Biomedical subjects

C A West

Publications and source records attributed to C A West.

At least 55 records · Page 3Linked to original sources

PubMed1988

Pitfalls in the use of brain slices.

In vitro brain slices are the preparation of choice for the detailed examination of local circuit properties in mammalian brain. However it is the investigator's responsibility to verify that the circuits under investigation are indeed confined within the boundaries of the functional region of the slice used. The medium in which the slice is maintained is under the full control of the investigator. This places the burden on the investigator to ensure that: (1) the properties of the medium are fully under control; (2) the effects of the medium on the slice are known; (3) the conditions under which the slice is being maintained bear some reasonable relation to those it enjoys (or endures) in vivo. Generalizations to in vivo conditions must be made with caution. If at all possible, similar studies (perhaps less extensive, due to the greater technical difficulties) should be done in vivo to provide a basis for comparison. Investigators using drugs should be aware of, and respect, the basic pharmacological principles cited in the text. In particular, the substantial freedom the investigator has in defining the extracellular medium should not be abused.

Animals↗

PubMed1988

Expression of human calmodulin cDNA in Escherichia coli and characterization of the protein.

A cDNA clone of human calmodulin, isolated from liver, was subcloned into the expression vector pKK233-2. The resulting expression plasmid, designated pCWCaM1, produced human calmodulin in Escherichia coli SG5. The cDNA was sequenced using novel primers designed for use in plasmid-sequencing protocols with pKK233-2 and pKK223-3. The expressed calmodulin was purified and subjected to NMR analysis which revealed a structure essentially the same as natural calmodulin isolated from human tissue. The activation of myosin light chain kinase by the genetically engineered human calmodulin and bovine brain calmodulin was studied and found to be comparable to a high degree. The expressed calmodulin appears to be comparable to normal calmodulin and can be used for site-directed mutagenesis and structure/function investigations.

Base Sequence↗

PubMed1988

The rat hippocampal slice preparation as an in vitro model of ischemia.

In vivo models of cerebral ischemia do not fully control for the interacting effects of many variables (e.g., anesthesia, temperature, cerebrovascular changes) and often do not clearly define the region affected. Numerous in vivo studies have indicated that hyperglycemia augments ischemic brain damage; this effect is often attributed to lactic acidosis. To separate the effects on neuronal tissue of ischemia from those due to actions on the cerebrovascular system, we used an in vitro blood-free system as an ischemic model. In our study we evaluated the effects of various combinations of oxygen and glucose levels on evoked synaptic activity in the CA1 region of the rat hippocampal slice preparation. A 50% inhibitory dose for both oxygen and glucose on neuronal synaptic function was determined. It is our intention to use this model for preliminary screening of antihypoxic/anti-ischemic drugs.

Animals↗

PubMed1988

Evidence for receptor-mediated uptake of adenosine deaminase in rabbit kidney.

We investigated the subcellular location of adenosine deaminase-complexing protein in the proximal renal tubules of rabbit kidney and its interaction with intravenously infused monomeric calf adenosine deaminase. Cortical tissue from non-infused animals, stained in suspension by the peroxidase-antiperoxidase method for complexing protein and embedded in resin, was examined by transmission electron microscopy. Positive staining indicated the presence of complexing protein on the surface of microvilli in the proximal tubules. Sections (1 micron) of resin-embedded cortex from infused rabbits, stained first for complexing protein and then for adenosine deaminase, were examined by light microscopy. After staining for complexing protein by indirect immunofluorescence, the sections were photographed and then immersed in buffer containing 6 M guanidine hydrochloride plus 2-mercaptoethanol for 3 hr at 60 degrees C to remove bound antibodies. The sections were then stained by the peroxidase-antiperoxidase method for infused enzyme. Vesicle-like apical structures, the basal membrane area and, as previously reported, the brush border of proximal tubule cells were positive for complexing protein. Vesicle-like structures and brush borders positive for complexing protein were also stained for adenosine deaminase. The basal membrane area did not stain. These results support the hypothesis that complexing protein can act as a receptor for adenosine deaminase.

Adenosine Deaminase↗

PubMed1987

Increased glucose improves recovery of neuronal function after cerebral hypoxia in vitro.

