Anti-glycated activity prediction of polysaccharides from two guava fruits using artificial neural networks

High-efficiency ultrasonic treatment was used to extract the polysaccharides of Psidium guajava (PPG) and Psidium littorale (PPL). The aims of this study were to compare polysaccharide activities from these two guavas, as well as to investigate the relationship between ultrasonic conditions and anti-glycated activity. A mathematical model of anti-glycated activity was constructed with the artificial neural network (ANN) toolbox of MATLAB software. Response surface plots showed the correlation between ultrasonic conditions and bioactivity. The optimal ultrasonic conditions of PPL for the highest anti-glycated activity were predicted to be 256 W, 60 °C, and 12 min, and the predicted activity was 42.2%. The predicted highest anti-glycated activity of PPG was 27.2% under its optimal predicted ultrasonic condition. The experimental result showed that PPG and PPL possessed anti-glycated and antioxidant activities, and those of PPL were greater. The experimental data also indicated that ANN had good prediction and optimization capability.     

Mechanism for multiple-substrates recognition of alpha-aminoadipate aminotransferase from Thermus thermophilus

Alpha-aminoadipate aminotransferase (AAA-AT), a homolog of mammalian kynurenine aminotransferase II (Kat II), transfers an amino group to 2-oxoadipate to yield alpha-aminoadipate in lysine biosynthesis through the alpha-aminoadipate pathway in Thermus thermophilus. AAA-AT catalyzes transamination against various substrates, including AAA, glutamate, leucine, and aromatic amino acids. To elucidate the structural change for recognition of various substrates, we determined crystal structures of AAA-AT in four forms: with pyridoxal 5'-phosphate (PLP) (PLP complex), with PLP and leucine (PLP/Leu complex), with N-phosphopyridoxyl-leucine (PPL) (PPL complex), and with N-phosphopyridoxyl-alpha-aminoadipate (PPA) at 2.67, 2.26, 1.75, and 1.67 A resolution, respectively. The PLP complex is in an open state, whereas PLP/Leu, PPL, and PPA complexes are in closed states with maximal displacement (over 7 A) of the alpha2 helix and the beta1 strand in the small domain to cover the active site, indicating that conformational change is induced by substrate binding. In PPL and PLP/Leu complexes, several hydrophobic residues on the alpha2 helix recognize the hydrophobic side chain of the bound leucine moiety whereas, in the PPA complex, the alpha2 helix rotates to place the guanidium moiety of Arg23 on the helix at the appropriate position to interact with the carboxyl side chain of the AAA moiety. These results indicate that AAA-AT can recognize various kinds of substrates using the mobile alpha2 helix. The crystal structures and site-directed mutagenesis revealed that intersubunit-electrostatic interactions contribute to the elevated thermostability of this enzyme.     

Distinct functions for the two PsbP-like proteins PPL1 and PPL2 in the chloroplast thylakoid lumen of Arabidopsis

PsbP, an extrinsic subunit of photosystem II (PSII), is a nuclear-encoded protein that optimizes the water-splitting reaction in vivo. In addition to PsbP, higher plants have two nuclear-encoded genes for PsbP homologs (PsbP-like proteins [PPLs]) that show significant sequence similarity to a cyanobacterial PsbP homolog (cyanoP); however, the function of PPLs in higher plants has not yet been elucidated. In this study, we characterized Arabidopsis (Arabidopsis thaliana) mutants lacking either of two PPLs, PPL1 and PPL2. Phylogenetic analysis suggests that PPL1 would be an ortholog of cyanoP, and PPL2 and PsbP may have a paralogous relationship with PPL1. Analysis on mRNA expression profiles showed that PPL1 expressed under stress conditions and PPL2 coexpressed with the subunits of chloroplast NAD(P)H dehydrogenase (NDH) complex. Consistent with these suggestions, PSII activity in a ppl1 mutant was more sensitive to high-intensity light than wild type, and the recovery of photoinhibited PSII activity was delayed in ppl1 plants. Therefore, PPL1 is required for efficient repair of photodamaged PSII. Furthermore, the stoichiometric level and activity of the chloroplast NDH complex in thylakoids were severely decreased in a ppl2 mutant, demonstrating that PPL2 is a novel thylakoid lumenal factor required for accumulation of the chloroplast NDH complex. These results suggest that during endosymbiosis and subsequent gene transfer to the host nucleus, cyanoP from ancient cyanobacteria evolved into PPL1, PPL2, and PsbP, and each of them has a distinct role in photosynthetic electron transfer in Arabidopsis.     

