Biomedical subjects

T K Smith

Publications and source records attributed to T K Smith.

At least 19 recordsLinked to original sources

PubMed1998

Nitric oxide modulates cholinergic reflex pathways to the longitudinal and circular muscle in the isolated guinea-pig distal colon.

1. The involvement of nitric oxide (NO) in enteric neural pathways underlying reflex responses of the longitudinal muscle (LM) and circular muscle (CM) layers activated by mucosal stimulation was examined in the isolated guinea-pig distal colon. 2. A segment of colon spanned two partitions (10 mm apart), which divided the organ bath into three chambers: a recording chamber where LM and CM tension was measured; a stimulation chamber where mucosal stimulation was applied; and a middle chamber separating them. 3. Brushing the mucosa anal and oral to the recording site evoked simultaneous oral contraction and anal relaxation of both the LM and CM. 4. N omega-nitro-L-argininel-NA; 100 microM) or N omega-nitro-L-arginine methyl ester (L-NAME; 100 microM) applied to the middle chamber or stimulation chamber decreased the oral contractile response of the LM and CM (by about 30-40 %), but increased the anal relaxation (> 600 %) and exposed an anal contraction (> 1000 % increase) of both muscles. The addition of L-NA to the recording chamber reduced the anal relaxation of the LM and CM and the anal contraction of the LM, but slightly increased the anal contraction of the CM. 5. S-Nitroso-N-acetylpenicillamine (SNAP; 10 microM), an NO donor, reversed the effects of L-NA in the middle or stimulation chambers. 6. 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ; 10 microM), a soluble guanylate cyclase inhibitor, mimicked the effects of L-NAin the middle chamber or stimulation chamber, but these effects were not reversed by SNAP. 7. The oral contractile responses, and the anal relaxation and contractile responses of the LM and CM produced by L-NA in the stimulation or middle chambers, were blocked by hexamethonium (300 microM) in any chamber. Atropine (1 microM) in the recording chamber reduced the contractile responses of the LM and CM. 8. In conclusion, endogenous NO facilitates and depresses release of acetylcholine from interneurons in ascending and descending nervous pathways, respectively. These NO effects are mediated through soluble guanylate cyclase in cholinergic interneurons

Animals↗

PubMed1998

Electrical characteristics and responses to jejunal distension of neurons in Remak's juxta-jejunal ganglia of the domestic fowl.

1. Remak's nerve is a ganglionated nerve trunk found only in birds that runs parallel to the gut from the duodenal-jejunal junction to the cloaca. We report the first electrophysiological characterization of these neurons and their responses to gut distension. 2. A segment of chicken jejunum with attached Remak's nerve was pinned in an electrophysiological chamber. Neurons in Remak's ganglia were impaled with microelectrodes. The adjacent segment of gut was distended with fluid. 3. One hundred and thirty neurons were characterized into three electrophysiological classes: (i) tonic neurons (74%) fired action potentials spontaneously (frequency 3.5 Hz) and continuously (up to 40 Hz) throughout a depolarizing current pulse; (ii) AD neurons (22%) fired a brief burst of action potentials (1-10), which were followed by a prolonged after-depolarization (AD) of duration 2.8 +/- 0.3 s; and (iii) phasic neurons (4%) fired an initial burst of action potentials followed by an after-hyperpolarization (duration, 520.0 +/- 32.0 ms). Tetrodotoxin (1 microM) abolished action potentials in tonic and AD neurons as well as the after-depolarization. 4. Spontaneous fast excitatory postsynaptic potentials (FEPSPs) occurred in all classes of neurons; they were not observed, however, in ganglia isolated from the jejunum. 5. Intracellular injection of biocytin revealed that neurons could be characterized into four morphological classes. Tonic neurons, which had long and extensive dendritic trees, were Remak's Type I, II and IV neurons. AD neurons also comprised Remak's type II neurons. Phasic neurons were Remak's Type III neurons. Most neurons had axons that projected orally along Remak's nerve. 6. Distension of the jejunum evoked FEPSPs and action potentials in tonic neurons, and repetitive bursts of action potentials (1-4) followed by an after-depolarization in AD neurons. All responses to distension were blocked by hexamethonium (300 microM) and tetrodotoxin (1 microM). 7. In conclusion, neurons in Remak's juxta-jejunal nerve appear to regulate gut motility. Three distinct electrophysiological classes of neurons were observed, all of which appear to be activated by distension sensitive cholinergic intestinofugal neurons in the jejunum.

