High Molecular Weight Bands Research Articles

A70 FECAL-ADHERENT MUCUS IS A NON-INVASIVE SOURCE OF PRIMARY HUMAN MUC2 FOR STRUCTURAL AND FUNCTIONAL CHARACTERIZATION IN HEALTH AND DISEASE

A70 FECAL-ADHERENT MUCUS IS A NON-INVASIVE SOURCE OF PRIMARY HUMAN MUC2 FOR STRUCTURAL AND FUNCTIONAL CHARACTERIZATION IN HEALTH AND DISEASE

Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

Antibodies against phosphorylated alpha-synuclein (aSyn) at S129 have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues of patients with Parkinson’s disease and other neurodegenerative diseases. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal post-translational modifications (PTMs) (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of pS129-aSyn species by pS129-aSyn antibodies. These observations prompted us to systematically reassess the specificity of the most commonly used pS129 antibodies against monomeric and aggregated forms of pS129-aSyn in mouse brain slices, primary neurons, mammalian cells and seeding models of aSyn pathology formation. We identified two antibodies that are insensitive to pS129 neighboring PTMs. Although most pS129 antibodies showed good performance in detecting aSyn aggregates in cells, neurons and mouse brain tissue containing abundant aSyn pathology, they also showed cross-reactivity towards other proteins and often detected non-specific low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or high molecular weight aSyn species. Our observations suggest that not all pS129 antibodies capture the biochemical and morphological diversity of aSyn pathology, and all should be used with the appropriate protein standards and controls when investigating aSyn under physiological conditions. Finally, our work underscores the need for more pS129 antibodies that are not sensitive to neighboring PTMs and more thorough characterization and validation of existing and new antibodies.

PRODUCTION OF ANTI-LACTOFERRIN ANTIBODIES AND THEIR APPLICATION IN ANALYSIS OF THE TEAR FLUID IN HEALTH AND CORNEAL INJURIES

Lactoferrin is a ubiquitous and multifunctional protein, which has antimicrobial and immunomodulatory activities. Lactoferrin plays an important role in the maintenance of ocular health. The aim of the study was to produce polyclonal antibodies against human lactoferrin in order to apply them in evaluation of lactoferrin levels in tear fluid collected from healthy eye and after corneal injury. Materials and methods. Affine chromatography on Protein A-sepharose was applied in order to isolate immunoglobulin G (IgG) fraction from the blood serum of lactoferrin-immunized rabbits. Each step of protein purification was monitored by denaturing gel electrophoresis (SDS-PAGE). Target antigen recognition by produced antibodies was established by western blot analysis with the use of diluted IgG fraction. Lactoferrin levels in the tear fluids collected from healthy individuals (n = 4) and patients with non-penetrating corneal injures (n = 6) were determined immunochemically with the use of purified antibodies. The results of western blot of lactoferrin levels in the tear fluids of healthy individuals and patients with corneal wounds were analysed using Mann-Whitney U-test. The difference between group mean values was considered significant at P<0.05. Results. Using affine chromatography on Protein A-sepharose, antibodies against human lactoferrin were purified as IgG fraction from blood serum of lactoferrin-immunized rabbits. Western blot analysis showed that obtained antibodies recognize the antigen as a 75-kDa band, which corresponds to the intact human lactoferrin polypeptide. The same major polypeptide band was visualized by western blot with enhanced chemiluminescence detection in the tear fluid samples. Densitometry analysis of 75-kDa lactoferrin band showed 3.2-fold decrease in lactoferrin level in the tear fluid samples obtained from patients with non-penetrating corneal traumas as compared with samples collected from healthy persons (P<0.05). Besides, tear fluid of patients with injured corneas contained large amounts of truncated lactoferrin immunoreactive polypeptides as well as high molecular weight bands, which could correspond to lactoferrin complexes with other proteins occurring during inflammation. Conclusions. According to our data, obtained anti-lactoferrin antibodies can be used as a valuable tool for development of advanced tests and procedures for diagnostics of eye diseases associated with the corneal lesions. Reduced lactoferrin concentration might represent a potential prognostic biomarker for diagnosis of ocular diseases including non-penetrating corneal injuries in a simple and non-invasive way.

Proteomic analysis of the excretory-secretory products from Strongyloides venezuelensis infective larvae: new insights for the immunodiagnosis of human strongyloidiasis.

