S-Sulfo-Cysteine is an Endogenous Amino Acid in Neonatal Rat Brain but an Unlikely Mediator of Cysteine Neurotoxicity

S-sulfo-cysteine (SSC) is an agonist of glutamate receptors which could be involved in cysteine-induced neurotoxicity. Here we analyzed SSC by HPLC and demonstrated that the concentration of SSC in cortex of cysteine-injected rats increased to 1.4 μM, about four times the value of control rats. The neurotoxic effect of SSC was evaluated in slice cultures of rat hippocampus and compared to NMDA and cysteine. The neurotoxicity threshold of SSC was well above the tissue concentration. Our results show that SSC increases in neonatal rat brain after cysteine injection but reaches a tissue concentration far below concentrations that induce neurotoxicity in vitro. Thus, even if all the tissue SSC after cysteine injection was extracellular it would be below the threshold for toxicity, indicating that SSC is not a main excitotoxin involved in cysteine toxicity. Special issue article in honor of Dr. Frode Fonnum. Part of this work has been submitted as an abstract and presented as a poster to the 7th Biennial Meeting of the Asian Pacific Society for Neurochemistry (APSN) 2–5 July 2006.     

The highly similar Arabidopsis homologs of trithorax ATX1 and ATX2 encode proteins with divergent biochemical functions

Gene duplication followed by functional specialization is a potent force in the evolution of biological diversity. A comparative study of two highly conserved duplicated genes, ARABIDOPSIS TRITHORAX-LIKE PROTEIN1 (ATX1) and ATX2, revealed features of both partial redundancy and of functional divergence. Although structurally similar, their regulatory sequences have diverged, resulting in distinct temporal and spatial patterns of expression of the ATX1 and ATX2 genes. We found that ATX2 methylates only a limited fraction of nucleosomes and that ATX1 and ATX2 influence the expression of largely nonoverlapping gene sets. Even when coregulating shared targets, ATX1 and ATX2 may employ different mechanisms. Most remarkable is the divergence of their biochemical activities: both proteins methylate K4 of histone H3, but while ATX1 trimethylates it, ATX2 dimethylates it. ATX2 and ATX1 provide an example of separated K4 di from K4 trimethyltransferase activity.     

Genetic engineering, expression, and activity of a chimeric monoclonal antibody-avidin fusion protein for receptor-mediated delivery of biotinylated drugs in humans

The genetic engineering, expression, and validation of a fusion protein of avidin (AV) and a chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR) is described. The 15 kDa avidin monomer was fused to the carboxyl terminus of the heavy chain of the HIRMAb. The fusion protein heavy chain reacted with antibodies specific for human IgG and avidin, and had the same affinity for binding to the HIR extracellular domain as the original chimeric HIRMAb. The fusion protein qualitatively bound biotinylated ligands, but was secreted fully saturated with biotin by COS cells, owing to the high level of biotin in tissue culture medium. Chinese hamster ovary (CHO) cells were permanently transfected with a tandem vector expressing the fusion protein genes, and high expressing cell lines were isolated by methotrexate amplification and dilutional cloning. The product expressed by CHO cells had high binding to the HIR, and migrated as a homogeneous species in size exclusion HPLC and native polyacrylamide gel electrophoresis. The CHO cells were adapted to a 4 week culture in biotin depleted medium, and the HIRMAb-AV fusion protein expressed under these conditions had 1 unoccupied biotin binding site per molecule, based on a [3H]-biotin ultrafiltration assay. The HIRMAb-AV increased biotin uptake by human cells >15-fold, and mediated the endocytosis of fluorescein-biotin, as demonstrated by confocal microscopy. In summary, the HIRMAb-AV fusion protein is a new drug targeting system for humans that can be adapted to monobiotinylated drugs or nucleic acids.     

Simultaneous voltammetric determination of norepinephrine, ascorbic acid and uric acid on polycalconcarboxylic acid modified glassy carbon electrode

A novel polycalconcarboxylic acid (CCA) modified glassy carbon electrode (GCE) was fabricated by electropolymerization and then successfully used to simultaneously determine ascorbic acid (AA), norepinephrine (NE) and uric acid (UA). The characterization of electrochemically synthesized Poly-CCA film was investigated by atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and voltammetric methods. It was found that the electrochemical behavior of the polymer-modified electrode depended on film thickness, i.e., the electropylmyerization time. Based on the electrochemical data, the charge transfer coefficient (alpha) and the surface coverage (Gamma) were calculated. This poly-CCA modified GCE could reduce the overpotential of ascorbic acid (AA), norepinephrine (NE) and uric acid (UA) oxidation in phosphate buffer solution (pH 6.0), while it increases the peak current significantly. The current peak separations of AA/NE, NE/UA and AA/UA on this modified electrode are 91mV, 256mV and 390mV in CV at 100mVs(-1), respectively. Therefore, the voltammetric responses of these three compounds can be well resolved on the polymer-modified electrode, and simultaneously determination of these three compounds can be achieved. In addition, this modified electrode can be successfully applied to determine AA and NE in injection and UA in urine samples without interferences.     

