Proteomics identification of overexpressed serum proteins in dogs with Babesiacanis infection.

Background and Aim: Canine babesiosis, caused by the protozoan parasite Babesia canis, is characterized by clinical manifestations, including hemolytic anemia, thrombocytopenia, multiple organ failure, and may result in death. This disease is detected using conventional blood smears, which are time-consuming and have low sensitivity. This study aimed to investigate a more rapid and sensitive method for detecting B. canis infection in dogs by examining the expressed serum protein profiles using proteomics. Materials and Methods: We collected six sera samples from three healthy and three B. canis-infected dogs diagnosed using blood smear and polymerase chain reaction. We analyzed the proteins using two-dimensional gel electrophoresis. The candidate spots from the gel were subjected to protein identification using a nano-liquid chromatography system coupled to an ion-trap mass spectrometer equipped with an electrospray ionization nano-sprayer. Results: We found that 10 protein spots were overexpressed in the serum samples from infected dogs compared with healthy dogs, which corresponded to three proteins: serotransferrin, serotransferrin isoforms X1, and hemopexin. Furthermore, analysis of the protein-protein interaction network confirmed that they strongly interacted with each other. Conclusion: This study suggests that high levels of serotransferrin and hemopexin are related to B. canis infection, making these proteins potential candidates for the development of diagnostic molecules or vaccines.

Peptidomic Analysis and Antimicrobial Activity of Serum Peptide from Hevea brasiliensis Clone BPM24.

BACKGROUND: Hevea brasiliensis is severely affected by the fungal disease caused by Phytophthora spp. Significant loss of rubber yield is widespread and extensive use of chemical fungicides has resulted in health and environmental problems. OBJECTIVE: This work aims to extract and identify the latex serum peptides from a disease tolerant clone of H. brasiliensis, and study the inhibitory efficacy against pathogenic bacteria and fungi. METHODS: Serum peptides were extracted from H. brasiliensis BPM24 using mixed lysis solution. Low molecular weight peptides were screened and fractionated by solid-phase extraction and then identified by tandem mass spectrometry. Total and fractionated serum peptides were assayed for bacterial and fungal inhibition using broth microdilution and poisoned food methods. An inhibitory control study in the greenhouse was also performed using susceptible clones for pre and postinfection with Phytophthora spp. RESULTS: Forty-three serum peptide sequences were successfully identified. Thirty-four peptides matched with the proteins associated with plant defense response signaling, host resistance, and adverse environmental factors. The inhibitory study of total serum peptides demonstrated antibacterial and anti-fungal properties. The greenhouse study exhibited disease inhibitory efficacy of 60% for the treatment of Phytophthora spp. in post-infected plants and 80% for pre-treated samples. CONCLUSION: Latex serum peptides from disease tolerant H. brasiliensis revealed several proteins and peptides associated with plant defense and disease resistance. The peptides play a vital role for defense against bacteria and fungi pathogens, including Phytophthora spp. Enhanced disease protection can be obtained when the extracted peptides were applied to the susceptible plants before exposure to the fungi. These findings provided an insight and may pave the way for the development of biocontrol peptides from natural resources.

Analysis and comparison of tear protein profiles in dogs using different tear collection methods.

BACKGROUND: Tear proteomic analysis has become an important tool in medical and veterinary research. The tear collection method could influence the tear protein profile. This study aims to evaluate the protein profiles of dog tears collected using microcapillary tubes (MT), Schirmer tear strips (ST), and ophthalmic sponges (OS). METHODS: The tear samples were collected using MT, ST, and OS. Tear protein profiles were analyzed using two-dimensional electrophoresis (2-DE) and the different protein spots' expression was compared. Fourteen protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Tear protein concentrations ranged from 2.80 to 4.03 µg/µL, with no statistically significant differences among collection methods. Protein expression in each collection method differed in terms of both the number and intensity of the spots. There were 249, 327, and 330 protein spots found from tears collected with MT, ST, and OS, respectively. The proteins albumin, haptoglobin, and lactoferrin identified from OS were found to have higher spot intensities than other methods of collection. The use of MT demonstrated the downregulation of nine proteins. CONCLUSIONS: The recent study supported that tear protein analysis is affected by different tear collection methods. Although ST is commonly used for tear collection, it provides insufficient information to study particular tear proteins.