Serodiagnosis of human strongyloidiasis is a practical alternative to parasitological methods due to its high sensitivity. However, cross-reactivity with other helminth infections limits its utility, and this problem is due to the use of homologous or heterologous somatic extracts of the parasite as an antigen source. Excretory-secretory (E/S) products from Strongyloides infective larvae can be used to improve the serodiagnosis. The combined use of western blot and proteomics became an interesting strategy to identify immunological markers for the serodiagnosis of strongyloidiasis. The present study describes the proteomic analysis of the antigenic components from E/S products of S. venezuelensis infective larvae that were recognized by IgG antibodies from patients with strongyloidiasis. Our results showed that IgG antibodies from patients with strongyloidiasis recognized between 15 and 16 antigenic bands in the E/S products from S. venezuelensis that were incubated in PBS or in RPMI culture medium, respectively. Overall, antigenic bands of low and high molecular weight were more specific than those of intermediate molecular weight, which were cross-reactive. A 36-kDa antigenic band was 93% sensitive and 100% specific (a probably arginine kinase of 37kDa), while other antigenic bands were highly sensitive but low specific. Proteomic analysis revealed differences between the protein profiles from E/S-RPMI and E/S-PBS since only one-third of all proteins identified were common in both types of E/S products. Bioinformatic analysis showed that more than 50% of the proteins from E/S products are secreted within extracellular vesicles and only a small percentage of them are actually released by the classical secretory pathway. Several components from the E/S products were identified as plasminogen-binding proteins, probably used as an immune evasion mechanism. The data provided here provide valuable information to increase understanding of E/S products from S. venezuelensis infective larvae. This may help us to find new targets for the immunodiagnosis of human strongyloidiasis.

Gelatin extracted from jundiá skin (Rhamdia quelen): An alternative to the discarded by-product

The production of gelatin from by-products of the fishing industry values ​​the discarded raw material and serves a part of the population that does not consume products originating from mammals. Therefore, the objective of the research was to use the jundiá skin (Rhamdia quelen) (JS) to obtain gelatin (GJS) and characterize this product, not yet studied until the present moment. Thus, the extraction process showed a yield of 7.3 % for JS and 18.2 % for GJS (in wet weight). Both JS and GJS presented, in their composition, high concentration of protein (26.3 and 88.1 %), low levels of fixed mineral residue (1.0 and 1.9 %), lipids (1.7 and 1.5 %) and hydroxyproline content (1.5 and 7.2 %), respectively. The GJS dispersion had a pH value of 4.7 and the color analysis indicated a snow effect with a white appearance. Fourier transform infrared spectroscopy (FTIR) showed amide bands commonly found in gelatin, gel electrophoresis (SDS-PAGE) showed high molecular weight bands, differential scanning calorimetry (DSC) revealed a denaturation temperature of 69.4 °C and scanning electron microscopy (SEM) showed a compact and non-porous structure. The emulsifying property was high when subjected to a temperature of 80 °C for 30 min, while the foaming capacity was significant at a concentration of 1 %. The highest dispersivity was observed at pH 2.0 and, in this condition, the viscosity was higher than that of other gelatin sources (25.5 cP). In view of the above, attention is drawn to the use of JS as a raw material for obtaining gelatin and for the various possibilities of application.

Tetraspanin CD53 controls Tcell immunity through regulation of CD45RO stability, mobility, and function.

T cells depend on the phosphatase CD45 to initiate Tcell receptor signaling. Although the critical role of CD45 in Tcells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in Tcell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and Tcell activation. CD53-negative Tcells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing Tcells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for Tcell immunity.

Pharmacological Characterization of Daboia russelii Venom and its Neutralization by Polyvalent Chicken Antibodies

Snake bite is considered as a socio-economic problem in tropical countries. Daboia russelii (Russell’s viper) is responsible for 30–40% of all snakebites and the most number of life-threatening bites. Antivenom is the only treatment available. Hyperimmune sera raised from horses were shown to produce adverse effects. In this study, chicken Ig Y was generated against the venom of Daboia russelii and its neutralization studies were carried out by in-vitro methods. Antivenom antibodies were generated in chicken and purified by DEAE cellulose column and the antibody titre was estimated in serum and egg yolk by ELISA which showed the increase in titres. Ig Y was subjected to protein estimation, and SDS- PAGE was carried out to study the banding pattern. High molecular weight band 180 KD was observed. Pharmacological characterization like coagulant activity, direct haemolytic assay, indirect haemolytic assay and proteolytic activity was carried out. Minimum coagulant dose was found to be 120 µg. Neutralization studies were carried out using column purified Ig Y antibodies. Current study suggests that Ig Y antivenoms are effective in neutralising the pharmacological activities induced by venom.

