Pyridoxal Phosphate Research Papers - Academia.edu (original) (raw)

Three inhibitors of glutamate decarboxylase (GAD), acting through different mechanisms, as well as pyridoxal phosphate (PLP), were microinjected unilaterally by stereotaxic procedures into the substantia nigra reticulata or the CA1 area... more

Three inhibitors of glutamate decarboxylase (GAD), acting through different mechanisms, as well as pyridoxal phosphate (PLP), were microinjected unilaterally by stereotaxic procedures into the substantia nigra reticulata or the CA1 area of the hippocampus of the rat. The ...

Background: Feedback inhibition of biosynthetic threonine deaminase (TD) from Escherichia coli provided one of the earliest examples of protein-based metabolic regulation. Isoleucine, the pathway end-product, and valine, the product of a... more

Background: Feedback inhibition of biosynthetic threonine deaminase (TD) from Escherichia coli provided one of the earliest examples of protein-based metabolic regulation. Isoleucine, the pathway end-product, and valine, the product of a parallel pathway, serve as allosteric inhibitor and activator, respectively. This enzyme is thus a useful model system for studying the structural basis of allosteric control mechanisms.

Chronic renal failure is often associated with a resistance to the biologically active form of vitamin D 3 , the nuclear hormone 1␣,25-dihydroxyvitamin D 3 (VD). The actions of VD are mediated by the vitamin D receptor (VDR), a... more

Chronic renal failure is often associated with a resistance to the biologically active form of vitamin D 3 , the nuclear hormone 1␣,25-dihydroxyvitamin D 3 (VD). The actions of VD are mediated by the vitamin D receptor (VDR), a ligand-dependent transcription factor that binds as a dimeric complex with the retinoid X receptor (RXR) to specific DNA binding sites in the promoter regions of primary VD responding genes, referred to as VD response elements (VDREs). It could be shown in this study that uremic solutions derived from ultrafiltrate from hemodialysis patients and dialysate from peritoneal dialysis patients had an inhibitory effect on the complex formation and ligand inducibility of VDR-RXR heterodimers on different VDRE types. This inhibition was attributed to the formation of Schiff bases between ''reactive aldehydes'' and lysine residues of the DNA binding domain (DBD) of the VDR, but point mutagenesis data of different lysine residues in this study could not confirm this idea. However, the inhibitory effect of uremic solutions could also be observed for the complex formation of other homo-or heterodimer forming nuclear receptors, whereas an as a monomer binding nuclear receptor did not appear to be affected. These results indicate that VDR is a target of substances in uremic solutions in vitro, but also to some extent other nuclear receptors (i.e., other endocrine signaling systems) may be affected by renal failure.

To examine the development and tracking of long-term vitamin B-6 status from infancy to early adolescence, measurements of erythrocyte pyridoxal 5'-phosphate concentration (EPLP), the erythrocyte aspartate transaminase (EAST)... more

To examine the development and tracking of long-term vitamin B-6 status from infancy to early adolescence, measurements of erythrocyte pyridoxal 5'-phosphate concentration (EPLP), the erythrocyte aspartate transaminase (EAST) stimulation test including measurements of basal activity (EASTo) and activation coefficient (alpha EAST), were made in a follow-up study of healthy children aged 2 (n = 139), 4 (n = 147), 6 (n = 157), 9 (n = 159) and 12 mo (n = 188) and 5 y (n = 148). The EAST stimulation test was repeated at 11 y (n = 153). Vitamin B-6 status, high during infancy, reached the adult level by 5 y of age. The 10th to 90th percentile ranges for EPLP values were 61-201 nmol/L at 4 mo, 49-101 nmol/L at 12 mo and 27-59 nmol/L at 5 y. The respective ranges for Easto were 16-24 microkat/L at 4 mo, 13-19 microkat/L at 12 mo, 9-14 microkat/L at 5 y and 25-39 microkat/L at 11 y of age. For alpha EAST values were 1.29-1.54 at 4 mo, 1.48-1.77 at 12 mo, 1.70-2.07 at 5 y and 2.00-2.57 at...

The three-dimensional structure of D-amino acid aminotransferase (D-AAT) in the pyridoxamine phosphate form has been determined crystallographically. The fold of this pyridoxal phosphate (PLP)containing enzyme is completely different from... more

The three-dimensional structure of D-amino acid aminotransferase (D-AAT) in the pyridoxamine phosphate form has been determined crystallographically. The fold of this pyridoxal phosphate (PLP)containing enzyme is completely different from those of any of the other enzymes that utilize PLP as part of their mechanism and whose structures are known. However, there are some striking similarities between the active sites of D-AAT and the corresponding enzyme that transaminates L-amino acids, L-aspartate aminotransferase. These similarities represent convergent evolution to a common solution of the problem of enforcing transamination chemistry on the PLP cofactor. Implications of these similarities are discussed in terms of their possible roles in the stabilization of intermediates of a transamination reaction. In addition, sequence similarity between D-AAT and branched chain L-amino acid aminotransferase suggests that this latter enzyme will also have a fold similar to that of D-AAT. / LY1145 k kelimmc pyridorminx md PYNVB(C FIGURE 1: Reaction catalyzed by the D-aminO acid aminotransferase. The cofactor is shown positioned as seen in the structure of D-AAT with the side facing solvent toward the viewer and the re side facing the protein. The incoming a-amino acid reacts with the internal aldimine between the active site lysine and pyridoxal phosphate (PLP) to give an external aldimine between the amino

The tautomeric equilibrium in a Schiff base, N-(3,5-dibromosalicylidene)-methylamine 1, a model for the hydrogen bonded structure of the cofactor pyridoxal-5′-phosphate PLP which is located in the active site of the enzyme, was measured... more

The tautomeric equilibrium in a Schiff base, N-(3,5-dibromosalicylidene)-methylamine 1, a model for the hydrogen bonded structure of the cofactor pyridoxal-5′-phosphate PLP which is located in the active site of the enzyme, was measured by means of 1 H and 15 N NMR and deuterium isotope effects on 15 N chemical shifts at variable temperature and in different organic solvents. The position of the equilibrium was estimated using the one-bond 1 J(OHN) and vicinal 3 J(HRCNH) scalar coupling constants. Additionally, DFT calculations of a series of Schiff bases, N-(R1-salicylidene)-alkyl(R2)amines, were performed to obtain the hydrogen bond geometries. The latter made it possible to investigate a broad range of equilibrium positions. The increase of the polarity of the aprotic solvent shifts the proton in the intramolecular OHN hydrogen bond closer to the nitrogen. The addition of methanol and of hexafluoro-2-propanol to 1 in aprotic solvents models the PLP-water interaction in the enzymatic active site. The alcohols, which vary in acidity and change the polarity around the hydrogen bond, also stabilize the equilibrium, so that the proton is shifted to the nitrogen.

The contractile responses of isolated Rana ridibunda frog sartorius muscle contractions evoked by electrical field stimulation (EFS) were studied at three temperature conditions of 17, 22 and 27 8C. Temperature-dependent increase of... more

The contractile responses of isolated Rana ridibunda frog sartorius muscle contractions evoked by electrical field stimulation (EFS) were studied at three temperature conditions of 17, 22 and 27 8C. Temperature-dependent increase of muscle contractility was found. ATP (10-100 AM) concentration dependently inhibited the electrical field stimulation-evoked contractions of sartorius muscle at all three temperatures; this effect was significantly more prominent at a temperature of 17 8C than at other two temperatures. Adenosine (100 AM) also caused inhibition of electrical field stimulation-evoked contractions which was statistically identical at all three temperature conditions tested. A P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2V,4V-disulphonic acid (PPADS, 10 AM) reduced the inhibitory effect of ATP at all three temperatures but did not affect inhibitory action of adenosine. In contrast, 8-( p-sulfophenyl)theophylline (8-SPT, 100 AM), a nonselective P1 receptor antagonist, abolished inhibitory effects of adenosine at all three temperature conditions but did not antagonize inhibition caused by ATP. In electrophysiological experiments, ATP (100 AM) and adenosine (100 AM) temperature dependently reduced end-plate currents recorded in sartorius neuromuscular junction by voltage-clamp technique. The inhibitory effects of both agonists were enhanced with the decrease of temperature. 8-SPT (100 AM) abolished the inhibitory effect of adenosine but not ATP on end-plate currents. Suramin (100 AM), a nonselective P2 receptor antagonist, inhibited the action of ATP but not adenosine, while PPADS (10 AM) had no influence on the effects of either ATP or adenosine. It is concluded from this study that the effectiveness of P2 receptor-mediated inhibition of frog skeletal muscle contraction in contrast to that of adenosine is dependent on the temperature conditions. D 2004 Published by Elsevier B.V.

