Band In SDS-PAGE Research Articles

Expression and characterization of a novel halophilic GH10 β-1,4-xylanase from Trichoderma asperellum ND-1 and its synergism with a commercial α-L-arabinofuranosidase on arabinoxylan degradation

Enzymatic hydrolysis of arabinoxylan is of cost-effective strategy to yield valuable macromolecules, e.g., xylooligosaccharides (XOS). A novel halophilic GH10 xylanase (TaXYL10) from Trichoderma asperellum ND-1 was over-expressed in Pichia pastoris and migrated as a single band (~36 kDa) in SDS-PAGE. TaXYL10 displayed >80 % activity in the presence of 4.28 M NaCl and 10 % ethanol. Moreover, TaXYL10 exhibited optimal activity at pH 6.0 and 55 °C, and remarkable pH stability (>80 % activity at pH 4.0–6.0). K+ and Al3+ could remarkably promote TaXYL10 activity, while the presence of 10 mM Fe2+, Zn2+, Cu2+ and Fe3+ decreased its activity. TaXYL10 possesses the highest catalytic activity towards beechwood xylan. TLC analysis revealed that it could rapidly degrade xylan and XOS with DP ≥ 3, yielding xylotriose and xylobiose. Site-directed mutagenesis indicated that Glu154 and Glu259 are crucial active residues for TaXYL10, while Asp295 and Glu69 played auxiliary roles in xylan hydrolysis. Additionally, TaXYL10 acted cooperatively with a commercial α-L-arabinofuranosidase (AnAra) towards arabinoxylan degradation (583.5 μg/mL), a greater synergy degree of 1.79 was obtained after optimizing enzymatic ratios. This work not only expands the diversity of Trichoderma GH10 xylanases, but also reveals the promising potential of TaXYL10 in various industrial applications.

Proteomic analysis and lethality of the venom of Aegaeobuthus nigrocinctus, a scorpion of medical significance in the Middle East

The scorpion Aegaeobuthus nigrocinctus inhabits areas in Turkey and the Levant region of the Middle East where severe/lethal envenomings have been reported. Previous research indicated its extreme venom lethality to vertebrates and distinct envenomation syndrome. We report on the composition of A. nigrocinctus venom from Lebanese specimens using nESI-MS/MS, MALDI-TOF MS, SDS-PAGE and RP-HPLC. Venom lethality in mice was also assessed (LD50 = 1.05 (0.19–1.91) mg/kg, i.p), confirming A. nigrocinctus venom toxicity from Levantine populations. Forty-seven peaks were resolved using RP-HPLC, 25 of which eluted between 20 and 40 % acetonitrile. In reducing SDS-PAGE, most predominant components were <10 kDa, with minor components at higher molecular masses of 19.6, 26.1, 46.3 and 57.7 kDa. MALDI-TOF venom fingerprinting detected 20 components within the 1,000–12,000 m/z range. Whole venom ‘shotgun’ bottom-up nLC-MS/MS approach, combined with in-gel tryptic digestion of SDS-PAGE bands, identified at least 67 different components belonging to 15 venom families, with ion channel-active components (K+ toxins (23); Na+ toxins (20); Cl− toxins (2)) being predominant. The sequence of a peptide (named α-KTx9.13) ortholog to Leiurus hebraeus putative α-KTx9.3 toxin was fully determined, which exhibited 81–96 % identity to other members of the α-KTx9 subfamily targeting Kv1.x and Ca2+-activated K+ channels. Chlorotoxin-like peptides were also identified. Our study underscores the medical significance of A. nigrocinctus in the region and reveals the potential value of its venom components as lead templates for biomedical applications. Future work should address whether available antivenoms in the Middle East are effective against A. nigrocinctus envenoming in the Levant area.

