30-kDa Band Research Articles

Isolation and Characterization of Submitochondrial Reticulum Complex Associated with Midpiece of Bovine Cauda Epididymal Sperm

The mitochondrial sheath of mammalian spermatozoa adheres to an underlying organized network of electron-dense material termed the submitochondrial reticulum (SMR). The objective of the present study is to characterize the structural components of the SMR. We have isolated SMR fractions from midpieces of bovine cauda sperm. SDS-PAGE of SMR fractions revealed four major polypeptides of 56kDa, 54kDa, 34kDa, and 31kDa. Proteomic analyses of the four polypeptides revealed that 56kDa and 54kDa polypeptides identified as glycerol kinase and 34kDa and 31kDa polypeptide identified as VDAC2 and VDAC3, respectively (Voltage-Dependent Anion-Selective Channel Protein 2/3). Western blots of midpiece stained with anti-56kDa, anti-54KDa, anti-34kDaVDAC2, and anti-31kDaVDAC3 antibodies demonstrated the presence of 56kDa, 54kDa, 34kDa, and 31kDa immunoreactive bands, respectively. Four polypeptides are localized to midpiece. Glycerol kinase activity was quantitively assayed in tails. We proposed that glycerol kinase and VDAC2/VDAC 3 polypeptides play an important role in sperm motility and in midpiece function.

Protein and antigen profiles of third-stage larvae of Gnathostoma spinigerum assessed with next-generation sequencing transcriptomic information

Gnathostomiasis is a food-borne zoonotic disease that can affect humans who eat improperly cooked meat containg infective third-stage larvae. Definitive diagnosis is through larval recovery. However, this is an invasive technique and is impractical if the larvae have encysted in inaccessible areas of the body. Antigen or antibody detection might be more interesting techniques for diagnosis. Proteomic could elucidate diagnostic markers and improve our understanding of parasite biology. However, proteomic studies on Gnathostoma spinigerum are hampered by the lack of a comprehensive database for protein identification. This study aimed to explore the protein and antigen profiles of advanced third-stage G. spinigerum larvae (aL3Gs) using interrogation of mass spectrometry data and an in-house transcriptomic database for protein identification. Immunoproteomic analysis found 74 proteins in 24-kDa SDS-PAGE bands, which is size-specific for the immunodiagnosis of gnathostomiasis. Moreover, 13 proteins were found in 2-DE 24-kDa bands. The data suggest that collagenase 3, cathepsin B, glutathione S-transferase 1, cuticle collagen 14, major antigen, zinc metalloproteinase nas-4, major egg antigen, peroxiredoxin, and superoxide dismutase [Cu–Zn] may be good candidates for novel human gnathostomiasis diagnostic assays. These findings improve our understanding of the parasite’s biology and provide additional potential targets for novel therapeutics, diagnostics, and vaccines.

Glycosylated Cathepsin V Serves as a Prognostic Marker in Lung Cancer.

Dysregulation of cysteine cathepsin protease activity is pivotal in tumorigenic transformation. However, the role of cathepsin protease in lung cancer remains unknown. Here, we analyzed GEO database and found that lung cancer presented high expression of cathepsin V (CTSV). We then performed immunohistochemistry assay in 73 paired lung cancer tissues and normal lung tissues and confirmed that CTSV is overexpressed in lung cancer and correlates with poor prognosis. The mass spectrometry experiment showed that the N-glycosylation locus of CTSV are N221 and N292, glycosylated CTSV (band 43 kDa) was particularly expressed in lung cancer samples and correlated with lymph node metastasis. Mechanistic studies showed that only glycosylated CTSV (43-kDa band) are secreted to extracellular matrix (ECM) and promoted the metastasis of lung cancer. Importantly, the Elisa detection in serum of 12 lung cancer patients and 12 healthy donors showed that the level of CTSV in serum distinguished lung cancer patients from healthy donors. Together, our findings reveal the clinical relevance of CTSV glycosylation and CTSV drives the metastasis of lung cancer, suggesting that the glycosylated CTSV in serum is a promising biomarker for lung cancer.

