Endometrial glycogen metabolism during early pregnancy in mice.

Glucose is critical during early pregnancy. The uterus can store glucose as glycogen but uterine glycogen metabolism is poorly understood. This study analyzed glycogen storage and localization of glycogen metabolizing enzymes from proestrus until implantation in the murine uterus. Quantification of diastase-labile periodic acid-Schiff (PAS) staining showed glycogen in the glandular epithelium decreased 71.4% at 1.5 days postcoitum (DPC) and 62.13% at DPC 3.5 compared to proestrus. In the luminal epithelium, glycogen was the highest at proestrus, decreased 46.2% at DPC 1.5 and 63.2% at DPC 3.5. Immunostaining showed that before implantation, glycogen metabolizing enzymes were primarily localized to the glandular and luminal epithelium. Stromal glycogen was low from proestrus to DPC 3.5. However, at the DPC 5.5 implantation sites, stromal glycogen levels increased sevenfold. Similarly, artificial decidualization resulted in a fivefold increase in glycogen levels. In both models, decidualization increased expression of glycogen synthase as determine by immunohistochemistry and western blot. In conclusion, glycogen levels decreased in the uterine epithelium before implantation, indicating that it could be used to support preimplantation embryos. Decidualization resulted in a dramatic increase in stromal glycogen levels, suggesting it may have an important, but yet undefined, role in pregnancy.

Crystalloids in FNA specimens of salivary gland lesions: A retrospective study in a single large institute.

BACKGROUND: Identifying crystalloids, which includes amylase and tyrosine crystalloids, is relatively uncommon in salivary gland fine-needle aspiration (FNA) cytology. Although it has been suggested that the presence of crystalloids favors a benign process, the full significance has not been well established. METHODS: The authors performed a review of slides from all salivary gland FNA cases received in their laboratory from January 2017 to September 2019 to identify cases with crystalloids (screened cohort). In addition, the departmental archives were searched retrospectively for all salivary gland FNA cases that had specifically reported crystalloids. Cytologic findings as well as correlation with surgical pathology and clinical follow-up were examined. RESULTS: There were 664 cases in the screened cohort. Crystalloids were present in 37 cases (incidence, 5.6%). Amylase crystalloids were the most commonly identified (n = 28; 75%), followed by tyrosine crystalloids (n = 4; 11%), and collagenous crystalloids (n = 1; 3%). Four cases with crystalloids could not be further classified because of low quantity (n = 4; 11%). An additional 54 cases were identified in the 10-year retrospective review. Diagnostic categorization for the total cohort (N = 91) was as follows: nondiagnostic, 30 cases (33%); nonneoplastic/benign, 42 cases (46%); neoplasm: benign, 10 cases (11%); and atypia of undetermined significance, 9 cases (10%). Twenty-six cases had subsequent resection findings, including oncocytic cyst/cystadenoma in 8 cases (31%), chronic sialadenitis/ductal obstructive change in 7 cases (27%), pleomorphic adenoma in 5 cases (27%), developmental cyst in 3 cases (12%), lymphoepithelial cyst in 2 cases (8%), and Warthin tumor in 1 case (4%). CONCLUSIONS: This cohort represents the largest FNA series of salivary gland crystalloids. All cases were associated with nonneoplastic or benign neoplastic lesions.

Oligonucleotide Cross-Linked Hydrogel for Recognition and Quantitation of MicroRNAs Based on a Portable Glucometer Readout.

A novel sensing platform for recognition and quantification of target microRNAs (miRNAs) was developed by combining an amylase-trapped DNA hydrogel, multicomponent nucleic acid enzymes (MNAzymes), and a portable glucometer (PGM) readout. First, the amylase was encapsulated inside the DNA hydrogel and physically separated from its substrate of amylose, which was in a solution outside the hydrogel. After addition of the target miRNA, the activity of the MNAzyme was restored, which cuts off the substrate linker strand. The active MNAzyme can catalytically act upon multiple substrate strands through diffusion, leading to the collapse of the hydrogel and the release of amylase, which catalyzes the hydrolysis of amylose to produce a large amount of glucose and generate a high PGM signal. The smart usage of the PGM enables simple portable detection of miR-21, with a detection limit as low as 0.325 fmol. Additionally, through the simple rational design of the target-binding sensor arms, the amylase-trapped DNA hydrogel sensing platform was successfully applied in the detection of multiple endogenous miRNAs (including miR-21, miR-335, miR-155, and miR-122) extracted from HeLa cells, HepG2 cells, MCF-7 cells, and L02 cells.

Nitration of Wheat Amylase Trypsin Inhibitors Increases Their Innate and Adaptive Immunostimulatory Potential in vitro.

