Protein Amino Research Articles

A new flow cell and chemometric protocol for implementing in‐line Raman spectroscopy in chromatography

On-line monitoring tools for downstream chromatographic processing (DSP) of biotherapeutics can enable fast actions to correct for disturbances in the upstream, gain process understanding, and eventually lead to process optimization. While UV/Vis spectroscopy is mostly assessing the protein's amino acid composition and the application of Fourier transform infrared spectroscopy is limited due to strong water interactions, Raman spectroscopy is able to assess the secondary and tertiary protein structure without significant water interactions. The aim of this work is to implement the Raman technology in DSP, by designing an in-line flow cell with a reduced dead volume of 80 μL and a reflector to increase the signal intensity as well as developing a chemometric modeling path. In this context, measurement settings were adjusted and spectra were taken from different chromatographic breakthrough curves of IgG1 in harvest. The resulting models show a small average RMSEP of 0.12 mg/mL, on a broad calibration range from 0 to 2.82 mg/mL IgG1. This work highlights the benefits of model assisted Raman spectroscopy in chromatography with complex backgrounds, lays the fundamentals for in-line monitoring of IgG1, and enables advanced control strategies. Moreover, the approach might be extended to further critical quality attributes like aggregates or could be transferred to other process steps.

Nickel tolerance and toxicity mechanisms in the halophyte Sesuvium portulacastrum L. as revealed by Ni localization and ligand environment studies.

Halophytes are able to tolerate relatively high concentrations of hazardous metals in a growing substrate, what makes them suitable candidates for phytoremediation of metal-contaminated soils. In this work, we aimed to study the physiological responses of the halophyte Sesuvium portulacastrum L. to Ni, with main focus on Ni localization, compartmentation and ligand environment, to decipher Ni tolerance and toxicity mechanisms. Seedlings were grown in hydroponic nutrient solution containing 0, 25, 50 and 100μM Ni as NiCl2 for 3weeks. Ni localization in leaves was assessed by micro-proton-induced X-ray emission (micro-PIXE). Ni ligand environment was studied by Ni K-edge X-ray absorption near edge structure (XANES). In addition, Ni-soluble, weakly bound/exchangeable and insoluble leaf tissue fractions were determined by sequential extraction. Results show that S. portulacastrum is able to tolerate up to ~ 500μgg-1 dry weight (DW) of Ni in the shoots without significant growth reduction. At higher Ni concentrations (> 50μM Ni in nutrient solution), chloroses were observed due to the accumulation of Ni in photosynthetically active chlorenchyma as revealed by micro-PIXE. Water storage tissue represented the main pool for Ni storage. Incorporation of Ni into Ca-oxalate crystals was also observed in some specimens, conferring tolerance to high leaf Ni concentrations. The majority of Ni (> 70%) was found in soluble tissue fraction. Ni K XANES revealed Ni bound mainly to O- (55%) and N-ligands (45%). Ni toxicity at higher Ni levels was associated with Ni binding to amino groups of proteins in cytosol of chlorenchyma and increased level of lipid peroxidation. Proline levels also increased at high Ni exposures and were associated with Ni-induced oxidative stress and alteration of water regime.

Protein Glycation by Glyoxal Promotes Amyloid Formation by Islet Amyloid Polypeptide

