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PubMed2006

Volumetric investigation of brain regions in patients with conversion disorder.

Preliminary evidence revealed a decrease of regional cerebral blood flow in the thalamus and basal ganglia contralateral to the deficit and suggested that hysterical conversion deficits might entail a functional disorder in striatothalamocortical circuits. However, there is no systematic structural magnetic resonance imaging (MRI) study in the literature in patients with conversion disorder (CD). Therefore, we aimed to perform structural MRI to evaluate the brain regions of interest in first applying patients with CD. Morphometric MRI was used to compare regional brain volumes in ten women with CD and same number of healthy comparison subjects. Intracranial volume (ICV), whole brain volume, gray and white matter volumes did not differ between the patient and control groups. Patients with CD had significantly smaller mean volumes of the left caudate nucleus, lentiform nucleus (p<0.01 for caudate nucleus and p<0.05 for lentiform nucleus) and right caudate nucleus and lentiform nucleus (p<0.05 for both structures). In patients, the right thalamus was significantly smaller, and the left thalamus rendered to be smaller compared to healthy controls. Age at onset showed a significant relation with left caudate, and a near-significant trend with right thalamus volumes. In conclusion, our findings suggest that patients with CD have significantly smaller mean volumes of the left and right basal ganglia and smaller right thalamus, with a trend toward to smaller left thalamus compared to healthy controls and that these findings provide novel constraints for a modern psychobiological theory of hysteria.

Adult↗
PubMed2003

Mutual interpersonal postural constraints are involved in cooperative conversation.

The research was designed to evaluate interpersonal coordination during conversation with a new measurement tool. The experiment uses an analysis based on recurrence strategies, known as cross recurrence quantification, to evaluate the shared activity between 2 postural time series in reconstructed phase space. Pairs of participants were found to share more locations in phase space (greater recurrence) in conditions where they were conversing with one another to solve a puzzle task than in conditions in which they convened with others. The trajectories of pairs of participants also showed less divergence when they conversed with each other than when they conversed with others well. This is offered as objective evidence of interpersonal coordination of postural sway in the context of a cooperative verbal task.

Adult↗
PubMed1996

Transcription factor PU.1 mediates induction of c-fms in vascular smooth muscle cells: a mechanism for phenotypic change to phagocytic cells.

The macrophage colony-stimulating factor receptor encoded by the c-fms gene is expressed in vascular intimal smooth muscle cells isolated from atherosclerotic lesions. A combination of platelet-derived growth factor-BB and epidermal growth factor induces stable expression of c-fms in normal vascular medial smooth muscle cells. The mechanism by which these growth factors induce c-fms expression has now been investigated in an attempt to gain insight into the events that underlie the phenotypic conversion of vascular smooth muscle cells in atherosclerosis. Deletion analysis of the c-fms promoter revealed that the region including a binding site for transcription factor PU.1 was required for transcriptional activity in human aortic medial smooth muscle cells. Mutation in the PU.1 binding site markedly reduced promoter activity. Northern (RNA) blot analysis demonstrated that growth factors induced the expression of PU.1 mRNA in vascular medial smooth muscle cells and that PU.1 mRNA was expressed in vascular intimal smooth muscle cells. PU.1 antisense oligonucleotides inhibited growth factor-induced c-fms expression and foam cell formation. These results suggest that transcription factor PU.1 plays an essential role in the phenotypic conversion of vascular smooth muscle cells to macrophagelike cells by mediating the induction of c-fms.

Aorta↗
PubMedDate not supplied

A theory-based approach to analysing conversation sequences.

AIMS: To assess the quality of communication generally two procedures are used: one defines categories of utterances and counts their frequency, the other uses global observer ratings. We investigated whether a sequence analysis of utterances yields results which more precisely reflect the process of a conversation. METHODS: We re-examined data from a randomised controlled intervention study in which residents' interviews with simulated patients were analysed with the Maastricht History and Advice Checklist (MAAS-R) and the Roter Interaction Analysis System (RIAS). Using the U-file of the RIAS we studied the effect of different types of physician questions (open, closed questions, facilitators, other physician actions) on the length of uninterrupted patients' speech and content of utterances. We investigated also whether reciprocity indices improve after a communication skills training, and whether they correlate with global scores form MAAS-R. RESULTS: Patients respond to a closed question with a mean of 1.78 (+/- 1.49) utterances as compared to 2.75 (+/- 2.72) utterances after an open question. The likelihood of a concern was more than 10 fold higher after an open question compared to closed questions. Reciprocal sequences make up less than 2 percent of the conversation, Still, they correlate with global items form MAAS-R. The 'empathy index' improves after the training.

