kDa Polypeptide Research Articles

Complement factor D homolog involved in the alternative complement pathway of rock bream (Oplegnathus fasciatus): Molecular and functional characterization and immune responsive mRNA expression analysis.

The complement system serves conventional role in the innate defense against common invading pathogens. Complement factor D (CfD) is vital to alternative complement pathway activation in cleaving complement factor B. This catalytic reaction forms the alternative C3 convertase that is crucial for complement-mediated pathogenesis. In this study, rock bream (Oplegnathus fasciatus) CfD (OfCfD) was characterized and OfCfD mRNA expression was investigated. OfCfD encodes 277amino acids (aa) for a 30-kDa polypeptide. A domain analysis of the deduced OfCfD aa sequence showed a single serine protease trypsin superfamily domain, a serine active region, three active sites, and three substrate-binding sites. Pairwise sequence comparisons indicated that OfCfD has the highest identity (84.5%) with Oreochromis niloticus CfD. The phylogenetic tree revealed a common ancestral origin of CfD members, with fish CfD distinct from other vertebrate orthologs. The structural arrangement of the OfCfD gene (2451bp) contained five exons interrupted by four introns. A spatial transcriptional analysis indicated that OfCfD transcripts constitutively expressed in all of the examined rock bream tissues, and that they were highest in the spleen and liver. In addition, OfCfD transcripts were immunologically upregulated by lipopolysaccharide (LPS) (12h p.i.), Streptococcus iniae (12h p.i.), rock bream iridovirus (RBIV) (6-12h p.i.), and poly I:C (6h p.i.) in spleen tissue. OfCfD is a trypsin protease and its recombinant protein showed strong protease activity similar to that of trypsin, indicating its catalytic function in the alternative pathway. Together, our findings suggest that OfCfD might be involved in immune responses in rock bream.

Identification and characterization of two cleavage fragments from the Aquareovirus nonstructural protein NS80.

Aquareovirus species vary with respect to pathogenicity, and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly, which can form viral inclusion bodies (VIBs) and recruit viral proteins to its VIBs in infected cells. NS80 consists of 742 amino acids with a molecular weight of approximately 80 kDa. Interestingly, a short specific fragment of NS80 has also been detected in infected cells. In this study, an approximately 58-kDa product of NS80 was confirmed in various infected and transfected cells by immunoblotting analyses using α-NS80C. Mutational analysis and time course expression assays indicated that the accumulation of the 58-kDa fragment was related to time and infection dose, suggesting that the fragment is not a transient intermediate of protein degradation. Moreover, another smaller fragment with a molecular mass of approximately 22 kDa was observed in transfected and infected cells by immunoblotting with a specific anti-FLAG monoclonal antibody or α-NS80N, indicating that the 58- kDa polypeptide is derived from a specific cleavage site near the amino terminus of NS80. Additionally, different subcellular localization patterns were observed for the 22-kDa and 58-kDa fragments in an immunofluorescence analysis, implying that the two cleavage fragments of NS80 function differently in the viral life cycle. These results provide a basis for additional studies of the role of NS80 played in replication and particle assembly of the Aquareovirus.

Effect of different doses of nitrogen on protein profiling, pasting and quality attributes of rice from different cultivars

Effect of different level (60, 120 and 180kgN/ha) of nitrogen (N) application on protein profiling, pasting and cooking quality characteristics of milled rice from different paddy cultivars was evaluated. N dose showed positive correlation with protein content and negative correlation with L*, whiteness and amylose content. N application significantly affected the protein profile, textural and pasting properties of different cultivars. All the cultivars expect PR120 and PAU201, showed an increase in the amount of accumulation of 60kDa polypeptide with increase in N application. Accumulation of prolamines (16 and 14kDa) and polypeptides of 38 and 35kDa increased in all the cultivars. Size exclusion chromatography revealed decrease in low molecular weight subunits and increase in medium molecular weight subunits in all the cultivars upon N application. However, high molecular weight subunits increased in IET21214 and decreased in PR120 and PAU20 upon N application. N application resulted in increase in glutelins and decrease in peak and breakdown viscosity. PAU201 and PR120 showed lower AAC due to low accumulation of 60kDa granule-bound starch synthase (GBSS), in response to N application. Gumminess and hardness of cooked rice increased with the increase in N dose and the increase was significant at 60kgN/ha.