Stereoselective transport and uptake of propranolol across human intestinal Caco‐2 cell monolayers

The transport and uptake of individual propranolol (PPL) enantiomers were studied in human intestinal Caco‐2 cell monolayers, and a reversed‐phase HPLC‐UV assay was used for quantitative analysis. S‐PPL and R‐PPL across Caco‐2 cell monolayers was determined in the concentrations range of 10–500 μM in both apical (AP) to basolateral (BL) and BL to AP directions. S‐PPL exhibited greater permeability than R‐PPL in the AP to BL direction, whereas in the BL to AP direction S‐enantiomer transported less than R‐enantiomer. Uptake of R‐PPL was significantly higher than that of S‐PPL either from AP side or from BL side. The statistically significant differences in uptake were observed at the concentrations range from 10 to 50 μM. Furthermore, the apparent Michaelis constant (K_m) and maximal velocity (V_max) also showed significant difference between the two enantiomers. Moreover, the AP to BL transport of PPL enantiomer was markedly decreased by lowering the pH of the apical side but it did not affect the stereoselectivity of PPL across Caco‐2 cell monolayers. The transport and uptake of PPL in the BL to AP direction was not influenced by several protein inhibitors. The results suggest that PPL enantiomers showed stereoselective transport and uptake across the Caco‐2 cell monolayers. A special transport mechanism capable of directing the PPL enantiomers might be present in the Caco‐2 monolayers. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Identification of various lipolytic enzymes in crude porcine pancreatic lipase preparations using covalent fluorescent inhibitors

We developed a specific method for determination and discrimination of lipo-/estero-lytic enzymes in crude lipase preparations. Here we study the composition of commercial porcine pancreatic lipase (PPL), since it is widely used for bioconversions of synthetic and natural substrates. Our method is based on incubation of enzyme samples with fluorescently labeled alkyl- or dialkylglyceryl-phosphonates in an appropriate solvent followed by protein separation by electrophoresis and fluorescence detection with a CCD camera. After incubation with short-chain alkylphosphonate solubilized by taurodeoxycholate, crude PPL preparations showed a very weak band at 50 kDa, which is indicative of low PPL concentrations in these samples. In addition, seven other fluorescent bands were detected. The band at the lowest molecular weight corresponded to alpha-chymotrypsin. Two intensive fluorescent bands were in the molecular weight range of chymotrypsinogen (26 kDa) and four weak bands were in the range 20-24 kDa. Long-chain dialkylglycerophosphonate labeled two protein bands in crude PPL: alpha-chymotrypsin and a very intensive band corresponding to the molecular weight of chymotrypsinogen. Detection of cholesterol esterase (98 kDa) in crude PPL preparations depended on addition of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) to the incubation mix, as demonstrated by spiking with cholesterol esterase. Thus, commercial crude PPL preparations contain a variety of estero-/lipo-lytic enzymes in addition to rather low amounts of active PPL, which should be considered when using crude PPL for bioconversions. Our method can also be used to show whether an isolated esterolytic activity corresponds to a single protein or isoenzymes. Here we confirm by 2D-electrophoretic separation of "pure" PPL that PPL exists as isoenzymes in different glycosylated forms.     

Development of an indirect method for measuring porcine pancreatic lipase in human duodenal fluid

Patients with exocrine pancreatic insufficiency are usually treated with porcine pancreatic enzymes but the bioavailability of these enzymes in the gut remains a matter of discussion. In order to determine the duodenal availability of porcine pancreatic lipase (PPL) present in pancreatic extracts (PE) taken orally, we developed a method for quantifying PPL in samples containing both PPL and human pancreatic lipase (HPL). Total pancreatic lipase activity measurements using the pH-stat technique and tributyrin as substrate were combined with an HPL-specific ELISA. Based on the known specific activity of the purified HPL, its activity was deduced from the ELISA measurements, and the PPL activity was obtained by subtracting the HPL activity from the total pancreatic lipase activity. This assay was established and validated using various samples containing pure PPL and recombinant HPL or PE, mixed or not with human duodenal juice. Samples collected in vivo from patients treated with PE were also tested. It was found that PPL did not affect the HPL ELISA, and the indirect PPL assay gave a measurement accuracy of 6.6% with the samples containing pure PPL and 10% with those containing PE. This assay was also used successfully to discriminate between PPL and the endogenous HPL present in the duodenal contents of patients with severe pancreatic insufficiency treated with PE. This method might provide a useful means of assessing the availability of PEs at their site of action, in the absence of a PPL-specific ELISA.     

