Gene Combination of CD44 rs187116, CD133 rs2240688, NF-κB1 rs28362491 and GSTM1 Deletion as a Potential Biomarker in Risk Prediction of Breast Cancer in Lower Northern Thailand.

Background: Biomarkers play an important role in oncology, including risk assessment, treatment prediction, and monitoring the progression of disease. In breast cancer, many genes are used as biomarkers. Since, several SNP variations of hallmark ­ related genes have been reported to be of value in risk prediction in various cancers and populations, some genetic polymorphism loci were combined and reported as biomarkers for use in the risk assessment of breast cancer in Thai people. Methods: Twelve cancer gene hallmarks (15 polymorphic loci) were selected and genotyped in 184 breast cancer patients and 176 healthy individuals in Phitsanulok, Thailand. Results: AA genotype of CD44 rs187116 (c.67+4883G>A), the C allele of CD133 rs2240688 (c.*667A>C), the *2 allele (4 bp deletion) of NF-κB1 rs28362491 and the homozygous null allele genotype of GSTM1 were significantly associated with an increased risk of breast cancer (p<0.05). A combination of these 4 significant loci showed that AA-AA-*1*1-homozygous null allele genotype has the greatest correlation with increased risk of breast cancer (OR = 21.00; 95% CI: 1.77 to 248.11; p = 0.015), followed by GA-AA-*2*2- homozygous null allele genotype (p = 0.037) and GG-AC-*1*2- homozygous null allele genotype (p = 0.028). Conclusion: These findings suggest that the polymorphisms of CD44 rs187116 (c.67+4883G>A), CD133 rs2240688 (c.*667A>C), NF-κB1 rs28362491 and GSTM1 homozygous null allele genotype might be associated with an increased risk of breast cancer, and this gene combination could possibly be used as biomarkers for risk prediction, which would be of benefit in planning health surveillance and cancer prevention.

A molecular dynamics study of catestatin docked on nicotinic acetylcholine receptors to identify amino acids potentially involved in the binding of chromogranin A fragments.

Catestatin, a cationic and hydrophobic 21-amino acid fragment of chromogranin A, is known to be a non-competitive nicotinic antagonist acting through nicotinic acetylcholine receptors (nAChRs) to inhibit catecholamine release. Since this receptor is the target of several neuronal and non-neuronal disorder prophylaxes and treatments, this study aims at the elucidation of the binding of human catestatin to the entire nAChR reconstructed in lipid bilayers by means of docking followed by full atomistic molecular dynamics simulations. The obtained results show that the minimum free energy for the binding of the peptide and the receptor attains minimal values for locations at the pore site and in the outer beta subunit. This result is consistent with previous studies showing that catestatin occludes the pore opening. A new finding is an additional even stronger binding seat at the beta subunit and that membrane presence could be an important factor. Specific amino acids involved in catestatin binding have been identified, indicating targets for point mutation studies. In addition to improving the understanding of the interaction between the peptide and muscle-type and even other nAChR subtypes, the results of this study provide directions for future peptidomimetic research.

Bacopa monnieri protects SH-SY5Y cells against tert-Butyl hydroperoxide-induced cell death via the ERK and PI3K pathways.

OBJECTIVE: Oxidative stress plays an important role in the pathological processes of various neurodegenerative diseases. Bacopa monnieri (BM) has a potent antioxidant property. Therefore, the purpose of this study was to evaluate the neuroprotective potential of BM against SH-SY5Y neuroblastoma cell death induced by the pro-oxidant insult, tert-Butyl hydroperoxide (TBHP), and to identify possible mechanisms related to its neuroprotective action. METHODS: The neuroprotective effect of BM was evaluated by the degree of protection against TBHP-induced cell death in human SH-SY5Y cells that was measured by calcein-AM assay. ERK1/2 and Akt phosphorylation was evaluated by immunoblotting. RESULTS: We found that BM exhibited protection against TBHP-mediated cytotoxicity. The neuroprotective effect of BM was abolished in the presence of either ERK1/2 or PI3K inhibitors. In addition, western blotting with anti-phospho-ERK1/2 and anti-phospho-Akt antibodies showed that BM increased both ERK1/2 and Akt phosphorylation. CONCLUSION: These results suggest that BM by activation of ERK/MAPK and PI3K/Akt signaling pathways protects SH-SY5Y cells from TBHP-induced cell death.

