Biomedical subjects
Publications and source records attributed to G Henderson.
A frame-based computer-aided learning system for use on microcomputers has been developed for teaching clinical chemistry. The system presents a case-study model that can be used by students to assess their ability to make a diagnosis, to select patterns of investigations and to institute treatment.
Angiotensin II (ANGII) (3-100 nM) facilitated the potassium-evoked (22.5 mM) release of [3H]-noradrenaline ([3H]NA) from slices of parietal cortex in a concentration-dependent manner, but did not significantly alter the release of [3H]NA evoked in a similar manner from locus coeruleus slices. The facilitatory action of ANGII was blocked by saralasin (0.1-3 microM). Neither nimodipine (10-30 microM) nor phenylmethylsulphonyl fluoride (1 mM) altered either [3H]NA release or the facilitatory action of ANGII in the parietal cortex. Carbachol (0.01-3 mM) and raised potassium (22.5 mM), but not ANGII (3-100 nM), stimulated the production of inositol phosphates in parietal cortex slices. The potassium-evoked increase in inositol phosphate production was unaffected by ANGII (3-100 nM). In the locus coeruleus, ANGII (3-100 nM) did not stimulate inositol phosphate production. The mechanism underlying the ANGII facilitation of [3H]NA release from the parietal cortex does not appear to involve either nimodipine-sensitive calcium channels, or, as far as we have been able to determine, the release of calcium from intracellular stores following the breakdown of phosphoinositides.
Protease A and protease B are extracellular proteins which are secreted by Streptomyces griseus. The genes encoding protease A (sprA) and protease B (sprB) were isolated from an S. griseus genomic library by using a synthetic oligonucleotide probe. Fragments containing sprA and sprB were characterized by hybridization and demonstration of proteolytic activity in Streptomyces lividans. Each DNA sequence contains a large open reading frame with the coding region of the mature protease situated at its carboxy terminus. The amino terminus of each reading frame appears to encode a 38-amino-acid signal peptide followed by a 76- or 78-amino-acid polypeptide, a propeptide, which is joined to the mature protease. Strong homology between the coding regions of the protease genes suggests that sprA and sprB originated by gene duplication.
We have used a large panel of thyroglobulins (Tg) prepared from a wide range of mammalian species to study the Tg autoantigenic epitopes recognized by populations of monoclonal and polyclonal murine T and B cells. This approach showed the existence of at least six different epitopes; three recognized by T cells (in association with I-Ak on antigen-presenting cells) and three by B cells (monoclonal antibodies). The majority of serum and monoclonal autoantibodies were found to be highly specific for mouse Tg, with some cross-reactive binding to rat Tg. In contrast, T-cell lines/clones and hybridomas recognized cross-reactive epitopes on Tg that were highly conserved throughout most of the mammalian orders. Moreover, two hybrid clones, which showed similar patterns of cross-reactivity, differed in their responsiveness to tryptic digests of human Tg. Thus, autoreactive T and B cells recognize distinct areas of the Tg molecule.
HSAG-1 is a cloned member of a heterogeneous middle repetitive family of genetic elements which is capable of eliciting a leukemia-related surface antigen detected with a monoclonal antibody after DNA transformation of mouse cells. HSAG-1 was originally isolated from a Chinese hamster-human leukemia hybrid cell gene library both by sib-selection for antigen producing activity and by hybridization with labelled human genomic human DNA. We show here that the human labelled site is at the right hand end of the insert, while the antigen-eliciting portion is included in a 1450 bp fragment at the left hand end of the insert. We also present the complete nucleotide sequence of the 3369 bp insert. The sequence contains 12 elements which bear a significant resemblance to accepted consensus sequences for Alu repetitive elements. The right hand end contains adjacent elements with close sequence similarity to portions of the human and hamster type I and type II Alu consensus sequences. All of the other Alu-related elements have diverged relative to the Alu consensus sequences by additions, long deletions and substitutions. The left hand portion of the insert which has the antigen-producing activity contains four of these diverged elements representing a relatively high proportion (26%) of the nucleotide sequence. The sequence is thus consistent with our previous observations of a repetitive family with biological function.