Venomics of Leiurus abdullahbayrami, the most lethal scorpion in the Levant region of the Middle East

The scorpion Leiurus abdullahbayrami has been associated with severe/lethal envenomings throughout the Levant region of the Middle East, encompassing Turkey, Syria, and Lebanon, and only scarce information is available on its venom composition, activity, and antigenicity. We report on the composition of L. abdullahbayrami venom collected from Lebanese specimens using nESI-MS/MS, MALDI-TOF MS, SDS-PAGE and RP-HPLC. Venom lethality, through LD50 determination in mice (intraperitoneal), was also assessed (0.75 (0.16–1.09) mg/kg), confirming L.abdullahbayrami venom vertebrate toxicity. Fifty-four peaks were detected using RP-HPLC, half of which eluted in the gradient region between 20 and 40% acetonitrile. In reducing SDS-PAGE, most predominant components were <10 kDa, with minor components at higher molecular masses of 24.4, 43.1, and 48.9 kDa. Venom mass fingerprint by MALDI-TOF detected 21 components within the 1000–12,000 m/z range. Whole venom ‘shotgun’ bottom-up nLC-MS/MS approach, combined with in-gel tryptic digestion of SDS-PAGE bands, identified at least 113 different components belonging to 15 venom families and uncharacterized proteins, with ion channel-active components (K+ channel toxins (28); Na+ channel toxins (42); Cl− channel toxins (4); Ca+2 toxins (2)) being predominant. A single match for a L. adbullahbayrami NaTx was found in the UniProt database with other congeneric species, toxin h3.1 from Leiurus hebraeus, suggesting this might be an indication of venom divergence within Leiurus, eventhough this warrants further investigation involving venom proteomics and transcriptomics of relevant species. Considering such potential interspecific venom variation, future work should address whether preparation of a specific anti-L. abdullahbayrami antivenom is justified.

Characterization of a novel acidophilic, ethanol tolerant and halophilic GH12 β-1,4-endoglucanase from Trichoderma asperellum ND-1 and its synergistic hydrolysis of lignocellulosic biomass

A novel acidophilic GH5 β-1,4-endoglucanase (TaCel12) from Trichoderma asperellum ND-1 was efficiently expressed in Pichia pastoris (a 1.5-fold increase). Deglycosylated TaCel12 migrated as a single band (26.5 kDa) in SDS-PAGE. TaCel12 was acidophilic with a pH optimum of 4.0 and displayed great pH stability (>80 % activity over pH 3.0–5.0). TaCel12 exhibited considerable activity towards sodium carboxymethyl cellulose and sodium alginate with Vmax values of 197.97 μmol/min/mg and 119.06 μmol/min/mg, respectively. Moreover, TaCel12 maintained >80 % activity in the presence of 20 % ethanol and 4.28 M NaCl. Additionally, Mn2+, Pb2+ and Cu2+ negatively affected TaCel12 activity, while the presence of 5 mM Co2+ significantly increased the enzyme activity. Analysis of action mode revealed that TaCel12 required at least four glucose (cellotetraose) residues for hydrolysis to yield cellobiose and cellotriose. Site-directed mutagenesis results suggested that Glu133 and Glu217 of TaCel12 are crucial catalytic residues, with Asp116 displaying an auxiliary function. Production of soluble sugars from lignocellulose is a crucial step in bioethanol development, and it is noteworthy that TaCel12 could synergistically yield fermentable sugars from corn stover and bagasse, respectively. Thus TaCel12 with excellent properties will be considered a potential biocatalyst for applications in various industries, especially for lignocellulosic biomass conversion.

Structural and functional analysis of a novel galactose-binding lectin derived from Chlorella sorokiniana MW769776

A freshwater green microalgal strain was isolated and the lectin was identified in it by a strong hemagglutination activity (HA) assay. Characterization of the algal strain was found to be Chlorella sorokiniana (MW769776). A single step affinity chromatographic technique was developed to purify Chlorella sorokiniana lectin (CSL) using guar gum as the affinity matrix. The precipitate showed a single active peak with a titer value of 1024 HU, with a concentration of 1111 U, and a purification fold of 9. The purified protein exhibited a single band in SDS-PAGE with a molecular weight of 16 kDa. Analysis by liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry (LC-ESI-Q-TOF-MS) of tryptic-digested purified lectin showed that it was a monomeric protein. A multiple sequence alignment analysis revealed that the peptide sequences of CSL exhibited similarity with the H-type lectin domain of Micractinium conductrix. The structure of CSL was studied by FTIR and homology modeling methods, indicating the presence of α-helix as well as β-sheet in its secondary structure. Whereas the 3D structure exhibited the similarity with the core protein of light-harvesting reaction center complex of photosystem I. The significance of this study suggests that the characteristics of CSL are consistent with its identification as a hemagglutinin, a type of novel lectin, which suggests its candidature for various biological purposes. Communicated by Ramaswamy H. Sarma