Structures of jacalin‐related lectin PPL3 regulating pearl shell biomineralization

The nacreous layer of pearl oysters is one of the major biominerals of commercial and industrial interest. Jacalin‐related lectins, including PPL3 isoforms, are known to regulate biomineralization of the Pteria penguin pearl shell, although the molecular mechanisms are largely unknown. The PPL3 crystal structures were determined partly by utilizing microgravity environments for 3 isoforms, namely, PPL3A, PPL3B, and PPL3C. The structures revealed a tail‐to‐tail dimer structure established by forming a unique inter‐subunit disulfide bond at C‐termini. The N‐terminal residues were found in pyroglutamate form, and this was partly explained by the post‐translational modification of PPL3 isoforms implied from the discrepancy between amino acid and gene sequences. The complex structures with trehalose and isomaltose indicated that the novel specificity originated from the unique α‐helix of PPL3 isoforms. Docking simulations of PPL3B to various calcite crystal faces suggested the edge of a β‐sheet and the carbohydrate‐binding site rich in charged residues were the interface to the biomineral, and implied that the isoforms differed in calcite interactions.     

Enhancing the catalytic properties of porcine pancreatic lipase by immobilization on SBA-15 modified by functionalized ionic liquid

The mesoporous silica SBA-15 was modified by carboxyl-functionalized ionic liquid (COOH-IL-SBA). The prepared support was used to immobilize porcine pancreatic lipase (PPL) by physical adsorption (PPL-COOH-IL-SBA) and covalent attachment (PPL-CON-IL-SBA). Enzymatic properties of the immobilized PPL were investigated in the triacetin hydrolysis reaction. It was found that carboxyl functionalized ionic liquid modification of the support surface was an effective method to improve the properties of immobilized PPL. Incorporating into the functionalized SBA-15 made PPL more resistant to temperature and pH changes, compared with PPL immobilized on parent SBA-15 (PPL-SBA). Especially, after the covalent attachment to a functionalized support, the stability of PPL was improved obviously, which retained 81.25% and 52.50% of the original activity after incubation for 20 days and four times recycling, respectively, whereas PPL-SBA exhibited only 58.80% and 27.78% of the original activity under the same conditions. In addition, physical and chemical properties of the supports and immobilized PPL were characterized by small-angle X-ray powder diffraction (SAXRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), nitrogen adsorption, nuclear magnetic resonance (NMR) and thermogravimetry (TG). The images and data confirmed chemical modification in SBA-15 and PPL immobilization on the tested support.     

A posterior probability of linkage-based re-analysis of schizophrenia data yields evidence of linkage to chromosomes 1 and 17

OBJECTIVE: Linkage analysis using 22 Canadian pedigrees identified a promising schizophrenia candidate region on 1q23 with a maximum 2-point HLOD under a recessive model of 5.8 [Brzustowicz et al. 2000]. In the current study, we revisited this data set using a Bayesian linkage analysis technique, namely the posterior probability of linkage (PPL). METHODS: The PPL has been developed as an alternative to traditional linkage analysis. It differs from both LOD scores and 'non-parametric' methods in that it directly measures the probability of linkage given the data, and incorporates prior genomic information. RESULTS: As expected, PPL results for 1q23 supported the previously observed linkage, with an estimated multipoint PPL of 99.7%. However, the PPL supported two further results: a second peak on chromosome 1 at 1p13 with a multipoint with PPL of 70% and a chromosome 17 marker (D17S784 at 17q25) with a multipoint PPL of 44%. CONCLUSIONS: The PPL-based analysis presented has the advantage over other likelihood-based linkage methods in that it avoids maximization and produces a less complex view of the strength of evidence for linkage.     

