Total Amount Of Protein Research Articles

Investigation of catalytic properties of glutathione reductase obtained by chromatographic methods from the liver of rats with paracetamol-induced liver damage upon administration of 6-hydroxy-2,2,2,4-trimethyl-1,2-dihydroquinoline

The aim of the present study was to analyze some catalytic and regulatory properties of the enzyme preparation of glutathione reductase (GR, EC 1.6.4.2.) obtained from rat liver using gel filtration and ion exchange chromatography under conditions of paracetamol-induced liver injury (PILI) and administration of 6-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline (DHQ). Laboratory animals (Rattus norvegicus rats of the Wistar line) were divided into 3 groups. The animals of the control group were orally administered vaseline oil. The second group consisted of rats with PILI induced by a single oral administration of paracetamol at a dose of 1000 mg/kg body weight dissolved in 1 cm3 of vaseline oil. Animals of the third group were modeled by PILI and orally administered with DHQ at a dose of 50 mg per 1 kg body weight dissolved in 1 cm3 of 1% starch, 1 hour and 12 hours after paracetamol administration. Liver and serum were collected 24 hours after paracetamol administration. GR activity was measured on a spectrophotometer at 340 nm wavelength. The enzyme was purified from rat liver by ammonium sulfate fractionation, gel filtration through Sephadex G-25, and ion exchange chromatography on DEAE-cellulose. The total amount of protein in the samples was measured using the BCA protein assay kit. GR was obtained from the liver of rats treated with DHQ on the background of PILI with a 32-fold purification degree. Using the method of double inverse Lineweaver-Burk coordinates it was shown that administration of DHQ to rats was accompanied by a decrease in the affinity of the enzyme for NADPH, as compared to the indicators at pathology. The introduction of isocitrate into the spectrophotometric medium resulted in a less pronounced increase in the activity of GR from the liver of animals treated with DHQ on the background of PILI, compared to the second group of rats. The enzyme from the liver of animals of the third group was characterized by a more significant decrease in activity when glucose-6-phosphate was introduced into the reaction medium. Citrate had a less pronounced regulatory effect on GR from the liver of rats treated with DHQ on the background of pathology, relative to the indicators of animals with PILI. The changes in enzyme properties demonstrated may be due to conformational changes in the molecule resulting from the inhibition effect of DHQ on the intensity of free radical-induced oxidation, which develops against the background of the toxic effect of paracetamol metabolites.

Biochemical parameters of dairy goats of the Zaanen breed when using a preparation based on bacillus Amyloliquefaciens

The effect of a drug based on B. amyloliquefaciens on the biochemical parameters of blood serum of dairy goats was studied. All biochemical parameters of the animals corresponded to the norm, but in the experimental groups during the experiment, the amount of total protein and albumin was higher on average by 8.39% (p≤0.05) and 16.5% (p≤0.05), respectively. The amount of cholesterol was 17.5% lower in the experimental groups (p≤0.01), the activity of the enzyme aspartate aminotransferase was 7.6% lower, the bilirubin content was 19.5% lower (p≤0.01) compared to the control parameters. The use of probiotic B. amyloliquefaciens optimizes the intensity of metabolic processes and improves liver function in animals. This contributed to an increase in the average milk yield per lactation in the experimental groups of goats by 60 and 51 kg relative to the control data. It is possible to recommend the drug probiotic B. Amyloliquefaciens as a biologically active supplement for goats during lactation in order to increase the milk yield of animals and receive conditionally additional profit in the amount of 2244.5 rubles per head on average.

Immunobiological indicators of anti-infection resistance of Karakul lambs

The study investigated immunobiological indicators of natural resistance in Karakul lambs. Indices of natural resistance to conditionally pathogenic microorganisms were measured to determine the general anti-infective resistance of Karakul lambs. The amount of total protein, their fractions and immunoglobulins in the perfusion fluid passed through the blood system of the gastrointestinal tract of lambs in the first 48 hours after birth and after 48 hours of postnatal life of Karakul lambs was determined. Also, the titer of antibodies against conditionally pathogenic microorganisms in blood serum of Karakul lambs of different ages was determined, their importance in formation of colostral and natural active immunities was analyzed.

