Biomedical subjects
Publications and source records attributed to D L Martin.
Granular cell tumour of the glans penis is a rare finding with only two other cases reported. This case highlights the importance of achieving the correct diagnosis, as differing excision margins are critical, particularly in this site.
A large number of techniques have been described for the correction of prominent ears to improve the cosmetic outcome and reduce the complication rates. The procedure favoured by the senior author brings together a number of refinements, notably, percutaneous anterior scoring using a modified green needle, control over the degree of fold created and a simple but effective dressing. 114 consecutive patients underwent the correction of 214 ears, with a mean follow up of 3 years and 11 months (9 months to 9 years and 6 months). The senior author performed 100 of these procedures and supervised a senior trainee for the remainder. The mean patient age was 18 years 3 months (3 to 66 years). 57 males and 57 females. 56 general anaesthetic and 58 local anaesthetic. Post-operative complications were; haemorrhage, one ear (required a dressing change); infection, four ears (treated with antibiotics); hypertrophic scarring, two ears which settled (no keloid); recurrence one ear (repeated surgery); continued prominence six ears (two had repeated surgery). No prominent sutures, no anterior skin necrosis, no visible irregularity of the anterior surface of the cartilage and no haematoma occurred. We feel that the low complication rate is due to maximising the advantages and minimising the disadvantages of the different techniques and refinements. We recommend this technique for the routine correction of prominent ears due to a poorly formed antihelical fold or deep conchal bowl.
gamma-Aminobutyric acid (GABA) synthesis in the brain is mediated by two major isoforms of glutamic acid decarboxylase, GAD(65) and GAD(67). The contribution of these isoforms to GABA synthesis flux (V(GAD)) is not known quantitatively. In the present study we compared V(GAD) in cortex of control and vigabatrin-treated rats under alpha-chloralose/70% nitrous oxide anesthesia, with total GAD activity and GAD isoform composition (GAD(65) and GAD(67)) measured by enzymatic assay and quantitative immunoblotting. V(GAD) was determined by re-analysis of 13C NMR data obtained ex vivo and in vivo during infusions of [1-13C]glucose using an extension of a model of glutamate-glutamine cycling that included a discrete GABAergic neuronal compartment with relevant interconnecting fluxes. V(GAD) was significantly lower in vigabatrin-treated rats (0.030-0.05 micromol/min per g, P<0.003) compared to the non-treated control group (0.10-0.15 micromol/min per g). The 67-70% decrease in V(GAD) was associated with a 13% decrease in total GAD activity (P=0.01) and a selective 44+/-15% decrease in GAD(67) protein (from 0.63+/-0.10 to 0.35+/-0.08 microg protein/mg tissue, P<0.05); GAD(65) protein was unchanged. The reduction in GAD(67) protein could account for a maximum of approximately 65% of the decrease in V(GAD) in vigabatrin-treated animals suggesting that inhibition of GAD(65) must have also occurred in these experiments, although product inhibition of GAD(67) by increased GABA could play a role. GAD(67) could account for 56-85% of cortical GABA synthesis flux under basal conditions and the entire flux after vigabatrin treatment.
Cofactor and tryptophan accessibility of the 65-kDa form of rat brain glutamate decarboxylase (GAD) was investigated by fluorescence quenching measurements using acrylamide, I-, and Cs+ as the quenchers. Trp residues were partially exposed to solvent. I- was less able and Cs+ was more able to quench the fluorescence of Trp residues in the holoenzyme of GAD (holoGAD) than the apoenzyme (apoGAD). The fraction of exposed Trp residues were in the range of 30-49%. In contrast, pyridoxal-P bound to the active site of GAD was exposed to solvent. I- was more able and Cs+ was less able to quench the fluorescence of pyridoxal-P in holoGAD. The cofactor was present in a positively charged microenvironment, making it accessible for interactions with anions. A difference in the exposure of Trp residues and pyridoxal-P to these charged quenchers suggested that the exposed Trp residues were essentially located outside of the active site. Changes in the accessibility of Trp residues upon pyridoxal-P binding strongly supported a significant conformational change in GAD. Fluorescence intensity measurements were also carried out to investigate the unfolding of GAD using guanidine hydrochloride (GdnHCl) as the denaturant. At 0.8-1.5 M GdnHCl, an intermediate step was observed during the unfolding of GAD from the native to the denatured state, and was not found during the refolding of GAD from the denatured to native state, indicating that this intermediate step was not a reversible process. However, at >1.5 M GdnHCl for holoGAD and >2.0 M GdnHCl for apoGAD, the transition leading to the denatured state was reversible. It was suggested that the intermediate step involved the dissociation of native dimer of GAD into monomers and the change in the secondary structure of the protein. Circular dichroism revealed a decrease in the alpha-helix content of GAD from 36 to 28%. The unfolding pattern suggested that GAD may consist of at least two unfolding domains. Unfolding of the lower GdnHCl-resisting domain occurred at a similar concentration of denaturant for apoGAD and holoGAD, while unfolding of the higher GdnHCl-resisting domain occurred at a higher concentration of GdnHCl for apoGAD than holoGAD.
