Identification of novel phospholipase A2 group IX members in metazoans

Sequence homologues of the bacterium Streptomyces violaceoruber and sea anemone Nematostella vectensis PLA₂ pfam09056 members were identified in several bacteria, fungi and metazoans illustrating the evolution of this PLA₂ sub-family. Comparison of their molecular structures revealed that bacteria and fungi members are part of the GXIV of PLA₂s while metazoan representatives are similar with GIX PLA₂ of the marine snail Conus magus. Members of GXIV and GIX PLA₂s show modest overall sequence similarity (21–35%) but considerable motif conservation within the putative Ca²⁺-binding, catalytic sites and cysteine residue positions which are essential for enzyme function. GXIV PLA₂s of bacteria and fungi typically contain four cysteine residues composing two intramolecular disulphide bonds. GIX PLA₂ homologues were identified in cnidarians and molluscs and in a single tunicate but appear to be absent from other metazoan genomes. The mature GIX PLA₂ deduced peptides contain up to ten cysteine residues capable of forming five putative disulphide bonds. Three disulphide bonds were identified in GIX PLA₂s, two of which correspond to those localized in GXIV PLA₂s. Phylogenetic analysis demonstrates that metazoan GIX PLA₂s cluster separate from the bacterial and fungal GXIV PLA₂s and both pfam09056 members form a group separate from the prokaryote and eukaryote GXIIA PLA₂ pfam06951. Duplicate PLA₂ pfam09056 genes were identified in the genomes of sea anemone N. vectensis and oyster Crassostrea gigas suggest that members of this family evolved via species-specific duplication events. These observations indicate that the newly identified metazoan pfam09056 members may be classified as GIX PLA₂s and support the idea of the common evolutionary origin of GXIV and GIX PLA₂ pfam09056 members, which emerged early in bacteria and were maintained in the genomes of fungi and selected extant metazoan taxa.     

Novel mammalian group XII secreted phospholipase A2 lacking enzymatic activity

An increasing number of mammalian secreted phospholipases A(2) (sPLA(2)s) has been identified over the past few years. Here, we report the identification and recombinant expression of a novel sPLA(2)-like protein in mouse and human species that has been called group XIIB (GXIIB). The mature protein has a molecular mass of 19.7 kDa and structural features similar to those of the previously identified GXII sPLA(2), now called GXIIA. Strikingly, the GXIIB sPLA(2) has a mutation in the active site, replacing the canonical histidine by a leucine, suggesting that this sPLA(2) is catalytically inactive. Recombinant expression of human (hGXIIB) and mouse (mGXIIB) sPLA(2)s in Escherichia coli indicates that GXIIB sPLA(2)s display no measurable lipolytic activity on various types of phospholipid substrates. Furthermore, these sPLA(2)-like proteins display relatively weak affinity to phospholipid vesicles. Binding experiments indicate that these proteins are also unable to bind to the well-known M-type sPLA(2) receptor. The RNA tissue distribution of GXIIB sPLA(2)s is distinct from that of other sPLA(2)s including the homologous GXIIA. Strong expression was observed in liver, small intestine, and kidney in both human and mouse species. Interestingly, the expression of the novel sPLA(2) is dramatically decreased in human tumors from the same tissues. The absence of enzymatic activity suggests that the GXIIB sPLA(2)-like proteins probably exert their biological roles by acting as ligands for as yet unidentified receptors.     

Biochemical and enzymatic characterization of two basic Asp49 phospholipase A2 isoforms from Lachesis muta muta (Surucucu) venom

Two basic phospholipase A₂ (PLA₂) isoforms were isolated from Lachesis muta muta snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-18 μ-Bondapack column and RP-HPLC on a C-8 column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of the two isoforms LmTX-I and LmTX-II was respectively measured as 14,245.4 and 14,186.2 Da. The pI was respectively estimated to be 8.7 and 8.6 for LmTX-I and LmTX-II, as determined by two-dimensional electrophoresis. The two proteins were sequenced and differentiated from each other by a single amino acid substitution, Arg₆₅ (LmTX-I) → Pro₆₅ (LmTX-II). The amino acid sequence showed a high degree of homology between PLA₂ isoforms from Lachesis muta muta and other PLA₂ snake venoms. LmTX-I and LmTX-II had PLA₂ activity in the presence of a synthetic substrate and showed a minimum sigmoidal behaviour; with maximal activity at pH 8.0 and 35–45 °C. Full PLA₂ activity required Ca²⁺ and was respectively inhibited by Cu²⁺ and Zn²⁺ in the presence and absence of Ca²⁺. Crotapotin from Crotalus durissus cascavella rattlesnake venom significantly inhibited (P < 0.05) the enzymatic activity of LmTX-I, suggesting that the binding site for crotapotin in this PLA₂ was similar to another in the basic PLA₂ of the crotoxin complex from C. durissus cascavella venom.     