Cloning, expression and characterization of C. crescentus xynA2 gene and application of Xylanase II in the deconstruction of plant biomass.

Biotechnology offers innovative alternatives for industrial bioprocesses mainly because it uses enzymes that biodegrade the hemicellulose releasing fermentable sugars. Caulobacter crescentus (C. crescentus) has seven genes responsible for xylanolytic cleavage, 5 to β-xylosidases (EC 3.2.1.37) and 2 for endoxylanases, like xynA2 (CCNA_03137) that encodes Xylanase II (EC 3.2.1.8) of the glycohydrolases-GH10 group. The xynA2 gene was amplified by PCR, cloned into the pTrcHisA vector e efficiently overexpressed in E. coli providing a His-tag fusion protein. Recombinant xylanase (XynA2) was purified by affinity chromatography using a nickel sepharose column and exhibited a single 43kDa band on SDS-PAGE gel. XynA2 showed an optimum alkaline pH (8) and stability at alkaline pH for 24h. Although C. crescentus is mesophilic, XynA2 has optimum temperature of 60°C and is thermo-resistance at 65°C. XynA maintains 66% of the enzymatic activity at high temperatures (90°C) without being denatured.The enzyme displayed a xylanolitic activity free of cellulase to xylan from beechwood and it was not inhibited in the presence of 50μmolmL-1 of xylose. In addition, dithiothreitol (DTT) induced XynA2 activity, as it improved its kinetic parameters by lowering the KM (5.78μmolmL-1) and increasing the KCat/KM ratio (1.63 U s-1). Finally, C. crescentus XynA2 efficiently hydrolyzed corn straw with high release of reducing sugars that can be applied in different branches of the industry.

Evaluation of Gamma Radiation-Attenuated Schistosoma mansoni Cercarial Antigens for Immunodiagnosis

DEVELOPMENT of sensitive and specific diagnostic tools for early diagnosis and treatment has an important necessity. This study focuses on identifying and evaluating immunodiagnostic potential of irradiated Schistosoma mansoni cercarial antigens by immunoblot analysis using infected human sera. Forty two serum samples were divided into: 14 sera from S. mansoni infected patients (Group I), 18 sera of patients with other parasitic diseases (Group II) and 10 sera from healthy individuals (Group III). Characterization of normal and 0.4Gy gamma radiation-attenuated cercarial antigens (Cer. Ags) was conducted using Gel Electrophoresis (SDS- PAGE). Normal Cer. Ags was resolved into six individual bands of 205, ≈160, 97, 66, 43 and ≈38kDa while irradiated Cer. Ags. showed same bands except 66kDa, in addition to two more bands which were ≈50 and 24kDa. Immunoblot analysis of irradiated cercarial antigens using pooled sera of patients with schistosomiasis mansoni identified four bands two like normal one, 97kDa and ≈32kDa, in addition to ≈50kDa and 43kDa bands. Total IgG immunoblotting using irradiated cercarial antigens with individual sera of patients with schistosomiasis and other parasites revealed that higher diagnostic accuracy was achieved by both 97kDa and 24kDa with 78.6%. Both 97 and 43kDa bands gave a higher sensitivity of 64.3% while 24kDa bands achieved the highest specificity of 89.3%. It could be concluded that total immunoblotting using irradiated cercarial antigens at 97kDa and 24kDa proved to be valuable as diagnostic tests for human schistosomiasis.

Identification and enzymatic characterization of clip domain serine protease in the digestive fluid of the sea hare, Aplysia kurodai.

