Polypeptide Subunits Research Articles

The alternative enzymes-bearing tunicates lack multiple widely distributed genes coding for peripheral OXPHOS subunits

The respiratory chain alternative enzymes (AEs) NDX and AOX from the tunicate Ciona intestinalis (Ascidiacea) have been xenotopically expressed and characterized in human cells in culture and in the model organisms Drosophila melanogaster and mouse, with the purpose of developing bypass therapies to combat mitochondrial diseases in human patients with defective complexes I and III/IV, respectively. The fact that the genes coding for NDX and AOX have been lost from genomes of evolutionarily successful animal groups, such as vertebrates and insects, led us to investigate if the composition of the respiratory chain of Ciona and other tunicates differs significantly from that of humans and Drosophila, to accommodate the natural presence of AEs. We have failed to identify in tunicate genomes fifteen orthologous genes that code for subunits of the respiratory chain complexes; all of these putatively missing subunits are peripheral to complexes I, III and IV in mammals, and many are important for complex-complex interaction in supercomplexes (SCs), such as NDUFA11, UQCR11 and COX7A. Modeling of all respiratory chain subunit polypeptides of Ciona indicates significant structural divergence that is consistent with the lack of these fifteen clear orthologous subunits. We also provide evidence using Ciona AOX expressed in Drosophila that this AE cannot access the coenzyme Q pool reduced by complex I, but it is readily available to oxidize coenzyme Q molecules reduced by glycerophosphate oxidase, a mitochondrial inner membrane-bound dehydrogenase that is not involved in SCs. Altogether, our results suggest that Ciona AEs might have evolved in a mitochondrial inner membrane environment much different from that of mammals and insects, possibly without SCs; this correlates with the preferential functional interaction between these AEs and non-SC dehydrogenases in heterologous mammalian and insect systems. We discuss the implications of these findings for the applicability of Ciona AEs in human bypass therapies and for our understanding of the evolution of animal respiratory chain.

High-pressure processing (HPP) of phycocyanin extract solutions: Enhancing stability through molecular interactions

C-Phycocyanin (PC) has been proposed as a replacement for synthetic colors in foods. However, C-PC stability is adversely affected during food processing due to exposure to high temperature, pressure, oxygen concentration or extreme pHs. This study assessed the degradation mechanisms of C-PC under a nonthermal pasteurization technology, i.e., High-Pressure Processing (HPP), and identified potential processing protocols to arrest C-PC instability at low pH. Aqueous C-PC solutions (pH:4–6) were subject to HPP (500–600 MPa) for 1–5 min. C-PC stability was monitored based on changes in the photophysical properties of its fluorophores (phycocyanobilin chromophore and aromatic amino acid residues) and particle size. pH significantly affected C-PC's stability towards HPP (<stability at < pH). Sharper hypsochromic (blue) shifts, lower intensity, and higher anisotropy were observed as pressure and treatment time increased, which suggested conformational changes in C-PC's polypeptides, followed by aggregation. pH adjustment after HPP treatments resulted in a significant reduction in color loss and aggregation, possibly due to partial stabilization of the polypeptide subunits' conformation.

Detection of NCF-2 Gene Expression in Egyptian Children with Chronic Granulomatous Disease

Abstract Background Chronic granulomatous disease (CGD) is a rare inherited primary immunodeficiency disorder (PID) which is characterized by enhanced susceptibility to severe bacterial and fungal infections. It results from defects in one of the five polypeptide subunits of the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) complex; CYBB (gp91-phox), NCF-1 (p47phox), CYBA (p22-phox), NCF-2 (p67-phox), or NCF-4 (p40-phox) with a resulting failure of the phagocytes in generating a variety of microbicidal reactive oxygen radicals during respiratory burst. Objectives This work intended to diagnose autosomal recessive (AR)-type of CGD by detection of NCF-2 gene expression using real time RT-PCR as a cheaper diagnostic method for CGD subtypes categorization among CGD children. Subjects and methods This case-control study was conducted on 15 children provisionally diagnosed as CGD patients by the use of dihydrorhodamine (DHR) stimulation index (Group I) in addition to 12 mothers and 8 fathers of the studied patients (Group IIa and IIb respectively) to detect the genetic mutations in carriers, if any, and 14 apparently healthy children as a control group (Group III). In the present study, cases with a fold change of NCF-2 gene expression less than 0.67 were considered defective for NCF-2 gene expression. At this cut-off value, from our molecularly studied subjects, 2 CGD cases and one mother showed underexpression of NCF-2 gene. In conclusion, defect in the NCF-2 gene, which encodes gp67-phox of the oxidase enzyme could be done by RT-PCR as a cheaper diagnostic method for CGD without the need to use complex and expensive methodologies such as northern blot, or genomic DNA sequencing.