The importance of stimulators in cultivating a non-traditional plant-indigofera Tinctoria L

The article depicts the duration and norms of the use of various stimulants in the cultivation of the non-traditional leguminous plant Indigofera (Indigofera tinctoria L.) and its effect on seed yield, root and stem residues, and the amount of total proteins grown in the conditions of the meadow alluvial soils of the Khorezm, the northern region of the Republic of Uzbekistan. To get a high and quality seed yield from Indigofera, it should be sown at 3 kg per hectare in the third ten days of April, and a geohumat stimulator is applied at a rate of 1.0 l/ha along with sowing, 1.6 l/ha during the period of 3–4 true leaf appearances, and 1.6 l/ha in the flowering season of the vegetation. When applied at the rate of, a higher seed yield of 18.3 t/ha was obtained compared to the control option without stimulant use, 6.8 t/ha, and 2.9 t/ha compared to the option where Uzgumi as a chosen option was used only with planting, and it is based on the fact that 1.6 t/ha of additional seed yield can be obtained compared to the option where Geohumat stimulator was used only with planting. Also, it has been scientifically justified that the amount of total proteins increased up to 12.960% in the plant leaf, 30.980% in the seed, and 6.945% in the root when the geohumat stimulator was used at acceptable rates along with planting, during the 3–4 true leaves of the growing season, and during the flowering period. It indicates that the result is 1.249, 1.47, and 1.307% more proportionally than options grown without the stimulator.

One-Dimensional Acrylamide Gel Electrophoresis for Analysis of Plant Samples.

Polyacrylamide gel electrophoresis (PAGE) is a widely used technique for separating proteins from complex plant samples. Prior to the analysis, proteins must be extracted from plant tissues, which are rather complex than other types of biological material. Different protocols have been applied depending on the protein source, such as seeds, pollen, leaves, roots, and flowers. Total protein amounts must also be determined before conducting gel electrophoresis. The most common methodologies include PAGE under native or denaturing conditions. Both procedures are used consequently for protein identification and characterization via mass spectrometry. Additionally, various staining procedures are available to visualize protein bands in the gel, facilitating the software-based digital evaluation of the gel through image acquisition.

КОЛОСТРАЛЬНЫЙ ИММУНИТЕТ ТЕЛЯТ НА ФОНЕ ИММУНОКОРРЕКЦИИ ОРГАНИЗМА КОРОВ-МАТЕРЕЙ

The aim of this work was to evaluate the effect of immunostimulating drugs Salus-P-E and Bovistim-K on the quality of colostrum and the immune status of calves after drinking colostrum. Work was carried out on first-calf heifers of the Holstein breed. Three groups of deep-calving cows were selected according to the principle of analogue groups of 10 animals each. Cows of the 1st experimental group were injected intramuscularly in the middle third of the neck with Salus-P-E at a dose of 10 ml three times 60, 30 and 15 days before the expected date of calving, the 2nd experimental group – Bovistim-K at the same time and dose, in the control group, biological preparations were not used. Colostrum sampling was carried out twice: within 60 minutes after calving and 24 hours after calving. In the second series of experiments, we studied the clinical and physiological state and blood parameters of newborn calves after drinking colostrum. The colostrum of cows of the 1st and 2nd experimental groups contained more immunoglobulins than control samples by 23,8 and 27,67 g/l; total protein - by 3,08 and 3,32%; the level of caseins – by 0,34 and 0,22%, respectively. Against the background of immunocorrection of the organism of deep-calving mother cows, there is an increase in the amount of hemoglobin, total protein, reserve alkalinity and certain changes in the ratio of protein fractions of blood of newborn calves. In the first day of life, an increase in the blood serum of calves of the experimental groups was found compared to the control: albumin by 7,9 and 8,4%; α-globulins – by 18,6 and 15,7%, β-globulins – by 22,1 and 16,9%, γ-globulins by 24,4 and 21,4%, respectively. Drinking high-quality colostrum (Brix over 24) contributed to an increase in average daily gains in calves of the 1st and 2nd experimental groups by 18,2 and 20,2%. The use of immunostimulating drugs Salus-P-E and Bovistim-K increases the immunocompetent properties of colostrum, which contributes to the formation of a high level of colostral immunity in the body of newborn calves, reducing morbidity and improving growth and survival rates.

