Isolation and identification of restriction endonuclease BseCI.
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Bacillus↗
Biomedical subjects
D Clark
Publications and source records attributed to D Clark.
Physical and biochemical characterization of five commercial resins for immunoaffinity purification of factor IX.
The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation factor IX (FIX) to homogeneity using monoclonal antibodies (MAb) that bind FIX in the presence of divalent cations. The MAb is immobilized on Sepharose CL2B, a soft gel with a low pressure tolerance as well as poor large-scale performance characteristics, including low reusability, and resin crumbling and deterioration. In this study, we examined several commercially available resin supports. Aside from Sepharose CL2B, we studied two other cross-linked agaroses, as well as two synthetic polymer supports. Immobilization chemistries included cyanogen bromide activation of agarose, 2-fluoro-2 methylpyridinium toluene-4-sulfonate activation of one of the synthetic polymer as well as aldehyde group reduction by NaCNBH3 to form secondary amine linkages on one of the cross-linked agaroses. To determine the feasibility of using the resins in large-scale immunoaffinity chromatographic purification of FIX, we studied physical and biochemical properties of the resins. The physical characteristics studied included the crushability of the resins under pressure as well as ability to support increasing flow-rates at increasing pressures. The biochemical examination of the various resins focused on efficiency of antigen capture by the immobilized antibody ligand and the effect of flow-rate on MAb efficiency, where we found that very low flow-rates slightly increased the capacity of the MAb. The results demonstrate a straightforward method of assessing the feasibility of using particular resins in large-scale affinity purification.
Antibodies, Monoclonal↗
Neurokinin agonists differentially affect A9 and A10 dopamine cells in the rat.
The effect of selective neurokinin (NK) receptor agonists on the activity of A9 and A10 dopamine cells was assessed using extracellular recording. A higher proportion of A10 cells which were administered the NK1 receptor agonist GR73632 or the NK3 receptor agonist senktide showed an effect, whereas the NK2 receptor agonist GR64349 did not discriminate as clearly between the two cell groups. The most frequently encountered response in all cases was an increase in firing rate.
Animals↗
Some effects of different extracellular proteins on oxygen consumption and heat production in isolated rat hepatocytes.
When hepatocytes prepared from 24-h-fasted rats were washed, suspended and incubated in Krebs-Henseleit bicarbonate-buffered saline, the endogenous rates of O2 consumption and heat production were 2.13 +/- 0.13 mumol/min per g wet wt. and 1.00 +/- 0.05 J/min per g wet wt. respectively. The inclusion of 2.5% (w/v) defatted and dialysed bovine serum albumin in either the cell suspension (washing) buffer or the cell incubation buffer produced a 20-25% increase in O2 consumption and heat production: these rates were increased by an additional 20-25% when the albumin (2.5%) was present in both the cell suspension and the cell incubation buffers. There was an inverse relationship between the increases in O2 consumption and heat production and the leakage of lactate dehydrogenase from the isolated hepatocytes: the inclusion of purified bovine serum albumin decreased lactate dehydrogenase leakage from 40% to 15% of total enzyme content. The calorimetric-respirometric ratios for hepatocytes incubated both in the absence (-461 +/- 19 kJ/mol O2) and presence (-477 +/- 8 kJ/mol O2) of the purified protein are very similar to the theoretical, thermochemically derived oxycaloric equivalents.
Animals↗
Iontophoretically administered drugs acting at the N-methyl-D-aspartate receptor modulate burst firing in A9 dopamine neurons in the rat.
Extracellular single-unit recording and iontophoresis were used to examine the effect of N-methyl-D-aspartate (NMDA) and the competitive NMDA antagonist (+/-)-4-(3-phosphonopropyl)-2-piperazine carboxylic acid (CPP) on the firing rate and firing pattern of A9 dopamine (DA) neurons in the rat. Administration of NMDA produced a dose-dependent increase in firing rate (up to nearly 300% of baseline at the highest ejection current), which could be blocked by iontophoretic CPP. Low currents (less than 10 nA) were sufficient to induce apparent depolarisation inactivation in some neurons. In addition to this effect on firing rate, NMDA also caused a dramatic increase in burst firing, which was also dose dependent; cells made more bursts, and each burst consisted of more spikes. The only measured aspect of burst morphology that was not affected was the mean burst interspike interval. All nonbursting cells (n = 10) were converted to burst firing by the drug. CPP administered alone was found to reduce burst firing, without affecting the firing rate. These data suggest that a tonically active excitatory amino acid input to A9 DA neurons is responsible for inducing burst firing in vivo and that this input seems to operate via the NMDA receptor, possibly by virtue of its link to a Ca2+ ionophore.
Animals↗
Electrophysiological evidence that intrastriatally administered N-methyl-D-aspartate augments striatal dopamine tone in the rat.