Proteomic evidence of protein degradation and oxidation in brined bighead carp fillets during long-term frozen storage

Protein degradation and oxidation are two major alterations during the storage of processed bighead carp fillets. This study conducted a comparative analysis of degraded and oxidized products as well as oxidation sites in fresh, frozen and brined frozen bighead carp fillets. Frozen storage played a dominant role in protein degradation and oxidation, and brining promoted these changes. In brined frozen samples, the decreased SDS-PAGE band intensities for tropomyosin, troponin, and myosin light chain were mainly due to their degradation. Myosin heavy chain fast skeletal muscle was the most oxidized and degraded protein during storage, with modifications such as monooxidation, protein-lipid peroxidation adducts, and α-aminoadipic semialdehydes formation. Amino acids in the tail portion of myosin were prone to oxidation than the head portions. Our results provided comprehensive insights into protein degradation and oxidation in bighead carp during storage, helping to assess the specific fate of oxidative products in future dietary investigations.

Aspergillus clavatus UEM 04: An efficient producer of glucoamylase and α-amylase able to hydrolyze gelatinized and raw starch

The best amylolytic activity production by Aspergillus clavatus UEM 04 occurred in submersed culture, with starch, for 72 h, at 25 °C, and 100 rpm. Exclusion chromatography partially purified two enzymes, which ran as unique bands in SDS-PAGE with approximately 84 kDa. LC-MS/MS identified a glucoamylase (GH15) and an α-amylase (GH13_1) as the predominant proteins and other co-purified proteins. Zn2+, Cu2+, and Mn2+ activated the glucoamylase, and SDS, Zn2+, Fe3+, and Cu2+ inhibited the α-amylase. The α-amylase optimum pH was 6.5. The optimal temperatures for the glucoamylase and α-amylase were 50 °C and 40 °C, and the Tm was 53.1 °C and 56.3 °C, respectively. Both enzymes remained almost fully active for 28–32 h at 40 °C, but the α-amylase thermal stability was calcium-dependent. Furthermore, the glucoamylase and α-amylase KM for starch were 2.95 and 1.0 mg/mL, respectively. Still, the Vmax was 0.28 μmol/min of released glucose for glucoamylase and 0.1 mg/min of consumed starch for α-amylase. Moreover, the glucoamylase showed greater affinity for amylopectin and α-amylase for maltodextrin. Additionally, both enzymes efficiently degraded raw starch. At last, glucose was the main product of glucoamylase, and α-amylase produced mainly maltose from gelatinized soluble starch hydrolysis.

Structural Properties of Quinoa Protein Isolate: Impact of Neutral to High Alkaline Extraction pH.

In this work, we extracted proteins from white quinoa cultivated in the northeast of Qinghai-Tibet plateau using the method of alkaline solubilization and acid precipitation, aiming to decipher how extraction pH (7-11) influenced extractability, purity and recovery rate, composition, multi-length scale structure, and gelling properties of quinoa protein isolate (QPI). The results showed that protein extractability increased from 39 to 58% with the increment of pH from 7 to 11 whereas protein purity decreased from 89 to 82%. At pH 7-11, extraction suspensions and QPI showed the similar major bands in SDS-PAGE with more minor ones (e.g., protein fractions at > 55 or 25-37 kDa) in suspensions. Extraction pH had limited effect on the secondary structure of QPI. In contrast, the higher-order structures of QPI were significantly affected, e.g., (1) emission maximum wavelength of intrinsic fluorescence increased with extraction pH; (2) surface hydrophobicity and the absolute value of zeta-potential increased with increasing extraction pH from 7 to 9, and then markedly decreased; (3) the particle size decreased to the lowest value at pH 9 and then increased to the highest value at pH 11; and (4) denaturation temperature of QPI had a large decrease with increasing extraction pH from 7/8 to 9/10. Besides, heat-set QPI gels were formed by loosely-connected protein aggregates, which were strengthened with increasing extraction pH. This study would provide fundamental data for industrial production of quinoa protein with desired quality.

