Auxin homeostasis in maize (Zea mays) is regulated via 1-O-indole-3-acetyl-myo-inositol synthesis at early stages of seedling development and under abiotic stress.

MAIN CONCLUSION: Indole-3-acetyl-myo-inositol biosynthesis is regulated during maize seedling development and in response to drought and cold stress. The main purpose of this pathway is maintenance of auxin homeostasis. Indole-3-acetic acid (IAA) conjugation to myo-inositol is a part of a mechanism controlling free auxin level in maize. In this work, we investigated changes in the indole-3-acetyl-myo-inositol (IAInos) biosynthesis pathway in 3-d- and 6-d-old maize seedlings and germinating seeds as well as in seedlings subjected to drought and cold stress to evaluate a role of this pathway in maize development and stress response. In germinating seeds, activity of the enzymes involved in IAInos biosynthesis remains unchanged between 3-d- and 6-d-old material but increases in coleoptiles and radicles of the seedlings. Under cold stress, in germinating seeds and in coleoptiles, activity of the enzymes decreases and increases, respectively; however, it does not entail changes in auxin level. In drought-exposed germinating maize seeds, totally diminished activities of IAInos synthesis pathway enzymes resulted in almost twofold increase of free IAA content. Similar increase of auxin level was observed in radicles of drought-subjected seedlings together with lack of catalytic activity of the first enzyme of the pathway. Exogenous IAInos has no effect on the level of non-enzymatic antioxidant, ascorbate. It has also either no effect on the protein carbonylation and lipid peroxidation, or it affects it in a similar way as exogenously applied IAA and myo-inositol, which are products of IAInos hydrolysis. Thus, IAInos biosynthesis pathway acts in maize development and stress responses by regulation of free IAA concentration, as IAInos itself does not appear to have a distinct role in these processes.

Novel Antibacterial, Cytotoxic and Catalytic Activities of Silver Nanoparticles Synthesized from Acidophilic Actinobacterial SL19 with Evidence for Protein as Coating Biomolecule.

Silver nanoparticles (AgNPs) have potential applications in medicine, photocatalysis, agriculture, and cosmetic fields due to their unique physicochemical properties and strong antimicrobial activity. Here, AgNPs were synthesized using actinobacterial SL19 strain, isolated from acidic forest soil in Poland, and confirmed by UV-vis and FTIR spectroscopy, TEM, and zeta potential analysis. The AgNPs were polydispersed, stable, spherical, and small, with an average size of 23 nm. The FTIR study revealed the presence of bonds characteristic of proteins that cover nanoparticles. These proteins were then studied by using liquid chromatography with tandem mass spectrometry (LC-MS/ MS) and identified with the highest similarity to hypothetical protein and porin with molecular masses equal to 41 and 38 kDa, respectively. Our AgNPs exhibited remarkable antibacterial activity against Escherichia coli and Pseudomonas aeruginosa. The combined, synergistic action of these synthesized AgNPs with commercial antibiotics (ampicillin, kanamycin, streptomycin, and tetracycline) enabled dose reductions in both components and increased their antimicrobial efficacy, especially in the case of streptomycin and tetracycline. Furthermore, the in vitro activity of the AgNPs on human cancer cell lines (MCF-7, A375, A549, and HepG2) showed cancer-specific sensitivity, while the genotoxic activity was evaluated by Ames assay, which revealed a lack of mutagenicity on the part of nanoparticles in Salmonella Typhimurium TA98 strain. We also studied the impact of the AgNPs on the catalytic and photocatalytic degradation of methyl orange (MO). The decomposition of MO was observed by a decrease in intensity of absorbance within time. The results of our study proved the easy, fast, and efficient synthesis of AgNPs using acidophilic actinomycete SL19 strain and demonstrated the remarkable potential of these AgNPs as anticancer and antibacterial agents. However, the properties and activity of such particles can vary by biosynthesized batch.

EPIP as an abscission promoting agent in the phytohormonal pathway.

