Purification and primary structure determination of a galactose-specific lectin from Vatairea guianensis Aublet seeds that exhibits vasorelaxant effect

Vatairea guianensis seeds, a typical plant from the Brazilian Amazon region that belongs to the Dalbergieae tribe, possess a lectin that was isolated by precipitation with solid ammonium sulfate followed by guar gum affinity chromatography. This lectin was named VGL. The V. guianensis lectin strongly agglutinated rabbit erythrocytes and was inhibited by d-galactose and d-galactose-derived sugars, especially N-acetyl-d-galactosamine. VGL has been shown to be a stable protein, maintaining its hemagglutinating activity after incubation at a wide range of temperature and pH values and after incubation with ethylenediamine tetraacetic acid (EDTA). In a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the purified VGL exhibited an electrophoretic profile consisting of a major 30–32 kDa double band, which is termed the alpha-chain, and two minor components of 18 and 15 kDa, which are referred to as the beta- and gamma-chains, respectively. An analysis using electrospray ionization mass spectrometry also indicated that purified VGL contains a mixture of chains with molecular weights of 28,437 ± 2, 14,952 ± 2 and 12,332 ± 2. The complete amino acid sequence of VGL, as determined using tandem mass spectrometry, consists of 239 amino acid residues. VGL is a glycoprotein exhibiting high similarity in primary structure to other lectins from evolutionarily related plants, such as Vatairea macrocarpa lectin and lectins belonging to the Sophoreae tribe. VGL exhibits vasorelaxant activity in contracted rat aortas, an effect that is strictly dependent on the endothelium and involves nitric oxide and the lectin domain.     

Isolation and antiproliferative activity of Lotus corniculatus lectin towards human tumour cell lines

The objective of the study was to investigate the anti cancer activity of a lectin isolated from Lotus corniculatus seeds. A tetrameric 70 kDa galactose specific lectin was purified using two step simple purification protocol which involved affinity chromatography on AF-BlueHC650M and gel filtration on Sephadex G-100. The lectin was adsorbed on AF-BlueHC650M and desorbed using 1 M NaCl in the starting buffer. Gel filtration on Sephadex G-100 yielded a major peak absorbance that gave two bands of 15 kDa and 20 kDa in SDS PAGE. Hemagglutination activity was completely preserved, when the temperature was in the range of 20–60 °C. However, drastic reduction in activity occurred at temperatures above 60 °C. Full hemagglutination activity was retained at ambient pH 4–12. Thereafter no activity was observed above pH 13. Hemaglutination of the lectin was inhibited by d-galactose. The lectin showed a strong antiproliferative activity towards human leukemic (THP-1) cancer cells followed by lung cancer (HOP62) cells and HCT116 with an IC₅₀ of 39 μg/ml and 50 μg/ml and 60 μg/ml respectively. Flow cytometry analysis showed an increase in the percentage of cells in sub G0G1 phase confirming that Lotus corniculatus lectin induced apoptosis. Morphological observations showed that Lotus corniculatus lectin (LCL) treated THP-1 cells displayed apparent apoptosis characteristics such as nuclear fragmentation, appearance of membrane enclosed apoptotic bodies and DNA fragmentation. Lotus corniculatus lectin (LCL) effectively inhibits the cell migration in a dose dependent manner as indicated by the wound healing assay.     

Characterization of a thermophilic l-arabinose isomerase from Thermoanaerobacterium saccharolyticum NTOU1

l-Arabinose isomerase (EC 5.3.1.4, l-AI) mainly catalyzes the reversible aldose–ketose isomerization between l-arabinose and l-ribulose. l-AIs can also catalyze other reactions, such as the conversion of d-galactose to d-tagatose. In this study, the araA gene encoding l-AI was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. The recombinant l-AI was purified from the cell-free extract using nickel nitrilotriacetic acid metal-affinity chromatography. The purified enzyme showed an optimal activity at 70 °C and pH 7–7.5. The enzyme was stable at pHs ranging from 6.5 to 9.5 and the activity was fully retained after 2 h incubation at 55–65 °C. The low concentrations of divalent metal ions, either 0.1 mM Mn²⁺ or 0.05 mM Co²⁺, could improve both catalytic activity and thermostability at higher temperatures. The recombinant T. saccharolyticum NTOU1 l-AI has the lowest demand for metal ions among all characterized thermophilic l-AIs. This thermophilic l-AI shows a potential to be used in industry to produce d-tagatose from d-galactose.     