Clip domain serine proteases (CDSPs) participate in the extracellular signaling cascades of various biological processes such as innate immune responses in invertebrates. CDSP genes have been isolated from numerous invertebrates. Nevertheless, the enzymatic properties of mollusk CDSPs are poorly understood. In the present study, we demonstrated that the amino acid sequences of the trypsin-like serine protease purified from the digestive fluid of the sea hare, Aplysia kurodai resemble those of the unidentified CDSP-type protein (TPS3) of Aplysia californica predicted by genome analysis. The purified enzyme produced single 34 and 26.5 kDa bands on SDS-PAGE under non-reducing and reducing conditions, respectively. The 34-kDa band generated two amino-terminal sequences that were similar to the deduced sequences of the clip and catalytic domains of TPS3. The single amino-terminal sequence of the 26.5 kDa band showed a single sequence homologous to the catalytic domain. Thus, the purified enzyme consists of clip and catalytic domains bridged by disulfide linkage(s). The subsite specificity and inhibitor sensitivity of the purified enzyme were clearly distinct from those of horseshoe crab and silkworm CDSPs. A good substrate for the sea hare enzyme was pyroglutamyl-Arg-Thr-Lys-Arg-4-methyl-7-coumarylamide. The enzyme activity was strongly inhibited by aprotinin but not leupeptin. The physiological function of the enzyme in the digestive fluid remains to be determined.

Calcium, Fe, Cu, Zn, and Mg Fractionation in In Natura and Aged Beef Samples by Bioanalytical Methods

Proteins play different and essential roles in the human organism. Containing essential amino acids, proteins, and minerals, beef is considered the main source of protein in human nutrition. It is generally accepted that the protein profile is directly correlated to tenderness and beef pigmentation and is also related to its organoleptic properties. In the present work, it is demonstrated the changes in protein profile, differences of metal concentrations, and how metals bonded to proteins can vary during the ripening phase, evaluated over a 14-day beef aging period. The proposed extraction procedure indicated 85% efficiency, preserving the metal-protein structure. Seventeen protein bands were detected using SDS-PAGE, and a 43-kDa band was found to be the most intense. The arrangement of SDS-PAGE and SEC-ICP-MS results indicated the possible links between minerals and organic functional molecules, such as Ca to troponin, Cu and Zn to albumin, and Fe to myoglobin and hemoglobin.

Chilo iridescent virus encodes two functional metalloproteases.

The genome of Chilo iridescent virus (CIV) has two open reading frames (ORFs) with matrix metalloprotease (MMP) domains. The protein encoded by ORF 136R contains 178 amino acids with over 40% amino acid sequence identity to hypothetical metalloproteases of other viruses, and the protein 165R contains 264 amino acids with over 40% amino acid sequence identity to metalloproteases of a large group of organisms, primarily including a variety of Drosophila species. These proteins possess conserved zinc-binding motifs in their catalytic domains. In this study, we focused on the functional analysis of these ORFs. They were cloned into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf9 cells with an N-terminal His tag, and purified to homogeneity at 72 hours postinfection using Ni-NTA affinity chromatography. Western blot analyses of purified 136R and 165R proteins with histidine tags resulted in 24- and 34-kDa protein bands, respectively. Biochemical assays with the purified proteins, performed using azocoll and azocasein as substrates, showed that both proteins have protease activity. The enzymatic activities were inhibited by the metalloprotease inhibitor EDTA. Effects of these proteins were also investigated on Galleria mellonella larvae. Insecticidal activity was tested by injecting the larvae with the virus derived from the AcMNPV bacmid carrying 136R or 165R ORFs. The results showed that the baculoviruses harbouring the iridoviral metalloproteases caused early death of the larvae compared to control group. These data suggest that the CIV 136R and 165R ORFs encode functional metalloproteases. This study expands our knowledge about iridoviruses, describes the characterization of CIV matrix metalloproteinases, and might ultimately contribute to the use of this virus as a research tool.

Microtubules are reorganised and fragmented for uterine receptivity.

