Potency optimization of Huwentoxin-IV on hNav1.7: A neurotoxin TTX-S sodium-channel antagonist from the venom of the Chinese bird-eating spider Selenocosmia huwena

The spider venom peptide Huwentoxin-IV (HwTx-IV) 1 is a potent antagonist of hNa_v1.7 (IC₅₀ determined herein as 17 ± 2 nM). Na_v1.7 is a voltage-gated sodium channel involved in the generation and conduction of neuropathic and nociceptive pain signals. We prepared a number of HwTx-IV analogs as part of a structure–function study into Na_v1.7 antagonism. The inhibitory potency of these analogs was determined by automated electrophysiology and is reported herein. In particular, the native residues Glu¹, Glu⁴, Phe⁶ and Tyr³³ were revealed as important activity modulators and several peptides bearing mutations in these positions showed significantly increased potency on hNa_v1.7 while maintaining the original selectivity profile of the wild-type peptide 1 on hNa_v1.5. Peptide 47 (Gly¹, Gly⁴, Trp³³-HwTx) demonstrated the largest potency increase on hNa_v1.7 (IC₅₀ 0.4 ± 0.1 nM).     

Molecular docking,a tool to determine interaction of CuO and TiO2 nanoparticles with human serum albumin

BackgroundWe study the human serum albumin (HSA) protein-CuO nanoparticle interaction to identify the specific binding site of protein with CuO nanoparticles by molecular docking and compared it with HSA-TiO₂ nanoparticle interaction.MethodsThe protein structural data that was obtained using Autodock 4.2.ResultsIn case of CuO np-HSA interaction, the distances from the centre of Subdomain IIIA to Arg-472 is 2.113 Å and Lys 475, Glu 492, Ala 490, Cys 487, Ala 490 are the bound neighbouring residues with Lys 475, Glu 492 at aliphatic region. The binding energy generated was ?1.64 kcal mol^?1. However, for TiO₂ nanoparticle, the binding region is surrounded by Arg 257, Ala 258, Ser 287, His 288, Leu 283, Ala 254, Tyr 150 (subdomain II A) as neighbouring residue. Moreover, Glu 285, Lys 286 forms aliphatic grove for TiO₂-HSA, Ser-287 at the centre region form hydrogen bond with nanoparticle and Leu 283, Leu 284 forming hydrophopobic grove for TiO₂ nanoparticle-HSA interaction. The binding energy generated was ?2.47 kcal mol^?1.ConclusionsAnalysis suggests that CuO bind to suldow site II i.e subdomain III A of HSA protein where as TiO₂ nanoparticle bind to suldow site I i.e subdomain IIA of HSA protein.General significanceThe structural information that derives from this study for CuO and TiO₂ nanoparticles may be useful in terms of both high and low-affinity binding sites when designing these nanoparticles based drugs delivery system.     

Antibacterial and anticancer activity of a series of novel peptides incorporating cyclic tetra-substituted Cα amino acids

Eleven antimicrobial peptides (AMP) based on the incorporation of cyclic tetra substituted C^α amino acids, as well as other unnatural amino acids were designed, synthesized and screened for in vitro activity against 18 strains of bacteria as well as 12 cancer cell lines. The AMPs discussed herein are derived from the following peptide sequence: Ac-GF(X)G(X)B(X)G(X)F(X)G(X)GB(X)BBBB-amide, X = any one of the following residues, A5c, A6c, Tic or Oic and B = any one of the following residues, Arg, Lys, Orn, Dpr or Dab. A diversity of in vitro inhibitory activity was observed for these AMPs. Several analogs exhibited single digit μM activity against drug resistant bacteria including; multiple drug resistant Mycobacterium tuberculosis, extremely drug resistant Mycobacterium tuberculosis and MRSA. The physicochemical properties of the basic amino acid residues incorporated into these AMPs seem to play a major role in defining antibacterial activity. Overall hydrophobicity seems to play a limited role in defining antibacterial activity. The ESKAPE pathogens were used to compare the activity of these AMPs to another family of synthetic AMPs incorporating the unnatural amino acids Tic and Oic. In most cases similarly substituted members of both families exhibited similar inhibitory activity against the ESKAPE pathogens. In specific cases differences in activity as high as 15 fold were observed between analogs. In addition four of these AMPs exhibited promising IC₅₀ (<7.5 μM) values against 12 different and diverse cancer cell lines. Five other AMPs exhibited promising IC₅₀ (<7.5 μM) values against selected cancer cell lines.     

