PubMed Health⌕ Search

Biomedical subjects

R Griffiths

Publications and source records attributed to R Griffiths.

At least 19 recordsLinked to original sources
PubMed1992

Neuronal and glial plasma membrane carrier-mediated uptake of L-homocysteate is not selectively blocked by beta-p-chlorophenylglutamate.

The proposed action of beta-p-chlorophenylglutamate (chlorpheg) as a selective blocker of L-homocysteate uptake was studied in primary cultures of mouse brain neurons and astrocytes and in rat brain synaptosomes. The preparations were incubated with 1 microM to 10 mM L-homocysteate and D-aspartate in the absence and presence of 7.5 mM chlorpheg. In each preparation uptake of the two amino acids comprised a saturable uptake and a non-saturable (passive diffusion) component. L-Homocysteate was of at least 100-fold lower affinity than D-aspartate as a substrate for the amino acid transporter. Chlorpheg was shown to be essentially equieffective as a weak competitive inhibitor of only the saturable uptake of L-homocysteate and D-aspartate. It is concluded that chlorpheg is not a selective blocker of L-homocysteate uptake.

Amino Acid Transport Systems↗
PubMed1992

Simultaneous measurement by HPLC of the excitatory amino acid transmitter candidates homocysteate and homocysteine sulphinate supports a predominant astrocytic localisation.

Primary cultures of mouse cerebral cortex neurons, cerebellar granule cells and cortical astrocytes were maintained in vitro for respectively 8-10, 7-10 and 21-24 days. Following these times, amino acids were extracted from the cells by use of ice-cold 70% (v/v) ethanol and the extracts lyophilised. The lyophilised extracts when resuspended were subjected to reverse-phase high performance liquid chromatographic (HPLC) analysis for detection of free amino acids. Samples of cell culture growth medium and water blanks were treated in a similar manner. Identification of L-homocysteate (HCA) and L-homocysteine sulphinate (HCSA) was undertaken by matching retention times with regard to external standards and by 'spiking' cell extracts with authentic compounds. On this basis, HCA and HCSA were consistently detectable in astrocytes at levels of, respectively, 72.3 +/- 33.7 pmol/mg protein (n = 24) and 49.4 +/- 28.7 pmol/mg protein (n = 24). However, in neurons, a peak corresponding to HCSA could not be detected above the background noise, while the area of the peak corresponding to HCA was always greater than, but not significantly different from, that of the background noise present in water blanks. HCA and HCSA were not detectable in the serum used for preparation of the cell culture growth medium. Taken together, these findings indicate a predominant localisation of HCA and HCSA in astrocytes which, at least in culture, appear to possess the metabolic machinery necessary for synthesising and storing these amino acids without any neuronal influence.

Amino Acids↗
PubMed1992

Synaptosomal plasma membrane transport of excitatory sulphur amino acid transmitter candidates: kinetic characterisation and analysis of carrier specificity.

The transport kinetics of the excitatory sulphur-containing amino acid (SAA) transmitter candidates, L-cysteine sulphinate (L-CSA), L-cysteate (L-CA), L-homocysteine sulphinate (L-HCSA), and L-homocysteate (L-HCA), together with their plasma membrane carrier specificity, was studied in cerebrocortical synaptosome fractions by a sensitive high performance liquid chromatographic assay. A high affinity uptake system could be demonstrated for L-CSA (Km = 57 +/- 6 microM; Vmax = 1.2 +/- 0.1 nmol/min/mg protein) and L-CA (Km = 23 +/- 3 microM; Vmax = 3.6 +/- 0.1 nmol/min/mg protein), whereas L-HCSA (Km = 502 +/- 152 microM; Vmax = 6.1 +/- 1.3 nmol/min/mg protein) and L-HCA (Km = 1550 +/- 169 microM; Vmax = 10.3 +/- 1.1 nmol/min/mg protein) exhibited much lower affinity as transport substrates. In all cases, only a single, saturable Na(+)-dependent component of uptake could be identified, co-existing with a non-saturable, Na(+)-independent influx component. Plasma membrane carrier specificity of the SAAs was established following comparison with other high-affinity neurotransmitter systems. High-affinity L-CSA and L-CA transport and low-affinity L-HCSA and L-HCA transport demonstrate strong positive correlations in inhibition profiles when compared against each other or individually against the high-affinity transport of L-[3H]glutamate, L-[3H]aspartate, or D-[3H]aspartate. Moreover, the transport systems for the excitatory SAAs exhibited a negative correlation when compared in inhibition profiles with the high affinity transport of both [3H] gamma-aminobutyric acid (GABA) and [3H]taurine.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1992

