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Biomedical subjects

D E Evans

Publications and source records attributed to D E Evans.

At least 37 records · Page 2Linked to original sources
PubMed1997

Visualization of Golgi apparatus in methacrylate embedded conifer embryo tissue using the monoclonal antibody JIM 84.

Methacrylate embedding followed by resin removal has been used for the first time to visualize a membrane-associated antigen at the tissue level. Monoclonal antibody JIM 84 was used to stain the Golgi apparatus of gymnosperm (conifer) embryos by light microscope immunocytochemistry. Specificity of labelling was confirmed by electron microscope immunocytochemistry using LR-white resin. GA staining was evident in all stages of white spruce somatic embryo development from immature to mature. Some regions of the somatic embryos (e.g. root cap/suspensor region) stained more vigorously than other regions (hypocotyl/cotyledon end). GA also stained in roots of Monterey pine and Douglas fir. Unlike the situation in most angiosperms, JIM 84 antigen appears to be absent from the conifer plasma membrane. However, it appears to be present in representatives of both major classes of higher plants.

Antibodies, Monoclonal↗
PubMed1997

Expression of nitric oxide synthase isoforms in bone and bone cell cultures.

Recent work has shown that nitric oxide (NO) acts as an important mediator of the effects of proinflammatory cytokines and mechanical strain in bone. Although several bone-derived cells have been shown to produce NO in vitro, less is known about the isoforms of NO synthase (NOS), which are expressed in bone or their cellular distribution. Here we investigated the expression, cellular localization, and regulation of NOS mRNA and protein in cultured bone-derived cells and in bone tissue sections. We failed to detect inducible NOS (iNOS) protein in normal bone using immunohistochemical techniques, even though low levels of iNOS mRNA were detected by sensitive reverse transcribed polymerase chain reaction (RT-PCR) assays in RNA extracted from whole bone samples. Cytokine stimulation of bone-derived cells and bone explant cultures caused dramatic induction of iNOS mRNA and protein in osteoblasts and bone marrow macrophages, but no evidence of iNOS expression was seen in osteoclasts by immunohistochemistry or in situ hybridization. Endothelial NOS (ecNOS) mRNA was also detected by RT-PCR in whole bone, and immunohistochemical studies showed widespread ecNOS expression in bone marrow cells and trabecular lining cells in vivo. Related studies in vitro confirmed that ecNOS was expressed in cultured osteoblasts, stromal cells, and osteoclasts. Neuronal NOS mRNA was detected by RT-PCR in whole bone, but we were unable to detect nNOS protein in bone cells in vivo or in studies of cultured bone-derived cells in vitro. In summary, our data show that mRNAs for all three NOS isoforms are expressed in bone and provide evidence for differential expression and regulation of the enzymes in different cell types. These findings confirm the likely importance of the L-arginine-NO pathway as a physiological mediator of bone cell function and demonstrate that it may be possible to exert differential effects on osteoblast and osteoclast activity in vivo by differential targeting of constitutive and inducible NOS isoforms by selective NOS inhibitors.

Animals↗
PubMed1997

[Cloning and expression of cDNA for tobacco rab1, and structural-functional analysis of the protein].

rab1 cDNA coding for a small GTP binding protein Rab1 was isolated from cDNA library of tobacco (Nicotiana tabacum) leaves. The primary structure of this protein was deduced from the rab1 structure. Tobacco rab1 cDNA was expressed in Escherichia coli, and the product was purified and shown to exhibit GTPase activity. A set of Rab1 mutants with altered GTP binding and/or GTPase activities was obtained. Polyclonal antipeptide antibodies were raised against a sequence in the C-terminal region of the tobacco Rab1 capable of recognizing this protein.

Amino Acid Sequence↗
PubMed1997

[Genes of plant Ser/Thr protein phosphatases: detection of sequences related to PPT/rdgC].

An unknown sequence that may encode a fragment of the Ser/Thr protein phosphatase (designated PP6Zm) related to PPT/rdgC phosphatases was identified using PCR on maize genomic DNA. A dbEST search using a partial amino acid sequence of PP6Zm revealed a putative homolog of PP6Zm expressed in Arabidopsis thaliana (EMBL AT6726). A search of the SwissProt database indicated that the partial amino acid sequence of AT6726 has the highest identity (54.3%) to the rdgC phosphatase from Drosophila melanogaster. The maize phosphatase PP1Zm6, described previously as a PP1 isoform (EMBO J., 1993, vol. 12, p. 3497), was found by us to be plant homolog of mammalian PPT. In addition, six fragments of new (pseudo) genes homologous to the phosphatase genes encoding PP1, PP2A, and PPX isoforms were detected in the maize genome. The existence in maize of a multigene PP2A family, reported only for dicotyledons, and of a PP1 multigene family, found earlier in both di- and monocotyledons, was shown.