Action Potentials↗

PubMed1998

The roles of mitotic arrest and protein synthesis in induction of apoptosis and differentiation in neuroblastoma cells in culture.

Studies of the response of neural crest tumor cells to the DNA cleaving antimitotic agent, neocarzinostatin, have left unanswered the question of whether the DNA cleavage per se or the antimitotic effect is responsible for this response. Furthermore, they do not define the timeframe within which a cell commits to its fate. Using the reversible microtubule-active agent, vinblastine, we now demonstrate that mitotic arrest, even without DNA cleavage, results in the same cellular changes as those seen with neocarzinostatin treatment. The commitment of the cell to its fate occurs within a 15 min treatment with vinblastine, and requires new protein synthesis. The immediate early gene products, c-Fos and c-Jun, appear not to be determinants of this process.

Antineoplastic Agents↗

PubMed1998

Synchronous movements of the longitudinal and circular muscle during peristalsis in the isolated guinea-pig distal colon.

1. Peristalsis, which involves enteric nervous reflexes, is the co-ordinated movements of the longitudinal (LM) and circular (CM) muscle layers that propel intraluminal contents down the bowel. Although the movements of the CM during peristalsis are reasonably clear the relative movements of the LM are poorly understood. 2. We studied the oral and anal movements of the LM and CM during a peristaltic wave in isolated segments of guinea-pig distal colon. Dissection techniques were used to prevent mechanical interactions between the LM and CM; also, the colonic segment was passed through a partition to prevent mechanical disturbances created by a peistaltic wave in the bulk of the colon from influencing the end from which recordings were made. 3. Peristalsis was generated by slowly filling the lumen of the colon with fluid. At threshold, the LM and CM synchronously contracted oral (ascending excitation) to, and relaxed anal (descending inhibition) to, a peristaltic wave. The anal relaxation was followed by a contraction (descending excitation) of both muscle layers. 4. Atropine (1 microM) in the recording chamber reduced both the oral (LM by 40% and CM by 27%) and anal (LM by 36% and CM by 36%) contractile responses as well as the anal relaxation response in both muscle layers. Hexamethonium (300 microM) almost blocked the oral contractile responses of the LM and CM but had no affect on the anal responses of either muscle layer. 5. N omega-nitro-L-arginine (L-NA; 100 microM) reduced the oral contractile response of the LM and CM by 50%, the anal contractile response of the LM by 30%, and the anal relaxation response of the LM and CM by about 30%. The anal contractile response of the CM was unaffected by L-NA. 6. Apamin (0.5 microM) also reduced the evoked anal relaxation of both the LM and CM by about 50%. Further addition of L-NA nearly abolished the relaxation response in the LM, but did not cause any further reduction in the relaxation response of the CM observed in apamin alone. 7. It is concluded, that the LM and CM exhibit synchronous movements during peristalsis in the colon. Also, peristalsis consists of activation of ascending excitatory, and descending inhibitory and excitatory nervous pathways to the LM and CM, which are cholinergic and non-cholinergic, respectively. Nitric oxide is an important neuromodulator within the intrinsic nervous pathways.

Anal Canal↗

PubMed1998

Inhibition of dimethylhydrazine-induced aberrant crypt foci and induction of apoptosis in rat colon following oral administration of the glucosinolate sinigrin.