Inherited disorders of neurotransmitters are a group of neurometabolic syndromes attributable to a primary disturbance of neurotransmitter metabolism or transport. This is an enlarging group of recognized disorders requiring specialized... more

Inherited disorders of neurotransmitters are a group of neurometabolic syndromes attributable to a primary disturbance of neurotransmitter metabolism or transport. This is an enlarging group of recognized disorders requiring specialized diagnostic procedures for detection. This review considers clinical disorders of biopterin, catecholamines, serotonin, glycine, pyridoxine, and GABA metabolism. Newly described syndromes such as cerebral folate deficiency and pyridoxal-5-phosphate dependency are included. The disorders of the metabolic pathways of biopterin, catecholamines, and serotonin are linked due to their common synthetic components. Glycine encephalopathy represents an enlarging phenotype related to abnormalities of the glycine degradative cleavage system. Both pyridoxine and pyridoxal-5-phosphate dependency need to be considered in refractory neonatal seizures. The most common disorder of GABA metabolism is SSADH deficiency, which has a broad phenotype of mental retardation, epilepsy, ataxia, and hyporeflexia and which invokes the combined problems of elevated brain GABA and GHB.

Vitamin B6 has been known to possess antiemetic effects since 1942. This water soluble compound has several forms in the circulation including pyridoxine, pyridoxal, and pyridoxal phosphate. The active antiemetic form of vitamin B6 is... more

Vitamin B6 has been known to possess antiemetic effects since 1942. This water soluble compound has several forms in the circulation including pyridoxine, pyridoxal, and pyridoxal phosphate. The active antiemetic form of vitamin B6 is unknown. This was a pre-specified substudy of a randomized, placebo-controlled trial comparing the antiemetic effect of the doxylamine-vitamin B6 combination (Diclectin 1 ) (n ¼ 131) to placebo (n ¼ 126) in women with nausea and vomiting of pregnancy. Serum concentrations of pyridoxine, pyridoxal, and pyridoxal 5 0 phosphate (PLP) and doxylamine were measured on Days 4, 8, and 15. With Diclectin 1 exhibiting a significant antiemetic effect in pregnancy, serum concentrations of pyridoxine were unmeasurable in almost all patients and those of pyridoxal were undetectable in half of patients. In contrast, PLP was measurable at sustained, stable steady-state levels in all patients. Our data suggest that there is a correlation between PLP levels and PUQE score of morning sickness symptoms when pyridoxine and pyridoxal levels are undetectable, and hence they might be prodrugs of PLP, which may be the active antiemetic form of vitamin B6.

The current study investigated whether ethanol alters ATP activation of purinergic type 2 receptors (P2Rs) in the ventral tegmental area (VTA). The VTA is a key region of the brain that has been implicated in the development of alcohol... more

The current study investigated whether ethanol alters ATP activation of purinergic type 2 receptors (P2Rs) in the ventral tegmental area (VTA). The VTA is a key region of the brain that has been implicated in the development of alcohol addiction. We investigated the effects of ATP and ethanol on spontaneous inhibitory postsynaptic currents (sIPSCs) and the spontaneous firings in the VTA dopaminergic neurons, obtained using an enzyme-free procedure. These neurons preserved some functional GABA-releasing terminals after isolation. We found that ATP (1-200 M) either increased or decreased the frequency of sIPSCs and the activity of VTA dopaminergic neurons. The effects of ATP on sIPSC frequency inversely correlated with its effects on dopaminergic neuron activity. The ATP-induced changes in sIPSC frequency were blocked by tetrodotoxin (a sodium channel blocker) and by suramin (a nonselective P2R antagonist). Furthermore, ␣,␤-methylene ATP, a selective P2X 1 and P2X 3 receptor agonist, increased sIPSC frequency, whereas adenosine 5Ј-[␤-thio]diphosphate, a preferential agonist of P2Y receptors, decreased sIPSC frequency. In experiments testing the effects of ethanol (10 and 40 mM) on sIPSCs, we found that ethanol significantly attenuated ATP-induced increase and enhanced ATP-induced decrease in sIPSC frequency. Taken together, the results demonstrate that multiple subtypes of P2Rs exist on GABA-releasing terminals that make synapses on VTA dopaminergic neurons. It seems that ATP increases sIPSC frequency involving P2X 1 and/or P2X 3 receptors, and ATP decreases sIPSC frequency involving P2YRs. These findings are also consistent with the notion that P2Rs at GABA-releasing terminals on VTA dopaminergic neurons are important targets for ethanol action.

We show here that children with pyridoxine-dependent seizures (PDS) have mutations in the ALDH7A1 gene, which encodes antiquitin; these mutations abolish the activity of antiquitin as a D 1 -piperideine-6-carboxylate (P6C)-a-aminoadipic... more

We show here that children with pyridoxine-dependent seizures (PDS) have mutations in the ALDH7A1 gene, which encodes antiquitin; these mutations abolish the activity of antiquitin as a D 1 -piperideine-6-carboxylate (P6C)-a-aminoadipic semialdehyde (a-AASA) dehydrogenase. The accumulating P6C inactivates pyridoxal 5¢-phosphate (PLP) by forming a Knoevenagel condensation product. Measurement of urinary a-AASA provides a simple way of confirming the diagnosis of PDS and ALDH7A1 gene analysis provides a means for prenatal diagnosis.

We present a patient with severe pyridox(am)ine 5′-phosphate oxidase deficiency and homozygosity for a novel nonsense-mutation, p.A174X, in the PNPO gene who died with pyridoxal phosphate (PLP) treatment despite initial clinical recovery.... more

We present a patient with severe pyridox(am)ine 5′-phosphate oxidase deficiency and homozygosity for a novel nonsense-mutation, p.A174X, in the PNPO gene who died with pyridoxal phosphate (PLP) treatment despite initial clinical recovery. He presented neonatally, with the classical clinical symptoms of the disease. Increase of urinary vanillactate was the first biochemical factor of alert. Amino acid and neurotransmitter analysis in CSF indicated reduced activity of several PLP-dependent enzymes. The diagnosis was confirmed by mutational studies. From this and the other reported patients it may be concluded that the administration of PLP should not be delayed until the complete biochemical evidence is obtained.

Vitamin B6 metabolites and their potential correlates to urinary oxalate excretion in idiopathic calcium stone formers (ICSF) compared with healthy subjects were investigated. This clinical study was performed in a population of male ICSF... more

Vitamin B6 metabolites and their potential correlates to urinary oxalate excretion in idiopathic calcium stone formers (ICSF) compared with healthy subjects were investigated. This clinical study was performed in a population of male ICSF with (Hyperoxalurics, n=55) or without hyperoxaluria (Normooxalurics, n=57) as well as in 100 healthy male control subjects. Pyridoxal 5Õ-phosphate serum concentration (S-pyridoxal 5ÕP) and 24-h urinary excretion of 4-pyridoxic acid (U-4pyridoxic acid) were measured using HPLC; 24-h urinary excretion of oxalate (U-oxalate) was measured concurrently. A subgroup of subjects (40 Hyperoxalurics, 15 Normooxalurics and 50 controls) underwent the same measurements before and after 7day pyridoxine loading per os (pyridoxine hydrochloride, 300 mg/d). Under usual conditions, U-4pyridoxic acid was similar in the three groups, whereas mean S-pyridoxal 5ÕP was significantly lower (p<0.0001) in the Hyperoxalurics (59.6±21.2 nmol/L) and in the Normooxalurics (64.9± 19.7 nmol/L) than in the controls (86.0±31.0 nmol/L). No correlation could be found between U-oxalate and U-4pyridoxic acid or S-pyridoxal 5ÕP. After B6 loading, S-pyridoxal 5ÕP was still significantly lower in the Hyperoxalurics (415±180 nmol/L, p<0.001) and in the Normoox-alurics (429±115 nmol/L, p=0.036) than in the controls (546±180 nmol/L), although there was no difference between groups for U-4pyridoxic acid. No correlation in any group could be found between changes in U-oxalate and changes in U-4pyridoxic acid or S-pyridoxal 5ÕP. Although there is no vitamin B6 deficiency in ICSF with or without hyperoxaluria, these patients, on average, have lower levels of S-pyridoxal 5ÕP than healthy subjects. However, this slight decrease does not seem to account for idiopathic hyperoxaluria.

