Milligram Of Protein Research Articles

Gene Cloning, Heterologous Expression, and In Silico Analysis of Chitinase B from Serratia marcescens for Biocontrol of Spodoptera frugiperda Larvae Infesting Maize Crops.

Spodoptera frugiperda, the fall armyworm (FAW), is a highly invasive polyphagous insect pest that is considered a source of severe economic losses to agricultural production. Currently, the majority of chemical insecticides pose tremendous threats to humans and animals besides insect resistance. Thus, there is an urgent need to develop new pest management strategies with more specificity, efficiency, and sustainability. Chitin-degrading enzymes, including chitinases, are promising agents which may contribute to FAW control. Chitinase-producing microorganisms are reported normally in bacteria and fungi. In the present study, Serratia marcescens was successfully isolated and identified from the larvae of Spodoptera frugiperda. The bacterial strain NRC408 displayed the highest chitinase enzyme activity of 250 units per milligram of protein. Subsequently, the chitinase gene was cloned and heterologously expressed in E. coli BL21 (DE3). Recombinant chitinase B was overproduced to 2.5-fold, driven by the T7 expression system. Recombinant chitinase B was evaluated for its efficacy as an insecticidal bioagent against S. frugiperda larvae, which induced significant alteration in subsequent developmental stages and conspicuous malformations. Additionally, our study highlights that in silico analyses of the anticipated protein encoded by the chitinase gene (ChiB) offered improved predictions for enzyme binding and catalytic activity. The effectiveness of (ChiB) against S. frugiperda was evaluated in laboratory and controlled field conditions. The results indicated significant mortality, disturbed development, different induced malformations, and a reduction in larval populations. Thus, the current study consequently recommends chitinase B for the first time to control FAW.

Sucraid (sacrosidase) is the only treatment for CSID: should there be a better option?

Sucraid (sacrosidase) is the only treatment for CSID: should there be a better option?

Micro-Scale Vacuum Compression Molding as a Predictive Screening Tool of Protein Integrity for Potential Hot-Melt Extrusion Processes.

Hot-melt extrusion (HME) is used for the production of solid protein formulations mainly for two reasons: increased protein stability in solid state and/or long-term release systems (e.g., protein-loaded implants). However, HME requires considerable amounts of material even at small-scale (>2 g batch size). In this study, we introduced vacuum compression molding (VCM) as a predictive screening tool of protein stability for potential HME processing. The focus was to identify appropriate polymeric matrices prior to extrusion and evaluation of protein stability after thermal stress using only a few milligrams of protein. The protein stability of lysozyme, BSA, and human insulin embedded in PEG 20,000, PLGA, or EVA by VCM was investigated by DSC, FT-IR, and SEC. The results from the protein-loaded discs provided important insights into the solid-state stabilizing mechanisms of protein candidates. We demonstrated the successful application of VCM for a set of proteins and polymers, showing, in particular, a high potential for EVA as a polymeric matrix for solid-state stabilization of proteins and the production of extended-release dosage forms. Stable protein-polymer mixtures with sufficient protein stability after VCM could be then introduced to a combination of thermal and shear stress by HME and further investigated with regard to their process-related protein stability.

Experience of measuring glutathione peroxidase activity in surgically induced endometrial-like lesions in rats