Manipulation of protein structure and bonding pattern to improve the gelling and textural quality of surimi gels from silver carp: An incorporation of mosambi (Citrus limetta) peel extract.

Present investigation focused on mosambi peels extract (MPE) fortification (at 0% to 1.50%, w/w) in silver carp surimi to improve its gelling, textural and other physicochemical properties. The peels were extracted in ethanol (40-100% concentrations, v/v) and water. Out of which, 100% ethanol had significantly (p<0.05) higher yield and total phenolic, flavonoid, and tannin content. The fortification of MPE at optimum level (0.75%) improved the breaking force (55.1%) and gel strength (89.9%), significantly (p<0.05) compared to 0% MPE gel samples. Moreover, 0.75% MPE-fortified gels had higher hydrogen and hydrophobic bonds, water holding capacity and lower sulfhydryl groups and free amino groups. MHC bands in SDS-PAGE were disappeared completely in the MPE-fortified gels. The MPE-fortification affects the secondary structures of protein as resulted in shifting of peaks were observed in FTIR-spectra. SEM images showed relatively organized finer and denser gel network in MPE treated gels. The surimi gels fortified with 0.75% MPE demonstrated improved gelling properties, with an overall higher acceptability than that of unfortified the gels (0% MPE). Further, the fortified gels got enriched with bioactive polyphenols which are generally not present in surimi. Moreover, this study will provide an efficient way to utilize mosambi peel to develop functional surimi and surimi-based products with improved gel ability This article is protected by copyright. All rights reserved.

Anatomizing extracellular polymer of Calothrix desertica with its anti-oxidation and anti-nutrient profiling

Calothrix desertica having a semilunar apical heterocyst is proficient at excreting 1.2 g/L of extracellular polymers (EPsC) in 45 days. The refined EPsC constitutes 430 mg/g of glycogen, 390 mg/g of protein, and 14.6 mg/g of glycoproteins (GPs). The glycoprotein estimation of EPsC was performed by two step hydrolysis methods with H2SO4. The peak between 10 mAU to 20 mAU in HPLC, 1400 cm-1 to 1700 cm-1 in FTIR, and 40kDa- 35kDa bands in SDS-PAGE authenticates the presence of glycoproteins in the EPsC. The EPsC agglomerate of 1000 nm to 3000 nm size with a Zeta potential of -20 mV to 5 mV was determined using DLS. Further EPsC of nanosizes of 30 nm to 150 nm in 50,000 X and 20 nm to 40 nm in 60,000 X was measured using FE- SEM. The DPPH assay and H2O2 scavenging assay showed 73.1% and 70.8% of anti-oxidant activity in EPsC, which is coequally efficient as standard gallic acid. EPsC biopolymer can also be used as a potential reducing agent, as per the anti-nutrient activity studies.

Influence of hydrophilic polysaccharide fat replacers on the in vitro digestibility of protein in emulsion-type sausage

Hydrophilic polysaccharides have been widely applied as fat replacers in meat products, but their effects on the digestibility of meat proteins has seldom been studied. Replacement of backfat in emulsion-type sausage with konjac gum (KG), sodium alginate (SA) and xanthan gum (XG) were found to reduce the released amino group (–NH2) during simulated gastric digestion and initial intestinal digestion. The suppressed gastric digestibility of protein was verified by the denser structures of protein gastric digests and reduced generation of peptides in gastric digestion when a polysaccharide was added. After the whole gastrointestinal digestion, high level of SA and XG resulted in larger digests and a more obvious SDS-PAGE band between 5 and 15 kDa, and KG and SA significantly reduced the total release of –NH2. Additional of KG, SA and XG were found to the increase the viscosity of the gastric digests mixture, which could account for the reduced hydrolysis efficiency of pepsin during the gastric digestion, as evidenced in the pepsin activity study (decreased by 12.2–39.1%). This work highlights the influence of polysaccharide fat replacer on the digestibility of meat protein by changing the matrix characteristics.