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To investigate ATP as a potential factor in the regulation of brain glutamate decarboxylase (GAD), the impact of ATP on the enzyme conformation and holoenzyme formation was investigated. ATP at 100 microM quenches fluorescence emission intensity of the holoenzyme of GAD (holoGAD) by 18% after a correction for the inner filter effect and enhances fluorescence steady-state polarization from 0.158 to 0. 183 when excited at 280 or 295 nm. These findings suggest that ATP moderately changes the microenvironment of one or more tryptophan or tyrosine residues in holoGAD and alters these residues from a more mobile state to a less mobile one. A moderate ATP-induced conformational change in holoGAD is also supported by the observations that ATP increases the thermal denaturation temperature of holoGAD by 2 degrees C, as derived from temperature-dependent fluorescence spectra, and decreases the alpha-helical content of holoGAD by 8-10%, as determined by circular dichroism. Moreover, ATP does not affect the keto-enol tautomerization of holoGAD and has little or no direct effect on its activity, implying that the ATP interacting domain in holoGAD is not at the active site. Kinetics studies, as demonstrated by stopped-flow fluorescence and UV/visible spectroscopy, demonstrate that formation of holoGAD involves two steps: a fast reaction forming an apoGAD-cofactor intermediate complex, followed by a slow reaction involving the conformational change in the intermediate complex. ATP reduces the rate constant of the fast step to one-third and decreases the rate of the slow step and the intermediate complex formation constant to 60% of their original values. The present data suggest that ATP may regulate the interconversion between apoGAD and holoGAD by interacting with apoGAD rather than holoGAD. By slowing down the rate of intermediate complex formation, ATP reduces the amount of holoGAD formed.
Several aspects of pyridoxine-dependent seizure (PDS) suggest a mutation affecting glutamate decarboxylase (GAD) as a possible cause. To examine the possibility of GAD linkage with PDS, the authors performed genotype analyses of three families using polymorphic markers near the GAD genes (GAD1 and GAD2). In each family, the affected siblings exhibited different genotypes for the GAD2 gene; in two families the GAD1 genotype was disparate. These findings suggest that a mutation of GAD is not directly involved in all cases of PDS.
The injection of Schistosoma mansoni eggs into the footpads of mice results in a localized Th2 cytokine response and tissue eosinophilia. We examined whether treatment with CD40-activating Abs would block the development of Th2 cytokine responses and eosinophilic tissue pathology in this model. Seven days after C57BL/6 mice were injected with eggs and the FGK45 anti-CD40 Ab, Ag-specific synthesis of IL-4, IL-5, and IL-13 in lymph node culture was reduced (>10-fold) relative to control mice treated with eggs and rat IgG. In contrast, IFN-gamma and IL-12 were increased in both culture supernatants and in the serum. Similar changes in lymph node cytokine mRNA were observed in vivo, and tissue eosinophilia was reduced nearly 20-fold. Th2 cytokine responses in anti-CD40-treated IFN-gamma-/- and IL-12 p40-/- C57BL/6 mice were unaffected, although anti-CD40 induced high levels of systemic and local IFN-gamma production in both wild-type and IL-12 p40-/- mice. We conclude that CD40-activating treatments strongly reverse the immune phenotype generated in response to a classic, Th2-biasing stimulus and stimulate IFN-gamma through a novel IL-12-independent pathway. This model for Th1-deviating immune therapy may have relevance to the treatment of Th2-dependent diseases in general.