Wiggle and Shake: Managing and Exploiting Conformational Dynamics during Proteasome Biogenesis.

The 26S proteasome is the largest and most complicated protease known, and changes to proteasome assembly or function contribute to numerous human diseases. Assembly of the 26S proteasome from its ~66 individual polypeptide subunits is a highly orchestrated process requiring the concerted actions of both intrinsic elements of proteasome subunits, as well as assistance by extrinsic, dedicated proteasome assembly chaperones. With the advent of near-atomic resolution cryo-electron microscopy, it has become evident that the proteasome is a highly dynamic machine, undergoing numerous conformational changes in response to ligand binding and during the proteolytic cycle. In contrast, an appreciation of the role of conformational dynamics during the biogenesis of the proteasome has only recently begun to emerge. Herein, we review our current knowledge of proteasome assembly, with a particular focus on how conformational dynamics guide particular proteasome biogenesis events. Furthermore, we highlight key emerging questions in this rapidly expanding area.

Serum sCD25 is an indicator for rheumatoid arthritis-associated interstitial lung disease.

CD25 (IL-2Rα) is one of IL-2 receptor's polypeptide subunits, and its soluble form is increased in patients with various inflammatory or autoimmune diseases. This study aimed to evaluate the clinical correlation of serum soluble CD25 (sCD25) with interstitial lung disease (ILD) in rheumatoid arthritis (RA) patients. 294 RA patients, including 72 in the discovery cohort (15 patients with ILD, 57 patients without ILD), 222 in the validation cohort (41 patients with ILD and 181 patients without ILD), and 58 healthy controls (HCs) were recruited. High-resolution computed tomography (HRCT) scan provided evidence and patterns of RA-ILD. Serum sCD25 concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Clinical and laboratory data were recorded and the association with sCD25 was also analysed. In the discovery cohort, 16 RA-related molecules including cytokines, chemokines and functional soluble cell surface proteins were investigated. The results showed that sCD25 was significantly higher in RA-ILD than in RA-no-ILD group (p=0.004). ROC analysis also showed RA-ILD was discriminated with RA-no-ILD by sCD25 (AUC=0.695, 95% CI=0.541-0.849). Logistics regression demonstrated that sCD25 was one of the risk factors of RA-ILD. This result was further confirmed in validation cohort (p<0.001). According to the cut-off value in the discovery cohort, the sensitivity and specificity of sCD25 in RA-ILD were 51.2%, 77.3%, respectively. Compared with RA-no-ILD, serum level of sCD25 was also higher in different HRCT patterns including UIP, NSIP and RA-ILA. The ROC curves revealed sCD25 as diagnostic marker in UIP, NSIP and RA-ILA (with AUCs of 0.730, 0.761, and 0. 694, respectively, p<0.05). The result indicated that sCD25 was a biomarker for RA-ILD subtypes. Although sCD25 was not correlated with HRCT scores, it was significantly higher in consolidation pattern by HRCT. sCD25 was significantly elevated in RA-ILD (including UIP, NSIP and RA-ILA) compared to RA-no-ILD and HCs, which supports their value as a potential biomarker in RA-ILD screening and assessment.

Coronavirus disease 2019 vaccines: challenges of using global mass vaccination to achieve herd immunity.

Coronavirus disease 2019 vaccines: challenges of using global mass vaccination to achieve herd immunity.