BACKGROUND: Endometriosis is known to be linked with altered activities of antioxidant enzymes and with their gene polymorphisms. Progestins are known to induce glutathione peroxidase activity in the endometrium and promote reduction of endometrial lesions. It could be useful to estimate the correlation between the activity of glutathione peroxidase within endometrial lesions and their degree of reduction.
 AIM: The present study was aimed at estimating glutathione peroxidase activity in surgically induced endometrial-like lesions of different degree of reduction in rat model of endometriosis.
 MATERIALS AND METHODS: The method for determining glutathione peroxidase activity using hydrogen peroxide as a substrate and 5,5-dithiobis(2-nitrobenzoic acid) for estimation of residual reduced glutathione was applied for quantitative analysis of the enzyme activity in endometriotic foci, surgically induced in female Wistar rats. An assay of glutathione peroxidase activity in tissue homogenates was performed at 37C in a reaction medium containing Tris-HCl buffer supplemented with tetrasodium ethylenediaminetetraacetate and sodium azide (pH 8.5) in the presence of 0.55 mM reduced glutathione and 0.192 mM hydrogen peroxide. Before adding trichloroacetic acid, 40-second incubation was used. The correlation between the specific activity of the enzyme and protein amount in endometriotic foci was estimated.
 RESULTS: In a rat model of endometriosis, there was a high, well-determined glutathione peroxidase activity in endometriotic foci. For the same endometriotic tissue sample, the enzymatic activity was proportional to the amount of protein in the reaction mixture. The range of specific glutathione peroxidase activity was 2.436.45 micromoles of consumed glutathione per minute per milligram of protein (n = 7). In most reduced endometriotic foci (with the minimum amount of endometriotic tissue), the highest specific activity of glutathione peroxidase was found (the Spearmans rho of 0.93 with p = 0.0067).
 CONCLUSIONS: The method for determining glutathione peroxidase activity using hydrogen peroxide and 5,5-dithiobis(2-nitrobenzoic acid) is convenient for working with the endometriotic tissue in a rat model of endometriosis. We can accept, with p 0.01, that weight of endometriotic foci is negatively linked with specific glutathione peroxidase activity within their tissue. The results are analogous to the previously obtained data on catalase activity and suggest the involvement of both antioxidant enzymes in reduction of endometrial lesions.

Peroxidase Enzyme Fractions as Markers of Somatic Embryogenesis Capacities in Olive (Olea europaea L.).

As part of the search for biochemical markers of somatic embryogenesis in tissue cultures of olive (Olea europaea L.), peroxidases (POXs) in both the soluble and ionically wall-bound fractions were studied in two reputed olive cultivars (cvs.): “Picholine Marocaine” and “Dahbia”. In order to carry out embryogenesis induction, proximal cotyledons were cultured in modified olive medium (OMc) supplemented with 25 μM indole-3-butylic acid (IBA) and 2.5 μM 2-isopentenyladenine (2iP), while distal leaf fragments (somatic explants) were cultured in OMc supplemented with 4.56 µM zeatin riboside (ZR) and 10.25 µM 1-naphthaleneacetic acid (NAA). Regarding embryogenic potentials, the zygotic explants (cv. Picholine Marocaine: 43.39%; cv. Dahbia: 53.41%) were more regenerative than the somatic explants (cv. Picholine Marocaine: 13.05%; cv. Dahbia: 19.51%). The enzyme assay showed a higher POX activity in embryogenic calluses (ECs) than in nonembryogenic calluses (NECs) for the zygotic explants in both studied cultivars. When expressed as units per milligram of proteins (U mg−1 proteins), the highest total POXs activities (soluble POXs + ionically wall-bound POXs) were found in the ECs derived from the zygotic explants; for cv. Dahbia, 65% of the enzyme activities came from the ionically wall-bound fractions. Polyacrylamide gel electrophoresis showed that the ECs of the highly active cv. Dahbia were characterized by highly active isoperoxidases that were revealed in four migration zones, particularly a doublet in the A4 zone (Rf 0.70–0.73) present in the ionically wall-bound POXs. The fast-moving anodic POXs of the ionically wall-bound fractions could be adopted as an early electrophoretic test to determine the embryogenesis capacities in olive tissue culture materials. As biochemical markers, the POX enzyme and its profile in fractions, i.e., as soluble POXs and ionically wall-bound POXs, can offer a valuable tool for improving the tissue culture of olive via somatic embryogenesis.