Liver ferrochelatase from normal and hexachlorobenzene porphyric rats. Mechanism of drug action

1. The action of hexachlorobenzene (HCB) on hepatic ferrochelatase was investigated. 2. A direct action of HCB, pentachlorophenol, porphyrins and haem on this enzyme activity was discarded. 3. In HCB porphyric liver there is probably an activator tightly bound to the enzyme. 4. Pyridoxal phosphate (PPL) may be a cofactor of ferrochelatase from both normal and porphyric rats. 5. The PPL would be involved in the binding site of Fe2+ or at least in the approaching of Fe2+ to the active site of the enzyme. 6. The differences found between normal and porphyric preparations could be attributed to conformational changes elicited by the HCB.     

Stereoselective Accumulation of Propranolol Enantiomers in K562 and K562/ADR Cells

The stereoselective uptake of propranolol enantiomers was investigated by using the K562 and K562 adriamycin‐resistant cell line (K562/ADR) as a model. An enantioselective RP‐HPLC method was applied to determine the accumulation of propranolol (PPL) stereoisomers in K562 and K562/ADR cells. The concentration, time and temperature dependent studies showed that the accumulation of S‐(?)‐PPL was higher than R‐(+)‐PPL in K562 cells and uptake of R‐(+)‐PPL was significantly higher than that of S‐(?)‐PPL in K562/ADR cells. The results indicate the enantioselective accumulation of propranolol enantiomers in K562 and K562 / ADR cells. Chirality 25:361–364, 2013. © 2013 Wiley Periodicals, Inc.     

Interface-mediated inactivation of pancreatic lipase by a water-reactive compound: 2-sulfobenzoic cyclic anhydride

2-Sulfobenzoic cyclic anhydride (SBA) rapidly and selectively inactivates porcine pancreatic lipase (PPL) only when added during the hydrolysis of an emulsified ester such as tributyrin or dodecyl acetate. The present data suggest that the inactivation of PPL occurs preferentially at the oil/water interface and not in the aqueous phase, since colipase and bile salt were found to adversely affect the inhibition process. Moreover, it is shown that at a molar ratio of SBA to pure PPL of 1, 40% of the lipase activity was already irreversibly lost. Complete inactivation was observed at SBA to pure PPL molar ratios of 120. A 60% inactivation occurred when 0.5 mol of 3H-labeled SBA was attached per mole of PPL. The SBA-inactivated PPL competes for binding to the dodecyl acetate/water interface as efficiently as the native enzyme. Larger SBA concentrations are required when crude lipase preparations are used as well as with pure PPL in the presence of bile salts and colipase. Lipases were found to have variable sensitivities to SBA inactivation, depending on their origin. In the presence of bile salts and tributyrin at pH 6.0, human gastric lipase activity was not affected by the presence of a 10(6) molar excess of SBA.     

A new method for computing the multipoint posterior probability of linkage

The posterior probability of linkage (PPL) is a Bayesian statistic which directly measures the probability of linkage between a trait locus and a marker (in the 2-point case) or a genomic region (in the multipoint case). It has several benefits, including ease of interpretation, the ability to incorporate prior genomic information, and a mathematically rigorous and robust procedure for accumulating linkage information across multiple heterogeneous datasets. To date, the majority of work on the PPL has focused on the development of the 2-point statistic, with only preliminary attempts at the development of an equivalent multipoint version. In this paper we present a new way of computing of the multipoint PPL. This new version imputes to each genomic point an estimate of the 2-point PPL we would have obtained from a fully informative marker giving similar evidence for linkage. This version, which we call the imputed PPL, is shown to be superior to previously developed versions.     