Amylase trypsin inhibitors (ATI) can be found in all gluten containing cereals and are, therefore, ingredient of basic foods like bread or pasta. In the gut ATI can mediate innate immunity via activation of the Toll-like receptor 4 (TLR4) on immune cells residing in the lamina propria, promoting intestinal, as well as extra-intestinal, inflammation. Inflammatory conditions can induce formation of peroxynitrite (ONOO-) and, thereby, endogenous protein nitration in the body. Moreover, air pollutants like ozone (O3) and nitrogen dioxide (NO2) can cause exogenous protein nitration in the environment. Both reaction pathways may lead to the nitration of ATI. To investigate if and how nitration modulates the immunostimulatory properties of ATI, they were chemically modified by three different methods simulating endogenous and exogenous protein nitration and tested in vitro. Here we show that ATI nitration was achieved by all three methods and lead to increased immune reactions. We found that ATI nitrated by tetranitromethane (TNM) or ONOO- lead to a significantly enhanced TLR4 activation. Furthermore, in human primary immune cells, TNM nitrated ATI induced a significantly higher T cell proliferation and release of Th1 and Th2 cytokines compared to unmodified ATI. Our findings implicate a causative chain between nitration, enhanced TLR4 stimulation, and adaptive immune responses, providing major implications for public health, as nitrated ATI may strongly promote inhalative wheat allergies (baker's asthma), non-celiac wheat sensitivity (NCWS), other allergies, and autoimmune diseases. This underlines the importance of future work analyzing the relationship between endo- and exogenous protein nitration, and the rise in incidence of ATI-related and other food hypersensitivities.

Preparation and characterization of an amylase-triggered dextrin-linked graphene oxide anticancer drug nanocarrier and its vascular permeability.

We synthesized a dextrin (DEX)-conjugated graphene oxide (GO) nanocarrier (GO100-DEX) as a potential drug delivery system to respond to a tumor-associated stimulus, α-amylase, that has high permeability through the fenestrated endothelial barrier to the tumor site. At acidic pH and in the presence of α-amylase to simulate tumor conditions, GO100-DEX released a 1.5-fold higher amount of doxorubicin (DOX) than of GO100. Under the same conditions, the cytotoxic effects of GO100-DEX/DOX were 2-fold greater than those of free DOX and 2.9-fold greater than those of GO100/DOX. Employing an in vitro biomimetic microfluidic blood vessel model lined with human umbilical vein endothelial cells, we evaluated the tumor vasculature endothelial permeation of GO100-DEX and GO100 using dextrans of 10 and 70kDa for comparison and as standards to validate the microfluidic blood vessel model. The results showed that the permeabilities of GO100-DEX and GO100 were 4.3- and 4.9-fold greater than that of 70kDa dextran and 2.7- and 3.1-fold higher than that of 10kDa dextran, thus demonstrating the good permeability of the GO-based nanocarrier through the fenestrated endothelial barrier.

Portuguese Honeys from Different Geographical and Botanical Origins: A 4-Year Stability Study Regarding Quality Parameters and Antioxidant Activity.

Portuguese honeys (n = 15) from different botanical and geographical origins were analysed regarding their quality parameters (diastase activity, hydroxymethylfurfural content, moisture and pH), colour (L*, a*, b*) and antioxidant profile (total phenolics content, total flavonoids content, DPPH• scavenging activity, and ferric reducing power). The samples were analysed fresh and after 4-years of storage (at 25 °C and protected from light). The hydroxymethylfurfural content and diastase activity of the fresh samples were in accordance with the recommended values described in the legislation. In general, the antioxidant activity of the samples correlated more with the bioactive compounds content than with colour. The storage affected differently each individual sample, especially regarding the antioxidant profile. Nevertheless, although in general the lightness of the samples decreased (and the redness increased), after 4 years, 11 samples still presented acceptable diastase activity and hydroxymethylfurfural values.

Combined small angle X-ray solution scattering with atomic force microscopy for characterizing radiation damage on biological macromolecules.

BACKGROUND: Synchrotron radiation facilities are pillars of modern structural biology. Small-Angle X-ray scattering performed at synchrotron sources is often used to characterize the shape of biological macromolecules. A major challenge with high-energy X-ray beam on such macromolecules is the perturbation of sample due to radiation damage. RESULTS: By employing atomic force microscopy, another common technique to determine the shape of biological macromolecules when deposited on flat substrates, we present a protocol to evaluate and characterize consequences of radiation damage. It requires the acquisition of images of irradiated samples at the single molecule level in a timely manner while using minimal amounts of protein. The protocol has been tested on two different molecular systems: a large globular tetremeric enzyme (ß-Amylase) and a rod-shape plant virus (tobacco mosaic virus). Radiation damage on the globular enzyme leads to an apparent increase in molecular sizes whereas the effect on the long virus is a breakage into smaller pieces resulting in a decrease of the average long-axis radius. CONCLUSIONS: These results show that radiation damage can appear in different forms and strongly support the need to check the effect of radiation damage at synchrotron sources using the presented protocol.

Novel ciliate lipases for enzyme replacement during exocrine pancreatic insufficiency.

AIM AND OBJECTIVES: Exocrine pancreatic insufficiency caused by inflammation or pancreatic tumors results in nutrient malfunction by a lack of digestive enzymes and neutralization compounds. Despite satisfactory clinical results with current enzyme therapies, a normalization of fat absorption in patients is rare. An individualized therapy is required that includes high dosage of enzymatic units, usage of enteric coating, and addition of gastric proton pump inhibitors. The key goal to improve this therapy is to identify digestive enzymes with high activity and stability in the gastrointestinal tract. METHODS: We cloned and analyzed three novel ciliate lipases derived from Tetrahymena thermophila. Using highly precise pH-STAT-titration and colorimetric methods, we determined stability and lipolytic activity under physiological conditions in comparison with commercially available porcine and fungal digestive enzyme preparations. We measured from pH 2.0 to 9.0, with different bile salts concentrations, and substrates such as olive oil and fat derived from pig diet. RESULTS: Ciliate lipases CL-120, CL-130, and CL-230 showed activities up to 220-fold higher than Creon, pancreatin standard, and rizolipase Nortase within a pH range from pH 2.0 to 9.0. They are highly active in the presence of bile salts and complex pig diet substrate, and more stable after incubation in human gastric juice compared with porcine pancreatic lipase and rizolipase. CONCLUSIONS: The newly cloned and characterized lipases fulfilled all requirements for high activity under physiological conditions. These novel enzymes are therefore promising candidates for an improved enzyme replacement therapy for exocrine pancreatic insufficiency.

Clinical applications of amylase: Novel perspectives.

BACKGROUND: Amylase was the first enzyme to be characterized, and for the previous 200 years, its clinical role has been restricted to a diagnostic aid. Recent interface research has led to a substantial expansion of its role into novel, viable diagnostic, and therapeutic applications to cancer, infection, and wound healing. This review provides a concise "state-of-the-art" overview of the genetics, structure, distribution, and localization of amylase in humans. METHOD: A first-generation literature search was performed with the MeSH search string "Amylase AND (diagnost∗ OR therapeut$)" on OVIDSP and PUBMED platforms. A second-generation search was then performed by forward and backward referencing on Web of Knowledge™ and manual indexing, limited to the English Language. RESULTS: "State of the Art" in amylase genetics, structure, function distribution, localisation and detection of amylase in humans is provided. To the 4 classic patterns of hyperamylasemia (pancreatic, salivary, macroamylasemia, and combinations) a fifth, the localized targeting of amylase to specific foci of infection, is proposed. CONCLUSIONS: The implications are directed at novel therapeutic and diagnostic clinical applications of amylase such as the novel therapeutic drug classes capable of targeted delivery and "smart release" in areas of clinical need. Future directions of research in areas of high clinical benefit are reported.

Existence of hydroxylated MWCNTs demotes the catalysis effect of amylases against starch degradation.

Possible interaction between amylase and Multi Walled Carbon Nanotubes (MWCNTs) has been elucidated with spectroscopic methods. Hyperchromism of the UV-visible spectra of amylase-CNT conjugates suggested ground state complex formation between them. On contrary, the decreasing fluorescence emission spectra revealed the fate of quenching mechanism to be static. Stoke shift observed from the synchronous and 3D spectra suggested the possibilities of disturbances to the aromatic micro-environment of amylases by OH-MWCNTS. FTIR and FT-Raman spectra showed alteration in the amide I band, that corresponds to their effect on alpha-helical structures. Loss of alpha-helical structures and alteration in the dichroic band again revealed possible conformational change and effect towards the stability of polypeptide backbone structures. In addition, the shift observed in the SPR band and FTIR peaks of CNTs-amylase conjugates suggested possible alteration in their optical and structural properties. On the functional aspect, amylase activity on starch degradation and hydrolysis were found to be decreased in the presence of CNTs.