A new chitin-binding lectin from rhizome of Setcreasea purpurea with antifungal,antiviral and apoptosis-inducing activities

A 48 kDa, chitin-binding lectin with antifungal, antiviral and apoptosis-inducing activities was isolated from the rhizomes of Setcreasea purpurea Boom, a member of family Commelinaceae. Setcreasea purpurea lectin (designated as SPL) is a homotetrameric protein consisting of 12031.9 Da subunits linked by non-covalent bonds as determined by SDS-PAGE, gel filtration and MS. The N-terminal 25 amino-acid sequence of SPL, NVLGRDAYCGSQNPGATCPGLCCSK was determined and homology analysis suggested that SPL belongs to the family of chitin-binding plant lectins composed of hevein domains. The lectin exhibited strong hemagglutinating activity towards rabbit erythrocytes at 0.95 μg/ml and the activity could be reversed exclusively by chitin hydrolysate (oligomers of GlcNAc). Its hemagglutinating activity was stable in pH range of 2.0–9.0 and it showed excellent thermal tolerance. SPL showed antifungal activity against Rhizoctonia solani, Sclerotinia sclerotiorum, Penicillium italicum and Helminthosporiun maydis. It also exhibited inhibitory effect on HIV-1 (IIIB) and HIV-2 (ROD), with an EC₅₀ of 13.8 ± 1.3 and 57.1 ± 15 μg/ml, respectively. Subsequently, MTT method, cell morphological analysis and LDH activity-based cytotoxicity assays demonstrated that SPL was highly cytotoxic to CNE-1 cells and induced apoptosis in a dose-dependent manner. Moreover, due to the caspase inhibitors analyses, caspase was also found to play an important role in the potential apoptotic mechanism of SPL.     

Molecular cloning,expression and functional characterization of the 40-kDa heat shock protein,DnaJ, from Bacillus halodurans

In the present study, we identified, cloned and expressed a 40-kDa heat shock protein, DnaJ, from Bacillus halodurans. The open reading frame of the cloned gene contained 1116 bp and encoded 371 amino acid residues. The purified recombinant DnaJ contained a His-tag at the C-terminus and showed a single band at approximately 41-kDa on SDS-PAGE gel. The 3D structures of DnaJ obtained by I-TASSER showed that the overall structures of DnaJ from B. halodurans Guj1 and E. coli are very similar, with 45% sequence similarity. The present study revealed that the DnaJ protein from B. halodurans inhibits the heat-induced aggregation of insulin in a concentration-dependent manner as aggregation of the insulin B-chain was reduced by approximately 50% at 40 °C in the presence of 0.1 mg/ml of purified recombinant DnaJ. The overexpression of DnaJ improved thermotolerance properties in E. coli transformed with pET-28a + DnaJ. Salt resistance experiments indicated that the survival of E. coli transformed with DnaJ was enhanced 1.85-fold compared to that of the control cells in the presence of 0.5 M NaCl for 72 h. According to the results obtained, DnaJ from B. halodurans can potentially be used for improving the functional properties of enzymes and proteins in various applications.     

Simultaneous quantification of rosiglitazone and its two major metabolites,N-desmethyl and p-hydroxy rosiglitazone in human plasma by liquid chromatography/tandem mass spectrometry: Application to a pharmacokinetic study

