Minor Polypeptides Research Articles

Physio-biochemical responses and crop performance analysis in chickpea upon botanical priming

Chickpea is a highly nutritious protein-rich source and one of the major crops to alleviate global malnutrition, but poor seed quality affects its productivity. Seed quality is essential for better crop establishment and higher yields, particularly in the uncertain climate change. The present study investigated the impact of botanical priming versus hydropriming and bavistin seed treatment on chickpea seeds. A detailed physiological (germination percentage, root and shoot length, vigour index) and biochemical (amylase, protease, dehydrogenase, phytase, and lipid peroxidation) analysis was carried out in order to assess the effect of priming treatments. Turmeric-primed seeds showed better germination rate (94.5%), seedling length, enzyme activity, and lower malondialdehyde (MDA) content. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the expression of minor polypeptides of albumin and globulin in the primed seeds. Moreover, field experiments indicated increased crop growth, vigour, days to 50% flowering, yield and its attributing traits in turmeric-primed seeds. Botanical priming can increase chickpea yield by up to 16% over the control group. This low-cost and eco-friendly technique enhances seed and crop performance, making it a powerful tool for augmenting chickpea growth. Therefore, chickpea growers must adopt botanical priming techniques to enhance the quality of seed and crop performance. Moreover, this approach is environmentally sustainable and can help conserve natural resources in the long term. Therefore, this new approach must be widely adopted across the agricultural industry to ensure sustainable and profitable farming practices.

Chromatography-Independent Fractionation and Newly Identified Molecular Features of the Adzuki Bean (Vigna angularis Willd.) β-vignin Protein.

Adzuki seed β-vignin, a vicilin-like globulin, has proven to exert various health-promoting biological activities, notably in cardiovascular health. A simple scalable enrichment procedure of this protein for further nutritional and functional studies is crucial. In this study, a simplified chromatography-independent protein fractionation procedure has been optimized and described. The electrophoretic analysis showed a high degree of homogeneity of β-vignin isolate. Furthermore, the molecular features of the purified protein were investigated. The adzuki bean β-vignin was found to have a native size of 146 kDa, and the molecular weight determined was consistent with a trimeric structure. These were identified in two main polypeptide chains (masses of 56–54 kDa) that are glycosylated polypeptides with metal binding capacity, and one minor polypeptide chain with a mass 37 kDa, wherein these features are absent. The in vitro analysis showed a high degree of digestibility of the protein (92%) and potential anti-inflammatory capacity. The results lay the basis not only for further investigation of the health-promoting properties of the adzuki bean β-vignin protein, but also for a possible application as nutraceutical molecule.

Metabolics and ionomics responses of tea leaves (Camellia sinensis (L.) O. Kuntze) to fluoride stress

Tea plant (Camellia sinensis (L.) O. Kuntze) is known to accumulate high concentrations of fluoride (F) in its leaves; however, the underlying mechanism of F accumulation remains unclear. The main objective of this study was to investigate the homeostatic self-defense mechanisms of tea leaves to F supplementation (0, 5, 20, and 50 mgL−1) by metabolomics and ionomics. We identified a total of 96 up-regulated and 40 down-regulated metabolites in tea leaves treated with F. Of these different compounds, minor polypeptides, carbohydrates and amino acids played valuable roles in the F-tolerating mechanism of tea plant. After F treatments, the concentrations of sodium (Na), ferrum (Fe), manganese (Mn), and molybdenum (Mo) were significantly increased in tea leaves, whereas the aluminum (Al) was decreased. These findings suggest that the ionic balance and metabolites are attributable to the development of F tolerance, providing new insight into tea plant adaptation to F stress.