The firing rate and terminal excitability of identified nigrostriatal dopamine (DA) neurons was determined before, and over a 10-15 min period following, direct intrastriatal administration of the glutamate (GLU) agonist NMDA, or saline. NMDA (0.025 and 0.075 mumol) produced a short latency increase in DA cell firing rate. In 7/8 cases, this increase in firing rate was accompanied by a profound reduction in terminal excitability. The decrease in excitability usually outlasted the increase in firing rate (sometimes by more than 8 min), and was superseded at a later stage by a marked increase in excitability. None of these effects were seen with saline (n = 5), and they could all be blocked by preadministration of the competitive NMDA antagonist AP-7 (0.025 mumol; n = 6). The sequence of events leading to the observed results is argued to be as follows; NMDA initially excites striatal efferents to the DA cell, which through disinhibition and direct stimulation increase DA cell firing rate. Increased firing rate leads to enhanced striatal DA release. Dopamine's inhibitory influence pre-empts any effect NMDA itself may have on the terminals of nigrostriatal neurons, and counteracts NMDA's stimulatory effect on striatal output cells. Furthermore, the marked reduction in terminal excitability suggests that DA becomes the dominant influence in the striatum for a time. Hence, the net outcome of the injection is augmented striatal DA tone. Later, the effect of residual NMDA becomes predominant once more.
2-Amino-5-phosphonovalerate↗
Relapsing polychondritis--a study of four cases.
Four case reports of relapsing polychondritis, (RP), are presented, together with a literature review and management suggestions. There are approximately 211 reported cases in world literature making RP an uncommon condition associated with high morbidity and mortality rates. The key to the management of RP is based on accurate and early diagnosis though the ideal medical regimen has yet to be elucidated.
Aged↗
Differences in energy metabolism between normal weight 'large-eating' and 'small-eating' women.
Nine 'large-eating' (approximately 12 MJ/d) and nine 'small-eating' (approximately 5.3 MJ/d) women were selected from the population on the basis of diet and activity diaries. At rest and in the post-absorptive state the rate of oxygen consumption (VO2)/kg fat-free mass (FFM) and rate of carbon dioxide production (VCO2)/kg FFM were 9-17% higher (P < 0.05) in the 'large-eaters' than in the 'small-eaters'. As energy expenditure was increased by walking at 2.4, 3.9 and 5.4 km/h the differences between the two experimental groups for both VO2/kg FFM and VCO2/kg FFM were decreased to negligible values, but energy expended on a body-weight basis (MJ/kg per min) remained significantly higher (5-10%) in 'large-eaters'. Oral temperature was also consistently higher (up to 0.5 degrees) in this group both at rest and during sitting, standing and walking activities. Although the average thermic effect of a standardized liquid meal tended to be higher (27%; not significant) in the 'small-eaters', the other results demonstrate that the 'large-eating' females had a markedly higher rate of energy expenditure at rest and during light physical activities.
Adult↗
Monoclonal antibody to Pneumocystis carinii. Comparison with silver stain in bronchial lavage specimens.
Monoclonal 3F6 anti-Pneumocystis carinii antibody (MAB-3F6) was used to stain cell blocks from 164 bronchial lavage specimens from patients with the acquired immune deficiency syndrome (AIDS) and AIDS-related complex and compared with slides stained with Grocott's modification of the Gomori methenamine silver stain. Pneumocystis organisms were present in 83 of 164 cases using MAB-3F6 stain, whereas Grocott's modified silver stain demonstrated Pneumocystis organisms in 48. MAB-3F6 demonstrated Pneumocystis organisms in 38 cases with negative silver stains, whereas silver stain identified Pneumocystis organisms in only three MAB-3F6-negative cases. Of 70 patients with clinical Pneumocystis pneumonia at the time of the specimen was obtained, 59 had MAB-3F6-positive specimens, whereas 39 had organisms detected using Grocott's modified silver stain. Of 37 patients without clinically apparent Pneumocystis pneumonia any time in their course, 4 had abundant organisms and 33 had negative stains with MAB-3F6. MAB-3F6 detected Pneumocystis organisms in 22 of 31 cases of Pneumocystis pneumonia that had no organisms identified using Grocott's silver stain (X2 = 5.76, P = 0.016). MAB-3F6 immunochemical staining is a more sensitive method than Grocott's modified silver stain to detect Pneumocystis organisms.
Antibodies, Monoclonal↗
Developmental regulation of globin gene expression.
We have used the globin family of genes in chicken to study developmental regulation of gene expression, both at the level of individual interaction of trans-acting factors with local promoters and enhancers, and at the level of chromatin structure. Regulation of all members of the alpha- and beta-globin clusters is affected by the erythroid regulatory factor GATA-1. Separate mechanisms exist for regulation of individual members of the family. As an example, we describe the control mechanisms that play a role in the expression of the rho-globin gene, which is expressed only in primitive lineage erythroid cells. In addressing the involvement of chromatin structure in gene activation, we have examined the role of locus control elements, and also considered the way in which RNA polymerase molecules might accommodate to the presence of nucleosomes on transcribed genes.
Animals↗
Establishing a Writer's Academy.
