Protein Fractions Research Articles

CK1δ/ε kinases regulate TDP-43 phosphorylation and are therapeutic targets for ALS-related TDP-43 hyperphosphorylation

Hyperphosphorylated TAR DNA-binding protein 43 (TDP-43) aggregates in the cytoplasm of neurons is the neuropathological hallmark of amyotrophic lateral sclerosis (ALS) and a group of neurodegenerative diseases collectively referred to as TDP-43 proteinopathies that includes frontotemporal dementia, Alzheimer's disease, and limbic onset age-related TDP-43 encephalopathy. The mechanism of TDP-43 phosphorylation is poorly understood. Previously we reported casein kinase 1 epsilon gene (CSNK1E gene encoding CK1ε protein) as being tightly correlated with phosphorylated TDP-43 (pTDP-43) pathology. Here we pursued studies to investigate in cellular models and in vitro how CK1ε and CK1δ (a closely related family sub-member) mediate TDP-43 phosphorylation in disease. We first validated the binding interaction between TDP-43 and either CK1δ and CK1ε using kinase activity assays and predictive bioinformatic database. We utilized novel inducible cellular models that generated translocated phosphorylated TDP-43 (pTDP-43) and cytoplasmic aggregation. Reducing CK1 kinase activity with siRNA or small molecule chemical inhibitors resulted in significant reduction of pTDP-43, in both soluble and insoluble protein fractions. We also established CK1δ and CK1ε are the primary kinases that phosphorylate TDP-43 compared to CK2α, CDC7, ERK1/2, p38α/MAPK14, and TTBK1, other identified kinases that have been implicated in TDP-43 phosphorylation. Throughout our studies, we were careful to examine both the soluble and insoluble TDP-43 protein fractions, the critical protein fractions related to protein aggregation diseases. These results identify CK1s as critical kinases involved in TDP-43 hyperphosphorylation and aggregation in cellular models and in vitro, and in turn are potential therapeutic targets by way of CK1δ/ε inhibitors.

Effect of Milk Sources on Whey Protein and Fractions of Casein

Aim: Milk from Cow, Buffalo and goat was used for the study of isolation and fractionation of casein and whey proteins. Buffalo and goat milk were subjected to physicochemical attribute studies and a detailed protein profile namely total protein, casein fractions α-, β- and κ casein and whey protein were separated by urea fractionation method. The aim of this study was to compare various casein fractions isolated from cows, Buffalo and Goat milk. The method of separating casein into its fractions is based on the solubility of the individual components in urea the solution. The separation of casein fractions was carried out in dissolving molar concentration of urea and the yield of fractions and total protein percentages were calculated. Buffalo and goat milk have comparatively higher total protein and whey protein content as compared to cow milk. Maximum whey proteins found in goat milk (20.58%) whereas cow milk had the lowest whey protein (19.29%). Among casein fractions, α fraction (16.64%) was found maximum in Buffalo milk followed by cow (14.92%) and goat milk (5.42%). The goat milk showed the highest level of β casein (17.81%) whereas, the lowest concentration of β casein was found in cow milk (9.38%). Study Design: A significant contribution to the total milk production of India comes from buffalo milk and goat milk. Despite that, Buffalo milk and goat milk are not being utilized for many products because of difference in composition of different species milk and their inherent problems associated in the production of products. Place and Duration of Study: Karnataka Veterinary Animal and Fisheries Sciences University (KVAFSU), Dairy Science College, Hebbal, Bangalore Karnataka, India Materials: The Goat milk samples were collected from Sinchana Goat and Sheep farm, Marenahalli village (Bengaluru Rural Dist) and Buffalo milk was obtained from Country Delight Pvt. Ltd., J. P. Nagar, Bengaluru, Karnataka. Cow milk used in this investigation was collected from SEDP, Dairy Science College, Hebbal, Bengalore.. All the glassware used was soaked in chromic acid solution, repeatedly washed with water, rinsed with distilled water and dried before use. For microbiological analysis dried test tubes, conical flasks, pipettes were cotton plugged and sterilized in a hot air oven. The chemicals and reagents used in this study were mainly of analytical grade procured from Prince Laboratory Company Pvt. Limited, Bangalore. The protein molecular weight markers used for the electrophoretic study were procured from Bangalore Genei Pvt. Ltd. Methodology: Standard urea fractionation method adopted by Hipp et al was used for isolation and fractionation of caseins. Skim milk was prepared by subjecting whole milk for centrifugation, the skim milk obtained was acidified using 1 % hydrochloric acid at 200 C to obtain casein precipitate. Obtained whole casein was dissolved in 3.3 M urea at pH 7.5 and adjusted to pH 4.6 which precipitates the bulk of α casein and leaving k-caseins into soluble filtrates. Again the supernatant was adjusted to pH 4.9 diluted to 1.0 M urea and warmed to 300 C precipitating the β-caeisn. Further, addition of ammonium sulphate was used to isolate k-casein from the precipitate. Results: The sources of protein had a significant influence on the total casein, casein fractions and whey protein content of different species. The highest quantity of total caseins (34.30g/l) and whey protein (8.87 g/l) were observed in buffalo milk followed by cow (28.52 g/l) and lowest was observed in goat milk (28.45 g/l). Total protein, casein and whey protein contents were greatly affected by the source of milk. Conclusion: The significant effect of source of milk on total protein, casein, its fractions and their yield was observed. highest yield of casein (54.31%) was observed in cow milk than buffalo milk (48.95 %) and lower yield was found in goat milk. Amongst the three species, highest per cent of β casein was noted in goat milk (54.05 %) followed by buffalo (36.03%) and cow milk (34.14%). Recommendation: Rasagulla will be consumed universally by all age groups globally. Thus, the consumption of Rasagulla helps in providing overall nutritional requirements. However, the study has been conducted on a pilot scale. Moreover, commercial production may necessitate huge cost on enzymatic modification of proteins and separation peptides for value addition.

