Spectrofluorometric and Molecular Docking Studies on the Binding of Curcumenol and Curcumenone to Human Serum Albumin

Curcumenol and curcumenone are two major constituents of the plants of medicinally important genus of Curcuma, and often govern the pharmacological effect of these plant extracts. These two compounds, isolated from C. zedoaria rhizomes were studied for their binding to human serum albumin (HSA) using the fluorescence quench titration method. Molecular docking was also performed to get a more detailed insight into their interaction with HSA at the binding site. Additions of these sesquiterpenes to HSA produced significant fluorescence quenching and blue shifts in the emission spectra of HSA. Analysis of the fluorescence data pointed toward moderate binding affinity between the ligands and HSA, with curcumenone showing a relatively higher binding constant (2.46 × 10⁵ M⁻¹) in comparison to curcumenol (1.97 × 10⁴ M⁻¹). Cluster analyses revealed that site I is the preferred binding site for both molecules with a minimum binding energy of −6.77 kcal·mol⁻¹. However, binding of these two molecules to site II cannot be ruled out as the binding energies were found to be −5.72 and −5.74 kcal·mol⁻¹ for curcumenol and curcumenone, respectively. The interactions of both ligands with HSA involved hydrophobic interactions as well as hydrogen bonding.     

Preparation and Characterization of a Chloroperoxidase-like Catalytic Antibody

The small molecule, meso-tetra(α,α,α,α-o-phenylacetamidophenyl) porphyrin (Mr1147.0) was used as complete antigen to elicit MAb through the immunization and cell fusion techniques. The MAb 1F2 obtained was demonstrated to be very pure by MALDI/TOFMS. The subtype of MAb 1F2 is IgG2a, which has a relative molecular weight of 156,678.8 Da.No significant change in the intensity of absorption peaks in UV and CD spectra was observed over a pH range between 6 and 12. The high stability of the abzyme and the tight binding between Fe porphyrin and antibody were also demonstrated. V_max, K_m, κcat, κcat/K_m for abzyme are 5.18 × 10⁻⁸ Ms⁻¹, 1.50 × 10⁻⁸ M, 0.518 s⁻¹, 3.45 × 10⁷ M⁻¹s⁻¹, respectively. The data obtained indicate that catalytic antibody has high catalytic activity. The chloroperoxidase activity of MAb 1F2-Fe porphyrin complex is stable from 10 °C to 60 °C.     

A High Redox Potential Laccase from Pycnoporus sanguineus RP15: Potential Application for Dye Decolorization

Laccase production by Pycnoporus sanguineus RP15 grown in wheat bran and corncob under solid-state fermentation was optimized by response surface methodology using a Central Composite Rotational Design. A laccase (Lacps1) was purified and characterized and the potential of the pure Lacps1 and the crude culture extract for synthetic dye decolorization was evaluated. At optimal conditions (eight days, 26 °C, 18% (w/w) milled corncob, 0.8% (w/w) NH₄Cl and 50 mmol·L⁻¹ CuSO₄, initial moisture 4.1 mL·g⁻¹), the laccase activity reached 138.6 ± 13.2 U·g⁻¹. Lacps1 was a monomeric glycoprotein (67 kDa, 24% carbohydrate). Optimum pH and temperature for the oxidation of 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) were 4.4 and 74.4 °C, respectively. Lacps1 was stable at pH 3.0–8.0, and after two hours at 55–60 °C, presenting high redox potential (0.747 V vs. NHE). ABTS was oxidized with an apparent affinity constant of 147.0 ± 6.4 μmol·L⁻¹, maximum velocity of 413.4 ± 21.2 U·mg⁻¹ and catalytic efficiency of 3140.1 ± 149.6 L·mmol⁻¹·s⁻¹. The maximum decolorization percentages of bromophenol blue (BPB), remazol brilliant blue R and reactive blue 4 (RB4), at 25 or 40 °C without redox mediators, reached 90%, 80% and 60%, respectively, using either pure Lacps1 or the crude extract. This is the first study of the decolorization of BPB and RB4 by a P. sanguineus laccase. The data suggested good potential for treatment of industrial dye-containing effluents.     

Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) exhibits significant antitumor activity. However, the mechanism of its pharmacological interaction with human serum albumin (HSA) and DNA remains poorly understood. Here, we aimed to elucidate the interactions of Dp44mT with HSA and DNA using MTT assays, spectroscopic methods, and molecular docking analysis. Our results indicated that addition of HSA at a ratio of 1:1 did not alter the cytotoxicity of Dp44mT, but did affect the cytotoxicity of the Dp44mT-Cu complex. Data from fluorescence quenching and UV-VIS absorbance measurements demonstrated that Dp44mT could bind to HSA with a moderate affinity (K_a = approximately 10⁴ M⁻¹). CD spectra revealed that Dp44mT could slightly disrupt the secondary structure of HSA. Dp44mT could also interact with Ct-DNA, but had a moderate binding constant (K_EB = approximately 10⁴ M⁻¹). Docking studies indicated that the IB site of HSA, but not the IIA and IIIA sites, could be favorable for Dp44mT and that binding of Dp44mT to HSA involved hydrogen bonds and hydrophobic force, consistent with thermodynamic results from spectral investigations. Thus, the moderate binding affinity of Dp44mT with HSA and DNA partially contributed to its antitumor activity and may be preferable in drug design approaches.     

Graphene oxide-based SPR biosensor chip for immunoassay applications

This work develops a highly sensitive immunoassay sensor for use in graphene oxide sheet (GOS)-based surface plasmon resonance (SPR) chips. This sensing film, which is formed by chemically modifying a GOS surface, has covalent bonds that strongly interact with the bovine serum albumin (BSA), explaining why it has a higher sensitivity. This GOS film-based SPR chip has a BSA concentration detection limit that is 100 times higher than that of the conventional Au-film-based sensor. The affinity constants (K_A) on the GOS film-based SPR chip and the conventional SPR chip for 100 μg/ml BSA are 80.82 × 10⁶ M^-1 and 15.67 × 10⁶ M^-1, respectively. Therefore, the affinity constant of the GOS film-based SPR chip is 5.2 times higher than that of the conventional chip. With respect to the protein-protein interaction, the SPR sensor capability to detect angle changes at a low concentration anti-BSA of 75.75 nM on the GOS film-based SPR chip and the conventional SPR chip is 36.1867 and 26.1759 mdeg, respectively. At a high concentration, anti-BSA of 378.78 nM on the GOS film-based SPR chip and the conventional SPR chip reveals two times increases in the SPR angle shift. Above results demonstrate that the GOS film is promising for highly sensitive clinical diagnostic applications.     

The interaction between bovine serum albumin and the self-aggregated nanoparticles of cholesterol-modified O-carboxymethyl chitosan

The interaction between bovine serum albumin (BSA) and self-aggregated nanoparticles of cholesterol-modified O-carboxymethyl chitosan (CCMC) with different degrees of substitution (DS) of cholesterol moiety was studied by transmission electron microscopy (TEM), fluorescence quenching method and circular dichroism (CD) measurement. This interaction was started at the disaggregation of CCMC self-aggregated nanoparticles and reached equilibrium after 3-4 h. The apparent quenching constant (K_q) between BSA and CCMC self-aggregated nanoparticles calculated by the modified Stern-Volmer plot increased from 4.14 × 10⁴ to 1.95 × 10⁵ M⁻¹ with DS of cholesterol moiety increasing from 3.2% to 9.8%, whereas the fraction of tryptophan residues in BSA molecule involved in the interaction decreased at the same time. Compared with free BSA, the relative α-helix content of BSA decreased and the unfolding of BSA by a denaturant such as urea was largely suppressed upon interaction with CCMC self-aggregated nanoparticles. DS of cholesterol moiety significantly affected the interaction between BSA and CCMC self-aggregated nanoparticles.     

Synthesis,DNA Binding,and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 10⁴ to 1.0 × 10⁶ M⁻¹ and quenching constants from −0.2 × 10⁴ to 2.18 × 10⁴ M⁻¹ indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.     

