A Calcium Ion-Dependent Dimeric Bean Lectin with Antiproliferative Activity Toward Human Breast Cancer MCF-7 Cells

In this study, a 60.8-kDa dimeric lectin was isolated from the Phaseolus vulgaris cv. jade bean and characterized. The lectin was bound on Blue Sepharose 6 and Q Sepharose and was finally purified by size exclusion chromatography on Superdex 200. Its hemagglutinating activity toward rabbit erythrocytes was dependent on divalent cations, especially calcium ions. Various carbohydrates tested were devoid of any effect on the hemagglutinating activity. The lectin was stable at pH between 4.5 and 9.4 and temperatures between 30 and 70 °C. It did not exert antifungal activity toward Valsa mali, Setosphaeria turcica, Mycosphaerella arachidicola, Fusarium oxysporum and Bipolaris maydis. The IC₅₀ of the antiproliferative activity of the lectin toward MCF-7 human breast cancer cells was 174 μM. It did not inhibit proliferation of WRL-68 human normal embryonic hepatocytes. The lectin was dependent on calcium ions for hemagglutinating activity and possessed a blocked N-terminus. These two characteristics make the lectin unique among Phaseolus lectins.     

A lectin with antifungal and mitogenic activities from red cluster pepper (<Emphasis Type="Italic">Capsicum frutescens</Emphasis>) seeds

A monomeric mannose/glucose-binding lectin, with a molecular mass of 29.5 kDa and an N-terminal sequence GQRELKL showing resemblance to that of the lectin-like oxidized low-density lipoprotein receptor from the rabbit, has been isolated from the seeds of red cluster pepper Capsium frutescens L. var. fasciculatum. The protocol involved anion exchange chromatography on diethylamino ethanol-cellulose and Q-Sepharose and fast protein liquid chromatography on Mono Q. Its hemagglutinating activity toward rabbit erythrocytes was inhibited by d-mannose and glucose, specifically. The activity was stable from 0 to 40°C, reached a maximum at pH 7 and 8, and was potentiated by Ca²⁺ and Mn²⁺ ions. The lectin showed strong mitogenic activity toward spleen cells isolated from BALB/c mice. The mitogenic activity, which reached a peak at a lectin concentration of 0.27 μM, was inhibited specifically by d(+)-mannose. The lectin was capable of inhibiting the germination of Aspergillus flavus and Fusarium moniliforme spores and hyphal growth in the two fungi.     

A mannose-specific tetrameric lectin with mitogenic and antibacterial activities from the ovary of a teleost,the cobia (<Emphasis Type="Italic">Rachycentron canadum</Emphasis>)

A tetrameric lectin, with hemagglutinating activity toward rabbit erythrocytes and with specificity toward d-mannosamine and d(+)-mannose, was isolated from the ovaries of a teleost, the cobia Rachycentron canadum. The isolation protocol comprised ion exchange chromatography on CM-cellulose and Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q, and finally gel filtration by FPLC on Superose 12. The lectin was adsorbed on all ion exchangers used. It exhibited a molecular mass of 180 kDa in gel filtration on Superose 12 and a single 45-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a tetrameric protein. The hemagglutinating activity of the lectin was stable up to 40°C and between pH 4 and pH 10. All hemagglutinating activity disappeared at 60°C and at pH 1 and pH 13. The hemagglutinating activity was doubled in the presence of 0.1 μM FeCl₃. The lectin exerted antibacterial activity against Escherichia coli with 50% inhibition at 250 μg. There was no antifungal activity toward Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, and Rhizoctonia solani at a dose of 300 μg. The lectin exhibited maximal mitogenic response from mouse splenocytes at a concentration of 14 μM.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

Novel fluorine‐containing chiral hydrazide‐hydrazones: Design,synthesis, structural elucidation,antioxidant and anticholinesterase activity,and in silico studies