Background: Hyperhomocysteinemia (.15 mmol/L) is highly prevalent in South Asian populations including Pakistan. In order to investigate the genetic determinants of this condition, we studied 6 polymorphisms in genes of 3... more

Background: Hyperhomocysteinemia (.15 mmol/L) is highly prevalent in South Asian populations including Pakistan. In order to investigate the genetic determinants of this condition, we studied 6 polymorphisms in genes of 3 enzymesmethylenetetrahydrofolate reductase (MTHFR; C677T; A1298C), methionine synthase (MS; A2756G), cystathionine-bsynthase (CBS; T833C/844ins68, G919A) involved in homocysteine metabolism and investigated their interactions with nutritional and environmental factors in a Pakistani population.

In this study we report the coupling of nucleotide receptors to GSK-3 signalling, a relevant survival pathway in cerebellar granule neurons. P2X7 agonist BzATP induced a 3–4-fold increase in GSK-3 phosphorylation, which is reported to be... more

In this study we report the coupling of nucleotide receptors to GSK-3 signalling, a relevant survival pathway in cerebellar granule neurons. P2X7 agonist BzATP induced a 3–4-fold increase in GSK-3 phosphorylation, which is reported to be associated with the catalytic activity inhibition. This effect was dependent on extracellular calcium and PKC, and independent of PI3-K (phosphatidyl-inositol-3-kinase)/Akt, the main survival route of neurotrophins. BzATP also prevented the apoptosis of granule neurons induced by the pharmacological inhibition of the PI3-K signalling. Both effects, BzATP-mediated GSK-3 phosphorylation and neuroprotection, were abolished by P2X7 receptor antagonists, BBG, PPADS and A-438079. We found that BzATP prevented the progressive GSK-3 dephosphorylation and caspase-3 activation occurring under conditions of sustained PI3-K inhibition. These results reveal that P2X7 receptor activation could provide a relevant survival route alternative to classical neurotrophic factors.

Metanx is a product containing L-methylfolate, pyridoxal 59-phosphate, and methylcobalamin for management of endothelial dysfunction. Metanx ingredients counteract endothelial nitric oxide synthase uncoupling and oxidative stress in... more

Metanx is a product containing L-methylfolate, pyridoxal 59-phosphate, and methylcobalamin for management of endothelial dysfunction. Metanx ingredients counteract endothelial nitric oxide synthase uncoupling and oxidative stress in vascular endothelium and peripheral nerve. This study evaluates Metanx on diabetic peripheral neuropathy in ZDF rats, a model of type 2 diabetes. Metanx was administered to 15-week-old ZDF and ZDF lean rats at either 4.87 mg $ kg 21 $ day 21 (a body weight-based equivalent of human dose) or 24.35 mg $ kg 21 $ day 21 by oral gavage two times a day for 4 weeks. Both doses alleviated hind limb digital sensory, but not sciatic motor, nerve conduction slowing and thermal and mechanical hypoalgesia in the absence of any reduction of hyperglycemia. Low-dose Metanx increased intraepidermal nerve fiber density but did not prevent morphometric changes in distal tibial nerve myelinated fibers. Metanx treatment counteracted endothelial nitric oxide synthase uncoupling, inducible nitric oxide synthase upregulation, and methylglyoxal-derived advanced glycation end product, nitrotyrosine, and nitrite/nitrate accumulation in the peripheral nerve. In conclusion, Metanx, at a body weight-based equivalent of human dose, increased intraepidermal nerve fiber density and improved multiple parameters of peripheral nerve function in ZDF rats. Clinical studies are needed to determine if Metanx finds use in management of diabetic peripheral neuropathy. Diabetes

To determine whether a combination of L-methylfolate, methylcobalamin, and pyridoxal-5=phosphate (LMF-MC-PLP [Metanx; Pamlab LLC, Covington, La]) improves sensory neuropathy. RESEARCH DESIGN AND METHODS: This multicenter, randomized,... more

To determine whether a combination of L-methylfolate, methylcobalamin, and pyridoxal-5=phosphate (LMF-MC-PLP [Metanx; Pamlab LLC, Covington, La]) improves sensory neuropathy. RESEARCH DESIGN AND METHODS: This multicenter, randomized, double-blind, placebo-controlled trial involved 214 patients with type 2 diabetes and neuropathy (baseline vibration perception threshold [VPT]: 25-45 volts), who were randomly assigned to 24 weeks of treatment with either L-methylfolate calcium 3 mg, methylcobalamin 2 mg, and pyridoxal-5=-phosphate 35 mg or placebo. The primary end point was effect on VPT. Secondary end points included Neuropathy Total Symptom Score (NTSS-6) and Short Form 36 (SF-36), as well as plasma levels of folate, vitamins B 6 and B 12 , methylmalonic acid (MMA), and homocysteine. RESULTS: There was no significant effect on VPT. However, patients receiving LMF-MC-PLP consistently reported symptomatic relief, with clinically significant improvement in NTSS-6 scores at week 16 (P ϭ .013 vs placebo) and week 24 (P ϭ .033). Improvement in NTSS scores was related to baseline MMA and inversely related to baseline PLP and metformin use. Quality-of-life measures also improved. Homocysteine decreased by 2.7 Ϯ 3.0 mol/L with LMF-MC-PLP versus an increase of 0.5 Ϯ 2.4 mol/L with placebo (P ϭ .0001). Adverse events were infrequent, with no single event occurring in Ն2% of subjects. CONCLUSIONS: LMF-MC-PLP appears to be a safe and effective therapy for alleviation of peripheral neuropathy symptoms, at least in the short term. Additional long-term studies should be conducted, as the trial duration may have been too short to show an effect on VPT. In addition, further research on the effects in patients with cobalamin deficiency would be useful.

Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5P-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) and those of free PLP, and the PLP-L-valine Schiff base have been measured to gain... more

Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5P-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) and those of free PLP, and the PLP-L-valine Schiff base have been measured to gain insight into the photophysics of PLP bound to OASS. Exciting at 330 nm, free coenzyme exhibits a band at 415 nm, whereas PLP-valine and OASS (also when excited at their absorbance maxima) exhibit a structured emission with a peak at 420 nm and shoulders at 490 and 530 nm. The emission bands at 420 and 490 nm are attributed to the enolimine and ketoenamine tautomers of the internal aldimine, respectively, while the 530 nm emission might arise from a dipolar species formed upon proton dissociation in the excited state. Time-resolved fluorescence of OASS (PLP-valine), excited at 412 nm (415 nm) and collected at V s 470 nm, indicates the presence of two components characterized by lifetimes (d) of 0.6 (0.08) and 3.8 (1.55) ns with equal fractional intensity (f). In the presence of acetate the slow component dominates OASS emission with f of 0.98. Excitation at 350 nm as a function of emission wavelengths (400^560 nm) shows at least three components. The f of the slow component increases from 400 to 440 nm, then decreases, whereas the f of the intermediate and fast components behave in the opposite way. Results indicate that: (i) the fast component is associated with the emission at 530 nm; (ii) the slow component is associated with the emission at 420 nm; (iii) a fast additive component, characterized by a very short lifetime, is present on the blue side of the emission spectrum; (iv) the intermediate component results from overlapping contributions, including the emission of the band at 490 nm, that could not be resolved;

Background. Accurate preoperative radiologic imaging is essential to assess the vascular and biliary anatomy of right-lobe living donors and to ensure their safety. Volumetric magnetic resonance cholangiography (MRCP) using Mangafodipir... more