Self-organizing nanodot structures on InP surfaces evolving under low-energy ion irradiation: analysis of morphology and composition

Surfaces of InP were bombarded by 1.9 keV Ar⁺ ions under normal incidence. The total accumulated ion fluence Φ the samples were exposed to was varied from 1 × 10¹⁷ cm⁻² to 3 × 10¹⁸ cm⁻², and ion fluxes f of (0.4 − 2) × 10¹⁴ cm⁻² s⁻¹ were used. The surface morphology resulting from these ion irradiations was examined by atomic force microscopy (AFM). Generally, nanodot structures are formed on the surface; their dimensions (diameter, height and separation), however, were found to depend critically on the specific bombardment conditions. As a function of ion fluence, the mean radius r, height h, and spacing l of the dots can be fitted by power-law dependences: r ∝ Φ^0.40, h ∝ Φ^0.48, and l ∝ Φ^0.19. In terms of ion flux, there appears to exist a distinct threshold: below f ~ (1.3 ± 0.2) × 10¹⁴ cm⁻² s⁻¹, no ordering of the dots exists and their size is comparatively small; above that value of f, the height and radius of the dots becomes substantially larger (h ~ 40 nm and r ~ 50 nm). This finding possibly indicates that surface diffusion processes could be important. In order to determine possible local compositional changes in these nanostructures induced by ion impact, selected samples were prepared for atom probe tomography (APT). The results indicate that APT can provide analytical information on the composition of individual InP nanodots. By means of 3D APT data, the surface region of such nanodots evolving under ion bombardment could be examined with atomic spatial resolution. At the InP surface, the values of the In/P concentration ratio are distinctly higher over a distance of approximately 1 nm and amount to 1.3 to 1.7.     

Novel Mechanism and Kinetics of Tetramethrin Degradation Using an Indigenous Gordonia cholesterolivorans A16

Tetramethrin is a pyrethroid insecticide that is commonly used worldwide. The toxicity of this insecticide into the living system is an important concern. In this study, a novel tetramethrin-degrading bacterial strain named A16 was isolated from the activated sludge and identified as Gordonia cholesterolivorans. Strain A16 exhibited superior tetramethrin degradation activity, and utilized tetramethrin as the sole carbon source for growth in a mineral salt medium (MSM). High-performance liquid chromatography (HPLC) analysis revealed that the A16 strain was able to completely degrade 25 mg·L⁻¹ of tetramethrin after 9 days of incubation. Strain A16 effectively degraded tetramethrin at temperature 20–40 °C, pH 5–9, and initial tetramethrin 25–800 mg·L⁻¹. The maximum specific degradation rate (q_max), half-saturation constant (K_s), and inhibition constant (K_i) were determined to be 0.4561 day⁻¹, 7.3 mg·L⁻¹, and 75.2 mg·L⁻¹, respectively. The Box–Behnken design was used to optimize degradation conditions, and maximum degradation was observed at pH 8.5 and a temperature of 38 °C. Five intermediate metabolites were identified after analyzing the degradation products through gas chromatography–mass spectrometry (GC-MS), which suggested that tetramethrin could be degraded first by cleavage of its carboxylester bond, followed by degradation of the five-carbon ring and its subsequent metabolism. This is the first report of a metabolic pathway of tetramethrin in a microorganism. Furthermore, bioaugmentation of tetramethrin-contaminated soils (50 mg·kg⁻¹) with strain A16 (1.0 × 10⁷ cells g⁻¹ of soil) significantly accelerated the degradation rate of tetramethrin, and 74.1% and 82.9% of tetramethrin was removed from sterile and non-sterile soils within 11 days, respectively. The strain A16 was also capable of efficiently degrading a broad spectrum of synthetic pyrethroids including D-cyphenothrin, chlorempenthrin, prallethrin, and allethrin, with a degradation efficiency of 68.3%, 60.7%, 91.6%, and 94.7%, respectively, after being cultured under the same conditions for 11 days. The results of the present study confirmed the bioremediation potential of strain A16 from a contaminated environment.     