THE USE OF AGENTS BASED ON THE BACTERIUM BACILLUS THURINGIENSIS VAR. ISRAELENSIS FOR MOSQUITO CONTROL

Mosquito control is necessary to improve the epidemic and, consequently, the sanitary and hygienic situation in human settlements. At the same time, the safest and more environmentally friendly way of controlling is not the fight against adult mosquitoes, but the treatment of reservoirs with microbiological larvicides based on entomopathogenic, aerobic, spore-forming, saprophytic bacteria Bacillus thuringiensis (de Barjac) (Bti). A new serotype of the bacterium B. thuringiensis was found in Israel in the Negev desert. This serotype being more active against larvae of blood-sucking and non-blood-sucking mosquitoes and midges than previously known serotypes, was named israelensis. Bti endotoxin is a typical insecticide with intestinal type of action for different mosquito species. For example, Bti H14 is highly insecticidal to the larvae of Aedes aegypti and Ae. albopictus at very low concentrations. The parasporal body (endotoxin crystal), a crystalline protein consisted of four main polypeptides and two minor polypeptides, possesses of a larvicidal action. Larvicidal activity is associated with a synergistic effect in a combination of four polypeptides. The possibility of development of resistance to products based on Bti and Bacillus sphaericus in populations of mosquitoes (Culicidae) was investigated. The use of domestic microbiological formulations based on Bti («Baktitsid», «Larviol-pasta», and «Antinat») was shown an eradication the larvae of bloodsucking mosquitoes and midges to be possible and rational, since they are not generated resistant populations of mosquitoes. This is confirmed by more than 30 years of the use of such formulations.

Effect of high-speed shearing treatment on dehulled walnut proteins

To understand more clearly about true characteristics of walnut protein, dehulled walnut kernels were used to prepare proteins, thus influences of polyphenols on proteins were minimized. Based on solubility, glutelin was found to be the predominant walnut protein (67.64% of total walnut proteins), meanwhile albumin and globulin accounted for 32.29%. SDS-PAGE and two-dimensional electrophoresis results showed that polypeptide profiles for albumin, globulin and glutelin exhibited significant differences. Walnut protein contained major polypeptides of MWs 30–35 kDa (pI 5.5–7.5) and 17–22 kDa (pI 9.0–10.0), along with minor polypeptides of MWs 50–73 kDa (pI 6.0–7.5) and 11–13 kDa (pI about 6.3). Disulfide bonds-linked polypeptides in walnut protein were determined with diagonal electrophoresis, and intermolecular disulfide bonds among polypeptides with MWs 30–34 kDa and 17–22 kDa were dominant. Further analysis by MALDI-TOF/TOF-MS showed that main components of walnut proteins were vicilin and legumin-like proteins. Through high-speed shearing (15,000 rpm) treatment at 60 °C for 5 min, walnut protein solubility were greatly increased from 24.33% to 62.81% at pH 8.0 and from 50.93% to 76.65% at pH 9.0. High-speed shearing treatment, especially at basic pH, could be a better way to improve protein solubility, with decreased particle sizes and more globulin and glutelin solubilized.

Isolation and Characterization of a Novel ATPase-Photosystem I Reaction Center Complex in the Chloroplast Thylakoid Membrane

A novel 1 MDa intra-membrane ‘super-complex’ dominated by the chloroplast ATP synthase and the photosystem I (PSI) reaction-center complex has been isolated from spinach thylakoid membranes. This super-complex has been characterized by assay of ATPase activity, negative stain electron microscopy, tandem mass spectrometry, and Western assay of subunits. High-resolution Orbitrap mass spectrometry was used to quantitatively analyze the presence and relative amount of the multiple major and minor polypeptide components in the super-complex. This is the first isolation of a higher order complex involving intact ATP synthase and Photosystem I to be identified and documented by negative stain electron microscopy. The importance of the ATPase-PSI super-complex in the thylakoid domain may imply efficient channeling of substrates and cofactors utilized for localized CO2 fixation. Studies supported by NIHGMS-038323 and the Henry Koffler Distinguished Professorship (WAC), and NIHDK-063491 (JPW).

Negative-sense virion RNA of segment 8 (NS) of influenza a virus is able to translate in vitro a new viral protein.

It was shown that full-length virion RNA of segment 8 of influenza virus A/Aichi/2/68 (H3N2) can initiate the synthesis of two major polypeptides with molecular weights of 23 and 13 kD and a minor polypeptide with a molecular weight of 19 kDa, which specifically reacted with the antibodies to the 30-membered peptide of the central part of the NSP protein of influenza A virus. Thus, the genomic-polarity RNA of segment 8 of influenza virus A has a translational template function. These data provide further confirmation of the concept of the bipolar (ambisens) strategy of functioning of the influenza A virus genome.

Structural modeling of the phycobilisome core and its association with the photosystems.

The phycobilisome (PBS) is a major light-harvesting complex in cyanobacteria and red algae. To obtain the detailed structure of the hemidiscoidal PBS core composed of allophycocyanin (APC) and minor polypeptide components, we analyzed all nine available 3D structures of APCs from different photosynthetic species and found several variants of crystal packing that potentially correspond to PBS core organization. Combination of face-to-face APC trimer crystal packing with back-to-back APC hexamer packing suggests two variants of the tricylindrical PBS core. To choose one of these structures, a computational model of the PBS core complex and photosystem II (PSII) dimer with minimized distance between the terminal PBS emitters and neighboring antenna chlorophylls was built. In the selected model, the distance between two types of pigments does not exceed 37 Å corresponding to the Förster mechanism of energy transfer. We also propose a model of PBS and photosystem I (PSI) monomer interaction showing a possibility of supercomplex formation and direct energy transfer from the PBS to PSI.