Chemical salivary composition and its relationship with periodontal disease and dental calculus

Aim: To determine the relationship between the chemical composition of saliva, periodontal disease and dental calculus. Methods: An observational analytical cross-sectional study was conducted with patients over 55 years of age. Ethical principles of autonomy and risk protection were applied according to the international standards. Sociodemographic and diagnosis variables (presence of dental calculus and periodontal status) were considered to measure salivary concentrations of glucose (by the glucose oxidase/peroxidase method, amylase (by the colorimetric test), urea (by the amount of indophenol), total protein (by the Bradford method) and albumin (by the nephelometric method). Patients chewed a sterile rubber band and 3 mL of stimulated saliva were collected. The samples were stored at -5 °C, centrifuged at 2,800 rpm for 10 min, and the supernatant was removed and stored at -20 °C. Data were presented as frequencies and proportions for qualitative variables and measures of central tendency and dispersion for quantitative variables. Data were analyzed by either analysis of variance or Kruskal Wallis test . A p value <0.05 was considered statistically significant. Results: Significant relationships were observed between the concentration of salivary urea and periodontal status (p = 0.03) and the presence of dental calculus and urea (p = 0.04) was demonstrated. Conclusions: A relationship between the salivary urea concentration and the presence of periodontal disease and dental calculus is suggested.

Overproduction of laccase from a newly isolated Ganoderma lucidum using the municipal food waste as main carbon and nitrogen supplement.

A strain of Ganoderma lucidum was separated and identified according to its morphological characteristics and phylogenetic data. The fungus is a laccase producer and it can secrete laccase using the municipal food waste (FW) as carbon and nitrogen supplement. After the statistic optimization, a laccase activity of 42,000 ± 600 U/l was obtained at 500 ml flask level and the activity is 12,000 U/l higher than that obtained by fermenting glucose and peptone, indicating that the use of FW to produce laccase not only reduces production cost, but also improves laccase activity. In 15 l bioreactor, FW is also suitable for laccase production and the maximum laccase activity reached 54,000 U/l. Moreover, some details of laccase overproduction using FW were investigated. The G. lucidum consumes FW by secreting a series of hydrolases and proteases and the improvement of laccase activity is because FW induces over-expression of three isoenzymes by polyacrylamide gel electrophoresis analysis.

Facile synthesis of multilayered polysaccharidic vesicles.

In this study, we developed facile synthesis method of multilayered polysaccharidic vesicles (hereafter termed 'mPSVs') using polysaccharides such as starch, hyaluronate (HA), and glycol chitosan (GC) via simple chemistry and using enzymatic reactions among polysaccharides. The enzymatic degradation of the HA shell by hyaluronidase (HYAL) enzyme contributed to accelerate the release of protein/peptide from the mPSVs. The mPSVs containing folate ligand and apoptotic cell death-inducing D-(KLAKLAK)2 peptide were effectively accumulated in in vivo KB tumor cells, primarily owing to passive tumor penetration via the enhanced permeability and retention (EPR) effect and active targeting via specific binding to folate receptors expressed on KB tumor cells. These mPSVs resulted in a significant increase in the in vivo tumor inhibition. This vesicle system is expected to exhibit great potential as an advanced platform technology for biomedical applications involving small molecular drugs with protein/gene targets.

Preparation and characterization of starch crosslinked with sodium trimetaphosphate and hydrolyzed by enzymes.

Crosslinked porous starch samples were produced by first crosslinking corn starch with sodium trimetaphosphate (STMP) and then partially hydrolyzing it with a mixture of α-amylase and glucoamylase. The granule morphology, porosity, swelling power, adsorption capacity, crystalline nature, molecular structure, melting and viscometric properties of these starch samples were measured and analyzed. The results showed that the porous starch which was crosslinked with 6% (w/w) STMP (ScPS-6) possessed remarkable superiority in terms of thermal and shear resistance among all the starch samples tested. The ScPS-6 also had the highest porosity and largest average pore diameter values. The swelling power of crosslinked porous starch was 56.3% lower than that of uncrosslinked porous starch. First order reaction kinetics equation was found to excellently (R(2) ≥ 0.99, average error = 6.03%) predict the experimental adsorption kinetics data of methylene blue for the crosslinked porous starch samples.

The effect of molar mass and degree of hydroxyethylation on the controlled shielding and deshielding of hydroxyethyl starch-coated polyplexes.