Protein glycation, also known as nonenzymatic glycosylation, is a spontaneous post-translational modification that would change the structure and stability of proteins or hormone peptides. Recent studies have indicated that glycation plays a role in type 2 diabetes (T2D) and neurodegenerative diseases. Over the last two decades, many types of advanced glycation end products (AGEs), formed through the reactions of an amino group of proteins with reducing sugars, have been identified and detected in vivo. However, the effect of glycation on protein aggregation has not been fully investigated. In this study, we aim to elucidate the impact of protein glycation on islet amyloid polypeptide (IAPP, also known as amylin) aggregation, which was strongly associated with T2D. We chemically synthesized glycated IAPP (AGE-IAPP) to mimic the consequence of this hormone peptide in a hyperglycemia (high blood sugar) environment. Our data revealed that AGE-IAPP formed amyloid faster than normal IAPP, and higher-molecular-weight AGE-IAPP oligomers were also observed in the early stage of aggregation. Circular dichroism spectra also indicated that AGE-IAPP exhibited faster conformational changes from random coil to its β-sheet fibrillar states. Moreover, AGE-IAPP can induce normal IAPP to expedite its aggregation process, and its fibrils can also act as templates to promote IAPP aggregation. AGE-IAPP, like normal IAPP, is capable of interacting with synthetic membranes and also exhibits cytotoxicity. Our studies demonstrated that glycation modification of IAPP promotes the amyloidogenic properties of IAPP, and it may play a role in accumulating additional amyloid during T2D progression.

Accurate and efficient amino acid analysis for protein quantification using hydrophilic interaction chromatography coupled tandem mass spectrometry

BackgroundMethods used to quantify protein from biological samples are often inaccurate with significant variability that requires care to minimize. The errors result from losses during protein preparation and purification and false detection of interfering compounds or elements. Amino acid analysis (AAA) involves a series of chromatographic techniques that can be used to measure protein levels, avoiding some difficulties and providing specific compositional information. However, unstable derivatives, that are toxic and can be costly, incomplete reactions, inadequate chromatographic separations, and the lack of a single hydrolysis method with sufficient recovery of all amino acids hinder precise protein quantitation using AAA.ResultsIn this study, a hydrophilic interaction chromatography based method was used to separate all proteinogenic amino acids, including isobaric compounds leucine and isoleucine, prior to detection by multiple reaction monitoring with LC–MS/MS. Through inclusion of commercially available isotopically labeled (13C, 15N) amino acids as internal standards we adapted an isotopic dilution strategy for amino acid-based quantification of proteins. Three hydrolysis methods were tested with ubiquitin, bovine serum albumin, (BSA), and a soy protein biological reference material (SRM 3234; NIST) resulting in protein estimates that were 86–103%, 82–94%, and 90–99% accurate for the three protein samples respectively. The methane sulfonic acid hydrolysis approach provided the best recovery of labile amino acids including: cysteine, methionine and tryptophan that are challenging to accurately quantify.ConclusionsAccurate determination of protein quantity and amino acid composition in heterogeneous biological samples is non-trivial. Recent advances in chromatographic phases and LC–MS/MS based methods, along with the availability of isotopic standards can minimize difficulties in analysis and improve protein quantitation. A robust method is described for high-throughput protein quantification and amino acid compositional analysis. Since accurate measurement of protein quality and quantity are a requirement for many biological studies that relate to crop improvement or more generally, our understanding of metabolism in living systems, we envision this method will have broad applicability.