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.

Introduction to molecular biology of influenza a viruses.

This minireview presents an overview of current knowledge on virion structure, genome organization and basic events in the development of influenza A virus. The processes of entry, transcription/replication and viral release are described. In this context, the roles of viral proteins (including recently discovered minor polypeptides) in the subsequent stages of viral development are also discussed.

Bacillus thuringiensis subsp. israelensis and Its Dipteran-Specific Toxins

Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major (of 134, 128, 72 and 27 kDa) and at least two minor (of 78 and 29 kDa) polypeptides encoded respectively by cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba, all mapped on the 128 kb plasmid known as pBtoxis. These six δ-endotoxins form a complex parasporal crystalline body with remarkably high, specific and different toxicities to Aedes, Culex and Anopheles larvae. Cry toxins are composed of three domains (perforating domain I and receptor binding II and III) and create cation-selective channels, whereas Cyts are composed of one domain that acts as well as a detergent-like membrane perforator. Despite the low toxicities of Cyt1Aa and Cyt2Ba alone against exposed larvae, they are highly synergistic with the Cry toxins and hence their combinations prevent emergence of resistance in the targets. The lack of significant levels of resistance in field mosquito populations treated for decades with Bti-bioinsecticide suggests that this bacterium will be an effective biocontrol agent for years to come.

Effect of thermal processing on protein solubility of green gram (Phaseolus aureus) legume cultivars.

Green gram legume cultivars were analyzed for their protein solubility profile by fractionation in the raw form and also after heat processing. The results indicated that globulin fractions, which are present in major amounts that ranged from 79.5 to 85.4% significantly decreased after the heat treatment. This decrease was accompanied by a significant increase in the glutelin-3 fractions. The prolamine contents did not vary considerably after processing. The protein and non-protein nitrogen contents ranged from 22.6 to 26.2% and 2.3 to 2.7% in the legume cultivars, respectively. The antinutritional factors like total polyphenol and phytic phosphorous were also determined. The accumulation of polyphenols was in the seed coat portion of the legume where as that of phytic phosphorus was in the cotyledons. SDS - PAGE profiles of all the three green gram cultivars had five major polypeptides (molecular weight 15, 18, 20, 45 and 60kDa) in the total protein composition. Wide variation in electrophoresis pattern was observed after heat processing. Thermal treatment increased the insoluble protein fractions and eliminated the minor polypeptide bands below 14.3kDa in the green gram cultivars.

Sequence variation at multiple loci influences red cell hemoglobin concentration

AbstractA substantial genetic contribution underlies variation in baseline peripheral blood counts. We performed quantitative trait locus/loci analyses to identify chromosome regions harboring genes influencing red cell hemoglobin concentration using the cell hemoglobin concentration mean (CHCM), a directly measured parameter analogous to the mean cell hemoglobin concentration. Fourteen significant loci (gene symbols Chcmq1-Chcmq14) were detected. Seven of these influenced CHCM in a sex-specific fashion, and 2 showed significant interactive effects (epistasis). For quantitative trait locus/loci detected in multiple crosses, confidence intervals were narrowed using statistical and bioinformatic approaches. Two strong candidate genes emerged and were further analyzed: adult β-globin (Hbb) for Chcmq3 on Chr 7, and transferrin (Trf) for Chcmq2 on Chr 9. High and low allele parental strains in crosses detecting Chcmq3 segregate 100% with the known ancestral haplotype blocks, hemoglobin (Hb) diffuse (Hbbd) and Hb single (Hbbs), respectively. Hbbd consists of nonidentical major and minor polypeptides and exhibits an increased positive charge relative to Hbbs due to the net loss of 2 negative residues in the Hbbdminor polypeptide, resulting in a pI of 7.85 versus 7.13. Thus, as shown in human erythrocytes, positively charged Hbs are associated with cell dehydration and increased CHCM in mouse erythrocytes.