We present a simple, rapid, and sensitive liquid chromatography (LC)–tandem mass spectrometry (MS/MS) method for the simultaneous quantification of rosiglitazone and its two major metabolites via CYP2C8/9, N-desmethyl and p-hydroxy rosiglitazone, in human plasma. The procedure was developed and validated using rosiglitazone-d₃ as the internal standard. Plasma samples (0.1 ml) were prepared using a simple deproteinization procedure with 0.2 ml of acetonitrile containing 40 ng/ml of rosiglitazone-d₃. Chromatographic separation was carried out on a Luna C₁₈ column (100 mm × 2.0 mm, 3-μm particle size) using an isocratic mobile phase consisting of a 60:40 (v/v) mixture of acetonitrile and 0.1% formic acid_(aq). Each sample was run at 0.2 ml/min for a total run time of 2.5 min per sample. Detection and quantification were performed using a mass spectrometer in selected reaction-monitoring mode with positive electrospray ionization at m/z 358.1 → 135.1 for rosiglitazone, m/z 344.2 → 121.1 for N-desmethyl rosiglitazone, m/z 374.1 → 151.1 for p-hydroxy rosiglitazone, and m/z 361.1 → 138.1 for rosiglitazone-d₃. The linear ranges of concentration for rosiglitazone, N-desmethyl rosiglitazone, and p-hydroxy rosiglitazone were 1–500, 1–150, and 1–25 ng/ml, respectively, with a lower limit of quantification of 1 ng/ml for all analytes. The coefficient of variation for assay precision was less than 14.4%, and the accuracy was 93.3–112.3%. No relevant cross-talk and matrix effect were observed. This method was successfully applied to a pharmacokinetic study after oral administration of a 4-mg rosiglitazone tablet to healthy male Korean volunteers.     

Racemization study on different N-acetylamino acids by a recombinant N-succinylamino acid racemase from Geobacillus kaustophilus CECT4264

N-Succinylamino acid racemase (NSAAR) with N-acylamino acid racemase (NAAAR) activity together with a d- or l-aminoacylase allows the total transformation of N-acetylamino acid racemic mixtures into optically pure d- or l-amino acids, respectively. In this work we have cloned and expressed the N-succinylamino acid racemase gene from the thermophilic Bacillus-related species Geobacillus kaustophilus CECT4264 in Escherichia coli BL21 (DE3). G. kaustophilus NSAAR (GkNSAAR) was purified in a one-step procedure by immobilized cobalt affinity chromatography and showed an apparent molecular mass of 43 kDa in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of about 150 kDa, suggesting that the native enzyme is a homotetramer. Optimum reaction conditions for the purified enzyme were 55 °C and pH 8.0, using N-acetyl-d-methionine as substrate. GkNSAAR showed a gradual loss of activity at preincubation temperatures over 60 °C, suggesting that it is thermostable. As activity was greatly enhanced by Co²⁺, Mn²⁺ and Ni²⁺ but inhibited by metal-chelating agents, it is considered a metalloenzyme. The Co²⁺-dependent activity profile of the enzyme was studied with no detectable inhibition at higher metal ion concentrations. GkNSAAR showed activity towards both aliphatic and aromatic N-acetylamino acids such as N-acetyl-methionine and N-acetyl-phenylalanine, respectively, with k_cat/K_m values ranging from 1 × 10³ to 9 × 10³ s^?1 M^?1. Kinetic parameters were better for N-acetyl-d-amino acids than for N-acetyl-l-specific ones.     

Directional selection by termites at a branching node created by a ballpoint pen

Subterranean termites excavate complex underground tunnels for foraging. Most tunnels comprise primary and secondary tunnels. Tunnels originating from the nest are called primary and those branching from the primary tunnels are named secondary tunnels; tertiary and quaternary tunnels are rarely observed. During foraging, termites may thus encounter a considerable number of tunnel-branching nodes. Directional selection at such a node is likely correlated to tunnel-growth activity because tunnels containing more termites have a higher probability of growth. In this study, we investigated how termites select the direction of movement at an artificially-designed branching node, by making chemical trails on filter paper, drawing lines using a ballpoint pen which contained the chemical substance that induces the termite to follow trails. The trails consisted of two lines: straight and branching. The branching line was drawn from the center of the straight line at an angle θ (10°, 20°,…, 90°). We then calculated the ratio of the directional selection as r = N_s/N_b, where N_s and N_b represent the number of straight and branching tunnels selected, respectively. The values of r were statistically classified into three groups based on the angle of the branching trail, as follows: 10° ≤ θ ≤ 20°, 30° ≤ θ ≤ 60°, and 70° ≤ θ ≤ 90°. Our paper briefly discusses the underlying mechanisms of the experimental results.     