Changes in tear protein profile in dogs with keratoconjunctivitis sicca following topical treatment using cyclosporine A.

BACKGROUND AND AIM: Keratoconjunctivitis sicca (KCS) is a chronic inflammatory ocular disease that occurs in many dog breeds worldwide. This study aimed to investigate the tear protein pattern of healthy dogs, KCS dogs, and KCS dogs after treatment with cyclosporine A (CsA). MATERIALS AND METHODS: Twenty-eight dogs of any breed were enrolled in the study. The subjects were divided into three groups: Healthy, KCS, and CsA-treated dogs. Tear samples were collected using Schirmer strips. Tear proteins extracted from the strips were analyzed using two-dimensional electrophoresis. For the first dimension, total protein from tears was separated by isoelectric focusing. The second dimension was performed using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel images were analyzed and the protein spots of differential expression were manually cut for protein annotation using mass spectrometry. RESULTS: In total, 12 protein spots were excised and subjected to protein identification. Associated with KCS, six protein spots were a downregulated protein, namely, lysozyme. The other six protein spots were upregulated in KCS dogs, consisting of heat shock protein beta-1, protein S100-A12, and keratin type II cytoskeletal 1 and 5. After treatment with CsA for 45 days, the lysozyme protein was still decreasing and the inflammation protein (S100-A12) was not identified. CONCLUSION: Inflammatory tear proteins and proteins involved in cellular stress were present in KCS dogs and appeared to be reduced in medicated eyes. Treatment with topical CsA in the short term may not improve the activity of antibacterial proteins. Changes in the expression patterns of these four proteins might be useful for disease severity and progression assessment, as well as for exploring a novel method for dry eye management in dogs.

Caspase-3, a shrimp phosphorylated hemocytic protein is necessary to control YHV infection.

By using immunohistochemistry detection, yellow head virus (YHV) was found to replicate in granule-containing hemocytes including semi-granular hemocytes (SGC) and granular hemocytes (GC) during the early phase (24 h post injection) of YHV-infected shrimp. Higher signal of YHV infection was found in GC more than in SGC. Comparative phosphoproteomic profiles between YHV-infected and non-infected GC reveal a number of phosphoproteins with different expression levels. The phosphoprotein spot with later on identified as caspase-3 in YHV-infected GC is most interesting. Blocking caspase-3 function using a specific inhibitor (Ac-DEVD-CMK) demonstrated high replication of YHV and consequently, high shrimp mortality. The immunohistochemistry results confirmed the high viral load in shrimp that caspase-3 activity was blocked. Caspase-3 is regulated through a variety of posttranslational modifications, including phosphorylation. Analysis of phosphorylation sites of shrimp caspase-3 revealed phosphorylation sites at serine residue. Taken together, caspase-3 is a hemocytic protein isolated from shrimp granular hemocytes with a role in anti-YHV response and regulated through the phosphorylation process.

Proteomic analysis and white spot syndrome virus interaction of mud crab (Scylla olivacea) revealed responsive roles of the hemocytes.

White spot disease (WSD) is a highly virulent viral disease in shrimps. Clinical signs and high mortality of WSD is generally observed after a few days of infection by White Spot Syndrome virus (WSSV). Mud crabs are the major carrier and persistent host for the WSSV. However, an elucidation of viral interaction and persistent mode of WSSV infection in mud crab is still limited. We investigated the defensive role of mud crab (Scylla olivacea) hemocytes against WSSV infection by using comparative proteomic analysis coupled with electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS). The proteomic maps of expressed proteins obtained from WSSV infected hemocytes revealed differential proteins related to various biological functions, including immune response, anti-apoptosis, endocytosis, phosphorylation signaling, stress response, oxygen transport, molting, metabolism, and biosynthesis. Four distinctive cell types of crab hemocytes: hyaline cells (HC), small granular cells (SGC), large granular cells (LGC) and mixed granular cells (MGC) were found susceptible to WSSV. However, immunohistochemistry analysis demonstrated a complete replication of WSSV only in SGC and LGC. WSSV induced apoptosis was also observed in HC, SGC and MGC except for LGC. These results suggested that HC and MGC may undergo apoptosis prior to a complete assembly of virion, while SGC is more susceptible showing higher amplification and releasing of virion. In contrast, WSSV may inhibit apoptosis in infected LGC to stay in latency. This present finding provides an insight for the responsive roles of crustacean hemocyte cells involved in molecular interaction and defense mechanism against WSSV.