Glucosinolates are sulphur compounds that occur as glycosides in brassica vegetables. In response to tissue disruption they are degraded by thioglucosidase, releasing a range of highly reactive breakdown products, including the isothiocyanates, which we have previously shown to be selectively cytotoxic to undifferentiated colorectal tumour cells (HT29). In the present study we explored the effect of sinigrin on the intestinal mucosa of rats previously treated with dimethylhydrazine (DMH). In the first experiment, a semisynthetic feed containing sinigrin (400 microg/g diet) was provided 6 h after the second of two injections of DMH. The level of apoptosis was measured by morphological assessment of intact microdissected crypts obtained at 18, 24, 38, 48 and 72 h after injection, and compared with control groups given DMH only, or a sham-injection. Higher numbers of apoptotic nuclei were present in colonic tissue from both groups of DMH-treated rats compared with the controls, and the level was significantly higher in DMH-treated rats fed sinigrin compared with those given DMH only (P < 0.02). In a second experiment, rats were given sinigrin (400 microg/g diet) 22 h after the second of two injections of DMH; the level of apoptosis was measured after 48 h and the numbers of aberrant crypt foci (ACF) were measured after 42 days. The level of apoptosis was significantly higher in DMH-treated rats given sinigrin compared with controls (P < 0.05), and the numbers of ACF were significantly lower in sinigrin-treated rats (P < 0.001). There was no statistically significant induction of apoptosis in animals fed sinigrin alone. Sinigrin administered after DMH suppresses induction of ACF. This may be due to increased apoptotic deletion of damaged stem cells in the crypts of animals fed sinigrin.

Animals↗

PubMed1998

Distension-related responses in circular and longitudinal muscle of the human esophagus: an ultrasonographic study.

Both circular muscles (CM) and longitudinal muscles (LM) of the esophagus participate in peristalsis. Various measurement techniques have yielded conflicting information as to the temporal correlation between contraction in the two muscle layers. High-frequency intraluminal ultrasound (HFIUS) is a novel technique to detect contraction of LM and CM of the esophagus. We investigated the temporal correlation between the CM and LM contraction during ascending excitatory and descending inhibitory reflexes using HFIUS. A manometric catheter equipped with two balloons and a 12.5-MHz ultrasound transducer catheter was used to study 10 normal healthy subjects. The changes in muscle thickness and pressure, proximal and distal to esophageal distension, were recorded at 5 and 10 cm above the lower esophageal sphincter (LES). The esophageal distension induced an increase in pressure and an increase in muscle thickness of both CM and LM layers proximal to the distension site. The onset of increase in muscle thickness and peak muscle thickness in two layers occurred at the same time. There was a close temporal correlation between the changes in pressure and changes in muscle thickness. Atropine inhibited the distension-related pressure and muscle thickness increase in both layers. Distal to the esophageal distension, there was no change in pressure but a decrease in the thickness of the two muscle layers. The decrease in muscle thickness of the two layers occurred at the same time. The responses of the two muscle layers to distension were similar at 5- and 10-cm sites above the LES. HFIUS is a relatively noninvasive technique to study the LM layer response during peristalsis in vivo. Our data indicate that the two muscle layers may contract and relax together during distension-related peristaltic reflexes in the esophagus.

Adult↗

PubMed1997

Parasite and mammalian GPI biosynthetic pathways can be distinguished using synthetic substrate analogues.

Glycosylphosphatidylinositol (GPI) structures are attached to many cell surface glycoproteins in lower and higher eukaryotes. GPI structures are particularly abundant in trypanosomatid parasites where they can be found attached to complex phosphosaccharides, as well as to glycoproteins, and as mature surface glycolipids. The high density of GPI structures at all life-cycle stages of African trypanosomes and Leishmania suggests that the GPI biosynthetic pathway might be a reasonable target for the development of anti-parasite drugs. In this paper we show that synthetic analogues of early GPI intermediates having the 2-hydroxyl group of the D-myo-inositol residue methylated are recognized and mannosylated by the GPI biosynthetic pathways of Trypanosoma brucei and Leishmania major but not by that of human (HeLa) cells. These findings suggest that the discovery and development of specific inhibitors of parasite GPI biosynthesis are attainable goals. Moreover, they demonstrate that inositol acylation is required for mannosylation in the HeLa cell GPI biosynthetic pathway, whereas it is required for ethanolamine phosphate addition in the T.brucei GPI biosynthetic pathway.

Animals↗

PubMed1997

Substrate specificity of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol membrane anchor biosynthesis in African trypanosomes and human cells.

