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Biomedical subjects

D J Clarke

Publications and source records attributed to D J Clarke.

144 records · Page 8Linked to original sources
PubMed1983

A type of aspiny neuron in the rat neostriatum accumulates [3H]gamma-aminobutyric acid: combination of Golgi-staining, autoradiography, and electron microscopy.

Light microscopic autoradiography was used to identify cells in the neostriatum that became labelled after the local injection of [3H]gamma-aminobutyrate (GABA). The GABA-accumulating cells comprised up to 15% of the total population of neurons. Thirty-seven of these cells were examined in the electron microscope and it was found that they all had similar cytological characteristics, i.e., prominent nuclear indentations, a moderate volume of cytoplasm, rich in organelles, and sparse synaptic input to the perikaryon. Nine of the cells that had accumulated GABA were also impregnated following Golgi staining. These Golgi-impregnated neurons were of medium size and all had dendrites that were aspiny, often varicose, and that occasionally followed a recurving path. After gold toning, the Golgi-impregnated, GABA-accumulating neurons were examined in the electron microscope and were found to receive boutons forming symmetrical or asymmetrical synaptic contacts on their somata and dendrites; the symmetrical synapses were most common on the cell body and proximal dendrites, while the distal dendrites mainly received boutons forming asymmetrical contacts. We conclude that one type of GABAergic neuron in the neostriatum is a type of medium-sized aspiny neuron and that this neuron is likely to receive synaptic input both from neurons within the striatum and from neurons in distant brain regions. We suggest that this neuron is a local circuit neuron in the neostriatum since its morphological features are quite distinct from those of identified projecting neurons.

Animals↗
PubMed1982

Butyricin 7423 and the membrane H+ -ATPase of Clostridium pasteurianum.

The bacteriocin butyricin 7423 inhibited the activity of the membrane H+ -ATPase (BFoF1) of vegetative cells of Clostridium pasteurianum but not that of its soluble BF1 component. In vitro studies with the H+-ATPases of mutant strains selected for diminished sensitivity sensitivity (a) to butyricin 7423 and (b) to dicyclohexylcarbodi-imide, confirmed that butyricin 7423 interacts with the BFo component of this enzyme complex. Even so, certain other mutant strains displaying decreased sensitivity to butyricin 7423 possessed H+-ATPases which in vitro showed undiminished sensitivity to inhibition by the bacteriocin. Furthermore, from the changes in intracellular ATP concentration and in the rates and net extent of efflux of intracellular 86Rb+ ions that were provoked by exposure of the parent and several of the mutant strains to butyricin 7423, it was concluded that its primary bactericidal action was not attributable to stoichiometric inhibition of the membrane H+-ATPase. High extracellular concentrations of K+ ions enabled Cl. pasteurianum to survive exposure to low concentration of this membrane active bacteriocin.

Adenosine Triphosphatases↗
PubMed1982

On the mode of action of the bacteriocin butyricin 7423. Effects on membrane potential and potassium-ion accumulation in Clostridium pasteurianum.

1. The apparent transmembrane bulk-phase electrical potential (delta psi) of Clostridium pasteurianum was determined from the distribution ratio of the membrane-permeable cation butyltriphenylphosphonium (BuPh3P+). In glycolysing cells the highest value of delta psi, calculated on the assumption that there was no energy-dependent binding of BuPh3P+ to the organisms, was recorded in media containing only 2-3 mM K+ ions and, even so, was only 100-110 mV. 2. Efrapeptin, a BF1-directed inhibitor of the membrane H+-ATPase of Cl. pasteurianum, abolished the membrane potential (delta psi) and caused complete efflux of actively-transported K+ ions. Thus protonmotive hydrolysis of ATP generated by substrate level phosphorylation is the sole means of membrane energisation in this anaerobe. 3. At low (sublethal) concentrations, butyricin 7423 stimulated K+ efflux from Cl. pasteurianum without measurably affecting its membrane potential. At lethal and supralethal concentrations of this bacteriocin, both delta psi and active K+ uptake were abolished. 4. Whilst the addition of valinomycin to cells of Cl. pasteurianum suspended in media of low K+ concentration generated a diffusion potential to which BuPh3P+ would respond, addition of butyricin 7423 in place of valinomycin caused no such effect. Also, unlike valinomycin, butyricin 7423 did not increase the rate of K+ efflux from non-glycolysing cells of Cl. pasteurianum. Valinomycin stimulated, but butyricin 7423 inhibited, the uptake of 86Rb+ ions by glycolysing cells of Cl. pasteurianum. 5. A mutant strain of Cl. pasteurianum (viz. strain DC3) which possessed a H+-ATPase with diminished sensitivity both to N,N'-dicyclohexylcarbodiimide and to butyricin 7423, exhibited a negligible decrease in delta psi and in K+ accumulation ratio in response to concentrations of butyricin 7423 that were bactericidal to the wild-type, parent organism. Even so, the bactericidal action of butyricin 7423 on Cl. pasteurianum is not adequately explained by its ability in vitro to inhibit the membrane H+-ATPase of this organism. 6. Bactericidal concentrations of butyricin 7423 neither provoked efflux of Na+ ions from Cl. pasteurianum nor exhibited any protonophorous activity. However, at artificially high concentration, butyricin 7423 catalysed the passage of Na+ ions as well as of K+ ions through multilayer lipid membranes. 7. As a non-protonophorous uncoupler, butyricin 7423 appears to act in a similar manner to that of the membrane-active colicins. Yet no evidence was obtained that butyricin 7423 at its minimum lethal concentration might form a gated ion channel in the cytoplasmic membrane of the target cell, or act as a classic ionophore.

