Medicines associated with folate-homocysteine-methionine pathway disruption.

Folate is vital for cell development and growth. It is involved in one-carbon transfer reactions essential for the synthesis of purines and pyrimidines. It also acts in conjunction with cobalamin (vitamin B12) as a fundamental cofactor in the remethylation cycle that converts homocysteine to methionine. A deficiency in folate or vitamin B12 can lead to elevated homocysteine level, which has been identified as an independent risk factor in several health-related conditions. Adequate folate levels are essential in women of childbearing age and in pregnant women, and folate deficiency is associated with several congenital malformations. Low folate levels can be caused by dietary deficiencies, a genetic predisposition or treatment with medicines that affect folate concentration. Women who are pregnant or of child-bearing age commonly use medicines, so it is important to identify the basic biochemical mechanisms by which medicines interfere with the folate-homocysteine-methionine pathway. This review focuses on prescription medicines associated with folate disruption. It also summarizes their undesirable/toxic effects. Recommendations regarding folate supplementation during medical therapy are also reviewed.

Common polymorphism in the glycine N-methyltransferase gene as a novel risk factor for cleft lip with or without cleft palate.

The objective of this study was to identify new environmental and genetic risk factors for orofacial clefts that arise during early foetal development. In this retrospective, case-control, mother-child pair study, 172 orofacial clefts cases and 199 healthy controls, and their respective mothers, were genotyped for common variants in relevant genes obtained by text and database mining using STRING 10.0. Exposure to environmental risk factors was evaluated using questionnaires. Variant glycine N-methyltransferase (odds ratio (OR) 2.1, 95% confidence interval (95% CI) 1.0-4.4) and dihydrofolate reductase (OR 2.4, 95% CI 1.3-4.5) genotypes were identified as risk factors for cleft lip with or without cleft palate formation. Furthermore, synergy was detected between variant glycine N-methyltransferase and dihydrofolate reductase genotypes in promoting cleft lip with or without cleft palate formation (OR 7, 95% CI 2-23). This study is novel in finding that common glycine N-methyltransferase variant genotypes increase the risk of cleft lip with or without cleft palate.

Oxidatively altered IgG with increased immunoreactivity to ß2-glycoprotein I and its peptide clusters influence human coronary artery endothelial cells.

Oxidative stress has been shown to play a role in modifying antibodies in favor of higher auto-immunoreactivity. We studied the immunoreactivity of oxidized IgG (oxIgG) to ß2-glycoprotein I (ß2GPI), six peptide sequences corresponding to amino acid clusters on its different domains, to determine their effects on human coronary artery endothelial cells (HCAEC). Human IgG was purified from seven donors, electro-oxidized and checked for immunoreactivity and avidity to ß2GPI and to peptides by ELISA. Conformational stability and antibody-antigen complex formation of oxIgG was analyzed by fluorescence spectroscopy and dynamic light scattering. Resting and activated sub-confluent HCAEC were stimulated with oxIgG or IgG. Secreted cytokines were measured by ELISA. Immunoreactivity of seven oxIgG samples increased to 7.5-fold against ß2GPI and to 3.8-fold against six peptides as compared to IgG. oxIgG showed low avidity "properties." Conformational changes and exposure of protein hydrophobic regions were confirmed by an elevation in fluorescence (2.4- to 5.0-fold) on bis-ANS dye binding to oxIgG. oxIgG significantly elevated the release of GROα and IL-8 in resting and activated states of HCAEC. Oxidation alters IgG in favor of autoreactivity toward whole ß2GPI and corresponding peptides on different domains of ß2GPI and could lead to dysfunction of arterial endothelium by upregulation of chemokines.

Thiopurine S-methyltransferase pharmacogenetics: genotype to phenotype correlation in the Slovenian population.