Breaking the Mucin Barrier: A New Affinity Chromatography-Mass Spectrometry Approach to Unveil Potential Cell Markers and Pathways Altered in Pseudomyxoma Peritonei

BackgroundPseudomyxoma peritonei (PMP) is a rare peritoneal mucinous carcinomatosis with largely unknown underlying molecular mechanisms. Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy is the only therapeutic option; however, despite its use, recurrence with a fatal outcome is common. The lack of molecular characterisation of PMP and other mucinous tumours is mainly due to the physicochemical properties of mucin.ResultsThis manuscript describes the first protocol capable of breaking the mucin barrier and isolating proteins from mucinous tumours. Briefly, mucinous tumour samples were homogenised and subjected to liquid chromatography using two specific columns to reduce mainly glycoproteins, albumins and immunoglobulin G. The protein fractions were then subjected to mass spectrometry analysis and the proteomic profile obtained was analysed using various bioinformatic tools. Thus, we present here the first proteome analysed in PMP and identified a distinct mucin isoform profile in soft compared to hard mucin tumour tissues as well as key biological processes/pathways altered in mucinous tumours. Importantly, this protocol also allowed us to identify MUC13 as a potential tumour cell marker in PMP.ConclusionsIn sum, our results demonstrate that this protein isolation protocol from mucin will have a high impact, allowing the oncology research community to more rapidly advance in the knowledge of PMP and other mucinous neoplasms, as well as develop new and effective therapeutic strategies.

Composition and functionality differences of oat protein concentrates: Potential of less refined concentrates obtained by wet milling

The objective of this study was to determine differences in composition and functionality for oat protein concentrates. This was achieved by comparing preparations extracted at different pH levels using either a twin screw press or alkaline extraction followed by isoelectric precipitation. Extraction by twin screw technology provided a higher protein yield compared to isoelectric precipitation. Analysis by SDS-PAGE revealed similar protein composition regardless of the extraction method or extraction pH except for extraction at pH 6. Extraction at a higher pH led to a higher protein purity and yield, while extraction closer to the isoelectric point of the protein resulted in less pure concentrates. All extracts were freeze-dried, and their composition and functionality were compared. Thermal analysis of the protein concentrates extracted by twin screw pressing showed a higher denaturation enthalpy compared to protein isolates prepared by isoelectric precipitation, suggesting that the native structure of the proteins is preserved to a higher extent. All protein fractions exhibited the typical U-shaped solubility profile, except for the concentrate extracted at pH 6, which showed an increase in solubility with increasing pH. Oat protein concentrates prepared by twin screw pressing had a higher affinity for binding to oil than to water, and this was also higher than for isoelectric precipitated protein extracts. Overall results demonstrate a potential for twin screw technology as a viable alternative to conventional wet extraction methods for producing less refined fractions from oats with tunable functional properties.