Adenosine Triphosphatases↗
PubMed1980

The mother-cell-membrane adenosine triphosphatase of sporulating Clostridium pasteurianum.

1. Sporulation of Clostridium pasteurianum effects several changes in its proton-translocating cell-membrane H(+)-ATPase. Notable among these are the acquisition of susceptibility to activation by trypsin and a changed protein subunit composition. 2. A protein was isolated from the mother-cell membrane that inhibited the ATP phosphohydrolase activity of purified vegetative-cell-membrane H(+)-ATPase [BF(0)F(1) complex, which consists of soluble ATPase (BF(1)) and the proton-channel component (BF(0))] and rendered it susceptible to trypsin activation. 3. This trypsin-sensitive inhibitor protein had a molecular weight of 10000 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was indistinguishable from the novel protein subunit e of the mother-cell-membrane ATPase 4. In bacteriorhodopsin-containing everted membrane vesicles, the specific ATP synthetase activity of the mother-cell-membrane ATPase was significantly greater than that of the vegetative-cell-membrane ATPase. 5. Treatment with trypsin-sensitive inhibitor protein of artificial proteoliposomes containing bacteriorhodopsin and vegetative-cell-membrane H(+)-ATPase (BF(0)F(1)) significantly increased the specific ATP synthetase activity of this enzyme. 6. The ATP synthetase activity of crude cell-membrane preparations from cultures of Clostridium pasteurianum increased during that period in the course of sporulation when the membrane ATP phosphohydrolase was both most rapidly decreasing in specific activity and acquiring its susceptibility to activation by trypsin.

Adenosine Triphosphatases↗
PubMed1980

The effect of haematin and catalase on Streptococcus faecalis var. zymogenes growing on glycerol.

Streptococcus faecalis var. zymogenes was grown aerobically on a complex medium containing glycerol as the carbon source. Addition of hematin or bovine liver catalase to the growth medium resulted in a small increment in growth yield. Suspensions of bacteria that had been grown in the presence of haematin or catalase, respectively, translocated 0.83 to 1.98 and 1.33 to 2.53 protons per oxygen atom consumed in glycerol oxidation. Bacterial grown without haematin or catalase had nil or little respiratory-induced proton translocation during glycerol oxidation. Inclusion of haematin in the growth medium caused the bacterium to form a cyanide- and azide-sensitive catalase. Superoxide dismutase activity was similar whether or not haematin was added to the growth medium.

Catalase↗
PubMed1979

The proton-translocating adenosine triphosphatase of the obligately anaerobic bacterium Clostridium pasteurianum. 1. ATP phosphohydrolase activity.