PEGylation is currently the gold-standard in shielding cationic DNA-polyplexes against non-specific interaction with blood components. However, it reduces cellular uptake and transfection, in what is known as the "PEG-dilemma". In an approach to solve this problem we developed hydroxyethyl starch (HES)-shielded polyplexes which get deshielded under the action of alpha amylase (AA). In this study, the effect of molar mass and degree of hydroxyethylation on the shielding and deshielding of the polyplexes as well as their in vivo performance were investigated. For this purpose, a battery of HES-polyethylenimine (PEI) conjugates was synthesized, and their rate and extent of biodegradation were investigated using asymmetric flow-field flow fractionation (AF4) and quartz-crystal microbalance with dissipation (QCM-D). Additionally, the transfection efficiency of the polyplexes was tested in Neuro2A cells and tumor-bearing mice. AF4 and QCM results show a rapid degradation for HES with lower degrees of hydroxyethylation. Meanwhile, in vitro transfection experiments showed a better shielding for higher HES molar masses, as well as deshielding with a significant boost in transfection upon addition of AA. Finally, in vivo experiments showed that the biodegradable HES markedly reduced the non-specific lung transcription of the polyplexes, but maintained gene expression in the tumor, contrary to the non-degradable HES and PEG controls, which reduced both tumor and lung expression. This study shows that by controlling the molecular characteristics of HES it is possible to engineer the shielding and deshielding properties of the polyplexes for more efficient gene delivery.

Human milk sIgA molecules contain various combinations of different antigen-binding sites resulting in a multiple binding specificity of antibodies and enzymatic activities of abzymes.

In the classic paradigm, immunoglobulins are monospecific molecules that have stable structures and two or more identical antigen-binding sites. However, we show here for the first time that the sIgA pool of human milk contains, depending on the donor, only 35±5% λ-sIgAs, 48±7% κ-sIgAs, and 17±4% of chimeric λ-κ-sIgAs. sIgA preparations contained no traces of canonical enzymes. However, all sIgA fractions eluted from several specific affinity sorbents under the conditions destroying even strong immune complexes demonstrated high catalytic activities in hydrolysis of ATP, DNA, and oligosaccharides, and phosphorylation of proteins, lipids, and oligosaccharides. Sequential re-chromatographies of the sIgA fractions with high affinity to one affinity sorbents on the second, third and then fourth affinity sorbents bearing other immobilized antigens led to the distribution of Abs and all catalytic activities all over the profiles of these chromatographies; in all cases some fractions eluted from affinity sorbents only under the conditions destroying strong immune complexes. In vitro, only an addition of reduced glutathione and milk plasma containing no Abs to two sIgA fractions with different affinity for DNA-cellulose led to a transition of up to 11-20% of Ab from one fraction to the other. Our data are indicative of the possibility of half-molecule exchange between different IgA and sIgA molecules. In addition, it cannot be excluded that during the penetration of IgAs through the specific milk barrier, the secretory component (S) and the join chain (J) can combine molecules of dimeric H(2)L(2) λ-IgAs and κ-IgAs against different antigens forming many different variants of H(4)L(4)SJ sIgA molecules. Therefore, some chimeric molecules of sIgA can contain from two to four HL-fragments to various antigens interacting with high affinity with different sorbents and catalyzing various chemical reactions. Our data essentially expand the ideas concerning explanation of the phenomenon of polyspecificity and cross-reactivity of Abs.

Peptide mixtures can self-assemble into large amyloid fibers of varying size and morphology.

Peptide mixtures spontaneously formed micrometer-sized fibers and ribbons from aqueous solution. Hydrolyzed gliadin produced short, slightly elliptical fibers while hydrolyzed wheat gluten, a mixture of gliadin and glutenin, formed round fibers of similar size. Mixing hydrolyzed gliadin with increasing molar amounts of myoglobin or amylase resulted in longer, wider fibers that transitioned from round to rectangular cross section. Fiber size, morphology, and modulus were controlled by peptide mixture composition. Fourier transform infrared (FT-IR) spectroscopy results showed that peptides experienced α to ß transitions forming an elementary cross-ß peptide secondary structure, indicative of amyloids. Large fiber formation was observed to be dependent on hydrophobic packing between constituent peptides. A model was developed to show how the fiber morphology was influenced by the peptides in the mixture.

Subcellular distribution of okadaic acid in the digestive gland of Mytilus galloprovincialis: first evidences of lipoprotein binding to okadaic acid.

The subcellular distribution of okadaic acid, the main diarrhetic shellfish poisoning (DSP) toxin, in the cells of the digestive gland of the mussel Mytilus galloprovincialis was studied. By means of differential centrifugation, ultrafiltration and extraction with methanol, it was found that most okadaic acid was stored in the cytosol. Notwithstanding only a small proportion of the total toxin was found to be in free form, being most of it bound to a soluble cellular compound with a molecular mass which ranged from 30 to 300 kDa. A series of fractionations of samples digested with a protease, a lipase, and amylase suggested that the component to which okadaic acid is bound is a high density lipoprotein. A new fractionation after digestion with a protein lipase additionally supports the previous conclusion.