Analysis of the components of Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) and its regulation of γδ T-cell function

BackgroundMycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) is a peptide antigen released from the mycobacterial cytoplasm into the supernatant of Mycobacterium tuberculosis (Mtb) attenuated H37Ra strain after autoclaving at 121 °C for 20 min. Mtb-HAg can specifically induce γδ T-cell proliferation in vitro. However, the exact composition of Mtb-HAg and the protein antigens that are responsible for its function are currently unknown.MethodsMtb-HAg extracted from the Mtb H37Ra strain was subjected to LC‒MS mass spectrometry. Twelve of the identified protein fractions were recombinantly expressed in Escherichia coli by genetic engineering technology using pET-28a as a plasmid and purified by Ni–NTA agarose resin to stimulate peripheral blood mononuclear cells (PBMCs) from different healthy individuals. The proliferation of γδ T cells and major γδ T-cell subset types as well as the production of TNF-α and IFN-γ were determined by flow cytometry. Their proliferating γδ T cells were isolated and purified using MACS separation columns, and Mtb H37Ra-infected THP-1 was co-cultured with isolated and purified γδ T cells to quantify Mycobacterium viability by counting CFUs.ResultsIn this study, Mtb-HAg from the attenuated Mtb H37Ra strain was analysed by LC‒MS mass spectrometry, and a total of 564 proteins were identified. Analysis of the identified protein fractions revealed that the major protein components included heat shock proteins and Mtb-specific antigenic proteins. Recombinant expression of 10 of these proteins in by Escherichia coli genetic engineering technology was used to successfully stimulate PBMCs from different healthy individuals, but 2 of the proteins, EsxJ and EsxA, were not expressed. Flow cytometry results showed that, compared with the IL-2 control, HspX, GroEL1, and GroES specifically induced γδ T-cell expansion, with Vγ2δ2 T cells as the main subset, and the secretion of the antimicrobial cytokines TNF-α and IFN-γ. In contrast, HtpG, DnaK, GroEL2, HbhA, Mpt63, EsxB, and EsxN were unable to promote γδ T-cell proliferation and the secretion of TNF-α and IFN-γ. None of the above recombinant proteins were able to induce the secretion of TNF-α and IFN-γ by αβ T cells. In addition, TNF-α, IFN-γ-producing γδ T cells inhibit the growth of intracellular Mtb.ConclusionActivated γδ T cells induced by Mtb-HAg components HspX, GroES, GroEL1 to produce TNF-α, IFN-γ modulate macrophages to inhibit intracellular Mtb growth. These data lay the foundation for subsequent studies on the mechanism by which Mtb-HAg induces γδ T-cell proliferation in vitro, as well as the development of preventive and therapeutic vaccines and rapid diagnostic reagents.

Evaluation of cytotoxicity, acute toxicity, genotoxicity and antioxidant and antigenotoxicity activities of the sarcotesta fraction of punica granatum L. rich in lectin (PgTel)

ABSTRACT Punica granatum, popularly known as pomegranate, is a fruit tree with wide worldwide distribution, containing numerous phytochemicals of great medicinal value. The aim of the present study was to determine the phytochemical profile and antioxidant potential of a protein fraction (PF) derived from P. granatum sarcotesta which is rich in lectin. In addition, the acute oral toxicity, genotoxicity and antigenotoxicity of this protein fraction (PF) from P. granatum sarcotesta was measured. The phytochemical profile of PF was determined using HPLC. The in vitro antioxidant effect was assessed using the methods of total antioxidant capacity (TAC) and DPPH and ABTS+ radical scavenging. Acute oral toxicity was determined in female Swiss mice administered a single dose of 2000 mg/kg. This PF was examined for genotoxicity and antigenotoxicity at doses of 500, 1000 and 2000 mg/kg, utilizing mouse peripheral blood cells. Phytochemical characterization detected a high content of ellagic acid and antioxidant capacity similar to that of ascorbic acid (positive control). PF was not toxic (LD50 >2000 mg/kg) and did not exert a genotoxic effect in mice. PF protected the DNA of peripheral blood cells against damage induced by cyclophosphamide. In conclusion, this PF fraction exhibited significant antioxidant activity without initiating toxic or genotoxic responses in mice.