De-N-acetylation of N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) is the second step of glycosylphosphatidylinositol (GPI) membrane anchor biosynthesis in eukaryotes. This step is a prerequisite for the subsequent mannosylation of glucosaminyl-phosphatidylinositol (GlcN-PI) which leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum. The substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells were studied with respect to the N-acyl groups (R) that could be removed from a series of GlcNR-PI substrates, where R=acetyl (Ac), propionyl (Pr), butyryl (Bu), isobutyryl (iBu), pentanoyl (Pen) or hexanoyl (Hex). The data show that the trypanosomal and HeLa enzymes had similar specificities and that the turnover of GlcNR-PIs by the trypanosomal enzyme was in the order GlcNAc-PI>GlcNPr-PI>>GlcNBu - PI approximately GlcNiBu - PI approximately GlcNPen - PI>>GlcNHex - PI. The trypanosome and HeLa de-N-acetylases were unable to de-N-acetylate mannosylated GlcNAc-PI intermediates, which explains why de-N-acetylation must precede mannosylation in the GPI biosynthetic pathway.

Amidohydrolases↗

PubMed1997

Early steps in glycosylphosphatidylinositol biosynthesis in Leishmania major.

A cell-free system based on washed Leishmania major membranes was labelled with GDP-[3H]Man in the presence of synthetic glucosaminyl-phosphatidylinositol (GlcN-PI) and N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI). In both cases, the major radiolabelled products were Man alpha 1-4GlcN alpha 1-6myo-inositol1-HPO4- (sn-1, 2-dipalmitoylglycerol) and Man alpha 1-4GlcN alpha 1-6myo-inositol1-HPO4- (sn-1-palmitoyl-2-lyso-glycerol), to which an additional d-mannose residue was added when a chase with an excess of GDP-Man was performed. The L. major cell-free system can therefore be used to observe the actions of four enzymes, namely GlcNAc-PI de-N-acetylase, Dol-P-Man-GlcN-PI alpha 1-4-mannosyltransferase, a phospholipase A2-like activity and a second alpha-mannosyltransferase activity. The substrate specificities of the first two of these enzymes were studied using a series of substrate analogues. GlcNAc-PI de-N-acetylase was tested against a variety of N-acylated GlcN-PI substrates and was able to cleave N-acetyl and N-propyl groups but not larger groups such as N-butyl, N-isobutyl, N-pentyl and N-hexyl. The Dol-P-Man-GlcN-PI alpha1-4-mannosyltransferase activity required the amino group of the glucosamine residue and the d-configuration of the myo-inositol residue of the GlcN-PI acceptor substrate.

Amidohydrolases↗

PubMed1997

Basal release of nitric oxide induces an oscillatory motor pattern in canine colon.

1. The consequences of intrinsic, basal nitric oxide release on electrical and contractile activity of canine proximal colon were examined. Membrane potential and contraction were simultaneously recorded from the circular muscle in the presence of drugs to block adrenergic and cholinergic responses. 2. Electrical slow waves were recorded from muscle cells near the submucosal surface of the circular layer. Spontaneous contractions were initiated by each slow wave. Contractile amplitude increased 1.9-fold when nerves were blocked with tetrodotoxin (TTX, 1 microM). 3. Muscle cells near the myenteric surface displayed myenteric potential oscillations (MPOs) averaging 16 cycles per minute (c.p.m.) in frequency and 10 mV in amplitude. Twenty-five per cent of muscles displayed an additional slow, neurogenic oscillation (mean frequency, 1 c.p.m.; amplitude, 14 mV) superimposed upon the MPO rhythm. 4. The nitric oxide (NO) synthase inhibitor N omega -nitro-L-arginine (L-NA, 100 microM; n = 16) abolished neurogenic oscillations, depolarized cells, and increased MPO upstroke velocity, amplitude and frequency. The actions of L-NA were mimicked by N omega-nitro-L-arginine methylester (L-NAME, 100 microM) and oxyhaemoglobin (3%). 5. Spontaneous contractions were increased 2.3-fold by L-NA, and TTX had no effect on contractions after addition of L-NA. 6. The NO-donor sodium nitroprusside (SNP, 1 microM) reversed the electrical and mechanical effects of L-NA and initiated slow oscillations similar to the neurogenic oscillations. Slow oscillations were also evoked with S-nitroso-N-acetylpenicillamine (SNAP, 1 microM). The effects of NO donors were blocked by oxyhaemoglobin. 7. Slow electrical oscillations could not be elicited by SNP after removal of a thin strip of circular muscle along the myenteric edge. 8. These data suggest that the spontaneous electrical and contractile activity of the proximal colon is tonically suppressed by basal release of NO. Basal NO causes an oscillatory pattern of electrical and mechanical activity. This activity does not require patterned firing of nerves; rather a continuous, low level release of NO would be capable of producing the neurogenic oscillatory behaviour. The slow oscillatory activity depends upon the presence of the myenteric region of the circular muscle layer, which contains cell bodies of enteric neurons and interstitial cells of Cajal.