Investigation of the effect of different culture conditions on recombinant protein production

After the COVID-19 pandemic, vaccine production technologies have become the focus of attention of researchers. As a matter of fact, recombinant protein-based antigen production, which is one of them, has taken its place in the first place. Proteins obtained by recombinant DNA technology are used in many industrial areas, especially vaccine applications, due to their reliability. Therefore, it is very important to produce targeted recombinant proteins in large quantities. This study, for the high amounts production of Omp25 protein, which is used as a vaccine candidate against brucellosis, in laboratory conditions, is aimed to reveal the effects of conditions that are the pre-culturing process, inoculation in LB or TB media, denatured or native purification, culturing with/without IPTG. All the results were analyzed by SDS-PAGE, confirmed Western Blot, and the total protein amounts were measured Bradford method. According to the results, Omp25 protein could not be obtained under native purification conditions in both cultures without induction, but it was observed under denatured conditions. This result can be explained that the protein in the cell is either misfolded or incorporated into the membrane. The amount of protein appears to be much higher in the presence of the inducer in both media inoculated with the starter pre-culture compared to the overnight pre-culture; 8.79 mg and 39.4 mg from 1 L culture, respectively. Additionally, as expected, the addition of IPTG increased the amount of protein, approximately one-and-a-half-fold for LB and about three-fold for TB. Finally, it was observed that TB medium provided higher protein production than LB, which can be explained by the presence of glycerol and high yeast extract in the medium. Although our study contains results that will attract the attention of vaccine industry, it should be kept in mind that all process should always be optimized depending on the structure of the targeted protein and thus the production amount can be further increased.

MILK PRODUCTIVITY OF COWS OF THE UKRAINIAN RED-AND-WHITE DAIRY BREED DEPENDS ON THE ORIGIN AND COMPATIBILITY OF THE BULLS

We studied the effectiveness of the bulls selection to the cows of the Ukrainian Red-and-White dairy breed (n = 490 mother-daughter pairs) different bloodlines and related groups. The level of milk productivity of first-borns cows (n = 490 mother-daughter pairs) for 305 days of the first lactation, obtained as a result of the compatibility of the bulls. The parents of the first-borns cows were four breeding bulls (bloodline Regal 352882, RG Valiant 1650414 and R. Sovereign 198998) of the Ukrainian Red-and-White dairy breed and one bull (Jornado Red 114386106, RG Chief 1427381) of the Holstein breed, and their mothers – from 11 bulls of three origin bloodlines (Dynemik 359742, Leader 1926780 and Hanover 1629391) and 4 related groups. A total of 29 combinations of interbloodline selection were investigated. The highest level of milk productivity was characterized by the daughters of bull Viter 4444 of the bloodline Regal 352882 (7191.5 ± 75.90), Jornado Red 114386106 of the Holstein breed of foreign selection, which belonged to the related group Chief 1427381 (the milk yield for 305 days of the first lactation was 7188.7 ± 31.05 kg), and Artek 344 related group Valiant 1650414 (the yield for 305 days of the first lactation was 7052.5 ± 75.51 kg). A statistically significant difference was established between milk yield indicators for 305 days of the first lactation between the groups of cows of the bull Jornado Red (related group Chief 14273381) and Luchnov 471 (r. g. R. Sovereign 198998) – +380 kg (td = 4.22 p < 0.001) in favor Jornado Red. Between cows of the bull Viter 4444 (bloodline Regal 352882) and Luchnov 471 (R. Sovereign 198998) – +383 kg (td = 3.38 p < 0.001) in favor of Viter 4444. Animals of the bulls Kanzler and Viter 4444 have the highest fat content in milk (3.71%), and cows of the related groups Valiant 1650414 characterized by the lowest fat content in milk (3.68%). A statistically significant difference was established between the amount of total milk fat and protein in the milk of cows of different bulls.
 A total 20 successful combinations of bulls were found, the first-borns cows of which were characterized by an increased milk yield on average across all successful compatibility by +96 kg (1.5%), milk fat yield – by +6,1 kg (2.3%) and total protein – by +5.3 kg (2.3%) from the average level of phenotypic manifestation of milk productivity traits of the daughters of all 5 evaluated bulls. The increasing of daughters milk yield of some successful combinations was from +42 kg (0.6%, sire Jornado 114386106 RG Chief 1427381) up to +296 kg (4.1%, sire Viter 4444,), milk fat from +2 kg (0.7%, sire Artek 344, RG Valiant) up to +8 kg (3%, sire Viter 4444) and protein from +1 kg (0.4%, sire Artek 344) up to +12 kg (5%, sire Viter 4444). The data, which we received, confirm the influence of the origin of cows by father on the level of their milk productivity and the high combining ability of individual improver bulls with the mothers of other genealogical formations.