For the development of uterine receptivity, many morphological and molecular changes occur in the apical surface of luminal uterine epithelial cells (UECs) including an increase in vesicular activity. Vesicular movements for exocytosis and endocytosis are dependent on microtubules; however, changes in microtubules in UECs during early pregnancy have received little attention. β-tubulin, one of the main component of microtubules, is distributed throughout the cytoplasm of UECs on day 1 (non-receptive) of pregnancy in the rat. On day 5.5, β-tubulin is concentrated above the nuclei and by day 6 (receptive), β-tubulin is concentrated in a band-like fashion above the nucleus. Western blot analysis of isolated UECs found two bands (50 and 34kDa) for β-tubulin in UECs during early pregnancy. The intensity of the 34kDa band was significantly higher on day 6 compared to day 1. The increase in the 34kDa band may be due to higher proteolytic activity associated with microtubule polymerisation during the receptive state. Transmission electron microscopy showed fragmented microtubules at the time of receptivity in UECs. This is the first study to show that microtubules are reorganised during uterine receptivity. This re-organisation likely facilitates vesicular movement and promotes the reorganisation of the apical plasma membrane for uterine receptivity.

Expression and Purification of gp40/15 Antigen of Cryptosporidium parvum Parasite in Escherichia coli: an Innovative Approach in Vaccine Production

Background: Cryptosporidium is a protozoan parasite that has medical and veterinary importance, and causes diarrhea and vomiting in a vast range of vertebrates. Some surface antigens, such as gp40/15, play important roles in adhesion and invasion of the parasite to host cells and consequently stimulate immune responses. Cloning and expression of the gp40/15 gene to provide recombinant proteins of the parasite antigens is valuable. Objectives: This study aimed at cloning and expression of the gp40/15 gene in Escherichia coli. Methods: In this experimental study, the gp40/15 gene sequence was extracted from GenBank (No. AF155624) and cloned in the PET28a+ plasmid. Colony polymerase chain reaction (PCR) and enzyme digestion methods by restricting enzymes, including BamHI and XhoI, were applied for verification. The recombinant plasmid was transferred to the Escherichia coli and the protein expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and enzyme linked immunosorbent assay (ELISA) techniques using serum containing antibodies against Cryptosporidium parvum. The protein was further purified by column chromatography. Results: The gp40/15 gene was successfully cloned in the PET28a+ plasmid. The colony PCR and enzymatic digestion methods showed a 921-bp fragment. Furthermore, expression of pEgp40/15 in Escherichia coli demonstrated a 43-kDa band. Antibody titrations in sera of test groups were significantly (P < 0.0001) higher than that of the control groups. Furthermore, antibody titration in test groups with four injections was significantly higher than that of the three injections (P < 0.05). Conclusions: The gp40/15 gene, which was cloned in the PET28a+, was successfully expressed and produced in E. coli. Therefore, this protein can be used in future studies to develop recombinant vaccines and diagnostic kits.

Cloning, expression and characterization of Brugia malayi abundant larval protein transcript-2 (BmALT-2) expressed in Pichia pastoris

ABSTRACTBesides mass drug administration, successful elimination of human lymphatic filariasis, a mosquito-borne tropical parasitic disease, requires developing other suitable prophylactic agents such as vaccines. The Brugia malayi abundant larval transcript-2 (BmALT-2) protein has been identified as one possible candidate. So far, it (E-ALT-2) has been expressed as a 24-kDa non-glycosylated protein in Escherichia coli. Easy and low-cost downstream purification of secreted BmALT-2 in Pichia pastoris may be a vital option to inexpensive large-scale production. This study focused on expression and molecular characterization of BmALT-2 (P-ALT-2) in P. pastoris. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that some P. pastoris colonies produced 27 and 24 kDa bands and few colonies produced a 24-kDa band. Preliminary studies confirmed glycosylation of 27 kDa P-ALT-2. The ratio of glycosylated and non-glycosylated P-ALT-2 was 20:80–70:30 in various colonies. The maximum yield of glycosylated and non-glycosylated P-ALT-2 was measured as 7.99 ± 1.12 and 9.18 ± 1.35 mg L−1 compared to 6.5 ± 1.2 mg L−1 of E-ALT-2 in shake flasks. Their overall expression was about 25% and 35% higher than that in E. coli. The glycosylated P-ALT-2 exhibited about 15% higher immunoreactivity with human endemic normal sera. The enhanced secreted production by P. pastoris may lead to cost-effective large-scale production of BmALT-2.

Purification of Regucalcin from the Seminal Vesicular Fluid: A Calcium Binding Multi-Functional Protein.