Animals↗

PubMed1997

The quality of health data obtained in a new survey of elderly Americans: a validation study of the proposed Medicare Beneficiary Health Status Registry (MBHSR).

The Medicare Beneficiary Health Status Registry (MBHSR) is a proposed new survey program that would collect health status indicators annually from large probability samples of Medicine beneficiaries. For reasons of economy, the MBHSR would use mail survey procedures with telephone follow-up of nonrespondents. Because of concerns about response rates and the validity and reliability of the data obtained by such methods, a large-scale (N = 1,922) field test was conducted. The field test assessed the validity of MBHSR survey reports of past medical treatment and conditions by comparing those reports with Medicare claims data. It assessed the (internal) reliability of MBHSR survey responses by comparing responses with logically related survey questions from the MBHSR. Analyses indicate that the MBHSR survey procedures using a combination of mail data collection with telephone follow-up of nonrespondents produced relatively high levels of sensitivity and specificity in identifying medical treatments and procedures previously recorded in Medicare claims data. In addition, the MBHSR Field Test obtained, in general, relatively high levels of internal consistency in survey reports.

Aged↗

PubMed1997

Effect of feeding blends of Fusarium mycotoxin-contaminated grains containing deoxynivalenol and fusaric acid on growth and feed consumption of immature swine.

Experiments were conducted to determine the effect of feeding diets containing combinations of the Fusarium metabolites deoxynivalenol (DON) and fusaric acid (FA) to starter swine. In all experiments, pigs of approximately 8.2 kg initial weight were fed diets containing blends of mycotoxin-contaminated corn, wheat, and barley for 21 d with growth and feed consumption determined weekly. In the first experiment, diets were determined to contain 0 microgram DON/g + 58.9 micrograms FA/g (control), 4.4 micrograms DON/g + 57.1 micrograms FA/g, 6.0 micrograms DON/g + 48.6 micrograms FA/g, and 7.5 micrograms DON/g + 57.4 micrograms FA/g. The feeding of all diets containing DON caused significant linear depressions in growth and feed intake after only 1 wk. Lower concentrations of DON and FA were fed in the second experiment with diets containing 0 microgram DON/g + 16.3 micrograms FA/g (control), .5 microgram DON/g + 14.3 micrograms FA/g, 1.1 micrograms DON/g + 14.1 micrograms FA/g, and 1.9 micrograms DON/g + 13.6 micrograms FA/g. There was a significant linear reduction in feed intake after 1 wk with increasing levels of dietary DON. Weight gains declined significantly only after 3 wk. Increasing amounts of FA combined with relatively constant amounts of DON were fed in the third experiment. By analysis, diets contained .5 micrograms DON/g + 2.9 micrograms FA/g (control), 2.2 micrograms DON/g + 12.2 micrograms FA/g, 2.5 micrograms DON/g + 15.6 micrograms FA/g, and 2.4 micrograms DON/g + 15.9 micrograms FA/g. In the 1st wk, the feeding of increasing amounts of fusaric acid combined with a relatively constant amount of DON caused a significant linear depression in weight gain. We concluded that a toxicological synergism exists between DON and FA when fed to immature swine and that FA concentrations in feeds should be determined whenever DON analysis is conducted.

Aging↗

PubMed1996

Substrate specificity of the dolichol phosphate mannose: glucosaminyl phosphatidylinositol alpha1-4-mannosyltransferase of the glycosylphosphatidylinositol biosynthetic pathway of African trypanosomes.