Polysiphonia morrowii Harvey'nin Biyokimyasal ve Fizyolojik Özellikleri Üzerinde Etkili Bir Parametre: Tuzluluk Stresi

In recent years, rising temperatures due to global climate change can constitute a decrease in salinity in the oceans by causing the ice to melt early and freeze late in its annual cycle and increased precipitation. On the other hand, high temperatures can increase the salinity of seawater locally by enhancing evaporation. It is expected that the changes in salinity of sea water would affect communities of seaweeds. In this study, Polysiphonia morrowii Harvey samples were collected from Altıntaş station in the Gulf of Gemlik in April 2013, and cultured in mediums with four different salt concentrations (10‰, 23‰, 33‰, 42‰). The changes of this species in the amounts of total protein, total phenol, phycocyanin (PC), phycoerythrin (PE), chlorophyll-a (Chl a), water-soluble antioxidant, oil-soluble antioxidant, total solid organic matter and the activity of the carbonic anhydrase were determined. The study clearly showed that studied biochemical and physiological properties of the species were affected by salinity changes. It is also detected that P. morrowii is a tolerant species that can adapt to changing salinity conditions with various defense strategies.

Plectasin: from evolution to truncation, expression, and better druggability.

Non-computational classical evolution analysis of plectasin and its functional relatives can especially contribute tool value during access to meet requirements for their better druggability in clinical use. Staphylococcus aureus is a zoonotic pathogen that can infect the skin, blood, and other tissues of humans and animals. The impact of pathogens on humans is exacerbated by the crisis of drug resistance caused by the misuse of antibiotics. In this study, we analyzed the evolution of anti-Staphylococcus target functional sequences, designed a series of plectasin derivatives by truncation, and recombinantly expressed them in Pichia pastoris X-33, from which the best recombinant Ple-AB was selected for the druggability study. The amount of total protein reached 2.9 g/L following 120 h of high-density expression in a 5-L fermenter. Ple-AB was found to have good bactericidal activity against gram-positive bacteria, with minimum inhibitory concentration (MIC) values ranging between 2 and 16 μg/mL. It showed good stability and maintained its bactericidal activity during high temperatures, strong acid and alkali environments. Notably, Ple-AB exhibited better druggability, including excellent trypsin resistance, and still possessed approximately 50% of its initial activity following exposure to simulated intestinal fluids for 1 h. In vitro safety testing of Ple-AB revealed low hemolytic activity against mouse erythrocytes and cytotoxicity against murine-derived macrophages. This study successfully realized the high expression of a new antimicrobial peptide (AMP), Ple-AB, in P. pastoris and the establishment of its oral administration as an additive form with high trypsin resistance; the study also revealed its antibacterial properties, indicating that truncation design is a valuable tool for improving druggability and that the candidate Ple-AB may be a novel promising antimicrobial agent.

Hsf1 and the molecular chaperone Hsp90 support a 'rewiring stress response' leading to an adaptive cell size increase in chronic stress.

Cells are exposed to a wide variety of internal and external stresses. Although many studies have focused on cellular responses to acute and severe stresses, little is known about how cellular systems adapt to sublethal chronic stresses. Using mammalian cells in culture, we discovered that they adapt to chronic mild stresses of up to two weeks, notably proteotoxic stresses such as heat, by increasing their size and translation, thereby scaling the amount of total protein. These adaptations render them more resilient to persistent and subsequent stresses. We demonstrate that Hsf1, well known for its role in acute stress responses, is required for the cell size increase, and that the molecular chaperone Hsp90 is essential for coupling the cell size increase to augmented translation. We term this translational reprogramming the 'rewiring stress response', and propose that this protective process of chronic stress adaptation contributes to the increase in size as cells get older, and that its failure promotes aging.

Hsf1 and the molecular chaperone Hsp90 support a ‘rewiring stress response’ leading to an adaptive cell size increase in chronic stress

Cells are exposed to a wide variety of internal and external stresses. Although many studies have focused on cellular responses to acute and severe stresses, little is known about how cellular systems adapt to sublethal chronic stresses. Using mammalian cells in culture, we discovered that they adapt to chronic mild stresses of up to two weeks, notably proteotoxic stresses such as heat, by increasing their size and translation, thereby scaling the amount of total protein. These adaptations render them more resilient to persistent and subsequent stresses. We demonstrate that Hsf1, well known for its role in acute stress responses, is required for the cell size increase, and that the molecular chaperone Hsp90 is essential for coupling the cell size increase to augmented translation. We term this translational reprogramming the ‘rewiring stress response’, and propose that this protective process of chronic stress adaptation contributes to the increase in size as cells get older, and that its failure promotes aging.

