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Biomedical subjects

G K Smith

Publications and source records attributed to G K Smith.

At least 91 records · Page 5Linked to original sources
PubMed1986

Synthesis, utilization, and structure of the tetrahydropterin intermediates in the bovine adrenal medullary de novo biosynthesis of tetrahydrobiopterin.

The biosynthesis of two tetrahydropterin intermediates (H4pterin-1 and H4pterin-2), their conversion to tetrahydrobiopterin, and their overall chemical structures are described. A new high performance liquid chromatographic separation of these and other tetrahydropterins is also described. The biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate proceeds in the presence of the bovine adrenal medullary biosynthetic enzymes, Mg2+, NADPH. The biosynthesis of H4pterin-2 occurs under identical conditions, and the compound accumulates in the presence of 1 to 10 microM of N-acetylserotonin, an inhibitor of sepiapterin reductase. At higher concentrations of the inhibitor, the synthesis of H4pterin-2 is also inhibited, and H4pterin-1 accumulates. H4pterin-1 also accumulates in the absence of NADPH. In the presence of NADPH the biosynthetic enzymes convert both intermediates to tetrahydrobiopterin at rates which are greater than the rate of conversion of dihydroneopterin triphosphate to tetrahydrobiopterin. Electrochemical, UV/VIS, oxidation, and ionization properties identify the compounds as tetrahydropterins. The side chain structures of the compounds were determined by a combination of chemical means. The structures of the compounds are 6R-(1',2'-dioxopropyl)-tetrahydropterin (H4pterin-1) and 6R-(L-1'-hydroxy-2'-oxopropyl)-tetrahydropterin (H4pterin-2). The data indicate that the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate proceeds in three steps: 1) formation of H4pterin-1 in the presence of Mg2+, 2) NADPH-dependent conversion of H4pterin-1 to H4pterin-2, and 3) NADPH-dependent conversion of H4pterin-2 to tetrahydrobiopterin.

Adrenal Medulla↗
PubMed1986

The use of thoracic impedance for determining thoracic blood volume changes in man.

In these studies, strong inferential evidence is provided which suggests that thoracic impedance provides reliable estimates of thoracic blood volume changes in man. There were 24 volunteers studied in 4 different experiments. The results of these studies are as follows: Impedance derived blood volume changes in the calf of man correlate closely with standard estimates of calf blood volume changes made with strain gauge plethysmography. There is a close linear relationship between the increase of thoracic impedance and the increase of calf blood volume during head-up tilt. Volunteers who develop syncope during head-up tilt (presumably due to excessive decreases of central blood volume) demonstrate exaggerated increases of thoracic impedance. Decreases in central venous pressure produced by lower body negative pressure are significantly correlated to thoracic impedance increases.

Adult↗
PubMed1985

Epileptiform burst responses in ventral vs dorsal hippocampal slices.

Field potentials from area CA1 evoked by stimulation of the Schaffer collaterals were compared in dorsal and ventral hippocampal slices of rat brain. Responses were categorized into 5 response types on the basis of their morphology, ranging from simple (single spike component) to complex (multiple spike components). A higher percentage of ventral slices, compared to dorsal slices, responded with a complex morphology under normal and increased K+ concentrations. Thus, there was a significantly greater tendency for cells within the ventral hippocampus to generate burst responses.

Animals↗
PubMed1985

Fibular head transposition for repair of cruciate-deficient stifle in the dog.

Fibular head transposition (FHT) was introduced as a surgical technique to reconstruct the cruciate-deficient stifle in the dog. Surgically moving the fibular head into a cranial position alters the orientation of the lateral collateral ligament, thereby preventing cranial drawer motion and minimizing internal rotation of the tibia. The technique was found to be safe, mechanically sound, and clinically effective. Comparison of clinical results for this technique with those for the fascial strip technique (using a uniform grading system) demonstrated an overall superior performance of FHT. Factors found to adversely influence clinical results were heavy body weight and meniscal damage. The interval between stifle injury and reconstructive surgery or the presence of associated osteoarthritic disease bore no relationship to clinical outcome. It was concluded that the clinical performance after FHT was equal or superior to other surgical methods for reconstructing the cruciate-deficient stifle in the dog.