Biochemical characterization and kinetic properties of alanine aminotransferase homologues partially purified from wheat (Triticum aestivum L.)

Four homologues of alanine aminotransferase have been isolated from shoots of wheat seedlings and purified by saline precipitation, gel filtration, preparative electrophoresis and anion exchange chromatography on Protein-Pak Q 8HR column attached to HPLC. Alanine aminotransferase 1 (AlaAT1) and 2 (AlaAT2) were purified 303- and 452-fold, respectively, whereas l-glutamate: glyoxylate aminotransferase 1 (GGAT1) and 2 (GGAT2) were purified 485- and 440-fold, respectively. Consistent inhibition of AlaAT (EC 2.6.1.2) and GGAT (EC 2.6.1.4) activities by p-hydroxymercuribenzoate points on participation of cysteine residues in the enzyme activity. The molecular weight of AlaAT1 and AlaAT2 was estimated to be 65 kDa and both of them are monomers in native state. Nonsignificant differences between K_m using alanine as substrate and catalytic efficiency (k_cat/K_m) for l-alanine in reaction with 2-oxoglutarate indicate comparable kinetic constants for AlaAT1 and AlaAT2. Similar kinetic constants for l-alanine in reaction with 2-oxoglutarate and for l-glutamate in reaction with pyruvate for all four homologues suggest equally efficient reaction in both forward and reverse directions. GGAT1 and GGAT2 were able to catalyze transamination between l-glutamate and glyoxylate, l-alanine and glyoxylate and reverse reactions between glycine and 2-oxoglutarate or pyruvate. Both GGATs also consisted of a single subunit with molecular weight of about 50 kDa. The estimated K_m for GGAT1 (3.22 M) and GGAT2 (1.27 M) using l-glutamate as substrate was lower in transamination with glyoxylate than with pyruvate (9.52 and 9.09 mM, respectively). Moreover, distinctively higher values of catalytic efficiency for l-glutamate in reaction with glyoxylate than for l-glutamate in reaction with pyruvate confirm involvement of these homologues into photorespiratory metabolism.     

Corrélations entre l'évolution morphologique des membres inférieurs et la récupération fonctionnelle de sujets hémiplégiques

ObjectiveThe aim is to quantify the help provided by recent morphological indicators for hemiplegics patients, after a cerebral vascular accident, during functional rehabilitation. The correlations between functional recovery and the morphological evolution of the muscles and the segments of the lower limb are studied using a MRI system.Material and methodTen adult subjects suffering from vascular hemiplegia have been studied at the ELAN rehabilitation center in Wattrelos. Bobath rehabilitation technique was used to treat patients. Muscular and external volumes of the lower limbs segments, obtained through medical imagery, as well as functional tests (Functional Independence Measure (FIM), Bourges's score and indices of motricity of the pathological limb), were assessed when patients were admitted in and as they left from the rehabilitation centre. Spearman's rho coefficient was used in this study.ResultsThis non-parametric test was considered significant when P < 0.05. It demonstrated a relationship between the evolution of thigh's external and muscular volumes and changes in Bourges's score in seated position (r_s = 0.730 and r_s = 0.764, respectively). It also showed a relationship between pathologic knee motricity index and pathologic leg segment volume evolution (r_s = 0.675).Discussion–conclusionStatistical analysis demonstrated that pathologic thigh volume changes is beneficial to balance during seating as no volume evolution seems to influence balance during standing.     

