Biomedical subjects
A Robinson
Publications and source records attributed to A Robinson.
Duchenne muscular dystrophy, cystic fibrosis, myotonic dystrophy: gene to protein.
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Sickle cell disease, the thalassemias, Tay-Sachs disease: protein to gene.
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Learning the secrets of human chromosomes: Canada's role in an international project.
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Anxiolytic effects of serotonergic interventions in the shock-probe burying test and the elevated plus-maze test.
Although serotonergic neural systems have been implicated in the control of anxiety for a number of years, evidence in favour of this role is controversial. The present experiments were designed to further characterize the putative role of serotonin (5-HT) in anxiety, using two pharmacologically validated animal models: the elevated plus-maze and the shock-probe burying tests. If the integrity of 5-HT neural systems is necessary for the expression of 'anxious' behaviors, then disruption of 5-HT systems should produce effects in the plus-maze and shock-probe tests that are similar to those of anxiolytic drugs. In the present experiments, serotonergic function was disrupted in rats, either by chemical depletion using the synthesis inhibitor p-CPA, by inhibitory autoreceptor activation using the selective 5-HT1A receptor ligand 8-OH-DPAT, or by electrolytic lesions of the serotonin-containing, dorsal raphe nucleus. p-CPA and dorsal raphe lesions produced robust anxiolytic effects in the elevated plus-maze and the shock-probe burying tests, whereas 8-OH-DPAT produced anxiolytic effects only in the shock-probe burying test, and 'anxiogenic' effects in the elevated plus-maze test. Although these results generally support the view that serotonin plays a role in the expression of 'anxious' behavior, the opposite effects of 8-OH-DPAT in the two behavioral paradigms suggest that the 5-HT1A receptor subtype exerts differential control over different types of experimental anxiety.
Localisation of a receptor-recognition domain on the S3 subunit of pertussis toxin by peptide mapping.
Overlapping 10-amino-acid peptides, which consecutively span the amino acid sequence of the S3 subunit of pertussis toxin, were synthesised on polyethylene pins and screened for their ability to bind the glycoprotein fetuin. Fetuin binding was localised to a single peptide comprising amino acids 46-55. A free peptide, (E)S3c, of longer sequence (S3 amino acids 44-58) was also found to bind alpha-1-acid glycoprotein, mixed brain gangliosides and fetuin. (E)S3c also recognised asialofetuin but with a lower apparent affinity relative to fetuin. The single tryptophan residue of the peptide yielded a fluorescence-emission maximum of 355 nm. In the presence of either ganglioside or the phospholipid L-alpha-lysolecithin, but not N-acetylneuramin-lactose or lactosylceramide, the emission intensity of (E)S3c was enhanced and the emission maximum blue-shifted to 340 nm by ganglioside, or to 345 nm by L-alpha-lysolecithin. Monosialogangliosides, disialogangliosides, and trisialogangliosides, when fluorescence-titrated, were each found to bind the peptide with a similar dissociation constant of 4.4 +/- 2.8 microM. These findings demonstrate that region 44-58 of the pertussis-toxin S3 subunit is likely to be involved in the recognition of both glycosylated and phospholipid constituents of target-cell membranes.
Report calls for major changes in drug-approval process, but will they be implemented?
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Molecules, cancer and sticking together.
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Demonstration of lipooligosaccharide immunotype and capsule as virulence factors for Neisseria meningitidis using an infant mouse intranasal infection model.
Using an infant mouse intranasal infection model, we have compared the virulence of 17 epidemiologically related isolates of Neisseria meningitidis associated with an outbreak of meningococcal disease in Gloucestershire, UK, and one German isolate. The isolates were all of serotype 15 subtype P1:7, 16 and were identical by restriction fragment length polymorphism analysis, but differed in either (i) whether they were isolated from a case or a carrier, (ii) the presence or absence of group B capsule, or (iii) their lipooligosaccharide (LOS) immunotype. The results indicate that capsule is a major virulence determinant and is required for colonization and hence for invasion. In addition, the LOS L3,7,9 immunotype, when compared to the L1,8,10 immunotype, is a secondary virulence factor which enhances colonization of nasal passages and invasion of the blood stream by both case and carrier isolates. Two case isolates which were unusual in possessing the L1,8,10 immunotype, established invasive infection, but this was associated with a switch to the L3,7,9 immunotype. The results confirm that LOS is a virulence factor for N. meningitidis and that immunotype L3,7,9 is associated with invasive disease.