Understanding the mechanisms underlying the activation of the abscission zone (AZ) responsible for organ separation from plant body in crop species will help improve their yielding and economic importance. Special attention has been given recently to the role of the INFLORESCENCE DEFICIENT IN ABSCISSION protein, particularly its functional fragment, EPIP peptide. Its stimulatory effect on abscission in different crops has been demonstrated. Recently we described the role of EPIP in the redox, lipid, and pectin-related events taking place in AZ of Lupinus luteus flowers, which undergo massive abscission in natural conditions. To further examine EPIP contribution in AZ functioning, here, we analyze its impact on the ultrastructural changes, synthesis of two hormonal abscission stimulators - abscisic acid (ABA) and ethylene (ET), and the appearance of phosphoproteins. As our results show, the response of flower AZ to exogenous EPIP involves the induction of distinct modifications related to the one hand with upregulation of cell activity but on the other hand degradation processes and possible autophagy. Furthermore, the EPIP stimulated biosynthesis pathways of ABA and ET precisely in AZ cells. In addition, progressive phosphorylation of proteins has been observed under EPIP influence. The highly accumulated ones were identified as those, related to primary metabolism and reactive oxygen species homeostasis, and their role in abscission has been discussed. To summarizing, the presented detailed description of EPIP action in AZ cells in combination with our previous data offers new insights into its regulatory function and provides opportunities to counteract excessive flower abscission in lupine.

The GH3 amidosynthetases family and their role in metabolic crosstalk modulation of plant signaling compounds.

The Gretchen Hagen 3 (GH3) genes encoding proteins belonging to the ANL superfamily are widespread in the plant kingdom. The ANL superfamily consists of three groups of adenylating enzymes: aryl- and acyl-CoA synthetases, firefly luciferase, and amino acid-activating adenylation domains of the nonribosomal peptide synthetases (NRPS). GH3s are cytosolic, acidic amidosynthetases of the firefly luciferase group that conjugate auxins, jasmonates, and benzoate derivatives to a wide group of amino acids. In contrast to auxins, which amide conjugates mainly serve as a storage pool of inactive phytohormone or are involved in the hormone degradation process, conjugation of jasmonic acid (JA) results in biologically active phytohormone jasmonyl-isoleucine (JA-Ile). Moreover, GH3s modulate salicylic acid (SA) concentration by conjugation of its precursor, isochorismate. GH3s, as regulators of the phytohormone level, are crucial for normal plant development as well as plant defense response to different abiotic and biotic stress factors. Surprisingly, recent studies indicate that FIN219/JAR1/GH3.11, one of the GH3 proteins, may act not only as an enzyme but is also able to interact with tau-class glutathione S-transferase (GSTU) and constitutive photomorphogenic 1 (COP1) proteins and regulate light and stress signaling pathways. The aim of this work is to summarize our current knowledge of the GH3 family.

Evaluating the Influence of Visual Attentional Tracking on Pointing Movement Precision.

Multiple object tracking (MOT) and goal-directed movements are both based on attentional processes. This study focused on the instant effect of attentive tracking with respect to pointing performance. We measured the precision of pointing to the targets of an MOT task immediately after the tracking period when all the objects were still in motion, and to the precision of pointing to moving objects in the task without attentional tracking. The results demonstrated that an increase in the attentional tracking load was the primary factor that decreased pointing precision, although visual load may also contribute to this influence. We also manipulated the relationship between the number of targets and the density of the MOT display (the total number of objects displayed), which affected the MOT accuracy but not the pointing precision.

Green Synthesized Silver Nanoparticles: Antibacterial and Anticancer Activities, Biocompatibility, and Analyses of Surface-Attached Proteins.