Background. Accurate preoperative radiologic imaging is essential to assess the vascular and biliary anatomy of right-lobe living donors and to ensure their safety. Volumetric magnetic resonance cholangiography (MRCP) using Mangafodipir trisodium (Mn-DPDP) contrast has been recently proposed to evaluate the biliary anatomy of living donor candidates.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5′-phosphate (PLP) dependent enzyme catalyzing the opening of the cyclopropane ring of ACC to give R-ketobutyric acid and ammonia as the products. This ring cleavage... more

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5′-phosphate (PLP) dependent enzyme catalyzing the opening of the cyclopropane ring of ACC to give R-ketobutyric acid and ammonia as the products. This ring cleavage reaction is unusual because the substrate, ACC, contains no abstractable R-proton and the carboxyl group is retained in the product. How the reaction is initiated to generate an R-carbanionic intermediate, which is the common entry for most PLP-dependent reactions, is not obvious. To gain insight into this unusual ring-opening reaction, we have solved the crystal structures of ACC deaminase from Pseudomonas sp. ACP in complex with substrate ACC, an inhibitor, 1-aminocyclopropane-1-phosphonate (ACP), the product R-ketobutyrate, and two D-amino acids. Several notable observations of these structural studies include the following: (1) a typically elusive gem-diamine intermediate is trapped in the enzyme complex with ACC or ACP; (2) Tyr294 is in close proximity (3.0 Å) to the pro-S methylene carbon of ACC in the gem-diamine complexes, implicating a direct role of this residue in the ring-opening reaction; (3) Tyr294 may also be responsible for the abstraction of the R-proton from D-amino acids, a prelude to the subsequent deamination reaction; (4) the steric hindrance precludes accessibility of active site functional groups to the L-amino acid substrates and may account for the stereospecificity of this enzyme toward D-amino acids. These structural data provide evidence favoring a mechanism in which the ring cleavage is induced by a nucleophilic attack at the pro-S -methylene carbon of ACC, with Tyr294 as the nucleophile. However, these observations are also consistent with an alternative mechanistic possibility in which the ring opening is acid-catalyzed and may be facilitated by charge relay through PLP, where Tyr294 functions as a general acid. The results of mutagenesis studies corroborated the assigned critical role for Tyr294 in the catalysis. † This work was supported in part by grants from the National Institutes of Health (GM63689 to H.Z. and GM40541 to H.-w.L.). ‡ The atomic coordinates and the structure factors for Pseudomonas ACC deaminase complexes have been deposited in the Protein Data Bank (PDB; http://www.rcsb.org/pdb/). The accession codes are 1tyz (native enzyme), 1tz2 (complex with ACC), 1tzm (complex with -Cl-D-Ala), 1tzj (complex with D-vinylglycine), and 1tzk (complex with R-ketobutyric acid).

In order to analyse the effects of arginine-vasopressin on the vascular contraction to sympathetic nerve stimulation during cooling, the isometric response of isolated, 2-mm segments of the rabbit central ear (cutaneous) artery to... more

In order to analyse the effects of arginine-vasopressin on the vascular contraction to sympathetic nerve stimulation during cooling, the isometric response of isolated, 2-mm segments of the rabbit central ear (cutaneous) artery to electrical field stimulation (1–8 Hz) was recorded at 37 and 30°C.Electrical stimulation (37°C) produced frequency-dependent arterial contraction, which was reduced at 30°C and potentiated by vasopressin (10 pM, 100 pM and 1 nM). This potentiation was greater at 30 than at 37°C and was abolished at both temperatures by the antagonist of vasopressin V1 receptors d(CH2)5 Tyr(Me)AVP (100 nM). Desmopressin (1 μM) did not affect the response to electrical stimulation.At 37°C, the vasopressin-induced potentiation was abolished by the purinoceptor antagonist PPADS (30 μM), increased by phentolamine (1 μM) or prazosin (1 μM) and not modified by yohimbine (1 μM), whilst at 30°C, the potentiation was reduced by phentolamine, yohimbine or PPADS, and was not modified by prazosin.The Ca2+-channel blockers, verapamil (10 μM) and NiCl2 (1 mM), abolished the potentiating effects of vasopressin at 37°C whilst verapamil reduced and NiCl2 abolished this potentiation at 30°C. The inhibitor of nitric oxide synthesis, L-NOARG (100 μM), or endothelium removal did not modify the potentiation by vasopressin at 37 and 30°C.Vasopressin also increased the arterial contraction to the α2-adrenoceptor agonist BHT-920 (10 μM) and to ATP (2 mM) at 30 and 37°C, but it did not modify the contraction to noradrenaline (1 μM) at either temperature.These results suggest that in cutaneous (ear) arteries, vasopressin potentiaties sympathetic vasoconstriction to a greater extent at 30 than at 37°C by activating vasopressin V1 receptors and Ca2+ channels at both temperatures. At 37°C, the potentiation appears related to activation of the purinoceptor component and, at 30°C, to activation of both purinoceptor and α2-adrenoceptor components of the sympathetic response.In order to analyse the effects of arginine-vasopressin on the vascular contraction to sympathetic nerve stimulation during cooling, the isometric response of isolated, 2-mm segments of the rabbit central ear (cutaneous) artery to electrical field stimulation (1–8 Hz) was recorded at 37 and 30°C.Electrical stimulation (37°C) produced frequency-dependent arterial contraction, which was reduced at 30°C and potentiated by vasopressin (10 pM, 100 pM and 1 nM). This potentiation was greater at 30 than at 37°C and was abolished at both temperatures by the antagonist of vasopressin V1 receptors d(CH2)5 Tyr(Me)AVP (100 nM). Desmopressin (1 μM) did not affect the response to electrical stimulation.At 37°C, the vasopressin-induced potentiation was abolished by the purinoceptor antagonist PPADS (30 μM), increased by phentolamine (1 μM) or prazosin (1 μM) and not modified by yohimbine (1 μM), whilst at 30°C, the potentiation was reduced by phentolamine, yohimbine or PPADS, and was not modified by prazosin.The Ca2+-channel blockers, verapamil (10 μM) and NiCl2 (1 mM), abolished the potentiating effects of vasopressin at 37°C whilst verapamil reduced and NiCl2 abolished this potentiation at 30°C. The inhibitor of nitric oxide synthesis, L-NOARG (100 μM), or endothelium removal did not modify the potentiation by vasopressin at 37 and 30°C.Vasopressin also increased the arterial contraction to the α2-adrenoceptor agonist BHT-920 (10 μM) and to ATP (2 mM) at 30 and 37°C, but it did not modify the contraction to noradrenaline (1 μM) at either temperature.These results suggest that in cutaneous (ear) arteries, vasopressin potentiaties sympathetic vasoconstriction to a greater extent at 30 than at 37°C by activating vasopressin V1 receptors and Ca2+ channels at both temperatures. At 37°C, the potentiation appears related to activation of the purinoceptor component and, at 30°C, to activation of both purinoceptor and α2-adrenoceptor components of the sympathetic response.British Journal of Pharmacology (1999) 126, 785–793; doi:10.1038/sj.bjp.0702345

Serine hydroxymethyltransferase (SHMT) plays an important role in both amino acid and nucleotide metabolism by providing one-carbon units for the biosynthesis of purines, thymidylate, methionine and choline [1]. SHMT is also considered to... more

Serine hydroxymethyltransferase (SHMT) plays an important role in both amino acid and nucleotide metabolism by providing one-carbon units for the biosynthesis of purines, thymidylate, methionine and choline [1]. SHMT is also considered to be an important target for cancer chemotherapy [2]. It catalyses the

The opportunistic pathogen Legionella pneumophila replicates in human lung macrophages and in free-living amoebae. To accommodate the transfer between host cells, L. pneumophila switches from a replicative to a transmissive phase. L.... more