Inhibition of phospholipase A2 increases Tau phosphorylation at Ser214 in embryonic rat hippocampal neurons

BackgroundArachidonic acid is released from cellular membranes by the action of phospholipase A₂ (PLA₂) and is implicated in microtubule-associated protein Tau phosphorylation. Tau hyperphosphorylation affects its ability to stabilize microtubules.ObjectiveTo determine the effect of PLA₂ inhibition on the phosphorylation state of Tau phosphoepitopes in primary cultures of hippocampal neurons.Methods4 DIC neurons were incubated at different concentrations of methyl-arachidonylfluorophosphonate (MAFP), an irreversible inhibitor of cPLA₂ and iPLA₂. Changes on Tau phosphorylation were determined by Western blotting with a panel of anti-Tau antibodies (C-terminal, Ser199/202, Ser202/205, Ser396 and Ser214).ResultsThe Ser214 site was hyperphosphorylated upon MAFP treatment. Significant differences were observed with 10 μM (p=0.01), 50 μM (p=0.01) and 100 μM (p=0.05) of MAFP. Less-intense changes were found in other phosphoepitopes.ConclusionThe present findings indicate that the phosphorylation of Ser214 is regulated by c- and/or iPLA₂, whereas other phosphoepitopes primarily regulated by GKS3b were not affected.     

Cloning,expression, and characterization of a thermostable β-xylosidase from thermoacidophilic Alicyclobacillus sp. A4

A β-xylosidase gene (xylA4) was identified in the genome sequence of thermoacidophilic Alicyclobacillus sp. A4. The deduced amino acid sequence was highly homologous with the β-xylosidases of family 52 of the glycoside hydrolases (GH). The full-length gene consisted of 2097 bp and encoded 698 amino acids without a signal peptide. The gene product was successfully expressed in Escherichia coli with an activity of 564.9 U/mL. Recombinant XylA4 was purified by Ni²⁺-NTA affinity chromatography with a molecular mass of 78.5 kDa. The enzyme showed optimal activity at pH 6.0 and 65 °C, and remained stable over the pH range of 5.0–9.0. The thermostability of XylA4 is noteworthy, retaining almost all of the activity after 1 h incubation at 65 °C. Using p-nitrophenyl-β-d-xylopyranoside (pNPX) as the substrate, XylA4 had the highest specific activity (261.1 U/mg) and catalytic efficiency (601.5/mM/s) known so far for GH52 xylosidases. The enzyme displayed high tolerance to xylose, with a K_i value of approximately 88.7 mM. It also had synergy with xylanase XynBE18 from Paenibacillus sp. E18 in xylan degradation, releasing more xylose (up to 1.43 folds) than XynBE18 alone. Therefore, this thermostable xylose-tolerant β-xylosidase may have a great application potential in many industrial fields.     

Absence of an effect of vitamin E on protein and lipid radical formation during lipoperoxidation of LDL by lipoxygenase

Low-density lipoprotein (LDL) oxidation is the primary event in atherosclerosis, and LDL lipoperoxidation leads to modifications in apolipoprotein B-100 (apo B-100) and lipids. Intermediate species of lipoperoxidation are known to be able to generate amino acid-centered radicals. Thus, we hypothesized that lipoperoxidation intermediates induce protein-derived free radical formation during LDL oxidation. Using DMPO and immuno-spin trapping, we detected the formation of protein free radicals on LDL incubated with Cu²⁺ or the soybean lipoxidase (LPOx)/phospholipase A₂ (PLA₂). With low concentrations of DMPO (1 mM), Cu²⁺ dose-dependently induced oxidation of LDL and easily detected apo B-100 radicals. Protein radical formation in LDL incubated with Cu²⁺ showed maximum yields after 30 min. In contrast, the yields of apo B-100 radicals formed by LPOx/PLA₂ followed a typical enzyme-catalyzed kinetics that was unaffected by DMPO concentrations of up to 50 mM. Furthermore, when we analyzed the effect of antioxidants on protein radical formation during LDL oxidation, we found that ascorbate, urate, and Trolox dose-dependently reduced apo B-100 free radical formation in LDL exposed to Cu²⁺. In contrast, Trolox was the only antioxidant that even partially protected LDL from LPOx/PLA₂. We also examined the kinetics of lipid radical formation and protein radical formation induced by Cu²⁺ or LPOx/PLA₂ for LDL supplemented with α-tocopherol. In contrast to the potent antioxidant effect of α-tocopherol on the delay of LDL oxidation induced by Cu²⁺, when we used the oxidizing system LPOx/PLA₂, no significant protection was detected. The lack of protection of α-tocopherol on the apo B-100 and lipid free radical formation by LPOx may explain the failure of vitamin E as a cardiovascular protective agent for humans.     