Changes in levels of pancreatic endoplasmic reticulum proteins that function in translocation and maturation of secretory proteins in response to cholecystokinin.
Two pathways operate to target newly-synthesised proteins to the endoplasmic reticulum. In one, the signal recognition particle attaches to the signal sequences of nascent chains on ribosomes and slows or stops translation until contact is made with the docking protein at the membrane. The second operates via molecular chaperons. The pathways converge at the level of a 43 kDa signal binding protein integrated into the membrane, where translocation through a proteinaceous pore is initiated. In the lumen, proteins fold and disulphide formation is catalysed by the enzyme protein disulphide isomerase. The heavy chain binding protein may attach to unassembled or unfolded proteins and prevent their exit from the ER to the Golgi. Cholecystokinin (CCK) treatment increases the biosynthesis and secretion of pancreatic proteins, increases the levels of PDI and the 43 kDa binding protein, and reduces levels of BiP. These proteins may be possible targets for genetic manipulation to improve processing of heterologous proteins from cultured mammalian cells.
Adjuvant action of cholera toxin and pertussis toxin in the induction of IgA antibody response to orally administered antigen.
The ability of cholera toxin B-subunit to bind to intestinal epithelium and in particular the dome epithelium of the Peyer's patch can account for its potency as an immunogen. However, pure B-subunit is a less effective immunogen than whole cholera toxin. The immunogenicity of B-subunit may be restored by the addition of either traces of whole toxin or by pertussis toxin. Cholera toxin and pertussis toxin were both able to stimulate a response when fed in conjunction with keyhole limpet haemocyanin, whereas recombinant B- subunit of heat-labile toxin from Escherichia coli had no effect, demonstrating that the adjuvant action is a property of the enzymically active A-subunits. The adjuvant activity of both pertussis toxin and cholera toxin may be due to their ability to cause an increase in the activity of adenylate cyclase via their action on GTP-binding regulatory proteins. However, feeding of forskolin, a direct activator of adenylate cyclase, had no effect on the mucosal immune response, indicating a role for cholera and pertussis toxin which is independent of enhancement of adenylate cyclase activity in the regulation of the immune response. Antibody to pertussis toxin was not detected, which was attributed to inadequate absorption of pertussis toxin.
Long-term oral etoposide in metastatic breast cancer: clinical and pharmacokinetic results.
To evaluate the activity of long-term, single-agent oral etoposide against advanced breast cancer, this study employed etoposide 50 mg/day and 100 mg/day (given over 14 days) in previously treated and chemotherapy-naive patients with histologically confirmed, recurrent or metastatic breast cancer. Of 38 patients, 24 had had chemotherapy, 34 had prior radiotherapy, and 31 had received hormone therapy. Etoposide courses in both treatment groups were repeated every 4 weeks for at least two courses; delays were instituted when patients' total leukocyte nadir fell to or below 3.0 x 10(9)/l. Dose reductions were made in the 100-mg group (to 50 mg/day) if World Health Organization leukopenia grade 3 or higher was present. Plasma pharmacokinetic profiles were measured in selected patients to assess inter- and intrapatient variability in etoposide's oral bioavailability. No complete responses were achieved among the 36 patients evaluable for response, but eight patients had a partial response. Responses were more frequent at the 100-mg dose and in previously untreated patients (seven of eight partial responders had not had previous chemotherapy). Median duration of response was 16 weeks (range, 7 to 46). Myelosuppression (variable and unpredictable) and alopecia (universal) were the notable toxicities. Pharmacokinetic analyses of oral bioavailability revealed significant interpatient variability, but much less intrapatient variability when successive etoposide courses in individual patients were evaluated. Despite the relatively small number of patients in this study, the responses achieved by previously untreated patients suggest etoposide's value against breast cancer. Further trials should use pharmacokinetic studies to assess bioavailability as well as to help define 'target' etoposide doses, based on plasma etoposide levels, during treatment.