Expression and Extracellular Secretion of Endo-glucanase and Xylanase by Zymomonas mobilis.

Recombinant Zymomonas mobilis (pGEX-4T-3 BI 120-2) was constructed to encode endo-glucanase (CelA) and endo-xylanase (Xyn11) from Z. mobilis ZM4 (ATCC 31821) and an uncultured bacterium. The recombinant was genetically engineered with the N-terminus of a predicted SecB-dependent (type II) secretion signal from phoC of Z. mobilis to translocate the enzymes extracellularly. Both the enzymes were characterized regarding their functional optimum pH and temperature, with the highest multi-enzyme activities at pH 6.0 and a temperature of 30 °C, which approximates the optimum conditions for ethanol production by Z. mobilis. The crude intracellular and extracellular fractions of the recombinant were characterized in terms of substrate specificity using carboxymethyl cellulose (CMC), beechwood xylan, filter paper, Avicel, and pretreated rice straw. The crude extracellular and intracellular enzymes with cellulolytic and xylanolytic activities were more robustly produced and secreted from the recombinant strain compared to the wild-type and ampicillin-sensitive strains, using CMC and beechwood xylan as the substrates. Ethanol production by the recombinant strain was greater than the production by the wild-type strain when pretreated rice straw was used as a sole carbon source.

Yellow head virus infection in black tiger shrimp reveals specific interaction with granule-containing hemocytes and crustinPm1 as a responsive protein.

Yellow head virus (YHV) causes acute infections and mass mortality in black tiger shrimp culture. Our study aims to investigate molecular interaction between YHV and circulating hemocytes of Penaeus monodon at early infection. Total shrimp hemocytes were isolated by Percoll gradient centrifugation and identified by flow cytometric analysis. At least three types of hemocyte cells were identified as hyaline, semi-granular, and granular hemocytes. Experimental infection of YHV in shrimp culture demonstrated drastic changes in total and each hemocyte cell counts. Immunohistochemistry analysis demonstrated interaction and replication of YHV mainly with the granule-containing hemocytes and little to none in hyaline cell. These granule-containing hemocytes are proposed to be YHV targets providing the first line of defense to viral infection. Protein expression profiling of granule-containing hemocytes revealed several immune-responsive proteins including antimicrobial protein crustins (crustinPm1 and crustinPm4), alpha-2-macroglobulin, and kazal-type serine proteinase inhibitor. During an early phase of YHV infection at 6 hpi crustinPm1 illustrated a significant increase of mRNA and protein expression level in plasma. The results suggest that an antimicrobial crustinPm1 may participate in shrimp defense mechanism against YHV, especially on the granule-containing hemocytes.

Comparative proteome analysis of rubber latex serum from pathogenic fungi tolerant and susceptible rubber tree (Hevea brasiliensis).