Biochemical and molecular characterization of mitochondrial membrane-bound arginase in Heteropneustes fossilis.

The two predominant forms of arginase, cytosolic Arginase-I and mitochondrial Arginase-II, catalyze hydrolysis of arginine into ornithine and urea. Based on presence of arginase activity in extracts using potassium chloride (KCl), mitochondrial membrane-bound arginase has also been suggested. However, the activity of arginase in fractions obtained after KCl-treatment may be either due to leakage of mitochondrial arginase or release of adhered cytosolic arginase to cell organelles having altered net charge. Therefore, it has been intended to analyse impact of KCl on ultra-structural properties of mitochondria, and biochemical analysis of mitochondrial membrane-bound proteins and arginase of Heteropneustes fossilis. Liver of H. fossilis was used for isolating mitochondria for analysis of ultrastructural properties, preparing cytosolic, mitochondrial, and mitochondrial-membrane bound extracts after treatment of KCl. Extracts were analysed for arginase activity assay, protein profiling through SDS-PAGE and MALDI MS/MS. The KCl-mediated modulation in polypeptides and arginase were also evaluated by PANTHER, MitoProt and IPSORT servers. The effects of KCl on ultra-structural integrity of mitochondria, activity of arginase, modulation on mitochondrial proteins and enzymes including arginase were observed. The 48 kDa polypeptide of mitochondrial fraction, that showed KCl-dependent alteration matched with Myb binding protein and 30 kDa bands resembles to arginase after MALDI MS/MS analysis. Results indicate KCl-dependent ultrastructural changes in mitochondria and release of mitochondrial arginase. The proposed membrane bound mitochondrial arginase could be mitochondrial arginase-II or altered form of cytosolic arginase-I contributing to KCl-induced arginase activity in H. fossilis.

Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein

Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome reaction, whereas the IZUMO1 and lactadherin polypeptides remain associated to the particulate fraction. Almost entire population of bovine sperm IZUMO1 relocates to the equatorial segment during the LPC-induced acrosome reaction. We propose that the interaction of OMC32 matrix polypeptide with detergent-soluble acrosomal proteins regulates the release of hydrolases/other acrosomal protein(s) during the acrosome reaction.

Effects of high temperature stress on enzymatic and nonenzymaticantioxidants and proteins in strawberry plants

The mechanism of tolerance to high temperatures was investigated in two strawberry (Fragaria x ananassa Duch) cultivars, 'Redlands Hope' ('R. Hope', heat tolerant) and 'Cal. Giant 3' ('CG3', heat sensitive). Leaves were collected from plants that were exposed to gradual heat stress and heat-shock stress separately. The contents of nonenzymatic antioxidants such as ascorbic acid (AsA) and glutathione (GSH) and the activities of enzymatic antioxidants such as ascorbate peroxidase (APX) (EC 1.11.1.11), catalase (CAT) (EC 1.11.1.6), and glutathione reductase (GR) (EC. 1.6.4.2) were measured followed by heat treatments. Additionally, proline content was determined, and heat shock proteins (HSPs) were analyzed with an immunoblotting method to investigate protein markers involved in the heat-stress tolerance of strawberry plants. The contents of AsA and GSH did not change depending on heat stress type, temperatures, or cultivars. While APX and CAT activities increased with high temperatures, GR activity was almost unchanged. The proline content of the cultivars increased in both treatments. Anti-HSP60 immunoblots revealed that a 23 kDa polypeptide was detected during the heat acclimation of strawberry cultivars. The intensity of the heat shock protein in 'R. Hope' plants was more than in 'CG3' plants. Thus, the accumulation of 23 kDa heat shock protein was correlated with the heat tolerance of the cultivars. In conclusion, strawberry leaf tissues of 'R. Hope' were found to enhance the structural stability of cellular membranes under high temperature by increasing both the activity of such enzymes as CAT and APX to activate the antioxidative systems and the expression of 23 kDa HSP.