The increasing number of multi-drug-resistant bacteria and cancer cases, that are a real threat to humankind, forces research world to develop new weapons to deal with it. Biogenic silver nanoparticles (AgNPs) are considered as a solution to this problem. Biosynthesis of AgNPs is regarded as a green, eco-friendly, low-priced process that provides small and biocompatible nanostructures with antimicrobial and anticancer activities and potential application in medicine. The biocompatibility of these nanoparticles is related to the coating with biomolecules of natural origin. The synthesis of AgNPs from actinobacterial strain was confirmed using UV-Vis spectroscopy while their morphology, crystalline structure, stability, and coating were characterized using, transmission electron microscopy (TEM), X-ray diffraction (XRD), Zeta potential and Fourier transform infrared spectroscopy (FTIR). Antibacterial activity of biogenic AgNPs was evaluated by determination of minimum inhibitory and minimum biocidal concentrations (MIC and MBC) against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. The potential mechanism of antibacterial action of AgNPs was determined by measurement of ATP level. Since the use of AgNPs in biomedical applications depend on their safety, the in vitro cytotoxicity of biosynthesized AgNPs on MCF-7 human breast cancer cell line and murine macrophage cell line RAW 264.7 using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, cell lactate dehydrogenase (LDH) release and measurement of reactive oxygen species (ROS) level were assessed. The nanoparticle protein capping agent that can be involved in reduction of silver ions to AgNPs and their stabilization was identified using LC-MS/MS. Nanoparticles were spherical in shape, small in size (mean 13.2 nm), showed crystalline nature, good stability (-18.7 mV) and presence of capping agents. They exhibited antibacterial activity (MIC of 8-128 µg ml-1, MBC of 64-256 µg ml-1) and significantly decreased ATP levels in bacterial cells after treatment with different concentrations of AgNPs. The in vitro analysis showed that the AgNPs demonstrated dose-dependent cytotoxicity against RAW 264.7 macrophages and MCF-7 breast cancer cells but higher against the latter than the former. Cell viability decrease was found to be 42.2-14.2 and 38.0-15.5% while LDH leakage 14.6-42.7% and 19.0-45.0%, respectively. IC50 values calculated for MTT assay was found to be 16.3 and 12.0 µg ml-1 and for LDH assay 102.3 and 76.2 µg ml-1, respectively. Moreover, MCF-7 cells released a greater amount of ROS than RAW 264.7 macrophages during stimulation with all tested concentrations of AgNPs (1.47-3.13 and 1.02-2.58 fold increase, respectively). The SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis revealed the presence of five protein bands at a molecular weight between 31.7 and 280.9 kDa. These proteins showed the highest homology to hypothetical proteins and porins from E. coli, Delftia sp. and Pseudomonas rhodesiae. Based on obtained results it can be concluded that biogenic AgNPs were capped with proteins and demonstrated potential as antimicrobial and anticancer agent.

Comparative Study of Structural Changes of Polylactide and Poly(ethylene terephthalate) in the Presence of Trichoderma viride.

Plastic pollution is one of the crucial global challenges nowadays, and biodegradation is a promising approach to manage plastic waste in an environment-friendly and cost-effective way. In this study we identified the strain of fungus Trichoderma viride GZ1, which was characterized by particularly high pectinolytic activity. Using differential scanning calorimetry, Fourier-transform infrared spectroscopy techniques, and viscosity measurements we showed that three-month incubation of polylactide and polyethylene terephthalate in the presence of the fungus lead to significant changes of the surface of polylactide. Further, to gain insight into molecular mechanisms underneath the biodegradation process, western blot hybridization was used to show that in the presence of poly(ethylene terephthalate) (PET) in laboratory conditions the fungus produced hydrophobin proteins. The mycelium adhered to the plastic surface, which was confirmed by scanning electron microscopy, possibly due to the presence of hydrophobins. Further, using atomic force microscopy we demonstrated for the first time the formation of hydrophobin film on the surface of aliphatic polylactide (PLA) and PET by T. viride GZ1. This is the first stage of research that will be continued under environmental conditions, potentially leading to a practical application.

Biochemical Characterization of Recombinant UDPG-Dependent IAA Glucosyltransferase from Maize (Zea mays).

Here, we report a biochemical characterization of recombinant maize indole-3-acetyl-ß-d-glucose (IAGlc) synthase which glucosylates indole-3-acetic acid (IAA) and thus abolishes its auxinic activity affecting plant hormonal homeostasis. Substrate specificity analysis revealed that IAA is a preferred substrate of IAGlc synthase; however, the enzyme can also glucosylate indole-3-butyric acid and indole-3-propionic acid with the relative activity of 66% and 49.7%, respectively. KM values determined for IAA and UDP glucose are 0.8 and 0.7 mM, respectively. 2,4-Dichlorophenoxyacetic acid is a competitive inhibitor of the synthase and causes a 1.5-fold decrease in the enzyme affinity towards IAA, with the Ki value determined as 117 µM, while IAA-Asp acts as an activator of the synthase. Two sugar-phosphate compounds, ATP and glucose-1-phosphate, have a unique effect on the enzyme by acting as activators at low concentrations and showing inhibitory effect at higher concentrations (above 0.6 and 4 mM for ATP and glucose-1-phosphate, respectively). Results of molecular docking revealed that both compounds can bind to the PSPG (plant secondary product glycosyltransferase) motif of IAGlc synthase; however, there are also different potential binding sites present in the enzyme. We postulate that IAGlc synthase may contain more than one binding site for ATP and glucose-1-phosphate as reflected in its activity modulation.