Amsacta moorei entomopoxvirus encodes a protein kinase with dual activity and a broad substrate spectrum including two putative cellular substrates.

Amsacta moorei entomopoxvirus (AMEV) is a poxvirus that can only infect insects. This virus is an attractive research material because it is similar to smallpox virus. AMEV is one of many viruses that encode protein kinases that drive the host's cellular mechanisms, modifying immune responses to it, and regulating viral protein activity. We report here the functional characterization of a serine/threonine (Ser/Thr) protein kinase (PK) gene (ORF AMV197) of AMEV. Expression of the AMV197 gene in baculovirus expression system yielded a ~ 35.5kDa protein. PK activity of expressed AMV197 was shown by standard PK assay. Substrate profiling of AMV197 protein by peptide microarray indicated that the expressed protein phosphorylated 81 of 624 substrates which belong to 28 families of PK substrates. While the hypothetical AMV197 protein phosphorylates Ser/Thr only, we demonstrated that the expressed PK also phosphorylates probes with tyrosine residues on the array which is a rare property among PKs. Pull-down assay of the AMV197 protein with the subcellular protein fractionations of Ld652 cells showed that it is using two cellular proteins (18 and 42kDa) as novel putative substrates. Our results suggest that AMEV can regulate cellular mechanisms by phosphorylating cellular proteins through AMV197 PK. However, further experiments are needed to identify the exact role of this PK in the replication of AMEV.

Dominant Circulating Cell-free Mycobacterial Proteins in in-use Machining Fluid and their Antigenicity Potential.

Occupational exposure to industrial Metalworking Fluid (MWF) colonized by Mycobacterium immunogenum (MI) has been associated with immune lung disease hypersensitivity pneumonitis (HP) in machinists. This warrants regular fluid monitoring for early detection of mycobacterial proteins, especially those with antigenic potential. To detect and identify dominant MI proteins and antigens directly from the field-drawn in-use MWF using an integrated immunoproteomic and immunoinformatic approach. An MI-positive MWF selected by DNA-based screening of several field-drawn MWF samples were cultured to isolate the colonizing strain and profiled for dominant circulating cell- free (ccf) MI proteins, including antigens using an integrated immunoproteomic (1D- and 2Dgel fractionation of seroreactivity proteins combined with shotgun proteomic analysis using LC-MS/ MS) and immunoinformatic strategy. A new MI strain (MJY-27) was identified. The gel fractionated MI protein bands (1Dgel) or spots (2D-gel) seroreactive with anti-MI sera probes (Rabbit and Patient sera) yielded 86 MI proteins, 29 of which showed peptide abundance. T-cell epitope analysis revealed high (90-100%) binding frequency for HLA-I& II alleles for 13 of the 29 proteins. Their antigenicity analysis revealed the presence of 6 to 37 antigenic determinants. Interestingly, one of the identified candidates corresponded to an experimentally validated strong B- and T-cell antigen (AgD) from our laboratory culture-based studies. This first report on dominant proteins, including putative antigens of M. immunogenum prevalent in field in-use MWF, is a significant step towards the overall goal of developing fluid monitoring for exposure and disease risk assessment for HP development in machining environments.