Communication↗
PubMed1983

A modular program for analysis of electrophysiological data on a laboratory minicomputer.

In the study of electrophysiological phenomena, the signals to be investigated are often reduced to sampled time series or point processes. Nowadays, the measuring equipment frequently includes one or more micro- and/or minicomputers which perform the data acquisition (including, e.g., A-D conversion and interval measurements) and the subsequent data processing and analysis. This paper describes the structured organization of data measurement and analysis of electrophysiological data performed by a modular software program implemented on a laboratory minicomputer. The program is used in our clinical laboratory by a number of research teams with different experimental objectives. The input data structure for the program has a standardized format, comprising multiplexed data channels preceded by a 256 entry parameter block containing general processing information and specific information concerning the analyses performed. The tasks carried out by the program are the calculation of distributions and correlations, digital filtering and estimation of spectra. The program can easily be extended by writing new software modules.

Computers↗
PubMed1985

Disease-associated loss of erythrocyte complement receptors (CR1, C3b receptors) in patients with systemic lupus erythematosus and other diseases involving autoantibodies and/or complement activation.

Although surface membrane density of complement receptor type one (CR1) on erythrocytes (E) is probably an inherited trait among normal individuals, recent evidence from our laboratories suggests that the reduced number of CR1 per E observed in patients with systemic lupus erythematosus (SLE) results from acquired as well as genetic factors. In the present investigation, the number of CR1 per E was quantitated with 125I-monoclonal anti-CR1 and was found to vary inversely with disease activity in patients with SLE who were followed serially for as long as 14 mo. Although evidence for E surface-bound immune complexes or fixed C3b/iC3b was not obtained, periods of disease activity and low amounts of CR1 per E correlated with the presence of 100 to 800 molecules per E of fixed C3dg fragments (less than 100 C3dg per E in normal subjects). Reduced CR1 and excess fixed C3dg on E also were observed in patients with other disorders associated with complement activation, including chronic cold agglutinin disease, autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria (PNH), Sjögren's syndrome, and mycoplasma pneumonia. A significant negative correlation (r = -0.498) between CR1/E and fixed C3dg/E was demonstrable in 255 individual assays evaluated by regression analysis. CR1 decreased and fixed C3dg increased during active disease; the converse was obtained during remission. In patients with active SLE, both serum complement activity and E CR1 decreased, whereas fixed C3dg fragments increased. By piecewise linear regression analysis, the appearance of 100 to 400 C3dg molecules on patients' E corresponded to a 27 to 60%, reduction in the number of CR1 per E (p less than 0.0002), confirming that fixation of C3 to E was correlated with a loss of CR1. In patients with PNH, low values for CR1 were observed on moderately complement-sensitive PNH type II E in association with increased fixed C3 fragments; however, the markedly complement-sensitive PNH type III E had essentially normal amounts of CR1 and bore little fixed C3. The addition of soluble DNA/anti-DNA immune complexes to normal blood generated levels of fixed C3dg fragments on E comparable to those observed on E from patients with SLE. Kinetic experiments indicated that C3b was fixed to E during the process of immune complex binding and release from E CR1, and that this fixed C3b was subsequently degraded rapidly to fixed iC3b and more slowly to fixed C3dg without the loss of CR1 that occurs in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Anemia, Hemolytic↗
PubMed1995

The yeast MER2 gene is required for chromosome synapsis and the initiation of meiotic recombination.

Mutation of the MER2 gene of Saccharomyces cerevisiae confers meiotic lethality. To gain insight into the function of the Mer2 protein, we have carried out a detailed characterization of the mer2 null mutant. Genetic analysis indicates that mer2 completely eliminates meiotic interchromosomal gene conversion and crossing over. In addition, mer2 abolishes intrachromosomal meiotic recombination, both in the ribosomal DNA array and in an artificial duplication. The results of a physical assay demonstrate that the mer2 mutation prevents the formation of meiosis-specific, double-strand breaks, indicating that the Mer2 protein acts at or before the initiation of meiotic recombination. Electron microscopic analysis reveals that the mer2 mutant makes axial elements, which are precursors to the synaptonemal complex, but homologous chromosomes fail to synapse. Fluorescence in situ hybridization of chromosome-specific DNA probes to spread meiotic chromosomes demonstrates that homolog alignment is also significantly reduced in the mer2 mutant. Although the MER2 gene is transcribed during vegetative growth, deletion or overexpression of the MER2 gene has no apparent effect on mitotic recombination or DNA damage repair. We suggest that the primary defect in the mer2 mutant is in the initiation of meiotic genetic exchange.