Regucalcin is a multi-functional protein having roles in calcium homeostasis as well as in anti-apoptotic, anti-prolific and anti-oxidative functions. Recently, it has been reported from the male reproductive tract, but its role in male reproduction needs further investigation; for which the native regucalcin of reproductive origin will be more appropriate. The gel exclusion chromatography followed by diethyl aminoethane cellulose chromatography and two-dimentional cellulose acetate membrane electrophoresis used for its purification are time consuming and less specific. Here, the regucalcin gene from buffalo testis has been cloned, expressed and purified in recombinant form, and subsequently used for raising hyper-immune serum. The Western blot of seminal vesicular fluid probed with anti-regucalcin polyclonal and monoclonal antibodies showed the presence of 28 and 34kDa bands specific to regucalcin. Further, an affinity matrix has been prepared using anti-regucalcin polyclonal antibodies. An immuno-affinity chromatography method has been standardized to isolate regucalcin from seminal vesicular fluid. The initial complexity of the protein mixture in the seminal vesicular fluid has been reduced by a heat coagulation step. The purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band at 68kDa that has been further confirmed as regucalcin by Liquid chromatography-mass spectrometry/mass spectrometry. The RGN purified from seminal vesicular fluid will be more appropriate for studying its possible role in male reproduction, especially sperm cell capacitation, hyperactivation, acrosome reaction and cryopreservation. The study can be applied in purifying regucalcin from different tissues or species with minor modifications in the methodology.

Comparative study of muscle proteins in relation to the development of yake in three tropical tuna species yellowfin (Thunnus albacares), big eye (Thunnus obesus) and skipjack (Katsuwonus pelamis)

Burnt tuna (BT), or yake-niku, is a quality flaw of the muscle characterised by a pale colour and grainy and exudative texture. Cathepsin-L, water soluble and total protein components from normal and BT muscles, from three tropical tuna species – yellowfin (YFT, Thunnus albacares), bigeye (BET, Thunnus obesus) and skipjack (SKJ, Katsuwonus pelamis) – were compared by electrophoretic and western blot analyses to identify biomarkers for BT. As expected, SDS–PAGE patterns were species-specific but differences, due to BT, were observed only between some low ionic strength extracts of BET and YFT. Protein oxidation and cell proliferation analysed by immunoblotting did not show differences between BT and normal muscles. Gelatine zymography revealed different gelatinase activity patterns that, although not linked to BT, may affect the final texture of the muscle. A 43kDa band, identified as creatine kinase by proteomic analysis, showed the potential to be a good indicator for BT in BET and YFT.

Effect of Aging on the Physicochemical, Textural, Microbial and Proteome Changes in Emu (D romaius novaehollandiae ) Meat Under Different Packaging Conditions

The effect of aging on the physicochemical, textural, microbial and proteome characteristics of emu (Dromaius novaehollandiae) meat was studied under aerobic packaging (AP) and vacuum packaging (VP) conditions at 4 ± 1C for 9 and 15 days, respectively. Improvement (P < 0.05) in water-holding capacity, myofibrillar fragmentation index and protein extractability with aging was observed in emu meat cubes under both AP and VP conditions. Reduction (P < 0.05) in Warner-Bratzler shear force values was observed on the 6th and 15th day of aging compared with the 0th day in the AP and VP samples, respectively. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis revealed the appearance of 30-kDa protein bands, indicating extensive proteolysis on the 6th day and 9th day of aging in the AP and VP samples, respectively. Proteome analysis using two-dimensional gel electrophoresis revealed significant (P < 0.01) changes in the number of differentially expressed protein spots in the AP and VP samples during aging. Practical Applications Emus are classified as ratites along with ostriches and rheas and are becoming increasingly popular as a source of low-fat, low-cholesterol, iron-rich red meat alternative. The emu industry is looking for science-based information in order to successfully produce and market emu meat. The present work provides information on emu meat quality, aging process, ultrastructure, proteome characteristics and microbial changes. This paper also discusses about the different packaging methods and the shelf life of emu meat. Considering the increasing demand for new source of animal proteins around the world, this paper provides significant information to all ostrich, rhea and emu meat processors.