The opportunistic pathogen Legionella pneumophila replicates in human lung macrophages and in free-living amoebae. To accommodate the transfer between host cells, L. pneumophila switches from a replicative to a transmissive phase. L. pneumophila harbors a gene cluster homologous to the Vibrio cholerae cqsAS quorum sensing system, encoding a putative autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a response regulator (lqsR). LqsR is an element of the L. pneumophila virulence regulatory network, which promotes pathogen-host cell interactions and inhibits entry into the replicative growth phase. Here, we show that lqsA functionally complements a V. cholerae cqsA autoinducer synthase deletion mutant and, upon expression in L. pneumophila or Escherichia coli, produces the diffusible signaling molecule LAI-1 (Legionella autoinducer-1). LAI-1 is distinct from CAI-1 (Cholerae autoinducer-1) and was identified as 3-hydroxypentadecan-4-one using liquid chromatography coupled to high resolution tandem mass spectrometry. The activity of both LqsA and CqsA was abolished upon mutation of a conserved lysine, and covalent binding of the cofactor pyridoxal 5-phosphate to this lysine was confirmed by mass spectrometry. Thus, LqsA and CqsA belong to a family of pyridoxal 5-phosphate-dependent autoinducer synthases, which produce the ␣-hydroxyketone signaling molecules LAI-1 and CAI-1. Legionella pneumophila is a ubiquitous bacterium that persists in biofilms and replicates within environmental predators including amoebae (1, 2). Upon inhalation of aerosols from contaminated water sources, the Gram-negative bacteria replicate within macrophages and may cause the severe pneumonia Legionnaires disease, which was first recognized 30 years ago

To compare the diagnostic accuracy of MnDPDP MR imaging and diffusion-weighted imaging (DWI), alone and in combination, for detecting colorectal liver metastases in patients with suspected metastatic disease. Thirty-three consecutive... more

To compare the diagnostic accuracy of MnDPDP MR imaging and diffusion-weighted imaging (DWI), alone and in combination, for detecting colorectal liver metastases in patients with suspected metastatic disease. Thirty-three consecutive patients with suspected colorectal liver metastases underwent MR imaging. Three image sets (MnDPDP, DWI and combined MnDPDP and DWI) were reviewed independently by two observers. Lesions were scored on a five-point scale for malignancy and the areas (Az) under the receiver operating characteristic curves were calculated for each observer and image set. The sensitivity and specificity for lesion detection were calculated for each image set and compared. There were 83 metastases, 49 cysts and 1 haemangioma. Using the combined set resulted in the highest diagnostic accuracy for both observers (Az = 0.94 and 0.96), with improved averaged sensitivity of lesion detection compared with the DWI set (p = 0.01), and a trend towards improved sensitivity compared with the MnDPDP set (p = 0.06). There was no difference in the averaged specificity using any of the three image sets (p > 0.5). Combination of MnDPDP MR imaging and DWI resulted in the highest diagnostic accuracy and can increase sensitivity without loss in specificity.

The Toll signalling pathway, which is crucial for innate immunity, is transduced in insect haemolymph via a proteolytic cascade consisting of three serine proteases. The proteolytic cascade is downregulated by a specific serine protease... more

The Toll signalling pathway, which is crucial for innate immunity, is transduced in insect haemolymph via a proteolytic cascade consisting of three serine proteases. The proteolytic cascade is downregulated by a specific serine protease inhibitor (serpin). Recently, the serpin SPN48 was found to show an unusual specific reactivity towards the terminal serine protease, Spä tzle-processing enzyme, in the beetle Tenebrio molitor. In this study, the mature form of SPN48 was overexpressed in Escherichia coli and purified. The purified SPN48 protein was crystallized using 14% polyethylene glycol 8000 and 0.1 M 2-(N-morpholino)ethanesulfonic acid pH 6.0 as the precipitant. The crystals diffracted X-rays to 2.1 Å resolution and were suitable for structure determination. The crystals belonged to space group P2 1 . The crystal structure will provide information regarding how SPN48 achieves its unusual specificity for its target protease.

Multiple brainstem sites are proposed to contribute to central respiratory chemosensitivity, however, the underlying molecular mechanisms remain unknown. P2X2 subunit-containing ATP receptors, which mediate pH-sensitive currents, appear... more

Multiple brainstem sites are proposed to contribute to central respiratory chemosensitivity, however, the underlying molecular mechanisms remain unknown. P2X2 subunit-containing ATP receptors, which mediate pH-sensitive currents, appear to contribute to central chemosensitivity in vivo [J. Physiol. 523 (2000) 441]. However, recent data from P2X2 knockout mice [J. Neurosci. 23 (2003) 11315] indicate that they are not essential. To further explore the role of P2 receptors in central chemosensitivity, we examined the effects of P2 receptor agonists/antagonists on respiratory-related activity and CO2-sensitivity of rhythmically-active in vitro preparations from neonatal rat. Our main findings: (i) that putative chemosensitive regions of the ventrolateral medulla are immunoreactive for the P2X2 subunit; (ii) that ATP potentiates respiratory frequency in a dose-dependent, and PPADS-sensitive (P2 receptor antagonist), manner; and (iii) that the increase in burst frequency produced by increasing CO2 is unaffected by PPADS, indicate that ATP is a potent modulator of respiratory activity, but that P2 receptors do not contribute to central chemosensitivity in vitro.

Low vitamin B-6 status, based on plasma concentrations of pyridoxal-5-phosphate (PLP), has been identified in inflammatory diseases, including cardiovascular disease, rheumatoid arthritis, inflammatory bowel disease, and diabetes. Our... more

Low vitamin B-6 status, based on plasma concentrations of pyridoxal-5-phosphate (PLP), has been identified in inflammatory diseases, including cardiovascular disease, rheumatoid arthritis, inflammatory bowel disease, and diabetes. Our objective was to examine the association between plasma PLP and multiple markers of inflammation in a communitybased cohort [n = 2229 participants (55% women, mean age 61 6 9 y)]. We created an overall inflammation score (IS) as the sum of standardized values of 13 individual inflammatory markers. Multivariable-adjusted regression analysis was used to assess the associations between the IS and plasma PLP. Geometric mean plasma PLP concentrations were lower in the highest tertile category of IS relative to the lowest (61 vs. 80 nmol/L; P-trend , 0.0001). Similarly, the prevalence of PLP insufficiency was significantly higher for participants in the highest compared with the lowest tertiles for IS categories. These relationships persisted after accounting for vitamin B-6 intake. Also, there were significant inverse relationships between plasma PLP and 4 IS based on functionally related markers, including acute phase reactants, cytokines, adhesion molecules, and oxidative stress. In addition, secondary analyses revealed that many of the individual inflammatory markers were inversely associated with plasma PLP after adjusting for plasma C-reactive protein concentration. This study, in combination with past findings, further supports our hypothesis that inflammation is associated with a functional deficiency of vitamin B-6. We discuss 2 possible roles for PLP in the inflammatory process, including tryptophan metabolism and serine hydroxymethyltransferase activity.

Simple and selective extractive spectrophotometric methods for the determination of quetiapine hemifumarate (QF) were developed and validated. The methods were based on the formation of yellow ion-pair complexes between QF and acidic dyes... more

Simple and selective extractive spectrophotometric methods for the determination of quetiapine hemifumarate (QF) were developed and validated. The methods were based on the formation of yellow ion-pair complexes between QF and acidic dyes namely bromcresol purple (BCP) and bromcresol green (BCG) at room temperature in phosphate buffer (pH 3.0). The formed complexes were extracted with chloroform and the absorbances were measured at 406.5 nm for BCP and at 416 nm for BCG complexes. The compositions of the ion-pairs were found as 1:1 by mole-ratio method. The reaction conditions such as concentration, pH, color formation time, temperature and chromogen stability were optimized. Good linear relationship was obtained between the absorbance and the concentration of QF in the range of 0.5 -20 Hg/mL for both BCP and BCG (r > 0.9974). LOD values were found as 0.12 and 0.16 \ig/mLfor BCP and BCG complexes, respectively. Intra-day precisions were found less than 1 % in the methods. The developed methods were applied successfully to the determination of QF in tablets marketed in Turkey.

Using purified enzymes of human origin and patients' sera, we examined factors influencing the in vitro association of pyridoxal phosphate with aspartate aminotransferase (EC 2.6.1.1). The rate of association was markedly retarded by... more

Using purified enzymes of human origin and patients' sera, we examined factors influencing the in vitro association of pyridoxal phosphate with aspartate aminotransferase (EC 2.6.1.1). The rate of association was markedly retarded by phosphate buffer in comparison with tris(hydroxymethyl)aminomethane or six other buffers. Pyridoxal phosphate at an incubation concentration of 130 mumol/liter reactivated the entire apoenzyme portion of an apoenzyme/holoenzyme mixture within 5 min in tris(hydroxymethyl)aminomethane; in contrast, less than 20% was associated during 15 min in phosphate. Activity measured in tris(hydroxymethyl)aminomethane-buffer without exogenous pyridoxal phosphate was 4% greater than that in phosphate and was slightly increased by increasing the pH of the assay mixture from 7.5 to 8.0. Aspartate in the incubation medium did not retard the stimulation in tris(hydroxymethyl)aminomethane buffer. While the magnitude of stimulation varied greatly among sera, a consisten...