In this study, a series of fluorine‐containing chiral hydrazide‐hydrazone derivatives [III‐XII] from ?‐cysteine ethyl ester hydrochloride was synthesized as new antioxidant and anticholinesterase agents. The antioxidant activity of these derivatives was evaluated by ABTS^+· and DPPH^· scavenging and CUPRAC assays and the anticholinesterase activity by the Ellman method spectrophotometrically. The results of the antioxidant assay showed that compounds V , IX , and X exhibited higher activity than BHT and α‐tocopherol used as positive standards. Among the synthesized derivatives, compound IX (IC₅₀: 2.3 ± 1.6 μM) exhibited higher acetylcholinesterase inhibitory activity than galantamine (IC₅₀: 4.5 ± 0.8 μM). Compounds XI (IC₅₀: 9.6 ± 1.0 μM), IX (IC₅₀: 12.5 ± 1.6 μM), III (IC₅₀: 16.0 ± 1.6 μM), X (IC₅₀: 17.2 ± 1.8 μM), VI (IC₅₀: 20.2 ± 0.8 μM), XII (IC₅₀: 21.5 ± 1.0 μM), and VII (IC₅₀: 24.6 ± 0.6 μM) displayed better butyrylcholinesterase inhibitory activity than galantamine (IC₅₀: 46.03 ± 0.14 μM). ADME‐Tox analysis was used to probe the drug‐like properties of the compounds. Molecular docking studies were also applied to understand the interactions between compounds and targets. The docking calculations were supported by the experimental data. In particular, compound IX , having better activity than galantamine against acetylcholinesterase and butyrylcholinesterase enzymes, was visualized using molecular docking.     

Purification,characterization, and biological activities of broccolini lectin

Plant lectins have displayed a variety of biological activities. In this study, for the first time, a 27 kDa arabinose‐ and mannose‐specific lectin from Broccolini (Brassica oleracea Italica × Alboglabra), named as BL (Broccolini lectin), was purified by an activity‐driven protocol. Mass spectrometry analysis and database search indicated that no matches with any plant lectin were found, but BL contained some peptide fragments (QQQGQQGQQLQQVISR, QQGQQQGQQGQQLQQVISR and VCNIPQVSVCPF QK). BL exhibited hemagglutinating activity against chicken erythrocytes at 4 µg/mL. BL retained full hemagglutinating activity at pH 7–8 and temperature 30–40°C, and had an optimal activity in Ca²⁺ solution. Bioactivity assay revealed that BL exhibited dose‐dependent inhibition activity on 5 bacterial species with IC₅₀ values of 178.82–350.93 μg/mL. Notably, 5‐fold reduction in IC₅₀ values was observed on normal L‐O2 vs cancerous HepG‐2 cells (924.35 vs. 178.82 μg/mL). This suggests that BL should be promising in food and medicine. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:736–743, 2015     

Inhibition of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> adhesion to Kato III cells by intact and low molecular weight acharan sulfate

We investigated the inhibitory activity of glycosaminoglycans (GAGs) in terms of growth, adhesion, and VacA vacuolation of Helicobacter pylori. Intact acharan sulfate (AS, MW:114 kDa) potently inhibited H. pylori adhesion to Kato III cells with IC₅₀ value of 1.4 mg/mL, while other GAGs did not show any inhibitory activity except for heparin which is a well-known inhibitor of H. pylori adhesion. To investigate whether low molecular weight acharan sulfate (LMWAS) can inhibit H. pylori adhesion, we performed chemical depolymerization of AS by radical reactions to obtain LMWAS. Its physicochemical properties were characterized by high-performance size exclusion chromatography (HPSEC), agarose gel electrophoresis, disaccharide compositional analysis after digestion with heparinase II, and ¹H-NMR spectroscopy. The most potent molecular size of LMWAS was 3 kDa with IC₅₀ value of 32 μg/mL, which is 44-fold more potent than intact AS. These results suggest that AS as well as other GAGs can be chemically depolymerized by free radicals and LMWAS compared to intact AS can be applied as a pharmaceutical candidate in order to inhibit H. pylori adhesion to Kato III cells.     