Pea GH3 acyl acid amidosynthetase conjugates IAA to proteins in immature seeds of Pisum sativum L. - A new perspective on formation of high-molecular weight conjugates of auxin.

Gretchen Hagen 3 (GH3) acyl acid amidosynthetases are encoded by early auxin-responsive genes and catalyze an ATP-dependent biosynthesis of IAA-amino acid conjugates. An amide conjugate of IAA, indole-3-acetyl-aspartate (IAA-aspartate, IAA-Asp), is a predominant form of bound auxin in immature seeds of pea. However, there is some evidence that IAA is also able to form high molecular weight amide conjugates with proteins in pea and other plant species. In this short study we report that recombinant PsGH3 IAA-amino acid synthetase, which exhibits a preference for the formation of IAA-Asp, can also conjugate IAA with the protein fraction from immature seeds of pea (S-10 fraction). We studied [14C]IAA incorporation to the S-10 protein fraction by two assays: TLC method and protein precipitation by trichloroacetic acid (TCA). In both cases, radioactivity of [14C]IAA in the protein fraction increases in comparison to the control (without PsGH3), about 9.3- and 3.17-fold, respectively. l-Asp, as a preferred substrate in the IAA conjugation catalyzed by PsGH3, down-regulates [14C]IAA conjugation to the proteins as shown by the TLC assay (∼2.8-fold decrease) and the TCA precipitation variant (∼2-fold decrease). Moreover, l-Trp that competes with Asp for the catalytic site of PsGH3 and inhibits activity of the enzyme, diminished radioactivity of [14C]IAA-proteins about 1.2- and 2.8-fold, respectively. Taking into account that amino group of an amino acid or a protein acts as an acceptor of the indole-3-acetyl moiety from IAA-AMP intermediate during GH3-dependent conjugation, we masked amine groups (α- and ε-NH2) of the S-10 protein fraction from pea seeds by reductive alkylation. The alkylated proteins revealed about 3- and 2.8-fold lower radioactivity of [14C]IAA than non-alkylated fraction for TLC and TCA precipitation variant, respectively. This is a first study demonstrating that formation of high molecular weight IAA conjugates with proteins is catalyzed by a GH3 acyl acid amidosynthetase.

Crystal Structure of Isoform CBd of the Basic Phospholipase A2 Subunit of Crotoxin: Description of the Structural Framework of CB for Interaction with Protein Targets.

Crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic ß-neurotoxin that exists in individual snake venom as a mixture of isoforms of a basic phospholipase A2 (PLA2) subunit (CBa2, CBb, CBc, and CBd) and acidic subunit (CA1-4). Specific natural mutations in CB isoforms are implicated in functional differences between crotoxin isoforms. The three-dimensional structure of two individual CB isoforms (CBa2, CBc), and one isoform in a crotoxin (CA2CBb) complex, have been previously reported. This study concerns CBd, which by interaction with various protein targets exhibits many physiological or pharmacological functions. It binds with high affinity to presynaptic receptors showing neurotoxicity, but also interacts with human coagulation factor Xa (hFXa), exhibiting anticoagulant effect, and acts as a positive allosteric modulator and corrector of mutated chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), implicated in cystic fibrosis. Thus, CBd represents a novel family of agents that have potential in identifying new drug leads related to anticoagulant and anti-cystic fibrosis function. We determined here the X-ray structure of CBd and compare it with the three other natural isoforms of CB. The structural role of specific amino acid variations between CB isoforms are analyzed and the structural framework of CB for interaction with protein targets is described.