Dimerisation of the Rhodobacter sphaeroides RC–LH1 photosynthetic complex is not facilitated by a GxxxG motif in the PufX polypeptide

In purple photosynthetic bacteria the initial steps of light energy transduction take place in an RC–LH1 complex formed by the photochemical reaction centre (RC) and the LH1 light harvesting pigment-protein. In Rhodobacter sphaeroides, the RC–LH1 complex assembles in a dimeric form in which two RCs are surrounded by an S-shaped LH1 antenna. There is currently debate over the detailed architecture of this dimeric RC–LH1 complex, with particular emphasis on the location and precise function of a minor polypeptide component termed PufX. It has been hypothesised that the membrane-spanning helical region of PufX contains a GxxxG dimerisation motif that facilitates the formation of a dimer of PufX at the interface of the RC–LH1 dimer, and more specifically that the formation of this PufX dimer seeds assembly of the remaining RC–LH1 dimer (J. Busselez et al., 2007). In the present work this hypothesis was tested by site directed mutagenesis of the glycine residues proposed to form the GxxxG motif. Mutation of these glycines to leucine did not decrease the propensity of the RC–LH1 complex to assemble in a dimeric form, as would be expected from experimental studies of the effect of mutation on GxxxG motifs in other membrane proteins. Indeed increased yields of dimer were seen in two of the glycine-to-leucine mutants constructed. It is concluded that the PufX from Rhodobacter sphaeroides does not contain a genuine GxxxG helix dimerisation motif.

Proteome of the Large Pseudomonas Myovirus 201φ2-1: DELINEATION OF PROTEOLYTICALLY PROCESSED VIRION PROTEINS

Pseudomonas chlororaphis phage 201 phi 2-1 produces a large structurally complex virion, including the products of 89 phage genes. Many of these proteins are modified by proteolysis during virion maturation. To delineate the proteolytic maturation process, 46 slices from an SDS-polyacrylamide gel were subjected to tryptic digestion and then HPLC-electrospray ionization-tandem mass spectrometry analysis. The scale of the experiment allowed high sequence coverage and detection of mass spectra assigned to peptides with one end produced by trypsin and the other end derived from a maturation cleavage (semitryptic peptides). Nineteen cleavage sites were detected in this way. From these sites, a cleavage motif was defined and used to predict the remaining cleavages required to explain the gel mobility of the processed polypeptide species. Profiling the gel with spectrum counts for specific polypeptide regions was found to be helpful in deducing the patterns of proteolysis. A total of 29 cleaved polypeptides derived from 19 gene products were thus detected in the mature 201 phi 2-1 virion. When combined with bioinformatics analyses, these results revealed the presence of head protein-encoding gene modules. Most of the propeptides that were removed from the virion after processing were acidic, whereas the mature domain remaining in the virion was nearly charge-neutral. For four of these processed virion proteins, the portions remaining in the mature virion were mutually homologous. Spectrum counts were found to overestimate the relative quantity of minor polypeptide species in the virion. The resulting sensitivity for minor species made it possible to observe a small amount of general proteolysis that also affected the virions.

Adenovirus Virion Stability and the Viral Genome: Size Matters

Ongoing investigations into the fundamental aspects of virus biology continue to provide crucial insights for the development of more effective virus-based therapeutics. Within the field of gene therapy, these discoveries have translated into virus vectors with improved tissue tropism, prolonged transgene expression, and enhanced safety. However, radical modifications to the viral genome or capsid constituents can have marked effects on virion stability. In particular, recent insights into the role of viral genomes in contributing to the physical stability of the virion highlight the need to reconsider this heretofore underappreciated aspect of vector design. Here we discuss the impact of the genome on virion stability, with particular emphasis on the development of adenovirus (Ad)-based vectors.

Purification, Cloning, and Identification of Two Thaumatin-like Protein Isoforms in Jelly Fig (Ficus awkeotsang) Achenes

Jelly curd used for a popular summer drink in Taiwan is prepared by extracting the pericarpial portion of jelly fig (Ficus awkeotsang Makino) achenes. The two most abundant proteins found in jelly curd have been identified as a pectin methylesterase and a chitinase. A method was developed to purify the next abundant protein by 40% ammonium sulfate precipitation and flowing through Mono Q chromatography. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, the purified protein migrated as a polypeptide of 20 kDa in the absence of beta-mercaptoethanol but split into a minor polypeptide of 20 kDa and a major polypeptide of 27 kDa in the presence of this reducing agent. Two cDNA fragments encoding precursor polypeptides of two putative thaumatin-like protein isoforms were obtained by polymerase chain reaction cloning and subsequently overexpressed in Escherichia coli to generate recombinant proteins for antibody preparations. Immunological detection and mass spectrometric analyses indicated that the two split polypeptides were thaumatin-like protein isoforms encoded by the two cloned cDNA fragments.