Design,synthesis and anticonvulsant activity of new hybrid compounds derived from N-phenyl-2-(2,5-dioxopyrrolidin-1-yl)-propanamides and -butanamides

The focused library of 21 new N-phenyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide, 2-(3-methyl-2,5-dioxopyrrolidin-1-yl)propanamide, and 2-(2,5-dioxopyrrolidin-1-yl)butanamide derivatives as potential new hybrid anticonvulsant agents was synthesized. These hybrid molecules were obtained as close analogs of previously described N-benzyl derivatives and fuse the chemical fragments of clinically relevant antiepileptic drugs such as ethosuximide, levetiracetam, and lacosamide. The initial anticonvulsant screening was performed in mice (ip) using the ‘classical’ maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests, as well as in the six-Hertz (6 Hz) model of pharmacoresistant limbic seizures. Applying the rotarod test, the acute neurological toxicity was determined. The broad spectra of activity across the preclinical seizure models in mice (ip) displayed compounds 4, 5, 11, and 19. The most favorable anticonvulsant properties demonstrated 4 (ED₅₀ MES = 96.9 mg/kg, ED₅₀ scPTZ = 75.4 mg/kg, ED₅₀ 6 Hz = 44.3 mg/kg) which showed TD₅₀ = 335.8 mg/kg in the rotarod test that yielded satisfying protective indexes (PI MES = 3.5, PI scPTZ = 4.4, PI 6 Hz = 7.6). Consequently, compound 4 revealed comparable or better safety profile than model antiepileptic drugs (AEDs): ethosuximide, lacosamide, and valproic acid. In the in vitro assays, compound 4 was observed as relatively effective binder to the neuronal voltage-sensitive sodium and diltiazem site of L-type calcium channels.     

Toxic isolectins from the mushroom Boletus venenatus

Ingestion of the toxic mushroom Boletus venenatus causes a severe gastrointestinal syndrome, such as nausea, repetitive vomiting, diarrhea, and stomachache. A family of isolectins (B. venenatus lectins, BVLs) was isolated as the toxic principles from the mushroom by successive 80% ammonium sulfate-precipitation, Super Q anion-exchange chromatography, and TSK-gel G3000SW gel filtration. Although BVLs showed a single band on SDS–PAGE, they were further divided into eight isolectins (BVL-1 to -8) by BioAssist Q anion-exchange chromatography. All the isolectins showed lectin activity and had very similar molecular weights as detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Among them, BVL-1 and -3 were further characterized with their complete amino acid sequences of 99 amino acids determined and found to be identical to each other. In the hemagglutination inhibition assay, both proteins failed to bind to any mono- or oligo-saccharides tested and showed the same sugar-binding specificity to glycoproteins. Among the glycoproteins examined, asialo-fetuin was the strongest inhibitor. The sugar-binding specificity of each isolectin was also analyzed by using frontal affinity chromatography and surface plasmon resonance analysis, indicating that they recognized N-linked sugar chains, especially Galβ1 → 4GlcNAcβ1 → 4Manβ1 → 4GlcNAcβ1 → 4GlcNAc (Type II) residues in N-linked sugar chains. BVLs ingestion resulted in fatal toxicity in mice upon intraperitoneal administration and caused diarrhea upon oral administration in rats.     

Evaluation of the cold-active Pseudoalteromonas haloplanktis β-galactosidase enzyme for lactose hydrolysis in whey permeate as primary step of d-tagatose production