The rat hippocampal slice preparation was used to evaluate the effect of increasing glucose levels in the perfusion medium on the recovery of synaptic function after a standardized hypoxic insult. Slices exposed to low glucose (5 mM) did not recover from a standard hypoxic insult (10 min of 95% N2/5% CO2 atmosphere). Following the same insult, 39% of the control (10 mM glucose) slices recovered their synaptic function, while 93% of the slices provided with high glucose level (20 mM) exhibited recovery of synaptic function. Thus, a dose-dependent effect of glucose on recovery of neuronal function following an intermediate period (10 min) of oxygen deprivation was found. The high-glucose-treated slices could tolerate a severe hypoxic insult of 15 min or even 20 min from which 94% and 81% of them recovered, respectively. Only 21% of the control (10 mM glucose) slices recovered their synaptic activity following 15 min of hypoxia, and none survived 20 min of that insult. The adverse effects of hyperglycemia reported in vivo were not seen in our study. This may be due to the sustained perfusion of the brain slice preparation, which could limit accumulation of lactic acid during hypoxia. However, treatment of slices with lactic acid prior to and during the hypoxic insult did not worsen the outcome. Alternatively, glucose may protect against the damaging effects of oxygen free radicals formed during reoxygenation. Nevertheless, the antihypoxic effect of glucose appears to be a metabolic one, since L-glucose (the non-metabolic analog of D-glucose) was innocuous in this respect.

Animals↗

PubMed1987

Taurine improves the recovery of neuronal function following cerebral hypoxia: an in vitro study.

Rat hippocampal slices were used in the present study to assess the effect of a pretreatment with the amino acid taurine on their ability to recover synaptic function following a standardized hypoxic insult. After 10 min hypoxia, 47% of all control (untreated) slices exhibited recovery of synaptic function (orthodromically evoked CA1 population spike). Of slices pretreated with 0.5, 1.0 or 2.0 mM taurine, 63, 88 and 97% recovered from the same hypoxic insult. This dose-dependent protective effect was biphasic, as 5.0 mM taurine produced no protection. When hypoxia was extended to 15 min, only 20% of the untreated slices recovered, while 88% of slices treated with 1.0 mM taurine recovered their population spike. The same pretreatment attenuated the fall in the population spike amplitude upon Ca2+ depletion. We hypothesize that taurine plays an important role in an endogenous antihypoxic mechanism through the attenuation of Ca2+ movement across the neuronal membrane.

Animals↗

PubMed1987

Comparison of O antigen-specific urinary immunoglobulins to Escherichia coli in normal women and women prone to Escherichia coli cystitis.

The enzyme-linked immunosorbent assay (ELISA) measured urine and serum levels of O antigen-specific IgA against Escherichia coli in five women prone to recurrent Esch. coli cystitis and in 20 normal women. Serial urine and serum samples were taken from the five women during Esch. coli infections and during uninfected intervals, while a single urine and serum sample was obtained from the 20 normal women. Elevation of any class of serum immunoglobulins for patients with uncomplicated Esch. coli cystitis was absent. A local antigen-specific immunological response, predominantly of the IgA class, was present during Esch. coli cystitis. There was no significant difference (P greater than 0.1) in the urinary or serum levels of any class of immunoglobulins between the recurrent cystitis group when uninfected and the normal women. There was a significant increase (P less than 0.001) in the levels of urinary IgA and secretory IgA during Esch. coli cystitis as compared with the same women when uninfected or with normal women. A deficiency in the local immunological response of women prone to recurrent Esch. coli cystitis is not supported by our data.

Adolescent↗

PubMed1987

Localization of adenosine deaminase and adenosine deaminase complexing protein in rabbit brain.

Adenosine deaminase and adenosine deaminase complexing protein have been localized in rabbit brain. Brains fixed in paraformaldehyde or in Clarke's solution were blocked coronally. Blocks from brains fixed in paraformaldehyde were either frozen in liquid nitrogen or embedded in paraffin. Tissue fixed in Clarke's solution was embedded in paraffin. Sections from each block were stained by the peroxidase-antiperoxidase method for adenosine deaminase or complexing protein using affinity-purified goat antibodies. Adenosine deaminase and complexing protein did not co-localize. Adenosine deaminase was detected in oligodendroglia and in endothelial cells lining blood vessels, whereas complexing protein was concentrated in neurons. The subcellular location and appearance of the peroxidase reaction product associated with individual cells was also quite distinctive. The cell bodies of adenosine deaminase-positive oligodendroglia were filled with intense deposits of peroxidase reaction product. In contrast to oligodendroglia, the reaction product associated with most neurons stained for complexing protein was concentrated in granular-appearing cytoplasmic deposits. In some instances, these deposits were clustered about the nuclear membrane. Staining of neurons in the granular layer of cerebellum was an exception. Granule cells were lightly outlined by peroxidase reaction product. Cerebellar islands, also referred to as glomeruli, were stained an intense uniform brown. These results raise the possibility that oligodendroglia and blood vessel endothelia, through the action of adenosine deaminase, might play a role in controlling the concentration of extracellular adenosine in brain. They do not, however, support the suggestion that complexing protein aids in adenosine metabolism by positioning adenosine deaminase on the plasma membrane.