A novel allelic donkey β-lactoglobulin I protein isoform generated by a non-AUG translation initiation codon is associated with a nonsynonymous SNP

β-Lactoglobulin I (β-LG I) is one of the most important whey proteins in donkey milk. However, to our knowledge, there has been no study focusing on the full nucleotide sequences of this gene (BLG I). Current investigation of donkey BLG I gene is very limited with only 2 variants (A and B) characterized so far at the protein level. Recently, a new β-LG I variant, with a significantly higher mass (+1,915 Da) than known variants has been detected. In this study, we report the whole nucleotide sequence of the BLG I gene from 2 donkeys, whose milk samples are characterized by the β-LG I SDS-PAGE band with a normal electrophoretic mobility (18,514.25 Da, β-LG I B1 form) the first, and by the presence of a unique β-LG I band with a higher electrophoretic mobility (20,428.5 Da, β-LG I D form) the latter. A high genetic variability was found all over the 2 sequenced BLG I alleles. In particular, 16 polymorphic sites were found in introns, one in the 5' flanking region, 3 SNPs in the 5' untranslated region and one SNP in the coding region (g.458G > A) located at the 40th nucleotide of exon 2 and responsible for the AA substitutions p.Asp28 > Asn in the mature protein. Two SNPs (g.920-922CAC > TGT and g.1871G/A) were genotyped in 93 donkeys of 2 Italian breeds (60 Ragusana and 33 Amiatina, respectively) and the overall frequencies of g.920-922CAC and g.1871A were 0.3065 and 0.043, respectively. Only the rare allele g.1871A was observed to be associated with the slower migrating β-LG I. Considering this genetic diversity and those found in the database, it was possible to deduce at least 5 different alleles (BLG I A, B, B1, C, D) responsible for 4 potential β-LG I translations. Among these alleles, B1 and D are those characterized in the present research, with the D allele of real novel identification. Haplotype data analysis suggests an evolutionary pathway of donkey BLG I gene and a possible phylogenetic map is proposed. Analyses of mRNA secondary structure showed relevant changes in the structures, as consequence of the g.1871G > A polymorphism, that might be responsible for the recognition of an alternative initiation site providing an additional signal peptide. The extension of 19 AA sequence to the mature protein, corresponding to the canonical signal peptide with an additional alanine residue, is sufficient to provide the observed molecular weight of the slower migrating β-LG I encoded by the BLG I D allele.

Detoxification of Meghalayan cherry (Prunus nepalensis) kernel and its effect on structural and thermal properties of proteins

Valorizing food wastes and by-products can improve economic and environmental sustainability of the food production chain. In this regard, Meghalayan cherry kernels are a good source of proteins, but the presence of toxic compounds like amygdalin, makes them underutilized. Therefore, the present study was focused on detoxifying Meghalayan cherry kernel using thermal, soaking and ultrasound treatments and studying their impact on the structural and thermal characteristics of protein isolate. The results showed that all three treatments significantly reduced amygdalin content, with complete detoxification achieved after 30 and 60 min at 70 °C and 60 °C, respectively, in ultrasound, and after 90 and 120 min at 70 °C and 60 °C, respectively, in soaking + thermal treatment. The detoxification treatments significantly affected the protein content and weight-loss of Meghalayan cherry kernel. Fluorescence spectroscopy and FTIR showed alterations in Meghalayan cherry kernel protein isolate (MCKPI) secondary and tertiary structure. The fluorescence intensity was observed at 340 nm for native and detoxified protein isolate, and the lowest peak for MCKPI-US depicts conformational changes. The fading of bands in SDS-PAGE confirms structural changes due to thermal and sonication effects. SEM images demonstrated that more cracks and porous structures were seen in treated MCKPI than native MCKPI. Detoxification treatment increased thermal stability, resulting in lesser weight loss and higher denaturation temperature than native MCKPI. In this study, ultrasound treatment demonstrated the most pronounced effects on the detoxification of MCKPI and its thermal and structural properties, suggesting that Meghalayan cherry kernel is one of the most promising substrates for zero-waste bioprocess development.