d-Tagatose is an innovative natural low-calorie bulk sweetener with a broad potential for low-calorie and low-glycaemic foods and drinks. Production of this healthy sweetener is realized through enzymatic d-galactose isomerization. d-Galactose needs to be produced in situ due to its limited availability. Whey permeate contains a substantial amount of lactose, which is an interesting source for d-galactose production through enzymatic lactose hydrolysis. In this context, the cold-active β-galactosidase from the psychrophile Pseudoalteromonas haloplanktis was studied. Optimal parameters for efficient lactose hydrolysis in whey permeate have been deduced, viz. optimal incubation temperature, pH and lactose concentration. Hydrolysis efficiencies above 96.0% were realized within 24 h at 23 °C and pH 7.0 in whey permeate with a maximum dry matter content of 10.0% (w/w). In addition, the effect of the presence of d-glucose and d-galactose was investigated up to concentrations of 100 g l⁻¹. d-Glucose inhibited lactose hydrolysis more strongly compared to d-galactose. Also, the operational stability of the cold-active β-galactosidase was studied. Hydrolysis efficiencies above 90.0% were maintained during 7 subsequent hydrolysis cycles.     

Purification,structural analysis and hydroxyl radical-scavenging capacity of a polysaccharide from the fruiting bodies of Russula virescens

In this study, one water-soluble polysaccharide (RVP) was isolated from the fruiting bodies of Russula virescens. Its molecular weight (Mw) was ∼3.9 ×10⁴ Da, as determined by high-performance size-exclusion chromatography (HPSEC). The RVP was composed of d-galactose and d-mannose in a ratio of 2:1, as detected by gas chromatography (GC). The structural features of RVP were investigated by partial hydrolysis with acid, periodate oxidation and Smith degradation, acetylation, methylation analysis, and NMR spectroscopy (¹H, ¹³C). The results revealed that RVP had a backbone consisting of (1 → 6)-linked-α-d-galactopyranosyl and (1 → 2, 6)-linked-α-d-galactopyranosyl residues that terminated in a single non-reducing terminal (1 → )-α-d-mannopyranosyl residue at the O-2 position of each (1 → 2, 6)-linked-α-d-galactopyranosyl residues along the main chain in the ratio of 1:1:1. Preliminary, in vitro antioxidant activity tests showed that RVP could dose-dependently enhance the scavenging effect on hydroxyl radicals at concentrations from 20 to 160 μg/ml. These results indicated that RVP was mainly composed of water-soluble galactomannan, and it had a significant effect on scavenging of hydroxyl radicals in vitro.     

Purification,primary structure and potential functions of a novel lectin from Bauhinia forficata seeds

A new lectin, BfL, was purified from Bauhinia forficata seeds by ammonium sulfate fractionation, DEAE-Sephadex ion exchange chromatography, Sepharose-4B and chitin affinity chromatographies and Superdex 75 size exclusion chromatography. The molecular homogeneity and purity of BfL were assessed by reversed-phase HPLC. BfL appeared as a single band of approximately 27.0 kDa on SDS-PAGE under non-reducing and reducing conditions, and its molecular weight was determined to be 27,850 Da by LC/ESI-MS. BfL is a glycoprotein with a carbohydrate content of 6.24% determined by the phenol–sulfuric acid method. Fetuin, asialofetuin, thyroglobulin and azocasein inhibited the hemagglutinating activity of BfL, whereas saccharides did not. BfL hemagglutinating activity was stable at 100 °C for 30 min, pH-dependent, with the highest activity at pH 6.0, and metal-independent. The primary structure of BfL shows similarity with other lectins from the genus Bauhinia. Deconvolution of the BfL circular dichroism (CD) spectrum indicated the presence of α-helix and β structures. BfL increases coagulation time, but this effect is not related to human plasma kallikrein or human factor Xa inhibition. BfL also inhibits ADP- and epinephrine-induced platelet aggregation in a dose-dependent manner and is the only currently described lectin from Bauhinia that exhibits anticoagulant and antiplatelet aggregating properties.     