Ischemia-Reperfusion Injury with Pringle's Maneuver Induces Increasing of Unusually Large Von Willebrand Factor Multimers after Hepatectomy

Background/Aims: Von Willebrand factor (VWF) is exclusively synthesized in vascular endothelial cells and released into the plasma as unusually large VWF multimers (UL-VWFM). ADAMTS13 is a metalloproteinase produced primarily in hepatic stellate cells and specifically cleaves the VWF. Since UL-VWFM have a strong activity of platelet aggregation, increase of UL-VWFM leads to platelet clumping and/or thrombus formation, resulting in microcirculatory disturbance. On the other hand, ischemia-reperfusion injury of the liver is an imperative event in liver surgery due to compression of the liver and/or Pringle's maneuver. Many previous studies have indicated that vascular endothelial cell is a major target of ischemia-reperfusion injury of the liver. Thus, we hypothesized UL-VWFM increases markedly by stimulating vascular endothelial cells with ischemia-reperfusion injury. However, little information is available regarding the alterations in VWF, ADAMTS13 and especially in UL-VWFM after hepatectomy. The aim of this study is to clarify the effect of hepatectomy on UL-VWFM.Methods: A total 35 patients who underwent a hepatectomy were eligible for the study. Blood samples were obtained for analysis at the following time points: preoperation, intraoperative period, and on the first, second, third, fifth, seventh postoperative days. Plasma levels of ADAMTS13 activity and VWF antigen were measured by the ELISA method. Multimer analysis of plasma VWF was performed according to the method of Ruggeri and Zimmerman. High molecular weight bands that were not detected in normal plasma were defined as UL-VWFM. UL-VWFM was quantified from images of VWF multimer analysis by NIH image. We calculated the UL-VWFM index, proposed in this study, which was defined as the ratio of the UL-VWFM area of a patient to that of normal plasma. All UL-VWFM indexes of 40 healthy volunteers were less than 1.00%. Therefore, UL-VWFM positive was defined as a UL-VWFM index of more than 1.00%. The patients were categorized and analyzed with respect to the positive or negative of UL-VWFM after hepatectomy. The values were expressed as median (range).Results: Plasma levels of ADAMTS13 activity were significantly decreased from 61.0% (27.7-126.2%) in preoperation to 37.4% (20.2-71.4%) in postoperative day 7 after hepatectomy (p <0.001). Plasma levels of VWF antigen were significantly increased from 172.1% (80.5-412.8%) in preoperation to 361.0% (154.7-745.8%) in postoperative day 2 after hepatectomy and remained continuously high until postoperative day 7 (p <0.001). The intraoperative plasma levels of ADAMTS13 activity, VWF antigen were not significantly altered. The UL-VWFM index were significantly increased from 0.2% (0.0-7.8%) in preoperation to 4.2% (0.1-16.3%) in postoperative day 7 after hepatectomy (p <0.001). The patients were divided into 2 groups with respect to the positive or negative of UL-VWFM after operation. Seven patients remained UL-VWFM negative after operation. UL-VWFM index were changed from negative in preoperation to positive after operation in 22 patients. Preoperative characteristics including ADAMTS13 activity, VWF antigen levels and liver functional reserve were similar in the two groups of UL-VWFM positive and negative. In surgery-related factors, the time of duration of Pringle's maneuver in UL-VWFM positive group were longer than those in UL-VWFM negative group (p=0.001). In addition, the volume of blood loss in UL-VWFM positive group was greater than those in UL-VWFM negative group (p=0.003). Multivariable analysis revealed that the time of duration of Pringle's maneuver [OR, 1.049 95%CI, 1.001-1.098; p=0.043] was significantly associated with increasing UL-VWFM after hepatectomy. Moreover, UL-VWFM index was significantly correlated with time of duration of Pringle's maneuver (r=0.444, p=0.017).Conclusion: In this study, we demonstrated that plasma UL-VWFM increases after hepatectomy due to ischemia-reperfusion injury with Pringle's maneuver. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interleukin-6 Drives Multiple Myeloma Progression through Upregulating of CD147/Emmprin Expression and Its Sialylation

Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation of plasma cells in the bone marrow (BM). CD147, known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. With regard to MM, it was recently documented that the aberrantly elevated expression of CD147 has been tightly correlated with MM cell colonization and proliferation. During malignant transformation, many glycoproteins undergo a wide range of glycosylation alterations, especially increased sialylation, have been associated with malignant transformation and metastasis. However, it is still unclear the mechanisms of regulating CD147 and its glycosylation in MM. In this study, we found that CD147 and its sialylation can be up-regulated by interleukin-6 (IL-6), which is derived from either autocrine or paracrine sources and plays an essential role in the malignant progression of MM. When serum-starved MM cell lines RPMI8226, MM1.S and NCI-H929 were stimulated with 20ng/mL of IL-6, we found that the expression of CD147 on MM cell membrane was significantly upregulated, as determined by both flow cytometric analysis and Western blotting. Importantly, the results of Western blotting also clearly show that CD147 is a glycoprotein with higher molecular weight bands than core CD147. By using PNGase F and sialidase to digest the protein samples, we further demonstrated that CD147 is typical N-linked glycoprotein with high level of sialylation. Moreover, we found that the expression of α-2,3 and α-2,6 sialic acid on the cell surface were significantly upregulated by IL-6 analyzed by flow cytometry using MAA and SNA lectin binding assay. Next, we investigated the signaling pathway involved in the IL-6 mediated upregulation of CD147 and its sialylation in MM cells. By using Real-Time PCR, we confirmed that CD147 was transcriptionally upregulated by IL-6. Simultaneously, several sialyltransferases like ST3GAL3, ST3GAL6 and ST6GAL1 were also transcriptionally upregulated by IL-6. By using Western blotting, we further confirmed that IL-6 can activate the JAK/STAT3 signaling pathway by inducing the phosphorylation of STAT3 at Tyrosine 705 in MM cells. Further bioinformatics and ChIP analysis demonstrated that the existing of transcription factor STAT3 binding sites in the promoter of CD147 and sialyltransferase (ST3GAL3, ST3GAL6 and ST6GAL1) genes.To further validate the role of JAK/STAT3 in regulating CD147 and its sialylation, we used a specific STAT3 inhibitor Cryptotanshinone to treat the MM cells and found that the expression of CD147 and its sialylation in MM cells was accordingly decreased. Furthermore, we validated the roles of high level of CD147 and its sialylation in MM biology by knocking-down CD147 or inhibiting its sialylation using 3Fax-Peracetyl Neu5Ac, which is a specific sialyltransferase inhibitor. We found that both siRNA mediated knock-down of CD147 and sialylation inhibition did not affect the proliferation in RPMI8226 and MM1.S cells, measured by EDU incorporation assay using flow cytometry. However, knock-down of CD147 and sialylation inhibition were found to significantly reduce the adhesion to BMSCs and HUVECs, and the migration to BMSC-conditioned media in vitro. Finally, to further validate the influence of IL-6 and the related sialyltransferase genes in MM patients, we assessed the effect of expression on survival of patients using GEP data from the CoMMpass trial (n=664), and noted a significantly reduced PFS (progression-free survival) for patients with high levels of expression of IL-6 and ST3GAL6 (P <0.0001). Taken together, our data provide evidence that IL-6 can up-regulate CD147 expression and its sialylation in a STAT3-dependent manner, and offer a compelling rationale for exploring this axis as a therapeutic target for MM. DisclosuresO'Dwyer:GlycoMimetics Inc: Research Funding; Onkimmune Ltd: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Janssen: Consultancy, Honoraria, Research Funding.

Verteporfin disrupts multiple steps of autophagy and regulates p53 to sensitize osteosarcoma cells