Sulfonamide inhibition profile of the γ-carbonic anhydrase identified in the genome of the pathogenic bacterium Burkholderia pseudomallei the etiological agent responsible of melioidosis

A new γ-carbonic anhydrase (CA, EC 4.1.1.1) was cloned and characterized kinetically in the genome of the bacterial pathogen Burkholderia pseudomallei, the etiological agent of melioidosis, an endemic disease of tropical and sub-tropical regions of the world. The catalytic activity of this new enzyme, BpsCAγ, is significant with a k_cat of 5.3 × 10⁵ s^?1 and k_cat/K_m of 2.5 × 10⁷ M^?1 × s^?1 for the physiologic CO₂ hydration reaction. The inhibition constant value for this enzyme for 39 sulfonamide inhibitors was obtained. Acetazolamide, benzolamide and metanilamide were the most effective (K_Is of 149–653 nM) inhibitors of BpsCAγ activity, whereas other sulfonamides/sulfamates such as ethoxzolamide, topiramate, sulpiride, indisulam, sulthiame and saccharin were active in the micromolar range (K_Is of 1.27–9.56 μM). As Burkholderia pseudomallei is resistant to many classical antibiotics, identifying compounds that interfere with crucial enzymes in the B. pseudomallei life cycle may lead to antibiotics with novel mechanisms of action.     

A neuromedin-pyrokinin-like neuropeptide signaling system in Caenorhabditis elegans

Neuromedin U (NMU) in vertebrates is a structurally highly conserved neuropeptide of which highest levels are found in the pituitary and gastrointestinal tract. In Drosophila, two neuropeptide genes encoding pyrokinins (PKs), capability (capa) and hugin, are possible insect homologs of vertebrate NMU. Here, the ligand for an orphan G protein-coupled receptor in the nematode Caenorhabditis elegans (Ce-PK-R) was found using a bioinformatics approach. After cloning and expressing Ce-PK-R in HEK293T cells, we found that it was activated by a neuropeptide from the C. elegans NLP-44 precursor (EC₅₀ = 18 nM). This neuropeptide precursor is reminiscent of insect CAPA precursors since it encodes a PK-like peptide and two periviscerokinin-like peptides (PVKs). Analogous to CAPA peptides in insects and NMUs in vertebrates, whole mount immunostaining in C. elegans revealed that the CAPA precursor is expressed in the nervous system. The present data also suggest that the ancestral CAPA precursor was already present in the common ancestor of Protostomians and Deuterostomians and that it might have been duplicated into CAPA and HUGIN in insects. In vertebrates, NMU is the putative homolog of a protostomian CAPA-PK.     

Novel acidic phospholipase A2 from Porthidium hyoprora causes inflammation with mast cell rich infiltrate

Phospholipases A₂ (PLA₂) are a group of enzymes that hydrolyze phospholipids at the sn-2 position, being present in all nature. In venomous animals, these proteins assume a special role, being able to exert diverse pharmacological effects. In this work, authors identified a new isoform of PLA₂ in the venom of Porthidium hyoprora, which was isolated through sequential chromatographic steps and named PhTX-III. The enzyme was characterized biochemically and structurally. Structural studies using mass spectrometry confirmed an acidic secretory PLA₂, family IIA, with molecular mass of 13,620.9 Da and identification of 86% of its primary sequence. PhTX-III did not exhibit myotoxic, anticoagulant or antibacterial effects, often present in this class of enzymes. Although, it was capable of initiate inflammatory response, with local edema and release of cytokines IL-1α, IL-6 and TNF-α, probably due to mast cell degranulation.     

Identification and biochemical analysis of a novel pectate lyase 3 gene in Bursaphelenchus xylophilus

A novel pectate lyase 3 (Bx-PEL3) gene from the stage-specifically expressed sequence tag library of Bursaphelenchus xylophilus was cloned. Bx-PEL1 and Bx-PEL2 were relatively predominant to Bx-PEL3 in quantitative real-time PCR analysis. The variation of expression level among Bx-PELs according to different life stages suggests that each Bx-PEL may play different biochemical roles in pathogenesis. Recombinant Bx-PEL3 showed activity against polygalacturonic acid and its optimized physical conditions for pH and Ca^2 + concentrations were 9.0 and 0.5 mM, respectively. Homology modeling revealed that Bx-PELs were structurally similar to PEL from the Bacillus strain KSM P-15 and shared essential residues for calcium binding and catalytic activity. Bx-PEL3 was expressed near the esophageal gland as shown in other Bx-PELs, indicating that Bx-PEL3 may be involved in the degradation of the cell wall after secretion from the stylet. Our data suggest that the novel PEL3 gene is a putative pathogenic factor which is biochemically functional.     