Proteolytic Changes During Ripening Of Kučki Cheese

Summary Production of various traditional dairy products, particularly cheese, has a long history in Montenegro. White-brined cheeses are the most well-known. One of them is Kučki cheese, with distinct flavor and higher level of proteolysis. The aim of this research was to analyze the proteolysis process during ripening of Kučki cheese. The cheese is produced using traditional technology. FTIR Spectrophotometry was used to calculate the amount of total protein in cheese (IDF141C: 2000). The degree of proteolysis was determined by SDS PAGE (Laemmli, 1970). The cheese was examined for their water-soluble nitrogen (WSN) content using Kuchroo and Fox’s method from 1982, as well as their 5% phosphotungstic acid soluble nitrogen (PTAN) content using Stadhouser’s method from 1960. The results were expressed as percentages of WSN and PTAN of the total nitrogen matter (WSN/TN and PTAN/TN), as well as PTAN as a percentage of WSN (PTAN/WSN). All analyses were done on 10th, 20th and 30th day of cheese ripening. Four samples of cheese were analyzed for each ripening period. The content of proteins increased during the ripening period. The parameters that determine the process of proteolysis increased during the first 30 days of ripening. The WSN/TN ranged from 13.33 to 44.32%. Also, PTAN/WSN varied from 3.79 to 21.57%. The initial results show that uneven ripening conditions have a direct impact on how proteolytic changes develop throughout ripening. Due to the absence of established cheese-making procedures, Kučki cheese has a considerable degree of heterogeneity in their protein content and proteolysis parameters. However, it is not possible to strictly define the optimal values of the degree of ripening for Kučki cheese. In order to reach certain conclusions, it is necessary to carry out further studies focusing on analyzing a larger number of samples.

Structural Features of Connective Tissue Formed around Resin Implants Subcutaneously Embedded in Dairy Cows

Foreign body reactions (FBRs) are inadvertently observed in invading or artificially embedded materials, triggering inflammation and subsequent fibrotic processes to occur in situ. Here, we assessed the spatiotemporal formation of connective tissue around implanted materials to establish a technique using connective tissue formed by FBRs as xenografts. An acrylic resin implant, comprising a columnar inner rod and a tubular outer cylinder (OC) with several slits, was embedded in adult dairy cows. Tissues formed in the inner rod and OC groups were histologically analyzed at weeks 2, 4, 8, and 12. Edematous tissues with non-collagenous fibers formed for 2 weeks and showed increased cellularity after 4 weeks. The weight, thickness, amounts of total protein, collagen, DNA, and quantitative scores of α-smooth muscle actin-positive myofibroblasts or elastic fibers notably increased after 8 weeks, with condensed collagen fibers showing orientation. Inflammatory cells were primarily localized in tissues close to the OC, and their numbers increased, with the count of CD204+ cells peaking at 8 weeks and declining at 12 weeks. The count of Ki67+ proliferating cells slightly increased in tissues close to the OC; however, the number and lumen of CD31+ vessels increased. These results may help understand FBR-related tissue remodeling.

Discovery of Unexpected Phospholipid-Binding Proteins in Antiphospholipid Syndrome Patients