Aminotransferase activities were measured in the serum of two- to three-year-old Thoroughbred fillies and colts during a four week period of peak training for flat racing. Aspartate aminotransferase (AspAT, EC 2.6.1.1), mitochondrial... more

Aminotransferase activities were measured in the serum of two- to three-year-old Thoroughbred fillies and colts during a four week period of peak training for flat racing. Aspartate aminotransferase (AspAT, EC 2.6.1.1), mitochondrial aspartate aminotransferase (m-AspAT) and alanine aminotransferase (AlaAT, EC 2.6.1.2) activities in serum were measured and the relative proportions of apoenzyme and holoenzyme were determined. The aminotransferase activities were increased only slightly immediately following exercise. This small and immediate post exercise increase in activity did not vary greatly over the period of peak training. Measured in the presence of exogenous pyridoxal 5’-phosphate, mean enzyme activities (iu/litre at 30°C) before exercise were: AspAT, 291; m-AspAT, 13; AlaAT, 18. After exercise they were: AspAT, 317; m-AspAT, 16; AlaAT, 23. Nearly all of the AspAT activity was present in the holoenzyme form (94 per cent holoenzyme) indicating excellent vitamin B6 status in these animals. Paradoxically, the AlaAT in serum from the same highly trained Thoroughbred horses was poorly saturated with pyridoxal phosphate, with nearly half of the AlaAT in most horses present in the inactive apoenzyme form (61 per cent that of holoenzyme). It is critical therefore, that exogenous pyridoxal phosphate be included in aminotransferase assays to determine the amounts of enzyme release into the peripheral circulation.

Abstract— The kinetic behavior of glutamate decarboxylase from mouse brain was analyzed in a wide range of glutamate and pyridoxal 5′-phosphate concentrations, approaching three limit conditions: (I) in the absence of glutamate-pyridoxal... more

Abstract— The kinetic behavior of glutamate decarboxylase from mouse brain was analyzed in a wide range of glutamate and pyridoxal 5′-phosphate concentrations, approaching three limit conditions: (I) in the absence of glutamate-pyridoxal phosphate Schiff base; (II) when all glutamate is trapped in the form of Schiff base; (III) when all pyridoxal phosphate is trapped in the form of Schiff base. The experimental results in limit condition (I) are consistent with the existence of two different enzyme activities, one dependent and the other independent of free pyridoxal phosphate. The results obtained in limit conditions (II) and (III) give further support to this postulation. These data show that the free pyridoxal phosphate-dependent activity can be abolished when either all substrate or all cofactor are in the form of Schiff base. The free pyridoxal phosphate-independent activity is also abolished when all substrate is trapped as Schiff base, but it is not affected by the conversion of free pyridoxal phosphate into the Schiff base. A kinetic and mechanistic model for brain glutamate decarboxylase activity, which accounts for these observations as well as for the results of previous dead end-inhibition studies, is postulated. Computer simulations of this model, using the experimentally obtained kinetic constants, reproduced all the observed features of the enzyme behavior. The possible implications of the kinetic model for the regulation of the enzyme activity are discussed.

Abstract: The in vitro effects of nicotinic acid (10–1000 μM), pyridoxine (0.1–500 μM) and pyridoxal-5′-phosphate (0.1–500 μM) and the ex vivo effects of nicotinic acid (2500 mg orally during 12 h) and pyridoxine (600 mg orally daily for... more

Abstract: The in vitro effects of nicotinic acid (10–1000 μM), pyridoxine (0.1–500 μM) and pyridoxal-5′-phosphate (0.1–500 μM) and the ex vivo effects of nicotinic acid (2500 mg orally during 12 h) and pyridoxine (600 mg orally daily for seven days) on arachidonic acid metabolism were investigated in calcium ionophore A23187 (calcimycin)-stimulated human whole blood. In vitro nicotinic acid stimulated prostaglandin E2, thromboxane B2 and leukotriene E4 synthesis. Pyridoxine at all concentrations and pyridoxal-5′-phosphate at the highest concentration stimulated prostaglandin E2 and thromboxane B2 production, but had no effect on leukotriene E4 synthesis. Nicotinic acid treatment increased ex vivo prostaglandin E2, thromboxane B2 and leukotriene E4 synthesis to 185%, 165% and 175% of the initial values, respectively. In the pyridoxine-treated subjects, ex vivo prostaglandin E2, thromboxane B2 and leukotriene E4 synthesis was decreased after seven days to 75%, 65% and 45% of the initial values, respectively. In the present study the effects of nicotinic acid on the 5-lipoxygenase pathway in arachidonic acid metabolism were studied for the first time and the drug was found to stimulate this pathway in vitro and ex vivo. In vitro pyridoxine and pyridoxal-5′-phosphate had no effect on the 5-lipoxygenase pathway. The inhibition of leukotriene synthesis by pyridoxine ex vivo might be of therapeutic importance.

It was shown that the rate of reconstruction of muscle glycogen phosphorylase b (Phb) from apoenzyme and pyridoxal 5 -phosphate decreased under crowding conditions. The effect of crowding was counteracted by chaperones (␣-crystallin and... more

It was shown that the rate of reconstruction of muscle glycogen phosphorylase b (Phb) from apoenzyme and pyridoxal 5 -phosphate decreased under crowding conditions. The effect of crowding was counteracted by chaperones (␣-crystallin and proline). Sedimentation analysis shows that crowding stimulates the formation of high-molecular-weight associates at 25 • C, whereas chaperones stabilize small oligomers. The study of the kinetics of apoPhb aggregation at 37 • C showed that the anti-aggregation activity of chaperones decreased under crowding conditions. When studying the sedimentation behavior of the mixture of apoPhb and ␣-crystallin, the complexes between unfolded apoPhb and dissociated forms of ␣-crystallin were observed. It is assumed that these complexes are responsible for realization of the chaperone-like activity of ␣-crystallin under crowding conditions.

α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated excitotoxicity contributes to the selective motor neuron death in amyotrophic lateral sclerosis (ALS). In this study, we investigated the effect of P2... more

α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated excitotoxicity contributes to the selective motor neuron death in amyotrophic lateral sclerosis (ALS). In this study, we investigated the effect of P2 receptor-influencing substances on kainate-induced motor neuron death in an in vitro model for AMPA receptor-mediated excitotoxicity. Complete protection was found after preincubation of the motor neurons with ivermectin or Cibacron Blue 3G-A. Preincubation with both P2X 4 modulators did not influence the number or Ca 2+ permeability of the AMPA receptors and addition during kainate stimulation alone had no effect. Preincubation with a low concentration of ATP, the natural agonist of the P2X 4 receptor, also protected the motor neurons against a subsequent excitotoxic stimulation, while high concentrations of ATP were toxic. Moreover, ivermectin increased the toxicity of low ATP concentrations, indicating that ivermectin can potentiate the effect of ATP on its receptor. Ivermectin and ATP also protected against hypoxia/hypoglycemia. To further investigate the relevance of these findings for ALS, we treated SOD1(G93A)-mice, a transgenic animal model for familial ALS, with ivermectin. This resulted in an extension of the life span of these mice with almost 10%. We conclude that ivermectin induces a mechanism in motor neurons, in vivo and in vitro, that protects against subsequent excitotoxic insults. Our in vitro data indicate that this protective mechanism is due to the potentiation by ivermectin of an effect of ATP mediated by the P2X 4 receptor.