Identification of the Unique Properties of IgGs and their Heavy chains in Blood Serum of Multiple Sclerosis Patients

Monitoring of multiple sclerosis (MS) requires of molecular markers. Recently, using TCA-precipitation/ extraction and MALDI TOF/TOF mass-spectrometry, we identified in blood serum of multiple sclerosis (MS) patients earlier unknown 46 kDa form of human unconventional myosin IC isoform b (Myo1C) (Myronovskij et al., 2015). During SDS-electrophoresis of TCA-extracted proteins of blood serum of 28 MS patients, 3 additional polypeptides with 55, 50 and 25 kDa molecular masses were detected. Western-blot analysis using monospecific anti-human IgG rabbit HRP-conjugated antibodies showed that 55 and 25 kDa polypeptides belong to heavy and light chains of IgG molecules, while 50 kDa protein corresponds to free heavy chains of IgGs. None of these polypeptides was found in fractions of TCA extracted polypeptides isolated from blood serum of healthy human donors or patients with systemic lupus erythematosis and rheumatoid arthritis.

Polypeptide composition and functional properties of African yam bean seed (Sphenostylis stenocarpa) albumin, globulin and protein concentrate

In this study, the functional properties of African yam bean seed protein concentrate (64.21% protein) as well as albumin (59.51% protein) and globulin (91.68% protein) fractions were investigated. Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the albumin consisted of mostly polypeptides with sizes ≤26 kDa. The protein concentrate and globulin had similar polypeptide composition while some of the bigger size (46–61 kDa) polypeptides were not present in the albumin. The albumin and globulin fractions had higher fluorescence intensity values than the protein concentrate at all measured pH values. Gelling ability was significantly (p < 0.05) stronger for the albumin (probably due to the high carbohydrate content) with least gelation concentration of 8% when compared to the 10% for protein concentrate and 14% for globulin. In contrast, oil absorption capacity significantly (p < 0.05) decreased in the following order: albumin (8.10 g/g) > globulin (4.75 g/g) > protein concentrate (4.05 g/g). At pH 3.0–9.0, the albumin had significantly (p < 0.05) higher solubility than the protein concentrate and globulin. The albumin also had significantly (p < 0.05) higher foaming capacity but similar emulsion forming ability as the protein concentrate. Emulsion forming ability and stability of globulin formed emulsions were poorer (p < 0.05) than those of albumin and protein concentrate. While non-protein components could have influenced some of the properties, the results suggest that smaller size of the albumin polypeptides could be responsible for some of the observed superior functional properties when compared to the globulin and protein concentrate that contained bigger size polypeptides.

NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.

Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 μM) and association constants (kon = 500 M(-1) s(-1), koff = 0.2 s(-1)) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction between SME-1 β-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and β-arrestin-2, were rapid, reversible, and robust to temperature (21-37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.

Immunoassay‐based Techniques for Early and Specific Detection of Latent Postharvest Anthracnose in Mango

AbstractThe postharvest anthracnose pathogen Colletotrichum gloeosporioides inciting latent or quiescent infection of mango was detected in early stages using immunoassay methods. Twenty‐five pathotypes isolated from different agroclimatic zones of Tamil Nadu, Karnataka and Pondicherry, India, revealed the variation in protein profile analysis (SDS‐PAGE). The polyclonal antibodies (PCA) were raised against the unfractioned mycelial protein (UMP) and a 40‐kDa polypeptide present in all pathotypes. Standardization of antigen and antiserum dilutions revealed that an antigen dilution of 1 : 200 (protein concentration of 20 μg/ml) and antiserum dilution of 1 : 100 (protein concentration of 40 μg/ml raised against UMP) and 1 : 200 (protein concentration of 20 μg/ml raised against 40 kDa polypeptide) was found to be optimum for the detection of anthracnose pathogen. Both antisera detected the C. gloeosporioides antigen in enzyme‐linked immunosorbent assays (ELISAs), dot immunobinding assays (DIBAs) and Western blots. The specificity in reaction was compared by isolating other Colletotrichum spp. from various hosts viz., C. lindemuthianum (beans), C. falcatum (sugarcane), C. musae (banana), C. capsici (chillies) and Botryodiplodia theobromae (mango). The antisera generated against UMP revealed the cross‐reaction with other host isolates and mango stem end rot pathogen (B. theobromae). The PCA raised against 40‐kDa polypeptide exhibited the specific reaction with C. gloeosporioides isolates in all the immunoassay techniques. By utilizing both PCA, the presence of latent infection was observed in healthy‐looking leaves, flowers and fruits in orchard conditions. The fruit tissues recorded high absorbance values followed by flowers and leaves in all the detection methods. The ELISA technique was also useful in assessing the pathogen inoculum at various biocontrol formulations sprayed mango trees under field conditions. The fluorescent pseudomonad strains mixture (KFP1 + FP7) amended with chitin sprayed at 30‐day intervals revealed the significant reduction in pathogen load than other formulations and unsprayed control.