Development of C4 Biochemistry and Change in Expression of Markers for Photosystems I and II in the Single-Cell C4 Species, Bienertia sinuspersici

Bienertia sinuspersici is one of four identified terrestrial plants that perform C4 photosynthesis within a single chlorenchyma cell via the compartmentation of organelles and photosynthetic enzymes. The patterns of accumulation of key photosynthetic enzymes and transcripts in developing leaves were examined using immunolocalization and in situ hybridization. The polypeptides of Rubisco large subunit (RbcL) and pyruvate Pi dikinase (PPDK) accumulated equally in all chloroplasts before the formation of two intracellular cytoplasmic compartments: the central (CCC) and peripheral (PCC) cytoplasmic compartments. The differential accumulation of these enzymes was not completed until the leaf had reached maturity, indicating that the transition from C3 to C4 photosynthesis occurred during leaf maturation. In mature chlorenchyma cells, RbcL accumulated 20-fold higher in the CCC than in the PCC, while PPDK exhibited a concentration gradient that was the lowest in the chloroplasts in the central region of the CCC and the highest in PCC chloroplasts. The pattern of rbcL transcript accumulation followed that of its polypeptides in developing leaves, suggesting that the expression of this gene was likely controlled by transcriptional and/or post-transcriptional processes. Immunocytochemical results examining the distribution of photosystems I and II in the chloroplasts of chlorenchyma cells from mature leaves showed that PSII is more abundant in chloroplasts of the central compartment, whereas PSI is higher in those of the peripheral compartment. The quantitative real-time PCR results of rbcL, psbA, and psaB transcripts from the isolated chloroplasts of each compartment further supported this observation. Our results suggest that multiple levels of regulation play a role in controlling the differential accumulation of photosynthetic gene expression in the dimorphic chloroplasts of single-cell C4 species during leaf development.

The role of the γ subunit in the photosystem of the lowest-energy phototrophs

Purple phototrophic bacteria use a 'photosystem' consisting of light harvesting complex 1 (LH1) surrounding the reaction centre (RC) that absorbs far-red-near-infrared light and converts it to chemical energy. Blastochloris species, which harvest light >1000 nm, use bacteriochlorophyll b rather than the more common bacteriochlorophyll a as their major photopigment, and assemble LH1 with an additional polypeptide subunit, LH1γ, encoded by multiple genes. To assign a role to γ, we deleted the four encoding genes in the model Blastochloris viridis. Interestingly, growth under halogen bulbs routinely used for cultivation yielded cells displaying an absorption maximum of 825 nm, similar to that of the RC only, but growth under white light yielded cells with an absorption maximum at 972 nm. HPLC analysis of pigment composition and sucrose gradient fractionation demonstrate that the white light-grown mutant assembles RC-LH1, albeit with an absorption maximum blue-shifted by 46 nm. Wavelengths between 900-1000 nm transmit poorly through the atmosphere due to absorption by water, so our results provide an evolutionary rationale for incorporation of γ; this polypeptide red-shifts absorption of RC-LH1 to a spectral range in which photons are of lower energy but are more abundant. Finally, we transformed the mutant with plasmids encoding natural LH1γ variants and demonstrate that the polypeptide found in the wild type complex red-shifts absorption back to 1018 nm, but incorporation of a distantly related variant results in only a moderate shift. This result suggests that tuning the absorption of RC-LH1 is possible and may permit photosynthesis past its current low-energy limit.

Cell wall channels of Rhodococcus species: identification and characterization of the cell wall channels of Rhodococcus corynebacteroides and Rhodococcus ruber

The cell wall of Rhodococcus corynebacteroides formerly known as Nocardia corynebacteroides contains cell wall channels that are responsible for the cell wall permeability of this bacterium. Based on partial sequencing of the polypeptide subunits and a BLAST search, we identified one polypeptide of R. corynebacteroides (PorARc) and two polypeptides (PorARr and PorBRr) from the closely related bacterium Rhodococcus ruber. The corresponding genes, porARc (606 bp), porARr (702 bp), and porBRr (540 bp) are constituents of the known genome of R. corynebacteroides DSM-20151 and R. ruber DSM-43338, respectively. porARr and porBRr of R. ruber are possibly forming a common operon coding for the polypeptide subunits of the cell wall channel. The genes coding for PorARc and for PorARr and PorBRr without signal peptide were separately expressed in the porin-deficient Escherichia coli BL21DE3Omp8 strain and the proteins were purified to homogeneity. All proteins were checked for channel formation in lipid bilayers. PorARc formed channels with characteristics that were very similar to those of a previous study. The proteins PorARr and PorBRr expressed in E. coli could alone create channels in lipid bilayer membranes, despite the possibility that the two corresponding genes form a porin operon and that both subunits possibly form the cell wall channels in vivo. Based on amino acid sequence comparison of a variety of proteins forming cell wall channels in bacteria of the suborder Corynebacterineae, it seems very likely that PorARc, PorARr, and PorBRr are members of a huge family of proteins (PF09203) that form MspA-like cell wall channels.