1. The cell-membrane ATP phosphohydrolase of vegetatively grown Clostridium pasteurianum was specifically Mg2+-dependent, but demonstrated significant activity with GTP, CTP and UTP. It displayed approximate Michaelis-Menten kinetics only in the presence of certain effectors (e.g. phosphoenolpyruvate, fructose 1,6-bis-phosphate) which decreased the Km for ATP (to below 2 mM) but also V, whilst extending to pH 5.8 the effective pH range of activity of the enzyme. 2. ATP phosphohydrolase activity of the membrane ATPase (BF0F1) was inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan, efrapeptin, leucinostatin and quercetin, and to a lesser degree by aurovertin and citreoviridin. The enzyme was not inhibited by oligomycin, spegazzinine, tributyl tin, triethyl tin or venturicidin. The soluble ATPase (BF1) component differed in not being inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423 or leucinostatin. 3. The ATPase (BF0F1) complex and its soluble (BF1) component were separately purified. 4. Dodecylsulphate/polyacrylamide gel electrophoresis separated only four polypeptide components in the purified ATPase (BF0F1), with approximate molecular weights (+/- 10%) as follows: subunit a, 65 500; subunit c, 57 500; subunit da, 43 000; subunit fa, 15 000. The soluble (BF1 component contained only the three polypeptide subunits a, c and da. These were present in the BF0F1 preparation in the ratio 2 : 1 : 2; the contribution of subunit fa could not satisfactorily be quantified. 5. Subunit a was identified as the component binding 4-chloro-7-nitrobenzofurazan and subunit fa as the component binding N,N'-dicyclohexylcarbodiimide. The ATP phosphohydrolase activity of the membrane ATPase was not activated by trypsin treatment and the ATPase (BF0F1) contained no trypsin-sensitive inhibitor protein subunit. 6. Purified ATPase (BF0F1) was incorporated into artificial proteoliposomes which demonstrated ATP-dependent enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and ATP-dependent proton influx. These reactions were abolished by proton conductors (e.g. carbonylcyanide m-chlorophenylhydrazone) by valinomycin in the presence of a high external concentration of K+, or by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan or leucinostatin. Oligomycin, tributyl tin, triethyl tin and venturicidin were not inhibitory. 7. When stripped of the soluble BF1 component, such ATPase-proteoliposomes demonstrated nil ATP phosphohydrolase activity and did not display ATP-dependent enhancement of 8-anilino-naphthalene-1-sulphonate fluorescence or ATP-dependent protein influx. All of these activities were restored by incubation of the BF1-depleted proteoliposomes with a purified preparation of the soluble BF1 component.

Adenosine Triphosphatases↗
PubMed1976

Butyricin 7423: a bacteriocin produced by Clostridium butyricum NCIB7423.

Butyricin 7423 is a trypsin-sensitive bacteriocin produced by Clostridium butyricum NCIB7423 and active against some other species of Clostridium. It has a bactericidal but non-lytic action on growing cultures of Clostridium pasteurianum. The primary action of butyricin 7423 appears to be at the cell membrane. Thus treatment of C. pasteurianum with an excess of butyricin altered the permeability of its cell membrane, allowing the release of several metabolites and ions. Efflux of rubidium ions from organisms preloaded with 86Rb+ was particularly rapid and extensive. The action of butyricin 7423 on Clostridium species seems to differ from that of other clostridocins, such as the boticins and perfringocin 28, and more closely resembles the mode of action on susceptible (aerobic) organisms of bacteriocins such as staphylococcin 1580 or colicins A, E1, I and K.

Bacterial Proteins↗
PubMed1975

Purification of two Clostridium bacteriocins by procedures appropriate to hydrophobic proteins.

Two clostridocins distinguishable by their different modes of action on Clostridium pasteurianum have been isolated, namely, butyricin 7423 found in cultures of Clostridium butyricum NCIB 7423 and perfringocin 11105 produced by Clostridium perfringens type A, NCIB 11105. Both were trypsin-susceptible proteins which were soluble in concentrated aqueous ethanol and were able to bind large amounts of the nonionic detergent Triton X-100. In the presence of Triton X-100, butyricin 7423 behaved as a hydrophobic protein in being concentrated in the polyethylene glycol layer of a three-phase partition system of dextran-Ficoll-polyethylene glycol. Their capacity to bind Triton X-100 was exploited in a purification procedure applicable to both bacteriocins. After aqueous ethanol extraction of an ammonium sulfate-precipitated fraction (and, in the case of the perfringocin, a heat-treatment step), a bacteriocin-Triton X-100 adduct was purified by gel filtration through Sepharose 6B. The bacteriocin was then freed of Triton X-100 by chromatography on Sephadex LH-20. Samples of butyricin 7423 purified in this way from different sources contained variable amounts of carbohydrate. Yet sodium dodecyl sulfate-gel electrophoresis revealed the existence of a polypeptide component of 32,500 daltons (+/-10%), which displayed the biological activity of butyricin 7423 in the absence of any detectable associated carbohydrate (or lipid). Preparations of perfringocin 11105 contained no carbohydrate or lipid and migrated in sodium dodecyl sulfate-gel electrophoresis as a single protein component of 76,000 daltons (+/-10%). It was concluded that both bacteriocins behave as amphiphilic proteins, and some implications of this finding are considered.

Bacteriocins↗
PubMedDate not supplied

Synaptic relationships between cortical and dopaminergic inputs and intrinsic GABAergic systems within intrastriatal striatal grafts.