Amino Acid Sequence↗
PubMed1994

PM-type calcium pumps are associated with higher plant cell intracellular membranes.

The supposition that all eukaryotic cell types contain a plasma membrane (PM)-type Ca pump (i.e. a Ca pump which is directly-stimulated by calmodulin and located exclusively at the PM) has been questioned by recent data from higher plant cells. These studies suggest the presence of Ca pumps directly stimulated by calmodulin associated with an intracellular membrane (probably the endoplasmic reticulum, ER) in a variety of monocotelydonous and dicotelydonous species. Thus plants have a 'PM-type' Ca pump at an intracellular membrane. The evidence for this includes studies on isolated membranes, purification and functional reconstitution and phosphorylated intermediate formation. Plant cells also contain a homologue of the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) Ca pump, probably located at the ER. The implications of these new data for our appreciation of the structure, function and location of eukaryotic Ca pumps are discussed, together with recent data from the use of inhibitors specific to mammalian ER/SR Ca pumps.

Calcium-Transporting ATPases↗
PubMed1994

Genomic sequence of a calnexin homolog from Arabidopsis thaliana.

Calnexin is a membrane-bound protein of the ER in animal cells (Wada et al., 1991). It shows considerable similarity to the major calcium-sequestering protein of the ER lumen, calreticulin, with two calcium-binding regions--a high-affinity, low-capacity region in the ER lumen and a low-affinity, high-capacity region in the cytoplasm. The protein is postulated to act as a calcium-regulated chaperone during protein maturation (Ou et al., 1993). We have isolated a genomic sequence showing significant homology to the animal gene over the predicted coding sequence (Table I). A partial cDNA from Zea mays was isolated from an expression library made from 6-d coleoptiles (Clontech, Palo Alto, CA). The library was screened using a monoclonal antibody raised against a small number of microsomal proteins resulting from a partial purification of plasma membrane Ca2+ ATPase (Briars et al., 1988). The partial cDNA showed sequence homology to the calcium-binding region common to calreticulin and calnexin. The fragment was used to screen a genomic library constructed from Arabidopsis thaliana (cv Larasbonerecta), and a 15-kb fragment was isolated and subcloned and the relevant subfragments were sequenced. The coding region contains five introns, two in the N-terminal region and three in the C-terminal region. The predicted amino acid sequence shows a high level of homology with the animal calnexin, although the terminal highly acidic calcium-binding region is shorter. A cDNA for a putative homolog of calnexin was isolated from A. thaliana (cv Columbia) by Huang et al.(1993); our coding sequence shows 85% identity and 92% similarity determined by FASTA (Wisconsin Genetics Computer Group package); however, the differences are greater than would be expected between cultivars of the same species. A Southern blot probed with DNA from the central calcium-binding region shows multiple bands. This, combined with the sequence heterogeneity, suggests that calnexin belongs to a family of related genes.

Animals↗
PubMed1994

The calcium pumps of plant cell membranes.

Active calcium transport in higher plant cell membranes involves both H(+)-linked antiport (at the tonoplast) and direct (P-type) calcium pumping ATPases. Both systems act to remove calcium from the cytoplasm either by pumping it into intracellular stores or into the apoplast. This chapter considers recent advances in our knowledge of the calcium-pumping ATPases of the plant cell, located both at the plasma membrane and in intracellular membranes. Progress in characterising the types of Ca2+ pump in plant cells is particularly important as it becomes increasingly clear that designations applicable to other eukaryotic Ca2+ pumps ('PM-type' and 'SR/ER type') are much less relevant for plant cells. Responses of plant Ca2+ pumps to mammalian Ca2+ pump inhibitors and differences in estimated relative molecular mass also underline the differences between plant and animal Ca2+ pumps. Molecular cloning has resulted in the identification of an SR/ER type Ca2+ pump in plants strongly homologous to that of mammals. These advances are put into the context of research aims in characterising the function and mechanisms of the plant Ca2+ pumps, and their role not only in regulating cytosolic free calcium concentrations, but also in providing intracellular signalling pools and in the regulation of secretion is discussed.