Comparative Analysis of Type 1 and Type Z Protein Phosphatases Reveals D615 as a Key Residue for Ppz1 Regulation.

Type 1 Ser/Thr protein phosphatases are represented in all fungi by two enzymes, the ubiquitous PP1, with a conserved catalytic polypeptide (PP1c) and numerous regulatory subunits, and PPZ, with a C-terminal catalytic domain related to PP1c and a variable N-terminal extension. Current evidence indicates that, although PP1 and PPZ enzymes might share some cellular targets and regulatory subunits, their functions are quite separated, and they have individual regulation. We explored the structures of PP1c and PPZ across 57 fungal species to identify those features that (1) are distinctive among these enzymes and (2) have been preserved through evolution. PP1c enzymes are more conserved than PPZs. Still, we identified 26 residues in the PP1 and PPZ catalytic moieties that are specific for each kind of phosphatase. In some cases, these differences likely affect the distribution of charges in the surface of the protein. In many fungi, Hal3 is a specific inhibitor of the PPZ phosphatases, although the basis for the interaction of these proteins is still obscure. By in vivo co-purification of the catalytic domain of ScPpz1 and ScHal3, followed by chemical cross-linking and MS analysis, we identified a likely Hal3-interacting region in ScPpz1 characterized by two major and conserved differences, D566 and D615 in ScPpz1, which correspond to K210 and K259 in ScPP1c (Glc7). Functional analysis showed that changing D615 to K renders Ppz1 refractory to Hal3 inhibition. Since ScHal3 does not regulate Glc7 but it inhibits all fungal PPZ tested so far, this conserved D residue could be pivotal for the differential regulation of both enzymes in fungi.

Purification and structural characterization of lectin with antibacterial and anticancer properties from grubs of hide beetle, Dermestes frischii

Lectins or haemagglutinins are diverse classes of non-immune proteins; they bind to carbohydrates and are abundant in nature. In the present study, a coleopteran lectin from grubs of hide beetle, Dermestes frischii called DFL, was purified by glutaraldehyde (fixative-agent) fixed hen erythrocytes and characterized further for its functional properties. The purified DFL was stable between pH range 5 to 9 and heat-stable up to 50 °C. It was insensitive to EDTA and did not require any divalent cations. DFL native molecular mass was approximately 69 kDa with three different polypeptide subunits of 33 (pI ~4.4), 22 (pI ~6) and 14 (pI ~4.4) kDa. Haemagglutinating activity of DFL was highly inhibited by N-acetyl-D-glucosamine. DFL partial peptide sequences obtained from peptide mass fingerprinting experiments matched with amino acid sequences of lectins from different organisms confirmed its nature. Biological properties of purified DFL namely antibacterial and bacterial agglutination experiments revealed that DFL have both the effects against laboratory cultures of Aeromonas hydrophila, Enterococcus faecalis, Escherichia coli and habitat bacterial isolates of Staphylococcus cohnii and Bacillus cereus. In addition, the DFL exhibited substantial anticancer properties against HeLa cells. These results concluded that purified DFL could serve as a potent therapeutic agent for various biomedical applications.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis of Proteins.