INTRODUCTION: The antiphospholipid syndrome (APS), initially described nearly 4 decades ago, remains enigmatic with unclear pathophysiology. In addition to the lack of a mechanistic diagnostic test, APS exhibits considerable phenotypic heterogeneity, including the occurrence of “non-consensus” clinical manifestations (i.e. other than vascular thrombosis and pregnancy complications). Despite the evidence that phospholipid-binding factors are highly likely to be central to APS pathogenesis, the full complement of phospholipid-binding proteins has yet to be systemically assayed. Proteomic profiling by label-free quantitation (LFQ) may identify patterns of protein binding to phospholipid that are central to APS pathogenesis and thereby elucidate a basis for the heterogeneous clinical presentations of APS. METHODS: C18 silica beads were coated with phospholipid by incubation with a suspension of 30% phosphatidylserine/70% phosphatidylcholine. Plasmas from 4 groups: 1) APS patients (n=11), 2) APL positive patients who lacked clinical manifestations (“APL”) (n=11), 3) non-APL patients with thrombosis (“nonAPL”)(n=11), and 4) normal healthy controls (“normal”) (n=6) were incubated with the phospholipid-coated beads, and eluates were processed by gel electrophoresis followed by tryptic digestion. The resulting peptides were analyzed by LC-MS/MS on a Thermo Orbitrap Fusion Lumos. Protein identification, false discovery rate (FDR) estimation, and LFQ were performed using Proteome Discoverer 2.2 (Thermo). LFQ values were normalized by overall peptide abundance, and relative quantitation was obtained. Groupwise comparisons were performed using PD 2.2 in order to identify differentially enriched proteins. RESULTS: A total of 1,210 proteins were identified, with 875 proteins identified at high confidence (q<0.01). Based on gel electrophoresis, all samples had comparable amounts of total protein, and normalization of LFQ data on the basis of total proteins was therefore performed. Differentially expressed proteins between groups were identified using both fold-change and -log p value cutoffs (Figure 1a). Primary analysis focused on identifying proteins that were consistently preferentially bound from the APS plasmas compared to the normal controls. Novel findings included the following proteins related to coagulation, immune response, and to lipid binding that bound preferentially from APS plasmas compared to normal plasmas (APS:control ratios shown in parentheses): coagulation factor VII (10.4), heparin cofactor 2 (9.6), CD14 (6.1), apolipoprotein L1 (6.0), and apolipoprotein L2 (5.5). Additional proteins that preferentially bound from APS plasmas included OAF (13.7), BRSK1 (13.2), MINPP1 (12.5), fetuin B (10.5), SHBG (8.9), ADAMTS-like protein 4 (8.5), SERPIN A4 (7.6), alpha-2-HS glycoprotein (6.7) and inter-alpha-trypsin inhibitor heavy chains (H3, H4 and H1; 8.5, 6.6, 6.5 respectively). Consistent with prior knowledge of APS, and validating this LFQ approach immunoglobulins and complement proteins were also among the preferentially bound proteins and included: Ig heavy chain 1-3 (15.6), complement C9 (13.3), complement C4-B, (7.1), complement component C8 beta chain (5.6), Ig kappa variable 1D-8 (5.6), and Ig lambda-like polypeptide 1 (3.9). Interestingly, a number of identified proteins showed intermediate abundance (between APS and control) in the APL positive patients without clinical manifestations (“APL”) and APL negative patients with thrombosis (“nonAPL”) (see figure 1B-D for examples), suggesting a potential dose response effect. CONCLUSIONS: 1) These results are a first demonstration of concept for the ability of LFQ proteomics to address the complexity of APS through analysis of phospholipid-bound proteins. 2) A number of phospholipid-binding proteins, many of which have not been previously implicated in APS pathogenesis, are preferentially present in APS patient plasma 3) Some plasmas from APL and non-APL patients also show increased binding of some of these proteins compared to the normal controls, suggesting that LFQ may identify markers for subgroups of patients within these 2 categories.

Hematopoietic Stem/Progenitor Cell Intrinsic SAM Synthesis Is Required to Prevent p53 Pathway Activation By Maintaining DNA Stability