Cross antigenicity of immunodominant polypeptides of somatic antigen of Oesophagostomum columbianum with other helminths by western blotting.

Aim:Oesophagostomum columbianum in small ruminants in India is found as mixed infection commonly in sheep and goat. Haemonchus contortus, an abomasal nematode is found as concurrent infection with it. Eggs of Haemonchus and O. columbianum cannot be easily distinguished. Diagnosis of O. columbianum may only be possible if a non-cross antigenic polypeptide was available for immunodiagnosis.Materials and Methods:Somatic antigen (SoAg) of O. columbianum was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodominant polypeptides were identified by western blotting with homologous hyperimmune serum (HIS) and experimental sera of sheep or goat infected with other helminths.Results:SoAg of O. columbianum was immunoaffinity purified. Sharp polypeptide bands of 130, 72 and 68 KDa were observed along with several faint bands of lower molecular weight. Western blot of purified SoAg of O. columbianum with homologous HIS showed reaction with all the protein bands of 17, 28, 30, 32, 35, 38, 50, 68, 100, 130, 150, and 170 kDa. For identification of non-cross antigenic polypeptide, immunoaffinity purified SoAg of O. columbianum was reacted to heterologous HIS against H. contortus, Paramphistomum epiclitum, and Fasciola gigantica in western blotting utilizing completely dry method (i-blot). Among high molecular weight polypeptides 100 and 150 kDa were non-cross antigenic and among low molecular weight except 50 kDa polypeptide, 17, 30, 32, 35, and 38 kDa of O. columbianum were not cross antigenic with other helminths.Conclusions:Hence, polypeptides of 17, 30, 32, 35 and 38 kDa as well as 100 and 150 kDa polypeptides of O. columbianum may be exploited for immunodiagnosis of the infection in sheep and goat with extensive studies on cross antigenicity.

Study on morpho-physiological differences in sugarcane cultivars varying in germination efficiency

Good germination is a basic requirement for higher sugarcane and sugar productivity. It is necessary to understand the mechanism of sugarcane bud germination to maintain optimal plant population. Hence, the present study was undertaken to find out the influence of cultivars and seed sett parts on morpho-physiological traits, biochemical parameters, and germination efficiency at different days after planting (DAPs). Two sugarcane cultivars CoS 97261 (early sprouting) and CoS 96269 (late sprouting) were selected for the study. The experiment was laid out in a complete randomized design with three replications. Bud weights, lengths, and widths at 0, 7, 14, and 21 DAPs were recorded to observe the factors responsible for morphogenesis in germination. Total sugar and total soluble protein were estimated quantitatively and SDS-PAGE protein profiling performed in three portions, viz., top (immature), middle (maturing), and bottom (mature) of a cane. Total sugar content was increased at 7 DAPs, and it decreased at later stages. The total soluble protein content was observed to increase at 7 DAPs; however, it decreased during 14–21 DAPs. The magnitude was more pronounced in CoS 97261 than that in CoS 96269. Protein profiling showed appearances of 60, 40, 25, and 23.5 kDa polypeptides. The results suggest that factors like cultivars, seed sett, and DAPs influenced the utilization of sugars and proteins from sett reserves during the bud germination. Identified fractions of polypeptide also play a significant role throughout the bud germination. This new information might be utilized for the selection of fast-germinating sugarcane varieties in future.

Impact of the ageing process on the intensity of post mortem proteolysis and tenderness of beef from crossbreeds

Abstract The aim of the study was the evaluation of the effect of ageing on the extent of myofibrillar proteins degradation and tenderness of beef in different crossbreeds, BB × HF and SM × HF, from which the musculus semitendinosus was obtained. The pH value, basic composition of meat, and colour parameters were determined on the 3rd d post mortem. The Warner Bratzler shear force and the extent of protein degradation were evaluated in regard to the effect of ageing time. Meat of BB × HF crossbreed had a lower amount of intramuscular fat and higher protein content (P ≤ 0.05). The shear force decreased with ageing time in the case of both crossbreeds. However, the highest values were noted in SM × HF crossbreed on days 3 and 7 of ageing. The differences in proteolysis of myofibrillar proteins and polypeptides, determined by SDS-PAGE electrophoresis, were observed between crossbreeds and the ageing time. A significant decrease in desmin and increased levels of 49-46 kDa and 32-27 kDa polypeptides (products of proteolytic degradation) were observed with an increasing ageing time. In addition, the rate of increase in the amount of 32-27 kDa polypeptides was more significant in BB × HF crossbreed. The data obtained showed that tenderness and the extent of protein degradation are associated with ageing process and animals’ genotype.