Characterization of Ages Produced from CELLS Using SECM-ELISA

1. IntroductionAdvanced glycation end products (AGEs) are substances produced in the body by nonenzymatic reaction of reducing sugars and amino groups of proteins. They can be a factor in ageing and attracts attention in recent years due to its association in diabetes complications and arteriosclerosis1). There are few cases where the amount of AGEs produced at the cell level was measured. In our current study, we present our results of electrochemical activity evaluation of competitive ELISA using SECM for the purpose of highly sensitive detection of AGEs produced at the cellular level2). 2. Experimental2.1 Fabrication of wells for AGEs detection by the competitive methodCompetition method well formation was performed in the following steps [steps 1-8].(1) Mix 10 ug/mL of AGE conjugate and 1Xconjugate diluent at a 1: 1 ratio and take 40uL.(2) After incubating at 4 degrees for 24 hours, remove AGE conjugate coating solution and wash twice with phosphate buffer saline (PBS) [Sigma Aldrich, USA].(3) Add 50 uL of assay diluent and block for 1 hour at room temperature (RT).The process up to this point was regarded as a well for detecting AGEs.(4) After removing the blocking agent, 20uL of THP-1 cell suspension was added.(5) Following incubating at room temperature for 10 minutes, add 20uL of 1000-fold diluted Anti-AGE antibody.(6) Further, incubate for 1 hour at RT, remove the solution and wash 3 times with buffer(7) Add 40uL of 1000-fold diluted secondary antibody-HRP conjugate.(8) 3X washing with buffer [as in step 6]. 2.2 AGEs production evaluation of THP-1 cells by SECM-ELISA methodExperiments were conducted using a PBS (pH 7.4) mixed with 4 mM FcCH2OH and 2 mM H2O2 as a measurement solution. An Au electrode (d= 1.6 mm) was used as a working electrode, Pt wire was used as a counter electrode, an Ag/AgCl electrode was used as a reference electrode, and the applied voltage was held at +0.05 V vs. Ag/ AgCl. The HRP activity was characterized by scanning the Au electrode at a scan rate of 10 um/s in the direction of Z-axis 500 um and measuring the reduction current of Fc+CH2OH (Fig.1). Using the SECM-ELISA method, the difference between the initial current value and the peak current value was set ΔI and AGEs produced from THP-1 cells were characterized by three average peak current values. 3. Results and Discussion At THP-1 cell count 1.5 × 103 cells/ 20 uL, the average peak current value was observed to be 4.8 nA which was found to be at 4.0 nA for a cell density of 1.5 × 104 cells/20uL. It was recorded to further decrease to 3.1nA for a cell density of 1.5 × 105 cells/20uL. It can be observed that the current value was decreased with the increase in the number of THP-1 cells. In addition, the results of the AGE amount produced from THP-1 cells from the calibration curve of the AGE-BSA standard are shown in Fig. 2. An increase in the amount of AGEs produced as the number of THP-1 cells increased was observed. References1)A. Goldin, et al., Basic Science for Clinicians, Circulation. 114, 597-605 (2006).2)S. Kasai, et al., Analytica Chimica Acta. 566, 55-59 (2006). Figure 1

The effect of Chlorella suspension on productivity of chickens

The full feeding is one of the most important conditions that form the level of productivity in poultry industry. The value of feeding rations is known to depend not only on the presence of all essential substances in them, but also on the degree of its biological availability. Due to a wide range of vitamins, including fat-soluble, as well as macro- and micro elements in a biologically accessible form in Chlorella composition, Chlorella suspension can be become the useful component in feeding of poultry farming. Therefore, the aim of our study was to investigate the influence of the Chlorella water suspension on growth, daily weight increment and amount of eggs of chicken. The Chlorella suspension was produced by “Samvel Farm Enterprise” in Bilyaivka district of Odessa region, Ukraine. The investigation was carried out on layer chickens of “Brown” breed at the age of 360 days, which were divided into 3 experimental groups: one of them was control group and two were research groups with 300 heads of layer chickens in each. Despite of Chlorella suspension chickens were fed by ordinary animal feeding stuff. Most of the feed composition for all chicken groups consisted of wheat, barley and limestone. The nutritional value of this complete feed involved the presence of the crude protein, crude fiber and necessary amino acids including lysine, methionine. The second chicken group, throughout the experiment during 160 days was fed with a suspension of Chlorella in a concentration of 50 million, and in the 3d experimental group – 60 million cells in 1 milliliter of fluid. The results of research showed that the use of Chlorella suspension positively influences the raise of growth of chickens in the 2nd and 3rd experimental groups, which is characterized by the higher absolute, relative and average daily increments. The maximum result was achieved in 2nd experimental group, where the optimal cell concentration in the Chlorella suspension was 50 million cells in 1 milliliter of fluid and daily rate of increment was 30 grams per 1 head. Also after feeding with Chlorella suspension egg-laying increased by 12.4% compared to egg-laying of chickens in control group. Consequently, the inclusion of Chlorella suspension in chicken feeding allows obtaining poultry products with the maximum consumer qualities.

Relief from nitrogen starvation entails quick unexpected down-regulation of glycolytic/lipid metabolism genes in enological Saccharomyces cerevisiae.