Biogenic Silver Nanoparticles: Assessment of Their Cytotoxicity, Genotoxicity and Study of Capping Proteins.

The development of nanotechnology in the last two decades has led to the use of silver nanoparticles (AgNPs) in various biomedical applications, including antimicrobial, anti-inflammatory, and anticancer therapies. However, the potential of the medical application of AgNPs depends on the safety of their use. In this work, we assessed the in vitro cytotoxicity and genotoxicity of silver nanoparticles and identified biomolecules covering AgNPs synthesized from actinobacterial strain SH11. The cytotoxicity of AgNPs against MCF-7 human breast cancer cell line and murine macrophage cell line RAW 264.7 was studied by MTT assay, cell LDH (lactate dehydrogenase) release, and the measurement of ROS (reactive oxygen species) level while genotoxicity in Salmonella typhimurium cells was testing using the Ames test. The in vitro analysis showed that the tested nanoparticles demonstrated dose-dependent cytotoxicity against RAW 264.6 macrophages and MCF-7 breast cancer cells. Moreover, biosynthesized AgNPs did not show a mutagenic effect of S. typhimurium. The analyses and identification of biomolecules present on the surface of silver nanoparticles showed that they were associated with proteins. The SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis revealed the presence of 34 and 43 kDa protein bands. The identification of proteins performed by using LC-MS/MS (liquid chromatography with tandem mass spectrometry) demonstrated their highest homology to bacterial porins. Capping biomolecules of natural origin may be involved in the synthesis process of AgNPs or may be responsible for their stabilization. Moreover, the presence of natural proteins on the surface of bionanoparticles eliminates the postproduction steps of capping which is necessary for chemical synthesis to obtain the stable nanostructures required for application in medicine.

Artificial intelligence outperforms human students in conducting neurosurgical audits.

OBJECTIVES: Neurosurgical audits are an important part of improving the safety, efficiency and quality of care but require considerable resources, time, and funding. To that end, the advent of the Artificial Intelligence-based algorithms offered a novel, more economically viable solution. The aim of the study was to evaluate whether the algorithm can indeed outperform humans in that task. PATIENTS & METHODS: Forty-six human students were invited to inspect the clinical notes of 45 medical outliers on a neurosurgical ward. The aim of the task was to produce a report containing a quantitative analysis of the scale of the problem (e.g. time to discharge) and a qualitative list of suggestions on how to improve the patient flow, quality of care, and healthcare costs. The Artificial Intelligence-based Frideswide algorithm (FwA) was used to analyse the same dataset. RESULTS: The FwA produced 44 recommendations whilst human students reported an average of 3.89. The mean time to deliver the final report was 5.80 s for the FwA and 10.21 days for humans. The mean relative error for factual inaccuracy for humans was 14.75 % for total waiting times and 81.06 % for times between investigations. The report produced by the FwA was entirely factually correct. 13 out of 46 students submitted an unfinished audit, 3 out of 46 made an overdue submission. Thematic analysis revealed numerous internal contradictions of the recommendations given by human students. CONCLUSION: The AI-based algorithm can produce significantly more recommendations in shorter time. The audits conducted by the AI are more factually accurate (0 % error rate) and logically consistent (no thematic contradictions). This study shows that the algorithm can produce reliable neurosurgical audits for a fraction of the resources required to conduct it by human means.

Biosynthesis pathway of indole-3-acetyl-myo-inositol during development of maize (Zea mays L.) seeds.