Identification and characterization of a bovine sperm acrosomal matrix protein and its mechanism of interaction with acrosomal hydrolases.

Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads comprised 54, 50, 45, and 38-19 kDa polypeptides. The objective of this study is to identify and characterize the 45 kDa (OMC45) polypeptide, to define its role in binding acrosomal hydrolases, and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI-TOF-TOF yielded eight peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X-100-permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti-OMC45 and anti-TEKT3. The OMC45 polypeptide was solubilized by radio-immunoprecipitation assay buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti-OMC45 immunoprecipitation pellet. An identical blot stained with anti-TEKT3 exhibited the presence of TEKT3 polypeptide in the anti-OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide and that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N-linked and O-linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N-acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid membrane complex.

Impact of germination on flour, protein and starch characteristics of lentil (Lens culinari) and horsegram (Macrotyloma uniflorum L.) lines

Effect of germination on protein profiling, physicochemical, functional and pasting properties of lentil and horsegram lines was studied. Proportion of β-sheets and β-turns were the highest in both the pulses, followed by random coils and α-helix and proportion of antiparallel β-sheets was the lowest. Proportion of anti-parallel β-sheets and β-sheets increased whereas that of α-helix and β-turns decreased with germination. Flours with higher β-sheets and lower α-helix showed higher water absorption capacity and foaming capacity but lower emulsifying activity index. Germination increased protein content, foaming capacity, water absorption capacity and breakdown viscosity while decreased foaming stability, emulsifying activity index and paste viscosities. HMW polypeptides of 80, 69, 60, 58, 55, 45, 37 and 33 kDa in lentils and 87, 81, and 72 kDa in horsegram decreased and LMW polypeptides of 19 and 14 kDa in lentils; 25 and 22 kDa polypeptide in horsegram increased during germination. Polypeptides of 15, 27 and 29 kDa corresponding to trypsin inhibitors were not affected by germination. Amylose content decreased with germination. Uncooked starch showed decrease in proportion of large granules accompanied by increase in proportion of small granules.

Molecular Characterization of Trypanosoma cruzi Tc8.2 Gene Indicates Two Differential Locations for the Encoded Protein in Epimastigote and Trypomastigote Forms.

This report describes the molecular characterization of the Tc8.2 gene of Trypanosoma cruzi. Both the Tc8.2 gene and its encoded protein were analyzed by bioinformatics, while Northern blot and RT-PCR were used for the transcripts. Besides, immunolocalization of recombinant protein was done by immunofluorescence and electron microscopy. Analysis indicated the presence of a single copy of Tc8.2 in the T. cruzi genome and 2-different sized transcripts in epimastigotes/amastigotes and trypomastigotes. Immunoblotting showed 70 and 80 kDa polypeptides in epimastigotes and trypomastigotes, respectively, and a differential pattern of immunolocalization. Overall, the results suggest that Tc8.2 is differentially expressed during the T. cruzi life cycle.

Functional interpretation and structural insights of Arabidopsis lyrata cytochrome P450 CYP71A13 involved in auxin synthesis.

Cytochrome P450 CYP71A13 of Arabidopsis lyrata is a heme protein involved in biosynthesis of indole-3-acetonitrile which leads to the formation of indolyl-3-acetic acid. It catalyzes a unique reaction: formation of a carbon-nitrogen triple bond and dehydration of indolyl-3-acetaldoxime. Homology model of this 57 kDa polypeptide revealed that the heme existed between H-helix and J- helix in the hydrophobic pocket, although both helixes are involved in catalytic activity, where Gly305 and Thr308, 311 of H- helix were involved in its stabilization. The substrate indole-3-acetaldoxime was tightly fitted into the substrate pocket with the aromatic ring being surrounded by amino acid residues creating a hydrophobic environment. The smaller size of the substrate binding pocket in cytochrome P450 CYP71A13 was due to the bulkiness of the two amino acid residues Phe182 and Trp315 pointing into the substrate binding cavity. The apparent role of the heme in cytochrome P450 CYP71A13 was to tether the substrate in the catalysis by indole-3-acetaldoxime dehydratase. Since the crystal structure of cytochrome P450 CYP71A13 has not yet been solved, the modeled structure revealed mechanism of substrate recognition and catalysis.

