Development and evaluation of a protein microarray chip for diagnosis of hepatitis C virus

A protein chip diagnostic kit was developed for the diagnosis of hepatitis C virus (HCV) based on the protein chip technique and the immuno-concentration method. This kit was designed for low-density protein chips and also for the availability of multiple sample screening. Applicability of the chip was evaluated using 96 blood specimens and the results were compared to results of an anti-HCV enzyme immunoassay (EIA) test. With further development, the technology associated with the development of this chip could be applied to the simultaneous detection of multiple protein-protein, protein-ligand interactions.     

In vitro Activation of Protein Kinase C by β-N-oxalyl-l-α, β-diaminopropionic acid,the Lathyrus sativus Neurotoxin

-N-oxalyl-l-,-diaminopropionic acid (l-ODAP) toxicity has been associated with lathyrism; a spastic paraparesis caused by excessive dietary intake of the pulse Lathyrus sativus. We investigated the effect of Lathyrus neurotoxin l-ODAP on protein kinase C (PKC) activity under in vitro conditions. l-ODAP activated phosphorylation activity of purified chick brain PKC. Both lysine-rich (histone III-S) and arginine-rich (protamine sulfate) substrate phosphorylation was enhanced in the presence of l-ODAP. The activation is concentration dependent, and maximal activation is observed at 100 M concentration. Protamine sulfate phosphorylation was enhanced by 47%, whereas histone III-S phosphorylation was enhanced by 50% over PS/PDBu/Ca²⁺ dependent activity. The nontoxic d-isomer (d-ODAP) did not affect both histone III-S and protamine sulfate phosphorylation activity. These results indicate that l-ODAP taken up by neuronal cells could also contribute to PKC activation and so be associated with toxicity.     

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.     

Proteomic identification and characterization of secreted N‐glycosylated NPC2 following cross‐linking of the high‐affinity receptor for IgE on mast cells

Allergen‐mediated cross‐linking of the high‐affinity receptor for IgE on mast cells triggers the release of diverse preformed and de novo synthesized immunoregulatory mediators that further the allergic response. A proteomic screen applied to the detection of proteins secreted by the model rat mast cell line, RBL‐2H3 (rat basophilic leukaemia, subline 2H3.1), led to the identification of the cholesterol‐binding glycoprotein, NPC2/RE1 (Niemann–Pick Type C2/epididymal secretory protein 1). Glycosylated NPC2 is secreted early in response to an IgE‐mediated stimulus and co‐localizes with the lysosomal membrane marker, CD63. NPC2 belongs to the ML (MD‐2‐related lipid‐recognition) protein family (155 members), which includes the Toll‐like receptor co‐factors, MD‐1 and MD‐2, and perhaps most interestingly, seven major house dust mite allergens of unknown function (including Der p 2 and Der f 2). Possible role(s) for the protein in the allergic response and future applications of this approach are discussed.     

REPORT OF A NEW INVASIVE ALGA IN THE ATLANTIC UNITED STATES: “HETEROSIPHONIA” JAPONICA IN RHODE ISLAND1

Recent collections of tetrasporangiate “Heterosiphonia” japonica Yendo from Watch Hill to Point Judith, Rhode Island, represent the first report of this nonnative alga in the western Atlantic. Native to the Pacific Ocean, this species was unintentionally introduced into European waters by 1984 and has subsequently invaded the eastern Atlantic Ocean widely from France to Norway and south into the Mediterranean Sea. Thus far, all western Atlantic collections of this species are confined to the outer coast of Rhode Island, and at present are not found in Narragansett Bay or in Long Island Sound along the Connecticut coast. Molecular and morphological studies confirm the identity of this newly introduced invasive species.     