Biological Transport, Active↗
PubMed1993

A comparison between mivacurium and suxamethonium in children.

The neuromuscular effects of mivacurium were compared with those of suxamethonium in 69 children (aged 2-12 years), during nitrous oxide, oxygen and halothane anaesthesia in a randomised open study. Neuromuscular block was monitored by measuring the acceleration of the thumb caused by contraction of the adductor pollicis muscle after supramaximal stimulation of the ulnar nerve at the wrist using an Accelograph. End-tidal carbon dioxide was maintained at about 4 kPa in both groups. The mean times (95% confidence intervals) for T1:T0 ratio to decrease to 75%, 50%, 25% and 5% of control values were 50 (42-59), 62 (52-74), 83 (68-100) and 93 (46-108) s respectively for mivacurium and 18 (15-22), 26 (22-30), 32 (28-37) and 43 (38-49) s respectively for suxamethonium. The times for T1:T0 ratio to recover to 25%, 50% and 70% of control values were 615 (542-698), 769 (687-859) and 901 (820-993) s respectively for mivacurium and 196 (179-214), 216 (201-234) and 242 (216-259) s respectively for suxamethonium. The range of maximum block was similar for both drugs. The average time to reach maximum block was 143 s for mivacurium and 56 s for suxamethonium. Intubating conditions were similar in the two groups.

Child↗
PubMed1992

Reconstitution and Characterization of a Calmodulin-Stimulated Ca-Pumping ATPase Purified from Brassica oleracea L.

Purification and functional reconstitution of a calmodulin-stimulated Ca(2+)-ATPase from cauliflower (Brassica oleracea L.) is described. Activity was purified about 120-fold from a microsomal fraction using calmodulin-affinity chromatography. The purified fraction showed a polypeptide at 115 kD, which formed a phosphorylated intermediate in the presence of Ca(2+), together with a few polypeptides with lower molecular masses that were not phosphorylated. The ATPase was reconstituted into liposomes by 3-([cholamidopropyl]-dimethylammonio-)1-propanesulfonate (CHAPS) dialysis. The proteoliposomes showed ATP-dependent Ca(2+) uptake and ATPase activity, both of which were stimulated about 4-fold by calmodulin. Specific ATPase activity was about 5 mumol min(-1) (mg protein)(-1), and the Ca(2+)/ATP ratio was 0.1 to 0.5 when the ATPase was reconstituted with entrapped oxalate. The purified, reconstituted Ca(2+)-ATPase was inhibited by vanadate and erythrosin B, but not by cyclopiazonic acid and thapsigargin. Activity was supported by ATP (100%) and GTP (50%) and had a pH optimum of about 7.0. The effect of monovalent and divalent cations (including Ca(2+)) on activity is described. Assay of membranes purified by two-phase partitioning indicated that approximately 95% of the activity was associated with intracellular membranes, but only about 5% with plasma membranes. Sucrose gradient centrifugation suggests that the endoplasmic reticulum is the major cellular location of calmodulin-stimulated Ca(2+)-pumping ATPase in Brassica oleracea inflorescences.

Journal Article↗
PubMed1991

A method for bioassay of potency and effectiveness of inhaled bronchodilators in normal subjects.

1. Bioassay of inhaled bronchodilator drugs for potency and effectiveness in normal human subjects is rendered difficult and imprecise because bronchomotor tone is low. When attempting to determine log dose-effect relationships for inhaled, nebulised bronchodilator drugs and measuring increased specific airways conductance (sGaw), the coefficient of variation of each mean response is likely to be so large that an intermediate response is not significantly different from either the maximum or no response. 2. A method is described by which inhaled, nebulised bronchodilator drugs can be bioassayed for potency and effectiveness with high precision. The method involves the use of inhaled, nebulised bronchoconstrictor agents (methacholine or histamine) to provide a highly reproducible, near-constant, background reduction in airway conductance in normal human subjects. The activity of bronchodilators is assessed against this background reduction in airway conductance. 3. Log specific airway conductance (log sGaw) was chosen as the response metameter on grounds of normality of distribution and relative homoscedasticity. A central straight line segment of the bronchoconstrictor drug dose-effect curve extending over a 10-fold dose multiple could be found easily; the upper end of this segment occurred near 67-75% reduction in sGaw and was reproducible. 4. The effect of a dose bronchoconstrictor drug causing 67-75% reduction in sGaw waned linearly with time at first, over the effect segment corresponding to the linear segment of the drug's log dose-effect curve. The rate constant of local elimination of bronchoconstrictor drug from its site of action, in inhaled dose equivalents, was deduced from the ratio of the slopes of the linear segments of the log dose-effect and time-effect curves. 5. The maintenance dose rate to maintain the peak effect (67-75% reduction in sGaw) of the loading dose was calculated. The repeated administration of maintenance doses at regular short intervals, at this calculated dose rate, created a steady state of bronchoconstriction equal in extent to the effect of the loading dose. This steady state bronchoconstriction occurred without undue discomfort for the subject and was reproducible. 6. Against this background bronchoconstriction, inhaled, nebulised salbutamol produced a rapidly developing and slowly waning increase in sGaw. Reproducible log dose-effect curves for the bronchodilator drugs salbutamol and reproterol allowed measurement of their potency and effectiveness; the potency of reproterol relative to salbutamol was 0.082 with 95% confidence limits of 0.0504 and 0.131. 7. Estimation of the subject's pharmacodynamic and pharmacokinetic parameters for inhaled methacholine from just three dosage individualising sessions provides estimates accurate enough to be of practical value.