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Intracellular recordings were made from neurones in the rat locus coeruleus in a brain slice maintained in vitro. Phencyclidine and related psychotomimetic drugs, applied in known concentrations in the fluid bathing the slice, depressed responses to N-methyl-D-aspartic acid noradrenaline (in the presence of the uptake inhibitor desmethylimipramine) and [D-Ala2,MePhe4,Gly-ol5]enkephalin and also prolonged the action potential. The sensitivities of these responses to depression by phencyclidine was N-methyl-D-aspartic acid (IC50 0.4 microM) greater than noradrenaline (IC50 3.9 microM) greater than [D-Ala2,MePhe4,Gly-ol5]enkephalin (IC50 8.5 microM) greater than prolongation of the action potential (41% increase by 30 microM). Stereoselectivity was observed only in the depression of responses to N-methyl-D-aspartic acid where (+)-1-(1-phenylcyclohexyl)-3-methyl piperidine was 3.3-fold more potent in suppressing N-methyl-D-aspartic acid depolarizations than its (-) isomer. The responses to N-methyl-D-aspartic acid were also depressed by the structurally unrelated psychotomimetic (+/-)-N-allyl-N-normetazocine (IC50 0.9 microM). All of the effects of the psychotomimetic drugs examined were slow in onset and difficult to reverse following washout. No effect of phencyclidine (0.03-100 microM) or related drugs was observed on membrane potential, input resistance or spontaneous action potential firing rate of locus coeruleus neurones. The depression of responses to N-methyl-D-aspartic acid by phencyclidine was the most potent and the only stereoselective effect of those studied. The importance of this effect and of those not showing stereoselectivity in relation to the phencyclidine behavioural syndrome is discussed.
The genome of Chinese hamster ovary (CHO) cells contains a complex family of approximately 16 alpha-tubulin genes, many of which may be pseudogenes. We present here the complete cDNA sequences of three expressed alpha-tubulin genes; one of these genes has been identified only in CHO cells. The noncoding regions of these three CHO alpha-tubulin genes differed significantly, but their coding regions were highly conserved. Nevertheless, we observed differences in the predicted amino acid sequences for the three genes. A comparison of the CHO alpha-tubulin sequences with all of the sequences available for mammals allowed assignment of the alpha-tubulin genes to three classes. The proteins encoded by the members of two of these classes showed no class-specific amino acids among the mammalian species examined. The gene belonging to the third class encoded an isoprotein which was clearly distinct, and members of this class may play a unique role in vivo. Sequencing of the three alpha-tubulin genes was also undertaken in CMR795, a colcemid-resistant clonal CHO cell line which has previously been shown to have structural and functional alterations in its tubulin proteins. We found differences in the tubulin nucleotide sequence compared with the parental line; however, no differences in the alpha-tubulin proteins encoded in the two cell lines were observed.
The following studies compare developmental patterns of a hepatic monoxygenase system using (1) the aminopyrine (Ap) breath test, (2) in vivo Ap pharmacokinetics (clearances) and (3) in vitro Ap demethylation by hepatic microsomes. Together, the three methodological approaches indicate that hepatic metabolism of Ap increases from the fetal stage through 70 days of age with the male showing a significantly greater drug metabolizing capacity than the female. However, sex and developmental patterns derived by the three methodologies do not necessarily correlate quantitatively, due to a number of complex variables (i.e., liver size, drug volume of distribution, etc.) less likely encountered in the adult of fixed age and mature sexual development.
Catheter related sepsis (CRS) is the most serious complication of total parenteral nutrition. Frequently, however, low rates of CRS are associated with a high incidence of infection of the catheter tip, the clinical significance of which is unclear. The relationships between CRS, infection of the catheter tip and infection at the site of catheter insertion have been investigated in 283 catheters of 257 patients receiving total parenteral nutrition. CRS occurred in only ten patients (3.5 per cent) whereas organisms were isolated from 108 catheter tips (38.2 per cent). The most common organism isolated was Staphylococcus epidermidis (66.7 per cent). Eight catheter tips were colonized from a distant septic focus. Organisms were isolated from 90 catheter tips which were removed electively from patients who displayed no clinical evidence of sepsis. There was a poor correlation between infected catheter tips and infected catheter insertion sites. Asymptomatic infection of the catheter tip appears to be of little clinical relevance, resulting in no patient morbidity. Contamination of the catheter tip during or after removal seems to account for a significant proportion of these infections.
The cytology of nuclear division and septation in the yeast and hyphal phases of Candida albicans growing at 37 degrees C has been studied by fluorescence microscopy after staining of specimens with 4'6-diaminido-2-phenylindole (DAPI) and Calcofluor. Yeast and hyphal cells replicated their nuclei at about 18 min after the emergence of a bud or germ-tube. The site of nuclear division coincided with the future location of the septum in both forms. This occurred at the junction of the bud and parent yeast cell or 6.0 micron from the parent yeast in germ tubes which were formed in medium containing serum. The filamentous forms of a range of clinical and laboratory strains grown in a variety of germ tube-inducing media were all extensively vacuolated. Germ tube extension in all of these media was linear. It is suggested that there is little biosynthesis of cytoplasm during the initial stages of germ tube growth in this organism and that this accounts for the development of the large vacuoles and the linear growth kinetics.