The antimicrobial peptide, tilapia hepcidin 2-3, and PMA differentially regulate the protein kinase C isoforms, TNF-α and COX-2, in mouse RAW264.7 macrophages

The antimicrobial and immunomodulatory functions of the antimicrobial peptide, tilapia hepcidin (TH)2-3, were previously studied. Herein, we report the differential modulation of protein kinase C (PKC)-associated proteins by TH2-3, and the PKC activator, phorbol 12-myristate 13-acetate (PMA), in RAW264.7 macrophages. Treatment with TH2-3 at 40 or 80 μg/ml did not affect the cell morphology, but TH2-3 at 120 μg/ml produced morphological changes similar to those after treatment with PMA in RAW264.7 cells. The coexistence of the PKC inhibitor, Ro-31-8220, prevented morphological changes induced by either PMA or 120 μg/ml TH2-3 in RAW264.7 cells. Since PMA is known to induce expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, activation of the TNF-α promoter in response to TH2-3 and PMA treatments in lipopolysaccharide (LPS)-stimulated cells was compared. In LPS-stimulated RAW264.7 cells, TNF-α promoter activity was significantly suppressed by TH2-3, but not by PMA. In addition, PMA activated prostaglandin synthase-associated cyclooxygenase (COX)-2 proteins on the cell surface, while the presence of TH2-3 inhibited its expression. Western blotting demonstrated that the expressions of PKC-μ, phosphorylated (p)-PKCμ at serine (S)-744, and p-PKCδ were activated by PMA, but were suppressed by TH2-3. In addition, p-PKC at S-916 was activated by TH2-3 and inhibited by PMA. In conclusion, the differential regulation of PKC isoforms by PMA and TH2-3 may influence morphological changes and regulation of TNF-α in RAW264.7 cells.     

Tricin‐lignins: occurrence and quantitation of tricin in relation to phylogeny

Tricin [5,7‐dihydroxy‐2‐(4‐hydroxy‐3,5‐dimethoxyphenyl)‐4H‐chromen‐4‐one], a flavone, was recently established as an authentic monomer in grass lignification that likely functions as a nucleation site. It is linked onto lignin as an aryl alkyl ether by radical coupling with monolignols or their acylated analogs. However, the level of tricin that incorporates into lignin remains unclear. Herein, three lignin characterization methods: acidolysis; thioacidolysis; and derivatization followed by reductive cleavage; were applied to quantitatively assess the amount of lignin‐integrated tricin. Their efficiencies at cleaving the tricin‐(4′–O–β)‐ether bonds and the degradation of tricin under the corresponding reaction conditions were evaluated. A hexadeuterated tricin analog was synthesized as an internal standard for accurate quantitation purposes. Thioacidolysis proved to be the most efficient method, liberating more than 91% of the tricin with little degradation. A survey of different seed‐plant species for the occurrence and content of tricin showed that it is widely distributed in the lignin from species in the family Poaceae (order Poales). Tricin occurs at low levels in some commelinid monocotyledon families outside the Poaceae, such as the Arecaceae (the palms, order Arecales) and Bromeliaceae (Poales), and the non‐commelinid monocotyledon family Orchidaceae (Orchidales). One eudicotyledon was found to have tricin (Medicago sativa, Fabaceae). The content of lignin‐integrated tricin is much higher than the extractable tricin level in all cases. Lignins, including waste lignin streams from biomass processing, could therefore provide a large and alternative source of this valuable flavone, reducing the costs, and encouraging studies into its application beyond its current roles.     

Dynamics of Aboveground and Soil Carbon and Nitrogen Stocks and Cycling of Available Nitrogen along a Land-use Gradient in Rondônia,Brazil