Oral verapamil with chemotherapy for advanced non-small cell lung cancer: a randomised study.
To determine if the chemotherapy resistance of non-small cell lung cancer could be modified by oral verapamil, 72 patients were entered into a randomised trial of verapamil plus chemotherapy vs the same chemotherapy alone. Verapamil 480 mg day-1 was given for 3 days starting 24 h prior to chemotherapy which consisted of bolus vindesine 7 mg followed by ifosfamide/mesna 5 g m-2 over 24 h, followed by mesna alone for a further 8 h. Cycles were repeated every 3 weeks for up to six courses. Sixty-six patients were eligible for tumour response analysis and responses occurred in 41% of those randomised to chemotherapy plus verapamil and in 18% of those randomised to chemotherapy alone (P = 0.057). Median survival from start of treatment was significantly better in the verapamil arm (P = 0.02). Toxicity of the combination of chemotherapy plus verapamil was principally neurological and was manageable. Thus the addition of oral verapamil to vindesine/ifosfamide chemotherapy is feasible and in this study was associated with improved outcome. Further confirmation of these observations is required in non-small cell lung cancer, a tumour characterised by resistance to conventional chemotherapy.
Severe peripheral neuropathy and elevated plantar pressures causing foot ulceration in pituitary gigantism.
We report two patients with treated pituitary gigantism and peripheral neuropathy, one of whom has chronic foot ulceration. Detailed neurophysiological assessment was performed on both patients. The patient with foot ulceration had clinical and neurophysiological evidence of severe neuropathy, whereas the patient without ulceration had only neurophysiological abnormalities. The sweating response to acetylcholine was markedly impaired in the feet of both patients, suggesting pedal autonomic denervation. Neither patient had evidence of diabetes mellitus and detailed investigation failed to reveal an alternative cause of peripheral neuropathy. Optical pedobarography revealed abnormally high pressure (> 10 kg/cm2) under the metatarsal heads of both patients, one such area coinciding with the area of ulceration. Thus in pituitary gigantism elevated plantar pressures may contribute to the development of foot ulceration when severe peripheral neuropathy is present. Furthermore, as in diabetes mellitus, impaired sweating may also increase the risk of ulceration as the resultant dry skin may develop fissures.
Envelope structure and the development of new vaccines.
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Isolation of a putative fimbrial adhesin from Bordetella pertussis and the identification of its gene.
We report the purification of a minor Bordetella pertussis fimbrial subunit, designated FimD, and the identification of its gene (fimD). FimD could be purified from the bulk of major fimbrial subunits by exploiting the fact that major subunit-subunit interactions are more stable in the presence of SDS than minor-major subunit interactions. To locate the gene for FimD, internal peptides of FimD were generated, purified and sequenced. Subsequently, an oligonucleotide probe, based on the primary sequence of one peptide, was used to clone fimD. The primary structure of FimD, derived from the DNA sequence of its gene, showed homology with a number of fimbrial adhesins. Most pronounced homology was observed with MrkD, a fimbrial adhesin derived from Klebsiella pneumoniae. These observations suggest that FimD may represent a B. pertussis fimbrial adhesin. With a fimD-specific probe we detected the presence of a fimD homologue in Bordetella parapertussis and Bordetella bronchiseptica but not in Bordetella avium. Cloning and sequencing revealed that the B. parapertussis and B. bronchiseptica fimD product differed from the B. pertussis fimD product in 20 and 1 amino acid residues, respectively. Since B. bronchiseptica is normally not a human pathogen, but causes respiratory disease in a wide range of non-human mammalian species, this may suggest that FimD recognizes a receptor that is well conserved in mammalian species. An in-frame deletion in fimD completely abolished FimD expression and also affected the expression of the major subunits Fim2 and Fim3 suggesting that, in contrast to other adhesins that are minor components of fimbriae, FimD is required for formation of the fimbrial structure.