Chromosomes, Fungal↗
PubMed2001

Cytokinin overproducing ove mutants of Physcomitrella patens show increased riboside to base conversion.

Ove mutants in the moss Physcomitrella patens can arise from different recessive mutations. These mutants display a much larger number of buds than the wild type (wt) due to a dramatic overproduction of cytokinins (Cks), which are released into the culture medium (T.L. Wang, R. Horgan, D.J. Cove [1981] Plant Physiol 68: 735-738). The amounts of isopentenyladenine (iP) and isopentenyladenosine ([9R]iP) produced by chloronema of different ove mutants were measured. Levels of the major Ck iP in the culture medium of the mutants oveA78, oveA201, oveC200, and oveB300 (cultured at 21 degrees C) were 4-fold (oveA78) to 22-fold (oveB300) higher than for the wt. A new temperature-sensitive ove strain oveST25, which exhibits a strong ove phenotype at 25 degrees C, was also studied. It produced about 260 times more iP than the thiamine auxotrophic wt from which it was derived. To contribute to the physiological understanding of Ck overproduction, in vivo labeling experiments with (3)H-[9R]iP were performed. In all ove mutants analyzed, the rate of (3)H-[9R]iP conversion to (3)H-iP was higher as compared with the wt. In oveST25, the 3-fold increased riboside to base conversion was temperature inducible and correlated with the iP production. Analysis of Ck catabolism revealed no major differences between ove mutants and wt, thus indicating that ove mutants are unlikely to be degradation mutants. The data suggest that in ove mutants the increased riboside to base conversion is part of a generally up-regulated Ck biosynthetic pathway and may play an important role for the enhanced release of iP into the medium.

Bryopsida↗
PubMedDate not supplied

A fully automated system for analysis of pesticides in water: on-line extraction followed by liquid chromatography-tandem photodiode array/postcolumn derivatization/fluorescence detection.

A fully automated system for on-line solid phase extraction (SPE) followed by high-performance liquid chromatography (HPLC) with tandem detection with a photodiode array detector and a fluorescence detector (after postcolumn derivatization) was developed for analysis of many chemical classes of pesticides and their major conversion products in aquatic systems. An automated on-line-SPE system (Prospekt) operated with reversed-phase cartridges (PRP-1) extracts analytes from 100 mL acidified (pH = 3) filtered water sample. On-line HPLC analysis is performed with a 15 cm C18 analytical column eluted with a mobile phase of phosphate (pH = 3)-acetonitrile in 25 min linear gradient mode. Solutes are detected by tandem diode array/derivatization/fluorescence detection. The system is controlled and monitored by a single computer operated with Millenium software. Recoveries of most analytes in samples fortified at 1 microgram/L are > 90%, with relative standard deviation values of < 5%. For a few very polar analytes, mostly N-methylcarbamoyloximes (i.e., aldicarb sulfone, methomyl, and oxamyl), recoveries are < 20%. However, for these compounds, as well as for the rest of the N-methylcarbamates except for aldicarb sulfoxide and butoxycarboxim, the limits of detection (LODs) are 0.005-0.05 microgram/L. LODs for aldicarb sulfoxide and butoxycarboxim are 0.2 and 0.1 microgram, respectively. LODs for the rest of the analytes except 4-nitrophenol, bentazone, captan, decamethrin, and MCPA are 0.05-0.1 microgram/L. LODs for the latter compounds are 0.2-1.0 microgram/L. The system can be operated unattended.

Autoanalysis↗
PubMed1998

[The assessment of skin tumors with magnetic resonance with high resolution and a paramagnetic contrast medium].

