A hydrophilic interaction liquid chromatography - Tandem mass spectrometry method for the determination of phenylephrine in dried blood spots from preterm infants.

A sensitive and accurate hydrophilic interaction liquid chromatography - tandem mass spectrometry method (HILIC-MS/MS) was developed and validated for the determination of phenylephrine concentration in Dried Blood Spot (DBS) samples from preterm infants, after ocular administration of an ophthalmic solution with phenylephrine. Sample preparation involved the extraction of the analyte from an 85 µL DBS sample with methanol - acetonitrile (50:50, v/v). Chromatographic separation was achieved on an ACQUITY UPLC BEH AMIDE column, under isocratic conditions within a 5 min run. Detection was achieved with a triple quadrupole MS applying electrospray ionization in positive mode. The method was fully validated and proved precise and accurate with in a linear range of 0.59-3.53 ng/ml in blood. The method was developed to provide insights on the level of exposure of infant population to phenylephrine after ocular administration.

A novel p27 gene mutation in a case of unclassified myeloproliferative disorder.

P27 encodes a member of Cip/Kip family of cyclin dependent kinase inhibitors, the inactivation of which has been implicated in the pathogenesis of various hematological neoplasias. We report on a novel point mutation of this gene identified in a case of unclassified myeloproliferative syndrome consisting of a T --> C transversion at 821bp of p27 exon 1, resulting in a Ile --> Thr substitution at codon 119. The analysis of larger number of cases as well as the effect of this mutation on protein's function will help to clarify its significance in the pathogenesis of myeloproliferative syndromes.

Isolation of a mouse brain cDNA expressed in developing neuroblasts and mature neurons.

A previously uncharacterized 4.5-kb mouse cDNA clone, designated mc7, was isolated and found to be predominantly expressed in brain. This cDNA predicts a 1035-bp open reading frame that encodes for a 345-amino acid polypeptide especially rich in glutamic acid residues located in the region from residues 80 to 174. Computational analysis revealed among other features, putative zinc-finger motifs and coiled-coil regions. The corresponding mc7 gene is detected in mouse, rat, pig and human genomes. In mouse the mc7 mRNA is expressed predominantly in brain and to a much lesser extent in kidney, lung and spleen. In brain it is detectable as early as embryonic day 14 while it is retained in the adult. In situ hybridization studies revealed that mc7 mRNA is widely, albeit unevenly, expressed in neurons throughout the adult brain. Developmental in situ hybridization studies in the cerebellar cortex demonstrated that at postnatal day 5 mc7 mRNA is mainly expressed in neuroblasts of the external granular layer and in developing neurons of the internal granular layer. Some staining is also present in purkinje cells becoming particularly pronounced at postnatal day 10, the time of arborarization of their dendritic tree. In the adult cerebellar cortex expression is mainly confined in purkinje cells and to a lesser extent in granule neurons. The early expression of mc7 in neuroblasts and developing neurons as well as its retention in a wide variety of mature neurons suggest that it may play a role in the process of differentiation and maturation of these cells in the brain.

Early expression of the BM88 antigen during neuronal differentiation of P19 embryonal carcinoma cells.

Previous studies have shown that the BM88 antigen, a neuron-specific molecule, promotes the differentiation of mouse neuroblastoma cells [23] (Mamalaki A., Boutou E., Hurel C., Patsavoudi E., Tzartos S. and Matsas R. (1995) The BM88 antigen, a novel neuron-specific molecule, enhances the differentiation of mouse neuroblastoma cells. J. Biol. Chem. 270, 14201-14208). In particular, stably transfected with the BM88 cDNA, Neuro 2a cells over-expressing the BM88 antigen are morphologically distinct from their non-transfected counterparts; they exhibit enhanced process outgrowth and a slower rate of division. Moreover, they respond differentially to growth factors [10] (Gomez J., Boutou E., Hurel C., Mamalaki A., Kentroti S. , Vernadakis A. and Matsas R. (1998) Overexpression of the neuron-specific molecule BM88 in mouse neuroblastoma cells: Altered responsiveness to growth factors. J. Neurosci. Res. 51, 119-128). In order to further elucidate the role of the BM88 antigen in the differentiation of developing neurons we used the in vitro system of differentiating P19 cells which closely resembles early murine development in vivo. In this study, P19 cells were driven to the neuronal pathway with retinoic acid. We examined by immunofluorescence studies the expression of the BM88 antigen in these cells and we found that it correlates well with the expression of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) which characterizes early differentiating post-mitotic neurons. In contrast, very few of the BM88 antigen-positive/PSA-NCAM-positive cells expressed neurofilament protein, a marker of more mature neurons. Our findings, in accordance with previously reported data, strongly suggest that the BM88 antigen is involved in the early stages of differentiation of neuronal cells.