BackgroundOsteosarcoma (OS) is a malignant tumor of the bone mostly observed in children and adolescents. The current treatment approach includes neoadjuvant and adjuvant chemotherapy; however, drug resistance often hinders therapy in OS patients. Also, the post-relapse survival of OS patients is as low as 20%. We therefore planned to understand the molecular cause for its poor prognosis and design an appropriate therapeutic strategy to combat the disease.MethodsWe analyzed OS patient dataset from Gene Expression Omnibus (GEO) and identified the differentially expressed genes and the top deregulated pathways in OS. Subsequently, drugs targeting the major de-regulated pathways were selected and the following assays were conducted- MTT assay to assess cytotoxicity of drugs in OS cells; immunoblotting and immunostaining to analyze key protein expression and localization after drug treatment; LysoTracker staining to monitor lysosomes; Acridine Orange to label acidic vesicles; and DCFDA to measure Reactive Oxygen Species (ROS).ResultsThe differential gene expression analysis from OS patient dataset implicated the striking involvement of cellular processes linked to autophagy and protein processing in the development of OS. We therefore selected the FDA approved drugs, chloroquine (CQ) and verteporfin (VP) known for autophagy inhibitory and proteotoxic functions to explore against OS. Importantly, VP, but not CQ, showed an extensive dose-dependent cytotoxicity. It resulted in autophagy disruption at multiple steps extending from perturbation of early autophagic processes, inhibition of autophagic flux to induction of lysosomal instability. Interestingly, VP treated protein lysates showed a ROS-dependent high molecular weight (HMW) band when probed for P62 and P53 protein. Further, VP triggered accumulation of ubiquitinated proteins as well. Since VP had a pronounced disruptive effect on cellular protein homeostasis, we explored the possibility of simultaneous inhibition of the ubiquitin-proteasomal system (UPS) by MG-132 (MG). Addition of a proteasomal inhibitor significantly aggravated VP induced cytotoxicity. MG co-treatment also led to selective targeting of P53 to the lysosomes.ConclusionHerein, we propose VP and MG induce regulation of autophagy and protein homeostasis which can be exploited as an effective therapeutic strategy against osteosarcoma.

Optimization of DNA Isolation Protocol from Emerging Leaves of Black Pepper [Piper nigrum (L.)

Black pepper (Piper nigrum L.) is a major spice crop and exported worldwide. The extraction of DNA from leaves of black pepper is challenging due to the presence of a significant amount of polysaccharides, polyphenolic compounds and other chemical constituents. Hence, the present study was aimed to standardize and optimize the DNA isolation protocol from the young folded leaves of black pepper. The emerging folded leaves of seven pepper accessions were selected and an efficient DNA extraction protocol from freshly collected emerging leaves of pepper was developed with modified cetyl trimethyl ammonium bromide (CTAB) method, which is a collective strategy of 2-mercaptoethanol, PVP (poly vinyl pyrrolidone), 1% sodium metabisulfite and 5M NaCl, which was used for efficient removal of polyphenols and polysaccharides. This protocol was pertinent to isolate genomic DNA from emerging leaf tissues of pepper. The presence of DNA was checked by 0.8% agarose gel; results showed that thick intact single band of high molecular weight confirming good quality. The genomic DNA yielded from fresh emerging leaves of seven pepper accessions was ranged from 75 - 120µg/g.

Vasectomy and Photoperiodic Regimen Modify the Protein Profile, Hormonal Content and Antioxidant Enzymes Activity of Ram Seminal Plasma.

This work aimed to determine the contribution of the testis and epididymis and the effect of the photoperiodic regimen on ram seminal plasma (SP). Semen was collected from 15 mature rams located in an equatorial (Colombian Creole and Romney Marsh, eight intact and two vasectomized) or a temperate climate (Rasa Aragonesa, three intact and two vasectomized). SP proteins were analyzed by Bradford, SDS-PAGE and difference gel electrophoresis (DIGE). Melatonin and testosterone concentrations were quantified by ELISA, and activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase by enzymatic assays. Vasectomy increased protein concentration and the intensity of high molecular weight bands (p < 0.001), with no differences between breeds. DIGE revealed the absence of six proteins in vasectomized rams: angiotensin-converting enzyme, lactotransferrin, phosphoglycerate kinase, sorbitol dehydrogenase, epididymal secretory glutathione peroxidase and epididymal secretory protein E1. Vasectomy also decreased melatonin concentrations in seasonal rams, and testosterone in all of them (p < 0.001), but did not affect antioxidant enzyme activity. Equatorial rams showed lower melatonin and testosterone concentration (p < 0.01) and catalase, but higher GPx activity (p < 0.05). In conclusion, vasectomy modifies the protein profile and hormonal content of ram seminal plasma, whereas the exposure to a constant photoperiod affects hormonal concentration and antioxidant enzymes activity.

Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers.