Expression change of β-1,4 galactosyltransferase I, V mRNAs and Galβ1,4GlcNAc group in rat sciatic nerve after crush

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of β-1,4-galactosylation is performed by a family of β-1,4-galactosyltransferases (β-1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. β-1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides. In the present study, Real-time PCR revealed that the β-1,4-GalT I and V mRNAs reached peaks at 2 w after sciatic nerve crush. In situ hybridization showed that at 1 d after sciatic nerve crush, the expression levels of β-1,4-GalT I and V mRNAs were strong at the crush site, and decreased gradually from crush site to the distal segments. In addition, combined in situ hybridization for β1,4-GalT I and V mRNAs and immunohistochemistry for S100 showed that β1,4-GalT I and V mRNAs were mainly located in Schwann cells. Lectin blot showed that the expression of Galβ1,4GlcNAc group increased at 6 h immediately, reached a peak at 12 h and remained elevated up to 4 w after sciatic nerve crush. In conclusion, β1,4-GalT I and V might play important roles in the regeneration of the injuried sciatic nerve, and upregulation of Galβ1,4GlcNAc group might be correlated with the process of the sciatic nerve injury.     

The theoretical 3D structure of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry5Ba

Cry5Ba is a δ-endotoxin produced by Bacillus thuringiensis PS86A1 NRRL B-18900. It is active against nematodes and has great potential for nematode control. Here, we predict the first theoretical model of the three-dimensional (3D) structure of a Cry5Ba toxin by homology modeling on the structure of the Cry1Aa toxin, which is specific to Lepidopteran insects. Cry5Ba resembles the previously reported Cry1Aa toxin structure in that they share a common 3D structure with three domains, but there are some distinctions, with the main differences being located in the loops of domain I. Cry5Ba exhibits a changeable extending conformation structure, and this special structure may also be involved in pore-forming and specificity determination. A fuller understanding of the 3D structure will be helpful in the design of mutagenesis experiments aimed at improving toxicity, and lead to a deep understanding of the mechanism of action of nematicidal toxins.     

Autoantibody profiling of Chinese patients with autoimmune hepatitis using immunoproteomic analysis

In the present study, immunoproteomic analysis was utilized to systemically characterize global autoantibody profiles in autoimmune hepatitis (AIH). Sera from 21 patients with AIH and 15 healthy controls were analyzed for the antibody reactivity against the protein antigens of HepG2, a human hepatoma cell line. The lysates of HepG2 cells were separated by two-dimensional electrophoresis and then immunoblotted with each serum sample. Matrix-assisted laser desorption/ionization mass spectrometry or/and nanoelectrospray ionization MS/MS were then used to identify antigens, among which a bifunctional enzyme in mitochondrial, fumarate hydratase (FH), was further analyzed by ELISA using recombinant FH as a coating antigen. A total of 18 immunoreactive spots were identified as 13 proteins, 8 of which have not been reported in AIH. Immune reactivity to FH was detected in 66.67% of patients with AIH, 19.35% of patients with primary biliary cirrhosis (PBC), 12.31% of patients with chronic hepatitis B (CHB), 6.35% of patients with chronic hepatitis C (CHC), 11.32% of patients with systemic lupus erythematosus (SLE), and 3.57% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P<0.001). These data demonstrate the serological heterogeneity in AIH and suggest the diversity of the mechanisms underlying AIH. FH, recognized mainly in AIH rather than in viral hepatitis and other autoimmune diseases, may have utility in improved diagnosis of AIH.     

Role of gap junction, hemichannels, and connexin 43 in mineralizing in response to intermittent and continuous application of parathyroid hormone

Intermittent administration stimulates bone formation, whereas sustained elevation of parathyroid hormone (PTH) as in hyperparathyroidism stimulates bone resorption. Even though PTH(1-34) is the only anabolic agent clinically approved for the treatment of osteoporosis, the molecular mechanism whereby PTH mediates these opposing effects depending on timing of administration is not well understood. In this study, we sought to determine the involvement of gap junctions and hemichannels, and the protein that forms them, connexin 43 (Cx43), in the effect of PTH(1-34) on osteoblast mineralization. The osteoblast-like cell line MLO-A5 that rapidly mineralizes in culture was used. Intermittent PTH enhances mineralization, whereas continuous PTH inhibits this process. The mineralization was significantly inhibited by 18 beta-glycyrrhetinic acid, an inhibitor known to block gap junctions and hemichannels. When the cells were treated with PTH(1-34), gap junctional coupling was increased; however, the degree of stimulation was similar between intermittent and continuous treatment. The permeabilization to dye was not detected under various intermittent or continuous PTH treatments. On the other hand, the overall level of Cx43 protein increased in response to continuous PTH treatment. In contrast, when the cells were subjected to intermittent treatment overall level of Cx43 was unchanged, but there was an increase of connexons associated with an increase in Cx43 expression on the cell surface. Our results suggest that Cx43 overall expression, connexon formation and cell surface expression are differentially regulated by intermittent and continuous PTH(1-34), implying the involvement of Cx43 and Cx43-forming channels in mediating the effects of PTH on bone formation.     

Raf activation is regulated by tyrosine 510 phosphorylation in Drosophila

The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK) signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, we demonstrate that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf), which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. We further show that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, we show that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved.