Free Soluble Form Research Articles

Quantitative mapping of calcium cell reservoirs in cyanobacteria at the submicrometer scale

Calcium plays an important role in the physiology of bacterial cells and as a free soluble form inside the cells, it is tightly regulated at a low concentration. However, much higher amounts of stored calcium exist within cells and can be remobilized at diverse occasions. As a consequence, there has been a variety of techniques developed to quantitatively map the different forms of Ca in cells. Here we show how scanning transmission x-ray microscopy (STXM), a synchrotron-based microscopy technique, conducted in the soft x-ray range at the Ca L2,3 edges (340–360 eV) offers an original and informative quantitative view of different Ca reservoirs in bacterial cells at a spatial resolution better than 100 nm. To illustrate this, we analyzed mutants of the cyanobacterium Synechococcus elongatus PCC 7942, overexpressing different versions of a gene called ccyA from two phylogenetically distant cyanobacteria: Gloeomargarita lithophora and Synechococcus sp. PCC 6312. This gene is diagnostic of the capability of some cyanobacteria to form intracellular amorphous calcium carbonate (iACC) but its function remains unknown. Here we show that the overexpression of the ccyA gene in the iACC non-forming cyanobacterium S. elongatus PCC 7942 results in an increased Ca content, especially in some Ca-rich cells. Moreover, we show that STXM can discriminate four different reservoirs of Ca in cyanobacteria and provides quantitative assessment of their relative importance.

Immobilization of Rhizomucor miehei lipase on magnetic multiwalled carbon nanotubes towards the synthesis of structured lipids rich in sn-2 palmitic acid and sn-1,3 oleic acid (OPO) for infant formula use

Nowadays, breast milk is considered as the ideal food for infants owing to the most common oleic acid-palmitic acid-oleic acid (OA-PA-OA) fatty acid distribution of the human milk fat (HMF). This study reports the synthesis of 1,3-dioleoyl-2-palmotoylglycerol (OPO)-rich human milk fat substitutes in a two-step enzymatic acidolysis reaction with Rhizomucor miehei lipase (RML) immobilized on magnetic multi-walled carbon nanotubes(mMWCNTs). The immobilized RML (RML-mMWCNTs) showed better thermal and pH stability, convenient recovery and reusability than the free soluble form. Under optimized reaction conditions (1:8 tripalmitin (PPP)/OA, 10%wt. enzyme, 50 °C, 5 h), PA content at the sn-2 position and OA incorporation at the sn-1,3 positions reached 93.46% and 59.54%, respectively. Comparison tests have also showed that RML-mMWCNTs has better catalytic activity and reusability than the commercial lipase Lipozyme RM IM. The results suggest that RML-mMWCNTs is a promising biocatalyst for the synthesis of OPO-rich TAGs with potential use in infant formulas.

Teclistamab and talquetamab modulate levels of soluble B-cell maturation antigen in patients with relapsed and/or refractory multiple myeloma.