INTRODUCTION: The technologic improvement of surface coils in MRI has allowed better visualization of the skin and thus permitted the clinical use of this technique in dermatology. MRI allows to assess the depth and extent of skin tumors and to detect any malignant transformation. The MR differentiation between benign and malignant skin lesions relies on morphological criteria which however do not have an absolute diagnostic value. We investigated the role of paramagnetic contrast agents in the differentiation between benign and malignant skin lesions. MATERIAL AND METHODS: Forty-one patients, 33 with benign and 8 with malignant skin tumors, were submitted to MRI. All the examinations were performed with a 1.5 T superconductive unit, with a 2.5 cm surface coil. Axial T1- and T2-weighted SE images were acquired with 2 mm slice thickness. Paramagnetic contrast material was administered to all patients. The signal intensity of the skin lesions was calculated before and after paramagnetic contrast agent administration positioning a region of interest. A percentage ratio of contrast enhancement was calculated to quantify contrast agent uptake and the relative values were compared between benign and malignant lesions. A qualitative analysis was also performed rating the contrast enhancement of each lesion as high, intermediate, or absent. RESULTS: The quantitative analysis showed a statistically significant difference (p < .5) between the contrast enhancement values of benign and malignant lesions. In particular, malignancies had values ranging 117.3 (+/- 28.7) to 125 (+/- 32.4), while benign lesions had -20.6 to 99.8 (+/- 21.1). Conversely, no difference in contrast enhancement was found at qualitative analysis. CONCLUSIONS: MRI is a promising tool for characterizing skin tumors. Our preliminary results should be confirmed on larger series of patients with the use of high temporal resolution imaging sequences.

Adolescent↗
PubMed2005

Childhood leukaemia, polymorphisms of metabolism enzyme genes, and interactions with maternal tobacco, coffee and alcohol consumption during pregnancy.

Metabolic polymorphisms may influence the risk of childhood leukaemia related to maternal tobacco, coffee or alcohol consumption. The data were extracted from a case-control study including 280 cases of acute leukaemia and 288 controls. Blood sampling was obtained for a representative subset of 219 cases and 105 controls. Gene-environment interactions were estimated using both case-control and case-only analyses. The polymorphisms of CYP1A1, GSTM1, GSTP1, GSTT1 and NQO1 were not associated with the risk of leukaemia. The slow EPHX1 allele was negatively associated with childhood leukaemia while an inverse non-significant association was observed with the fast EPHX1 allele. Maternal smoking during pregnancy was not related to leukaemia, but an interaction was observed in the case-only analysis with CYP1A1*2A variant allele (odds ratio (OR) 2.2 [1.0-4.9]) and with GSTM1 deletion (OR 2.3 [1.2-4.4]). Conversely, coffee drinking interacted negatively with NQO1 polymorphism in the case-only analysis (OR 0.6 [0.3-1.2] and 0.4 [0.1-1.0] for light and heavy coffee consumptions, respectively). This study suggests that maternal smoking may be a risk factor for leukaemia in children who carry CYP1A1 or GSTM1 genotypes, which might increase reactive metabolites of polycyclic aromatic hydrocarbons.

Acute Disease↗
PubMed1990

Behavior of the [mi-3] mutation and conversion of polymorphic mtDNA markers in heterokaryons of Neurospora crassa.

We have examined the behavior of the [mi-3] mitochondrial mutation and two physical mtDNA markers in heterokaryotic cultures of Neurospora crassa. Previous workers showed that a 1.2-kilobase insertion in the larger polymorphic form of EcoRI-5 restriction fragment is a site of high frequency and rapid unidirectional gene conversion. We have confirmed this observation and determined by DNA sequence analysis that the insertion in the EcoRI-5 fragment corresponds precisely to an optional intron that contains a long open reading frame in the ND1 gene. Thus, the conversion of the short, intron-lacking, form of EcoRI-5 to the longer, intron-containing, form may be analogous to the unidirectional gene conversion events catalyzed by intron-encoded proteins in other organisms. The resolution of two polymorphic forms of the mtDNA EcoRI-9 restriction fragment in our heterokaryons differs from that observed previously and suggests that this locus is not a site of gene conversion in our heterokaryon pair. The size polymorphism of the EcoRI-9 fragments is due to a tandemly reiterated 78-base-pair sequence which occurs two times in the short form and three times in the long form. One copy of the repeat unit and 66 base pairs following it have been duplicated from the ND2 gene which is located about 30 kilobases distant on the mtDNA. In contrast to the [poky] mitochondrial mutant, which was completely dominant over wild-type mitochondria in heterokaryons, the [mi-3] mutant was recovered in only seven of twenty heterokaryons after ten cycles of conidiation and subculturing. The resolution of the [mi-3] or wild-type phenotype in heterokaryons may depend solely on random factors such as allele input frequency, drift, and segregation rather than specific dominant or suppressive effects.

Base Sequence↗
PubMed2004

Assay method for antitumor L-methionine gamma-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine.