S-adenosylmethionine (SAM) is a principle methyl group donor to maintain epigenetic status. SAM is synthesized from methionine and ATP via the enzymatic activity of Mat2a (Methionine adenosyltransferase 2a). It has been reported that SAM reduction by methionine restriction or Mat2a inhibition suppresses the proliferation of leukemia cells to attract attentions as a potential therapeutic target for leukemia. To develop the therapy targeting methionine metabolism in leukemia, it is essential to elucidate the significance of methionine metabolism in non-leukemic hematopoiesis. However, still little is known about the importance of SAM synthesis in hematopoiesis, including hematopoietic stem/progenitor cells (HSPCs), in vivo. By using Mx1-Cre mediated Mat2a gene deletion in mice ( Mat2a fl/fl;Mx1-Cre mice), we investigated the roles of Mat2a in hematopoiesis in vivo. Reduction of Mat2a activity in Mat2a knockout (KO) mice in bone marrow (BM) was confirmed by ultra-high performance liquid chromatography tandem mass spectrometry analysis. Mat2a KO mice displayed severe pancytopenia in peripheral blood (PB). Mature cell fractions (Myeloid cells, B cells and T cells) and HSPCs (HSCs, MPP2s, MPP3s and MPP4s) in BM were also significantly decreased in Mat2a KO mice, suggesting that Mat2a is essential for the maintenance of hematopoiesis from HSPCs in vivo. To exclude the possible contributions of the effects induced by Mat2a KO in non-hematopoietic cells, we performed BM transplantation analyses in which hematopoietic cell specific KO or non-hematopoietic cell specific KO were achieved. These analyses clearly confirmed that HSPC intrinsic SAM synthesis is inevitable for the maintenance of hematopoiesis. To reveal the mechanistic insights of the regulatory roles for Mat2a in HSPCs, we performed RNA sequencing analysis ( Mat2a WT vs KO in HSPCs). GSEA (Gene Set Enrichment Analysis) using the Hallmark gene sets showed that p53 pathway was most highly enriched in Mat2a KO. In depth, genes downstream of p53 ( Bax, Apaf1, Bbc3, Casp3, Casp8, Fas, Cdkn1a etc.) were up-regulated by Mat2a KO, causing cell cycle arrest and apoptosis in HSPCs. On the other hand, gene expressions of the upstream of p53 ( Atr, Atm, Chek1, Chek2 etc.) were unchanged, suggesting that p53 activity was not increased at mRNA expression levels. In line with this, mRNA expression levels of Trp53 was unchanged, but total protein amount of p53 was increased in Mat2a KO HSPCs. Since DNA stability is known as a critical regulator of p53 pathway, we analyzed gH2AX levels as DNA damage marker and found that gH2AX levels were increased in Mat2a KO HSPCs. To be noted, expressions of genes involved in Fanc pathway ( Fanca, Fancc, Fancd2, Fance, Fancg, Fancl, Fanci etc), which is responsible for the DNA maintenance, were broadly down-regulated in Mat2a KO HSPCs at transcriptomic levels. Therefore, the Fanc pathway repression by SAM synthesis inhibition could cause DNA damage and p53 pathway dependent loss of maintenance in Mat2a KO HSPCs. To further understand the importance of SAM synthesis under stress hematopoiesis. We analyzed Mat2a expression changes in HSPCs by irradiation (5.5 Gy/mouse). Intriguingly, Mat2a expression was up-regulated by irradiation. We also found that dietary methionine restriction (MR) in mice prevents its hematopoietic recovery from irradiation stress. To be noted, long term MR (24 weeks) did not show any particular compared to control diet. Therefore, methionine metabolism possesses significant roles at least in the situation where acute hematopoietic recovery is required. Collectively, in this study, we found that cell intrinsic SAM synthesis is required for the maintenance (proliferation and survival) of HSPCs in vivo. SAM might be required for the prevention of DNA damage and p53 pathway activation by supporting Fanc pathway gene expressions. Methionine and/or SAM availability might possess critical roles for the regeneration of non-leukemic hematopoiesis. Further investigation about the significance of SAM synthesis in non-leukemic hematopoiesis is required for the proper establishment of SAM synthesis targeting therapy in cancers.

Cadmium and copper-induced metabolic and proteomic changes in the root tip during early maize growth.

In this study, the metabolic adjustments performed by maize (Zea mays L.) seminal roots exposed to 25µM Cd2+ or 25µM Cu2+ at pre-emergence are compared, focusing on the proteomic changes after metal exposure. Root width was increased, and root length was decreased after 72h of metal treatment. Both metals induced H2O2 accumulation and lipid peroxidation in the root tip. These changes were accompanied by increases in lipoxygenase activity and 4-hydroxy-2-nonenal content. NMR spectroscopy revealed that the abundance of 38 water-soluble metabolites was significantly modified by Cd and Cu exposure; this set of metabolites comprised carboxylic acids, amino acids, carbohydrates, and unidentified phenolic compounds. Linoleic acid content significantly decreased in Cu-treated samples. The total amount of proteins detected in maize root apexes was 2,171. Gene ontology enrichment analysis of the differentially accumulated proteins was performed to detect pathways probably affected by metal additions. Both metals altered redox homeostasis, up-regulated oxylipins biosynthetic process, and shifted metabolism towards the oxidative pentose-phosphate in the root apexes. However, the methionine salvage pathway appears as a key metabolic module only under Cd stress. The integrative analysis carried out in this study suggests that most molecular features behind the reprogramming of maize root tips to cope with cadmium and copper toxicity are common, but some are not.