8047 Background: B-cell maturation antigen (BCMA, CD269) is a single transmembrane protein that is selectively expressed in the B-cell lineage and is a validated target for multiple myeloma. BCMA exists as both surface protein and as a free soluble form (sBCMA). γ-secretase activity at the transmembrane domain leads to a shed BCMA protein fragment of approximately 6 kilodalton that can exist as free circulating sBCMA in blood. Teclistamab and talquetamab are CD3 bispecific antibodies that have been developed to recruit CD3+ T-cells to BCMA+ or GPRC5D+ multiple myeloma (MM) cells, respectively. The objective of this work was to evaluate sBCMA in relapsed and/or refractory MM patients in response to treatment with teclistamab or talquetamab. Methods: Serum samples from relapsed and/or refractory MM patients in teclistamab and talquetamab phase 1 studies (64007957MMY1001 and 64407564MMY1001) were collected (at various timepoints between baseline and cycle 4 or end of treatment) and analyzed for sBCMA by an electrochemiluminescence ligand binding assay. Soluble BCMA data were quantitatively analyzed in reference to patient’s tumor burden and response, as well as pharmacokinetic data. Results: Teclistamab and talquetamab modulated levels of sBCMA in patients with high ( ≥ 50%) and low ( < 50%) frequency of tumor plasma cells (TPCs), as well as in high and low risk cytogenetic groups. In cycle 3, majority of the responders had reduction in sBCMA [88% (50 out of 57) for teclistamab and 98% (49 out of 50) for talquetamab] compared to baseline. On the contrary, non-responders (progressive disease, stable disease, or minimal response) seemed to show an increase in sBCMA [80% (33 out of 41) for teclistamab and 49% (24 out of 49) for talquetamab] from baseline. Patients with deep responses tend to have higher magnitude of sBCMA reduction compared to others. Based on few patients who responded to teclistamab or talquetamab and then relapsed, sBCMA seemed to have an initial reduction followed by an increase in the levels. Soluble BCMA corelated with % bone marrow TPCs. Majority of patients with plasmacytoma (limited data) seemed to have high sBCMA; suggesting sBCMA could be a comprehensive marker for tumor burden. Teclistamab preliminary population pharmacokinetic analysis showed that sBCMA did not appear to impact teclistamab exposure, suggesting that sBCMA was not acting as a sink for teclistamab. Conclusions: Teclistamab and talquetamab induced changes in levels of sBCMA that correlated with clinical activity, further supporting clinical development of these bispecific antibodies. Lastly, the results support that sBCMA is a potential surrogate marker of myeloma tumor burden, and as a valuable marker for response in MM patients. Clinical trial information: NCT03145181 and NCT03399799.

Optimal immobilization of trypsin from the spleen of albacore tuna (Thunnus alalunga) and its characterization

Trypsin purified from the spleen of albacore tuna was immobilized onto Octyl Sepharose CL-4B, glutaraldehyde activated silica and 5′-4,4′-dimethyltryptamine-thymidine-succinyl controlled pore glass. Trypsin was highly and efficiently immobilized onto Octyl Sepharose CL-4B, with the highest activity (6.26 U/g support) and specific activity (1.45 U/mg bound protein). The optimum conditions for trypsin immobilization onto Octyl Sepharose CL-4B were 40 mg/mL trypsin solution, pH 7 at 4 °C for 6 h of incubation time. The optimal temperature and pH for the hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) by the immobilized trypsin were 55 °C and 8.5, both of which were higher than that of the free form. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and organic solvents. The immobilized enzyme was less sensitive to inhibition by the soybean trypsin inhibitor compared with the free soluble form of the enzyme. According to the results, the immobilized trypsin and free enzyme retained 83% and 47% of their activity, respectively, when they were incubated with 1 μM of the soybean trypsin inhibitor. For the reusability study, the immobilized trypsin maintained 60% of its activity after 4 periods of activity, indicating that the immobilized trypsin had appropriate stability and could be reused.

Structure and Functions of Microtubule Associated Proteins Tau and MAP2c: Similarities and Differences

The stability and dynamics of cytoskeleton in brain nerve cells are regulated by microtubule associated proteins (MAPs), tau and MAP2. Both proteins are intrinsically disordered and involved in multiple molecular interactions important for normal physiology and pathology of chronic neurodegenerative diseases. Nuclear magnetic resonance and cryo-electron microscopy recently revealed propensities of MAPs to form transient local structures and long-range contacts in the free state, and conformations adopted in complexes with microtubules and filamentous actin, as well as in pathological aggregates. In this paper, we compare the longest, 441-residue brain isoform of tau (tau40), and a 467-residue isoform of MAP2, known as MAP2c. For both molecules, we present transient structural motifs revealed by conformational analysis of experimental data obtained for free soluble forms of the proteins. We show that many of the short sequence motifs that exhibit transient structural features are linked to functional properties, manifested by specific interactions. The transient structural motifs can be therefore classified as molecular recognition elements of tau40 and MAP2c. Their interactions are further regulated by post-translational modifications, in particular phosphorylation. The structure-function analysis also explains differences between biological activities of tau40 and MAP2c.

ELABORATION OF THE NEW METHOD OF CONSERVING VOLATILE AROMATIC SUBSTANCES OF SPICY VEGETABLES AT CREATING HEALTHY CRYO-SUPPLEMENTS

The aim of the work is to develop the new method of deep processing of spicy vegetables (ginger root, garlic, celery root, horseradish root) that gives a possibility to fully conserve volatile aromatic substances (ethereal oils, ketones, aldehydes, esters) and other biologically active substances (BAS) – phytocomponents of vegetable raw materials. At the same time this method gives a possibility to extract their hidden forms (bound in nanocomplexes with biopolymers, BAS) in the free soluble active form at receiving healthy semi-products (in puree form) and food products in the freely assimilated – nanosize form. For attaining this aim, cryogenic “shock” freezing and fine-dyspersated comminution using liquid and gas-like nitrogen with the modern equipment for cryo-processing and fine-dyspersated comminution was used as an innovation. There was developed the new method of getting healthy supplements and products of spicy vegetables (roots of ginger, celery, horseradish, garlic), cryo-processed using cryogenic liquid of liquid and gas-like nitrogen in the cryogenic fast-freezing apparatus and fine-dyspersately comminuted with the high content of aromatic phytosubstances, especially ethereal oils and other BAS, especially low-molecular phenol compounds, polyphenols, ascorbic acid and prebiotics. The new method of deep processing of spicy vegetables is based on the complex effect of processes of cryo-destruction, mechanodestruction and non-fermentative catalysis on raw materials at fine-dyspersated comminution. It was established, that at cryogenic “shock” freezing there takes place not only complete conservation of vitamins, aromatic substances, but their more complete extraction from the bound hidden form with biopolymers and BAS in the free one (1,7…2,5 times more that extracted from free vegetables). The quality of spicy vegetables 1,7…2,5 exceeds the quality of fresh vegetables and analogues. The essentially more effect of extracting hidden forms of aromatic substances and other BAS was revealed at low-temperature fine-dyspersated comminution of cryo-processed spicy vegetables. It was demonstrated, that at fine-dyspersated comminution there takes place 1,7…3,2 times more full extraction of hidden forms of low-molecular BAS than in fresh vegetables. Using new types of fine-dyspersated puree of spicy vegetables, there is developed the line of high-quality products for healthy nutrition with the stable texture and long storage life (juice nanodrinks, sauces-dressings, sauces-deeps, salted fillings for confectionary products, snacks, milk-vegetable products).

The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is a severe and often fatal systemic malignancy. Malignant cells are capable of escaping host immune surveillance by inactivating cytotoxic lymphoid cells. In this work we discovered a fundamental molecular pathway, which includes ligand-dependent activation of ectopically expressed latrophilin 1 and possibly other G-protein coupled receptors leading to increased translation and exocytosis of the immune receptor Tim-3 and its ligand galectin-9. This occurs in a protein kinase C and mTOR (mammalian target of rapamycin)-dependent manner. Tim-3 participates in galectin-9 secretion and is also released in a free soluble form. Galectin-9 impairs the anti-cancer activity of cytotoxic lymphoid cells including natural killer (NK) cells. Soluble Tim-3 prevents secretion of interleukin-2 (IL-2) required for the activation of cytotoxic lymphoid cells. These results were validated in ex vivo experiments using primary samples from AML patients. This pathway provides reliable targets for both highly specific diagnosis and immune therapy of AML.

Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility

BackgroundTEX101 is a cell membrane protein exclusively expressed by testicular germ cells and shed into seminal plasma. We previously verified human TEX101 as a biomarker for the differential diagnosis of azoospermia, and developed a first-of-its-kind TEX101 ELISA. To demonstrate the clinical utility of TEX101, in this work we aimed at evaluating ELISA performance in a large population of fertile, subfertile, and infertile men.MethodsMass spectrometry, size-exclusion chromatography, ultracentrifugation, and immunohistochemistry were used to characterize TEX101 protein as an analyte in seminal plasma. Using the optimized protocol for seminal plasma pretreatment, TEX101 was measured by ELISA in 805 seminal plasma samples.ResultsWe demonstrated that TEX101 was present in seminal plasma mostly in a free soluble form and that its small fraction was associated with seminal microvesicles. TEX101 median values were estimated in healthy, fertile pre-vasectomy men (5436 ng/mL, N = 64) and in patients with unexplained infertility (4967 ng/mL, N = 277), oligospermia (450 ng/mL, N = 270), and azoospermia (0.5 ng/mL, N = 137). Fertile post-vasectomy men (N = 57) and patients with Sertoli cell-only syndrome (N = 13) and obstructive azoospermia (N = 36) had undetectable levels of TEX101 (≤0.5 ng/mL). A cut-off value of 0.9 ng/mL provided 100% sensitivity at 100% specificity for distinguishing pre- and post-vasectomy men. The combination of a concentration of TEX101 > 0.9 ng/mL and epididymis-specific protein ECM1 > 2.3 μg/mL provided 81% sensitivity at 100% specificity for differentiating between non-obstructive and obstructive azoospermia, thus eliminating the majority of diagnostic testicular biopsies. In addition, a cut-off value of ≥0.6 ng/mL provided 73% sensitivity at 64% specificity for predicting sperm or spermatid retrieval in patients with non-obstructive azoospermia.ConclusionsWe demonstrated the clinical utility of TEX101 ELISA as a test to evaluate vasectomy success, to stratify azoospermia forms, and to better select patients for sperm retrieval.

Technology of healthy processed cheese products without melting salts with the use of freezing and non-fermentative catalysis

Authors studied comprehensive influence of the processes of non–fermentative catalysis – cryomechanolysis and freezing of solid rennet cheeses during their preparation for melting, which leads to the cryodestruction of low–soluble paracaseinatcalciumphosphate nanocomplexes into soluble gel form. It was established that there occurs their cryodestruction and transformation of their significant part to the nanoform (by 45…55 %). A nanotechnology of healthy processed products was developed. Mechanisms of the processes were revealed. It was established that during freezing and finely dispersed grinding of solid rennet cheeses before melting, there occurs cryomechanodestruction and non–fermentative cryocatalysis (destruction) of protein molecules to separate monomers – α–amino acids by 55… 60 %, that is a significant part of amino acid is transformed from the bound state to the free soluble form. A mechanism of the process was revealed; it was shown that in parallel with the destruction of nanocomplexes, nanoassociants of protein, its conformational changes take place: erasing molecules, decreasing in volume, shape, the ratio of hydrophobic and hydrophilic groups in a molecule, and filling the nucleus of a molecule with hydrophobic residues. Authors proposed and developed the cryogenic nanotechnology of manufacturing processed products based on solid rennet cheeses without melting salts, which includes an integrated influence of freezing and finely dispersed grinding, non–fermentative catalysis. It was established that products, produced by the nanotechnology (fillings for confectionery products PanCake, dressing sauces, dipping sauces, ball shaped snacks) and enriched with herbal additives, exceed the known analogs in chemical composition. In addition, a large part of substances (as BAS and biopolymers) in products is in the nanostructured form (55...60 % of protein) in the form of free amino acids. New technologies of healthy processed products have been tested under production conditions at a number of the Ukrainian enterprises (TOV VKG Lisova kazka, NVP FIPAR, NVP KRIAS–1). The regulatory documentation (TU, TI for cheese and vegetable fillings for confectionery products PanCake and cheese dressing sauces) was developed and approved.

The effect of cryomechanodestruction on activation of heteropolysaccaride-protein nanocomplexes when developing nanotechnologies of plant supplements