Nitrogen composition of the grape must has an impact on yeast growth and fermentation kinetics as well as on the organoleptic properties of the final product. In some technological processes, such as white wine/rosé winemaking, the yeast-assimilable nitrogen content is sometimes insufficient to cover yeast requirements, which can lead to slow or sluggish fermentations. Growth is nevertheless quickly restored upon relief from nutrient starvation, e.g. through the addition of ammonium nitrogen, allowing fermentation completion. The aim of this study was to determine how nitrogen repletion affected the transcriptional response of a Saccharomyces cerevisiae wine yeast strain, in particular within the first hour after nitrogen addition. We found almost 4800 genes induced or repressed, sometimes within minutes after nutrient changes. Some of these responses to nitrogen depended on the TOR pathway, which controls positively ribosomal protein genes, amino acid and purine biosynthesis or amino acid permease genes and negatively stress-response genes, and genes related to the retrograde response (RTG) specific to the tricarboxylic acid (TCA) cycle and nitrogen catabolite repression (NCR). Some unexpected transcriptional responses concerned all the glycolytic genes, carbohydrate metabolism and TCA cycle-related genes that were down-regulated, as well as genes from the lipid metabolism.

Oxindole-based chalcones: synthesis and their activity against glycation of proteins

Diabetes mellitus, a metabolic disorder, is characterized by a substantial hyperglycaemia. Prevalence of hyperglycaemia for longer period of time can cause nonenzymatic condensation of sugar in blood with amino group of protein and give rise to advanced glycation end products (AGEs). AGEs play a major role in the onset of late diabetic complications including diabetic retinopathy, nephropathy, neuropathy and cardiovascular diseases. There is a need to establish potential therapeutic regimens that can effectively inhibit the formation of AGEs. To this end a series of novel oxindole-based chalcones have been investigated for their antiglycation potential. Analogues 1 (IC50 = 155.22 ± 2.98 µM), 3 (IC50 = 195.95 ± 0.43 µM), 4 (IC50 = 289.47 ± 2.47 µM), 5 (IC50 = 222.44 ± 4.03 µM), 7 (IC50 = 251.27 ± 2.80 µM), and 20 (224.23 ± 1.93 µM) showed potent inhibitory activity against glycation compared to the reference Rutin (IC50 = 294.5 ± 1.5 µM). These results reveal that multiple hydroxyl substituents and their position on the aromatic ring play a key role in inhibitory effect due to their hydrogen bonding potential. The study also reveals the influence of substituents on the binding capabilities and in turn inhibitory potential of different analogues.

Effect of pyrogallic acid (1,2,3-benzenetriol) polyphenol-protein covalent conjugation reaction degree on structure and antioxidant properties of pumpkin (Cucurbita sp.) seed protein isolate

Pumpkin (Cucurbita sp.) seed protein isolate (PSPI) and pyrogallic acid (1,2,3-benzenetriol) covalent conjugation with various ratios was prepared at pH 9. The degree of covalent conjugates in the reaction system was determined by polyphenol bound equivalents and SDS-PAGE. When increasing the polyphenols concentration, the number of bound equivalents was increased from 14.30 ± 3.35 to 131.56 ± 4.47 nmol/mg, while SDS-PAGE showed all the bands of the protein-polyphenol slightly shifted to the higher molecular weight direction, indicating that the reaction could lead to cross-linking of PSPI and PA to increase its molecular weight. The results of FTIR spectra indicated that the amino group participated in the covalent conjugates, and the structure became closer by the formation of hydrogen bonds during the reaction. According to the established mechanism model, it could be speculated that the covalent interaction between PSPI and pyrogallic acid (PA) relied on free amino groups of protein. In addition, the thermal stability and antioxidant properties got significant improvement. These protein-polyphenol covalent conjugates had potential to be used as new antioxidants in the food industry. At the same time, the conclusion that the polyphenol loading could be controlled was proved.