Glycan-binding profile of a D-galactose binding lectin purified from the annelid, Perinereis nuntia ver. vallata

A lectin recognizing D-galactose was purified from the pacific annelid Perinereis nuntia ver. vallata (Polychaeta) by affinity chromatography. Hemagglutinating activity, with a very low titer suggesting the presence of lectin appeared in the supernatant from the homogenization of body with Tris-buffered saline. However, dialyzed supernatant from the precipitate homogenized by galactose in the buffer revealed strong hemagglutinating activity against human erythrocytes. The crude supernatant was applied onto lactosyl–agarose column, and only the supernatant eluted from precipitate with galactose was obtained a galactose-binding lectin with 32 kDa polypeptide was obtained from the supernatant of the precipitate, extracted in presence of galactose. It suggests that the lectin tightly binds with glycoconjugate as endogenous ligand(s) in the tissue. Hemagglutinating activity against trypsinized and glutaraldehyde-fixed human erythrocytes was specifically inhibited by D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and asialofetuin. Glycan-binding profile of the lectin analyzed by frontal affinity chromatography shows that the lectin recognizes branched complex type N-linked oligosaccharides and both type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) lactosamine. The surface plasmon resonance study of the lectin against asialofetuin showed the k_ass and k_diss values are 5.14 × 10⁴ M^− 1 s^− 1 and 2.9 × 10⁻³ s^− 1, respectively. The partial primary structure of the lectin reveals 182 amino acids with novel sequence.     

Purification and properties of extracellular phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl₂ and pH 6.0-9.5 and 60°C with 10 mM CaCl₂. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl₂. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The K _m value for sodium phytate was 50 M. Its activity was inhibited by EDTA and metal ions such as Ba²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, and Mn²⁺ ions.     

Oleanane-type triterpene oligoglycosides with pancreatic lipase inhibitory activity from the pericarps of Sapindus rarak

The methanolic extract from the pericarps of Sapindus rarak DC. was found to show pancreatic lipase inhibitory activity (IC₅₀ = ca. 614 μg/mL). From the extract, oleanane-type triterpene oligoglycosides, rarasaponins I–III (1–3), and raraoside A (4), were isolated together with 13 known saponins and four known sesquiterpene glycosides. Among them, several saponin constituents including rarasaponins I (1, IC₅₀ = 131 μM) and II (2, 172 μM), and raraoside A (4, 151 μM) inhibited pancreatic lipase activity, which were stronger than that of theasaponin E₁ (270 μM).     

Leishmanicidal and antitumoral activities of endophytic fungi associated with the Antarctic angiosperms Deschampsia antarctica Desv. and Colobanthus quitensis (Kunth) Bartl.

A total of 564 isolates of endophytic fungi were recovered from the plants Deschampsia antarctica and Colobanthus quitensis collected from Antarctica. The isolates were screened against parasites Leishmania amazonensis and Trypanosoma cruzi and against the human tumour cell lines. Of the 313 fungal isolates obtained from D. antarctica and 251 from C. quitensis, 25 displayed biological activity. Nineteen extracts displayed leishmanicidal activity, and six inhibited the growth of at least one tumour cell line. These fungi belong to 19 taxa of the genera Alternaria, Antarctomyces, Cadophora, Davidiella, Helgardia, Herpotrichia, Microdochium, Oculimacula, Phaeosphaeria and one unidentified fungus. Extracts of 12 fungal isolates inhibited the proliferation of L. amazonesis at a low IC₅₀ of between 0.2 and 12.5 μg ml⁻¹. The fungus Phaeosphaeria herpotrichoides displayed only leishmanicidal activity with an IC₅₀ of 0.2 μg ml⁻¹, which is equivalent to the inhibitory value of amphotericin B. The extract of Microdochium phragmitis displayed specific cytotoxic activity against the UACC-62 cell line with an IC₅₀ value of 12.5 μg ml⁻¹. Our results indicate that the unique angiosperms living in Antarctica shelter an interesting bioactive fungal community that is able to produce antiprotozoal and antitumoral molecules. These molecules may be used to develop new leishmanicidal and anticancer drugs.     