Identification and molecular characterization of drug targets of methicillin resistant Staphylococcus aureus

Antimicrobial resistance is a major world health concern and drug-resistant Staphylococcus aureus is a serious threat. Due to the emergence of multidrug-resistant bacterial strains, there is an urgent need to develop novel drug targets to meet the challenge of multidrug-resistant organisms. The main objective of the current study was to determine molecular targets against S. aureus using by computational approach. S. aureus was cultured in brain heart infusion broth medium and MRSA (Methicillin resistant S. aureus) protein was extracted acetone-sodium dodecyl sulfate method. The cell lysate was treated with various antibiotics and proteinase K stable proteins were analyzed. The molecular weight of Geninthiocin-targeted protein of interest in S. aureus ranged from 46 to 50 kDa. A prominent protein band in SDS-PAGE indicated that the protein corresponding 50 kDa was resistant against proteinase K. The SDS-PAGE separated sample was excised and trypsinated, and the peptides were characterized using Nano Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) analysis. Spectrum with clusters of molecular peptides and peptide fragments ranging from 110.0716 to 1002.7093 mass/charge ratio (m/z) were displayed against intensity or relative abundance in the excised gel band. The spectral data from nano LC-MS/MS was subjected to mascot search in the NCBIprot database (taxonomy-bacteria (eubacteria), resulting in seven bacterial proteins. Geninthiocin target proteins were determined against MRSA. To conclude, antibiotic target proteins were identified using a machine learning approach and these targets may have a lot of applications in developing a novel lead molecule against drug-resistant bacteria.

Tenebrio molitor Proteins-Derived DPP-4 Inhibitory Peptides: Preparation, Identification, and Molecular Binding Mechanism.

Inhibition of dipeptidyl peptidase-4 (DPP-4) is an effective way to control blood glucose in diabetic patients. Tenebrio (T.) molitor is an edible insect containing abundant protein. T. molitor protein-derived peptides can suppress the DPP-4 activity. However, the amino acid sequence and binding mechanism of these DPP-4 inhibitory peptides remain unclear. This study used the flavourzyme for T. molitor protein hydrolysis, identified the released peptides with DPP-4 inhibitory effect, and investigated the binding interactions of these peptides with DPP-4. The results showed that flavourzyme efficiently hydrolyzed the T. molitor protein, as demonstrated by the high degree of hydrolysis, disappearance of protein bands in SDS-PAGE, and changes to protein structure. The 4-h flavourzyme hydrolysates showed a good inhibitory effect on DPP-4 (IC50 value of 1.64 mg/mL). The fragment of 1000-3000 Da accounted for 10.39% of the total peptides, but showed the strongest inhibitory effect on DPP-4. The peptides LPDQWDWR and APPDGGFWEWGD were identified from this fraction, and their IC50 values against DPP-4 were 0.15 and 1.03 mg/mL, respectively. Molecular docking showed that these two peptides interacted with the DPP-4 active site via hydrogen bonding, hydrophobic interactions, salt bridge formation, π-cation interactions, and π-π stacking. Our findings indicated that T. molitor protein-derived peptides could be used as natural DPP-4 inhibitors.

Dibenzyl (benzo [d] thiazol-2-yl (hydroxy) methyl) phosphonate (DBTMP) showing anti-S. aureus and anti-biofilm properties by elevating activities of serine protease (SspA) and cysteine protease staphopain B (SspB).

Staphylococcus aureus biofilms are the pathogenic factor in the spread of infection and are more pronounced in multidrug-resistant strains of S. aureus, where high expression of proteases is observed. Among various proteases, Serine protease (SspA) and cysteine protease Staphopain B (SspB) are known to play a key role in the biofilm formation and removal of biofilms. In earlier studies, we have reported Dibenzyl (benzo [d] thiazol-2-yl (hydroxy) methyl) phosphonate (DBTMP) exhibits anti-S. aureus and anti-biofilm properties by elevating the expression of the protease. In this study, the effect of DBTMP on the activities of SspA, and SspB of S. aureus was evaluated. The SspA and SspB genes of S. aureus ATCC12600 were sequenced (Genbank accession numbers: MZ456982 and MW574006). In S. aureus active SspA is formed by proteolytic cleavage of immature SspA, to get this mature SspA (mSspA), we have PCR amplified the mSspA sequence from the SspA gene. The mSspA and SspB genes were cloned, expressed, and characterized. The pure recombinant proteins rSspB and rmSspA exhibited a single band in SDS-PAGE with a molecular weight of 40 and 30KD, respectively. The activities of rmSspA and rSspB are 32.33 and 35.45Units/mL correspondingly. DBTMP elevated the activities of rmSspA and rSspB by docking with respective enzymes. This compound disrupted the biofilms formed by the multidrug-resistant strains of S. aureus and further prevented biofilm formation. These findings explain that DBTMP possesses anti-S. aureus and anti-biofilm features.

