Biomedical subjects
Publications and source records attributed to M Robinson.
By inserting synthetic oligonucleotides into a highly expressed gene in E. coli it has been shown that unfavourable codon usage can reduce the maximum translation rate of a protein. However, in the case of the codon used (AGG), a significant effect on translation was only seen at very high transcription rates from a gene containing multiple copies of the unfavourable codon.
A novel method is described for the quantitative analysis of subclasses (alk-1-enylacyl, alkylacyl, and diacyl types) and molecular species within each subclass of glycerophosphatides. Diradylglycerols from phospholipase C hydrolysis of the phospholipids are converted to benzoate derivatives, the benzoates are separated into their respective subclasses by thin-layer chromatography, and quantitated by measuring absorbance at 230 nm. Molecular species within individual subclasses are separated using a combination of argentation thin-layer chromatography and reversed-phase high-performance liquid chromatography with direct, on-line quantitation at 230 nm. We applied the method to the analysis of ethanolamine phosphatides from beef brain and were able to quantitate the three diradylglycerol subclasses (alk-1-enylacyl, alkylacyl, and diacyl types) as well as ca. 29 molecular species within each of these subclasses. This new quantitative approach for the analysis of specific molecular species of glycerolipids should be applicable to studies involving a variety of biologically important lipids, such as phosphatidylcholine, phosphatidylinositol, platelet activating factor, plasmalogens, and neutral type glycerolipids including diacylglycerols.
A longitudinal study was carried out from 1975 to 1979 in a cohort of 405 secondary school children. At yearly intervals they underwent a series of tests of pulmonary function designed to monitor lung development; some of these tests are relatively sensitive indicators of early abnormalities. A self administered questionnaire provided details of smoking habits and respiratory symptoms. The prevalence of smoking increased with age; most of those smoking at 16 had already been smoking, at least experimentally, at 13. Taking up smoking was clearly associated with the early onset of cough, production of phlegm, and shortness of breath on exertion. After two years of smoking more than a few cigarettes a day the children who smoked appeared considerably less healthy than their non-smoking peers and showed some evidence of early obstruction of the airways.
Coal tar and petroleum asphalt paints are among the products used as coatings for water pipes and storage tanks to retard corrosion. Formulations of these coatings were tested in the Ames mutagenesis and the mouse skin carcinogenesis bioassays. To test the mutagenicity of the paints, six doses ranging from 0.005 to 10 microliters per plate were assayed. In the mouse skin bioassay, doses of the coal tar paints ranging from 0.2 to 200 microliters were administered topically to 30 SENCAR mice per group. These initiating doses were followed by applications of 1.0 micrograms of 12-o-tetradecanoyl-phorbol-13-acetate (TPA) in 0.2 ml acetone topically, three times weekly for 20 weeks. Petroleum asphalt paints were tested in groups of 40 animals at 200 and 600 microliters doses. All coal tar paints showed mutagenic activity after metabolic activation with S-9, with the highest response being in strains TA 98 and TA 100. None of the petroleum asphalt paints gave mutagenic responses. Both types of coatings resulted in positive responses in the initiation/promotion study. The coal tar paints gave rise to 1000-1800 times the tumor response observed with petroleum asphalt products. One coal tar product was positive when tested as a complete carcinogen in the mouse at 2 microliters per application once weekly for 30 weeks, whereas the asphalt paint was negative at 100 times the dose. The biological responses to the products were greater than expected from their polycyclic aromatic hydrocarbon (PAH) content. These findings suggest that the hazard posed by these coatings may not be fully explained by their PAH contents.
Despite evidence for calcium-induced damage in the postischemic myocardium, calcium remains a frequently used inotropic agent following cardiopulmonary bypass surgery with cardioplegic arrest. The purpose of this study was (1) to challenge the postischemic myocardium with incremental doses of ionized calcium, and (2) to relate postischemic calcium reperfusion concentration to final recovery of left ventricular contractile function. Rabbit hearts (N = 38) were perfused and equipped with a ventricular balloon to monitor developed pressure (DP) +/- dp/dt, and left ventricular end diastolic pressure (LVEDP). Hearts underwent 40 min of global ischemia. Hearts were then assigned to one of four groups to receive a variable calcium concentration (0.6, 1.2, 2.5, 5.0 mM) for the initial 5 min of reperfusion followed by 55 min of reperfusion (Ca+2 = 1.25 mM). No differences were found between groups for final recovery of DP +/- dp/dt, or final LVEDP. It was concluded that: (1) within the physiologic range, variable calcium infusions during the first 5 min of postischemic reperfusion do not impair final recovery of LV contractile function, (2) irreversible partial recovery of left ventricular function appears due to mechanisms other than mitochondrial or myofibrillar calcium loading during reperfusion, and (3) infused calcium is a safe inotropic agent even in the postischemic myocardium.