The selection of which high-pass filter cutoff to use in P300 studies continues to be a serious methodological consideration. To determine whether there was an optimal range of bandpass widths-a range in which P300 voltage and latency would not show statistically significant differences-the authors recorded P300 responses to the auditory "oddball" paradigm from Cz and Pz electrodes in a group of eight healthy males. The authors used high-pass filter cutoffs of 0.01, 0.1, 0.3, and 1.0 Hz with low-pass filter cutoffs of 30 and 100 Hz and measured both P300 peak voltages and P300 integrated mean voltages at 12 bandpass settings. There were statistically significant differences in 7 out of 12 bandpass comparisons for P300 peak voltages and in 7 out of 12 bandwidth comparisons for P300 integrated mean voltages. For P300 latencies, there were statistically significant differences in 9 out of 12 bandwidth comparisons. Based on these results, the best recommendation, therefore, is that the high-pass filter be set no higher than 0.3 Hz.
This article reports the first available human retrieval data following the use of a new fixation system for tumor prostheses. The compliant prestress (CPS) fixation system obviates the need for long intramedullary stems. The CPS was designed to provide a stable, high-pressure, motion-free bone-implant interface that would prevent aseptic loosening and allow osseointegration at the bone-implant interface. At 10 months, the fourth patient in the human trial required amputation. Backscatter electron microscopy revealed a buttress of new bone had formed along 70% of the bone-metal interface, with excellent bony ingrowth (average: 42%) into the transverse, porous-coated titanium interface.
The conformation of bovine serum albumin (BSA), a pyridoxal phosphate (pyridoxal-P) carrier, was investigated by using uv/visible spectrophotometry, fluorescence spectroscopy, circular dichroism, and differential scanning microcalorimetry. Upon interacting with pyridoxal-P, the uv/visible absorption spectrum of BSA exhibits peaks at 330 and 392 nm due to the formation of a Schiff base. Pyridoxal-P quenches the fluorescence emission intensity (excited at 295 or 280 nm) by 24% and enhances fluorescence steady-state polarization of BSA by 20%. These observations suggest a conformational change in BSA when it interacts with pyridoxal-P. However, this conformational change appears to be small since circular dichroism showed only a 2-4% decrease in the alpha-helical content of BSA and no change in the beta-sheet content, and differential scanning microcalorimetry yielded only a 10% change in the enthalpy of thermal unfolding of BSA. 2-Aminoethylisothiouronium bromide, an antioxidant, causes no effect on either uv/visible absorption spectrum or fluorescence emission intensity of BSA, suggesting that BSA lacks sensitive sulfhydryl groups. To help in understanding BSA as a carrier for pyridoxal-P, the results were compared with those for glutamate decarboxylase (GAD), a pyridoxal-P-dependent protein, which requires pyridoxal-P as the cofactor for activity. Although BSA and GAD exhibit comparable molecular weights (66430 versus 65300), numbers of amino acid residues (582 versus 585), and binding affinity (>10(6) M-1), distinct conformational alterations occur between the two proteins upon interacting with pyridoxal-P: a small conformational change for BSA versus a large conformational change for GAD. In contrast to the case of BSA, AET causes significant effects on both the uv/visible spectrum and fluorescence emission intensity of GAD, because GAD contains sensitive sulfhydryl groups. Factors such as disulfide bond and active site sequence were discussed to understand BAS as a carrier for pyridoxal-P and a pyridoxal-P-independent protein.
The levels of the two isoforms of glutamate decarboxylase (GAD) were measured in 12 regions of adult rat brain and three regions of mouse brain by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting with an antiserum that recognizes the identical C-terminal sequence in both isoforms from both species. In rat brain the amount of smaller isoform, GAD65, was greater than that of the larger isoform, GAD67, in all twelve regions. GAD65 ranged from 77-89% of total GAD in frontal cortex, hippocampus, hypothalamus, midbrain, olfactory bulb, periaqueductal gray matter, substantia nigra, striatum, thalamus and the ventral tegmental area. The proportion of GAD65 was lower in amygdala and cerebellum but still greater than half of the total. There was a strong correlation between total GAD protein and GAD activity. In the three mouse brain regions analysed (cerebellum, cerebral cortex and hippocampus) the proportion of GAD65 (35,47, and 51% of total GAD) was significantly lower than in the corresponding rat-brain regions. The amount of GAD67 was greater than the amount of GAD65 in mouse cerebellum and was approximately equal to the amount of GAD65 in mouse cerebral cortex and hippocampus.
