less than or equal to .05), and elevated subcutaneous fat content (P less than or equal to .01) relative to that of PL rats or CON rats. After a 12-week rest period, a significant increase in subcutaneous fat cell number was also observed in PNL rats. PL rats, on the other hand, have significantly elevated internal/subcutaneous fat ratios (P less than or equal to .05), both immediately after weaning and after the 12-week rest period. The significance of this change is not yet known.
The protein components that direct and activate accurate transcription by rat RNA polymerase I were studied in extracts of Novikoff hepatoma ascites cells. A minimum of at least two components, besides RNA polymerase I, that are necessary for efficient utilization of templates were identified. The first factor, rat SL-1, is required for species-specific recognition of the rat RNA polymerase I promoter and may be sufficient to direct transcription by pure RNA polymerase I. Rat SL-1 directed the transcription of templates deleted to -31, the 5' boundary of the core promoter element (+1 being the transcription initiation site). The second factor, rUBF, increased the efficiency of template utilization. Transcription of deletion mutants indicated that the 5' boundary of the domain required for rUBF lay between -137 and -127. Experiments using block substitution mutants confirmed and extended these observations. Transcription experiments using those mutants demonstrated that two regions within the upstream promoter element were required for optimal levels of transcription in vitro. The first region was centered on nucleotides -129 and -124. The 5' boundary of the second domain mapped to between nucleotides -106 and -101. DNase footprint experiments using highly purified rUBF indicated that rUBF bound between -130 and -50. However, mutation of nucleotides -129 and -124 did not affect the rUBF footprint. These results indicate that basal levels of transcription by RNA polymerase I may require only SL-1 and the core promoter element. However, higher transcription levels are mediated by additional interactions of rUBF, and possibly SL-1, bound to distal promoter elements.
The published amino acid sequence of pancreatic spasmolytic polypeptide (Thim, L., Thomsen, J., Christensen, M. and Jørgensen, K.H. et al. (1985) Biochim. Biophys. Acta 827, 410-418) has been checked by a combination of mass spectroscopy and Edman degradation. The pyroglutamyl blocking group was positively identified, and residue assignments at four positions were corrected: Lys48 (not Ser), Ser63 (not Lys), Cys68 (not Ser) and Ser74 (not Cys). The revised sequence exhibits greater similarity with pS2 peptide, a 60 residue polypeptide which is induced by oestrogen in the human breast cancer cell line MCF-7 and found in malignant but not in non-malignant breast tissue.
We describe the preparation of the modified chelator aminooxyacetyl-ferrioxamine, and the replacement of its iron atom by 67Ga at high specific activity. The aminooxy function of this compound was allowed to react with the aldehyde groups generated by the periodate oxidation of the oligosaccharide of a mouse IgG1 monoclonal antibody (MAb) directed against carcino-embryonic antigen (CEA). The use of the aminooxy group allowed a stable bond to be formed between the chelon and the antibody with no need for reduction. Iron was removed from the ferrioxamine moiety and replaced by 67Ga either before or after conjugation of the chelon to the antibody. In either case the labelled antibody was injected into nude mice bearing a human colon carcinoma having the appropriate antigenicity. Unoxidized antibody, labelled with 125I by conventional methods, was co-injected as an internal control. Additional control experiments were carried out with a non-immune IgG using the same 67Ga-labelled modified chelon as above. The in vivo distribution of the modified antibodies was evaluated at various times between 24 and 96 hr after injection. The methods used were gamma-camera imaging and, more quantitatively, gamma-counting of the various organs after dissection. Interestingly, with the metal-chelon-labelled antibody, the intensity and specificity of tumor labelling was comparable and in some cases superior to the results obtained with radio-iodinated antibody. In particular, there was almost no increase in liver and spleen uptake of radioactive metal relative to radio-iodine, contrary to what has been observed with most antibodies labelled with 111In after conjugation with DTPA.
Protein fragments have been activated by the addition of amino acid esters using proteolytic enzymes under conditions where the equilibria are shifted in the direction of synthesis. Because of the natural propensity of large protein fragments to form complexes approximating native conformation, these activated fragments have been induced to recombine by formation of the missing peptide bond. Although the incorporated ester is only weakly activating, the complex, mimicking an enzyme, provides proximity and orientation at the reacting termini, so that coupling yields are high. In other words, the protein catalyzes its own resynthesis. What distinguishes our technique from the rare natural examples of this phenomenon is that it operates at a variety of cleavage sites and with varying chain lengths. There seems to be no particular limitations on the amino acid esters that can be added, and serine proteases with a wide range of specificities can be used. It thus appears that we have a truly general method for the condensation of large fragments in protein synthesis, be they natural or the products of synthetic or genetic methods. This approach has the advantages over conventional methods of great specificity, high efficiency, and mild conditions of use. With our model protein, cytochrome c, we have used this approach to make analogues that illuminate structure-function relations. Both the highly conserved lysine 39 and the functionally invariant threonine 40 have been replaced by a range of substitutions. The results show how crucial these residues are to the structural and functional integrity of the bottom omega-loop of the protein.
An aminopeptidase specific for methionine (peptidase M) has been purified from wild-type and mutant Salmonella typhimurium strains. Recombinant peptidase M was also purified from Escherichia coli. These preparations were characterized with respect to their physicochemical properties using analytical ultracentrifugation, SDS/PAGE, isoelectric focusing, titration curve analysis, amino acid analysis, N-and C-terminal sequencing and various spectroscopic methods. Peptidase M activity is stimulated by Co2+, in agreement with previous studies using crude extracts of Salmonella. The purified preparations did not contain significant amounts of any metal. Enzymically important metal is loosely associated and lost during enzyme purification. Peptidase M was shown to contain seven free sulphydryl residues none of which are involved in either intra-or inter-molecular disulphide bonds. Most appear solvent-accessible as evidenced by their reactivity under native conditions. Limited modification of the sulphydryl residues with either iodoacetamide or 5,5'-dithiobis(2-nitrobenzoic acid) led to inactivation. Several cysteines were shown to be labelled to various degrees by peptide mapping of inactivated S-[14C]carboxymethylated protein. Whether cysteine modification affects enzymic activity directly (blocking an active site) or indirectly (by causing conformational change) remains to be established.