The biosynthesis of glycosylphosphatidylinositol (GPI) precursors in Trypanosoma brucei involves the D-mannosylation of D-GlcN alpha 1-6-D-myo-inositol-1-PO4-sn-1,2-diacylglycerol (GlcN-PI). An assay for the first mannosyltransferase of the pathway, Dol-P-Man:GlcN-PI alpha 1-4-mannosyltransferase, is described. Analysis of the acceptor specificity revealed (a) that the enzyme requires the myo-inositol residue of the GlcN-PI substrate have the D configuration; (b) that the enzyme requires the presence of the NH2 group of the D-GlcN residue; (c) that GlcNAc-PI is more efficiently presented to the enzyme than GlcN-PI, suggesting a degree of substrate channelling via the preceding GlcNAc-PI de-N-acetylase enzyme; (d) that the fatty acid and phosphoglycerol components of the phosphatidyl moiety are important for enhancing substrate presentation and substrate recognition, respectively; and (e) that D-GlcN alpha 1-6-D-myo-inositol is the minimum structure that can support detectable acceptor activity. Analysis of the donor specificity revealed that short chain (C5 and C15) analogues of dolichol phosphate can act as substrates for the trypanosomal dolichol-phosphomannose synthetase, whereas the corresponding mannopyranosides cannot act as donors for the Dol-P-Man:GlcN-PI alpha 1-4-mannosyltransferase.

Animals↗

PubMed1996

Morphology and projections of neurons in Remak's nerve of the domestic fowl revealed by intracellular injection of biocytin.

Micro-injections of biocytin were made into neurons in whole-mount preparations of Remak's nerve of the domestic fowl to visualise the morphology and projections of Remak's neurons. Remak's neurons were classified into four distinct morphological types. Remak type-I and -II neurons had a morphology resembling that of mammalian sympathetic neurons. Type-I neurons (found only in juxta-jejunal ganglia) had numerous primary dendritic processes (8-14) with large dendritic fields and extensive dendritic arborizations. Type-II neurons had 2-9 primary dendritic processes, large dendritic fields and sparse dendritic branching. These were found in similar numbers in juxta-rectal and juxta-jejunal ganglia. Remak type-III neurons were the most numerous cell type of juxta-rectal ganglia. They had small cell somata and short dendritic processes that branched infrequently. Remak type-IV neurons (found only in juxta-jejunal ganglia) had a morphology resembling that of invertebrate neurons in that they possessed a prominent long tapering axon from which most of the numerous long dendritic processes emerged. In juxta-jejunal ganglia, all type-IV and most type-I, -II and -III neurons projected orally, whereas axons of juxta-rectal neurons (types II and III) projected either orally or aborally, or projected directly into a lateral nerve bundle supplying the gut. These regional differences in neuron types and axonal projections suggest that different neural circuits exist between Remak's nerve and the small and large intestine.

Animals↗

PubMed1996

Effect of feeding raw soybeans on polyamine metabolism in chicks and the therapeutic effect of exogenous putrescine.

The inclusion of isolated soy protein in milk replacer diets for calves and neonatal pigs inhibits development of intestinal mucosal cells. Simultaneous administration of putrescine partially overcomes this effect. We therefore conducted experiments to determine the potential for dietary putrescine to overcome the toxicity of raw soybeans in chicks. In the first experiment, week-old chicks were fed either an isolated soy protein-based control diet or an isoenergetic and isonitrogenous diet containing 52% raw, ground soybeans for 14 d. The feeding of raw soybeans depressed (P < .001) growth and feed consumption, caused enlargement (P < .001) of the duodenum and pancreas, depressed (P < .001) activities of polyamine synthetic enzymes in the duodenum, and reduced (P < .01) duodenal tissue concentrations of putrescine. In the second experiment, the diet containing raw soybeans was fed with and without .2, .3, and .4% supplemental putrescine. The feeding of supplemental putrescine largely overcame the inhibition of growth due to the feeding of raw soybeans and increased intestinal putrescine concentrations. Putrescine supplementation had no effect, however, on pancreatic and intestinal enlargement in birds fed raw soybeans and tended to depress the activity of polyamine synthetic enzymes. The beneficial effects of putrescine supplementation were confirmed in the third experiment when up to 1.0% supplemental putrescine was fed. We conclude that the toxicity of raw soybeans to chicks can be overcome by feeding putrescine. These effects are likely due to improved nutrient uptake by overcoming the adverse effects of lectins in the intestinal tract and are not likely due to alleviation of the pancreatic enlargement caused by protease inhibitors.