Concerted involvement of cooperative proton–electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu_A and heme a₃/Cu_B are cooperatively linked to proton transfer at acid/base groups in the enzyme. H⁺/e⁻ cooperative linkage at Fe_a3/Cu_B is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H⁺/e⁻ linkage at heme a (and Cu_A). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H₂O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu_A and heme a₃/Cu_B in the soluble oxidase; (iii) H⁺ release in the external phase (i.e. H⁺ pumping) associated with the oxidative (R → O transition), reductive (O → R transition) and a full catalytic cycle (R → O → R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H⁺/e⁻ linkage at heme a/Cu_A and heme a₃/Cu_B with acid/base clusters, C₁ and C₂ respectively, and protonmotive steps of the reduction of O₂ to water are involved in proton pumping.     

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe_a3–CO and appearance of Cu_B–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba₃–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^? 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^? 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm^? 1, 1977 cm^? 1, 1981 cm^? 1, and 1985 cm^? 1, previously assigned to the Fe_a3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^? 1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO₂ at 2337 cm^? 1 and one from traces of CO at 2133 cm^? 1; while bands associated with CO bound to either Fe_a3 or to Cu_B in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe_a3–CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu_B atom; Fe_a3 and Cu_B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Expanding the pharmacological profile of κ-hefutoxin 1 and analogues: A focus on the inhibitory effect on the oncogenic channel Kv10.1

Peptide toxins, such as scorpion peptides, are interesting lead compounds in the search for novel drugs. In this paper, the focus is on the scorpion peptide κ-hefutoxin 1. This peptide displays a cysteine-stabilized helix-loop-helix fold (CSα/α) and is known to be a weak K_v1.x inhibitor. Due to the low affinity of κ-hefutoxin 1 for these channels, it is assumed that the main target(s) of κ-hefutoxin 1 remain(s) unknown. In order to identify novel targets, electrophysiological measurements and antifungal assays were performed. The effect of κ-hefutoxin 1 was previously evaluated on a panel of 11 different voltage-gated potassium channels. Here, we extended this target screening with the oncogenic potassium channel K_v10.1. κ-Hefutoxin 1 was able to inhibit this channel in a dose-dependent manner (IC₅₀ ∼ 26 μM). Although the affinity is rather low, this is the first peptide toxin ever described to be a K_v10.1 inhibitor. The structure-activity relationship of κ-hefutoxin 1 on K_v10.1 was investigated by testing eight κ-hefutoxin 1 variants using the two-electrode voltage clamp technique. Several important amino acid residues were identified; the functional dyad residues (Tyr⁵ and Lys¹⁹), N-terminal residues (Gly¹ and His²) and the amidated C-terminal residue (Cys²²). Since the CSα/α fold is also found in a class of antifungal plant peptides, the α-hairpinines, we investigated the antifungal activity of κ-hefutoxin 1. κ-Hefutoxin 1 showed low activity against the plant pathogen Fusarium culmorum and no activity against three other yeast and fungal species, even at high concentrations (∼100 μM).     

Design and synthesis of a β-lactamase activated 5-fluorouracil prodrug

An efficient synthesis of a 5-fluorouracil-cephalosporin prodrug is described for use against colorectal and other cancers in antibody and gene-directed therapies. The compound shows stability in aqueous media until specifically activated by β-lactamase (βL). The kinetic parameters of the 5-fluorouracil-cephalosporin conjugate were determined in the presence of Enterobacter cloacae P99 βL (ECl βL) revealing a K_m = 95.4 μM and V_max = 3.21 μMol min^?1 mg^?1. The data compare favorably to related systems that have been reported and enable testing of this prodrug against cancer cell lines in vitro and in vivo.     