Bombyx batryticatus Cocoonase Inhibitor Separation, Purification, and Inhibitory Effect on the Proliferation of SMCC-7721 HeLa-Derived Cells.

The present study was to isolate and purify Bombyx batryticatus cocoonase inhibitor (BBCI) and to evaluate its inhibitory effect on the proliferation of SMCC-7721 cells. BBCI was purified from the crude proteins of Bombyx batryticatus using affinity chromatography with cocoonase as the ligand, its N-terminal amino acid sequence was determined using the Edman degradation method, and its inhibiting activity on SMCC-7721 cell proliferation was detected in vitro using the MTT method and in vivo in tumor-bearing nude mice. The purified BBCI presented as a single band in SDS-PAGE, the molecular weight determined by time-of-flight mass spectrometry was 13,973.63 Da, and its N-terminal amino acid sequence was VRNKRQSNDD. BBCI was a noncompetitive cocoonase inhibitor with an average Michaelis constant of 76.50, and it inhibited cocoonase activity with an inhibition ratio of 1 : 1 (molar). BBCI could inhibit the proliferation of SMCC-7721 cells in vitro with the IC50 being about 260.52 μg/ml within 36 h of treatment and inhibit the SMCC-7721 tumor growth in nude mice by subcutaneous injection of BBCI around the tumor, where the tumor inhibitory effect was dose dependent. BBCI did not significantly influence the spleen coefficient of the mice. In conclusion, to the best of our knowledge, the present study is the first to report that BBCI, which was purified from Bombyx batryticatus, was a serine proteinase inhibitor with antitumor activity.

In vitro digestion of tofu with different textures using an artificial gastric digestive system

Two kinds of tofu with obvious differences in texture [“GDL” and “CaSO4”, standing for tofus made with the application of either glucono-δ-lactone (GDL) or calcium sulfate, with measured hardness 23.1 ± 3.3 g and 105.2 ± 25.1 g, respectively] were used as to investigate the in vitro progress and extent of tofu digestion, using an independently-developed artificial gastric digestion system (AGDS). The particle size distributions of both CaSO4 and GDL tofu shifted towards smaller particles as the digestion time increased, while the viscosity of the gastric digesta also increased. Tofu proteins were hydrolyzed in the simulated stomach, with GDL tofu showing a higher hydrolysis rate, based on the temporal evolution of SDS-PAGE bands, and had a higher amino acids accumulation than CaSO4 tofu at the end of gastric digestion. In the absence of peptic enzymes, the protein was acidically-hydrolyzed, but the degree of hydrolysis was much lower than in the presence of enzymes; these findings are in accord with the changes in microstructure observed by scanning electron microscopy. The results indicated that the in vitro extent of tofu digestion is related to its hardness, which is in turn related to its microstructure; they also indicated the potential of our developed in vitro dynamic stomach in studying semi-solid foods.

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

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

The Lysosome Origin of Biosilica Machinery in the Demospongiae Model Petrosia ficiformis (Poiret, 1789)

The silicification mechanism in sponges is a biologically controlled process where the complex and amazing shape of spicules is the result of the hierarchical assembly of silicon particles to form a composite structure with organic compounds, mainly constituted by proteins. In this work, using an integrated approach of transcriptomic and proteomic analysis, we describe the protein content of sponge spicules in the marine demosponge Petrosia ficiformis (Poiret, 1789). Proteins from spicules were obtained via an ammonium fluoride extraction procedure to remove the inorganic silica followed by SDS-PAGE electrophoresis. The resulting data of LC-MS/MS analysis of the extracted SDS-PAGE bands were then processed with the MASCOT software to search against a database consisting of transcripts and predicted proteins of P. ficiformis. The results revealed a very heterogeneous group of 21 proteins, including silicatein beta, different isoforms of cathepsins, proteins with strong homologies with enzymes like sulphatases, glycosidases, lipid-related proteins, phosphatases, and some others with unknown function. Most of the proteins found here have structures and domains attributable to lysosomes enzymes and for this reason it could be related to these cellular structures the evolutionary origin of the biosilica machinery in sponges.