Homocysteine-induced alterations in extracellular amino acids in rat hippocampus.

The effects of DL-homocysteine, and DL-homocysteate, on extracellular levels of amino acids in the rat hippocampus have been studied using brain microdialysis. Hippocampal electroencephalogram activity was monitored simultaneously using an electrode attached to the dialysis probe. DL-Homocysteine (1200 mg/kg; i.p. injection) produced epileptic activity in hippocampus in an inconsistent manner. Alterations in electroencephalogram activity were not observed in urethane anaesthetized animals, whereas 50% of Hypnorm anaesthetized animals exhibited epileptic activity. DL-Homocysteate (2 mu mol; i.c.v.) induced epileptic activity in a majority of animals anaesthetized using urethane. Dialysate levels of aspartate were significantly elevated by homocysteine in both groups of animals. Conversely, dialysis levels of GABA were reduced. Dialysate levels of other amino acids measured (glutamate, glutamine, taurine, alanine and valine) were not affected significantly. Dialysate levels of taurine were increased significantly in animals injected with homocysteate. These data suggest that the imbalance in excitatory:inhibitory neurotransmission in the hippocampus caused by these alterations in extracellular levels of neuroexcitatory (i.e. aspartate) and neuroinhibitory (i.e. GABA) transmitters could underly the epileptic effect of homocysteine.

Amino Acids↗
PubMed1992

Sulphur-containing excitatory amino acid-evoked Ca(2+)-independent release of D-[3H]aspartate from cultured cerebellar granule cells: the role of glutamate receptor activation coupled to reversal of the acidic amino acid plasma membrane carrier.

Sulphur-containing excitatory amino acid transmitter candidates (500 microM) stimulated the Ca(2+)-independent efflux of exogenously-supplied D-[3H]aspartate from primary cultures of cerebellar granule cells superfused continuously with HEPES-buffered saline containing CoCl2 (1 mM) in place of CaCl2. The stimulated release of D-[3H]aspartate was markedly attenuated by 200 microM 6,7-dinitroquinoxalinedione, a concentration at which the antagonist inhibits both non-N-methyl-D-aspartate and N-methyl-D-aspartate ionotropic excitatory amino acid receptors. The Ca(2+)-independent component of evoked release was also markedly attenuated and, in some cases, abolished by removing NaCl from the superfusion medium. Furthermore, when 700 microM dihydrokainate (demonstrated herein as a mixed/non-competitive inhibitor of the high-affinity dicarboxylic amino acid transporter in cultured granule cells) was included in the superfusion medium, stimulated efflux of D-[3H]aspartate was reduced by between 15-78% of the control response; the extent of inhibition varying with the agonist employed. In constrast, agents which act as competitive inhibitors of the plasma membrane carrier in granule cells, e.g. beta-methylene-D,L-aspartate, potentiated the release of D-[3H]aspartate in a synergistic manner. Taken together, these findings are consistent with a mechanism for the Ca(2+)-independent release of D-[3H]aspartate that is mediated predominantly by activation of excitatory amino acid receptors resulting in a reversal of the high-affinity dicarboxylic amino acid transport system. Although the physiological relevance of such non-vesicular release from the cytosol remains obscure and is still a matter of some debate, this mode of release may be of pathological significance.

Animals↗
PubMed1992

The identification of sex in the starling Sturnus vulgaris using a molecular DNA technique.