Comparison of isolation methods using commercially available kits for obtaining extracellular vesicles from cow milk

Extracellular vesicles (EV) are important for delivering biologically active substances to facilitate cell-to-cell communication. Milk-derived EV are widely known because of their potential for immune enhancement. However, procedures for isolating milk-derived EV have not been fully established. To obtain pure milk-derived EV and accurately reveal their function, such procedures must be established. The aim of the present study was to compare methods using commercially available kits for isolating milk-derived EV. Initially, we investigated procedures to remove casein, which is the major obstacle in determining milk-derived EV purity. We separated whey using centrifugation only, acetic acid precipitation, and EDTA precipitation. Then, we isolated milk-derived EV by ultracentrifugation, membrane affinity column, size exclusion chromatography (SEC), polymer-based isolation, or phosphatidylserine-affinity isolation. Using EV count per milligram of protein, which is a good indicator of purity, we determined that acetic acid precipitation was the best method for removing casein. Using nanoparticle tracking analysis, protein quantity analysis, and RNA quantity analysis, we comprehensively compared each isolation method for its purity and yield. We found that SEC-based qEV column (Izon Science) could collect purer milk-derived EV at higher quantities. Thus, a combination of acetic acid precipitation and qEV can effectively isolate high amounts of pure extracellular vesicles from bovine milk.

Development of 48-condition buffer screen for protein stability assessment.

The determination of a suitable buffer environment for a protein of interest is not an easy task. The requirements of advanced techniques, the demands on the biological material and the researcher time needed for buffer optimization, as well as personal inflexibility, lead frequently to the use of sub-optimal buffers. Here, we demonstrate the design of a 48-condition buffer screen that can be used to determine an appropriate environment for downstream studies. By the combination of several techniques (differential scanning fluorimetry, dynamic light scattering, and bio-layer interferometry), we are able to assess the protein stability, homogeneity and binding activity across the screen with less than half a milligram of protein in 1day. The application of this screen helps to avoid unsuitable conditions, to explain problems observed upon protein analysis and to choose the most suitable buffers for further research. The screen can be routinely used as a primary screen for buffer optimization in labs and facilities.

High-Level Production of Recombinant Eukaryotic Proteins from Mammalian Cells Using Lentivirus.

Mammalian protein expression systems are ideally suited for the high-level production of recombinant eukaryotic secreted and membrane proteins for structural biology applications. Here, we present genetic transduction of HEK293-derived cells using lentivirus as a robust and cost-efficient method for the rapid generation of stable expression cell lines. We describe the features of the lentiviral transfer plasmid pHR-CMV-TetO2, as well as detailed protocols for production of lentiviral particles, determination of functional lentiviraltiter, infection of expression cells, culture and expansion of the resulting stable cell lines, their adaptation to adherent and suspension growth, and constitutive or inducible milligram-scale protein production. The typical lead-time for a full production run is ~3-4weeks, with an anticipated yield of up to tens of milligrams of protein per liter of expression medium.

Impact of essential oils on Musca domestica larvae: oxidative stress and antioxidant responses

The housefly (Musca domestica) is incriminated as a vector of many detrimental diseases like diarrhoea, cholera, etc. To combat its population below threshold levels many types of insecticides including essential oils (EOs) have been tested from time to time. However the insect has often shown some resistance to proliferate and reproduce continuously. In view of this objective, present study highlights the antioxidative responses of M. domestica larvae to Bay, Lemongrass and Tea tree EOs during a short-term exposure (24 h) to respective compounds. After treatment with EOs, the housefly larvae showed increased levels of Malondialdehyde (MDA) indicating increased lipid peroxidation due to tested oils. The Tea tree essential oil induced substantial increase in superoxide dismutase and glutathione peroxidase activities as compared to lemongrass and bay EOs. Moreover, Lemongrass EO was found to significantly affect the catalase activity depicting a value of 0.0733 ± 0.006 units per milligram of protein (U/mg protein). In case of glutathione reductase activity, higher effect was induced by bay EO exhibiting 0.0150 ± 0.001 U/mg protein which was preceded by tea tree compound. The observations of the present study signpost that M. domestica has evolved antioxidant defence mechanism to combat reactive oxygen species generated by short term exposure of LC50 concentration of tested EOs. Therefore proper optimization of EO concentration together with time of exposure of the insect becomes imperative for formulating organic insecticides against house fly populations.