Indole-3-acetic acid (IAA) conjugation is one of the mechanisms responsible for auxin homeostasis. IAA ester conjugates biosynthesis has been studied during development of maize seeds where IAA-inositol (IAInos) and its glycosidic forms make up about 50 % of its ester conjugates pool. 1-O-indole-3-acetyl-ß-d-glucose (IAGlc) synthase and indole-3-acetyl transferase (IAInos synthase) are key enzymes in a two-step pathway of IAInos synthesis. In the first reaction, IAA is glucosylated to a high energy acetal, 1-O-indole-3-acetyl-ß-d-glucose by IAGlc synthase, whereas in the second step, IAInos synthase transfers IAA moiety to myo-inositol forming a stable auxin ester, indole-3-acetyl-myo-inositol (IAInos). It should be mentioned that IAGlc synthase catalyzes a reversible reaction with unfavourable equilibrium that delivers IAGlc for favourable transacylation to IAInos. This is the first study where IAGlc synthase and IAInos synthase are simultaneously analyzed by enzymatic activity assay and quantitative RT-PCR in maize seeds at four stages of development (13, 26, 39 and 52 Days After Flowering). Activity of IAGlc/IAInos synthases as well as their expression profiles during seed development were different. While both enzymatic activities and ZmIAIn expression were the highest in seeds at 26 DAF, the highest expression of ZmIAGlc was observed at 13 DAF. Protein gel blot analysis showed that IAInos synthase exists as a mixture of several isoforms at a similar protein level at particular stages of seed development. Neither of other ester conjugates of IAA (IAA-mannose) nor IAA-amino acids were detected at the stages studied. Catalytic activity of l-tryptophan aminotransferase involved in IAA biosynthesis as well as UDPG pyrophosphorylase, synthesizing UDPG as a substrate for IAGlc synthase, were also analyzed. l-tryptophan aminotransferase activity was the highest at 26 DAF. Changes in enzyme activity of UDPG pyrophosphorylase are difficult to interpret. Expression levels of ZmIPS and ZmIPP encoding two enzymes of myo-inositol biosynthesis pathway: inositol-x-phosphate synthase (IPS) and inositol-x-phosphate phosphatase (IPP), respectively, were analyzed. 26 DAF seeds displayed the highest expression level of ZmIPS, whereas transcription of ZmIPP was the highest at 13 DAF.

Spatio-temporal IAA gradient is determined by interactions with ET and governs flower abscission.

The abscission zone (AZ) is a specialized tissue that usually develops at the base of an organ and is highly sensitive to phytohormones, e.g., abscisic acid (ABA), ethylene (ET), and gibberellins (GAs). A current model of organ abscission assumes that the formation of an auxin gradient around the AZ area determines the time of shedding; however, that thesis is supported by studies that are primarily concerned with auxin transporters. To better understand the events underlying the progression of abscission, we focused for the first time on indole-3-acetic acid (IAA) distribution following AZ activation. We performed a series of immunolocalization studies in proximal and distal regions of floral AZ cells in yellow lupine, which is an agriculturally important legume. The examined phytohormone was abundant in natural active AZ cells, as well as above and below parts of this structure. A similar gradient of IAA was observed during the early steps of abscission, which was induced artificially by flower removal. Surprisingly, IAA was not detected in inactive AZ cells. This paper is also a consequence of our comprehensive studies concerning the phytohormonal regulation of flower abscission in yellow lupine. We present new data on interactions between IAA and ET, previously pointed out as a strong modulator of flower separation. The detailed analysis shows that disruption of the natural auxin gradient around the AZ area through the application of synthetic IAA had a positive effect on ET biosynthesis genes. We proved that these changes are accompanied by an accumulation of the ET precursor. On the other hand, exposure to ET significantly affected IAA localization in the whole AZ area in a time-dependent manner. Our results provide insight into the existence of a spatio-temporal sequential pattern of the IAA gradient related to the abscission process; this pattern is maintained by interactions with ET. We present new valuable evidence for the existence of conservative mechanisms that regulate generative organ separation and can help to improve the yield of agronomically significant species in the future.

Structure of the alimentary tract in the Atlantic mudskipper Periophthalmus barbarus (Gobiidae: Oxudercinae): anatomical, histological and ultrastructural studies.

The alimentary tract of oxudercine gobies is characterized by a lack of an anatomically distinct stomach, owing to which they are classified as stomachless. Since the environment, food requirements, and feeding habits have a significant impact on the anatomy of the alimentary tract of fish, it was assumed that predominantly carnivorous, semi-terrestrial mudskippers would have a stomach. In order to verify this hypothesis, anatomical, histological, histochemical and ultrastructural analysis of the alimentary tract of the Atlantic mudskipper Periophthalmus barbarus was performed. The results revealed that despite a lack of clear anatomical distinction within the alimentary tract, there were four well-distinguished sections visible at the histological level: oesophagus, stomach, intestine, and rectum. The division was enhanced by the presence of a pyloric sphincter and an ileorectal valve. The stomach contained tubular glands composed of oxynticopeptic cells. Gland cells had pepsinogen granules and a well-developed tubulovesicular network of smooth membranes, which indicates the secretion of gastric juice. The presence of neutral mucus in the apical region of surface epithelial cells as protection against hydrochloric acid as well as the presence of active pepsin also confirm gastric function. However, low pepsin activity seems to implies low protein digestion. The results of this study indicate that the Atlantic mudskipper P. barbarus has a functional stomach.