The Snow Flea Antifreeze Protein and Cryopreservation

Hypogastrura harveyi (snow fleas) produce a glycine‐rich antifreeze protein which enables it to survive in environments that would otherwise freeze its body fluid. Snow flea antifreeze protein (sfAFP) is a 6.5 kDa monomeric protein made of six alpha helices linked by hydrogen bonds and disulfide bridges. The protein's amino acid sequence has a frequent glycine‐x‐y amino acid pattern, where the x amino acid is also glycine 50% of the time and the y amino acid is either hydrophobic or hydrophilic. The hydrophobic side of sfAFP binds to the surface of seed ice crystals, breaking up the ice surface into smaller areas and preventing the further binding of water molecules due to the Kelvin effect. Other AFP's have been shown to be efficacious in improving the preservation of rat hearts, leading to the yet‐untested hypothesis that sfAFP's are also able to prolong the cryopreservation of organs. The Ashbury MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine structure‐function relationships of sfAFP when bonding to an ice lattice.Support or Funding InformationMSOE ‐ Center for Biomolecular Modelling ‐ co‐sponsor Diane MunzenmaierThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glycation profile of minor abundant erythrocyte proteome across varying glycemic index in diabetes mellitus

PurposeLong-term glycemic index in patients with diabetes mellitus (DM) is measured by glycated hemoglobin (HbA1c) besides blood glucose. In DM, the primary amino groups of proteins get glycated via non-enzymatic post-translational modification. This study aims at identifying and characterizing site-specific glycation of erythrocyte proteome across varying glycemic index in patients with DM. ExperimentsWe isolated the glycated erythrocyte proteome devoid of hemoglobin from control and diabetic samples using boronate affinity chromatography. Proteomic analysis was performed using nanoLC/ESI-MS proteomics platform. The site-specific modification on different proteins was deciphered using a customized database. ResultsWe report 37 glycated proteins identified and characterized from samples with HbA1c of 6%, 8%, 12%, and 16%. Our results show that both extent and site-specific modification of proteins increased with increasing HbA1c. The observed residue-specific modifications of catalase, peroxiredoxin, carbonic anhydrase, lactate dehydrogenase B and delta-aminolevulinic acid dehydratase were correlated with the literature report on their functional disorder in DM. Conclusionsand clinical relevance: 37 glycated erythrocyte proteins apart from hemoglobin were characterized from DM patient samples with varying HbA1c values. We correlated the site-specific glycation and associated functional disorder of five representative proteins. However, the clinical correlation with the observed modifications needs further investigation.

Lactobacillus plantarum ITM21B fermentation product and chickpea flour enhance the nutritional profile of salt reduced bakery products

The study aimed at improving the nutritional profile of yeast leavened salt reduced sliced bread and puccia type bread fortified with a wheat-based Lactobacillus plantarum ITM21B fermentation product (Bio21B). The protein content of bread made under laboratory conditions was increased by using: (i) chickpea flour (CF) (15% wt/wt flour) and Bio21B or (ii) the Bio21B containing a fungal protease to favour the gluten hydrolysis. Products showed increased protein and total amino acid content and improved protein digestibility. Moreover, the formula significantly affected the protein pattern of breads which, according to the results of the microfluidic two-dimensional electrophoresis (μ2DE) protein pattern, were discriminated as observed by the PCA plot. The use of CF was validated at industrial pilot plant producing salt reduced sliced bread and puccia type bread. The resulting products showed improved nutritional profile and a sensory quality comparable to the company’s products containing salt.