Regional Proteomic Quantification of Clinically Relevant Non-Cytochrome P450 Enzymes along the Human Small Intestine.

Current challenges in accurately predicting intestinal metabolism arise from the complex nature of the intestine, leading to limited applicability of available in vitro tools as well as knowledge deficits in intestinal physiology, including enzyme abundance. In particular, information on regional enzyme abundance along the small intestine is lacking, especially for non-cytochrome P450 enzymes such as carboxylesterases (CESs), UDP-glucuronosyltransferases (UGTs), and sulfotransferases (SULTs). We used cryopreserved human intestinal mucosa samples from nine donors as an in vitro surrogate model for the small intestine and performed liquid chromatography tandem mass spectrometry-based quantitative proteomics for 17 non-cytochrome P450 enzymes using stable isotope-labeled peptides. Relative protein quantification was done by normalization with enterocyte marker proteins, i.e., villin-1, sucrase isomaltase, and fatty acid binding protein 2, and absolute protein quantification is reported as picomoles per milligram of protein. Activity assays in glucuronidations and sequential metabolisms were conducted to validate the proteomics findings. Relative or absolute quantifications are reported for CES1, CES2, five UGTs, and four SULTs along the small intestine: duodenum, jejunum, and ileum for six donors and in 10 segments along the entire small intestine (A-J) for three donors. Relative quantification using marker proteins may be beneficial in further controlling for technical variabilities. Absolute quantification data will allow for scaling factor generation and in vivo extrapolation of intestinal clearance using physiologically based pharmacokinetic modeling. SIGNIFICANCE STATEMENT: Current knowledge gaps exist in intestinal protein abundance of non-cytochrome P450 enzymes. Here, we employ quantitative proteomics to measure non-cytochrome P450 enzymes along the human small intestine in nine donors using cryopreserved human intestinal mucosa samples. Absolute and relative abundances reported here will allow better scaling of intestinal clearance.

Short communication: The essential role of N-glycosylation in the transport activity of bovine peptide transporter 2

Bovine peptide transporter 2 (bPepT2), which mediates the absorption of di- and tripeptides in the bovine mammary gland, was predicted to contain multiple putative N-glycosylation sites of asparagine residues. N-Linked glycosylation is proven to be essential for the folding, stability, localization, and substrate binding of nutrient transporters and could therefore potentially have an essential role in the function of bPepT2. This study investigated the effect of mutagenesis of N-glycosylation sites on the transport function of bPepT2 in Chinese hamster ovary (CHO) cells. The bPepT2 cDNA was cloned and sequenced. BioXM (http://202.195.246.60/BioXM/) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) software were used to predict the AA composition and transmembrane domain of bPepT2, respectively. The AA sequence of bPepT2 was predicted to have 12 transmembrane domains, with a large extracellular loop between the ninth and tenth transmembrane domains. All 5 putative N-glycosylation sites in this loop were altered by site-directed mutagenesis, and the mutant construct was transfected into CHO cells for transport activity assay. Compared with the wild type, the bPepT2 mutant had significantly lower uptake activity of β-alanyl-l-lysyl-Nε-7-amino-4-methyl-coumarin-3-acetic acid (β-Ala-Lys-AMCA), a model dipeptide. Treatment with tunicamycin, an inhibitor of N-linked glycosylation, reduced the uptake of β-Ala-Lys-AMCA in CHO cells relative to the control group. Kinetic studies indicated that the Michaelis constant of bPepT2 was not affected by the mutation (98.03 ± 8.30 and 88.33 ± 4.23 µM for the wild type and the mutant, respectively), but the maximum transport activity was significantly reduced (40.29 ± 8.30 and 13.02 ± 2.95 pmol/min per milligram of protein for the wild type and the mutant, respectively). In summary, this study demonstrated that N-glycosylation is critical for the function of bPepT2.