First report of an anti-tumor, anti-fungal, anti-yeast and anti-bacterial hemolysin from Albizia lebbeck seeds

A monomeric 5.5-kDa protein with hemolytic activity toward rabbit erythrocytes was isolated from seeds of Albizia lebbeck by using a protocol that involved ion-exchange chromatography on Q-Sepharose and SP-Sepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on both Q-Sepharose and SP-Sepharose, but adsorbed on Phenyl-Sepharose. Its hemolytic activity was fully preserved in the pH range 0-14 and in the temperature range 0-100 °C, and unaffected in the presence of a variety of metal ions and carbohydrates. The hemolysin reduced viability of murine splenocytes and inhibited proliferation of MCF-7 breast cancer cells and HepG2 hepatoma cells with an IC₅₀ of 0.21, 0.97, and 1.37 μM, respectively. It impeded mycelial growth in the fungi Rhizoctonia solani with an IC₅₀ of 39 μM but there was no effect on a variety of other filamentous fungi, including Fusarium oxysporum, Helminthosporium maydis, Valsa mali and Mycosphaerella arachidicola. Lebbeckalysin inhibited growth of Escherichia coli with an IC₅₀ of 0.52 μM.     

Thymidylate synthase activity and the cell growth are inhibited by the β-carboline-benzoquinolizidine alkaloid deoxytubulosine

Employing thymidylate synthase (TS) (5, 10-CH₂-H₄PteGlu: dUMP C-methyltransferase, EC 2.1.1.45), a key target enzyme in chemotherapy, the biological activity of the β-carboline-benzoquinolizidine alkaloid deoxytubulosine (DTB) isolated from the Indian medicinal plant Alangium lamarckii has been evaluated and assessed for the first time. The TS employed in the present studies was purified from Lactobacillus leichmannii. The DTB was demonstrated to exhibit potent cytotoxicity and inhibited the cell growth of L. leichmannii, and DTB potently inhibited TS activity (IC₅₀ = 40 μM). The DTB concentrations >80 μM resulted in a total loss of the TS activity, thus suggesting that the β-carboline-benzoquinolizidine alkaloid is a promising potential antitumor agent. The DTB binding to TS appears to be irreversible and tight through a possible covalent linkage. Although DTB strongly binds to DNA, it is not known whether DTB binds to RNA associated with TS. Inhibition kinetics showed that TS has a K_i value of 7 × 10⁻⁶ M for DTB and that the inhibition is a simple linear “noncompetitive” type. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 167–173, 1998     

Desensitization of nicotinic acetylcholine receptors in central nervous system neurons of the stick insect (Carausius morosus) by imidacloprid and sulfoximine insecticides

Imidacloprid, sulfoxaflor and two experimental sulfoximine insecticides caused generally depressive symptoms in stick insects, characterized by stillness and weakness, while also variably inducing postural changes such as persistent ovipositor opening, leg flexion or extension and abdomen bending that could indicate excitation of certain neural circuits. We examined the same compounds on nicotinic acetylcholine receptors in stick insect neurons, which have previously been shown to desensitize in the presence of ACh. Brief U-tube application of 10⁻⁴ M solutions of insecticides for 1 s evoked currents that were much smaller than ACh-evoked currents, and depressed subsequent ACh-evoked currents for several minutes, indicating that the compounds are low-efficacy partial agonists that potently desensitize the receptors. Much lower concentrations of insecticides applied in the bath for longer periods did not activate currents, but inhibited ACh-evoked currents via desensitization of the receptors. Previously described fast- and slowly-desensitizing nACh currents, I_ACh1 and I_ACh2 respectively, were each found to consist of two components with differing sensitivities to the insecticides. Imidacloprid applied in the bath desensitized high-sensitivity components, I_ACh1H and I_ACh2H with IC₅₀s of 0.18 and 0.13 pM, respectively. It desensitized the low-sensitivity slowly desensitizing component, I_ACh2L, with an IC₅₀ of 2.6 nM, while a component of the fast-desensitizing current, I_ACh1L, was least sensitive, with an IC₅₀ of 81 nM I_ACh1L appeared to be insensitive to the three sulfoximines tested, whereas all three sulfoximines potently desensitized I_ACh1H and both slowly desensitizing components, with IC₅₀s between 2 and 7 nM. We conclude that selective desensitization of certain nAChR subtypes can account for the insecticidal actions of imidacloprid and sulfoximines in stick insects.