Adult↗
PubMed1990

Surface hydrophobicity and dispersal of Pseudomonas aeruginosa from biofilms.

A novel method of cell culture was employed to control the growth-rate of bacterial biofilms [1]. Cell-surface hydrophobicity increased progressively with growth rate for planktonic, chemostatgrown Pseudomonas aeruginosa and also for cells, resuspended from the biofilms. Dependence of surface hydrophobicity upon growth rate was greater for the planktonic cells. Newly-formed daughter cells, shed from the biofilms, were in all cases more hydrophilic than their adherent counterparts and demonstrated only slight growth rate dependency for this property.

Pseudomonas aeruginosa↗
PubMed1990

A Study of Phospholipids and Galactolipids in Pollen of Two Lines of Brassica napus L. (Rapeseed) with Different Ratios of Linoleic to Linolenic Acid.

The phospholipids and galactolipids of the pollen-coat and internal domains of two lines of Brassica napus, Wesroona and IXLIN, with different linoleic/linolenic acid ratios (18:2/18:3) have been characterized by normal phase silica high performance liquid chromatography and gas liquid chromatography. The polar lipids of the pollen-coat are similar to leaf lipids in the high proportion of galactolipids (almost 50%) and the fatty acids; 18:3, palmitic (16:0) and hexadecatrienoic (16:3). In contrast, the pollen internal domain, although rich in 18:3, 18:2 and 16:0, is composed primarily of phosphatidyl-choline, -ethanolamine, and -inositol whose 18:2/18:3 ratio is correlated with that of the seed generation. The difference between the two divergent 18:2/18:3 ratio lines is most evident in the internal domain phospholipids. The 18:2/18:3 ratio of the galactolipids of both pollen domains is not significantly effected by the line genotype. The results are interpreted in terms of the previously described ;prokaryotic' and ;eukaryotic' plant desaturation pathways (PG Roughan, CR Slack [1982] Annu Rev Plant Physiol 33: 97-132). We propose that the eukaryotic pathway is the major desaturation pathway providing polyunsaturated fatty acids to the haploid-specified internal domain in which the IXLIN genotype modifies the activity of the sn-2 linoleoyl phosphatidylcholine desaturase/s of the endoplasmic reticulum. In the diploid-specified pollen-coat, our evidence suggests that a combination of the prokaryotic and eukaryotic pathways contribute polyunsaturated fatty acids.

Journal Article↗
PubMed1990

Treatment of experimental cerebral air embolism with lidocaine and hyperbaric oxygen.

Experiments were performed to assess the combined therapeutic effects of hyperbaric oxygen (HBO) and i.v. lidocaine on neural function after ischemia induced by cerebral air embolism in anesthetized cats. Neural function was determined by measuring the somatosensory evoked potential (SEP) amplitude. Air was infused into the carotid artery in increments of 0.08 ml to maintain the SEP amplitude at 10% or less of baseline values for 15 min. Three groups were studied. A control group (n = 9) received no further treatment after SEP suppression. An HBO group (n = 8) was treated with oxygen at 2.8 atm abs for 130 min. A third group (n = 8) received an i.v. lidocaine infusion in addition to HBO. Air infusion suppressed the SEP amplitude to the same level in all groups. The control group recovered 27.4 +/- 5.5% (mean +/- SEM) of the baseline SEP amplitude, whereas the HBO group recovered 62.0% +/- 7.2%, and the HBO plus lidocaine group recovered 75.3 +/- 5.7%. The results show that both HBO and the combination of HBO and lidocaine promote a significant recovery of the SEP amplitude compared to no treatment. However, lidocaine therapy adds no benefit to HBO therapy alone.