Cadmium effects on embryo growth of pea seeds during germination: Investigation of the mechanisms of interference of the heavy metal with protein mobilization-related factors.

This work aims to give more insight into mechanisms of action of cadmium (Cd) on germinating pea seeds (Pisum sativum L. var. douce province), specifically the different ways by which Cd cations may interfere with the principal factors involved during germination process, notably storage proteins mobilization, amino acids freeing and proteolytic activities. Obtained results revealed that the process of hydrolysis of main storage proteins showed a significant disruption, which resulted in the decrease of the release of free amino acids, thus imposing a lack in nitrogen supply of essential nutrients to growing embryo under Cd stress. This hypothesis was evidenced by Cd-induced changes occurring in main purified protein fractions; Albumins, Legumins and Vicilins, during their breakdown. Besides, at enzymatic level, the activities of main proteases responsible for this hydrolysis were altered. Indeed, assays using synthetic substrates and specific protease inhibitors followed by protease activity measurements demonstrated that Cd inhibited drastically the total azocaseinolytic activity (ACA) and activities of different proteolytic classes: cysteine-, aspartic-, serine- and metallo-endopeptidases (EP), leucine- and proline-aminopeptidases (LAP and PAP, respectively), and glycine-carboxypeptidases (Gly-CP). The data here presented may suggest that the vulnerability of the embryonic axes towards Cd toxicity could be explained as a result of eventual disruption of metabolic pathways that affect mobilization of reserves and availability of nutrients. In vitro studies suggest that Cd cations may act either directly on the catalytic sites of the proteolytic enzymes, which may cause their deactivation, or indirectly via the generation of oxidative stress and overproduction of free radicals that can interact with enzymes, by altering their activity and structure.

A serine carboxypeptidase-like acyltransferase catalyzes synthesis of indole-3-acetic (IAA) ester conjugate in rice (Oryza sativa).

Indole-3-acetic acid (IAA) conjugation is one of mechanisms responsible for regulation of free auxin levels in plants. A new member of the serine carboxypeptidase-like (SCPL) acyltransferases family from Oryza sativa has been cloned and characterized. 1-O-indole-3-acetyl-ß-D-glucose (1-O-IAGlc): myo-inositol acyltransferase (IAInos synthase) is an enzyme of IAA ester conjugates biosynthesis pathway that catalyzes transfer of IAA moiety from 1-O-IAGlc to myo-inositol forming IA-myo-inositol (IAInos). The OsIAA-At cDNA has been cloned and expressed using yeast and bacterial expression systems. Proteins produced in Saccharomyces cerevisiae and Escherichia coli contained 483 and 517 amino acids, respectively. The enzyme functionally expressed in both expression systems exhibits 1-O-IAGlc-dependent acyltransferase activity. Analysis of amino acid sequence confirmed that rice IAInos synthase belongs to the SCPL protein family. Recombinant IAInos synthases produced in yeast and bacterial expression systems have been partially characterized and their properties have been compared to those of the native enzyme obtained from 6-days-old rice seedlings by biochemical approach. The oligosaccharide component of the protein enzyme is not necessary for its catalytic activity. The native enzyme showed the lowest specific activity of 5.01 nmol min-1 mg-1 protein, whereas the recombinant enzymes produced in yeast and bacteria showed specific activity of 18.75 nmol min-1 mg-1 protein and 18.09 nmol min-1 mg-1 protein, respectively. The KM values for myo-inositol were similar for all three forms of the enzyme: 1.38, 0.83, 1.0 mM for native, bacterial and yeast protein, respectively. Both recombinant forms of IAInos synthase and the native enzyme also have the same optimal pH of 7.4 and all of them are inhibited by phenylmethylsulfonyl fluoride (PMSF), specific inhibitor of serine carboxypeptidases.