The toxicity of thiopurine drugs has been correlated to the activity of thiopurine S-methyltransferase (TPMT), whose interindividual variation is a consequence of genetic polymorphisms. We have herein investigated the relevance of some genetic markers for the prediction of thiopurine-related toxicities and to determine the genotype to phenotype correlation in the Slovenian population. The most prevalent mutant allele in the Slovenian population is TPMT*3A (4.1%), followed by TPMT*3C (0.5) and TPMT*3B (0.3), while the TPMT*2 allele was not found in any of the examined samples. TPMT enzyme activity distribution in the subgroup sample was bimodal and as such correlated with genetic data. Using a cutoff value of 9.82 pmol/10(7) RBC per h, the genetic data correctly predicted TPMT enzyme activity in 91.6% of the examined individuals. Pharmacogenetic TPMT analyses have therefore proved to have significant clinical implications for prediction of individuals' responses to treatment with thiopurine drugs in order to avoid possible life-threatening therapy-related toxicities.

(99m)Tc-rituximab radiolabelled by photo-activation: a new non-Hodgkin's lymphoma imaging agent.

PURPOSE: Rituximab was the first chimeric monoclonal antibody to be approved for treatment of indolent B-cell non-Hodgkin's lymphoma (NHL). It is directed against the CD20 antigen, which is expressed by 95% of B-cell NHLs. The aim of this study was to explore the possibility of radiolabelling rituximab with (99m)Tc for use as an imaging agent in NHL for early detection, staging, remission assessment, monitoring for metastatic spread and tumour recurrence, and assessment of CD20 expression prior to (radio)immunotherapy. METHODS: Rituximab was purified from Mabthera solution (Roche), photo-activated at 302 nm by UV irradiation and radiolabelled with (99m)Tc. The effectiveness of the labelling method was evaluated by determination of the number of free thiol groups per photoreduced antibody, radiochemical purity and in vitro stability of (99m)Tc-rituximab. RESULTS: On average, 4.4 free thiol groups per photoreduced antibody were determined. Radiolabelling yields greater than 95% were routinely observed after storage of the photo-activated antibody at -80 degrees C for 195 days. The direct binding assay showed preserved ability of (99m)Tc-rituximab to bind to CD20, with an average immunoreactive fraction of 93.3%. The internalisation rate was proven to be low, with only 5.3% of bound (99m)Tc-rituximab being internalised over 4 h at 37 degrees C. CONCLUSION: Our results demonstrate that (99m)Tc-rituximab of high radiochemical purity and with preserved binding affinity for the antigen can be prepared by photoreduction and that the method shows good reproducibility. (99m)Tc-rituximab will be further explored as an imaging agent applicable in NHL for the purposes mentioned above.

B cell receptor signaling involves physical and functional association of FAK with Lyn and IgM.

B cell receptor (BCR) stimulation induces phosphorylation of a number of proteins, leading to functional activation of B lymphocytes. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase, involved in a variety of signaling pathways. In this study, we show that FAK is tyrosine-phosphorylated and activated following BCR stimulation. We also demonstrate constitutive association of FAK with the Src-family kinase Lyn and with components of the BCR. Association of Lyn with FAK which was not correlated with BCR-induced activation of both kinases, appeared to be mediated via the binding of Lyn to the COOH-terminal part of the FAK molecule. Our results indicate that FAK is a component of the BCR complex and that it participates in BCR signaling.

A double-edged kinase Lyn: a positive and negative regulator for antigen receptor-mediated signals.

B cells from young lyn-/- mice are hyperresponsive to anti-IgM-induced proliferation, suggesting involvement of Lyn in negative regulation of B cell antigen receptor (BCR)-mediated signaling. Here we show that tyrosine phosphorylation of FcgammaRIIB and CD22 coreceptors, which are important for feedback suppression of BCR-induced signaling, was severely impaired in lyn-/- B cells upon their coligation with the BCR. Hypophosphorylation on tyrosine residues of these molecules resulted in failure of recruiting the tyrosine phosphatase SHP-1 and inositol phosphatase SHIP, SH2-containing potent inhibitors of BCR-induced B cell activation, to the coreceptors. Consequently, lyn-/- B cells exhibited defects in suppressing BCR-induced Ca2+ influx and proliferation. Thus, Lyn is critically important in tyrosine phosphorylation of the coreceptors, which is required for feedback suppression of B cell activation.

Signaling capacity of the T cell antigen receptor is negatively regulated by the PTP1C tyrosine phosphatase.