Animals↗
PubMed1985

The C-6 proton of tetrahydrobiopterin is acquired from water, not NADPH, during de novo biosynthesis.

Tetrahydrobiopterin, the cofactor for the aromatic amino acid hydroxylases, is synthesized in mammals from GTP via a pathway involving both dihydropterin and tetrahydropterin intermediates. In this work, we have investigated the mechanism of conversion of the product formed from GTP, 7,8-dihydroneopterin triphosphate, into the tetrahydropterin intermediates. Tetrahydrobiopterin can be oxidized under conditions which yield pterin or pterin 6-carboxylate without exchange of the C-6 and C-7 protons. Using these techniques, a gas chromatography/mass spectrometry method was developed to determine that in the biosynthesis of tetrahydrobiopterin de novo, in preparations of bovine adrenal medulla, the C-6 proton of tetrahydrobiopterin is derived from water and not from NADPH. In contrast, the C-6 proton of tetrahydrobiopterin produced from sepiapterin (6-lactoyl-7,8-dihydropterin) comes from NADPH. The results are consistent with evidence for the formation of the first tetrahydropterin intermediate by a tautomerization without any requirement for NADPH.

Animals↗
PubMed1985

Single-cell activity and synchronous bursting in the rat hippocampus during waking behavior and sleep.

Eighty-one of eighty-five isolated neurons recorded from the rat dorsal hippocampus (Ammon's horn and area dentata) were classifiable into four types according to their firing patterns and behavioral correlates. Sixty-two percent of the total neurons were complex spike cells which fired slowly (less than 2/s) most of the time, but were most active during slow-wave sleep, slightly less active during awake immobility, and least active during awake movement and paradoxical sleep. Theta cells (21% of the total) generally fired much faster than complex spike cells and increased their discharge rate whenever rhythmical slow activity was present in the hippocampal EEG (i.e., during awake movement and paradoxical sleep). The behavioral correlates of seven putative granule cells were similar to those of theta cells. However, the behavior-dependent changes in firing frequency were much more pronounced in the former than in the latter. Three (fast) complex spike cells were observed which fired much faster than any other complex spike cell during all states and showed the behavioral correlates similar to those of theta cells. The complex spike cells, theta cells, and fast complex spike cells were found in both Ammon's horn and dentate hilus. A type of multiunit burst was recorded from the pyramidal cell layer. It occurred most frequently during slow-wave sleep, slightly less frequently during awake immobility, and was virtually absent during awake movement and paradoxical sleep. The multiunit burst was considered to represent a summed activity of synchronously bursting complex spike cells. Possible cellular mechanisms of the state-dependent activities in the different neuronal populations of the hippocampus are discussed.

Action Potentials↗
PubMed1985

Burst characteristics of hippocampal complex spike cells in the awake rat.

Burst firing of hippocampal complex spike cells was examined in the awake rat. The results indicated that each complex spike cell tended to show a unique distribution of the number of spikes in each burst. This distribution was stable over time. The mean number of spikes per burst in 21 cells ranged from 1.06 to 4.07. Each subdivision of the hippocampus (CA1, CA3, dentate) contained cells with a wide range of burst distributions.

Action Potentials↗
PubMed1984

The development of the interictal spike during kindling in the rat.

An attempt was made to determine the location of the generator(s) of interictal discharge in the kindled rat preparation. Animals were kindled by stimulation of hippocampal area CA3, fornix/fimbria, perforant path, amygdala or lateral olfactory tract. The development of interictal discharge was monitored in the intact preparation, and hippocampal slices were subsequently taken from both kindled and control animals. Contrary to our initial hypothesis, the hippocampus did not appear to generate interictal discharge (as determined by onset time) even when the animal was kindled in the hippocampus or one of its input pathways. Also, hippocampal slices taken from kindled animals did not appear to be significantly more prone to either evoked or spontaneous epileptiform responses, compared to control slices, in medium containing high K+ concentrations. Among the structures from which recordings were taken, the amygdala and/or pyriform cortex appeared to show the earliest onset spikes regardless of the site of stimulation. It is proposed that the generator of interictal discharge in kindled animals may reside in the pyriform lobe.

Action Potentials↗
PubMed1984

A sensitive high-performance liquid chromatographic-fluorometric assay for dihydrofolate reductase in adult rat brain, using 7,8-dihydrobiopterin as substrate.