Following synchronisation of oestrus using FGA and PMSG, ewes were inseminated by either the conventional cervical (CC) method or directly into the uterus by laparoscopy (LI). The CC method was carried out either at 48 and 60 hours following progestagen withdrawal with 480 x 10(6) spermatozoa per inseminate or once only at 56 hours with 600 x 10(6) spermatozoa. The laparoscopic method was performed at 52 hr using 48 x 10(6) spermatozoa per ewe. In the first two trials eggs were recovered at laparotomy. The egg recovery rate was significantly lower (P<0.05) for those ewes which had been inseminated by the LI method (74%) compared with those inseminated by the CC method (85%); fertilization rates were not significantly different (92% and 89% respectively). In the third trial 20 ewes were bled to determine their periovulatory LH concentrations and the timing of peak LH concentrations correlated with the outcome of each insemination. Ewes inseminated using laparoscopy did not conceive when their LH surge occurred >58 hr after progestagen withdrawal. In this and in the final experiment, the combined pregnancy rates and litter sizes (assessed radiographically) were 67% (n = 51) and 2.21 (n = 34) for the CC method and 75% (n = 48) and 1.97 (n = 36) for the LI method (P>0.05).
Intracellular recordings were made from locus coeruleus neurones in a slice of rat pons superfused in vitro. A single-electrode voltage-clamp amplifier was used to measure membrane currents. Superfusion of the slice with clonidine (3-100 nM) or noradrenaline (100 nM-100 microM), or brief application of noradrenaline from a pipette by a pressure pulse, caused dose-dependent membrane hyperpolarizations. Phenylephrine (10 microM) and isoprenaline (10 microM) were ineffective. The hyperpolarizations were accompanied by a decrease in neurone input resistance. The hyperpolarization evoked by pressure ejection of noradrenaline could be reversed by membrane polarization to -110 mV. Clonidine and noradrenaline caused a membrane current which was linearly related to membrane potential between -50 and -120 mV, being outward at resting levels and reversing at -110 mV. The concentration-response curves for clonidine and noradrenaline were shifted rightwards in a parallel manner by alpha 2-adrenoceptor antagonists. The antagonist KeS estimated from the degree of shift were: RX 781094 9 nM, yohimbine 14 nM, phentolamine 20 nM and piperoxane 49 nM. These experiments indicate that in locus coeruleus neurones an increase in potassium conductance results from activation of alpha 2-adrenoceptors similar to those characterized on peripheral neurones.
Chinese hamster ovary cells contain a complex family of ca. 16 unique alpha-tubulin sequences and a similar multiplicity of beta sequences. To examine which members of this multigene family are expressed, we constructed cDNA libraries from two Chinese hamster ovary cell lines according to the method of H. Okayama and P. Berg (Mol. Cell. Biol. 3:280-289, 1983). Each library consisted of 5.5 X 10(5) transformants and contained a high percentage of full-length tubulin clones. Three different alpha-tubulin genes were identified by sequence analysis of the 3' noncoding regions of these tubulin clones. The relative abundance of the transcripts corresponding to the three genes was estimated by gene-specific dot blotting of 96 cDNA alpha-tubulin clones and was found to be 71, 24, and 5%. There is little homology in the 3' noncoding sequences of these genes; however, a strong interspecies homology exists in this region for two of the Chinese hamster ovary genes with the two alpha-tubulin genes previously described in other systems. The third Chinese hamster ovary gene, with an expression frequency of 24%, is unique in that its 3' noncoding region is unlike that of the other mammalian alpha-tubulin genes. In addition, limited sequence data from the coding region of this gene indicates it codes for a unique alpha-tubulin protein.
We have analyzed the complex tubulin gene family in clonal lines of Chinese hamster ovary cells. There are approximately 16 alpha-tubulin genes and a similar multiplicity of beta-tubulin genes. The alpha-tubulin genes are not closely linked to each other nor to the beta-tubulin genes. A genomic library has been constructed in the vector lambda charon 4A containing insert sizes of 13-20 kilobases. The library has been screened with both inter- and intra-species alpha-tubulin probes. Eleven alpha-tubulin clones with different restriction patterns have been isolated and characterized. At least seven of these clones contain the complete gene coding region. One clone appears to represent the transcribed alpha-tubulin gene II. The sequence of an intron from this gene is compared with that from an equivalent gene in the rat.
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