We have synthesized a series of derivatives of the known P2 receptor antagonist PPADS (pyridoxal-5′-phosphate-6-azo-phenyl-2,4-disulfonate) and examined their ability to inhibit functional activity of the recombinant human P2Y 13... more

We have synthesized a series of derivatives of the known P2 receptor antagonist PPADS (pyridoxal-5′-phosphate-6-azo-phenyl-2,4-disulfonate) and examined their ability to inhibit functional activity of the recombinant human P2Y 13 nucleotide receptor expressed in 1321N1 human astrocytoma cells co-expressing Gα 16 protein (AG32). Analogues of PPADS modified through substitution of the phenylazo ring, including halo and nitro substitution, and 5′-alkyl phosphonate analogues were synthesized and tested. A 6-benzyl-5′-methyl phosphonate analogue was prepared to examine the effect of stable replacement of the azo linkage. The highest antagonistic potency was observed for 6-(3-nitrophenylazo) derivatives of pyridoxal-5′phosphate. The 2-chloro-5-nitro analogue (MRS 2211) and 4-chloro-3-nitro analogue (MRS 2603) inhibited ADP (100 nM)-induced inositol trisphosphate (IP 3 ) formation with pIC 50 values of 5.97 and 6.18, respectively, being 45-and 74-fold more potent than PPADS. The antagonism of MRS 2211 was competitive with a pA 2 value of 6.3. MRS2211 and MRS2603 inhibited phospholipase C (PLC) responses to 30 nM 2-methylthio-ADP in human P2Y 1 receptor-mediated 1321N1 astrocytoma cells with IC 50 values of >10 and 0.245 μM, respectively. Both analogues were inactive (IC 50 > 10 μM) as antagonists of human P2Y 12 receptor-mediated PLC responses in 1321N1 astrocytoma cells. Thus, MRS2211 displayed >20-fold selectivity as antagonist of the P2Y 13 receptor in comparison to P2Y 1 and P2Y 12 receptors, while MRS2603 antagonized both P2Y 1 and P2Y 13 receptors.

The origins of C−H activation in pyridoxal-5′-phosphate (PLP) Schiff bases and modulation of reaction specificity in PLPenzymes are still not completely understood. There are no available studies that compare the reactivity of C4′ carbons... more

The origins of C−H activation in pyridoxal-5′-phosphate (PLP) Schiff bases and modulation of reaction specificity in PLPenzymes are still not completely understood. There are no available studies that compare the reactivity of C4′ carbons in ketimine Schiff bases with that of Cα carbons in their aldimine counterparts, which is essential to unravel the mechanisms that govern the evolution of their common carbanionic intermediates. Second-order rate constants for phosphate-catalyzed proton/deuterium exchange reactions in D 2 O of C4′ carbons suffer a 10 5 -fold increase due to Schiff base formation (k B = 5.3 × 10 1 M −1 s −1 ) according to NMR measurements. The C4′ carbon acidity is also increased to pK a = 9.8, which is significantly higher than that of Cα in PLP-aldimines. DFT calculations reveal the role of each heteroatom in modulating the electrophilicity of C4′ and Cα carbons. Specifically, the protonation state of pyridine nitrogen is the main factor in determining the absolute carbon acidity in aldimines (pK a of Cα varies from ∼14 to ∼23) and ketimines (pK a of C4′ varies from ∼12 to ∼18), whereas the protonation state of both imine nitrogen and O3′ phenol oxygen modulates the relative acidities of Cα and C4′ from 1.5 to 7.5 pK a units. Our results provide an explanation to the modulation of reaction specificity observed in different PLP-enzymes based on the differences in the protonation state of the cofactor and Hbonding patterns in the active site.

molecular structure and physiology/pathology of the Glc-6-Pase system. ᭧ 1998 Academic Press S 3483, a synthetic derivative of chlorogenic acid Key Words: glucose-6-phosphatase; transport inhibi-(CHL), was found to be a reversible, linear... more

molecular structure and physiology/pathology of the Glc-6-Pase system. ᭧ 1998 Academic Press S 3483, a synthetic derivative of chlorogenic acid Key Words: glucose-6-phosphatase; transport inhibi-(CHL), was found to be a reversible, linear competitive tion; synthetic derivatives; chlorogenic acid; pyrophoinhibitor of the glucose-6-phosphatase (Glc-6-Pase) sphatase; liver; kidney; microsomes. system in rat renal microsomes and rat and human liver microsomes. The K i for S 3483 in rat liver microsomes (129 nM) is three orders of magnitude smaller than the K i for CHL. S 3483 up to 100 mM had no effect Glc-6-Pase 3 (EC 3.1.3.9) is a multicomponent system on the Glc-6-Pase enzyme activity or on the system inorganic pyrophosphatase activity (i.e., on T2, the P i / within the hepatic and renal endoplasmic reticulum inorganic pyrophosphate transporter). Thus, like (3-7). Steady-state hydrolysis of Glc-6-P involves the CHL, S 3483 appears to be a site-specific inhibitor of T1, function of at least three integral membrane proteins. the Glc-6-P transporter of renal and liver microsomes. Transport of the Glc-6-P across the membrane is facili-The potency of S 3483 was unaffected when the ratio tated by a specific transport protein, called T1 (4, 8, 9). V max(T1) :V max(enzyme) was altered over a 10-fold range by Hydrolysis is catalyzed by a less specific phosphohydroapplying enzyme inhibition and selective inactivation lase whose active site faces the lumen. A second transof T1. The absence of T1-imposed rate restrictions on porter, denoted T2, mediates efflux of the reaction prodthe potency of reversible T1 inhibitors contrasts markuct P i and influx of alternative substrates for the enedly with the response of reversible Glc-6-Pase enzyme zyme, such as PP i and carbamyl phosphate. Efflux of inhibitors, whose potency declines sharply as T1 beD glucose , the other hydrolytic product, appears to be comes more rate controlling. The potency of S 3483, an unmediated process (4), although this conclusion is but not of CHL, decreased as the microsomal protein controversial (5). concentration in the assay medium was increased. Because Glc-6-Pase catalyzes the terminal reactions This effect suggests that as the protein concentration of both glycogenolysis and gluconeogenesis (10), it is a was raised the concentration of T1 in the assay melogical target site for therapeutic agents intended, for dium approached the order of magnitude of the K i for S 3483. Thus, the microsomal content of T1 is likely to be on the order of 100 pmol/mg protein. S 3483 is the 3 Abbreviations used: Glc-6-Pase, glucose-6-phosphatase; HNB, most potent inhibitor of the Glc-6-Pase system re-2-hydroxy-5-nitrobenzaldehyde; IC 50 , the concentration of inhibiported to date. It and other tight-binding inhibitors of tor required for 50% inhibition; K is , slope inhibitor constant, i.e., the concentration of inhibitor needed to double the slope of the T1 will provide useful new tools for investigating the Lineweaver-Burk plots (1) of kinetic data (2); K ii , intercept inhibitor constant, i.e., the concentration of inhibitor needed to double the intercept of Lineweaver-Burk plots of kinetic data (2); Man-1 This research was supported in part by funds provided to W.J.A. 6-Pase, mannose-6-phosphatase; PP i ase, inorganic pyrophosphatase; PLP, pyridoxal phosphate; CHL, chlorogenic acid; S 3483, (1-by Hoechst Marion Roussel. 2 To whom correspondence should be addressed at 227 Savage Hall, [[2-(4-chlorophenyl)cyclopropyl]methoxy]-3,4-dihydroxy-5-[[3-(4hydroxyphenyl)-1-oxo-2-propenyl]oxy]cyclohexanecarboxylic

Objectives: We investigated the stability of 36 analytes related to clinical chemistry in a controlled storage study. Design and methods: Blood was collected from 11 subjects and was maintained for 45 min, 2.5 h, 5 h, or 24 h after... more

Objectives: We investigated the stability of 36 analytes related to clinical chemistry in a controlled storage study. Design and methods: Blood was collected from 11 subjects and was maintained for 45 min, 2.5 h, 5 h, or 24 h after phlebotomy before centrifugation. Results: Statistically significant changes were observed only for parathyroid hormone, osteocalcin, zinc, pyridoxal 5′-phosphate, and homocysteine. Conclusions: These studies indicate that many analytes in clinical chemistry are stable for 24 h before centrifugation.