Synaptic relationships between gamma-aminobutyric acid-ergic systems intrinsic to intrastriatal striatal grafts and inputs from host adult rat neocortex and substantia nigra were investigated using a variety of neuroanatomical techniques. The input from host frontal cortex was demonstrated using an anterograde degeneration technique, whilst a double immunocytochemical procedure, using the chromogens diaminobenzidine and benzidine dihydrochloride was utilized to visualize the tyrosine hydroxylase (TH)- and glutamate decarboxylase (GAD)-immunoreactive systems. Only areas receiving dense TH-immunoreactive innervation were examined for synaptic interactions since these areas were judged as being striatal in origin. Examples of synaptic interactions were observed between cortical and TH-immunoreactive inputs; between cortical input and GAD-immunoreactive neuronal elements within TH-immunoreactive inputs and a variety of GAD-immunoreactive neuronal elements within the striatal grafts. No interactions were seen between cortical input and GAD-immunoreactive neuronal perikarya or dendrites, possibly because of technical limitations since the GAD-immunoreactivity did not extend into the distal dendrites where cortical input is predominantly located, nor between all three systems. The results suggest that the formation of new synaptic connections in a pattern reminiscent of that seen in control neostriatum may be responsible, in part at least, for the behavioural recovery in motor skills seen in rats following intrastriatal striatal transplants. They also demonstrate that the host adult brain retains sufficient plasticity and may play an important role in the control of synaptic output from the transplant.

Animals↗
PubMedDate not supplied

Human neural graft function in rats treated with anti-interleukin II receptor antibody.

Prolonged immunosuppression with cyclosporin A allows survival of human xenografts in the rat Parkinsonian model but the drug has side effects. Ideally immunosuppression should be of short duration, to minimize the chance of infection, yet be capable of supporting long term survival of the transplanted tissue. We report that short term treatment with an anti-rat interleukin II receptor (IL2R) monoclonal antibody (MAb) resulted in apparently permanent survival of human fetal dopaminergic grafts in "Parkinsonian" rats. The recipients remained healthy and the excellent survival of the transplants suggests that the antibody injection strategy would almost certainly abrogate allograft rejection and raises the possibility that xenogenic dopaminergic neurones could be used as donor tissue in humans.

Animals↗
PubMedDate not supplied

Educational and occupational outcome in Prader-Willi syndrome.

An educational and occupational history was obtained for affected members of the Prader-Willi Syndrome Association (UK). Of 32 females and 29 males over 16 years of age (mean 23 years), over 45% attended mainstream infant schools, but by the time they reached junior and secondary schools the majority were in schools for children with learning disabilities. On reaching adulthood, 31% were attending adult training centres or day centres. Nearly one in six were in employment, usually of a sheltered type. The majority had no formal educational qualifications, but over 10% had at least one Certificate of Secondary Education and one person had three O levels. Nearly three-quarters of those with known intelligence quotients had IQs in the 70 to 100 range. The physical and behavioural characteristics of the study population closely resembled those reported in Prader-Willi syndrome by other authors, although it is possible that some members of the Prader-Willi Syndrome Association (UK) may not have Prader-Willi syndrome. The findings highlight the heterogeneity in educational achievements associated with Prader-Willi syndrome, and illustrate the importance of assessing individual educational strengths and needs.

Adolescent↗
PubMedDate not supplied

Survival of nigral grafts within the striatum of marmosets with 6-OHDA lesions depends critically on donor embryo age.

The study examined the importance of embryonic donor age for the survival of nigral grafts in 6-OHDA-lesioned marmosets. The issue as to whether donor age is critical for the survival of nigral grafts in primates is controversial, because several early reports suggested that relatively old tissue could survive transplantation and produce functional benefits in monkeys, in contrast to the restrictive time dependence observed in rodents. Embryonic marmoset donors embryos of three different ages were employed: 1) E74 (Carnegie stage 18-19); 2) E83-84 (Carnegie stage 23+); 3) E92-93 (foetal period). The nigral neurons derived from the ventral mesencephalon in the two older donor age groups did not survive well when grafted to the striatum of adult marmosets with unilateral 6-OHDA lesions. Although a few tyrosine hydroxylase (TH+) neurons could be identified by immunohistochemistry at graft sites in all recipients in older donor age groups, the numbers of surviving neurons in these were small, on average typically less than 100 TH+ cells. These small grafts were not sufficient to affect amphetamine-induced rotation. In contrast, many more TH+ cells typically survived transplantation in the recipients of graft tissue derived from the youngest donors and amphetamine-induced rotation was significantly reduced in this group alone. The time course and extent of the reduction in rotation was remarkably similar to that observed in previous marmoset nigral graft studies, confirming the utility of amphetamine-induced rotation as a sensitive and reliable indicator of nigral graft function in this species. Considering these results and other recent evidence from monkey to monkey, human to rat, and human to human graft studies, the survival of embryonic nigral tissues derived from primate donors transplanted into the striatum does appear to be critically dependent on the age of the donor tissue.

Animals↗