Many cultivated rubber trees (Hevea brasiliensis) are invaded by various Phytophthora species fungi, especially in tropical regions which result in crop yield losses. Comparative proteome analysis coupled with liquid chromatography electrospray/ionization (LC-ESI) mass spectrometry identification was employed to investigate the relative abundance of defense related proteins in Phytophthora sp. susceptible (RRIM600) and tolerant (BPM24) clones of rubber tree. Proteome maps of non-rubber constituent of these two model clones show similar protein counts, although some proteins show significant alterations in their abundance. Most of the differentially abundant proteins found in the serum of BPM24 illustrate the accumulation of defense related proteins that participate in plant defense mechanisms such as beta-1,3-glucanase, chitinase, and lectin. SDS-PAGE and 2-D Western blot analysis showed greater level of accumulation of beta-1,3-glucanase and chitinase in latex serum of BPM24 when compared to RRIM600. A functional study of these two enzymes showed that BPM24 serum had greater beta-1,3-glucanase and chitinase activities than that of RRIM600. These up-regulated proteins are constitutively expressed and would serve to protect the rubber tree BPM24 from any fungal invader. The information obtained from this work is valuable for understanding of defense mechanisms and plantation improvement of H. brasiliensis. BIOLOGICAL SIGNIFICANCE: Non-rubber constituents (latex serum) have almost no value and are treated as waste in the rubber agricultural industry. However, the serum of natural rubber latex contains biochemical substances. The comparative proteomics analysis of latex serum between tolerant and susceptible clones reveals that the tolerant BPM24 clone contained a high abundance of several classes of fungal pathogen-responsive proteins, such as glucanase and chitinase. Moreover, other proteins identified highlighted the accumulation of defensive-associated proteins participating in plant fungal immunity. The isolation of beta-1,3-glucanase, chitinase, and lectin from latex serum should be further investigated and may provide a therapeutic application. This investigation will lead to possible use of latex serum as a great biotechnological resource due to the large quantity of serum produced and the biochemicals contained therein.

Yellow head virus binding to cell surface proteins from Penaeus monodon hemocytes.

Our previous data revealed that viral particles of yellow head virus (YHV) specifically interacted with granule-containing hemocytes. After isolation of targeted hemocytes, biotinylation was performed using Biotin-NSH-LC. Biotinylated protein was extracted and separated by 2-D PAGE. Electro-transferred proteins on a nitrocellulose membrane were probed with streptavidin-HRP complex to detect biotinylated proteins. The data from 2-D PAGE combined with affinity pull down purification revealed 8 and 6 biotinylated proteins specific to hyaline and granule containing hemocytes, respectively. Four proteins were found in common for both two hemocytes. The majority of proteins detected in granular hemocytes are membrane-associated proteins and immune-related proteins such as alpha-2-macroglobulin (A2M), kazal-type serine protease inhibitor (SPI) and crustin. CrustinPm1 was found to bind to YHV as shown with biotinylation pull-down assay and confirmed with two-dimensional virus overlay protein binding assay (2-D VOPBA). The expression of crustinPm1 was observed in semigranular and granular hemocytes whereas very low or no expression occurred in hyaline hemocytes. CrustinPm1 appears to either be directly involved in cellular binding or mediating virus internalization into permissive hemocytes.

Phosphorylation is required for myosin regulatory light chain (PmMRLC) to control yellow head virus infection in shrimp hemocytes.

The cellular signal-transduction process is largely controlled by protein phosphorylation. Shrimp infected with yellow head virus show dramatic changes in their hemocyte phosphoproteomic patterns, and aberrant activation of phosphorylation-based signaling networks has been implicated in a number of diseases. In this study, we focused on phosphorylation of Penaeus monodon myosin regulatory light chain (PmMRLC) that is induced at an early hour post YHV infection and is concomitant with cellular actin remodeling. In shrimp cell cultures, this phosphorylation was inhibited by the myosin light chain kinase (MLCK) inhibitors ML-7 and ML-9, suggesting that PmMLC phosphorylation is MLCK pathway-dependent. Blocking PmMRLC phosphorylation resulted in increased replication of YHV and reduction of phagocytic activities of shrimp hemocytes called semigranular cells (SGC) and granular cells (GC). Injection of MLCK inhibitors prior to YHV challenge resulted in dose-dependent elevation in quantity of YHV-positive GC and cytoplasmic YHV protein, coincident with high shrimp mortality. Altogether, we demonstrated that PmMRLC phosphorylation increases after YHV infection in shrimp and that inhibition of the phosphorylation leads to increased YHV replication, reduced hemocyte phagocytic activity (probably through actin remodeling) and subsequent shrimp death. Thus, further studies on the MLCK activation pathway may lead to new strategies in development and implementation of therapy for YHV infections in shrimp.