NO Scavenging through Reductive Nitrosylation of Ferric Mycobacterium tuberculosis and Homo sapiens Nitrobindins

Ferric nitrobindins (Nbs) selectively bind NO and catalyze the conversion of peroxynitrite to nitrate. In this study, we show that NO scavenging occurs through the reductive nitrosylation of ferric Mycobacterium tuberculosis and Homo sapiens nitrobindins (Mt-Nb(III) and Hs-Nb(III), respectively). The conversion of Mt-Nb(III) and Hs-Nb(III) to Mt-Nb(II)-NO and Hs-Nb(II)-NO, respectively, is a monophasic process, suggesting that over the explored NO concentration range (between 2.5 × 10⁻⁵ and 1.0 × 10⁻³ M), NO binding is lost in the mixing time (i.e., ^NOk_on ≥ 1.0 × 10⁶ M⁻¹ s⁻¹). The pseudo-first-order rate constant for the reductive nitrosylation of Mt-Nb(III) and Hs-Nb(III) (i.e., k) is not linearly dependent on the NO concentration but tends to level off, with a rate-limiting step (i.e., k_lim) whose values increase linearly with [OH⁻]. This indicates that the conversion of Mt-Nb(III) and Hs-Nb(III) to Mt-Nb(II)-NO and Hs-Nb(II)-NO, respectively, is limited by the OH⁻-based catalysis. From the dependence of k_lim on [OH⁻], the values of the second-order rate constant k_OH− for the reductive nitrosylation of Mt-Nb(III)-NO and Hs-Nb(III)-NO were obtained (4.9 (±0.5) × 10³ M⁻¹ s⁻¹ and 6.9 (±0.8) × 10³ M⁻¹ s⁻¹, respectively). This process leads to the inactivation of two NO molecules: one being converted to HNO₂ and another being tightly bound to the ferrous heme-Fe(II) atom.     

Insight into Molecular Interactions of Two Methyl Benzoate Derivatives with Bovine Serum Albumin

The nature and mechanisms of interaction between two selected methyl benzoate derivatives (methyl o-methoxy p-methylaminobenzoate–I and methyl o-hydroxy p-methylaminobenzoate–II) and model transport protein bovine serum albumin (BSA) was studied using steady-state and time-resolved spectroscopic techniques. In order to understand the role of Trp residue of BSA in the I-BSA and II-BSA interaction, the effect of free Trp amino acid on the both emission modes (LE–locally excited (I and II) and ESIPT–excited state intramolecular proton transfer (II)) was investigated as well. Experimental results show that the investigated interactions (with both BSA and Trp) are mostly conditioned by the ground and excited state complex formation processes. Both molecules form stable complexes with BSA and Trp (with 1:1 stoichiometry) in the ground and excited states. The binding constants were in the order of 10⁴ M⁻¹. The absorption- and fluorescence-titration experiments along with the time-resolved fluorescence measurements show that the binding of the I and II causes fluorescence quenching of BSA through the static mechanism, revealing a 1:1 interaction. The magnitude and the sign of the thermodynamic parameters, ΔH, ΔS, and ΔG, determined from van’t Hoff relationship, confirm the predominance of the hydrogen-bonding interactions for the binding phenomenon. To improve and complete knowledge of methyl benzoate derivative-protein interactions in relation to supramolecular solvation dynamics, the time-dependent fluorescence Stokes’ shifts, represented by the normalized spectral response function c(t), was studied. Our studies reveal that the solvation dynamics that occurs in subpicosecond time scale in neat solvents of different polarities is slowed down significantly when the organic molecule is transferred to BSA cavity.     