Mannose-specific legume lectin from the seeds of Dolichos lablab (FRIL) stimulates inflammatory and hypernociceptive processes in mice

Lectins are proteins that specifically bind to carbohydrates and form complexes with molecules and biological structures containing saccharides. The FRIL (Flt3 receptor interacting lectin) is a dimeric two-chain (αβ)2 lectin presenting 67 kDa molecular mass. The interaction of FRIL with carbohydrates, as determined by molecular docking, showed that this lectin has highest affinity to the carbohydrate trimannoside (−135.618 MDS), followed by trehalosamine (−127.072 MDS) and αα-trehalose (−121.729 MDS). FRIL evoked dose-dependent paw edema, increasing animal paw volumes. The edematogenic effect of FRIL was paralleled by an increase in vascular permeability, about 10-fold higher compared to control. FRIL also significantly raised the animals flinch reaction in the first, third and fifth hours in response to mechanical stimulation. Injection of α-methyl-d-mannoside associated with FRIL inhibited edema and hypernociception. The histopathological analysis of animal paws showed a characteristically acute inflammatory process that included severe infiltration of mixed leukocytes, changes in cytoarchitecture, edema and focal areas of hemorrhage. In addition, in silico assays confirmed that FRIL preferentially interacts with trimannoside that makes up the core N-glycans cell. Therefore, our study supports the hypothesis that the lectin domain and the likely glycoconjugates containing α-d-methyl mannoside residues contribute to the inflammatory effects of FRIL.     

Antimycobacterial and herbicidal activity of ring-substituted 1-hydroxynaphthalene-2-carboxanilides

In this study, a series of 22 ring-substituted 1-hydroxynaphthalene-2-carboxanilides were prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Mycobacterium marinum, Mycobacterium kansasii and Mycobacterium smegmatis. The compounds were also tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Most of tested compounds showed the antimycobacterial activity against the three strains comparable or higher than the standard isoniazid. N-(3-Fluorophenyl)-1-hydroxynaphthalene-2-carboxamide showed the highest biological activity (MIC = 28.4 μmol/L) against M. marinum, N-(4-fluorophenyl)-1-hydroxynaphthalene-2-carboxamide showed the highest biological activity (MIC = 14.2 μmol/L) against M. kansasii, and N-(4-bromophenyl)-1-hydroxynaphthalene-2-carboxamide expressed the highest biological activity (MIC = 46.7 μmol/L) against M. smegmatis. This compound and 1-hydroxy-N-(3-methylphenyl)naphthalene-2-carboxamide were the most active compounds against all three tested strains. The PET inhibition expressed by IC₅₀ value of the most active compound 1-hydroxy-N-(3-trifluoromethylphenyl)naphthalene-2-carboxamide was 5.3 μmol/L. The most effective compounds demonstrated insignificant toxicity against the human monocytic leukemia THP-1 cell line. For all compounds, structure–activity relationships are discussed.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

TbGT8 is a bifunctional glycosyltransferase that elaborates N-linked glycans on a protein phosphatase AcP115 and a GPI-anchor modifying glycan in Trypanosoma brucei

The procyclic form of Trypanosoma brucei expresses procyclin surface glycoproteins with unusual glycosylphosphatidylinositol-anchor side chain structures that contain branched N-acetyllactosamine and lacto-N-biose units. The glycosyltransferase TbGT8 is involved in the synthesis of the branched side chain through its UDP-GlcNAc: βGal β1-3N-acetylglucosaminyltransferase activity. Here, we explored the role of TbGT8 in the mammalian bloodstream form of the parasite with a tetracycline-inducible conditional null T. brucei mutant for TbGT8. Under non-permissive conditions, the mutant showed significantly reduced binding to tomato lectin, which recognizes poly-N-acetyllactosamine-containing glycans. Lectin pull-down assays revealed differences between the wild type and TbGT8 null-mutant T. brucei, notably the absence of a broad protein band with an approximate molecular weight of 110 kDa in the mutant lysate. Proteomic analysis revealed that the band contained several glycoproteins, including the acidic ecto-protein phosphatase AcP115, a stage-specific glycoprotein in the bloodstream form of T. brucei. Western blotting with an anti-AcP115 antibody revealed that AcP115 was approximately 10 kDa smaller in the mutant. Enzymatic de-N-glycosylation demonstrated that the underlying protein cores were the same, suggesting that the 10-kDa difference was due to differences in N-linked glycans. Immunofluorescence microscopy revealed the colocalization of hemagglutinin epitope-tagged TbGT8 and the Golgi-associated protein GRASP. These data suggest that TbGT8 is involved in the construction of complex poly-N-acetyllactosamine-containing type N-linked and GPI-linked glycans in the Golgi of the bloodstream and procyclic parasite forms, respectively.     