Adenosylmethionine Decarboxylase↗

PubMed1995

Chemical modification of active site residues in gamma-glutamyl transpeptidase. Aspartate 422 and cysteine 453.

gamma-Glutamyl transpeptidase, an enzyme of central significance in glutathione metabolism, is inactivated by iodoacetamide, which esterifies an active site carboxyl group identified here as that of Asp-422. Treatment of the inactivated enzyme with hydroxylamine leads to deesterification and to restoration of enzymatic activity. N-Acetylimidazole, which also inactivates the enzyme, acetylates several amino acid residues. Acetylation exposes Cys-453, which is buried in the native enzyme, to reaction with iodoacetamide. Incubation of the acetylated enzyme with glutamine produces a stabilized gamma-glutamyl-enzyme form which is (a) located exclusively on the light subunit, (b) more labile to base than to acid, (c) destabilized by denaturation of the enzyme with guanidinium ions, and (d) reactive with hydroxylamine to form gamma-glutamylhydroxamate. Stabilization of the gamma-glutamyl-enzyme appears to be associated with acetylation of lysine residues (including Lys-99). These and other findings suggest that the alpha-amino group of the gamma-glutamyl substrate is linked electrostatically to Asp-422 so as to facilitate reaction of the gamma-carbonyl of the substrate with an enzyme hydroxyl group to form a gamma-glutamyl-enzyme.

Acetylation↗

PubMed1995

Different sites of acivicin binding and inactivation of gamma-glutamyl transpeptidases.

Acivicin is a potent inhibitor of gamma-glutamyl transpeptidase (EC 2.3.2.2), an enzyme of importance in glutathione metabolism. Acivicin inhibition and binding are prevented by gamma-glutamyl substrates and analogs (e.g., serine plus borate), consistent with the previous postulate that acivicin and substrates bind to the same enzyme site. Inactivation of rat kidney transpeptidase by acivicin leads to its binding as an ester to Thr-523. The pig enzyme, which has Ala-523 in place of Thr-523, is inhibited by acivicin with esterification at Ser-405. The human enzyme has Thr-524 (corresponding to Thr-523 in rat); its inactivation leads to esterification of Ser-406 (corresponding to Ser-405 in rat and pig). Hydroxylamine treatment of the acivicin-inactivated enzymes restores activity and releases the acivicin-derived threo-beta-hydroxyglutamate moiety. The findings indicate that there are significant structural differences between the active site region of the rat enzyme and the active site regions of the human and pig. Human mutant enzymes in which Thr-524 and Ser-406 were replaced by Ala, separately and together, are enzymatically active, indicating that these amino acid residues are not required for catalysis. However, esterification of these residues (and of another near the active site) effectively blocks the active site or hinders its function. Acivicin can bind at enzyme sites that are close to that at which gamma-glutamylation occurs; it may bind at the latter site and then be transesterified to another enzyme site.

Alanine↗

PubMed1995

On the cytotoxicity and genotoxicity of allyl and phenethyl isothiocyanates and their parent glucosinolates sinigrin and gluconasturtiin.

Four compounds commonly found in the human diet, allyl isothiocyanate (AITC), phenethyl isothiocyanate (PEITC) and their parent glucosinolates sinigrin and gluconasturtiin, were tested for cytotoxic and genotoxic effects in a Chinese hamster ovary cell line (CHO). The isothiocyanates were found to be more than one thousand times more cytotoxic than the glucosinolates, showing significant cytotoxic activity at concentrations below 1.0 microgram/ml. AITC was unable to induce either chromosome aberrations or sister chromatid exchanges (SCEs) even at highly cytotoxic doses. In contrast, PEITC was found to induce both aberrations and SCE at concentrations of 0.9-1.2 micrograms/ml whilst sinigrin and gluconasturtiin induced aberrations at concentrations above 2 mg/ml.

Animals↗