1,3‐Regiospecific ethanolysis of soybean oil catalyzed by crosslinked porcine pancreas lipase aggregates

The preparation of crosslinked aggregates of pancreatic porcine lipase (PPL‐CLEA) was systematically studied, evaluating the influence of three precipitants and two crosslinking agents, as well as the use of soy protein as an alternative feeder protein on the catalytic properties and stability of the immobilized PPL. Standard CLEAs showed a global yield (CLEA’ observed activity/offered total activity) of less than 4%, whereas with the addition of soy protein (PPL:soy protein mass ratio of 1:3) the global yield was approximately fivefold higher. The CLEA of PPL prepared with soy protein as feeder (PPL:soy protein mass ratio of 1:3) and glutaraldehyde as crosslinking reagent (10 μmol of aldehyde groups/mg of total protein) was more active mainly because of the reduced enzyme leaching in the washing step. This CLEA, named PPL‐SOY‐CLEA, had an immobilization yield around 60% and an expressed activity around 40%. In the ethanolysis of soybean oil, the PPL‐SOY‐CLEA yielded maximum fatty acid ethyl ester (FAEE) concentration around 12‐fold higher than that achieved using soluble PPL (34 h reaction at 30°C, 300 rpm stirring, soybean oil/ethanol molar ratio of 1:5) with an enzyme load around 2‐fold lower (very likely due to free enzyme inactivation). The operational stability of the PPL‐SOY‐CLEA in the ethanolysis of soybean oil in a vortex flow type reactor showed that FAEE yield was higher than 50% during ten reaction cycles of 24 h. This reactor configuration may be an attractive alternative to the conventional stirred reactors for biotransformations in industrial plants using carrier‐free biocatalysts. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:910–920, 2018     

Development and Characterization of Propranolol Selective Molecular Imprinted Polymer Composite Electrospun Nanofiber Membrane

Propranolol (PPL) imprinted microspheres (MIP) were successfully prepared via oil/water polymerization using a methyl methacrylate (MMA) monomer, PLL template, and divinylbenzene (DVB) cross-linker and favorably incorporated in a Eudragit-RS100 nanofiber membrane. A non-PPL imprinted polymer (NIP), without a template, was used as a control. The morphology and particle size of the beads were investigated using scanning electron microscopy. The results revealed that both MIP and NIP had a spherical shape with a micron size of approximately 50–100 μm depending on the amounts of DVB and PPL used. NIP2 (MMA/DVB, 75:2.5) and MIP8 (PPL/MMA/DVB, 0.8:75:2.5) were selected for reloading of PPL, and the result indicated that increasing the ratio of PPL to polymer beads resulted in increase PPL reloading (>80%). A total of 10–50% NIP2 or MIP8 was incorporated into a 40% (w/v) Eudragit-RS100 fiber membrane using an electrospinning technique. PPL could be bound to the 50% MIP8 composite fiber membrane with a higher extent and at a higher rate than the control (NIP2). Furthermore, the MIP8 composite fiber membrane showed higher selectivity to PPL than the other β-blockers (atenolol, metoprolol, and timolol). Thus, the MIP8 composite fiber membrane can be further developed for various applications in pharmaceutical and other affinity separation fields.Key words: membrane, molecularly imprinted, propranolol, selective molecular imprinting     

Biocatalysts for cascade reaction: porcine pancreas lipase (PPL)-catalyzed synthesis of bis(indolyl)alkanes

A cascade reaction between aldehydes and indole catalyzed by lipase from porcine pancreas Type II (PPL) in solvent mixture at 50 °C was reported for the first time. Some control experiments had been designed to demonstrate that the PPL was responsible for the cascade reaction. After the optimization of the stepwise process, a series of bis(indolyl)alkanes were prepared in moderate to excellent yields under the catalysis of PPL.     

Effects of insulin and transgenic overexpression of UDP-glucose pyrophosphorylase on UDP-glucose and glycogen accumulation in skeletal muscle fibers

UDP-glucose (UDP-Glc) and glycogen levels in skeletal muscle fibers of defined fiber type were measured using microanalytical methods. Infusing rats with insulin increased glycogen in both Type I and Type II fibers. Insulin was without effect on UDP-Glc in Type I fibers but decreased UDP-Glc by 35-40% in Type IIA/D and Type IIB fibers. The reduction in UDP-Glc suggested that UDP-Glc pyrophosphorylase (PPL) activity might limit glycogen synthesis in response to insulin. To explore this possibility, we generated mice overexpressing a UDP-Glc PPL transgene in skeletal muscle. The transgene increased both UDP-Glc PPL activity and levels of UDP-Glc in skeletal muscles by approximately 3-fold. However, overexpression of UDP-Glc PPL was without effect on either the levels of skeletal muscle glycogen or glucose tolerance in vivo. The transgene was also without effect on either control or insulin-stimulated rates of (14)C-glucose incorporation into glycogen in muscles incubated in vitro. The results indicate that UDP-Glc PPL activity is not limiting for glycogen synthesis.     