A lactose specific lectin from the sponge Cinachyrella apion: Purification,characterization, N-terminal sequences alignment and agglutinating activity on Leishmania promastigotes

Crude extract from the sponge Cinachyrella apion showed cross-reactivity with the polyclonal antibody IgG anti-CvL (Cliona varians lectin) and also a strong haemagglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglycosylated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA Purifier) gel filtration on a Superose 6 10/300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A erythrocytes. The haemagglutinating activity is independent of Ca²⁺, Mg²⁺ and Mn²⁺ ions, and it was strongly inhibited by the disaccharide lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass, determined by FPLC-gel filtration on a Superose 12 10/300 column and SDS gel electrophoresis, was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was heat-stable between 0 and 60 °C and pH-stable. The N-terminal amino acid sequence of CaL was also determined and a blast search on amino acid sequences revealed that the protein showed similarity only with a silicatein. Leishmania chagasi promastigotes were agglutinated by CaL and this activity was abolished by lactose, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the potential biotechnological application of CaL as diagnostic of pathogenic protozoa.     

Cloning,characterization and validation of inosine 5′-monophosphate dehydrogenase of Babesia gibsoni as molecular drug target

The inosine monophosphate dehydrogenase (IMPDH) enzyme has been characterized and validated as a molecular drug target in other apicomplexans but not in the genus Babesia. Subsequently, we cloned and expressed a Babesia gibsoni IMPDH (BgIMPDH) cDNA in Escherichia coli. We also determined the inhibitory effect of mycophenolic acid (MPA) on recombinant BgIMPDH (rBgIMPDH) activity and the Babesia-growths in vitro. The translated BgIMPDH peptide contained thirteen amino acid residues responsible for substrate and cofactor binding in its catalytic domain with Gly374 in BgIMPDH being replaced by Ser388 in mammalian IMPDH. The native BgIMPDH enzyme in the parasite was approximately 54-kDa a mass similar to His-tag rBgIMPDH protein. The K_m values of the rBgIMPDH were 8.18 ± 0.878 (mean ± standard error of the mean) μM and 360.80 ± 43.41 μM for IMP and NAD⁺, respectively. MPA inhibited the rBgIMPDH activity yielding a K_i value of 20.93 ± 1.83 μM with respect to NAD⁺. For Babesia growths, the IC₅₀s were 0.95 ± 0.21 and 2.88 ± 0.49 μM for B. gibsoni and B. bovis, respectively. Therefore, our results suggest that MPA may inhibit the replication of Babesia parasites by targeting IMPDH enzyme of the purine pathway.     

Enhanced T cell responsiveness to Mycobacterium bovis BCG r32-kDa Ag correlates with successful anti-tuberculosis treatment in humans

Th1 and Th2 cytokines play key role in protection from and pathogenesis of mycobacterial infection and their dynamic changes may predict clinical outcome of the patient. Patients with tuberculosis (TB) have a poorer cellular immune response to recombinant 32-kDa antigen (Ag) of Mycobacterium bovis (r32-kDa M. bovis) than do healthy volunteers. The basis for this observation was studied by evaluating the Th1 (gamma interferon [IFN-γ]) produced in response to the r32-kDa Ag M. bovis by peripheral blood mononuclear cells (PBMC) from patients with pulmonary TB (n = 20), extra-pulmonary TB (n = 13) and from healthy volunteers (n = 9). Recombinant 32-kDa M. bovis stimulated PBMC from TB patients produced significantly lower levels of IFN-γ at 0 month, and increased at 2–4, and 6 months of treatment and were highly significant (p < 0.000) compared to the responses in controls. The ratios of IFN-γ to IL-10 were low in patients newly diagnosed and improved both during and after treatment. The present study concludes that the levels of in vitro response to M. bovis BCG r32-kDa Ag leading to the specific release of IFN-γ increased after anti-tuberculosis treatment and seems to reflect the clinical status of the patient, thus reiterating the utility of this antigen in T cell based assays as a surrogate marker of cell mediated responses.