Analysis of Variance↗
PubMed1989

The calmodulin-stimulated ATPase of maize coleoptiles forms a phosphorylated intermediate.

The calmodulin-stimulated ATPase of maize coleoptiles is a 140,000 Mr polypeptide. In the present study, formation of a phosphorylated intermediate by the enzyme is demonstrated. Phosphorylation is sensitive to chasing with unlabelled ATP and to hydroxylamine; lanthanum enhances its intensity while calmodulin enhances phosphorylation in the presence of lanthanum but not in its absence. Ethyleneglycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) inhibits phosphorylation of the purified enzyme, but microsome preparations give a band of phosphorylation of 153,000 Mr in its presence. This latter phosphorylated band was not abolished after a variety of permeabilising treatments in the presence of Triton X-100; phosphorylation of the enzyme was absent when sodium deoxycholate was used as the solubilising detergent. The identity of the 153,000 Mr band is discussed.

Adenosine Triphosphatases↗
PubMed1989

Multiple-channel stimulation of the cochlear nucleus.

To further test the feasibility of a central nervous system auditory prosthesis, the characteristics of the electrically evoked middle latency response were studied in a series of acutely anesthetized pigmented guinea pigs, with multi-channel penetrating cochlear nucleus electrodes placed into the cochlear nucleus under direct visualization. These stimulating electrodes consisted of a silicone substrate, with five stimulating pads each, sputtered with iridium. Monopolar and bipolar stimulation were used. Threshold, latency, and input-output functions of the electrically evoked middle latency response were studied. Systematic differences were observed, depending on the site and parameters of stimulation. Principally, higher currents were required to produce waves of equal amplitude when the electrodes were closely spaced. For near electrode pairs, the maximum wave amplitudies obtainable within the limits of tissue safety were much lower than for distant electrode pairs. The slope of the growth function curve was steeper for widely spaced electrodes than for adjacent sites. Monopolar stimulation demonstrated maximum wave amplitudes with the lowest current intensity, implying current spread to the entire cochlear nucleus with this stimulation montage. In some cases, threshold differences were observed, higher thresholds being associated with closely spaced electrodes. These findings are consistent with simple models of the electric fields expected to be generated by these electrode arrays. The results support the hypothesis that activation of subpopulations of auditory brainstem neurons with multi-channel penetrating microelectrodes is possible.

Animals↗
PubMed1989

Effect of lidocaine after experimental cerebral ischemia induced by air embolism.

To investigate possible approaches to the treatment of neural damage induced by air embolism and other forms of acute cerebral ischemia, somatosensory evoked potentials (SEP's) were measured after cerebral air embolism in the anesthetized cat. Air was introduced into the carotid artery in increments of 0.08 ml until the SEP amplitude was reduced to approximately 10% or less of baseline values. Either a saline or lidocaine infusion was begun 5 minutes after inducing cerebral ischemia. In the saline-treated group, SEP amplitude was reduced to 6.7% +/- 1.6% (mean +/- standard error of the mean) of baseline, with a return to 32.6% +/- 4.7% of baseline over a 2-hour period. In the lidocaine-treated group, SEP amplitude was reduced to 5.9% +/- 1.5%, with a return to 77.3% +/- 6.2% over a 2-hour period. The results suggest that lidocaine administration facilitates the return of neural function after acute cerebral ischemia induced by air embolism.

Animals↗
PubMed1987

Reduction of experimental intracranial hypertension by lidocaine.

Previous animal studies in our laboratory revealed that acute cerebral ischemia induced by air embolism is accompanied by a sharp and pronounced rise in both systemic blood pressure and intracranial pressure. To investigate possible approaches to the treatment of severe intracranial hypertension, we produced cerebral air embolism in anesthetized, ventilated cats by the infusion of air into the vertebral artery. Untreated animals consistently experienced severe acute intracranial hypertension. Pretreating the animals with lidocaine greatly attenuated the intracranial hypertension caused by air embolism. Lidocaine given after the injury was also effective in reducing intracranial pressure. Our results suggest that lidocaine may be a useful agent for reducing intracranial hypertension associated with ischemic or traumatic injuries to the brain.

Acute Disease↗