The purpose of this study was to use near-infrared spectroscopy (NIRS) as a nondestructive technique to (a) differentiate three Avicel products (microcrystalline cellulose [MCC] PH-101, PH-102, and PH-200) in powdered form and in compressed tablets with and without 0.5% w/w magnesium stearate as a lubricant; (b) determine the magnesium stearate concentrations in the tablets; and (c) measure hardness of tablets compressed at several compression forces. Diffuse reflectance NIR spectra from Avicel powders and tablets (compression forces ranging from 0.2 to 1.2 tons) were collected and distance scores calculated from the second-derivative spectra were used to distinguish the different Avicel products. A multiple linear regression model was generated to determine magnesium stearate concentrations (from 0.25 to 2% w/w), and partial least squares (PLS) models were generated to predict hardness of tablets. The NIRS technique could distinguish between the three different Avicel products, irrespective of lubricant concentration, in both the powdered form and in the compressed tablets because of the differences in the particle size of the Avicel products. The percent error for predicting the lubricant concentration of tablets ranged from 0.2 to 10% w/w. The maximum percent error of prediction of hardness of tablets compressed at the various compression forces was 8.8% for MCC PH-101, 5.3% for MCC PH-102, and 4.6% for MCC PH-200. The NIRS nondestructive technique can be used to predict the Avicel type in both powdered and tablet forms as well as to predict the lubricant concentration and hardness.
Exogenous acetate is preferentially metabolized by astrocytes in the CNS, but the biochemical basis for this selectivity is unknown. We observed that rat cortical astrocytes produce 14CO2 from 0.2 mM [14C]acetate at a rate of 0.43 nmol/min per milligram of protein, 18 times faster than cortical synaptosomes. Subsequent studies examined whether this was attributable to cellular differences in the transport or metabolism of acetate. The activity of acetyl-CoA synthetase, the first enzymatic step in acetate utilization, was greater in synaptosomes than in astrocytes (5.0 and 2.9 nmol/min per milligram of protein), indicating that slower metabolism in synaptosomes cannot be attributed to lack of enzymatic activity. [14C]Acetate uptake in astrocytes is rapid and time-dependent and follows saturation kinetics (Vmax, 498 nmol/min per milligram of protein; Km, 9.3 mM). Uptake is inhibited stereospecifically by L-lactate as well as by pyruvate, fluoroacetate, propionate, and alpha-cyano-4-hydroxycinnamate (CHC). Preloading astrocytes with L-lactate or acetate, but not D-lactate, pyruvate, or glyoxylate, transaccelerates [14C]acetate uptake. Acetate uptake by astrocytes appears to be mediated by a carrier with properties similar to that of monocarboxylate transport. In contrast, studies with synaptosomes provided no evidence for time-dependent, saturable, transaccelerated, or CHC-inhibitable uptake of [14C]acetate. The high rate of transport in astrocytes compared with synaptosomes explains the rapid incorporation of [14C]acetate into brain glutamine over glutamate. These findings provide support for the use of acetate as a marker for glial metabolism and suggest that extracellular acetate in the brain generated from acetylcholine and ethanol metabolism is accumulated first by astrocytes.
Previous studies have demonstrated that the expression of one of the isoforms of glutamate decarboxylase, GAD67, is selectively reduced in cultured cortical neurons and in rat cerebral cortex when the concentration of GABA is elevated. We asked whether the expression of GAD67 was similarly affected by elevated GABA throughout the brain. The concentration of GABA in rat brain was increased by inhibiting GABA transaminase (GABA-T) with vigabatrin (gamma-vinylGABA, GVG), an antiepileptic drug and selective inhibitor of GABA-T. Rats were injected with saline or vigabatrin (150 mg/kg) daily for 5 days, and the effects of accumulated GABA on total GAD activity and the expression of GAD65 and GAD67 proteins were determined in twelve brain regions. The GABA concentration was significantly elevated in all regions except amygdala and olfactory bulb after vigabatrin treatment. Total GAD activity was significantly lower than controls in six regions: cerebellum, frontal cortex, thalamus, substantia nigra, ventral tegmentum, and the remaining midbrain. The decrease in GAD activity was largest in cerebellum and thalamus (33% and 29%), while the changes in the other four areas were 15-18%. Vigabatrin treatment significantly reduced GAD67 protein in all regions except olfactory bulb, whereas GAD65 protein decreased significantly only in cerebellum. The failure to detect significant changes in GAD activity in regions having a significant change in GAD67 levels is attributable to the small contribution of GAD67 to total GAD in those regions. It is evident that there are marked regional differences in the effects of tissue GABA levels on the expression of GAD67.