Overexpression of the neuron-specific molecule BM88 in mouse neuroblastoma cells: altered responsiveness to growth factors.

Previous studies have shown that the BM88 antigen, a novel neuron-specific molecule, promotes the differentiation of mouse neuroblastoma (Neuro 2a) cells. In particular, stably transfected, with the BM88 cDNA, Neuro 2a cells overexpressing the BM88 antigen (Neuro2a-BM88 cells) are morphologically distinct from the nontransfected Neuro 2a cells; they exhibit enhanced process outgrowth and a slower rate of division. In this study we used Neuro2a and the morphologically differentiated Neuro 2a-BM88 cells to compare their responsiveness to growth factors. The growth factors we used were nerve growth factor (NGF), basic-fibroblast growth factor (b-FGF), and glial cell-line derived neurotrophic factor (GDNF). In addition, we used glial conditioned medium derived from either newborn mouse cerebral cortex (NBCC) or aged mouse cerebral hemispheres (MACH), as a source of normal glial factors. Because these cells express the cholinergic phenotype, we used choline acetyltransferase (ChAT) activity as a biochemical marker for comparison. A differential responsiveness to these factors was observed between Neuro 2a and Neuro 2a-BM88. The presence of NGF, 25 ng/ml, in the culture medium did not affect ChAT activity in either cell type. In contrast to NGF, in the presence of b-FGF, 5 ng/ml, the transfected cells, Neuro 2a-BM88, responded with a marked increase in ChAT activity. On the other hand, with GDNF, 1 ng/ml, only Neuro 2a cells showed an increase in ChAT activity. Finally, we found no response to the glial conditioned media, although these media contain several growth factors, including b-FGF. In conclusion, our findings show that overexpression of the neuron-specific antigen BM88 in neuroblastoma cells modifies their properties with respect to growth factor sensitivity, and, hence, the Neuro 2a and Neuro 2a-BM88 are suitable cell models to examine the role of growth factors in neuronal differentiation.

Recognition of subsets of the mammalian A/B-type core heterogeneous nuclear ribonucleoprotein polypeptides by novel autoantibodies.

The structurally related A/B-type core heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptides of 34-39 kDa (A1, A2, B1 and B2) belong to a family of RNA-binding proteins that are major components of 40 S hnRNP complexes. By two-dimensional gel electrophoresis and peptide mapping analysis we compared each member of the A/B-type core proteins in the human and rat liver cells. This comparison revealed the unique presence in rat cells of major protein species, referred to as mBx polypeptides, that appeared as three charge isoforms at a position corresponding to the minor HeLa B1b protein spot. In addition, clear differences in the ratios of the A1 polypeptide to the A1b isoform were observed. The detection, in sera of patients with rheumatic autoimmune diseases, of two novel autoantibody specificities, one recognizing solely B2 protein and the second both the B2 and mBx polypeptides, helped to identify mBx proteins as new A/B-type hnRNP components, immunologically related to B2 protein. A common immunoreactive V8 protease peptide of approx. 17 kDa has been identified in B2 and mBx hnRNP polypeptides. mBx protein species are identified in cells of murine origin, and have a ubiquitous tissue distribution and developmental appearance.

The BM88 antigen, a novel neuron-specific molecule, enhances the differentiation of mouse neuroblastoma cells.

The BM88 antigen is a neuron-specific molecule widely distributed in the mammalian nervous system. It is a 22-kDa, apparently not glycosylated, integral membrane protein, which appears early during brain development and remains at high levels in the mature animal. Here, we describe the cDNA cloning of the porcine BM88 antigen and present evidence that this protein is involved in neuroblastoma cell differentiation. The deduced protein is a novel molecule consisting of 140 amino acids and bears a putative transmembrane domain at the COOH-terminal region. The mRNA of this protein is expressed only in neural tissues, where it is restricted to neurons. Stably transfected Neuro-2a cells overexpressing the BM88 antigen exhibited a significant change in morphology, reflected by enhanced process outgrowth, and a slower rate of division. Moreover, in the presence of differentiation agents, such as sucrose and retinoic acid, an accelerated differentiation of the transfected Neuro-2a cells was observed. Especially in the presence of sucrose, the consequent overexpression of the BM88 antigen in the transfected cells resulted in their enhanced morphological differentiation accompanied by the induction of neurofilament protein expression. Our results suggest that the BM88 antigen plays a role in the differentiation of neuroblastoma cells.