Adenosine Deaminase↗

PubMed1986

A simple rapid swim test to determine spatial preference in the rat.

A swim test is described for the evaluation of spatial preference in untreated male rats. The test is rapid, objective and simple to perform and was able to distinguish right-left preferences in 47 rats. It is encouraged that the swim test be compared to or used in conjunction with other spatial preference tests.

Animals↗

PubMed1986

Effect of electrical stimulation on the viability of the hippocampal slice preparation.

Continuous electrical stimulation of rat hippocampal slices at a frequency of 1 Hz brought about a 50% decline in the evoked population spike amplitude at a rate 5 times faster than that caused by very low frequency (1/600 Hz) stimulation. Within 2 hr after the high frequency stimulation began the evoked response totally disappeared. By contrast low frequency stimulated slices maintained an evoked response for at least 9 hr. Continuous electrical stimulation, especially at high frequency seems to facilitate the deterioration of the in vitro hippocampal slice preparation.

Animals↗

PubMed1986

Lidocaine depresses synaptic activity in the rat hippocampal slice.

The direct effect of the local anesthetic lidocaine was studied using the hippocampal slice preparation in order to assess the involvement of this structure in lidocaine-induced seizure activity. Changes in the evoked field potential amplitude and latency were used to measure the effect of the drug. A dose-dependent depression of the evoked field potentials was observed at lidocaine concentration of 10(-4)M and greater. No synchronized population bursting (seizures) was observed at any of the concentrations tested (10(-6)M to 10(-3)M). However, the hippocampal slice preparation is capable of producing seizure activity, as was demonstrated following application of penicillin G. The results suggest that the hippocampus is not the site of lidocaine-induced seizure activity.

Animals↗

PubMed1986

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : Changes in Enzyme Levels Induced by Fungal Infection and Intracellular Localization of the Pathway.

Casbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids.

Journal Article↗

PubMed1986

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : The Purification and Properties of Farnesyl Transferase from Elicited Seedlings.

FARNESYL TRANSFERASE (FARNESYL PYROPHOSPHATE: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 +/- 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg(2+) ion at 4 millimolar gave the greatest stimulation of activity; Mn(2+) ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (K(m) = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K(m) = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K(m) = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes-a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.

Journal Article↗

PubMed1985

Casbene synthetase: regulation of phytoalexin biosynthesis in Ricinus communis L. seedlings. Purification of casbene synthetase and regulation of its biosynthesis during elicitation.

Castor bean (Ricinus communis L.) seedlings responded to stress by producing the antifungal diterpene, casbene. Casbene synthetase, the enzyme catalyzing the production of casbene from geranylgeranyl pyrophosphate, was purified 4700-fold to a final specific activity of 4.2 nkat/mg protein by a combination of ion-exchange and dye-ligand chromatographic procedures. Approximately 500 micrograms of purified enzyme was recovered from 1600 seedlings that had been infected with the fungus, Rhizopus stolonifer. The purified enzyme showed a single protein band, by Ag staining, of Mr 59,000 +/- 1000 after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoretic analysis of the immunoprecipitate obtained from a crude enzyme extract and polyclonal rabbit antibodies raised against the purified enzyme revealed no contaminants or cross-reacting components. In vitro translation of polysomal RNA pools obtained from healthy castor bean seedlings and seedlings at various times after exposure to pectic fragment elicitors coupled with immunoprecipitation showed that healthy seedlings have nondetectable levels of casbene synthetase mRNA and that seedlings exposed to elicitor show a rapid increase in casbene synthetase mRNA which reaches a maximum after 6 h. Casbene synthetase activity increases to a maximum 10 h after elicitation under comparable conditions. These results show that increases in the activity of mRNA for casbene synthetase after elicitation by pectic fragments precede the appearance of casbene synthetase activity as would be expected if the enzyme were being synthesized de novo.