A computerized scheduling program was developed to provide hospital anaesthetic coverage and yet still satisfy the complex workstyles of 30 anaesthetists. Hospital commitments required ten anaesthetists available each weekday and four each weekend day for after-hours coverage of the intensive care units and operating rooms. The workstyles included part-time arrangements, limited calls by some individuals, and calls restricted to certain anaesthetists with specific areas of expertise. Rules were defined to limit the proximity of late calls to avoid fatigue and computer software was developed. For each scheduling period, the program assists in making daily call assignments based on each anaesthetist's availability and a priority ranking system. It is flexible enough to allow personal preferences. The number and distribution of all calls scheduled are counted. When the assignments are completed, differences are reconciled. For two years, this program has proved superior to previous manual systems for scheduling this group of anaesthetists with variable workstyles.
The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes which transform toluene and xylenes to benzoate and toluates. The genetic organization of the operon was characterized by cloning of the upper operon genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the upper operon contains at least five genes in the order of xylC-xylM-xylA-xylB-xylN. Between the promoter of the operon and xylC, there is a 1.7-kilobase-long space of DNA in which no gene function was identified. In contrast, most of the DNA between xylC and xylN consists of coding sequences. The xylC gene encodes the 57-kilodalton benzaldehyde dehydrogenase. The xylM and xylA genes encode 35- and 40-kilodalton polypeptides, respectively, which were shown by genetic complementation tests to be subunits of xylene oxygenase. The structural gene for benzyl alcohol dehydrogenase, xylB, encodes a 40-kilodalton polypeptide. The last gene of this operon is xylN, which synthesizes a 52-kilodalton polypeptide of unknown function.
We find, contrary to previous reports, that substantial cleavage of glucagon by insulin proteinase occurs at only one region, namely the double-basic sequence -Arg17-Arg18-. Cleavage takes place almost exclusively between these two residues, liberating fragments glucagon-(1-17) and glucagon-(18-29). Others have shown that the fragment glucagon-(19-29) is 1000-fold more efficient compared with intact glucagon, at inhibiting the Ca2+-activated and Mg2+-dependent ATPase activity and the Ca2+ pump of liver plasma membranes. We show that this fragment is not liberated in detectable quantities by our insulin proteinase preparation. On the other hand, others have shown that glucagon-(18-29), though less active than glucagon-(19-29), was still 100-fold more active than glucagon itself in the above-mentioned system. Our observations represent the first demonstration of the release by insulin proteinase of a hormone fragment having enhanced activity, although it has yet to be shown that the activity of this fragment is important in vivo. Since the formation of glucagon-(19-29) from glucagon-(18-29) would involve merely removal of Arg18, a second enzyme might exist to provide the more active fragment.
Human granulocyte colony-stimulating factor (G-CSF), and a mutant having a Ser for Cys substitution at residue 18 were produced in Escherichia coli strain W3110. About 60 mg of pure protein was obtained from 50 g of wet cells with a recovery of about 20%. The proteins were characterized physically and chemically, including determination of disulphide bonds, which were found to exist between residues 37-43 and 65-75. Cys-18 is not involved in disulphide bond formation and was substituted by Ser with no effects on gross protein conformation or biological activity. Both the wild-type and the mutant recombinant-derived proteins, although not glycosylated, possess colony-stimulating activities. In a bioassay using the murine myelomonocytic leukaemic cell line WEH1 3B D+, activities were obtained which were similar to those of natural G-CSF and of a glycosylated recombinant-derived human G-CSF produced in monkey cells.
A previously described technique [Rose, Simona, Offord, Prior, Otto & Thatcher (1983) Biochem. J. 215, 273-277] permits the identification of the C-terminal peptide of a protein as the only peptide that does not incorporate any 18O upon partial enzymic hydrolysis in 18O-labelled water. Formation of chemical derivatives followed by combined g.l.c.-m.s. was used in this earlier work. We now describe the isolation from protein digests, by reversed-phase h.p.l.c., of labelled and unlabelled polypeptides and their direct analysis by fast atom bombardment mass spectrometry. Under the conditions used, the 18O label is retained throughout the separation and analysis, thus permitting assignments of C-terminal peptides to be made. Enzyme-catalysed exchange of label into the terminal carboxy group was found to occur in some cases without hydrolysis of a peptide bond. This effect, which may be exploited to prepare labelled peptides, does not prevent application of the method (two separate digests must then be used). We have applied our method to the analysis of enzymic partial hydrolysates of glucagon, insulin and of several proteins produced by expression of recombinant DNA.
A method is described for the preparation of polypeptides activated uniquely at the C-terminus. The polypeptide is incubated in a concentrated solution of an amino acid active ester, the latter having its amino group free but adequately protected by protonation. The amino acid ester is coupled via its amino group to the C-terminus of the polypeptide by enzymic catalysis (reverse proteolysis). The resulting polypeptide C-terminal active ester is then isolated and coupled to a suitable amino component (generally a polypeptide) in a subsequent chemical coupling. The method appears to be generally applicable; fragments of horse heart cytochrome c, and porcine insulin, are used as examples. Two new analogues of cytochrome c have been prepared by using this method, with yields of up to 60% in the final coupling. Scope and limitations of the method are discussed.