A proteome reference map of the causative agent of melioidosis Burkholderia pseudomallei.

Burkholderia pseudomallei is the etiologic agent of melioidosis. Using 2DE and MALDI-TOF MS, we report here a proteome reference map constructed from early stationary phase, a bacterial adaptation process. We identified 282 protein spots representing 220 ORFs; many of them have been implicated in bacterial pathogenesis. Up to 20% of identified ORFs belong to post-translational modification and stress responses. The proteome reference map will support future analysis of the bacterial gene and environmental regulation and facilitate comparative proteomics with its sibling species.

Potential of an injectable chitosan/starch/beta-glycerol phosphate hydrogel for sustaining normal chondrocyte function.

An injectable hydrogel for chondrocyte delivery was developed by blending chitosan and starch derived from various sources with beta-glycerol phosphate (beta-GP) in the expectation that it would retain a liquid state at room temperature and gel at raised temperatures. Rheological investigation indicated that the system consisting of chitosan derived from crab shell and corn starch at 4:1 by weight ratio (1.53%, w/v of total polymers), and 6.0% (w/v) beta-GP (C/S/GP system) exhibited the sharpest sol-gel transition at 37+/-2 degrees C. The C/S/GP hydrogel was gradually degraded by 67% within 56 days in PBS containing 0.02 mg/ml lysozyme. The presence of starch in the system increased the water absorption of the hydrogel when compared to the system without starch. SEM observation revealed to the interior structure of the C/S/GP hydrogel having interconnected pore structure (average pore size 26.4 microm) whereas the pore size of the hydrogel without starch was 19.8 microm. The hydrogel also showed an ability to maintain chondrocyte phenotype as shown by cell morphology and expression of type II collagen mRNA and protein. In vivo study revealed that the gel was formed rapidly and localized at the injection site.

Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones.

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.

Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter.

Multistep proteolytic mechanisms are essential for converting proprotein precursors into active peptide neurotransmitters and hormones. Cysteine proteases have been implicated in the processing of proenkephalin and other neuropeptide precursors. Although the papain family of cysteine proteases has been considered the primary proteases of the lysosomal degradation pathway, more recent studies indicate that functions of these enzymes are linked to specific biological processes. However, few protein substrates have been described for members of this family. We show here that secretory vesicle cathepsin L is the responsible cysteine protease of chromaffin granules for converting proenkephalin to the active enkephalin peptide neurotransmitter. The cysteine protease activity was identified as cathepsin L by affinity labeling with an activity-based probe for cysteine proteases followed by mass spectrometry for peptide sequencing. Production of [Met]enkephalin by cathepsin L occurred by proteolytic processing at dibasic and monobasic prohormone-processing sites. Cellular studies showed the colocalization of cathepsin L with [Met]enkephalin in secretory vesicles of neuroendocrine chromaffin cells by immunofluorescent confocal and immunoelectron microscopy. Functional localization of cathepsin L to the regulated secretory pathway was demonstrated by its cosecretion with [Met]enkephalin. Finally, in cathepsin L gene knockout mice, [Met]enkephalin levels in brain were reduced significantly; this occurred with an increase in the relative amounts of enkephalin precursor. These findings indicate a previously uncharacterized biological role for secretory vesicle cathepsin L in the production of [Met]enkephalin, an endogenous peptide neurotransmitter.