Characterization of alkaline phosphatases activities in uterine secretion of buffaloes

Alkaline phosphatases is reported as a predominant protein in buffalo uterine luminal secretion during the late- luteal phase of the cycle. Since the biochemical properties of this important protein are not known, an experiment was designed to purify and characterize the alkaline phosphatase from buffalo uterine secretion. The enzyme could be purified from uterine luminal fluid by 2 chromatography steps using DEAE cellulose ion-exchange and Sephacryl S-200 HR gel-filtration columns. The molecular weight of the purified protein was 179 kDa by non-reducing SDS- PAGE. Reduction by ß-metcaptoethanol yielded 136kDa and 43kDa bands indicated presence of 2 separable subunits of this protein. Complete inhibition of activity by ß-mercaptoethanol and partial inhibition by iodoacetamide indicated that both the subunits are essential for bioactivity. Three isoforms with pI values 5.9, 6.0 and 6.2 were detected by iso-electric focusing. Purified enzyme showed optimum activities at 37°C in pH ranges 10.5 to 11.0. It is a temperature sensitive enzyme as evidenced by a loss of 91% enzyme activity within 28 h of room temperature storage. The loss of activity was slow (12% per day) at 4p C storage indicating importance of cold chain maintenance while processing. The SDS proved to be a better detergent compared to Triton X100 as less inhibition (46% vs 76%) of enzyme activities was observed with the former. Inhibition of activities by metal chelators confirmed that it was metallo- phosphatase. Further study would help exploring its role in buffalo uterine microenvironment.

Association between the presence of protein bands in ram seminal plasma and sperm tolerance to freezing

This study evaluated associations between the presence of protein bands in ram seminal plasma and the quality of sperm frozen with distinct extenders. Ejaculates were frozen in a Tris–egg yolk based extender, including either 5% glycerol or 100mM trehalose. Seminal plasma samples were submitted to unidimensional electrophoresis. Pre-freezing and post-thawing sperm quality was similar between extenders (P>0.05). A total of 26 bands were identified in ram seminal plasma. Pre-freezing sperm motility was increased when the 15, 19 and 80kDa bands were present in seminal plasma (P<0.05). The presence of an 11kDa band in seminal plasma was associated with reduced pre-freezing membrane integrity (P<0.05). After thawing, both sperm motility and membrane integrity were reduced when a 24kDa band was present in seminal plasma (P<0.05). Post-thawing acrosome integrity was greater in the presence of a 31kDa band in seminal plasma (P<0.05). Regardless of the cryoprotectant included in the freezing extender, these six bands may be potential markers for ram sperm tolerance to freezing.

Distribution of Secretory Phospholipase A2 XIIA in the Brain and its Role in Lipid Metabolism and Cognition

Phospholipases A(2) (PLA(2)) catalyze the hydrolysis of membrane phospholipids to produce free fatty acids and lysophospholipids, which have important functions in cell signaling. The present study elucidated differential expression of PLA(2) isoforms in the rat cortex by quantitative reverse transcription PCR and demonstrated that sPLA(2)-XIIA had greater messenger RNA expression than iPLA(2)-VI or cPLA(2)-IVA in different brain regions, or compared to other sPLA(2) isoforms in the prefrontal cortex (PFC) and hippocampus. Western blots identified a 24-kDa band in different regions of the adult brain, and high levels of sPLA(2)-XIIA protein expression were detected in the PFC, striatum, and thalamus. Electron microscopy showed that sPLA(2)-XIIA is present in axon terminals and dendrites. Injection of antisense oligonucleotide to sPLA(2)-XIIA in the PFC and lipidomic analysis showed increase in phospholipid but decrease in lysophospholipid species consistent with decreased catalytic activity of the enzyme, changes in arachidonic acid release, and alterations in sphingolipids. sPLA(2)-XIIA knockdown also resulted in shorter latency timings in the passive avoidance test, and higher number of errors in the attention set-shifting task, indicating deficits in working memory and attention. Together, the results show an important role of sPLA(2)-XIIA in lipid metabolism, prefrontal cortical function, and cognition.