Production of vinegar from kashar cheese whey and volatile component profile, antibacterial effect, and antioxidant potential of whey vinegar

Whey is a liquid by-product of cheese production and releasing this by-product to the environment as a waste causes environmental pollution, loss of nutritional value, and economic loss. In this study, kashar cheese whey (KCW) and whey supplemented with lactose (5%, 10%, and 15%, w/v) were employed to produce four whey vinegars (KCWV, KCWV 5%, KCWV 10%, and KCWV 15%). Kluyveromyces marxianus CECT 1442 and Acetobacter aceti CECT 298 were used for fermentation. The whey vinegars were analyzed for pH, titratable acidity, lactose and total protein contents, antibacterial activities (agar well diffusion, MIC, MTC, and MBC), antioxidant parameters (TOC, TAC, GSH, and CAT), and volatile component profile. According to the results, the titratable acidity of the vinegars were 5.92%–14.97% (w/v). The lactose amounts of KCW, KCWV, KCWV 5%, KCWV 10%, and KCWV 15% were determined as 32.912, 0.540, 0.615, 3.477, and 50.798 g/L, respectively and total protein amounts were determined as 0.948, 0.308, 0.423, 0.423, and 0.432 g/100 g, respectively. While KCW did not exhibit antibacterial activity against test bacteria, whey vinegars showed at different rates. KCWV exhibited a weaker antibacterial effect than the other whey vinegars. No TOC activity was found except for KCWV, and TAC was found at an average level in all samples. Additionally, ethanol and acetic acid were the dominant components in whey vinegars. The whey vinegar may be an important alternative to the valorization of whey which is a by-product of cheese production.

Determination of modes for safe application of selective micro-pulse individual retinal therapy of the macular zone using the modeling based on the theory of activated complex

Purpose: to make modeling of various modes of laser radiation, to assess the degree of damage to the retinal pigment epithelium and adjacent structures, and to determine modes and safety conditions of selective micro-pulse individual retinal therapy based on the theory of activated complex. Materials and methods. The authors have modeled the degree of damage to the chorioretinal complex and adjacent structures using the activated complex theory with the Eyring equation. The following definitions were used to determine the regime quality and safety conditions: micro-pulse mode is a mode with fixed values of micro-pulse duration, duty cycle and the number of pulses in the packet; power is a variable parameter. Efficiency is the ratio of the amount of denatured protein inside the retinal pigment epithelium layer to the total amount of protein in it. Selectivity is the ratio of the amount of denatured protein inside the retinal pigment epithelium layer to the total amount of denatured protein. Relative harmfulness is the ratio of denatured protein volume outside the retinal pigment epithelium to the volume of protein in it. Results and discussion. The quality of micro-pulse mode was found as depending on the selectivity value with efficiency equal to 90 %. With the efficiency value less than 50 %, the mode is non-selective, with more than 67 % – selective. Safety conditions for selective micro-pulse modes have been established, taking into account safety of the neurosensory retina and choroid in terms of relative harmfulness which should not exceed 1. Parameters at which micro-pulse modes have high values (more than 90 – 99) of efficiency and selectivity at the same time have been determined as well. It requires single pulse mode, micro pulse duration of 25 microseconds and power from 6 to 8 W. To expand the combination of parameters, it is necessary to be able to freely change them in increments of 1 pulse, 5 microseconds, 100 mW, with long interval and short duty cycle. These technical capabilities can be realized and implemented into clinical practice by manufacturers of laser devices. Conclusion. For selective micro-pulse individual retinal therapy of the macular zone with existing lasers, the following recommendation are to be observed: micro-pulse modes with pulse duration no more than 200 microseconds, short duty cycle less than 5 % and number of pulses in an envelope no more than 5; relative harmfulness less than one, radiation power selection, personalized patient’s characteristics.

RNA polymerase II dynamics and mRNA stability feedback scale mRNA amounts with cell size

A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of this is that global transcription rates increase in larger cells. Here, we identify RNA polymerase II (RNAPII) as the limiting factor scaling mRNA transcription with cell size in budding yeast, as transcription is highly sensitive to the dosage of RNAPII but not to other components of the transcriptional machinery. Our experiments support a dynamic equilibrium model where global RNAPII transcription at a given size is set by the mass action recruitment kinetics of unengaged nucleoplasmic RNAPII to the genome. However, this only drives a sub-linear increase in transcription with size, which is then partially compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to scale mRNA amounts with cell size.