The Relevance of Toxic AGEs (TAGE) Cytotoxicity to NASH Pathogenesis: A Mini-Review.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common feature of chronic liver disease. Non-alcoholic steatohepatitis (NASH) is a severe form of NAFLD, and one of its risk factors is hyperglycemia. The chronic ingestion of excessive amounts of high-fructose corn syrup is associated with an increased prevalence of fatty liver. Under hyperglycemic conditions, advanced glycation end-products (AGEs) are generated through a non-enzymatic glycation reaction between the ketone or aldehyde groups of sugars and amino groups of proteins. Glyceraldehyde (GA) is a metabolic intermediate of sugars, and GA-derived AGEs (known as toxic AGEs (TAGE)) have been implicated in the development of NASH. TAGE accumulates more in serum or liver tissue in NASH patients than in healthy controls or patients with simple steatosis. Furthermore, the TAGE precursor, GA, causes cell damage through protein dysfunctions by TAGE modifications and induces necrotic-type hepatocyte death. Intracellular TAGE may leak outside of necrotic-type cells. Extracellular TAGE then induce inflammatory or fibrotic responses related to the pathology of NASH in surrounding cells, including hepatocytes and hepatic stellate cells. This review focuses on the contribution of TAGE to the pathology of NASH, particularly hepatic cell death related to NASH.

Intrinsically Disordered Human T Lymphotropic Virus Type 1 p30 Protein: Experimental and Computational Evidence.

Human T lymphotropic virus type 1 (HTLV-1) causes adult T cell leukemia and lymphoma and other neuroinflammatory diseases. The pX region of HTLV-1 genome encodes an accessory protein p30 that is required for viral persistence and spread in the host. p30 regulates viral gene expression at the transcription level by competing with Tax for p300 binding and at posttranscriptional level by nuclear retention of tax/rex messenger RNA (mRNA). In addition, p30 modulates the host cellular environment by binding to various host proteins such as ATM, REGγ, and PRMT5. However, the low expression levels of p30 has been a major hurdle in studying its structure-function relationship in the context of HTLV-1 pathobiology, which is most likely due to its intrinsically disordered nature. To investigate the unstable nature of p30, flow cytometric analysis of p30-GFP fusion protein expressed in Escherichia coli was conducted and bioinformatics analysis of p30 was performed. The bacterial cells were green fluorescent protein (GFP) positive, indicating that p30-GFP was in the soluble fraction. Induction, particularly at higher temperature, reduced the expression of p30-GFP. Moreover, p30-GFP was detected exclusively in insoluble fraction upon cell lysis, suggesting its unstable and disordered nature. The bioinformatics analysis of p30 protein sequence and amino acid content revealed that p30 has highly disordered regions from amino acids 75-155 and 197-241. Furthermore, p30 has regions for macromolecular interactions that could stabilize it and these regions coincide with the unstable regions. Collectively, the study indicates that HTLV-1 p30 is an intrinsically disordered protein.

The impact of carbonylated proteins on the skin and potential agents to block their effects.

Carbonylated proteins (CPs) are synthesized by reactions between amino groups in proteins and reactive aldehyde compounds (RAC) yielded from lipid peroxidation initiated by reactive oxygen species (ROS). In the skin, CPs are detected in a higher frequency at sun-exposed sites of the skin in elderly subjects. Since CPs in the stratum corneum (SC) have been reported to correlate with skin water content and transepidermal water loss, it is considered that the accumulation of CPs in the SC involves the loss of skin moisture functions. However, the roles of CPs in the dermis on skin physiology are still unclear. The purpose of this study was to investigate the roles of CPs in the dermis during the progression of photoaged skin and to propose a method to prevent or reduce the synthesis of CPs. The exposure of human normal dermal fibroblasts to CPs increased intracellular ROS levels and the synthesis of intracellular CPs. In addition, CPs caused morphological changes of fibroblasts. Furthermore, CPs caused alterations of mRNA expression levels of dermal matrix-related proteins, such as upregulating MMP-1 and IL-8. These results indicated that CPs disrupt construction of the dermal matrix. On the other hand, α-tocopherol and β-carotene suppressed the synthesis of RAC during lipid peroxidation which resulted in the reduction of UVA-induced CPs in the SC. From these results, we propose that extracellular CPs increase intracellular ROS levels and contribute to alterations of the dermal matrix. To prevent the synthesis of CPs, the application of α-tocopherol or β-carotene could be effective.