Female birds can be identified through the presence of a W-chromosome. We describe a procedure for amplifying a W-linked DNA marker in the starling (Sturnus vulgaris) by the polymerase chain reaction (PCR) so allowing the diagnosis of sex in this species. The technique is sensitive, allowing even the smallest chicks to be sexed from a blood sample. The method possesses a positive internal control to ensure accuracy. It is also applicable to the spotless starling (S. unicolor) but not to two bird species outside the genus. The nucleotide sequence of the female-specific PCR product is given.

Animals↗
PubMed1992

Health service utilisation in the public and private sector by patients with diabetes mellitus aged less than 40 years.

This paper uses the Illawarra 0-39 years diabetes register to provide a data base of health service utilisation in the public and private sectors. Eligible patients from the register were divided into a 'stable group' comprising children and adults who had had diabetes for more than one year prior to review and a 'newly diagnosed' group who were followed for one year after diagnosis. The records of inpatient and selected outpatient services at public hospitals as well as visits to general practitioners, paediatricians and physicians were considered. Less than half of all 'newly diagnosed' patients were admitted to hospital for stabilisation and none required readmission in the 12 month review period. Less than one fifth of all 'stable' patients were admitted to hospital during the year. General practitioners were the most frequently used health resource being attended by 31/38 (82%) 'stable children', 122/133 (92%) 'stable adults' and 25/25 (100%) 'newly diagnosed patients'. Public hospital Accident and Emergency Services were the least used health facility being frequented by only 9/38 (24%), 23/133 (17%) and 7/25 (28%) respectively.

Adolescent↗
PubMed1992

Crosstransplantation of kidneys in normal and Hyp mice. Evidence that the Hyp mouse phenotype is unrelated to an intrinsic renal defect.

Although deranged phosphate transport is the fundamental abnormality in X-linked hypophosphatemic (XLH) rickets, it remains unknown if this defect is the consequence of an intrinsic kidney abnormality or aberrant production of a humoral factor. To discriminate between these possibilities, we examined phosphate homeostasis in normal and Hyp mice, subjected to renal crosstransplantation. We initially evaluated the effects of uninephrectomy on the indices of phosphate metabolism that identify the mutant biochemical phenotype. No differences were found in the serum phosphorus concentration, fractional excretion of phosphate (FEP), or tubular reabsorption of phosphate per milliliter of glomerular filtrate (TRP) in uninephrectomized normal and Hyp mice, compared with sham-operated controls. Subsequently, single kidneys from normal or Hyp mice were transplanted into normal and Hyp mouse recipients. Normal mice transplanted with normal kidneys and Hyp mice engrafted with mutant kidneys exhibited serum phosphorus, FEP, and TRP no different from those of uninephrectomized normal and Hyp mice, respectively. However, engraftment of normal kidneys in Hyp mice and mutant kidneys in normal mice affected neither serum phosphorus (4.69 +/- 0.31 and 8.25 +/- 0.52 mg/dl, respectively) nor FEP and TRP of the recipients. These data indicate that the Hyp mouse phenotype is neither corrected nor transferred by renal transplantation. Further, they suggest that the phosphate transport defect in Hyp mice, and likely X-linked hypophosphatemia, is the result of a humoral factor, and is not an intrinsic renal abnormality.

Animals↗
PubMed1991

The isolation of conserved DNA sequences related to the human sex-determining region Y gene from the lesser black-backed gull (Larus fuscus).

The sex-determining region gene (SRY) is a recently discovered candidate for the mammalian testis determining factor (TDF). The gene appears to form part of a family with several autosomal representatives. I have investigated the occurrence of SRY-related sequences in birds. The polymerase chain reaction was used to isolate six homologues of the conserved motif of the SRY gene from the lesser black-backed gull (Larus fuscus). Female unique copies of the motif are apparently absent. Sequence similarity and phylogenetic parsimony methods suggest that, irrespective of avian or mammalian origin, SRY-type sequences fall into two subfamilies. This is consistent with the presence of two archetypal genes in an ancestor common to both taxonomic classes.