[Enzymatic and non-enzymatic functions of secreted phospholipases A2]. / Enzymatyczne i nieenzymatyczne funkcje sekrecyjnych fosfolipaz A2.

Phospholipases catalyze enzymatic degradation of membrane lipids. The phospholipases are divided into four major groups: A1, A2, C, and D by the type of ester bond which is hydrolyzed. Phospholipases A2 (PLA2) hydrolyze membrane glycerophospholipids at sn-2 position releasing lysophospholipid and free fatty acid. The PLA2s are involved in biosynthesis of intracellular messengers (eicosanoids), endo- and exocytosis, and cytoskeleton reorganization. Moreover, secreted PLA2 play various functions which are not dependent on their enzymatic activity. An intriguing question is specific interaction of sPLA2 from snake venom with several protein acceptors (human coagulation factor Xa or CFTR). In this review, we describe classification of PLA, mechanism of catalytic action, as well as interactions of snake venom PLA2 with various human proteins.

Abiotic stress and phytohormones affect enzymic activity of 1-O-(indole-3-acetyl)-ß-d-glucose: myo-inositol indoleacetyl transferase from rice (Oryza sativa).

Indole-3-acetic acid (IAA) conjugation is a part of mechanism regulating free auxin concentration. 1-O-(indole-3-acetyl)-ß-d-glucose: myo-inositol indoleacetyl transferase (IAInos synthase) is an enzyme involved in IAA-ester conjugates biosynthesis. Biotic and abiotic stress conditions can modulate auxin conjugates formation in plants. In this study, we investigated effect of plant hormones (IAA, ABA, SA and 2,4-D) and abiotic stress (drought and salt stress: 150mM NaCl and 300mM NaCl) on expression level and catalytic activity of rice IAInos synthase. Enzymic activity assay indicated that all tested phytohormones affected activity of IAInos synthase, but only ABA had inhibiting effect, while IAA, SA and 2,4-D activated the enzyme. Drought and salt stress induced with lower NaCl concentration resulted in decreased activity of IAInos synthase, but 300mM NaCl had no effect on the enzyme. Despite observed differences in enzymic activities, no changes of expression level, tested by semiquantitative RT-PCR and Western blot, were detected. Based on our results it has been supposed that plant hormones and stress conditions affect IAInos synthase activity on posttranslational level.

Rattlesnake Phospholipase A2 Increases CFTR-Chloride Channel Current and Corrects ∆F508CFTR Dysfunction: Impact in Cystic Fibrosis.

Deletion of Phe508 in the nucleotide binding domain (∆F508-NBD1) of the cystic fibrosis transmembrane regulator (CFTR; a cyclic AMP-regulated chloride channel) is the most frequent mutation associated with cystic fibrosis. This mutation affects the maturation and gating of CFTR protein. The search for new high-affinity ligands of CFTR acting as dual modulators (correctors/activators) presents a major challenge in the pharmacology of cystic fibrosis. Snake venoms are a rich source of natural multifunctional proteins, potential binders of ion channels. In this study, we identified the CB subunit of crotoxin from Crotalus durissus terrificus as a new ligand and allosteric modulator of CFTR. We showed that CB interacts with NBD1 of both wild type and ∆F508CFTR and increases their chloride channel currents. The potentiating effect of CB on CFTR activity was demonstrated using electrophysiological techniques in Xenopus laevis oocytes, in CFTR-HeLa cells, and ex vivo in mouse colon tissue. The correcting effect of CB was shown by functional rescue of CFTR activity after 24-h ΔF508CFTR treatments with CB. Moreover, the presence of fully glycosylated CFTR was observed. Molecular docking allowed us to propose a model of the complex involving of the ABCß and F1-like ATP-binding subdomains of ΔF508-NBD1. Hydrogen-deuterium exchange analysis confirmed stabilization in these regions, also showing allosteric stabilization in two other distal regions. Surface plasmon resonance competition studies showed that CB disrupts the ∆F508CFTR-cytokeratin 8 complex, allowing for the escape of ∆F508CFTR from degradation. Therefore CB, as a dual modulator of ΔF508CFTR, constitutes a template for the development of new anti-CF agents.