Is Neovascularization a Leading Mechanism of Intra-plaque Hemorrhage and Carotid Plaque Destabilization?

Introduction: The accelerating carotid plaque growth can cause fibrous cap rupture followed by embolization to brain vessels. Important role in this process is ascribed to thrombotic factors and their inhibitors although another mechanism based on rupture of deficient vessels formed inside the plaque within the neovascularization process is also proposed. Interestingly, the second sequence of events provoke intra-plaque hemorrhage without a fibrous cap rupture. To determine whether the neovascularization is a main process responsible for carotid plaque destabilization the current study was conducted in asymptomatic and symptomatic patients with carotid stenosis. Methods: There were 121 individuals included to the study. Among them, study group consisted of 86 consecutive patients undergoing carotid endarterectomy (23 symptomatic, 63 asymptomatic, mean age 68±7,4 years) and 35 individuals without any symptoms and signs of atherosclerosis served as a control group. The concentration of vascular endothelial growth factor (VEGF) and its receptors, sVEGFR1, sVEGFR2, and tissue factor (TF) and tissue factor pathway inhibitor (TFPI) (ELISA immunoassay) in plaque homogenates and blood plasma was measured. In addition, activity of TF and TFPI (actichrome assay) were also measured. Concentration and activity results of all tested parameters were recounted for the milligram of protein contained in the plasma and plaques homogenates. Results: Analyzing patients with symptomatic and asymptomatic carotid stenosis the only statistically significant difference was found in the concentration of VEGF in plaque homogenates which was significantly higher in symptomatic plaques (302,9 vs 182,1 pg/ml; p=0,005 and 88,0 vs 67,9 pg/mg protein; p=0,05). The plaques homogenate’s TF level recounted for the milligram of protein was nearly 300-fold higher and TF activity was 5-fold higher in comparison with their plasma concertation and activity in the study group. The concentration and activity of TFPI was also higher in the plaque homogenates then in the plasma. The activity of TFPI in plaque homogenates recounted for the milligram of protein was 25-fold higher than in the plasma of the study patients. The VEGF concentration in plaque homogenates was significantly higher than in the plasma of study patients. There was no difference in VEGF concentration in the plasma of the study group and the control group. The lowest concentration of VEGF-R1 recounted for the milligram of protein was observed in the plasma of the control group. Higher concentration of VEGF-R1 recounted for the milligram protein was found in the plasma of the study group, but the distinctly highest concentration of VEGF-R1 (more than 10-fold higher) was observed in the study group plaque homogenates. Reverse characteristic showed the concentration of VEGF-R2, the lowest level was observed in the plaque homogenates and the highest in the plasma of the control group. Conclusion: The neovascularization process might be a leading mechanism of intra-plaque hemorrhage and carotid plaque destabilization. The plaque homogenates and blood plasma of patients with severe carotid stenosis have a strong prothrombotic profile. The plaque homogenates of patients with carotid stenosis in contrary to plasma possess signs of outspread angiogenetic activity.

Bacterial immunogenic α-galactosylceramide identified in the murine large intestine: dependency on diet and inflammation