The regularities and mechanisms of the effect of deep processing of plant raw materials were established, such as finely dispersed grinding in developing nanotechnology of obtaining frozen nanopuree and nanopowders on the transformation of bound amino acids of protein to free soluble form by меchanolysis of molecules of protein (by 45…55 % to separate α­amino acids). We discovered the mechanism of mechanodestruction of protein molecules and its nanocomplexes with other biopolymers and BAS, which is linked to mechanocracking. In the deep processing of plant raw materials, in particular, Jerusalem artichoke, which is based on the comprehensive action of cryogenic «shock» freezing, freeze drying and finely dispersed grinding processes when obtaining nanopowders, the processes of cryodestruction, mechanodestruction and mechanochemistry occur that lead to the fuller extraction of BAS from the raw material (by 1.8…2.3 times more than is in the original raw material) and destruction of biopolymers (inulin, proteins) to their monomers. It was found that the freezing and cryomechanodestruction lead to the transformation of chemical substances of Jerusalem artichoke (cryomechanochemistry) and transformation, in particular, conformational changes of protein molecules: reduction of radius of the volume of a protein molecule, radius of its nucleus, and also to a decrease in the indicator of filling the nucleaus with hydrophobic remains of amino acids. In addition, the shape of protein molecules changes. We proposed and designed cryogenic nanotechnology of finely dispersed frozen nanopuree and nanopowders from Jerusalem artichoke with prebiotic properties. It was shown that nanosupplements exceed the known world analogues in the content of BAS and dispersed composition. In addition, a large part of the substances (both BAS and biopolymers) is in the nanodimensional form.

Metabolism of Flavonoids in Novel Banana Germplasm during Fruit Development

Banana is a commercially important fruit, but its flavonoid composition and characteristics has not been well studied in detail. In the present study, the metabolism of flavonoids was investigated in banana pulp during the entire developmental period of fruit. ‘Xiangfen 1,’ a novel flavonoid-rich banana germplasm, was studied with ‘Brazil’ serving as a control. In both varieties, flavonoids were found to exist mainly in free soluble form and quercetin was the predominant flavonoid. The most abundant free soluble flavonoid was cyanidin-3-O-glucoside chloride, and quercetin was the major conjugated soluble and bound flavonoid. Higher content of soluble flavonoids was associated with stronger antioxidant activity compared with the bound flavonoids. Strong correlation was observed between antioxidant activity and cyanidin-3-O-glucoside chloride content, suggesting that cyanidin-3-O-glucoside chloride is one of the major antioxidants in banana. In addition, compared with ‘Brazil,’ ‘Xiangfen 1’ fruit exhibited higher antioxidant activity and had more total flavonoids. These results indicate that soluble flavonoids play a key role in the antioxidant activity of banana, and ‘Xiangfen 1’ banana can be a rich source of natural antioxidants in human diets.

Multiplex bead-based immunoassay for the free soluble forms of the HLA-G receptors, ILT2 and ILT4

Human leukocyte antigen (HLA)-G is an immune-inhibitory molecule that exerts its function via interaction with two main inhibitory receptors: ILT2 and ILT4. This interaction is considered to be an immune checkpoint. HLA-G can be found as a soluble molecule, but it is not known if its receptors can also be found as soluble molecules. In this work, we present a multiplex luminex-based assay to measure soluble ILT2 (sILT2) and soluble ILT4 (sILT4) molecules together. It is based on two antibody pairs, GHI/75 and HP-F1-PE for ILT2 and 27D6 and 42D1-PE for ILT4. The characterization of our method reveals that it specifically detects the free soluble forms of sILT2 and sILT4, and not those complexed to HLA Class I molecules such as their ligand of highest affinity HLA-G. A study on two small cohorts of cancer patients demonstrated that soluble ILT2 and ILT4 molecules were of low abundance in the plasma of healthy controls, but that elevated levels of plasmatic sILT2 were present in non-muscle-infiltrating bladder cancer patients. This demonstrated that the titration test is indeed working, and that soluble ILT2 molecules do exist in pathological contexts, which relevance may now be sought on larger cohorts and other pathologies.

Endothelial protein C receptor gene 6936A/G single-nucleotide polymorphism as a possible biomarker of thrombotic risk in acute myeloid leukemia.