Most analytical electrophoreses of proteins are achieved by separation in polyacrylamide gels under conditions that ensure dissociation of proteins into individual polypeptide subunits and minimize aggregation. Most commonly, the anionic detergent sodium dodecyl sulfate (SDS) is used in combination with a reducing agent (β-mercaptoethanol or dithiothreitol) and with heating to dissociate proteins before loading onto the gel. SDS binding denatures the polypeptides and imparts a negative charge that masks their intrinsic charge. The amount of SDS bound is generally sequence-independent and proportional to molecular weight; at saturation, approximately one SDS molecule is bound per two amino acids, or ∼1.4 g of SDS per gram of polypeptide. Therefore, the migration of SDS-polypeptide complexes in an electric field is proportional to the relative size of the polypeptide chain, and its molecular weight can be estimated by comparison to protein markers of known molecular weight. However, hydrophobicity, highly charged sequences, and certain posttranslational modifications such as glycosylation or phosphorylation may also influence migration. Thus, the apparent molecular weight of modified proteins does not always accurately reflect the mass of the polypeptide chain. This protocol describes preparation and running of SDS-PAGE gels, followed by staining to detect proteins using Coomassie Brilliant Blue. Finally, the stained SDS-PAGE gel may be scanned to an image or preserved by drying.

CCT6A may act as a potential biomarker reflecting tumor size, lymphatic metastasis, FIGO stage, and prognosis in cervical cancer patients.

ObjectiveChaperonin‐containing tailless complex polypeptide subunit 6A (CCT6A) is a critical regulator and newly identified clinical biomarker of several cancers, while its correlation with the clinical characteristics and prognosis of cervical cancer patients is unclear. Therefore, this study aimed to explore this issue.MethodsChaperonin‐containing tailless complex polypeptide subunit 6A expression in tumor and tumor‐adjacent tissues from 198 cervical cancer patients who underwent resection were detected by immunohistochemistry assay and reverse transcription‐quantitative polymerase chain reaction. Besides, the clinicopathological features and survival data of cervical cancer patients were collected.ResultsChaperonin‐containing tailless complex polypeptide subunit 6A protein and mRNA levels were both increased in tumor tissues compared with tumor‐adjacent tissues (both p < 0.001). Receiver operating characteristic curves showed that CCT6A protein (AUC: 0.774, 95% CI: 0.729–0.819) and mRNA levels (AUC: 0.904, 95% CI: 0.874–0.934) well discriminated tumor tissues from tumor‐adjacent tissues. Besides, correlation analyses found that CCT6A protein and mRNA levels were positively correlated with lymph node metastasis and FIGO stage (all p < 0.05), apart from which CCT6A mRNA level was also positively associated with tumor size (p = 0.032). In addition, CCT6A protein and mRNA levels were negatively correlated with accumulating disease‐free survival (both p < 0.05); meanwhile CCT6A mRNA level was negatively associated with accumulating overall survival as well (p = 0.010).ConclusionChaperonin‐containing tailless complex polypeptide subunit 6A is elevated in tumor tissues, and its high expression associates with larger tumor size, lymph node metastasis, higher FIGO stage, and worse prognosis in cervical cancer patients.

Homozygous ITGA3 Missense Mutation in Adults in a Family with Syndromic Epidermolysis Bullosa (ILNEB) without Pulmonary Involvement

Homozygous ITGA3 Missense Mutation in Adults in a Family with Syndromic Epidermolysis Bullosa (ILNEB) without Pulmonary Involvement

Towards improved understanding of intersubunit interactions in modular polyketide biosynthesis: Docking in the enacyloxin IIa polyketide synthase

Modular polyketide synthases (PKSs) are molecular-scale assembly lines comprising multiple gigantic polypeptide subunits. Faithful ordering of the subunits is mediated by extreme C- and N-terminal regions called docking domains (DDs). Decrypting how specificity is achieved by these elements is important both for understanding PKS function and modifying it to generate useful polyketide analogues for biological evaluation. Here we report the biophysical and structural characterisation of all six PKS/PKS interfaces in the unusual, chimaeric cis-AT/trans-AT PKS pathway responsible for biosynthesis of the antibiotic enacyloxin IIa in Burkholderia ambifaria. Taken together with previous work, our data reveal that specificity is achieved in the enacyloxin PKS by deploying at least three functionally orthogonal classes of DDs. We also demonstrate for the first time that cis-AT PKS subunits incorporate DDs with intrinsically disordered character, reinforcing the utility of such regions for achieving both medium affinity and high specificity at PKS intersubunit junctions. Overall, this work substantially increases the number of orthogonal DDs available for creating novel, highly-specific interfaces within cis- and trans-AT PKSs and their hybrids. It also reveals unexpected sequence/structure relationships in PKS DDs, identifying major current limitations to both accurately predicting and categorising these useful protein–protein interaction motifs.