Complete mitochondrial genome of Coelomactra antiquata (Mollusca: Bivalvia): The first representative from the family Mactridae with novel gene order and unusual tandem repeats

The complete mitochondrial genome plays an important role in the accurate inference of phylogenetic relationships among metazoans. Mactridae, also known as trough shells or duck clams, is an important family of marine bivalve clams in the order Veneroida. Here we present the complete mitochondrial genome sequence of the Xishishe Coelomactra antiquata (Mollusca: Bivalvia), which is the first representative from the family Mactridae. The mitochondrial genome of C. antiquata is of 17,384 bp in length, and encodes 35 genes, including 12 protein-coding, 21 transfer RNA, and 2 ribosomal RNA genes. Compared with the typical gene content of animal mitochondrial genomes, atp8 and tRNAS₂ are missing. Gene order of the mitochondrial genome of C. antiquata is unique compared with others from Veneroida. In the mitochondrial genome of the C. antiquata, a total of 2189 bp of non-coding nucleotides are scattered among 26 non-coding regions. The largest non-coding region contains one section of tandem repeats (99 bp × 11), which is the second largest tandem repeats found in the mitochondrial genomes from Veneroida. The phylogenetic trees based on mitochondrial genomes support the monophyly of Veneridae and Lucinidae, and the relationship at the family level: ((Veneridae + Mactridae) + (Cardiidae + Solecurtidae)) + Lucinidae. The phylogenetic result is consistent with the morphological classification. Meanwhile, bootstrap values are very high (BP = 94–100), suggesting that the evolutionary relationship based on mitochondrial genomes is very reliable.     

Characterization and directed evolution of BliGO,a novel glycine oxidase from Bacillus licheniformis

Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtilis. The optimal activity of BliGO was observed at pH 8.5 and 40 °C. Interestingly, BliGO retained 60% of the maximum activity at 0 °C, suggesting it is a cold-adapted enzyme. The kinetic parameters on glyphosate (K_m, k_cat and k_cat/K_m) of BliGO were 11.22 mM, 0.08 s⁻¹, and 0.01 mM⁻¹ s⁻¹, respectively. To improve the catalytic activity to glyphosate, the BliGO was engineered by directed evolution. With error-prone PCR and two rounds of DNA shuffling, the most evolved mutant SCF-4 was obtained from 45,000 colonies, which showed 7.1- and 8-fold increase of affinity (1.58 mM) and catalytic efficiency (0.08 mM⁻¹ s⁻¹) to glyphosate, respectively. In contrast, its activity to glycine (the natural substrate of GO) decreased by 113-fold. Structure modeling and site-directed mutation study indicated that Ser51 in SCF-4 involved in the binding of enzyme with glyphosate and played a crucial role in the improvement of catalytic efficiency.     

Purification and characterization of a novel thermostable α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp.

The purification and characterization of an extracellular α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp. was investigated in this report. The α-l-AFase was purified to homogeneity with a purification fold of 1030. The purified α-l-AFase had a specific activity of 20.6 U mg^?1. The molecular mass of the enzyme was estimated to be 52.9 kDa and 51.6 kDa by SDS–PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.0 and 70 °C, respectively. The enzyme was stable over a broad pH range of 4.0–10.0 and also exhibited excellent thermostability, i.e., the residual activities reached 75% after treatment at 60 °C for 1 h. The enzyme showed strict substrate specificity for the α-l-arabinofuranosyl linkage. The K_m and V_max values for p-nitrophenyl (pNP)-α-l-arabinofuranoside were calculated to be 1.43 mM and 68.3 μmol min^?1 mg^?1 protein, respectively. Furthermore, the gene encoding α-l-AFase was cloned and sequenced and found to contain a catalytic domain belonging to the glycoside hydrolase (GH) family 43 α-l-AFase. The deduced amino acid sequence of the gene showed the highest identity (67%) to the putative α-l-AFase from Neurospora crassa. This is the first report on the purification, characterization and gene sequence of an α-l-AFase from Chaetomium sp.     