Characterization of interspecific hybrid dikaryons of the oyster mushrooms, Pleurotus florida PAU-5 and P. sajor-caju PAU-3 (higher Basidiomycetes) from India.

Five Pleurotus hybrid dikaryons, developed through cross-breeding of P. florida PAU-5 (PF-5) and P. sajor-caju PAU-3 (PSC-3) were characterized with respect to textural properties, color, and enzymatic and genetic variability. Texture profile revealed significant differences in springiness, resilience, cohesiveness, and chewiness between all hybrids compared to the parents. Among the hybrid cultures, maximum whiteness was reported in hybrid 37, whereas hybrid 8 had minimum whiteness. Three hybrids (16, 37, 42) showed an increased linear growth rate in relation to PF-5, whereas no hybrid showed a higher growth rate than PSC-3. Maximum endoglucanase and xylanase activity was observed in hybrid 46, whereas minimum activity occurred in hybrid 42. Laccase and protease activity was higher in hybrid 37 and 46, respectively. Four hybrids (16, 37, 42, 46) showed increased peroxidase activity in relation to PF-5, whereas hybrid 46 showed activity higher than the parent PSC-3. Comparison of isozyme patterns confirmed the hybrid nature of hybrid 16. The large variation in the intensity of bands could be a result of recombination. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of extracellular enzymes revealed 60.3- and 43-KDa bands in all the hybrids. An additional 25-KDa band was reported in hybrids 37, 42, and 46 and the parent PF-5, indicating their close relatedness. Parental strains showed higher divergence in small-subunit ribosomal DNA region compared with the internal transcribed spacer region, indicating their significance in varietal discrimination. Hybrid 46 had a small-subunit ribosomal DNA region more similar to that of PSC-3 compared with PF-5, whereas the internal transcribed spacer region of hybrids 42 and 46 revealed close resemblance to that of PF-5 and PSC-3, respectively.

Biochemical Characterization of a Phospholipase A2 from Photobacterium damselae subsp. piscicida

Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of fish photobacteriosis (pasteurellosis) in cultured cobia (Rachycentron canadum) in Taiwan. A component was purified from the extracellular products (ECP) of the bacterium strain 9205 by fast protein liquid chromatography (FPLC) and identified as a phospholipase. An N-terminal sequence of 10 amino acid residues, QDQPNLDPGK, was determined by mass spectroscopy (MS) and found to be identical with that of another Phdp phospholipase (GenBank accession no. BAB85814) at positions 21 to 30. The corresponding gene sequence of the phospholipase (GenBank accession no. AB071137) was employed to design primers for amplification of the sequence by the polymerase chain reaction (PCR). The PCR products were transformed into Escherichia coli, and a recombinant protein product was obtained which was purified as a His-tag fusion protein by Ni-metal affinity chromatography. A single 43-kDa band was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Phosphatidylcholine was degraded by this protein to lysophosphatidylcholine and a fatty acid. These products were characterized by thin-layer (TLC) and gas chromatography (GC), respectively, allowing the identification of the protein as a phospholipase A2. The recombinant protein had maximum enzymatic activity between pH 4 and 7, and at 40 degrees C. The activity was inhibited by Zn(2+) and Cu(2+), activated by Ca(2+) and Mg(2+), and completely inactivated by dexamethasone and p-bromophenacyl bromide. A rabbit antiserum against the recombinant protein neutralized the phospholipase A2 activity in the ECP of Phdp strain 9205 and the recombinant protein itself. The recombinant protein was toxic to cobia of about 5 g weight with an LD50 value between 2 and 4 microg protein/g fish. The results revealed phospholipase A2 as a fish toxin in the ECP of Phdp strain 9205.

Identification of a Receptor for Neuropeptide VGF and Its Role in Neuropathic Pain

VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca(2+) levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca(2+) levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.