Real time structural search of the Protein Data Bank.

Detection of protein structure similarity is a central challenge in structural bioinformatics. Comparisons are usually performed at the polypeptide chain level, however the functional form of a protein within the cell is often an oligomer. This fact, together with recent growth of oligomeric structures in the Protein Data Bank (PDB), demands more efficient approaches to oligomeric assembly alignment/retrieval. Traditional methods use atom level information, which can be complicated by the presence of topological permutations within a polypeptide chain and/or subunit rearrangements. These challenges can be overcome by comparing electron density volumes directly. But, brute force alignment of 3D data is a compute intensive search problem. We developed a 3D Zernike moment normalization procedure to orient electron density volumes and assess similarity with unprecedented speed. Similarity searching with this approach enables real-time retrieval of proteins/protein assemblies resembling a target, from PDB or user input, together with resulting alignments (http://shape.rcsb.org).

The role of COL6A1and PD-1 expressions in renal cell carcinoma.

The COL6A1 is a gene encoding the alpha 1 polypeptide subunit of collagen 6 (COL6A1), an extracellular matrix protein subunit. Programmed cell death receptor-1 (PD-1) and its ligand, programmed cell death receptor ligand-1 (PD-L1) have been shown to have a prognostic significance in clear cell renal cell carcinomas (RCCs). In this study, we evaluated the expressions of COL6A1 and PD-1 in four different RCC subtypes. A total of 161 radical nephrectomy and nephron-sparing surgery cases with RCCs from five different health care centers were included in this study. Clinical data of the cases were taken from electronic records of the institutions. The pathological data were collected by an expert uropathologist and re-evaluated with slides obtained from paraffin blocks of the cases. The correlation of COL6A1 and PD-1 expression with sex, age, tumor type, lymphovascular invasion (LVI), World Health Organization/International Society of Urological Pathology (WHO/ISUP) grade, and tumor stage (pT) was analyzed with the Pearson chi-squared test. Patients with sarcomatoid RCC and clear cell RCC had significantly higher COL6A1 scores and intensities than in other types of RCC (p=0.004 and p=0.002, respectively). WHO/ISUP grade and, COL6A1 and PD-1 staining scores also showed positive correlation (r=0.230, p=0.004 and r=0.277, p=0.001, respectively for COL6A1 and r=0.191, p=0.018 and r=0.166, p=0.041, respectively for PD-1). The staining scores and intensities of COL6A1 and PD-1 were not different between the patients with positive and negative LVI (p>0.05). In high-grade RCCs, we found the relationship between immunohistochemical staining scores of COL6A1 and PD-1 proteins and clinical, demographic, and histopathological parameters. Our results proved that COL6A1 and PD-1 are really promising proteins as prognostic parameters and for targeted immunotherapy.

Effect of germination time on physico-chemical, functional, pasting, rheology and electrophoretic characteristics of chickpea flour

The aim of the study was to investigate the effect of germination time on physical and cooking properties of chickpea grain, and physico-chemical, functional, pasting, rheological, structural and electrophoretic properties of chickpea flour. The study showed that with increase in germination time, L* and b* values, bulk density, cooking time and gruel solid loss of chickpea grain decreased significantly (p ≤ 0.05) whereas a* value, dry matter loss, swelling capacity and water-uptake ratio of chickpea grain increased significantly (p ≤ 0.05). Moisture, protein, crude fiber, ash and total carbohydrate of chickpea flour varied significantly (p ≤ 0.05) with germination time from 8.60 to 11.27%, 21.94 to 24.19%, 2.45 to 3.91%, 3.66 to 2.84%, 3.91 to 3.30% and 60.05 to 53.48%, respectively. Functional properties (oil absorption capacity, water absorption capacity and solubility index), antioxidant activity and total phenolic compound of chickpea flour increased significantly with increase in germination time. Increase in germination time showed significant decrease in tannins (232.57 to 67.61 mg TAE/100 g), phytic acid (1064.54–453.72 mg/100 g), pasting and thermal properties of chick pea flour. All the germinated chickpea flour samples showed pseudoplastic behaviour and their mechanical spectra showed weak gel like behaviour (G′ > G″). FT-IR spectroscopy of chickpea flour samples did not showed any variations in spectra but change in intensity of bands was observed. The SDS-PAGE of chickpea flour samples showed polypeptide subunit from 126 to 10 KDa with varied intensity of bands due to increase in germination time. The process of germination improved the nutritional profile, functional and cooking properties of chickpea grain and flour thereof and could enhance its utilization in development of functional food products.