Studies of nonenzymatic electrophilic catalysis of carbon deprotonation of glycine show that pyridoxal 5'-phosphate (PLP) strongly enhances the carbon acidity of α-amino acids, but that this is not the overriding mechanistic imperative... more

Studies of nonenzymatic electrophilic catalysis of carbon deprotonation of glycine show that pyridoxal 5'-phosphate (PLP) strongly enhances the carbon acidity of α-amino acids, but that this is not the overriding mechanistic imperative for cofactor catalysis. Although the fully protonated PLP-glycine iminium ion adduct exhibits an extraordinary low α-imino carbon acidity (pKa = 6), the more weakly acidic zwitterionic iminium ion adduct (pKa = 17) is selected for use in enzymatic reactions. The similar α-imino carbon acidities of the iminium ion adducts of glycine with 5'deoxypyridoxal and with phenylglyoxylate shows that the cofactor pyridine nitrogen plays a relatively minor role in carbanion stabilization. The 5'-phosphodianion group of PLP likely plays an important role in catalysis by providing up to 12 kcal/mol of binding energy that may be utilized for transition state stabilization.

PurposeTo determine the diagnostic performance of functional magnetic resonance cholangiography (fMRC) for the evaluation of anatomic and functional biliary disorders.To determine the diagnostic performance of functional magnetic... more

PurposeTo determine the diagnostic performance of functional magnetic resonance cholangiography (fMRC) for the evaluation of anatomic and functional biliary disorders.To determine the diagnostic performance of functional magnetic resonance cholangiography (fMRC) for the evaluation of anatomic and functional biliary disorders.Materials and MethodsAt 1.5 T, 39 MR examinations with conventional MRC and mangafodipir trisodium-enhanced fMRC were retrospectively reviewed by three observers who recorded anatomic (duct dilation, stricture, filling defects) and functional (cholecystitis, obstruction) abnormalities in three modes: MRC alone, fMRC alone, and MRC and fMRC images together (combined-MRC). Performance was determined by comparing findings with each mode to findings of invasive cholangiography (IC) and surgery.At 1.5 T, 39 MR examinations with conventional MRC and mangafodipir trisodium-enhanced fMRC were retrospectively reviewed by three observers who recorded anatomic (duct dilation, stricture, filling defects) and functional (cholecystitis, obstruction) abnormalities in three modes: MRC alone, fMRC alone, and MRC and fMRC images together (combined-MRC). Performance was determined by comparing findings with each mode to findings of invasive cholangiography (IC) and surgery.ResultsAmong 75 biliary segments (correlated with IC), the sensitivity/specificity for diagnosing dilation (N = 41) with MRC was 95%/97%; with fMRC, 90%/100%; with combined-MRC, 100%/97%. For stricture (N = 7), the sensitivity/specificity of MRC was 86%/98%; of fMRC, 43%/100%; of combined-MRC, 86%/100%. For filling defects (N = 9), the sensitivity/specificity of MRC was 91%/98%; of fMRC, 82%/100%; of combined-MRC, 91%/100%. For diagnosing obstruction (N = 9), the sensitivity/specificity of MRC, fMRC, and combined-MRC were 89%/100%, 100%/100%, and 100%/100%, respectively. For surgically proven cholecystitis (N = 13), positive predictive values for diagnosing acute/chronic cholecystitis for MRC were 33%/40%; for fMRC, 100%/50%; for combined-MRC, 100%/50%.Among 75 biliary segments (correlated with IC), the sensitivity/specificity for diagnosing dilation (N = 41) with MRC was 95%/97%; with fMRC, 90%/100%; with combined-MRC, 100%/97%. For stricture (N = 7), the sensitivity/specificity of MRC was 86%/98%; of fMRC, 43%/100%; of combined-MRC, 86%/100%. For filling defects (N = 9), the sensitivity/specificity of MRC was 91%/98%; of fMRC, 82%/100%; of combined-MRC, 91%/100%. For diagnosing obstruction (N = 9), the sensitivity/specificity of MRC, fMRC, and combined-MRC were 89%/100%, 100%/100%, and 100%/100%, respectively. For surgically proven cholecystitis (N = 13), positive predictive values for diagnosing acute/chronic cholecystitis for MRC were 33%/40%; for fMRC, 100%/50%; for combined-MRC, 100%/50%.ConclusionAlthough single-shot fast spin echo (SSFSE)-MRC is valuable, the addition of fMRC increased diagnostic performance for functional biliary disorders. J. Magn. Reson. Imaging 2003;18:449–460. © 2003 Wiley-Liss, Inc.Although single-shot fast spin echo (SSFSE)-MRC is valuable, the addition of fMRC increased diagnostic performance for functional biliary disorders. J. Magn. Reson. Imaging 2003;18:449–460. © 2003 Wiley-Liss, Inc.

Background We assessed the role of mangafodipir-enhanced magnetic resonance (MR) cholangiography in the detection and location of bile duct leaks after laparoscopic cholecystectomy. Methods In a prospective study, 34 patients with... more

Background We assessed the role of mangafodipir-enhanced magnetic resonance (MR) cholangiography in the detection and location of bile duct leaks after laparoscopic cholecystectomy. Methods In a prospective study, 34 patients with clinical suspicion of bile duct leak after laparoscopic cholecystectomy underwent MR imaging. Our protocol included conventional heavily T2-weighted MR cholangiography and three-dimensional T1-weighted MR cholangiography after an intravenous bolus injection of mangafodipir trisodium. All studies were performed on a 1.5-T or 1-T scanner. Contrast-enhanced MR cholangiograms were evaluated for the presence and location of bile duct leaks. Correlation was obtained in all cases with surgery (n = 15), endoscopic retrograde cholangiography (n = 5), percutaneous drainage (n = 5), and clinical follow-up (n = 9). Results In 20 of 34 patients, bile duct leakage was proved by surgery, endoscopic retrograde cholangiography, or drainage. Contrast enhancement displayed the leakage in 19 of 20 patients and ruled out leaks in the other 14 patients (95% sensitivity, 100% specificity). The leak site was depicted in 14 patients and contrast-enhanced MR cholangiography successfully located the origin of the leak in 11 patients. Conclusions Contrast-enhanced MR cholangiography with intravenous mangafodipir trisodium can accurately diagnose the presence and location of bile duct leaks in patients who have undergone laparoscopic cholecystectomy.

The purpose of this study was to examine the feasibility of contrast-enhanced virtual MR cholangioscopy (CE VMRC). Intraluminal views of the extrahepatic biliary tree were generated in ten patients undergoing abdominal MRI post... more

The purpose of this study was to examine the feasibility of contrast-enhanced virtual MR cholangioscopy (CE VMRC). Intraluminal views of the extrahepatic biliary tree were generated in ten patients undergoing abdominal MRI post mangafodipir trisodium administration employing coronal 2.5-mm 3D fast low-angle shot (FLASH) images (TR 6.8 ms, TE 2.3 ms, matrix 195×512) with fat saturation and a commercially available software. Contrast-enhanced VMRC was compared with single-shot turbo spin-echo T2-weighted MR cholangiography (T2 MRC) in terms of ductal visualization and artifact presence, utilizing a five-point grading scale. Four anatomic segments were evaluated: the intra- and extra-pancreatic segment of the common bile duct (CBD), and the cystic duct and the area of hepatic duct bifurcation. Both CE VMRC and T2 MRC depicted 38 of 40 segments. There were no significant differences between CE VMRC and T2 MRC in ranking ductal segments visualization (p=0.27). The high contrast between intraluminal fluid and extraluminal tissues facilitated the generation of endoscopic views. Contrast-enhanced virtual MR cholangioscopy is a feasible technique providing endoscopic views of the CBD. Initial results show correlation of CE VMRC with projectional MR cholangiography.

Purpose: Worldwide efforts to understand developmental processes demand new high-resolution 3D imaging methods to detect the consequences of gene function in embryo development and diseases. Encouragingly, recent studies have shown that... more

Purpose: Worldwide efforts to understand developmental processes demand new high-resolution 3D imaging methods to detect the consequences of gene function in embryo development and diseases. Encouragingly, recent studies have shown that MRI contrast agents can highlight specific tissue structures in ex vivo adult mouse brains. MR imaging of mouse embryos is currently limited by a lack of tissue staining capabilities that would provide the flexibility and specificity offered by histological stains conventionally used for mouse embryo phenotyping.