Impact of genetic κ-casein variants (A, B, E) on chymosin-induced milk coagulation properties: Application of a new LC–MS-based genotyping method

This study evaluated the impact of κ-casein (κ-CN) protein phenotype (AA (n = 18), AB (n = 18), AE (n = 5), BB (n = 3)) in the raw milk of 44 individual Holstein cows from a single farm on coagulation properties using diffusion wave spectroscopy. κ-CN variants were analyzed using a newly developed LC–MS method in individual milk samples, and relative milk protein fractions were quantified by LabChip-analysis. κ-CN fractions expressed in the percentage of total milk protein, total casein, and β- and κ-CN were then calculated. The gross composition of the individual milk samples and variation between individual cows was analyzed and was rather large, as expected. Traits were analyzed using an ANCOVA model with pH as a covariate.Our results showed that mean percentages of κ-CN fractions increased for κ-CN phenotypes in the order AA < AE < AB < BB. Rennet coagulation time was slightly affected, but curd firmness was significantly affected by κ-CN phenotype (BB > AE > AB > AA) when controlling for pH.

Drought priming enhances wheat grain starch and protein quality under drought stress during grain filling

The impact of drought stress on crop yield and quality is substantial. Drought priming during the early growth stage of plants has been shown to improve tolerance to drought stress during the reproductive stage, although its effects on grain quality remains elusive. This study aimed to investigate the influence of drought priming on starch and protein levels in grains under drought stress during grain filling. Our results reveal that drought stress results in a reduction in starch content and its constituents, while simultaneously increasing glutenin macropolymers and protein fractons. Notably, drought primed plants under drought stress (PD) exhibit mitigated declines in starch content and its components, leading to improvements in starch swelling power and pasting properties. Additionally, PD results in a slight increase in protein fractions, limiting the overall rise in total protein content compared to drought stress alone. Collectively, our study underscores the efficacyof drought priming as a strategy to counteract the negative effects of drought stress on grain quality, particularly by minimizing starch losses and restraining protein content elevation.

CERVUS ELAPHUS: CULTIVATION IN THE KALININGRAD REGION, PROCESSING AND EVALUATION OF THE QUALITY AND SAFETY OF VENISON PRODUCTS

Recently, there has appeared a tendency to use non-traditional animal raw materials to expand the range of sausages. The purpose of the research is to determine the organoleptic and physico-chemical properties, as well as quality indicators of new sausages with a mass fraction of deer muscle tissue in the recipe (&gt; 60%). An organoleptic evaluation was carried out, then the study of physicochemical parameters, the mass fraction of protein, fat, salt, sodium nitrite, total phosphorus, and residual activity of acid phosphatase was performed. Next there were studied such microbiological indicators as QMAFAnM, BGKP (coliforms), sulfite-reducing clostridia, Staphylococcus aureus, and pathogenic microorganisms: Salmonella, Escherichia coli, Listeria monocytogenes. Safety indicators have been established: the content of toxic elements (mercury, arsenic, cadmium, lead) and quality indicators: the content of nitrosamines, pesticides. The quality control of packaging and labeling was carried out visually in accordance with the requirements of the standard. The temperature is measured inside the product with a glass liquid thermometer. The net weight of consumer packaging was determined gravimetrically by static weighing. It has been proved that all three types of the studied sausages «Zavtrak okhotnika», «Drezdenskiye» and «Oldenburgskiye» corresponded to the normative documentation in terms of organoleptic, physico-chemical properties, quality and safety indicators, nutritional value.

Technological Parameters of Microfiltration in the Production of Micellar Casein Concentrate

Background:: There are many different factors that influence the process of microfiltration, including membrane pore size, pressure, temperature, and membrane origin. Studying the joint influence of technological parameters in order to justify rational modes of microfiltration is relevant. Objective:: This research aimed to define the rational technological parameters of microfiltration, which allow to achieve the target parameters of retentate in the process of obtaining micellar casein concentrate. Methods:: Standard and modified methods and techniques have been used in the process of experimental research. The mathematical processing was carried out by methods of mathematical statistics according to several trials data at three stage sequence. Results:: As a result of experimental investigations, the applicability of ceramic membranes for microfiltration of skim milk has been shown. The rational pore size has been established as 0,1 μm. To obtain the micellar casein concentrate, it is advisably to carry out the microfiltration of skim milk to the mass fraction of dry solids in concentrate (13 ± 3) %, followed by two-phase diafiltration with a double volume of reverse osmosis water. The rational operating conditions, allowing to get the target value of protein fractions correlation, have been established, as described as follows: input pressure as 0,2 – 0,3 MPa, and temperature as 10 – 15°С. Conclusion:: The analysis of microfiltration concentrates and permeates composition of raw milk, including vitamins and minerals content, showed both their high nutritional and biological value and the viability of further processing into food ingredients, including the ingredients for the specialized food products.