Two short-term in vitro tests for mutagenicity (Salmonella reverse mutation and BHK21 cell transformation) were conducted on a series of fluorocarbons. Some of these materials (FC22, FC31, FC142b, FC143, and FC143a) were found to be positive in one or both of the tests and could therefore be considered as being potentially carcinogenic to animals. Such activity was not anticipated for what were previously considered inert materials and in consequence several examples of these fluorocarbons, which represented different combinations of short-term test results, were tested for carcinogenicity in limited in vivo bioassays. In these studies, rats were dosed for 1 year by gavage 5 days a week with either FC22, FC31, FC133a, FC134a, or FC143a dissolved in a corn-oil at a single dosage of 300 mg/kg body weight. The animals were then observed until week 125 with detailed necropsy at termination. The study revealed that FC31 was a potent carcinogen (to the rat stomach), a result which reflected the short-term test predictions, but FC133a, which gave a negative response in both the in vitro assays, induced a high incidence of reproductive tract tumors. The weak bacterial mutagens FC22 and FC143a did not induce tumors in this study, and the nonmutagenic FC134a was without overt carcinogenic activity. It is concluded that, while recognizing the limitations of the in vivo component of this study, the short-term tests were only partially successful in identifying potential carcinogens for this series of chemicals. Fluorocarbon 31 was a potent carcinogen which was first identified by bacterial mutation and cell transformation, whereas the equally potent carcinogen FC133a was not so identified. The lack of genotoxic activity with this particular compound leads us to believe that the carcinogenic activity may be due to mechanisms other than those which involve direct DNA interactions.
Doses of acrylamide ranging from 12.5 to 50 mg/kg were administered orally to female ICR-Swiss mice over 3 days for each of 2 weeks (total doses of 75, 150 and 300 mg/kg). Two weeks later some of the animals were started on a promotion schedule involving the application of 2.5 micrograms TPA/mouse 3 times weekly. Development of tumors was observed weekly in the skin, and in the lungs at 1 year. Acrylamide was found to initiate squamous cell adenoma and carcinomas in the skin and increased the yield of adenomas and carcinomas in the lung. Skin tumor development was dependent upon 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion whereas lung tumor induction was not. These data extend previous observations of carcinogenic activity of acrylamide in the skin of SENCAR mice and lungs of strain A/J mice to a third strain of mouse, the ICR-Swiss.
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Neuron-specific enolase (NSE) is present in many types of peptide-secreting neuroendocrine cells and in tumours derived from them, but little work has been done on the pituitary gland. Serial sections of normal rat (n = 9) and human (n = 7) pituitary gland, spontaneous rat pituitary tumours (n = 14) and human pituitary tumours, both hormonally active (n = 7) and inactive (n = 10), were immunostained for NSE and the 6 major anterior pituitary hormones. The neural lobe stained strongly and the intermediate lobe variably for NSE. In the anterior lobe, NSE immunoreactivity was present with variable intensity in the majority of hormone-producing cells of all six types. Cells with strong hormone immunoreactivity were usually only moderately stained for NSE. All the human and rat pituitary adenomas examined were positively stained for NSE, though to varying degrees. The pituitary gland is thus no exception to the rule that NSE is found in peptide-secreting neuroendocrine cells and their tumours.
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An experimental model is described which provides direct evidence of endocytosis of complexes of a charged colloid (dextran sulphate or DS) and of plasma fibronectin (FN) by reticulo-endothelial (RE) cells. This supports previous suggestions that plasma FN acts as a non-immunologically mediated opsonin which effects the clearance of various kinds of circulating colloids and particles by RE cells. DS of molecular weight 500 000 forms FN-containing complexes in rat plasma in vitro and lowers plasma FN levels acutely and in a dose-related fashion when given parenterally to rats. The plasma changes are accompanied by deposition of DS (shown histochemically as metachromatic material) and of FN (shown by specific immunofluorescence) in an identical distribution within RE cells in rat liver and spleen. The protein and polysaccharide components of the complex are disposed of by RE cells at markedly different rates. The model thus also offers a means of studying the dynamics of catabolism of plasma FN by this 'scavenger' pathway. Possible further extensions of the model for other purposes are also discussed.
Acrylamide structurally resembles vinyl carbamate, a proposed proximate carcinogenic form of ethyl carbamate. To test the hypothesis that acrylamide should possess carcinogenic properties, it was tested in the Salmonella-microsome assay for point mutation, as a skin tumor initiator in the Sencar mouse, and for its ability to induce lung adenomas in the A/J mouse. Acrylamide was found to be without activity as a mutagen in Salmonella strains TA 1535, TA 1537, TA 98, and TA 100 both in the presence and absence of rat liver microsomes using both the plate and liquid suspension assays. However, acrylamide was found to approximate ethyl carbamate in potency as a tumor initiator in the skin of the female Sencar mice. As with ethyl carbamate, acrylamide was more potent by systemic routes of administration relative to topical application. Acrylamide was also found to induce lung adenomas in male and female A/J mice using both the p.o. and i.p. routes of administration. Acrylamide was approximately one-seventh as potent as ethyl carbamate in the induction of lung adenomas. These data confirm the hypothesis that acrylamide possesses carcinogenic properties similar to ethyl carbamate.