The association of PTP1C deficiency with the multiplicity of lymphoid cell abnormalities manifested by motheaten (me) and viable motheaten (me(v)) mice suggests a pivotal role for this tyrosine phosphatase in the regulation of lymphocyte differentiation and function. To delineate the relevance of PTP1C to T cell physiology, we have examined me and me(v) T cells with regards to their capacity to transduce activating signals through the T cell antigen receptor (TCR). Although thymocyte maturation appeared normal in the mutant mice, both thymocytes and peripheral T cells from these animals exhibited proliferative response to TCR stimulation that were markedly increased relative to those elicited in normal cells. Compared to normal thymocytes, PTP1C-deficient thymocytes also showed increased constitutive tyrosine phosphorylation of the TCR complex and enhanced and prolonged TCR-induced tyrosine phosphorylation of the TCR-zeta and CD3-epsilon, as well as a number of cytosolic proteins, most notably a 38-kD phosphoprotein found to associate with the Grb2 adaptor SH2 domain in activated thymocytes. These latter phosphoproteins also associated with the Vav guanine nucleotide exchange factor upon TCR ligation, and were dephosphorylated by recombinant PTP1C in vitro. In conjunction with the finding of PTP1C-TCR association in unstimulated normal thymocytes, these results reveal the capacity of PTP1C to interact with and likely dephosphorylate resting and activated TCR complex components, as well as more distal signaling effectors that are normally recruited to the Vav and Grb2 SH2 domains after TCR stimulation. These data therefore strongly implicate PTP1C in the downregulation of TCR signaling capacity and, taken together with the aberrant prolongation of TCR-induced, mitogen-associated kinase (MAPK) activation observed in PTP1C-deficient thymocytes, these findings suggest that the inhibitory influence of PTP1C on TCR signal relay is realized through its effects on both the TCR complex and downstream signaling elements that couple the activated antigen receptor to the Ras/MAPK response pathway.

Increased expression of the stefin A cysteine proteinase inhibitor occurs in the myelomonocytic cell-infiltrated tissues of autoimmune motheaten mice.

Mice homozygous for the motheaten (me) and viable motheaten (me) mutations develop a progressively severe inflammatory disease in association with profound disruption of normal hematopoiesis. These mutant mice have been previously shown to manifest abnormally high expression in the bone marrow of cDNAs encoding three members of the stefin family of cysteine proteinase inhibitors. The data reported here reveal that increases in levels of both stefin transcripts and proteins occur in bone marrow, splenic and pulmonary tissues of me and mev mice, and correlate with the abnormal expansion of stefin A-producing myelomonocytic cells in these tissues. Increases in stefin expression are also apparent in me and mev skin and appear to reflect focal hyperplasia of stefin-producing epidermal cells as well as infiltration by stefin-expressing monocytic and granulocytic cells. Because the increases in stefin protein levels occur in the same tissues that are most adversely affected by the me mutation, it appears that overexpression of these proteins may distinguish the cell population responsible for disease pathogenesis and may be relevant to the severe tissue inflammation and damage observed in these mutant mice.

Expression and catalytic activity of the tyrosine phosphatase PTP1C is severely impaired in motheaten and viable motheaten mice.

Mutations in the gene encoding the phosphotyrosine phosphatase PTP1C, a cytoplasmic protein containing a COOH-terminal catalytic and two NH2-terminal Src homology 2 (SH2) domains, have been identified in motheaten (me) and viable motheaten (mev) mice and are associated with severe hemopoietic dysregulation. The me mutation is predicted to result in termination of the PTP1C polypeptide within the first SH2 domain, whereas the mev mutation creates an insertion or deletion in the phosphatase domain. No PTP1C RNA or protein could be detected in the hemopoietic tissues of me mice, nor could PTP1C phosphotyrosine phosphatase activity be isolated from cells homozygous for the me mutation. In contrast, mice homozygous for the less severe mev mutation expressed levels of full-length PTP1C protein comparable to those detected in wild type mice and the SH2 domains of mev PTP1C bound normally to phosphotyrosine-containing ligands in vitro. Nevertheless, the mev mutation induced a marked reduction in PTP1C activity. These observations provide strong evidence that the motheaten phenotypic results from loss-of-function mutations in the PTP1C gene and imply a critical role for PTP1C in the regulation of hemopoietic differentiation and immune function.