The conformation, stability, cofactor interactions, and activation of a recombinant 65-kDa form of rat brain glutamate decarboxylase (GAD65) were investigated by using UV/visible spectrophotometry, fluorescence spectroscopy, circular dichroism, and differential scanning microcalorimetry. The enzyme was prepared from Sf9 insect cells infected with a recombinant baculovirus containing the entire GAD65 coding region. The UV/visible absorption spectrum of purified holoenzyme (holoGAD) exhibits two peaks in the range of 300-450 nm, which are due to the formation of a Schiff base when pyridoxal phosphate (pyridoxal-P) binds to GAD. Fluorescence emission intensity (excited at 295 or 280 nm) was substantially enhanced when pyridoxal-P was removed from holoGAD and quenched when pyridoxal-P was added to the apoenzyme (apoGAD). These observations implied that a significant enzyme conformational change occurs during the formation of holoGAD. Circular dichroism provided additional evidence for a conformational change, as the ellipticity of both negative (202-242 nm) and positive (188-202 nm) bands decreased when pyridoxal-P was removed from holoGAD. Secondary structure determination estimated that holoGAD contains a higher content of alpha-helix (34% versus 24%) and a lower content of beta-sheet (18% versus 30%) than apo-GAD. Differential scanning microcalorimetry indicated that holoGAD exhibits a much larger enthalpy and a 3 degrees C higher temperature of thermal unfolding than apoGAD, suggesting that holoGAD has a much tighter conformation and greater stability than apoGAD. A model describing the interaction of pyridoxal-P with GAD is presented, which proposes that an intermediate complex involving ionic interaction between the phosphate group of pyridoxal-P and the positive, charged residues in the active site of GAD maintains the pyridoxal-P molecule in an appropriate position in the active center. Simultaneously, this complex formation is accompanied by a moderate enzyme conformational change, providing a favorable configuration that enables the epsilon-amino of the active-site lysine to react with the aldehyde group of pyridoxal-P. The formation of active holoGAD involves a large enzyme conformational change, which leads to increased stability.
The pyridoxal-P binding sites of the two isoforms of human glutamate decarboxylase (GAD65 and GAD67) were modeled by using PROBE (a recently developed algorithm for multiple sequence alignment and database searching) to align the primary sequence of GAD with pyridoxal-P binding proteins of known structure. GAD's cofactor binding site is particularly interesting because GAD activity in the brain is controlled in part by a regulated interconversion of the apo- and holoenzymes. PROBE identified six motifs shared by the two GADs and four proteins of known structure: bacterial ornithine decarboxylase, dialkylglycine decarboxylase, aspartate aminotransferase, and tyrosine phenol-lyase. Five of the motifs corresponded to the alpha/beta elements and loops that form most of the conserved fold of the pyridoxal-P binding cleft of the four enzymes of known structure; the sixth motif corresponded to a helical element of the small domain that closes when the substrate binds. Eight residues that interact with pyridoxal-P and a ninth residue that lies at the interface of the large and small domains were also identified. Eleven additional conserved residues were identified and their functions were evaluated by examining the proteins of known structure. The key residues that interact directly with pyridoxal-P were identical in ornithine decarboxylase and the two GADs, thus allowing us to make a specific structural prediction of the cofactor binding site of GAD. The strong conservation of the cofactor binding site in GAD indicates that the highly regulated transition between apo- and holoGAD is accomplished by modifications in this basic fold rather than through a novel folding pattern.
Adults express two isoforms of glutamate decarboxylase (GAD), GAD67 and GAD65, which are encoded by different independently regulated genes, a situation that differs from that of other neurotransmitters. In this article, J-J. Soghomonian and David Martin review current knowledge on the differences between these two isoforms. Both isoforms are present in most GABA-containing neurones in the CNS, but GAD65 appears to be targeted to membranes and nerve endings, whereas GAD67 is more widely distributed in cells. Both forms can synthesize transmitter GABA, but GAD67 might preferentially synthesize cytoplasmic GABA and GAD65 might preferentially synthesize GABA for vesicular release. Several lines of evidence suggest that the two forms have different roles in the coding of information by GABA-containing neurones.