Novel potent inhibitors of hepatitis C virus (HCV) NS3 protease with cyclic sulfonyl P3 cappings

Extensive SAR studies of the P3 capping group led to the discovery of a series of potent inhibitors with sultam and cyclic sulfonyl urea moieties as the P3 capping. The bicyclic thiophene-sultam or phenyl-sultam cappings were selected for further SAR development. Modification at the P3 side chain determined that the tert-butyl group was the best choice at that position. Optimization of P1 residue significantly improved potency and selectivity. The combination of optimal moieties at all positions led to the discovery of compound 33. This compound had the best overall profile in potency and PK profile: excellent K_i¹ of 5.3 nM and activity in replicon (EC₉₀) of 80 nM, extremely high selectivity of 6100, and a good rat PO AUC of 1.43 μM h.     

Atomic structure of the sweet-tasting protein thaumatin I at pH 8.0 reveals the large disulfide-rich region in domain II to be sensitive to a pH change

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at 50 nM. Although the sweetness remains when thaumatin is heated at 80 °C for 4 h under acid conditions, it rapidly declines when heating at a pH above 6.5. To clarify the structural difference at high pH, the atomic structure of a recombinant thaumatin I at pH 8.0 was determined at a resolution of 1.0 Å. Comparison to the crystal structure of thaumatin at pH 7.3 and 7.0 revealed the root-mean square deviation value of a Cα atom to be substantially greater in the large disulfide-rich region of domain II, especially residues 154–164, suggesting that a loop region in domain II to be affected by solvent conditions. Furthermore, B-factors of Lys137, Lys163, and Lys187 were significantly affected by pH change, suggesting that a striking increase in the mobility of these lysine residues, which could facilitate a reaction with a free sulfhydryl residue produced via the β-elimination of disulfide bonds by heating at a pH above 7.0. The increase in mobility of lysine residues as well as a loop region in domain II might play an important role in the heat-induced aggregation of thaumatin above pH 7.0.     

Mutations in algal and cyanobacterial Photosystem I that independently affect the yield of initial charge separation in the two electron transfer cofactor branches

In Photosystem I, light-induced electron transfer can occur in either of two symmetry-related branches of cofactors, each of which is composed of a pair of chlorophylls (ec2_A/ec3_A or ec2_B/ec3_B) and a phylloquinone (PhQ_A or PhQ_B). The axial ligand to the central Mg^2 + of the ec2_A and ec2_B chlorophylls is a water molecule that is also H-bonded to a nearby Asn residue. Here, we investigate the importance of this interaction for charge separation by converting each of the Asn residues to a Leu in the green alga, Chlamydomonas reinhardtii, and the cyanobacterium, Synechocystis sp. PCC6803, and studying the energy and electron transfer using time-resolved optical and EPR spectroscopy. Nanosecond transient absorbance measurements of the PhQ to F_X electron transfer show that in both species, the PsaA-N604L mutation (near ec2_B) results in a ~ 50% reduction in the amount of electron transfer in the B-branch, while the PsaB-N591L mutation (near ec2_A) results in a ~ 70% reduction in the amount of electron transfer in the A-branch. A diminished quantum yield of P₇₀₀⁺ PhQ^? is also observed in ultrafast optical experiments, but the lower yield does not appear to be a consequence of charge recombination in the nanosecond or microsecond timescales. The most significant finding is that the yield of electron transfer in the unaffected branch did not increase to compensate for the lower yield in the affected branch. Hence, each branch of the reaction center appears to operate independently of the other in carrying out light-induced charge separation.     

Synergistic interactions between the antinociceptive effect of Rhodiola rosea extract and B vitamins in the mouse formalin test

AimIn this study, the pharmacological interactions between a Rhodiola rosea ethanol extract and B-vitamins such as thiamine (B₁), riboflavine (B₂), pyridoxine (B₆), cyanocobalamin (B₁₂) and a mixture of vitamins B₁ + B₆ + B₁₂ was investigated in the mouse formalin test.MethodsIndividual dose response curves of the Rhodiola rosea ethanol extract, as well as B-vitamins alone or in a mixture were evaluated in mice in which nociception was induced with 2% formalin intraplantarly. The antinociceptive mechanisms of the Rhodiola rosea were investigated by exploring the role of the opioid and serotonin receptors and the nitric oxide pathway. Isobolographic analysis was used to evaluate the pharmacological interactions between the Rhodiola rosea ethanol extract and each B-vitamin individually or the mixture of vitamins B₁ + B₆ +B₁₂ by using the ED₃₀ and a fixed 1:1 ratio combination.ResultsAdministration of the Rhodiola rosea extract alone or in combination with all of the vitamins produced a significant and dose-dependent antinociceptive response. The antinociceptive effect of the Rhodiola rosea extract (ED₅₀ = 81 mg/kg, p.o.) was significant and reverted in the presence of antagonists of the 5-HT_1A, GABA/BDZs and opioid receptors and by blocking mediators of the nitric oxide/cGMP/K⁺ channels pathway. Isobolograms demonstrate that all of the combinations investigated in this study produced a synergistic interaction experimental ED₃₀ values were significantly smaller than those calculated theoretically.ConclusionsThese results provide evidence that a Rhodiola rosea ethanol extract in combination with B-vitamins produces a significant diminution in the nociceptive response in a synergistic manner, which is controlled by various mechanisms. These findings could aid in the design of clinical studies and suggest that these combinations could be applied for pain therapy.     