Importance of pressing temperature on protein enrichment by electrostatic separation of rapeseed press cakes

Electrostatic separation presents a promising method to concentrate protein from rapeseed. Previous research, with focus on meals neglected, the impact of de-oiling methods on the separation process and protein functionality. Therefore, this study describes the impact of processing history on the electrostatic protein enrichment from rapeseed press cakes pressed at varying temperatures on separation efficiency and hydration properties. A defatted meal was used as control. Pressing temperatures ranging from 60 to 130 °C resulted in varied oil removal, with most removal at 80 °C, yielding final oil and protein contents of 20 and 24 g/100 g dry matter, respectively. Subsequent milling and electrostatic separation produced protein fractions with protein enrichment levels of 17 %–30 % compared to the initial press cakes. Recovery of these fractions was influenced by residual oil content and heat-induced structural changes. The press cake with the least oil demonstrated protein yields comparable to rapeseed meal, recovering 0.4 g protein/g press cake protein. Hydration properties revealed that protein solubility was improved by protein enrichment. No significant effect on water holding capacity was noted due to protein enrichment. These findings demonstrate a promising approach for producing protein-rich ingredients from rapeseed press cakes using tribo-electrostatic separation.

Combined techniques for revealing the mechanism beneath the inhibition effects of pectin on gluten digestibility using static in vitro gastro-duodenal protocols.

In the current study, how pectin retards the digestibility of wheat gluten was investigated using a static in vitro gastric-duodenal model. The degree of protein hydrolysis was estimated using the o-phthaldialdehyde method, while the in vitro digestograms were mathematically fitted using a single first-order kinetics model. Peptides' profile, free amino acids compositions, gluten-pectin interactions and their effects on enzymatic activities of proteolytic enzymes as well as on the gluten secondary structures under digestive conditions were studied using combined techniques. Results showed that pectin could retard gluten digestibility through 1). preferential absorption to insoluble gluten aggregates by electrostatic interactions; 2). increasing the helix and reducing the β-sheet content of the solubilized gluten protein fractions in terms of their secondary molecular structures; 3). reducing pepsin activity by forming negatively charged pectin-gluten mixtures which then interacted with the positively charged pepsin molecules. The deeper insight into gluten-pectin interactions and their influences on gluten digestibility under gastrointestinal conditions provides important clues for developing effective forms of dietary fiber to improve the nutritional benefits of plant protein in individuals.

Crucial role of SWL1 in chloroplast biogenesis and development in Arabidopsis thaliana.

The disruption of the SWL1 gene leads to a significant down regulation of chloroplast and secondary metabolites gene expression in Arabidopsis thaliana. And finally results in a dysfunction of chloroplast and plant growth. Although the development of the chloroplast has been a consistent focus of research, the corresponding regulatory mechanisms remain unidentified. In this study, the CRISPR/Cas9 system was used to mutate the SWL1 gene, resulting in albino cotyledons and variegated true leaf phenotype. Confocal microscopy and western blot of chloroplast protein fractions revealed that SWL1 localized in the chloroplast stroma. Electron microscopy indicated chloroplasts in the cotyledons of swl1 lack well-defined grana and internal membrane structures, and similar structures have been detected in the albino region of variegated true leaves. Transcriptome analysis revealed that down regulation of chloroplast and nuclear gene expression related to chloroplast, including light harvesting complexes, porphyrin, chlorophyll metabolism and carbon metabolism in the swl1 compared to wild-type plant. In addition, proteomic analysis combined with western blot analysis, showed that a significant decrease in chloroplast proteins of swl1. Furthermore, the expression of genes associated with secondary metabolites and growth hormones was also reduced, which may be attributed to SWL1 associated with absorption and fixation of inorganic carbon during chloroplast development. Together, the above findings provide valuable information to elucidate the exact function of SWL1 in chloroplast biogenesis and development.