Primary sequence characterization of catestatin intermediates and peptides defines proteolytic cleavage sites utilized for converting chromogranin a into active catestatin secreted from neuroendocrine chromaffin cells.

Catestatin is an active 21-residue peptide derived from the chromogranin A (CgA) precursor, and catestatin is secreted from neuroendocrine chromaffin cells as an autocrine regulator of nicotine-stimulated catecholamine release. The goal of this study was to characterize the primary sequences of high molecular mass catestatin intermediates and peptides to define the proteolytic cleavage sites within CgA that are utilized in the biosynthesis of catestatin. Catestatin-containing polypeptides, demonstrated by anti-catestatin western blots, of 54-56, 50, 32, and 17 kDa contained NH(2)-terminal peptide sequences that indicated proteolytic cleavages of the CgA precursor at KK downward arrow, KR downward arrow, R downward arrow, and KR downward arrow basic residue sites, respectively. The COOH termini of these catestatin intermediates were defined by the presence of the COOH-terminal tryptic peptide of the CgA precursor, corresponding to residues 421-430, which was identified by MALDI-TOF mass spectrometry. Results also demonstrated the presence of 54-56 and 50 kDa catestatin intermediates that contain the NH(2) terminus of CgA. Secretion of catestatin intermediates from chromaffin cells was accompanied by the cosecretion of catestatin (CgA(344)(-)(364)) and variant peptide forms (CgA(343)(-)(368) and CgA(332)(-)(361)). These determined cleavage sites predicted that production of high molecular mass catestatin intermediates requires cleavage at the COOH-terminal sides of paired basic residues, which is compatible with the cleavage specificities of PC1 and PC2 prohormone convertases. However, it is notable that production of catestatin itself (CgA(344)(-)(364)) utilizes more unusual cleavage sites at the NH(2)-terminal sides of downward arrow R and downward arrow RR basic residue sites, consistent with the cleavage specificities of the chromaffin granule cysteine protease "PTP" that participates in proenkephalin processing. These findings demonstrate that production of catestatin involves cleavage of CgA at paired basic and monobasic residues, necessary steps for catestatin peptide regulation of nicotinic cholinergic-induced catecholamine release.

Novel chromaffin granule serpins, endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities.

Endopin 1 and endopin 2 represent two novel serpin protease inhibitors localized within chromaffin granules, secretory vesicles of adrenomedullary chromaffin cells that represent a model neuroendocrine cell for synthesis and secretion of peptide neurotransmitters. This chapter describes the molecular features of the primary sequences of endopin 1 and endopin 2 that provided prediction of their distinct target protease specificities. Endopin 1 inhibits trypsin that cleaves at basic residues. In contrast, endopin 2 possesses cross-class inhibition of papain and elastase that represent cysteine and serine proteases, respectively. Cell biological studies indicate that endopin 1 and endopin 2 are localized within chromaffin granules. These results implicate endopin 1 inhibition in vivo of trypsin-like proteases in secretory vesicles, and endopin 2 inhibition of papain- or elastase-like proteases. Indeed, endopin 2 inhibits the endogenous cysteine protease PTP (prohormone thiol protease), present in chromaffin granules, that participates in the proteolytic processing of proenkephalin. These findings indicate the presence of endogenous endopin 1 and endopin 2 in secretory vesicle function.

The local chromaffin cell plasminogen/plasmin system and the regulation of catecholamine secretion.

Chromaffin cells express components of the plasminogen/plasmin system, including its major activator, tissue plasminogen activator (t-PA), and high-affinity cellular receptors for plasminogen, which promote local concentration and activation of plasminogen at the cell surface. Our studies suggest that plasmin participates in local neuroendocrine prohormone processing and that perturbation of this system profoundly affects the secretory characteristics of the cells. These results suggest the presence of a local, functionally active, chromaffin cell plasminogen/plasmin system that plays a major role in the regulation of catecholamine release from catecholaminergic cells.

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles.

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.