High rates of deforestation in the Brazilian Amazon have the potential to alter the storage and cycling of carbon (C) and nitrogen (N) across this region. To investigate the impacts of deforestation, we quantified total aboveground biomass (TAGB), aboveground and soil pools of C and N, and soil N availability along a land-use gradient in Rondônia, Brazil, that included standing primary forest, slashed primary and secondary forest, shifting cultivation, and pasture sites. TAGB decreased substantially with increasing land use, ranging from 311 and 399 Mg ha^–1 (primary forests) to 63 Mg ha^–1 (pasture). Aboveground C and N pools declined in patterns and magnitudes similar to those of TAGB. Unlike aboveground pools, soil C and N concentrations and pools did not show consistent declines in response to land use. Instead, C and N concentrations were strongly related to percent clay content of soils. Concentrations of NO₃-N and NH₄-N generally increased in soils following slash-and-burn events along the land-use gradient and decreased with increasing land use. Increasing land use resulted in marked declines in NO₃-N pools relative to NH₄-N pools. Rates of net nitrification and N-mineralization were also generally higher in postfire treatments relative to prefire treatments along the land-use gradient and declined with increasing land use. Results demonstrate the linked responses of aboveground C and N pools and soil N availability to land use in the Brazilian Amazon; steady reductions in aboveground pools along the land-use gradient were accompanied by declines in inorganic soil N pools and transformation rates.     

Employment of 4‐(1,2,4‐triazol‐1‐yl)phenol as a signal enhancer of the chemiluminescent luminol–H2O2–horseradish peroxidase reaction for detection of hepatitis C virus in real samples

Highly sensitive detection of hepatitis C virus (HCV) in serum is a key method for diagnosing and classifying the extent of HCV infection. In this study, a p‐phenol derivative, 4‐(1,2,4‐triazol‐1‐yl)phenol (4‐TRP), was employed as an efficient enhancer of the luminol–hydrogen peroxide (H₂O₂)–horseradish peroxidase (HRP) chemiluminescence (CL) system for detection of HCV. Compared with a traditional enhancer, 4‐TRP strongly enhanced CL intensity with the effect of prolonging and stabilizing light emission. The developed CL system was applied to detecting HCV core antigen (HCV‐cAg) using a sandwich structure inside microwells. Our experimental results showed that there was good linear relationship between CL intensity and HCV‐cAg concentration in the 0.6–3.6 pg/mL range (R = 0.99). The intra‐ and inter‐assay coefficients of variation were 4.5–5.8% and 5.0–7.3%, respectively. In addition, sensitive determination of HCV‐cAg in serum samples using the luminol–H₂O₂–HRP–4‐TRP CL system was also feasible in clinical settings. Copyright © 2015 John Wiley & Sons, Ltd.     

Circulating miR‐145 is associated with plasma high‐sensitivity C‐reactive protein in acute ischemic stroke patients

Stroke is a major cerebrovascular disease threatening human health and life with high morbidity, disability and mortality. We aimed to find effective biomarkers for the early diagnosis on stroke. Nine previously reported stroke‐associated miRNAs (miR‐21, miR‐23a, miR‐29b, miR‐124, miR‐145, miR‐210, miR‐221, miR‐223 and miR‐483‐5p) were measured by quantitative real time‐PCR, and plasma high‐sensitivity C‐reactive protein (hs‐CRP) and serum interleukin 6 (IL‐6), the pro‐inflammation markers in brain injury, were examined by enzyme‐linked immunosorbent assay in 146 acute ischemic stroke patients and 96 healthy blood donors. We found that serum miR‐145 was significantly increased within 24 h after stroke onset and serum miR‐23a and miR‐221 were decreased in patients. Moreover, serum miR‐145 was strong positively correlated with plasma hs‐CRP and moderate positively correlated with serum IL‐6. Meanwhile, serum miR‐23a and miR‐221 were moderate negatively correlated with plasma hs‐CRP but not serum IL‐6. Importantly, the combination of hs‐CRP and serum miR‐145 gained a better sensitivity/spectivity for prediction of acute ischemia stroke (area under receiver operating characteristic curve from 0.794 to 0.896). Conclusively, our preliminary findings indicate that serum miR‐145 upregulated in acute ischemic stroke might be a new biomarker for acute ischemia stroke evaluation. Copyright © 2015 John Wiley & Sons, Ltd.