Development of an isotope dilution mass spectrometry assay for HbA1c based on enzyme-cleaved peptide analysis

The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli. HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points. HIC and ATPS results correlated well (R(2) = 0.89). Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity. A GluAsnAlaAspVal modification resulted in increased protein expression. In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution. Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed.     

Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye-ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000-7.0% dextran T500-0.1 M potassium phosphate buffers and 16% PEG 1000-12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000-potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6h with minimum loss of its enzymatic activity.     

Stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry for the measurement of 4-nonylphenol and 4-tert-octylphenol in human biological samples

Alkylphenols, 4-nonylphenol (NP) and 4-tert-octylphenol (OP), in human urine and plasma samples were analyzed using stir bar sorptive extraction (SBSE) in combination with thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). The method involved correction by stable isotopically labeled surrogate standards, 4-(1-methyl)octylphenol-d5 (m-OP-d5) and deuterium 4-tert-octylphenol (OP-d). A biological sample was extracted for 60 min at room temperature (25 degrees C) using a stir bar coated with a 500 microm thick polydimethylsiloxane (PDMS) layer. Then, the stir bar was analyzed by TD-GC-MS in the selected ion monitoring (SIM) mode without any derivatization step. The average recoveries in human urine and plasma samples spiked with NP and OP at levels of 0.5 and 10 ng ml-1 were between 95.8 and 99.8% with correction using the added surrogate standards. The limits of quantitation were 0.2 ng ml-1 for NP and 0.02 ng ml-1 for OP. We measured the background levels of NP and OP in five human urine and three human plasma samples from healthy volunteers. NP and OP were not detected in all human urine samples (N.D. < 0.2 ng ml-1 for NP, and N.D. < 0.02 ng ml-1 for OP). However, 0.2-0.3 ng ml-1 for NP and 0.1-0.2 ng ml-1 for OP in human plasma samples were observed by this method.     

Gender difference in QTc prolongation of people with mental disorders

BACKGROUND: We examined gender difference in QTc interval distribution and its related factors in people with mental disorders. METHODS: We retrospectively reviewed medical charts of patients discharged from a university psychiatric unit between November 1997 and December 2000. Subjects were 328 patients (145 males and 183 females) taking psychotropics at their admission. We examined patient characteristics, medical history, diagnosis, and medication before admission. RESULTS: Mean QTc interval was 0.408 (SD = 0.036). QTc intervals in females were significantly longer than those in males. QTc of females without comorbidity was significantly longer than that of males. CONCLUSION: The influence of gender difference on QTc prolongation in people with mental disorders merits further research.     

Extraabdominal fibromatosis in retroperitoneal space

Background  

Fibromatosis or desmoid tumor covers a broad spectrum of benign fibrous tissue proliferations. It is characterized by infiltrative growth and a tendency towards recurrence; however, unlike sarcoma, it never metastasizes.     

Vinyl polymers based on L-histidine residues. Part 1. The thermodynamics of poly(ampholyte)s in the free and in the cross-linked gel form

Amphiphilic vinyl polymers (in the free and cross-linked forms), carrying carboxyl and imidazole groups, were prepared by a radical polymerization of the purposely synthesized N-acryloyl-L-histidine. The protonation thermodynamic studies (at 25 degrees C in 0.15 M NaCl) showed high polyelectrolyte character of the soluble polymer. Unlike the linear decreasing trend of the basicity constant, over the whole range of alpha (degree of protonation), the enthalpy changes for the protonation of the imidazole nitrogen in the polymer showed a decreasing pattern only at alpha > 0.5. This was ascribed to the formation of hydrogen bonds between protonated and free neighboring monomer units. Viscometric data revealed a minimum hydrodynamic volume of the polymer at its isoelectric point (pH 5), whereas at higher or lower pHs, the macromolecule expanded greatly as a consequence of the charged sites formation. This produced a preferential solvation of the protonated imidazole and carboxylate ions, the latter being surrounded by more water molecules in the hydration shell. The peculiar hydration behavior was confirmed in the cross-linked polymer. The hydrogel showed an equilibrium degree of swelling (EDS), strongly dependent on pH, in a similar manner as viscometric data of the soluble polymer. A linear relationship between the reduced viscosity and the EDS was found. The polymer was non toxic against the RAW264 cell line.     

A temperature-sensitive dcw1 mutant of Saccharomyces cerevisiae is cell cycle arrested with small buds which have aberrant cell walls

Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a Deltadcw1 Deltadfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3 Deltadfg5). When DC61 cells were incubated at 37 degrees C, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37 degrees C. At 37 degrees C, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37 degrees C, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37 degrees C, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (Deltadcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.     