The catalytic properties of a 1440-fold purified preparation of lysosomal phospholipase A1 were examined. The preparation was at least 95% specific for the sn-1 position of neat phosphatidylethanolamine (PE). The apparent specificity of the enzyme toward substrates was affected by three factors: the physical arrangement of molecules in the substrate aggregate, the charge on the lipid-water interface and the chemical structure of the substrate as it relates to the active site of the enzyme. Of various phospholipids tested in the absence of detergent PE was the preferred substrate, phosphatidylcholine (PC) was hydrolyzed at one-fifth the rate of PE, while phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylglycerol (PG) were degraded very slowly. Triton WR1339 stimulated the hydrolysis of PC, PI, PS, and PG but inhibited the hydrolysis of PE, with PG the preferred substrate at a 6:1 Triton/phospholipid ratio. The preference for PC over PE in detergent mixtures was attributed to the active site fit of the chemical structures of the substrate molecules. The enzyme preferentially hydrolyzed neat PE containing palmitic and oleic acids at position 1. A negative surface charge was required for the hydrolysis of PC and PE. Ca2+ stimulated the hydrolysis of PI, PS, and PG but inhibited the hydrolysis of PE. The inhibition of PE hydrolysis by Ca2+ was the result of an alteration in the surface charge of the PE vesicle. Chromatography of phospholipase A1 on concanavalin A-Sepharose resulted in a loss of activity toward acidic phospholipids which could be restored with Ca2+. Plasmalogen PE was found to inhibit the hydrolysis of diacyl-PE at the level of interfacial binding but not by competition for the active site of the enzyme. These results suggest that the hexagonal structure of PE represents a preferred physical form for catalysis by phospholipase A1, while the bilayer form is less readily attacked. Dispersion of the substrate in the inert detergent enhanced the activity of those substrates normally forming bilayer structures. We demonstrate the importance of the "quality of interface" in regulating the activity of the enzyme.
8 patients with advanced prostatic carcinoma were treated with the luteinising-hormone releasing-hormone agonist, ICI 118630, for up to 3 months. Patients received subcutaneous injections of ICI 118630 (either 100 micrograms or 250 micrograms daily). At the higher dose level, plasma testosterone concentrations were significantly reduced by day 14 and approximated to those previously recorded in castrated or diethylstilboestrol-treated patients. Plasma concentrations of luteinising hormone and follicle-stimulating hormone were similarly reduced. Reduction in the dose, to 100 micrograms/day, similarly reduced plasma testosterone. ICI 118630 shows considerable potential for the management of patients with advanced carcinoma of the prostate.
Following various periods of strangulation, the fates of intestinal segments were predicted by standard clinical criteria and visual (Wood's lamp) and fluorometric (perfusion fluorometer) assessment of fluorescein distribution. With fluorometry, a means of quantifying fluorescence transmitted via a fiberoptic light guide, the delivery and removal of fluorescein were monitored and analyzed. If either was restricted significantly, tissue death was predicted. Analysis of computerized graphic patterns or simple interpretation of fluorometric readings at two time points predicted tissue fate with 98% accuracy and a 93% negative predictive value. Wood's lamp evaluation had only a 53% accuracy and a 33% negative predictive value, while standard clinical criteria had an 81% accuracy and a 53% negative predictive value. Fluorescein leakage in segments which suffered significant endothelial damage provided staining patterns that incorrectly suggested viability. By monitoring elimination as well as uptake of dye, fluorometry provided much greater discrimination than did Wood's lamp inspection in this setting. In addition, fluorometry was readily repeatable within minutes, as fluorescence remaining from a previous injection could be subtracted from new, postinjection values.
6-p-Dimethylaminophenylazobenzothiazole (6BT) administered at a dose of 10 mg/kg by gavage to Sprague-Dawley and Wistar-derived rats for 2 months produced marked cellular changes in the liver, including proliferation of oval cells and the formation of regenerative/hyperplastic nodules. Cellular change continued after stopping dosing at 2 months such that liver tumours were first observed after only 4 months into the study, and animals examined between 4 months and termination at 6 months showed a 75% and 85% incidence of hepatocellular carcinoma for the Sprague-Dawley and Wistar strains, respectively. This study establishes 6BT as a potent hepatocarcinogen in the rat when administered by gavage, and confirms and extends an initial brief report by Brown and Sanchorawala in 1968 of its carcinogenicity following dietary administration.
A randomized clinical trial was performed on 308 patients with stage T1 carcinoma of the bladder to compare the efficacy of transurethral resection alone or followed by bladder instillations of thiotepa or VM-26 (teniposide) for 1 year. With the recurrence rate as the primary end point of interest the data from this trial were used to assess the prognostic importance of the following factors at entry into the study: number of tumors, prior recurrence rate, tumor size, grade, age, treatment group assigned and, finally, the interval between transurethral resection at entry into the study and the start of intravesical treatment. Using multivariate statistical techniques we found that the number of tumors at presentation was the most important prognostic factor followed by, in order of importance, the recurrence rate at entry and the size of the largest tumor. Of particular note was the discovery that patients with less than 1 recurrence per year at entry had a prognosis similar to patients with primary tumors, while those with a higher recurrence rate did uniformly poorly. These results show that patients with stage T1 carcinoma of the bladder form a heterogeneous group and that more aggressive therapy should be considered for patients with a poor prognosis.