Mitigating base-catalysed degradation of periodate-oxidized capsular polysaccharides: Conjugation by reductive amination in acidic media

Reductive amination coupling an aldehyde-containing polysaccharide, generated by periodate oxidation, with the amino groups in protein has been widely used in the synthesis of glycoconjugate vaccines. The conjugation is often achieved under slightly basic conditions via a Schiff’s base intermediate followed by its reduction with sodium cyanoborohydride. We observed that oxidized capsular polysaccharides such as Streptococcus pneumoniae type 6B (Pn-6B) and Haemophilus influenzae type a (HiA) underwent significant degradation during the conjugation in slightly basic media leading to sub-optimal glycoconjugates. Further study on oxidized Pn-3, Pn-6A, Pn-6C, Pn-2 polysaccharides and dextran provided evidence that the degradation is a result of base-catalysed β-elimination. In contrast to HiA, Pn-2, Pn-3, Pn-6B polysaccharides and dextran, oxidized Pn-6A and Pn-6C polysaccharides were stable under basic conditions due to lack of the leaving group at the β-position of the aldehyde. By performing conjugation of oxidized polysaccharides to bovine serum albumin (BSA) in phosphate buffer at pH 6.0, 6.8, 7.2 and 8.0, we concluded that the reductive amination proceeds best in slightly acidic media, particularly with those β-elimination susceptible polysaccharides.

Glycation interferes with natural killer cell function

One hallmark of molecular aging is glycation, better known as formation of so-called advanced glycation end products (AGEs), where reactive carbonyls react with amino-groups of proteins. AGEs accumulate over time and are responsible for various age-dependent diseases and impairments. Two very potent dicarbonyls to generate AGEs are glyoxal (GO) and methylglyoxal (MGO). The plasma level of such dicarbonyls is higher in aging and age-related diseases. Natural killer (NK) cells are cells of the innate immune system and provide a major defense against tumor cells and virus infected cells. They are able to kill modified or infected cells and produce different cytokines to modulate the function of other immune cells. Here we investigated the effect of GO- and MGO-induced glycation on the function of NK cells. Using the human NK cell line NK-92, we could demonstrate that both GO and MGO lead to glycation of cellular proteins, but that MGO interferes much stronger with NK cell function (cytotoxicity) than GO. In addition, glycation of NK cell targets, such as K562 tumor cells, also interferes with their lysis by NK cells. From this data we conclude that glycation acts negatively on NK cells function and reduces their cytotoxic potential towards tumor cells.

Platelet and Red Blood Cell Counts, as well as the Concentrations of Uric Acid, but Not Homocysteinaemia or Oxidative Stress, Contribute Mostly to Platelet Reactivity in Older Adults.

Purpose The goal of this study was to estimate the hierarchical contribution of the most commonly recognized cardiovascular risk factors associated with atherogenesis to activation and reactivity of blood platelets in a group of men and women at ages 60-65. Methods Socioeconomic and anthropometric data were taken from questionnaires. Blood morphology and biochemistry were measured with standard diagnostic methods. Plasma serum homocysteine was measured by immunochemical method. Plasma concentrations of VCAM, ICAM, total antioxidant status, and total oxidant status were estimated with commercial ELISA kits. Markers of oxidative stress of plasma and platelet proteins (concentrations of protein free thiol and amino groups) and lipids (concentrations of lipid peroxides) and generation of superoxide anion by platelets were measured with colorimetric methods. Platelet reactivity was estimated by impedance aggregometry with arachidonate, collagen, and ADP as agonists. Expression of selectin-P and GPIIb/IIIa on blood platelets was tested by flow cytometry. Results Platelet aggregation associated significantly negatively with HGB and age and significantly positively with PLT, MPV, PCT, PDW, and P-LCR. When platelet reactivity (“cumulative platelet reactivity_aggregation”) was analyzed in a cumulated manner, the negative association with serum concentration of uric acid (Rs = −0.169, p = 0.003) was confirmed. Multivariate analysis revealed that amongst blood morphological parameters, platelet count, plateletcrit, and number of large platelets and uric acid are the most predictive variables for platelet reactivity. Conclusions The most significant contributors to platelet reactivity in older subjects are platelet morphology, plasma uricaemia, and erythrocyte morphology.