Sulfanylphthalonitrile analogues as selective and potent inhibitors of monoamine oxidase B

It has recently been reported that nitrile containing compounds frequently act as potent monoamine oxidase B (MAO-B) inhibitors. Modelling studies suggest that this high potency inhibition may rely, at least in part, on polar interactions between nitrile functional groups and polar moieties within the MAO-B substrate cavity. In an attempt to identify potent and selective inhibitors of MAO-B and to contribute to the known structure–activity relationships of MAO inhibition by nitrile containing compounds, the present study examined the MAO inhibitory properties of series of novel sulfanylphthalonitriles and sulfanylbenzonitriles. The results document that the evaluated compounds are potent and selective MAO-B inhibitors with most homologues possessing IC₅₀ values in the nanomolar range. In general, the sulfanylphthalonitriles exhibited higher binding affinities for MAO-B than the corresponding sulfanylbenzonitrile homologues. Among the compounds evaluated, 4-[(4-bromobenzyl)sulfanyl]phthalonitrile is a particularly promising inhibitor since it displayed a high degree of selectivity (8720-fold) for MAO-B over MAO-A, and potent MAO-B inhibition (IC₅₀ = 0.025 μM). Based on these observations, this structure may serve as a lead for the development of therapies for neurodegenerative disorders such as Parkinson’s disease.     

Differential mode of attack on membrane phospholipids by an acidic phospholipase A2 (RVVA-PLA2-I) from Daboia russelli venom

An acidic phospholipase A₂ (RVVA-PLA₂-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA₂-I was non‐lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA₂ enzyme. RVVA-PLA₂-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA₂-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24 h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA₂-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA₂s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA₂-I suggested the correlation between catalytic and membrane damaging activities of this PLA₂ enzyme. Our study advocates that the presence of a large number of PLA₂-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA₂ enzyme.     

Which bovine endometrial cells are the source of and target for lysophosphatidic acid?

The objective of the study was to examine which cultured endometrial cells are the source and which are the target for lysophosphatidic acid (LPA) in the bovine uterus. LPA concentration as well as mRNA and protein expressions of the enzymes responsible for LPA synthesis (phospholipase A2: PLA₂, autotaxin: AX) were greater in epithelial than in stromal cells (P < 0.05). In turn, mRNA and protein expression of LPA receptor (LPAR1) was lower in epithelial than in stromal cells (P < 0.05). We suggest that LPA in bovine endometrium is produced mainly by epithelial cells and affects mostly stromal cells acting via LPAR1.     

Improving the catalytic performance of porcine pancreatic lipase in the presence of [MMIm][MeSO4] with the modification of functional ionic liquids

Porcine pancreatic lipase (PPL) was chemically modified with various functional ionic liquids (ILs) to increase its catalytic performance in water-miscible IL. Catalytic activity and thermostability were tested with a p-nitrophenyl palmitate (pNPP) hydrolysis reaction. The native enzyme lost 18% of its initial activity in 0.4 M [MMIm][MeSO₄], whereas the activities of all the modified enzymes increased. The [HOOCBMIm][Cl] modification led to a 2-fold increase in activity in 0.3 M [MMIm][MeSO₄] than in aqueous. All the modified enzymes exhibited higher thermostability compared with the native enzyme at high temperature. In particular, the [HOOCBMIm][Cl] modification led to a 6-fold increase in thermostability at 60 °C. Conformational changes were confirmed by fluorescence spectroscopy and circular dichroism spectroscopy to elucidate the mechanism of catalytic performance alteration.     

Anion inhibition profiles of α-, β- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae

Among the numerous metalloenzymes known to date, carbonic anhydrase (CA, EC 4.2.1.1) was the first zinc containing one, being discovered decades ago. CA is a hydro-lyase, which catalyzes the following hydration–dehydration reaction: CO₂ + H₂O ⇋ HCO₃⁻ + H⁺. Several CA classes are presently known, including the α-, β-, γ-, δ-, ζ- and η-CAs. In prokaryotes, the existence of genes encoding CAs from at least three classes (α-, β- and γ-class) suggests that these enzymes play a key role in the physiology of these organisms. In many bacteria CAs are essential for the life cycle of microbes and their inhibition leads to growth impairment or growth defects of the pathogen. CAs thus started to be investigated in detail in bacteria, fungi and protozoa with the aim to identify antiinfectives with a novel mechanism of action. Here, we investigated the catalytic activity, biochemical properties and anion inhibition profiles of the three CAs from the bacterial pathogen Vibrio cholera, VchCA, VchCAβ and VchCAγ. The three enzymes are efficient catalysts for CO₂ hydration, with k_cat values ranging between (3.4 − 8.23) × 10⁵ s⁻¹ and k_cat/K_M of (4.1 − 7.0) × 10⁷ M⁻¹ s⁻¹. A set of inorganic anions and small molecules was investigated for inhibition of these enzymes. The most potent VchCAγ inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_I values ranging between 44 and 91 μM.