In vitro toxicological characterisation of the antifungal compound soybean toxin (SBTX)

Soybean toxin (SBTX) is a protein isolated from soybean seeds and composed of two polypeptide subunits (17 and 27 kDa). SBTX has in vitro activity against phytopathogenic fungi such as Cercospora sojina, Aspergillus niger, and Penicillium herguei, and yeasts like Candida albicans, C. parapsilosis, Kluyveromyces marxiannus, and Pichia membranifaciens. The present study aimed to analyze in vitro whether SBTX causes any side effects on non-target bacterial and mammalian cells that could impede its potential use as a novel antifungal agent. SBTX at 100 μg/mL and 200 μg/mL did not hinder the growth of the bacteria Salmonella enterica (subspecies enterica serovar choleraesuis), Bacillus subtilis (subspecies spizizenii) and Staphylococcus aureus. Moreover, SBTX at concentrations up to 500 μg/mL did not significantly affect the viability of erythrocytes, neutrophils, and human intestinal Caco-2 cells. To study whether SBTX could induce relevant alterations in gene expression, in vitro DNA microarray experiments were conducted in which differentiated Caco-2 cells were exposed for 24 h to 100 μg/mL or 200 μg/mL SBTX. SBTX up-regulated genes involved in cell cycle and immune response pathways, but down-regulated genes that play a role in cholesterol biosynthesis and platelet degranulation pathways. Thus, although SBTX did not affect bacteria, nor induced cytotoxity in mammalian cells, it affected some biological pathways in the human Caco-2 cell line that warrants further investigation.

Molecular characterization of yolk proteins in the female crab Neptunus pelagicus (A. Milne-Edwards, 1861) from the Mediterranean Sea of Alexandria, Egypt

This study aimed to characterize the morphological changes in the ovary of the female crab Neptunus pelagicus and to identify specific fractions of vitelloginin and vitelline molecules during primary and secondary vitellogenesis. Samples of the blue crab were collected from the Mediterranean Sea of Alexandria monthly during 2017. Ovaries and oocytes in primary and secondary vitellogenesis were detached and treated for histological test. Native polyacrylamide gel electrophoresis (PAGE) Bis-Tris Gels was applied to identify vitelloginin (VN) and vitelline (VL) molecules. Protein Analyses were done by PAGE-SDS. The initial degenerate primers were built regarding the conserved amino acid domains of the yolk proteins. Primary and secondary vitellogeneses consisted of 8 phases. Lipoprotein fraction with molecular weight 550 kDa was identified in the hemolymph in secondary vitellogenesis. Two protein fractions (VLI & VLII) were identified in secondary vitellogenic oocytes. The electrophoresis performed with extract of stage I oocyte showed two protein fractions with molecular weights 550 kDa and 460 kDa. In stage II and III oocyte, 4 subunits were presented of 180, 195, 140 and 120 kDa in VLI and 2 subunits with molecular weight of 110 kDa and 95 kDa in VLII. Another two fractions in stage V oocyte presented with molecular weights of 380 kDa and 360 kDa. Western blot analysis proved that both fractions were of four major polypeptide subunits with molecular weight of 180, 125, 90 and 85 kDa in each of the two VLs. The hybridization signal obtained by the Northern blot was detected in the hepatopancreas during ovarian cycle and in the ovary during secondary vitellogenesis. The result of the reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the mRNA that encodes the C-terminal region of the VN cDNA was found in the ovary in secondary vitellogenesis and in the hepatopancreas.