L-Methionine gamma-lyase (EC 4.4.1.11) is a pyridoxal 5'-phosphate-dependent multifunctional enzyme. Measuring the initial velocity of alpha-ketobutyrate production by alpha,gamma-elimination of L-methionine catalyzed by L-methionine gamma-lyase is not very feasible, because the enzyme simultaneously catalyzes both gamma-replacement and alpha,gamma-elimination. To develop an accurate enzyme assay, the comprehensive enzyme kinetics needed to be elucidated by progress curve analysis on the basis of a reaction model for conversion of L-methionine to alpha-ketobutyrate, methanethiol, and ammonia with pyridoxal 5'-phosphate as a cofactor. Kinetic parameters were determined by linear transformation using an approximation of a Maclaurin series from the whole velocity of alpha-ketobutyrate production including alpha,gamma-elimination and gamma-replacement. The significance of gamma-replacement was revealed both theoretically and practically by the kinetic analysis. The enzyme activity was standardized and represented as the Vmax value taking into consideration gamma-replacement in the presence of L-methionine at 37 degrees C and pH 8.0. The novel method that we proposed is accurate, sensitive, reproducible, and linear over a wide range for the determination of L-methionine gamma-lyase activity.

Animals↗
PubMed2000

An evaluation of the safety and effectiveness of telephone triage as a method of patient prioritization in an ophthalmic accident and emergency service.

Service changes in the accident and emergency service at Manchester Royal Eye Hospital in England resulted in a telephone triage-based referral service for health care professionals. It became clear that this service needed evaluation in order to assure both providers and users of the service that this referral strategy, based on experienced nurse practitioners making decisions about patient priority, was safe and effective. The evaluation was extended to encompass the other area of the service where telephone referrals tend to be directly from patients. A mixed method was used. Information gained within the telephone triage conversation was compared with a final diagnosis through retrospective analysis of secondary data; the documentation of those patients who were not given access to the service initially was followed-up to ensure that this decision was safe and a number of nurses were questioned about telephone information gathering, using partially structured interviews. The study showed that nurse practitioners within the accident and emergency service were able to elicit accurate information from the telephone triage conversation on which to base a decision about patient access in most cases (76% over the whole service). This resulted in the appropriate prioritization of patients. No patient who needed urgent access to the service was denied it. The decisions made to deny urgent appointments to a number of patients were safe in all cases. It appears that one of the problem areas in the gathering of information for prioritization purposes is in the nurses' telephone triage discussions with some, but by no means all, doctors. Some general practitioners seemed unwilling to discuss the patient and give accurate information to a nurse and this is an area which appears to need some further work.

Emergencies↗
PubMed2006

Mathematical tools in analytical mass spectrometry.

Over the last few decades, mass spectrometry has become a powerful tool for exploring various aspects of molecular processes occurring in biological systems. Such exploration is leading to a greater understanding of various complex life processes; unraveling these processes poses the greatest challenge to contemporary bioscience. With due respect to sample preparation, data analysis is rapidly becoming a major obstacle to the conversion of experimental knowledge into valid conclusions. It is interesting to note that many problems related to mass spectrometry can be solved using techniques from computer science, graph theory and discrete mathematics. The aim of this manuscript is to recollect several essays that demonstrate the power and the need to apply such skills to mass spectrometry data interpretation. Special attention is paid to situations where traditional chemical analysis reaches its limits but mathematical reasoning can still allow us to reach valid conclusions.

Journal Article↗
PubMed1995

Cytochrome P450IIIA activity and cytokine-mediated synthesis of nitric oxide.

BACKGROUND: Although nitric oxide synthase (NOS) is a cytochrome P450-like hemoprotein with additional sequence homology to cytochrome P450 reductase, the role of the cytochrome P450 system in cytokine-mediated NO synthesis is unknown. METHODS: To characterize the role of the P450 system in the synthesis of NO, NO production, NOS enzyme activity, and steady state NOS mRNA and protein expression were characterized in the setting of P450 isoform activity inhibition by using a model of isolated rat hepatocytes in primary culture. Cimetidine (0 to 10 mmol/L) was chosen as a specific inhibitor of P450IIIA activity. NO production was induced by interleukin-1 (50 ng/ml) and tumor necrosis factor (500 units/ml) and quantified by measurement of its metabolite, nitrite, in the culture medium. Steady state NOS mRNA and protein expression were determined by reverse-transcriptase polymerase chain reaction and immunoblot analysis, respectively. NOS enzyme activity was measured by the conversion of tritiated-L-arginine to tritiated-L-citrulline. RESULTS: Inhibition of P450IIIA activity was associated with a concentration-dependent decrease in cytokine-mediated NO production. Levels of NOS mRNA and protein were not altered. The NOS enzyme assay was notable for stable concentrations of intermediate, N-OH-L-arginine, and decreased production of the final end product, L-citrulline. Dixon plot kinetic analysis of cimetidine-mediated inhibition of NOS yielded an inhibition constant Ki = 1.76 mmol/L. CONCLUSIONS: These results indicate that cytochrome P450IIIA isoform may play a posttranslational role in cytokine-mediated NO synthesis in this model of isolated rat hepatocytes in primary culture.