β‐arrestins (βarr1 and βarr2) are ubiquitous regulators of G protein‐coupled receptor (GPCR) signaling. Available data suggest that β‐arrestins dock to different receptors in different ways. However, the structural characterization of GPCR‐arrestin complexes is challenging and alternative approaches to study GPCR‐arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non‐canonical amino acids for photo‐ and chemical crosslinking into βarr1 and βarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin‐like), the corticotropin‐releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin‐like). We show that each receptor leaves a unique footprint on arrestins, whereas the two β‐arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell.

Assessment of high yielding local Pakistani bread wheat genotypes for improved chapati making quality

The experiment was conducted to classify the maximum glutenin protein possessed Pakistani bread wheat genotype for superlative chapati making quality by ten yield-related parameters. The studied germplasm was acquired from NARC, Pakistan, and planted in randomized-complete-block-design with four replicates at the screen house of the Genetics Department. Data were assessed via Duncan’s test, correlation analysis, SDS-PAGE, and cluster analysis. Duncan’s test conceded that Pirsabak-85 had the highest plant height, flag leaf area, biomass, grain yield plantˉ1, harvest index, and protein content. While, the correlation studies showed that plant height, tillers plantˉ1 (r = 0.649), fertile tillers plantˉ1 (r = 0.713),biomass (r = 0.861), spike length (LS), thousand-grain weight and harvest index had a positive higher significant association with grain yield plantˉ1. The SDS-PAGE analysis resolved 30 diverse high and low molecular weight bands, ranging from 200 kDa to 28 kDa glutenin subunits. Among genotypes, Pirsabak-85 showed maximum protein content and 10 Glu-1 scores. The dendrogram analysis revealed that Pirsabak-85 associated with cluster-II, which was a major and most diverged cluster. The Pirsabak-85 can be utilized to enhance bread wheat production and better chapati making quality

Characterization of hydrolysates of collagen from mechanically separated chicken meat residue

Abstract Obtaining collagen and hydrolysates from mechanically separated chicken meat residue is an excellent way to add value to this waste. The aims of this study were to obtain collagen hydrolysates from chicken MSM residue using Alcalase® and Flavourzyme® enzymes, as well characterizing the functional, structural and thermal stability properties. The highest degree of hydrolysis was obtained using Alcalase®, 36.11%, while using Flavourzyme® resulted in 12.02%. The DSC analysis of the collagen indicated a denaturation temperature of 46.47 °C. The FTIR spectra of the crude collagen showed absorption peaks that were characteristic of amide bands A, B, I, II and III. In the spectra of the hydrolysed the area of ​​the amide bands was reduced, and some peaks appeared between 800 and 1,200 cm-1. The disappearance of high molecular weight bands in the SDS-PAGE analysis also confirmed the hydrolysis of collagen. After the hydrolysis, the collagen presented reduced viscosity, the capacity to form foam, and foam stability. The emulsifying activity index was high in the hydrolysates in relation to the crude collagen. Thus, the use of Alcalase® and Flavourzyme® to obtain hydrolysates from collagen derived from chicken MSM residue was shown to be viable, and potentially useful for industrial applications.

Suppressing PARylation by 2',5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death.

High expression of 2',5'-oligoadenylate synthetase 1 (OAS1), which adds AMP residues in 2',5' linkage to a variety of substrates, is observed in many cancers as a part of the interferon-related DNA damage resistance signature (IRDS). Poly(ADP-ribose) (PAR) is rapidly synthesized from NAD+ at sites of DNA damage to facilitate repair, but excessive PAR synthesis due to extensive DNA damage results in cell death by energy depletion and/or activation of PAR-dependent programmed cell death pathways. We find that OAS1 adds AMP residues in 2',5' linkage to PAR, inhibiting its synthesis invitro and reducing its accumulation in cells. Increased OAS1 expression substantially improves cell viability following DNA-damaging treatments that stimulate PAR synthesis during DNA repair. We conclude that high expression of OAS1 in cancer cells promotes their ability to survive DNA damage by attenuating PAR synthesis and thus preventing cell death.

Characterization of macrophage stimulating compound in glycated whey protein concentrate.

Whey, a by-product of cheese making, is a collection of several milk proteins and has functional and nutritional values. Whey protein concentrate (WPC) exhibits various functional effects by glycation. Studies to find sugar-binding sites in a protein having a functional effect are reported. However, it is rarely clear whether it confirms that glycated single protein exhibits the same effect of protein cluster. This study confirmed which protein sites are responsible for the effect of glycated WPC (G-WPC). β-Lactoglobulin (LG) was the major protein of G-WPC and glycated with lactose. The glycated LG increased the nitric oxide and cytokine secretion similar to G-WPC and peptide sequences of active compound was confirmed using the high molecular weight band of G-WPC. The K151 and K157 residues of LG were modified by glycation with sugar in common with G-WPC. These residues of glycated LG potentially contribute to the immune-modulation effect of G-WPC.

Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase.

A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslinked following treatment with 1.5 mM chlorpyrifos oxon. High molecular weight bands were visualized on an SDS gel. Proteins in the gel bands were digested with trypsin, separated by liquid chromatography and analyzed in an Orbitrap mass spectrometer. MSMS files were searched for crosslinked peptides using the Batch-Tag program in Protein Prospector. MSMS spectra were manually evaluated for the presence of ions that supported the crosslinks. The crosslink between Lys544 in VLEMTGNIDEAEWEWK544AGFHR and Glu542 in VLEMTGNIDEAEWE542WK satisfied our criteria including that of spatial proximity. Distances between Lys544 and Glu542 were 7.4 and 9.5 Å, calculated from the cryo-EM (electron microscopy) structure of the HuBChE tetramer. Paraoxon ethyl, diazoxon, and dichlorvos had less pronounced effects as visualized on SDS gels. Our proof-of-principle study provides evidence that OP have the ability to crosslink proteins. If OP-induced protein crosslinking occurs in the brain, OP exposure could be responsible for some cases of neurodegenerative disease.

Physicochemical state and in vitro digestibility of heat treated water-soluble protein from Pacific oyster (Crassostrea gigas)

Oyster water-soluble protein (OWSP) was extracted from the Pacific oyster (Crassostrea gigas) and the impact of heat treatment on the physicochemical properties, structural changes and in vitro digestibility (proteolysis using pepsin, trypsin and α-chymotrypsin) of OWSP at different temperatures (35, 45, 55, 65, 75, 85, 95, 100 °C) were studied. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the intensity of low molecular weight bands decreased while the intensity of high molecular weight bands increased with increased temperature, indicating the bonding of proteins. The differential scanning calorimetry curve showed that the denaturation temperature and enthalpy of OWSP were 39.3 °C and 4.42 J/g, respectively. Heating resulted in significantly increased surface hydrophobicity and electronegativity, and decreased protein solubility of OWSP (p < 0.05). However, the dynamic light scattering, confocal microscopy and Nile red fluorescence showed that protein aggregates repelled each other and dispersed more evenly in solution due to the increase of surface hydrophobicity and electronegativity at 65–95 °C. The secondary structure determined using circular dichroism spectrometer showed that the α-helix content decreased and β-sheet content increased with heating. In vitro digestion showed that the proteolysis of OWSP increased with the denaturation of proteins at lower heating temperatures (45, 55 °C), while protein aggregation and the formation of a more compact structure inhibited the digestion of OWSP at higher heating temperatures (65–100 °C).

Tau Phosphorylation and Aggregation in the Developing Human Brain.

Tau hyperphosphorylation, mostly at serine (Ser) or threonine (Thr) residues, plays a key role in the pathogenesis of Alzheimer disease (AD) and other tauopathies. Rodent studies show similar hyperphosphorylation in the developing brain, which may be involved in regulating axonal growth and plasticity, but detailed human studies are lacking. Here, we examine tau phosphorylation by immunohistochemistry and immunoblotting in human fetal and adult autopsy brain tissue. Of the 20 cases with sufficient tissue preservation, 18 (90%) showed positive staining for S214 (pSer214), with the majority also positive for CP13 (pSer202), and PHF-1 (pSer396/pSer404). AT8 (pSer202/pThr205) and RZ3 (pThr231) were largely negative while PG5 (pSer409) was negative in all cases. Immunoblotting showed tau monomers with a similar staining pattern. We also observed phospho-tau aggregates in the fetal molecular layer, staining positively for S214, CP13, and PHF1 and negative for thioflavin S. These corresponded to high-molecular weight (∼150 kD) bands seen on Western blots probed with S214, PHF1, and PG5. We therefore conclude that fetal phosphorylation overlaps with AD in some residues, while others (e.g. T231, S409) appear to be unique to AD, and that tau is capable of forming nontoxic aggregates in the developing brain. These findings suggest that the fetal brain is resilient to formation of toxic aggregates, the mechanism for which may yield insights into the pathogenesis of tau aggregation and toxicity in the aging brain.