Phosphorylation of Thyroid Hormone Receptor-associated Nuclear Receptor Corepressor Holocomplex by the DNA-dependent Protein Kinase Enhances Its Histone Deacetylase Activity

It is well documented that unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of specific cellular target genes by acting in concert with a corepressor complex harboring histone deacetylase (HDAC) activity. To fully explore the cofactors that interact with the transcriptionally repressive form of TR, we biochemically isolated a multiprotein complex that assembles on a TR.retinoid X receptor (RXR) heterodimer in HeLa nuclear extracts and identified its polypeptide components by mass spectrometry. A subset of TR.RXR-associated polypeptides included NCoR, SMRT, TBL1, and HDAC3, which represent the core components of a previously described NCoR/SMRT corepressor complex. We also identified several polypeptides that constitute a DNA-dependent protein kinase (DNA-PK) enzyme complex, a regulator of DNA repair, recombination, and transcription. These polypeptides included the catalytic subunit DNA-PKcs, the regulatory subunits Ku70 and Ku86, and the poly(ADP-ribose) polymerase 1. Density gradient fractionation and immunoprecipitation analyses provided evidence for the existence of a high molecular weight TR.RXR.corepressor holocomplex containing both NCoR/SMRT and DNA-PK complexes. Chromatin immunoprecipitation studies confirmed that unliganded TR.RXR recruits both complexes to the triiodothyronine-responsive region of growth hormone gene in vivo. Interestingly, DNA-PKcs, a member of the phosphatidylinositol 3-kinase family, was found to phosphorylate HDAC3 when the purified TR.RXR.corepressor holocomplex was incubated with ATP. This phosphorylation was accompanied by a significant enhancement of the HDAC activity of this complex. Collectively, our results indicated that DNA-PK promotes the establishment of a repressive chromatin at a TR target promoter by enhancing the HDAC activity of the receptor-bound NCoR/SMRT corepressor complex.

The Ectodomain of the Viral Receptor YueB Forms a Fiber That Triggers Ejection of Bacteriophage SPP1 DNA

The irreversible binding of bacteriophages to their receptor(s) in the host cell surface triggers release of the naked genome from the virion followed by transit of viral DNA to the host cell cytoplasm. We have purified, for the first time, a receptor from a Gram-positive bacterium that is active to trigger viral DNA ejection in vitro. This extracellular region ("ectodomain") of the Bacillus subtilis protein YueB (YueB780) was a 7 S elongated dimer forming a 36.5-nm-long fiber. YueB780 bound to the tail tip of bacteriophage SPP1. Although a stable receptor-phage interaction occurred between 0 and 37 degrees C, complete blocking of phage DNA release or partial ejection events were observed at temperatures below 15 degrees C. We also showed that the receptor was exposed to the B. subtilis surface. YueB differed structurally from phage receptors from Gram-negative bacteria. Its properties revealed a fiber spanning the full length of the 30-nm-thick peptidoglycan layer. The fiber is predicted to be anchored in the cell membrane through transmembrane segments. These features, highly suitable for a virus receptor in Gram-positive bacteria, are very likely shared by a large number of phage receptors.

Biochemical characterization of a pollen-specific cDNA encoding polygalacturonase in Lilium longiflorum

A pollen-specific transcript, designated LLP-A1.1, in lily ( Lilium longiflorum Thunb. cv. Snow Queen) plants was isolated and characterized. The full-length cDNA encodes polygalacturonase (PG) having a sequence of 413 amino acids, a calculated molecular mass of 44 kDa, and a calculated p I of 8.1. Assessment of the hydropathy shows that the protein contains a hydrophobic segment at the N-terminus, indicating the presence of a putative signal peptide. The LLP-PG sequence displays significant resemblance to known pollen PG from various species and a group of major allergens including Phl p 13 of timothy grass and Pla a 2 of London planetree. Antiserum was raised against the overexpressed rLLP-PG protein in E. coli. Affinity-purified antibodies were prepared from antiserum to investigate the specificity and distribution of the protein during development. Immunoblot analyses of total protein from floral and vegetative organs confirmed that LLP-PG accumulated to detectable levels only in a discrete stage of anther development. It is a heterogeneous glycoprotein, consisting of four major and two minor polypeptides. Premature drying of developing anthers indicated that the lily LlpPG gene expression was not induced by desiccation that naturally occurs in pollen prior to anthesis.