Antibody Specificity↗

PubMed1985

Plasmid profiles and transfer of plasmid-encoded antibiotic resistance in Lactobacillus plantarum.

Plasmids were visualized in strains of Lactobacillus plantarum by use of a rapid method. Plasmids pIP501 and pAM beta 1 were transferred by conjugation from Streptococcus strains to Lactobacillus plantarum, and recipient strains were shown to act as donors in crosses to S. lactis. Attempts to transfer these plasmids between strains of L. plantarum were not successful.

Anti-Bacterial Agents↗

PubMed1985

Adenosine deaminase complexing proteins are localized in exocrine glands of the rabbit.

Adenosine deaminase complexing proteins have been localized in four exocrine glands of the rabbit by immunoperoxidase staining employing affinity-purified goat anti-rabbit complexing protein immunoglobulin as the primary antibody. In pancreatic acinar cells and in serous cells of Brunner glands (duodenal glands), staining was concentrated in granular appearing deposits between the nucleus and cell apex. Bile canaliculi, components of the exocrine liver, were also positive for complexing protein. In submaxillary glands, staining was localized in serous demilunes and striated ducts. In each instance staining was blocked by preincubating the primary antibody with complexing protein purified from rabbit kidney.

Adenosine Deaminase↗

PubMed1984

Metabolism of different molecular forms of adenosine deaminase intravenously infused into the rabbit.

High-Mr and monomeric adenosine deaminase were injected intravenously into the rabbit. The rates of clearance and sites of uptake of the enzymes were compared. Calf intestinal mucosa served as the source of monomeric adenosine deaminase. High-Mr enzymes were assembled in vitro from the calf enzyme and adenosine deaminase-complexing proteins isolated from rabbit plasma or kidney. An immunoassay specific for calf adenosine deaminase was used to determine which organs took up the injected enzyme. The enzymes were cleared from circulation in the following order: monomeric adenosine deaminase greater than high-Mr enzyme prepared with kidney complexing protein greater than high-Mr enzyme prepared with plasma complexing protein. High-Mr enzyme assembled with kidney complexing protein was taken up primarily by the liver. Complexing protein and adenosine deaminating activity were cleared from circulation at similar rates. This and other evidence indicate that kidney complexing protein and calf adenosine deaminase are taken up as a unit by the liver. In contrast, adenosine-deaminating activity was cleared more quickly from circulation than complexing protein in rabbits injected with enzyme prepared with plasma complexing protein. Immunoassay results indicated that calf adenosine deaminase was taken up principally by the kidney cortex and liver. Kidney was the major site of uptake of monomeric adenosine deaminase. Indirect immunoperoxidase staining was used to localize the calf enzyme in glomeruli and proximal renal tubules, the same areas in which rabbit complexing protein is localized. These results support the hypothesis that complexing proteins play a role in the clearance of adenosine deaminase from plasma [W.P. Schrader and P.J. Bryer (1982) Arch. Biochem. Biophys. 215, 107-115].

Adenosine Deaminase↗

PubMed1984

Kaurene Synthetase Activity in Helianthus annuus L. : Increases in Enzyme Activity after Storage of Seedlings in Liquid Nitrogen.

In previous studies, the conversion of geranylgeranyl pyrophosphate to ent-kaurene (kaurene synthetase AB activity) could not be detected readily in crude extracts of sunflower (Helianthus annuus L.) seedlings (Shen-Miller, West 1982 Plant Physiol 69: 637-641). These investigations also revealed the presence of inhibitors for Marah macrocarpus kaurene synthetase AB activity in crude extracts of sunflower seedlings. It has now been found that crude extracts prepared from intact sunflower seedlings stored in liquid N(2) for several days have greatly enhanced AB activity in comparison with frozen, but not stored, controls. The levels of activity for the conversion of copalyl pyrophosphate to ent-kaurene (kaurene synthetase B activity) are affected only slightly by storage of intact seedlings in liquid N(2). Extracts from intact seedlings that had been stored in liquid N(2) also showed less inhibitory activity for Marah macrocarpus endosperm kaurene synthetase AB activity.

Journal Article↗