Cloning,expression, characterisation and mutational analysis of l-aspartate oxidase from Pseudomonas putida

l-Amino acid oxidases (LAAOs) are useful catalysts for the deracemisation of racemic amino acid substrates when combined with abiotic reductants. The gene nadB encoding the l-aspartate amino acid oxidase from Pseudomonas putida (PpLASPO) has been cloned and expressed in E. coli. The purified PpLASPO enzyme displayed a K_M for l-aspartic acid of 2.26 mM and a k_cat = 10.6 s⁻¹, with lower activity also displayed towards l-asparagine, for which pronounced substrate inhibition was also observed. The pH optimum of the enzyme was recorded at pH 7.4. The enzyme was stable for 60 min at up to 40 °C, but rapid losses in activity were observed at 50 °C. A mutational analysis of the enzyme, based on its sequence homology with the LASPO from E. coli of known structure, appeared to confirm roles in substrate binding or catalysis for residues His244, His351, Arg386 and Arg290 and also for Thr259 and Gln242. The high activity of the enzyme, and its promiscuous acceptance of both l-asparagine and l-glutamate as substrates, if with low activity, suggests that PpLASPO may provide a good model enzyme for evolution studies towards AAOs of altered or improved properties in the future.     

[Lys(DOTA)4]BVD15, a novel and potent neuropeptide Y analog designed for Y1 receptor-targeted breast tumor imaging

We substituted a truncated neuropeptide Y (NPY) analog, [Pro³⁰, Tyr³², Leu³⁴]NPY(28-36)NH₂ also called BVD15, at various positions with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7-10-tetraacetic acid) and evaluated the effect of the coupling position with the binding affinity for NPY Y₁ receptors (NPY1R). Our data suggest that [Lys(DOTA)⁴]BVD15 (K_i = 63 ± 25 nM vs. K_i = 39 ± 34 nM for BVD15) is a potent NPY analog suitable for radiolabeling with metallo positron emitters for PET imaging of breast cancer.     

In vitro and in vivo ACE inhibitory of pistachio hydrolysates and in silico mechanism of identified peptide binding with ACE

The ACE inhibitory activity of pistachio (Pistacia vera L.) kernel's hydrolysates by gastrointestinal enzymes was studied. Results indicated that hydrolysate successively hydrolyzed by pepsin and trypsin, Pe–Tr–H, presented in vitro ACE inhibitory activity as IC₅₀ 0.87 ± 0.04 mg/ml. The Pe–Tr–H can in vivo decrease around 22 mmHg in systolic blood pressure (SBP) and 16 mmHg in the diastolic blood pressure (DBP) at 4 h after the oral administration, however the pistachio kernel powder can slightly lower SBP and DBP. The Pe–Tr–H with the highest activity was then separated by ultrafiltration membrane of 3 kDa, size exclusion chromatography on Sephadex G-15 and G-10 columns and reversed phase high-performance liquid chromatography (RP-HPLC) consecutively. A novel ACE inhibitory peptide, ACKEP, with the IC₅₀ value of 126 μM, was identified by MALDI–TOF/TOF system. ACKEP has the same C-terminal residue as Lisinopril and Enalapril, which plays a key role in binding with ACE. The binding mechanism was explored at a molecular basis by docking experiments, which revealed that seven residues from ACE active site (His383, His387, Glu384, Arg522, Asp358, Ala356 and Asn70) and two atoms of ACKEP (O5, H60) greatly contributed to the combinative stabilization.     