Isolation, Identification and in-Silico Characterization of Bioactive Peptide from the Venom Sac of Conus inscriptus

Nature has been the source of new medications since ancient times, and at least half of all commercialised pharmaceuticals have been made from renewable resources to treat various illnesses, including pain, inflammation, and infection. In recent years, interest has risen in bioprospecting of new bioactive peptides, especially for medical and health-related applications. Therefore, the production of functional meals is starting to get recognition for its potential to enhance quality of life when combined with a healthy lifestyle. In view of this, the current work aimed to isolate, purify, and identify bioactive peptides from Conus inscriptus venom sac protein extracts, with an emphasis on antioxidant and anti-inflammatory properties. The findings reveal that the DPPH radical scavenging activity, albumin denaturation, and HRBC membrane stabilisation inhibition activities of Conus inscriptus venom sac crude protein extracts are 23.76±2.4, 66.23±3.1, and 57.32±2.6% respectively. Secondary screening of purified protein fractions obtained through sephadex G100 gel filtration exhibited increased anti-inflammatory efficacy from fractions 19 to 23. Electrophoresis and PMF analysis revealed that the purified anti-inflammatory peptide was homogenous with a molecular weight of 12 KDa, and it was identified as a conotoxin MI15b precursor. The 3D model of conotoxin exhibits the highest similarity with template c6nk9A with a confidence of 54.1%, and its function was predicted as a potassium ion channel inhibitor. The findings of the Insilco characterisation of the conotoxin MI15b precursor concluded that this protein might serve as an anti-inflammatory agent and may be responsible for therapeutic actions in the medical management of many inflammatory-related disorders.

Evaluating The Cytotoxic Activity of Lactobacillus plantarum IIA-1A5 Against MCF-7 Human Breast Cancer Cells and Identifying Its Surface Layer Protein Gene

Breast cancer is a serious global health concern, with a high mortality rate worldwide. Using natural compounds as potential cancer therapies is a promising approach to address this issue. Previous research has shown that probiotic lactic acid bacteria (LAB) and their metabolites, such as surface layer protein (slp), have a positive impact on a variety of health disorders, including cancer. The purpose of this study was to evaluate the ability of Lactobacillus plantarum IIA-1A5 to suppress the growth of the MCF-7 breast cancer cell line and detect the presence of the slp gene. Intracellular and extracellular protein fractions were isolated from L. plantarum IIA-1A5 cultures. The protein concentrations and molecular weights of the extracts were measured. The anticancer activity of the extracts was assessed using the MTT cytotoxicity test, and IC50 values were calculated. The slp gene was identified through polymerase chain reaction (PCR) amplification and nucleotide sequencing. The results demonstrated that L. plantarum IIA-1A5 had a concentration-dependent inhibitory effect on MCF-7 breast cancer cells, with IC50 values of 6.831 and 12.35 μg/mL for intracellular extracts and extracellular extracts, respectively. Additionally, PCR amplification and nucleotide sequencing confirmed the presence of the slp gene, which may contribute to the strain’s anticancer abilities. These findings suggest the potential of L. plantarum IIA-1A5 as a natural anticancer agent against MCF-7 breast cancer cells. Further research is warranted to elucidate the underlying mechanisms of L. plantarum IIA-1A5 in breast cancer treatment.

EFFECT OF RICE STRAW ON SOME BLOOD BIOCHEMICAL PARAMETERS OF IRAQI BUFFALO (Bubalus Bubalis)