A liquid chromatographic-fluorometric assay has been developed to study the role of dihydrofolate reductase in adult rat brain since low levels of the enzyme preclude measurement by current spectrophotometric procedures. This method involves in vitro incubation of desalted, cell-free brain extracts with 7,8-dihydrobiopterin, NADPH, and an NADPH-regenerating system. The tetrahydrobiopterin formed is quantitatively converted to pterin using alkaline iodine oxidation, and the pterin formed is separated by liquid chromatography and detected fluorometrically. The method is linear from 100 fmol to greater than or equal to 1 nmol of product, and the sensitivity is at least 100 times greater than that of existing spectrophotometric assays. Enzyme activity of desalted brain extracts is linear with both time (to 100 min) and protein (from 50 to 620 micrograms). The enzyme shows an absolute requirement for NADPH, does not use NADH, and is completely inhibited by 10 nM methotrexate. The Km of the enzyme for NADPH was found to be 7.5 microM, while the Km for 7,8-dihydrobiopterin was 88 microM. Since brain dihydrobiopterin reductase has the same properties as dihydrofolate reductase, this fluorometric procedure can serve as a sensitive assay for dihydrofolate reductase.

Animals↗
PubMed1984

Two new tetrahydropterin intermediates in the adrenal medullary de novo biosynthesis of tetrahydrobiopterin.

Isolation of two kinetically competent tetrahydropterin intermediates on the de novo biosynthetic pathway to tetrahydrobiopterin is reported. The compounds were detected in HPLC chromatograms by electrochemical oxidation at 270 mv. The hydrodynamic voltamograms of the compounds are indistinguishable from those of tetrahydropterins . Both require Mg+ + for biosynthesis from dihydroneopterin triphosphate. One compound is produced in the absence of NADPH; the other requires NADPH for synthesis. Enzymatic conversion of the former into the latter intermediate requires NADPH. Conversion of either intermediate into tetrahydrobiopterin also requires NADPH. Mg+ +, which is required for the biosynthesis of the intermediates, is not needed for their conversion to tetrahydrobiopterin. Neither compound coelutes with 6-lactoyl tetrahydropterin, the tetrahydropterin analog of sepiapterin.

Adrenal Medulla↗
PubMed1984

Effects of maternal isolation on the development of activity rhythms in infant rats.

Motor activity of infant rats was measured continuously from day 3 to day 18 of post-natal age. Mother reared pups maintained on a 12 hour light/12 hour dark cycle (MRLD) show a clear predominance of nocturnal activity on day 31 post conception (post-natal day 9 or 10), 4 days before eye opening. Nocturnal partitioning increases progressively to the time of weaning. Rats reared artificially and in isolation from maternal and sibling contact but in the same light/dark regimen (ARLD) fail to develop clear nocturnal partitioning of motor activity.

Animals↗
PubMed1983

Effects of pulsed electric fields on mouse spleen lymphocytes in vitro.

The effects of pulsed electric fields on cell membranes were investigated. In vitro exposure of mouse splenocytes to a single high-voltage pulse resulted in an increase in membrane permeability that was dependent on both the electric field strength and the pulse duration. Exposure to a 2 microseconds, 3.0 kV/cm pulse resulted in the induction of a 1.26 V transmembrane potential, and elicited a 50% loss of intracellular K+. These results are in agreement with previous studies of the effects of pulsed electric fields on erythrocytes and microorganisms. The effect of pulsed electric fields on the functional integrity of lymphocytes was investigated by measuring [3H]thymidine incorporation by cells cultured in the presence and absence of various mitogens following exposure to an electrical pulse. No statistically significant effects on the response of mouse spleen lymphocytes to concanavalin A, phytohemagglutinin or lipopolysaccharide were observed following exposure to 2 microseconds electric pulses at amplitudes of up to 3.5 kV/cm. Exposure to a single 10 microseconds pulse of 2.4-3.5 kV/cm produced a statistically significant reduction in the response of lymphocytes to lipopolysaccharide stimulation that was attributed to cell death.

Animals↗
PubMed1983

Tetrahydrobiopterin is synthesized by separate pathways from dihydroneopterin triphosphate and from sepiapterin in adrenal medulla preparations.