Alteration of plankton communities and biogeochemical cycles by harmful Cochlodinium polykrikoides (Dinophyceae) blooms

Cochlodinium polykrikoides is a globally distributed, ichthyotoxic, bloom-forming dinoflagellate. Blooms of C. polykrikoides manifest themselves as large (many km²) and distinct patches with cell densities exceeding 10³ ml⁻¹ while water adjacent to these patches can have low cell densities (<100 cells ml⁻¹). While the effect of these blooms on fish and shellfish is well-known, their impacts on microbial communities and biogeochemical cycles are poorly understood. Here, we investigated plankton communities and the cycling of carbon, nitrogen, and B-vitamins within blooms of C. polykrikoides and compared them to areas in close proximity (<100 m) with low C. polykrikoides densities. Within blooms, C. polykrikoides represented more than 90% of microplankton (>20 μm) cells, and there were significantly more heterotrophic bacteria and picoeukaryotic phytoplankton but fewer Synechococcus. Terminal restriction fragment length polymorphism analysis of 16S and 18S rRNA genes revealed significant differences in community composition between bloom and non-bloom samples. Inside the bloom patches, concentrations of vitamin B₁₂ were significantly lower while concentrations of dissolved oxygen were significantly higher. Carbon fixation and nitrogen uptake rates were up to ten times higher within C. polykrikoides bloom patches. Ammonium was a more important source of nitrogen, relative to nitrate and urea, for microplankton within bloom patches compared to non-bloom communities. While uptake rates of vitamin B₁ were similar in bloom and non-bloom samples, vitamin B₁₂ was taken up at rates five-fold higher (>100 pmol⁻¹ L⁻¹ d⁻¹) in bloom samples, resulting in turn-over times of hours during blooms. This high vitamin demand likely led to the vitamin B₁₂ limitation of C. polykrikoides observed during nutrient amendment experiments conducted with bloom water. Collectively, this study revealed that C. polykrikoides blooms fundamentally change microbial communities and accelerate the cycling of carbon, some nutrients, and vitamin B₁₂.     

The effect of glycerol mixed substrate on the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system

A recombinant Rhizopus oryzae lipase producing Mut^s Pichia pastoris strain was used as a model organism to study the effect of mixed substrates (glycerol and methanol) on the specific product productivity. Different fed-batch cultivations were performed under three constant specific growth rates (0.02, 0.05 and 0.1 h⁻¹), maintaining a constant methanol concentration of 2 g l⁻¹.At the lowest μ tested (0.02 h⁻¹), the specific productivity was 1.23 and 1.61 fold higher and the specific methanol consumption rate (q_sMeOH) was 3 and 3.5 fold higher than values obtained when μ was 0.05 and 0.1 h⁻¹, respectively. This implies a relation between the q_sMeOH and the specific productivity, yielding higher specific productivities whenever the consumption of methanol is higher. Although glycerol was maintained under limiting conditions in all μ tested, when the relation between the μ_Gly and μ_MeOH was larger than 4, an important decrease on the maximal activity value was observed.Finally, a comparison under the same conditions using glycerol or sorbitol as co-substrates was also performed, obtaining better specific productivity when sorbitol was used. In addition, protease activity was detected when glycerol was used as co-substrate.     

Anion inhibition profiles of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum

We have cloned, purified and investigated the catalytic activity and anion inhibition profiles of a full catalytic domain (358 amino acid residues) carbonic anhydrase (CA, EC 4.2.1.1) from Plasmodium falciparum, PfCAdom, an enzyme belonging to the η-CA class and identified in the genome of the malaria-producing protozoa. A truncated such enzyme, PfCA1, containing 235 residues was investigated earlier for its catalytic and inhibition profiles. The two enzymes were efficient catalysts for CO₂ hydration: PfCAdom showed a k_cat of 3.8 × 10⁵ s⁻¹ and k_cat/K_m of 7.2 × 10⁷ M⁻¹ × s⁻¹, whereas PfCA showed a lower activity compared to PfCAdom, with a k_cat of 1.4 × 10⁵ s⁻¹ and k_cat/K_m of 5.4 × 10⁶ M⁻¹ × s⁻¹. PfCAdom was generally less inhibited by most anions and small molecules compared to PfCA1. The best PfCAdom inhibitors were sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, which showed K_Is in the range of 9–68 μM, followed by bicarbonate, hydrogensulfide, stannate and N,N-diethyldithiocarbamate, which were submillimolar inhibitors, with K_Is in the range of 0.53–0.97 mM. Malaria parasites CA inhibition was proposed as a new strategy to develop antimalarial drugs, with a novel mechanism of action.     