The glycosphingolipid, α-galactosylceramide (αGalCer), when presented by CD1d on antigen-presenting cells, efficiently activates invariant natural killer T (iNKT) cells. Thereby, it modulates immune responses against tumors, microbial and viral infections, and autoimmune diseases. Recently, the production of αGalCer by Bacteroidetes from the human gut microbiome was elucidated. Using hydrophilic interaction chromatography coupled to MS2, we screened murine intestinal tracts to identify and quantify αGalCers, and we investigated the αGalCer response to different dietary and physiologic conditions. In both the cecum and the colon of mice, we found 1-15 pmol of αGalCer per milligram of protein; in contrast, mice lacking microbiota (germ-free mice) and fed identical diet did not harbor αGalCer. The identified αGalCer contained a β(R)-hydroxylated hexadecanoyl chain N-linked to C18-sphinganine, which differed from what has been reported with Bacteroides fragilis Unlike β-anomeric structures, but similar to αGalCers from B. fragilis, the synthetic form of the murine αGalCer induced iNKT cell activation in vitro. Last, we observed a decrease in αGalCer production in mice exposed to conditions that alter the composition of the gut microbiota, including Western type diet, colitis, and influenza A virus infection. Collectively, this study suggests that αGalCer is produced by commensals in the mouse intestine and reveals that stressful conditions causing dysbiosis alter its synthesis. The consequences of this altered production on iNKT cell-mediated local and systemic immune responses are worthy of future studies.

Effect of sleep deprivation and exercise on reaction threshold in adults with peanut allergy: A randomized controlled study

Peanut allergy causes severe and fatal reactions. Current food allergen labeling does not address these risks adequately against the burden of restricting food choice for allergic patients because of limited data on thresholds of reactivity and the influence of everyday factors. We estimated peanut threshold doses for a United Kingdom population with peanut allergy and examined the effect of sleep deprivation and exercise. In a crossover study, after blind challenge, participants with peanut allergy underwent 3 open peanut challenges in random order: with exercise after each dose, with sleep deprivation preceding challenge, and with no intervention. Primary outcome was the threshold dose triggering symptoms (in milligrams of protein). Primary analysis estimated the difference between the nonintervention challenge and each intervention in log threshold (as percentage change). Dose distributions were modeled, deriving eliciting doses in the population with peanut allergy. Baseline challenges were performed in 126 participants, 100 were randomized, and 81 (mean age, 25years) completed at least 1 further challenge. The mean threshold was 214mg (SD, 330mg) for nonintervention challenges, and this was reduced by 45% (95% CI, 21% to 61%; P=.001) and 45% (95% CI, 22% to 62%; P=.001) for exercise and sleep deprivation, respectively. Mean estimated eliciting doses for 1% of the population were 1.5mg (95% CI, 0.8-2.5mg) during nonintervention challenge (n=81), 0.5mg (95% CI, 0.2-0.8mg) after sleep, and 0.3mg (95% CI, 0.1-0.6mg) after exercise. Exercise and sleep deprivation each significantly reduce the threshold of reactivity in patients with peanut allergy, putting them at greater risk of a reaction. Adjusting reference doses using these data will improve allergen risk management and labeling to optimize protection of consumerswith peanut allergy.

Effects of Protein-Derived Amino Acid Modification Products Present in Infant Formula on Metabolic Function, Oxidative Stress, and Intestinal Permeability in Cell Models.

Proteins present in infant formulas are modified by oxidation and glycation during processing. Modified amino acid residues released from proteins may be absorbed in the gastrointestinal tract, and pose a health risk to infants. In this study, the markers of glycation furosine (1.7-3.5 μg per milligram of protein) and Nε-(carboxymethyl)lysine (28-81 ng per milligram of protein) were quantitated in infant formulas. The effects of these species, and other amino acid modifications, at the levels detected in infant formulas, on 3T3-L1 (murine preadipocyte) and Caco-2 (human intestinal epithelial) cells were assessed. Incubation of 3T3-L1 cells for 48 h with amino acid side chain oxidation and glycation products (1 and 10 μM) resulted in a loss (up to 40%, p < 0.05) of cell thiols and decreased metabolic activity compared with those of the controls. In contrast, Caco-2 cells showed a stimulation (10-50%, p < 0.05) of cellular metabolism on exposure to these products for 24 or 48 h. A 28% ( p < 0.05) increase in protein carbonyls was detected upon incubation with 200 μM modified amino acids for 48 h, although no alteration in transepithelial electrical resistance was detected. Oxidation products were detected in the basolateral compartments of Caco-2 monolayers when modified amino acids were applied to the apical side, consistent with limited permeability (up to 3.4%) across the monolayer. These data indicate that modified amino acids present in infant formulas can induce effects on different cell types, with evidence of bioavailability and induction of cellular stress. This may lead to potential health risks for infants consistently exposed to high levels of infant formulas.