This study was aimed to show the changes of some blood parameters by the effect of biological and chemical treatment of rice straw ,It was conducted at The Ruminant Research Station in Abu Ghraib / Directorate of / Agricultural Researches / Ministry of Agriculture, for the period from June 1, 2021 until August 27, 2021 (preceded by the preliminary period 14 days), The study included 12 Iraqi buffalo calves aged 5 - 6 months. the calves were randomly divided into three equal groups (4 calves / group), kept in individual pens and distributed among group.T1 (the control group) in which the calves were fed on untreated rice straw T2 the calves were fed on rice straw treated with urea at a rate of 3% T3 the calves were fed on rice straw treated with Trichoderma harzianum at a rate of 0.1%. The triglycerides concentration were decreased significantly (p≤0.05) in the T2 (11.28 mg/dL) and T3 (11.32 mg/dL) compared with T1 (20.79 mg/dL). similarly, the level of very low-density lipoprotein (vLDL) was decreased 2.25, 2.26, 4.16 mg/dL for T2, T3, and T1, respectively. The creatinine concentration in T2(87.19 mmol/L) was significantly decrease than that of T1(177.8 mmol/L) and T3(176.75 mmol/L). Also, ALP concentration increased in T2 (238.93 IU/liter) compared with T1. (151.82 IU/liter), while it did not differ significantly with T3( 205.83 IU/liter) Also T3 did not differ significantly from T1. The differences in the concentration of lipid profile, protein fractions and enzymes activity among groups lacked significance , feeding of rice straw treated biologically and chemically had no negative effect on buffalo calves health.

Extraction of Protein and Bioactive Compounds from Mediterranean Red Algae (Sphaerococcus coronopifolius and Gelidium spinosum) Using Various Innovative Pretreatment Strategies.

In this study, the release of proteins and other biomolecules into an aqueous media from two red macroalgae (Sphaerococcus coronopifolius and Gelidium spinosum) was studied using eight different cell disruption techniques. The contents of carbohydrates, pigments, and phenolic compounds coextracted with proteins were quantified. In addition, morphological changes at the cellular level in response to the different pretreatment methods were observed by an optical microscope. Finally, the antioxidant capacity of obtained protein extracts was evaluated using three in vitro tests. For both S. coronopifolius and G. spinosum, ultrasonication for 60 min proved to be the most effective technique for protein extraction, yielding values of 3.46 ± 0.06 mg/g DW and 9.73 ± 0.41 mg/g DW, respectively. Furthermore, the highest total contents of phenolic compounds, flavonoids, and carbohydrates were also recorded with the same method. However, the highest pigment contents were found with ultrasonication for 15 min. Interestingly, relatively high antioxidant activities like radical scavenging activity (31.57-65.16%), reducing power (0.51-1.70, OD at 700 nm), and ferrous iron-chelating activity (28.76-61.37%) were exerted by the different protein extracts whatever the pretreatment method applied. This antioxidant potency could be attributed to the presence of polyphenolic compounds, pigments, and/or other bioactive substances in these extracts. Among all the used techniques, ultrasonication pretreatment for 60 min appears to be the most efficient method in terms of destroying the macroalgae cell wall and extracting the molecules of interest, especially proteins. The protein fractions derived from the two red macroalgae under these conditions were precipitated with ammonium sulfate, lyophilized, and their molecular weight distribution was determined using SDS-PAGE. Our results showed that the major protein bands were observed between 25 kDa and 60 kDa for S. coronopifolius and ranged from 20 kDa to 150 kDa for G. spinosum. These findings indicated that ultrasonication for 60 min could be sufficient to disrupt the algae cells for obtaining protein-rich extracts with promising biological properties, especially antioxidant activity.

Isolation and characterization of four protein fractions of gluten by conventional methods

Many gluten components have been reported, but their physicochemical properties are rarely reported due to the small amount of preparation. In this research, ω-gliadin, αβγ-gliadin, alkali-soluble and urea-soluble glutenin (ASG and USG) fractions were isolated by conventional methods and characterized by molecular weights, disulfide bond contents, amino acid compositions, Ultraviolet (UV) absorption, FTIR and 13C Solid-state NMR Spectroscopy. The results showed that purified ω-gliadin, αβγ-gliadin, ASG and USG could be obtained by conventional methods on a large scale (approximately 9.67 %, 12.43 %, 6.00 % and 18.26 %, respectively). The UV absorption of gluten components in the helical and randomly coiled state could not be detected. The molecular weights of purified ω-gliadin, αβγ-gliadin, ASG and USG were all ∼38,500 g/mol and their disulfide bond contents were 0.076, 0.374, 0.086 and 0.576 μmol/g, respectively. USG is characterized by free Glu and redundant Met, Phe and Ile. The distinctively diffraction angles of the gluten component monomers were 2θ at 22 °, 24 °, 29 ° and 35 °. This study will be of great reference value for the industrial application of these newly isolated proteins in gluten production.