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Academies and Institutes↗
In vitro fertilisation in immunological infertility.
Fertilisation occurs through a complex and orderly sequence of changes in both the oocyte and the spermatozoon. Although mechanisms have evolved to protect spermatozoa from recognition by the immune system, there is evidence that a breakdown in protection can occur resulting in homologous or heterologous antisperm antibodies which can immobilise or agglutinate spermatozoa leading to infertility. Antisperm antibodies may affect the reproductive process by interfering with sperm survival and motility and sperm transport through cervical mucus. Fertilisation is also affected, and those oocytes that do fertilise go on to develop poorly. Antisperm antibodies appear to inhibit sperm attachment to and penetration of the zona pellucida. Immune recognition of sperm antigens, which are normally present on the surface of the embryo once fertilisation has occurred, may lead to abortion after implantation. Despite the adverse effects of antisperm antibodies on fertilisation and embryo development, acceptable pregnancy rates can be achieved with assisted reproductive techniques using sperm-washing to remove antibody and using a protein source other than female serum. Further study is necessary to understand the mechanism of immunological infertility and to evaluate newer techniques using micromanipulation of gametes to achieve fertilisation.
Antibodies↗
General practitioners in the front line of suicide prevention.
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Cause of Death↗
Results of the multicenter evaluation of the CEDIA cortisol assay.
The present paper describes the multicenter evaluation of the CEDIA Cortisol test for total cortisol. The observed linearity of the test was between 1.2 and 50 micrograms/dL cortisol. The limit of detection was calculated as 1.2/dL. Imprecision studies covering the diagnostically relevant range (5-20 micrograms/dL cortisol) yielded coefficients of variation between 1.7-8.9% (within-run) and 2.7-10.5% (between-day). An interlaboratory survey using 41 human samples and three control sera demonstrated that the new CEDIA Cortisol assay has a good interlaboratory transferability. Method comparison studies between the CEDIA Cortisol test and EIA, FIA, FPIA, and various RIAs yielded an acceptable level of agreement and concordant results in most cases. Low cross-reactivity of the antibody used in the new cortisol assay was observed with precursors or metabolites of cortisol. Especially, dexamethasone did not cross-react. However, prednisolone, 6-methylprednisone, and corticosterone showed cross-reactivities. No limitation by endogenous interferences was observed. The CEDIA Cortisol assay permits the precise, fast and sufficiently specific determination of cortisol. Furthermore, it offers the advantages of a non-radioactive assay and can be performed conveniently on Boehringer Mannheim/Hitachi analyzers in combination with routine clinical chemistry.
Humans↗
Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.
The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.
Antibodies, Monoclonal↗
Role of dopamine D1 and D2 receptors in mediating the d-amphetamine discriminative cue.
The role of D1 and D2 dopamine (DA) receptors in mediating the discriminative cue produced by d-amphetamine (0.5 mg/kg) in rats has been assessed by using compounds which exert strong selectivity for each of these DA receptor subtypes. The D2 agonists quinpirole and RU 24213 substituted completely for d-amphetamine, while the D1 agonists SKF 38393 and SKF 81297 failed to exert such effects. On the other hand, the D2 antagonists raclopride and YM 09151-2, and D1 antagonists SCH 23390 and SKF 83566, all completely blocked d-amphetamine discrimination. The D2 antagonists produced more pronounced inhibitory effects on response rate than did D1 antagonists. Quinpirole substitution for d-amphetamine was blocked by YM 09151-2, but not by SCH 23390, while the locomotor stimulatory effect of quinpirole was inhibited by both drugs. The present findings confirm that D2 receptors play a primary role in the d-amphetamine discriminative cue, while the precise role of D1 receptors remains to be disclosed.
Animals↗
N-methyl-D-aspartate increases the excitability of nigrostriatal dopamine terminals.
The terminal excitability of nigrostriatal dopamine cells was measured before and after i.v. administration of N-methyl-D-aspartate (NMDA; 1 or 4 mg/kg), the competitive NMDA antagonist AP-7 (2-amino-7-phosphonoheptanoic acid) (4 mg/kg) or saline. NMDA produced a dose-dependent increase in terminal excitability, in the absence of an effect on the somal membrane, whereas AP-7 and saline were without effect. These data provide convergent in vivo evidence that glutamate can enhance striatal dopamine release via the NMDA receptor.
2-Amino-5-phosphonovalerate↗
Localization of [3H]GABA-labelled nerve fibre networks in the rat intestinal mucosa.
Laminar preparations of the rat colonic mucosa were treated for [3H]GABA (5 x 10(-8) M) autoradiography. Under conditions specific for high-affinity labelling of neuronal elements, a network of fibres overlying the base of the mucosal crypts and a second network coursing in close association with the crypts, were intensely labelled. Along the course of the overlying network of fibres, were dense accumulations of silver grains reminiscent of junctions for fibres projecting into the mucosa. All labelling could be prevented by the specific neuronal uptake inhibitor, L-DABA (10(-3) M).
Aminobutyrates↗