Activation of a Dab1/CrkL/C3G/Rap1 pathway in Reelin-stimulated neurons

During brain development, many neurons migrate long distances before settling and differentiating. These migrations are coordinated to ensure normal development. The secreted protein Reelin controls the locations of many types of neurons, and its absence causes the classic "Reeler" phenotype. Reelin action requires tyrosine phosphorylation of the intracellular protein Dab1 by Src-family kinases. However, little is known about signaling pathways downstream of Dab1. Here, we identify several proteins in embryonic brain extract that bind to tyrosine-phosphorylated, but not non-phosphorylated, Dab1. Of these, the Crk-family proteins (CrkL, CrkI, and CrkII ), bind significant quantities of Dab1 when embryonic cortical neurons are exposed to Reelin. CrkL binding to Dab1 involves two tyrosine phosphorylation sites, Y220 and 232, that are critical for proper positioning of migrating cortical plate neurons. CrkL also binds C3G, an exchange factor (GEF) for the small GTPase Rap1 that is activated in other systems by tyrosine phosphorylation. We report that Reelin stimulates tyrosine phosphorylation of C3G and activates Rap1. C3G and Rap1 regulate adhesion of fibroblasts and other cell types. Regulation of Crk/CrkL, C3G, and Rap1 by Reelin may be involved in coordinating neuron migrations during brain development.     

Mnd1 is required for meiotic interhomolog repair

BACKGROUND: While double-strand break (DSB) repair is vital to the survival of cells during both meiosis and mitosis, the preferred mechanism of repair differs drastically between the two types of cell cycle. Thus, during meiosis, it is the homologous chromosome rather than the sister chromatid that is used as a repair template. RESULTS: Cells attempting to undergo meiosis in the absence of Mnd1 arrest in prophase I due to the activation of the Mec1 DNA-damage checkpoint accumulating hyperresected DSBs and aberrant synapsis. Sporulation of mnd1Delta strains can be restored by deleting RED1 or HOP1, which permits repair of DSBs by using the sister chromatid as a repair template. Mnd1 localizes to chromatin as foci independently of DSB formation, axial element (AE) formation, and synaptonemal complex (SC) formation and does not colocalize with Rad51. Mnd1 does not preferentially associate with hotspots of recombination. CONCLUSIONS: Our results suggest that Mnd1 acts specifically to promote DSB repair by using the homologous chromosome as a repair template. The presence of Rec8, Red1, or Hop1 renders Mnd1 indispensable for DNA repair, presumably through the establishment of interhomolog (IH) bias. Localization studies suggest that Mnd1 carries out this function without being specifically recruited to the sites of DNA repair. We propose a model in which Mnd1 facilitates chromatin accessibility, which is required to allow strand invasion in meiotic chromatin.     

A highly bioactive lignophenol derivative from bamboo lignin exhibits a potent activity to suppress apoptosis induced by oxidative stress in human neuroblastoma SH-SY5Y cells

Approaches to protection against neurodegenerative diseases, in which oxidative stress and inflammation are implicated, should be based on the current concept on the etiology of these diseases. Recently, a new therapeutic strategy has been proposed to protect neurons from cell death by attenuating the apoptotic signal transduction. Lignin, a durable aromatic network polymer second to cellulose in abundance, was able to be converted into highly active lignophenol derivatives with antioxidant activity by using our newly developed phase-separation technique. These lignophenol derivatives were found to show the potent neuroprotective activity against oxidative stress. Among the compounds examined, a lignocresol derivative from bamboo (lig-8) exhibited the most potent neuroprotective activity against hydrogen peroxide (H(2)O(2))-induced apoptosis in human neuroblastoma cell line SH-SY5Y by preventing the caspase-3 activation via either caspase-8 or caspase-9. Furthermore, it was found that lig-8 exerted the antiapoptotic effect by inhibiting dissipation of the mitochondrial membrane permeability transition induced by H(2)O(2) or by the peripheral benzodiazepin receptor ligand PK11195. Lig-8 was also shown to be potent in the antioxidant activity in the cells exposed to H(2)O(2), as assessed by flow cytometry using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and in vitro reactive oxygen species-scavenging potency. These data suggest that lig-8 is a promising neuroprotector, which affects the signaling pathway of neuronal cell death and that it would be of benefit to delay the progress of neurodegenerative diseases.