ISOLASI PROTEIN DAN PRODUKSI KONSENTRAT PROTEIN DAUN (KPD) SEBAGAI SUPLEMEN PAKAN TERNAK

The research aim to determine the effect of extraction using various combination of pH alkaline-acid to protein and amino acid content of Lamtoro Protein Concentrate Leaf (LPCL). Completely Randomized Design (CRD) with 3 treatments and 5 replications. The Lamtoro Leaf Meal (LLM) (50 gram) was blended in NaOH (200 ml) for 15 menit and centrifuged to separate the supernatant from extracted LLM. The extracted LLM was re-extracted with buffer acetat pH 4,6 and sentrifuged again to separate supernantant from extracted LLM. Both supernatant from NaOH and buffera acetat pH 4,6 extraction were mixed and precipitated with buffer phospat until isoelectric point. The treatments were tested consisted of extracted with NaOH pH 8, NaOH pH 9 and NaOH pH 10. The parameters measured; isoelectric point at precipited, crude protein, total amino acid (TAA), total essential amino acid (TEAA) and individual amino acid. The results showed that the precipited obtained isoelectric point pH 7 was 2.66; 1.03 and 2.58 gram at the extraction treatments using NaOH at pH 8, 9 and 10, respectively. LPCL extracted using NaOH at pH 9 and 10 contained a high crude protein of 54.82 and 52.60% higher than the extraction using NaOH at pH 8 of 46.98%. The total amount of amino acid and essential amino acid (AAE) of LPCL were highest in treatment using NaOH at pH 10 of 26,58; 13.31 %, respectively. Combination extraction LLM using pH buffer 10 followed by re-extract using acetate buffer pH 4,6 contains crude protein and good essential amino acid, especially leucine. Conclution of the research that the combination extraction using NaOH at pH 10 and followed by re-extract using acetate buffer at pH 4.6 is the best treatment contain crude protein and essential amino acid at LPCL.
 Keywords : Crude protein, essential amico acid, extraction.

Prediction of Structural and Functional Effects of Single Nucleotide Polymorphisms in <i>NAT2</i>gene, a Computational Analysis

N-acetyltransferase 2 (encoded by NAT2) is a phase II enzyme that detoxifies and metabolizes xenobiotics and drugs components. It is a critical enzyme in clinical pharmacology. It has remarkable genetic polymorphisms, which is associated with the risk of developing cancer due to the change of normally fast acetylation of substrates to slow acetylation. This study assessed single nucleotide polymorphisms (SNPs) in the coding region and (3ʹUTR) of NAT2. Computational approaches were used in this study for functional and structural effects of NAT2 gene. SNPs. Were retrieved from NCBI SNPsdatabase. TheNAT2 protein sequence and amino acid change were used as an input to the SIFT, PolyPhen-2, PhD-SNP, SNPs& GO, SNPAnalyzer, I-Mutant 3.0 and PMut to determine the deleterious and SNPs conditions. Other software for predication of the structural change were Mutation3D, Chimera and Project HOPE. GeneMANIA software was used to show gene –gene interaction. PolymiRTs was used to investigate the disruption or creation of SNPs of miRNA region. In Homosapiens182 were nonsynonymous SNPs (nsSNPs), 60 synonymous SNPs, 48 3ʹUTRSNPs and 19 5ʹUTR SNPs. A total, 65 of thosensSNPs were predicted to be highly damaging with 3-6 score rates when analyzed with six software. Re-computation of results with I-Mutant 3.0 showed adecrease in the effective stability of the protein due to 55 nsSNPs. Consequent structural changes were shown using Project HOPE and Chimera. NAT2 is a highly polymorphic gene; the majority of deleterious NAT2SNPs are nsSNPs that alter the physiochemical and structural properties of the protein, possibly leading to the loss or distortion of the protein's ability to detoxify and metabolize xenobiotic and aromatic amine compounds. There were three SNPs at the 3ʹUTR that changed the miRNA binding sites, which might affect the gene regulation.