Amino Acid Sequence↗
PubMed1991

Glutamate uptake into synaptic vesicles--inhibition by sulphur amino acids.

In an attempt to investigate the apparent absolute selectivity of the synaptic vesicle L-glutamate carrier, L- and D-enantiomers of excitatory sulphur-containing amino acid (SAA) transmitter candidates (which are close structural analogues of L-glutamate) were tested for their capacity to compete for vesicular L-[3H]-glutamate uptake. All SAAs inhibited, to varying degrees (52-86%), the vesicular uptake of L-[3H]-glutamate. A similar level of inhibition was exerted by SAAs with either a shorter or equal carbon chain length to L-glutamate. Moreover, inhibition was stereospecific in favour of the D-enantiomers. These studies indicate an appreciable interaction of the SAAs with the recognition site of the vesicular L-glutamate carrier. Further investigations are required to establish the substrate potential of the SAAs.

Animals↗
PubMed1991

Stimulation of gamma-[3H]aminobutyric acid release from cultured mouse cerebral cortex neurons by sulphur-containing excitatory amino acid transmitter candidates: receptor activation mediates two distinct mechanisms of release.

In primary cultures of mouse cerebral cortex neurons, sulphur-containing excitatory amino acids (SAAs; namely, L-cysteine sulphinate, L-cysteate, L-homocysteine sulphinate, L-homocysteate, S-sulphocysteine) at concentrations ranging from 0.1 microM to 1 mM evoked a saturable release of gamma-[3H]aminobutyric acid ([3H]GABA) in the absence of any other depolarizing agent. All SAAs exhibited essentially similar potency (EC50, 100-150 microM) in releasing [3H]GABA although a variable profile of maximal stimulatory effect was observed when compared with basal release. The intracellular accumulation of the lipophilic cation, [3H]tetraphenylphosphonium, was significantly reduced in the presence of all SAAs, thus verifying a depolarization of the neuronal plasma membrane. SAA-stimulated release of [3H]GABA was shown to comprise two distinct components, calcium-dependent and calcium-independent, which occur after activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Thus, all SAA-evoked responses were antagonized by the selective, competitive NMDA-receptor antagonist, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (IC50 range, greater than 50 microM) and the non-NMDA-receptor antagonist, 6,7-dinitroquinoxalinedione (IC50 range, 5-50 microM). Removal of magnesium ions from the superfusion medium caused a significant potentiation of SAA-evoked responses without having any effect on basal levels of [3H]GABA efflux, a result consistent with an involvement of NMDA-receptor activation. Calcium-independent release (i.e., that release remaining in the presence of 1 mM cobalt ions) was a distinct component but of smaller magnitude. Using 500 microM excitatory amino acid agonist concentrations, this component of release was (1) markedly attenuated by 15 microM SKF-89976-A, a non-transportable inhibitor of the GABA carrier, and (2) abolished when choline ions replaced sodium ions in the superfusion medium or when in the presence of excitatory amino acid receptor antagonists. These observations are clearly consistent with a receptor-mediated, depolarization-induced reversal of the GABA carrier.

Amino Acids↗
PubMed1990

A novel avian W chromosome DNA repeat sequence in the lesser black-backed gull (Larus fuscus).

The phenol emulsion reassociation technique was used to isolate and clone a female specific, repetitive DNA sequence from Larus fuscus. The repeat, designated P2000-17, is restricted to the W chromosome, although related sequences occur elsewhere in the genome of L. fuscus. Similar sequences were detected in the genome of six other bird species from outside the genus Laridae, but the sequence occurs less frequently and to a similar extent in both sexes. The 298 bp DNA sequence of P2000-17 was determined and found to have extensive sequence identity to the rabbit dihydropyridine (DHP) receptor calcium channel. P2000-17 is represented once within a larger 8.6 kb tandem repeat (LfW-1), which has a complex internal DNA sequence. LfW-1 is highly conserved between repeat motifs and may comprise 3% of the female genome. The possible evolutionary origin of LfW-1 is discussed in relation to the repeat types found on the W and Y chromosomes of other species.

Animals↗