Protein C (PC) is a natural anticoagulant, which interacts with the endothelial PC receptor (EPCR). EPCR single-nucleotide polymorphism (SNP) 6936A/G results in high levels of a free soluble form of EPCR (sEPCR) and may affect the risk of coagulation. The objective of this study was to assess whether the 6936A/G SNP of the EPCR gene is involved in the procoagulant activity displayed by hematological malignancies. EPCR 6936A/G polymorphism analysis was performed in 205 patients with hematological malignancies and in 63 healthy controls. All the subjects were genotyped for the EPCR 6936A/G SNP (AA, AG and GG genotypes). The 6936A/G polymorphism distribution was similar between healthy donors and patients. The association between EPCR 6936A/G SNP and thrombosis was investigated in 110 patients. The disease-wise break-up revealed that 55 of the patients suffered from acute myeloid leukemia (AML). In AML patients, the incidence of thrombosis was 28.3% and significantly higher in the 6936AG compared with that in the 6936AA genotype (50 vs. 22%, respectively). In conclusion, this study revealed a significant association of the 6936AG genotype of EPCR with thrombotic events in AML. Therefore, the presence of the 6936AG genotype in AML patients may be considered as a risk indicator of thrombosis.

Nitrogen fertilization and maturity influence the phenolic concentration of wheat grain (Triticum aestivum)

AbstractThis work investigated the influence of N fertilization and grain maturity on total phenolic concentration (TPC) of wheat caryopses. A pot experiment was conducted, using soft spring wheat (Triticum aestivum cv. Thasos) which was treated with four different amounts of nitrogen (0.25–2.00 g N pot−1) and harvested at three different development stages (medium milk stage, late milk stage, and dough maturity). Phenolic compounds were extracted and analyzed as total phenolic concentration in three discrete fractions: free soluble, conjugated soluble and insoluble bound forms. TPC of free phenolic compounds rose with increasing N supply while TPC of conjugated soluble phenolics decreased at the same time. Insoluble phenolics were less affected by N treatment. Total phenolic concentration also changed with the development stage of caryopses and reached a peak at the late milk stage.

Thiolated nanocarriers for oral delivery of hydrophilic macromolecular drugs.

It was the aim of this study to investigate the effect of unmodified as well as thiolated anionic poly(acrylic acid) (PAA) and cationic chitosan (CS) utilized in free-soluble form and as nanoparticulate system on the absorption of the hydrophilic compound FD4 across intestinal epithelial cell layer with and without a mucus layer. Modifications of these polymers were achieved by conjugation with cysteine to PAA (PAA-Cys) and thioglycolic acid to CS (CS-TGA). Particles were prepared via ionic gelation and characterized based on their amount of thiol groups, particle size and zeta potential. Effects on the cell layer concerning absorption enhancement, transepithelial electrical resistance (TEER) and cytotoxicity were investigated. Permeation enhancement was evaluated with respect to in vitro transport of FD4 across Caco-2 cells, while mucoadhesion was indirectly examined in terms of adsorption behaviour when cells were covered with a mucus layer. Lyophilized particles displayed around 1000 μmol/g of free thiol groups, particle sizes of less than 300 nm and a zeta potential of 18 mV (CS-TGA) and -14 mV (PAA-Cys). Cytotoxicity studies confirmed that all polymer samples were used at nontoxic concentrations (0.5% m/v). Permeation studies revealed that all thiolated formulations had pronounced effects on the paracellular permeability of mucus-free Caco-2 layers and enhanced the permeation of FD4 3.0- to 5.3-fold. Moreover, polymers administered as particles showed a higher permeation enhancement than their corresponding solutions. However, the absorption-enhancing effect of each thiolated formulation was significantly (p<0.05) reduced when cells were covered with mucus layer. In addition, all formulations were able to decrease the TEER of the cell layer significantly (p<0.05). Therefore, both thiolated polymers as nanoparticulate delivery systems represent a promising tool for the oral administration of hydrophilic macromolecules.