NO-SYNTHASE ACTIVITY OF PERIPHERAL BLOOD LEUKOCYTES IN PATIENTS WITH ACNE VULGARIS

The aim is to study changes in the activity of distinct NO-synthase isoforms of blood lymphocytes in persons with acne vulgaris, for those individuals with disease accompanied by seeding of film-forming and planktonic staphylococcus forms. Enzyme activity was determined both before and after treatment. Methods. 44 patients with acne vulgaris were studied, for that persons the cultures of filmforming and planktonic forms of Staphylococcus aureus and Staphylococcus epidermidis were isolated from purulent pustules. Separation of lymphocytes from fresh peripheral heparinized blood of patients (before and after treatment) and individuals in the control group was done in the ficol triumbras (ρ=1.08 g/cm3 ). NO-synthase activity was expressed as citrulline production in picomoles per 1 milligram of protein per 1 minute. Results. Slightly higher rates of leukocyte іNOS were detected with the participation of biofilm staphylococci for S. aureus and S. epidermidis species compared to the planktonic forms of these staphylococcal species. For the acne caused both by S. aureus and S. epidermidis (planktonic and film-forming form) it was shown the significant increase ofendothelial NO-synthase activity (3.8–5.2 times) and inducible NO-synthas activity (52.5–55.1 times) ofperiferial blood leukocytes compared to control. The activity of the inducible isoform NO synthase significantly decreases relative to the control level after the course of acne treatment complicated by the proliferation of S. aureus (planktonic and film-forming form). Conclusions. The increase in the activity level of iNOS was observed with the participation of biofilm staphylococci of S. aureus and S. epidermidis species. In both forms of acne caused by S. aureus and S. epidermidis (planktonic and film-forming), the activity of NO-synthase isoforms increases with respect to control values. The activity of the inducible isoform NO-synthase can serve as a biomarker for monitoring treatment efficacy.

Functional Characterization of Peptide Transporters in Bovine Mammary Epithelial Cells.

The objective of this study was to characterize the expression profile, transport kinetics, and regulation of peptide transporters in bovine mammary epithelial cells (BMECs). Quantitative reverse-transcription real-time PCR, Western blotting, and immunofluorescence staining were used to investigate the expression of peptide transporters in bovine mammary tissues. The effects of time, pH, concentration, and specific inhibitors on β-alanyl-l-lysyl- Nε-7-amino-4-methyl-coumarin-3-acetic acid (β-Ala-Lys-AMCA) uptake in BMECs were also studied. The results showed that the peptide transporters PepT2 and PhT1 are both expressed in bovine mammary glands. The optimal pH for the uptake of β-Ala-Lys-AMCA in BMECs was 6.5. The transport-kinetics study suggested that the uptake of β-Ala-Lys-AMCA in BMECs is saturable over the tested concentration, with a Km value of 82 ± 18 μM and a Vmax of 124 ± 11 pmol/min per milligram of protein. Other dipeptides, including Gly-Sar, Met-Gly, and Met-Met, competitively inhibited β-Ala-Lys-AMCA uptake in BMECs. However, histidine had no effect on β-Ala-Lys-AMCA uptake. Furthermore, knocking down PepT2 could significantly reduce β-Ala-Lys-AMCA uptake, but PhT1 interference had no effect on peptide uptake in BMECs. The inhibition of PI3K and Akt decreased the uptake of β-Ala-Lys-AMCA. The above results revealed functional characteristics of peptide transporters and demonstrated that PepT2 may play a major role in β-Ala-Lys-AMCA uptake in BMECs. Moreover, the PI3K-Akt signaling pathway may regulate the uptake of β-Ala-Lys-AMCA in BMECs.