Amino Acid Oxidoreductases↗
PubMed2005

Factors that predict conversion in 69 consecutive patients undergoing laparoscopic ileocecal resection for Crohn's disease: a prospective study.

PURPOSE: This prospective study was designed to identify factors that could predict conversion in patients undergoing first laparoscopic ileocecal resection for Crohn's disease. METHODS: Between 1998 and 2004, 69 consecutive patients (32 males; mean age, 32 +/- 9 years) who had undergone a first laparoscopic ileocecal resection for Crohn's disease were included in a prospective study. Twenty-one patients (30 percent) were converted into laparotomy. Possible factors for conversion were analyzed by both univariate and multivariate analyses. RESULTS: No patient died. Four patients (9 percent; 2 in each group) required five reoperations because of intraperitoneal hemorrhage (n = 1), anastomotic fistula (n = 3), and small-bowel obstruction (n = 1). Mean hospital stay was significantly increased in converted compared with laparoscopic patients (9 +/- 4 vs. 7 +/- 3 days; P < 0.05). On univariate analysis, more than three episodes of acute flare of Crohn's disease (P = 0.02), male gender (P = 0.03), preoperative immunosuppressive drugs (P = 0.04), intra-abdominal abscess or fistula at the time of laparoscopy (P = 0.02), and resection of other intestinal segment (P = 0.02) were factors that predicted conversion. On multivariate analysis, recurrent medical episodes of Crohn's disease (odds ratio, 2; 95 percent confidence interval, 1-4), and intra-abdominal abscess or fistula at the time of laparoscopy (odds ratio, 15; 95 percent confidence interval, 4-78) were the two independent risk factors for conversion. CONCLUSIONS: This prospective study demonstrated that the severity of the disease increased significantly the conversion rate of the first laparoscopic ileocecal resection. Knowledge of these risk factors for conversion could be helpful in preoperative preparation and counseling of patients.

Abscess↗
PubMed2006

Defining the role of phosphomethylethanolamine N-methyltransferase from Caenorhabditis elegans in phosphocholine biosynthesis by biochemical and kinetic analysis.

In plants and Plasmodium falciparum, the synthesis of phosphatidylcholine requires the conversion of phosphoethanolamine to phosphocholine by phosphoethanolamine methyltransferase (PEAMT). This pathway differs from the metabolic route of phosphatidylcholine synthesis used in mammals and, on the basis of bioinformatics, was postulated to function in the nematode Caenorhabditis elegans. Here we describe the cloning and biochemical characterization of a PEAMT from C. elegans (gene, pmt-2; protein, PMT-2). Although similar in size to the PEAMT from plants, which contain two tandem methyltransferase domains, PMT-2 retains only the C-terminal methyltransferase domain. RNA-mediated interference experiments in C. elegans show that PMT-2 is essential for worm viability and that choline supplementation rescues the RNAi-generated phenotype. Unlike the plant and Plasmodium PEAMT, which catalyze all three methylations in the pathway, PMT-2 catalyzes only the last two steps in the pathway, i.e., the methylation of phosphomonomethylethanolamine (P-MME) to phosphodimethylethanolamine (P-DME) and of P-DME to phosphocholine. Analysis of initial velocity patterns suggests a random sequential kinetic mechanism for PMT-2. Product inhibition by S-adenosylhomocysteine was competitive versus S-adenosylmethionine and noncompetitive versus P-DME, consistent with formation of a dead-end complex. Inhibition by phosphocholine was competitive versus each substrate. Fluorescence titrations show that all substrates and products bind to the free enzyme. The biochemical data are consistent with a random sequential kinetic mechanism for PMT-2. This work provides a kinetic basis for additional studies on the reaction mechanism of PEAMT. Our results indicate that nematodes also use the PEAMT pathway for phosphatidylcholine biosynthesis. If the essential role of PMT-2 in C. elegans is conserved in parasitic nematodes of mammals and plants, then inhibition of the PEAMT pathway may be a viable approach for targeting these parasites with compounds of medicinal or agronomic value.

Amino Acid Sequence↗