Are protease inhibitors required for gel-based proteomics of kidney and urine?

Proteolysis is one of the major problems in collection and storage of biological samples for proteome analysis, particularly when the samples undergo freeze-thaw cycles. The use of protease inhibitors for prevention of such proteolysis in some samples is debated because protease inhibitors may interfere with proteome analysis and whether protease inhibitors are useful for renal and urinary proteomics remains unclear. We therefore performed a systematic evaluation of the use of protease inhibitors in gel-based renal and urinary proteomics. Renal proteins were extracted from porcine kidney tissue and stored at -30 or -70 degrees C without protease inhibitors. After 0, 2, 4, 6, 8, 10, and 12 freeze-thaw cycles, the 2-D proteome profile was examined. Differential spot analysis and ANOVA with Tukey posthoc multiple comparisons revealed significantly quantitative changes in intensity levels of 12 and 7 renal proteins that were stored at -30 and -70 degrees C, respectively, after >or=4 freeze-thaw cycles. Additionally, there were qualitative changes (vertical elongation or streak) in 6 and 1 renal proteins that were stored at -30 and -70 degrees C, respectively. All these changes could be successfully prevented by the addition of 1% (v/v) protease inhibitors cocktail prior to storage. In contrast, neither quantitative nor qualitative changes were observed in urine samples that were stored without protease inhibitors and processed as for kidney samples. From these data, the addition of protease inhibitors is highly recommended for gel-based renal proteomics, but no longer recommended for gel-based urinary proteomics.

C-terminal hemocyanin from hemocytes of Penaeus vannamei interacts with ERK1/2 and undergoes serine phosphorylation.

To understand molecular immune response of Penaeus vannamei during Taura syndrome virus (TSV) infection, expression and functional proteomics studies were performed on hemocyanin, which is a major abundant protein in shrimp hemocytes. Two-dimensional electrophoresis (2-DE) revealed up-regulation of several C-terminal fragments of hemocyanin, whereas the N-terminal fragments were down-regulated during TSV infection. 2-D Western blot analysis showed that the C-terminal hemocyanin fragments had more acidic isoelectric points (pI), whereas the N-terminal fragments had less acidic pI. Further analysis by NetPhos showed a greater number of serine phosphorylation sites in the C-terminal hemocyanin. Additionally, motif scan using Scansite revealed ERK D-domain, which is required for activation of ERK1/2 effector kinase, as a kinase-binding site at the 527th valine in the C-terminal hemocyanin, whereas neither motif nor functional domain was found in the N-terminus. Co-immunoprecipitation confirmed the interaction between the C-terminal hemocyanin and ERK1/2. 1-D Western blot analysis showed that ERK1/2 was also up-regulated during TSV infection. Our findings demonstrate for the first time that ERK1/2 signaling pathway may play an important role in molecular immune response of P. vannamei upon TSV infection through its interaction with the C-terminal hemocyanin.

Proteomic analysis of altered proteins in lymphoid organ of yellow head virus infected Penaeus monodon.

A comparative proteomic analysis was employed to identify altered proteins in the yellow head virus (YHV) infected lymphoid organ (LO) of Penaeus monodon. At 24 h post-infection, the infected shrimps showed obvious signs of infection, while the control shrimps remained healthy. Two-dimensional electrophoresis of proteins extracted from the LO revealed significant alterations in abundance of several proteins in the infected group. Protein identification by MALDI-TOF MS and nanoLC-ESI-MS/MS revealed significant increase of transglutaminase, protein disulfide isomerase, ATP synthase beta subunit, V-ATPase subunit A, and hemocyanin fragments. A significant decrease was also identified for Rab GDP-dissociation inhibitor, 6-phosphogluconate dehydrogenase, actin, fast tropomyosin isoform, and hemolymph clottable protein. Some of these altered proteins were further investigated at the mRNA level using real-time RT-PCR, which confirmed the proteomic data. Identification of these altered proteins in the YHV-infected shrimps may provide novel insights into the molecular responses of P. monodon to YHV infection.