Using Escherichia coli guanosine triphosphate cyclohydrolase, dihydroneopterin triphosphate was synthesized from guanosine triphosphate and was compared with sepiapterin as a substrate for tetrahydrobiopterin formation in bovine adrenal medulla extracts. The dihydrofolate reductase inhibitor, methotrexate, blocks the formation of tetrahydrobiopterin from sepiapterin but not from dihydroneopterin triphosphate. Reduced nicotinamide adenine dinucleotide phosphate and a divalent metal ion are required in partially purified preparations (gel filtration of 40-60% ammonium sulfate fraction on Ultrogel ACA-34) for the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. Sepiapterin was converted only to dihydrobiopterin in the same fractions since dihydrofolate reductase was removed. The evidence indicates that both dihydroneopterin triphosphate and sepiapterin are good precursors of tetrahydrobiopterin but they are not on the same pathway, contrary to previous proposals.

Adrenal Medulla↗
PubMed1982

Direct transfer of one-carbon units in the transformylations of de novo purine biosynthesis.

It is shown that the transfer of formyl units in the de novo purine biosynthetic pathway as catalyzed by glycinamide ribonucleotide (GAR) transformylase and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase probably proceeds through a direct displacement mechanism involving only formyl donor (10-CHO-H4folate) and formyl acceptor (GAR or AICAR). The inability to observe enzyme-catalyzed solvent oxygen incorporation or uncoupling by hydroxylamine of 1:1 stoichiometry between formylated acceptor [formylglycinamide ribonucleotide or 5-(formylamino)imidazole-4-carboxamide ribonucleotide] and deformylated donor implies the absence of an amidine intermediate and suggests that either a formylated enzyme-bound intermediate is not formed or such an intermediate is not accessible to hydroxylamine.

Acyltransferases↗
PubMed1982

Identification and quantification of n-acetylserotonin (NAS) in the developing hippocampus of the rat.

1. A specific anti-NAS antibody and fluorescein-labelled second antibody were employed to investigate the presence of NAS in the dentate gyrus of the hippocampus as a function of age. 2. Immunoreactive NAS (INAS) was present in the granular cell layer of the dentate gyrus as early as 20 days postconception. 3. INAS appears to be present primarily in cell bodies. 4. Cross-reactivity and inhibition experiments confirm the positive identification of INAS. 5. INAS is age-dependent and increases with age reaching adult levels by day 30 post-conception/17 days after birth. 6. The appearance and subsequent increase in INAS correlates with the development of dentate granular cells and their subsequent synapse development suggesting a role for NAS in the normal functioning of these cells.

Animals↗
PubMed1981

L(-)-10-Formyltetrahydrofolate is the cofactor for glycinamide ribonucleotide transformylase from chicken liver.

It is shown that L(-)-10-formyltetrahydrofolate serves as the cofactor for glycinamide ribonucleotide transformylase from chicken liver. The utilization of L(-)-10-formyl-H4folate was not previously recognized, because L-(+)-10-formyl-H4folate is an excellent competitive inhibitor of the enzyme, Ki = 0.75 +/- 0.07 microM, and historically the transformylase assay was carried out with a mixture of diastereomers. The results are discussed in relation to the utilization of L(+)-5,10-methenyl-H4folate.

Acyltransferases↗
PubMed1981

On the cofactor specificity of glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase from chicken liver.

Tests of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and glycinamide ribonucleotide (GAR) transformylase cofactor specificity were conducted with 5-and/or 8-deazafolate analogues formylated at N-10. Several of these compounds were found to serve as cofactors for both the enzymes. The finding that 10-formyl-8-deazafolate can be used by AICAR transformylase eliminates those mechanisms requiring cyclization to a methenyl derivative prior to carbon unit transfer for this transformylase. Surprisingly, a similar analogue, 10-formyl-5,8-deazafolate, is very effective as a cofactor for GAR transformylase in the presence or absence of the trifunctional protein which is required for 5,10-methenyl-H4-folate activity with this transformylase. This finding suggests that the trifunctional protein modulates GAR transformylase cofactor specificity by supplying the active cofactor as the N10-formyl species, possibly through a transport process that avoids its dissociation into solution.

Acyltransferases↗