Gamma-Glutamyltransferase Fractions in Human Plasma and Bile: Characteristic and Biogenesis

Total plasma gamma-glutamyltransferase (GGT) activity is a sensitive, non-specific marker of liver dysfunction. Four GGT fractions (b-, m-, s-, f-GGT) were described in plasma and their differential specificity in the diagnosis of liver diseases was suggested. Nevertheless fractional GGT properties have not been investigated yet. The aim of this study was to characterize the molecular nature of fractional GGT in both human plasma and bile.Plasma was obtained from healthy volunteers; whereas bile was collected from patients undergoing liver transplantation. Molecular weight (MW), density, distribution by centrifugal sedimentation and sensitivity to both detergent (deoxycholic acid) and protease (papain) were evaluated. A partial purification of b-GGT was obtained by ultracentrifugation.Plasma b-GGT fraction showed a MW of 2000 kDa and a density between 1.063–1.210 g/ml. Detergent converted b-GGT into s-GGT, whereas papain alone did not produce any effect. Plasma m-GGT and s-GGT showed a MW of 1,000 and 200 kDa, and densities between 1.006-1.063 g/ml and 1.063–1.210 g/ml respectively. Both fractions were unaffected by deoxycholic acid, while GGT activity was recovered into f-GGT peak after papain treatment. Plasma f-GGT showed a MW of 70 kDa and a density higher than 1.21 g/ml. We identified only two chromatographic peaks, in bile, showing similar characteristics as plasma b- and f-GGT fractions.These evidences, together with centrifugal sedimentation properties and immunogold electronic microscopy data, indicate that b-GGT is constituted of membrane microvesicles in both bile and plasma, m-GGT and s-GGT might be constituted of bile-acid micelles, while f-GGT represents the free-soluble form of the enzyme.

Immobilization of Bovine Trypsin onto Controlled Pore Glass

Bovine trypsin was immobilized onto controlled pore glass (CPG) beads by covalent binding using glutaraldehyde as a cross-linking reagent. Increasing the concentration of the enzyme solution resulted in an increase in the amount of enzyme protein bound to the support material. The effects of pH and incubation time on immobilization were studied as well. For the pH range (pH 3.0 to pH 11.0), the stability of the immobilized enzyme was shifted to a relatively higher pH value (pH 9) compared with the free soluble form of the enzyme (pH 3 and 5). The immobilized enzyme was evaluated for its capacity to extract carotenoproteins from shrimp shell. After 11 reuses, the immobilized enzyme retained about 77% of its initial activity, and the total yield of the product from the same immobilized trypsin of 11 reuses was 4.3 times higher than a single use of the same amount of the free enzyme. Practical Applications Enzyme immobilization technology provides a means for recovering and reusing enzymes to reduce food processing cost for the food industry. Trypsins and other proteases have received a lot of attention by the food industry because they are used extensively in food processing. For example, approximately 50% of the enzymes used as processing aids in food industry are protein hydrolases. The study reported here is intended to serve as a model for use to study fish trypsin immobilization.

Abscisic acid has contrasting effects on salt excretion and polyamine concentrations of an inland and a coastal population of the Mediterranean xero-halophyte species Atriplex halimus

Different populations of the Mediterranean xerohalophyte species Atriplex halimus exhibit different levels of resistance to salt and osmotic stress depending on the nature of the osmocompatible solute they accumulate. There is, however, no conclusive description of the involvement of abscisic acid (ABA) in the plant response to NaCl or osmotic stress in this species. Seedlings issued from an inland water-stress-resistant population (Sbikha) and from a coastal salt-resistant one (Monastir) were exposed in nutrient solutions to NaCl (40 or 160 mm) or to 15 % PEG for 1 d and 10 d in the presence or absence of 50 microm ABA. Plants from Sbikha accumulated higher amounts of ABA in response to osmotic stress than those of Monastir, while an opposite trend was recorded for NaCl exposure. Exogenous ABA improved osmotic stress resistance in Monastir through an improvement in the efficiency of stomatal conductance regulation. It also improved NaCl resistance in Sbikha through an increase in sodium excretion through the external bladders. It is suggested that polyamines (spermidine and spermine) are involved in the salt excretion process and that ABA contributes to polyamine synthesis as well as to the conversion from the bound and conjugated to the free soluble forms of polyamine. Proline accumulated in response to osmotic stress and slightly increased in response to ABA treatment while glycinebetaine accumulated in response to salinity and was not influenced by ABA. It is concluded that ABA is involved in both salt and osmotic stress resistance in the xerohalophyte species Atriplex halimus but that it acts on different physiological cues in response to those distinct environmental constraints.

Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells

BackgroundAggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis. Increasing evidence points to a link between periodontitis and cardiovascular diseases, however, the underlying mechanisms are poorly understood. This study investigated the pathogenic potential of free-soluble surface material, released from live planktonic and biofilm A. actinomycetemcomitans cells.ResultsBy employing an ex vivo insert model (filter pore size 20 nm) we demonstrated that the A. actinomycetemcomitans strain D7S and its derivatives, in both planktonic and in biofilm life-form, released free-soluble surface material independent of outer membrane vesicles. This material clearly enhanced the production of several proinflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, MIP-1β) in human whole blood, as evidenced by using a cytokine antibody array and dissociation-enhanced-lanthanide-fluorescent-immunoassay. In agreement with this, quantitative real-time PCR indicated a concomitant increase in transcription of each of these cytokine genes. Experiments in which the LPS activity was blocked with polymyxin B showed that the stimulatory effect was only partly LPS-dependent, suggesting the involvement of additional free-soluble factors. Consistent with this, MALDI-TOF-MS and immunoblotting revealed release of GroEL-like protein in free-soluble form. Conversely, the immunomodulatory toxins, cytolethal distending toxin and leukotoxin, and peptidoglycan-associated lipoprotein, appeared to be less important, as evidenced by studying strain D7S cdt/ltx double, and pal single mutants. In addition to A. actinomycetemcomitans a non-oral species, Escherichia coli strain IHE3034, tested in the same ex vivo model also released free-soluble surface material with proinflammatory activity.ConclusionA. actinomycetemcomitans, grown in biofilm and planktonic form, releases free-soluble surface material independent of outer membrane vesicles, which induces proinflammatory responses in human whole blood. Our findings therefore suggest that release of surface components from live bacterial cells could constitute a mechanism for systemic stimulation and be of particular importance in chronic localized infections, such as periodontitis.

Permeation enhancer effect of chitosan and chitosan derivatives: Comparison of formulations as soluble polymers and nanoparticulate systems on insulin absorption in Caco-2 cells

In this study four quaternized derivatives of chitosan: trimethyl chitosan (TMC), dimethylethyl chitosan (DMEC), diethylmethyl chitosan (DEMC) and triethyl chitosan (TEC) with degree of substitution of approximately 50 ± 5% were synthesized and their effect on the permeability of insulin across intestinal Caco-2 monolayers was studied and compared with chitosan both in free-soluble form and in nanoparticulate systems. Transepithelial electrical resistance (TEER) studies revealed that all four chitosan derivatives in free-soluble forms were able to decrease the TEER value in the following order TMC > DMEC > DEMC = TEC > chitosan, indicating their abilities to open the tight junctions. Recovery studies on the TEER showed that the effect of the polymers on Caco-2 cell monolayer is reversible and proves the viability of cells after incubation with all polymers. A similar rank order was also observed when measuring the zeta-potentials of the various polymers in solution form. Transport studies of insulin together with the soluble polymers across Caco-2 cell layers showed the following ranking: TMC > DMEC > DEMC > TEC > chitosan which is in agreement with the strength of the cationic charge of the polymer. In comparison to the free-soluble polymers, the nanoparticles prepared by ionic gelation of the chitosan and its quaternized derivatives had much lower effect on decreasing the TEER by opening of the tight junctions. This can be explained by the reduced available amount of positive charge at the surface of the nanoparticles. In accordance with these results, the insulin loaded nanoparticles showed much less permeation across the Caco-2 cell monolayer in comparison to the free-soluble polymers. Mass balance transport studies revealed that a substantial amount of the nanoparticles has been entrapped into the Caco-2 monolayer or attached to the cell surface. It can thus be stated that while free-soluble polymers can reversibly open the tight junctions and increase the permeation of insulin, the nanoparticles had basically only a low effect on the opening of the tight junction and the paracellular transport of insulin across the Caco-2 cell monolayer. These data convincingly show that nanoparticles consisting of chitosan and its quaternary ammonium derivatives loaded with insulin are less effective in facilitating paracellular transport across Caco-2 cell monolayers than the corresponding free polymers.