Role of murine Peyer's patch lymphocytes against primary and challenge infections with Cryptosporidium parvum

In order to determine the role of Peyeros patch lymphocytes (PPL) in self-clearing of Cryptosporidium parvum infection in murine models, changes in PPL subsets, their cytokine expression, and in vitro IgG1 and IgA secretions by PPL were observed in primary- and challenge-infected C57BL/6 mice. In primary-infected mice, the percentages of CD4+ T cells, CD8+ T cells, sIgA+ B cells, IL-2+ T cells, and IFN-gamma+ T cells among the PPL, increased significantly (P < 0.05) on day 10 post-infection (PI). Secretion of IgG1 and IgA in vitro by PPL also increased on day 10 PI. However, all these responses, with the exception of IgG1 and IgA secretions, decreased in challenge-infected mice on day 7 post-challenge (= day 13 PI); their IgG1 and IgA levels were higher (P > 0.05) than those in primaryinfected mice. The results suggest that murine PPL play an important role in self-clearing of primary C. parvum infections through proliferation of CD4+, CD8+, IL-2+, and IFN-gamma+ T cells, and IgG1 and IgA-secreting B cells. In challenge infections, the role of T cells is reduced whereas that of B cells secreting IgA appeared to be continuously important.     

Immobilization of porcine pancreatic lipase on poly-hydroxybutyrate particles for the production of ethyl esters from macaw palm oils and pineapple flavor

Poly-hydroxybutyrate particles (PHB) were used as support to immobilize porcine pancreatic lipase (PPL). The biocatalysts prepared were tested in the synthesis of pineapple flavor by esterification of butanol and butyric acid in heptane medium, and in the synthesis of ethyl esters by transesterification of macaw palm pulp (MPPO) and macaw palm kernel (MPKO) oils with ethanol in solvent-free systems. The effect of protein loading on the biocatalyst activity was assessed in olive oil hydrolysis. Maximum hydrolytic activity of 292.8 ± 8.60 IU/g was observed. Langmuir isotherm model was applicable to the adsorption of PPL on PHB particles. Maximum immobilized protein amount was 24.3 ± 1.70 mg/g. The optimal pH and temperature in hydrolysis reaction for the immobilized PPL were at pH 8.5 and 50 °C, while for the crude PPL extract were at pH 8.0 and 45 °C. Immobilized PPL exhibited full hydrolytic activity after 2 h of incubation in non-polar solvents. In esterification reaction, optimal conversion was around 93% after 2 h of reaction. After six esterification cycles, the biocatalyst retained 63% of its initial activity. The biocatalyst prepared attained transesterification yield of 50% after 48 h of reaction for MPKO and 35% after 96 h of reaction for MPPO.     

Novel Matrix Proteins of Pteria penguin Pearl Oyster Shell Nacre Homologous to the Jacalin-Related β-Prism Fold Lectins

Nacreous layers of pearl oyster are one of the major functional biominerals. By participating in organic compound-crystal interactions, they assemble into consecutive mineral lamellae-like photonic crystals. Their biomineralization mechanisms are controlled by macromolecules; however, they are largely unknown. Here, we report two novel lectins termed PPL2A and PPL2B, which were isolated from the mantle and the secreted fluid of Pteria penguin oyster. PPL2A is a hetero-dimer composed of α and γ subunits, and PPL2B is a homo-dimer of β subunit, all of which surprisingly shared sequence homology with the jacalin-related plant lectin. On the basis of knockdown experiments at the larval stage, the identification of PPLs in the shell matrix, and in vitro CaCO₃ crystallization analysis, we conclude that two novel jacalin-related lectins participate in the biomineralization of P. penguin nacre as matrix proteins. Furthermore, it was found that trehalose, which is specific recognizing carbohydrates for PPL2A and is abundant in the secreted fluid of P. penguin mantle, functions as a regulatory factor for biomineralization via PPL2A. These observations highlight the unique functions, diversity and molecular evolution of this lectin family involved in the mollusk shell formation.