Glycopeptide dendrimer colchicine conjugates targeting cancer cells

Screening of a 65,536-member one-bead-one-compound (OBOC) combinatorial library of glycopeptide dendrimers of structure ((βGal)_n + 1X⁸X⁷X⁶X⁵)₂DapX⁴X³X²X¹(β-Gal)_m (βGal = β-galactosyl-thiopropionic acid, X^8–1 = variable amino acids, Dap = l-2,3-diaminopropionic acid, n, m = 0, or 1 if X⁸ = Lys resp. X¹ = Lys) for binding of Jurkat cells to the library beads in cell culture, resynthesis and testing lead to the identification of dendrimer J1 (βGal-Gly-Arg-His-Ala)₂Dap-Thr-Arg-His-Asp-CysNH₂ and related analogues as delivery vehicles. Cell targeting is evidenced by FACS with fluorescein conjugates such as J1F. The colchicine conjugate J1C is cytotoxic with LD₅₀ = 1.5 μM. The β-galactoside groups are necessary for activity, as evidenced by the absence of cell-binding and cytotoxicity in the non-galactosylated, acetylated analogue AcJ1F and AcJ1C, respectively. The pentagalactosylated dendrimer J4 βGal₄(Lys-Arg-His-Leu)₂Dap-Thr-Tyr-His-Lys(βGal)-Cys) selectively labels Jurkat cell as the fluorescein derivative J4F, but its colchicine conjugate J4C lacks cytotoxicity. Tubulin binding assays show that the colchicine dendrimer conjugates do not bind to tubulin, implying intracellular degradation of the dendrimers releasing the active drug.

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Structural elucidation and biological activity of a novel polysaccharide by alkaline extraction from cultured Cordyceps militaris

A novel polysaccharide named CBP-1 was isolated from the fruiting body of cultured Cordyceps militaris by alkaline extraction as well as anion-exchange and gel-permeation chromatography. Its structural features were investigated by a combination of chemical and instrumental analysis approaches, including partial hydrolysis, methylation analysis, HIO₄ oxidation-Smith degradation, GC–MS, ¹³C NMR, HPAEC-PAD and FT-IR. The results indicated that CBP-1 has a backbone of (1 → 4)-α-d-mannose residues which occasionally branches at O-3. The branches were mainly composed of (1 → 4)-α-d-glucose residues and (1 → 6)-β-d-galactose residues, and terminated with β-d-galactose residues. In the in vitro antioxidant assay, CBP-1 was found to possess the hydroxyl radical-scavenging activity with an IC₅₀ value of 0.638 mg/ml.     

Mn2+ and Mg2+ improved sphingomyelinase production by Lactobacillus rhamnosus FTDC 8313 and binding affinity to sphingomyelin for generation of ceramides

The present research aims to optimize the sphingomyelinase (SMase) activity produced by Lactobacillus rhamnosus FTDC 8313 using divalent metal ions via response surface methodology and to further study the effects of the divalent metal ions on SMase activity using molecular modeling approach. This study also aimed to assess the possibility of increasing ceramide levels in vitro on cultured keratinocytes upon treatment with the extracellular extract of the optimized L. rhamnosus FTDC 8313. Using a central composite design, an optimum point of SMase activity (6.54 mU ml^?1) was produced from a combination of 0.65% (w/v) MnSO₄ and 0.82% (w/v) MgSO₄. 3D response surface indicated that the altered availability of the two ions (Mn²⁺ and Mg²⁺) reduced their effects on SMase activity. In addition, the treatment of the HaCaT cells with optimized extracellular extract of L. rhamnosus FTDC8313 significantly increased (P < 0.05) the conversion of sphingomyelin to ceramide as compared to the control. Molecular docking demonstrated that the addition of Mn²⁺ and Mg²⁺ into the active site of SMase improved the binding affinity between the SMase and sphingomyelin based on its free energy of binding as well as the interaction distances between the important catalytic residues Glu53 and His296.