Substrate Selectivity Check of the Ergothioneine Transporter.

The candidate vitamin ergothioneine (ET) is a unique antioxidant. Expression of the ET transporter (ETT) (gene symbol SLC22A4) in distinct cells is thought to signal intracellular ET activity, since we have previously shown that the ETT is highly selective for ET. Unfortunately, some continue to hold the ETT as a relevant drug transporter, using the misleading functional name OCTN1, novel organic cation transporter. The present study was provoked by two recent reports in which new ETT substrates were declared. Astonishingly, the transport efficiencies (TEs) of ETT for saracatinib and some nucleoside drugs were as high as the TE for ET. Here we examined, based on regulated expression of ETT from human and rat in 293 cells and liquid chromatography-mass spectrometry quantification, the transport of several drugs. With the nucleosides cytarabine, gemcitabine, 2'-deoxycytidine, and 2'-deoxyadenosine, and the drugs saracatinib, ipratropium, metformin, and oxaliplatin, the uptake into cells expressing ETT was not increased over control cells. ETT-mediated uptake of gabapentin was detectable, but the TE was approximately 100-fold lower than the TE for ergothioneine (50-200 µl/min per milligram of protein). In conclusion, the ETT remains highly specific for its physiologic substrate ergothioneine. Our results contradict several reports on additional substrates. The ETT does not provide multiple substrate specificities, and it is not a transporter of cationic drugs. Only compounds that are related to ET in substructure-for example, gabapentin, carnitine, and TEA-can be transported, but with very low efficiency. Thus, ETT persists as a specific molecular indicator of ET activity.

Bacterial cyclic β-(1,2)-glucans sequester iron to protect against iron-induced toxicity.

Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron-restricted conditions while also limiting the potential for the production of iron-induced reactive oxygen species (ROS). Here, we report that membrane-derived oligosaccharide (mdo) glucan, an intrinsic component of Gram-negative bacteria, sequesters the ferrous form of iron. Iron-binding, uptake, and localization experiments indicated that both secreted and periplasmic β-(1,2)-glucans bind iron specifically and promote growth under iron-restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of β-(1,2)-glucan accumulate low amounts of intracellular iron under iron-restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron-replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram-negative bacteria.

Catalase, superoxide dismutase, glutathione peroxidase and oxygen radical absorbance capacity in the gut of juvenile pacu Piaractus mesopotamicus and dourado Salminus brasiliensis fed bovine first milk secretion

Besides the immunological benefits of colostrum consumption, this lacteal secretion has a great concentration of biological molecules that can affect gut physiology and fish healthy. This study evaluated the antioxidant potential in the gut of juvenile pacu, Piaractus mesopotamicus , and dourado, Salminus brasiliensis , fed twice a day with diets containing 0, 10 or 20% of lyophilized bovine colostrum (LBC). The whole medium and posterior intestine was collected after 30 or 60 experimental days and the activity of catalase, superoxide dismutase and glutathione peroxidase was determined as one international unit per milligram of protein, and the oxygen radical absorbance capacity as µM of equivalent Trolox per milligram of protein. Only the juvenile pacu were affected by the diet containing LBC ( P < 0.05). The juvenile fed 10% LBC showed higher oxygen absorbance capacity than the juveniles fed 20% LBC ( P < 0.05). Interaction between diet and period was observed to superoxide dismutase (SOD) activity ( P < 0.05), juvenile pacu fed 0% LBC and 10% LBC did not change enzyme activity at 30 and 60 days, whereas juvenile fed 20% LBC showed higher value at 60 days compared to 30 days ( P < 0.05). The present result reveal that the consumption of diet containing LBC improved SOD activity in the gut of juvenile pacu indicating a possible protective action of this lacteal secretion in an omnivorous fish.