A member of the HSP90 family from ovine Babesia in China: molecular characterization, phylogenetic analysis and antigenicity.

Heat shock protein 90 (HSP90) is a key component of the molecular chaperone complex essential for activating many signalling proteins involved in the development and progression of pathogenic cellular transformation. A Hsp90 gene (BQHsp90) was cloned and characterized from Babesia sp. BQ1 (Lintan), an ovine Babesia isolate belonging to Babesia motasi-like group, by screening a cDNA expression library and performing rapid amplification of cDNA ends. The full-length cDNA of BQHsp90 is 2399 bp with an open reading frame of 2154 bp encoding a predicted 83 kDa polypeptide with 717 amino acid residues. It shows significant homology and similar structural characteristics to Hsp90 of other apicomplex organisms. Phylogenetic analysis, based on the HSP90 amino acid sequences, showed that the Babesia genus is clearly separated from other apicomplexa genera. Five Chinese ovine Babesia isolates were divided into 2 phylogenetic clusters, namely Babesia sp. Xinjiang (previously designated a new species) cluster and B. motasi-like cluster which could be further divided into 2 subclusters (Babesia sp. BQ1 (Lintan)/Babesia sp. Tianzhu and Babesia sp. BQ1 (Ningxian)/Babesia sp. Hebei). Finally, the antigenicity of rBQHSP90 protein from prokaryotic expression was also evaluated using western blot and enzyme-linked immunosorbent assay (ELISA).

The first trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae was found in Dendrobium pendulum: purification, characterization, and effects of stress factors.

Trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae with two conformational forms was first studied in Dendrobium pendulum . It was highly expressed by stress factors. Using mannan-agarose column chromatography, a mannose-binding protein was purified from Dendrobium pendulum Roxb. pseudobulb. After heating in the presence of sodium dodecyl sulfate (SDS) with or without 2-mercaptoethanol, the protein showed one band with molecular mass of 14.0 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Without heating, three bands were found at positions of 14.0, 39.4, and 41.5 kDa, but a higher amount of 39.4 and 41.5 kDa protein bands were seen in the presence of 2-mercaptoethanol. Liquid chromatography-tandem mass spectrometry and database search indicated that the 14.0 kDa protein band contained three peptide fragments identical to parts of a lectin precursor from Dendrobiu m findleyanum Parish & Rchb.f. Native-PAGE and Ferguson plot showed that the purified protein had two native forms with molecular masses of 44.2 and 45.3 kDa, indicating three 14.0 kDa polypeptide subunits. The purified protein exhibited the agglutination activity with trypsinized chicken erythrocytes. It was then recognized as a Galanthus nivalis agglutinin-related lectin and named D. pendulum agglutinin (DPA). Using reverse transcription-polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of DPA precursor showed the highest homology (96.4%) with a lectin precursor of D. findleyanum and contained three mannose-binding sites. Greater amounts of DPA were found when the pseudobulbs were treated with stress factors including ultraviolet light, abscisic acid, hydrogen peroxide, and acetylene gas.

A nontoxic polypeptide oligomer with a fungicide potency under agricultural conditions which is equal or greater than that of their chemical counterparts.

There are literally hundreds of polypeptides described in the literature which exhibit fungicide activity. Tens of them have had attempted protection by patent applications but none, as far as we are aware, have found application under real agricultural conditions. The reasons behind may be multiple where the sensitivity to the Sun UV radiation can come in first place. Here we describe a multifunctional glyco-oligomer with 210 kDa which is mainly composed by a 20 kDa polypeptide termed Blad that has been previously shown to be a stable intermediary product of β-conglutin catabolism. This oligomer accumulates exclusively in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination. Blad-oligomer reveals a plethora of biochemical properties, like lectin and catalytic activities, which are not unusual per si, but are remarkable when found to coexist in the same protein molecule. With this vast range of chemical characteristics, antifungal activity arises almost as a natural consequence. The biological significance and potential technological applications of Blad-oligomer as a plant fungicide to agriculture, its uniqueness stems from being of polypeptidic in nature, and with efficacies which are either equal or greater than the top fungicides currently in the market are addressed.