Secondary Metabolic Profile as a Tool for Distinction and Characterization of Cultivars of Black Pepper (Piper nigrum L.) Cultivated in Pará State,Brazil

This study evaluated the chemical compositions of the leaves and fruits of eight black pepper cultivars cultivated in Pará State (Amazon, Brazil). Hydrodistillation and gas chromatography–mass spectrometry were employed to extract and analyze the volatile compounds, respectively. Sesquiterpene hydrocarbons were predominant (58.5–90.9%) in the cultivars “Cingapura”, “Equador”, “Guajarina”, “Iaçará”, and “Kottanadan”, and “Bragantina”, “Clonada”, and “Uthirankota” displayed oxygenated sesquiterpenoids (50.6–75.0%). The multivariate statistical analysis applied using volatile composition grouped the samples into four groups: γ-Elemene, curzerene, and δ-elemene (“Equador”/“Guajarina”, I); δ-elemene (“Iaçará”/“Kottanadan”/“Cingapura”, II); elemol (“Clonada”/“Uthirankota”, III) and α-muurolol, bicyclogermacrene, and cubebol (“Bragantina”, IV). The major compounds in all fruit samples were monoterpene hydrocarbons such as α-pinene, β-pinene, and limonene. Among the cultivar leaves, phenolics content (44.75–140.53 mg GAE·g⁻¹ FW), the enzymatic activity of phenylalanine-ammonia lyase (20.19–57.22 µU·mL⁻¹), and carotenoids (0.21–2.31 µg·mL⁻¹) displayed significant variations. Due to black pepper’s susceptibility to Fusarium infection, a molecular docking analysis was carried out on Fusarium protein targets using each cultivar’s volatile components. F. oxysporum endoglucanase was identified as the preferential protein target of the compounds. These results can be used to identify chemical markers related to the susceptibility degree of black pepper cultivars to plant diseases prevalent in Pará State.     

Novel Tertiary Amino Containing Blinding Composite Membranes via Raft Polymerization and Their Preliminary CO2 Permeation Performance

Facile synthesis of poly (N,N-dimethylaminoethyl methacrylate) (PDMAEMA) star polymers on the basis of the prepolymer chains, PDMAEMA as the macro chain transfer agent and divinyl benzene (DVB) as the cross-linking reagent by reversible addition-fragmentation chain transfer (RAFT) polymerization was described. The RAFT polymerizations of DMAEMA at 70 °C using four RAFT agents with different R and Z group were investigated. The RAFT agents used in these polymerizations were dibenzyl trithiocarbonate (DBTTC), s-1-dodecyl-s''-(α,α''-dimethyl-α-acetic acid) trithiocarbonate (MTTCD), s,s''-bis (2-hydroxyethyl-2''-dimethylacrylate) trithiocarbonate (BDATC) and s-(2-cyanoprop-2-yl)-s-dodecyltrithiocarbonate (CPTCD). The results indicated that the structure of the end-group of RAFT agents had significant effects on the ability to control polymerization. Compared with the above-mentioned RAFT agents, CPTCD provides better control over the molecular weight and molecular weight distribution. The polydispersity index (PDI) was determined to be within the scope of 1.26 to 1.36. The yields, molecular weight, and distribution of the star polymers can be tuned by changing the molar ratio of DVB/PDMAEMA-CPTCD. The chemical composition and structure of the linear and star polymers were characterized by GPC, FTIR, ¹H NMR, XRD analysis. For the pure Chitosan membrane, a great improvement was observed for both CO₂ permeation rate and ideal selectivity of the blending composite membrane upon increasing the content of SPDMAEMA-8. At a feed gas pressure of 37.5 cmHg and 30 °C, the blinding composite membrane (Cs: SPDMAEMA-8 = 4:4) has a CO₂ permeation rate of 8.54 × 10⁻⁴ cm³ (STP) cm⁻²∙s⁻¹∙cm∙Hg⁻¹ and a N₂ permeation rate of 6.76 × 10⁻⁵ cm³ (STP) cm⁻²∙s⁻¹∙cm∙Hg⁻¹, and an ideal CO₂/N₂ selectivity of 35.2.     

Interactions of Chemically Synthesized Ferrihydrite Nanoparticles with Human Serum Transferrin: Insights from Fluorescence Spectroscopic Studies

Human serum transferrin (HST) is a glycoprotein involved in iron transport that may be a candidate for functionalized nanoparticles to bind and target cancer cells. In this study, the effects of the simple and doped with cobalt (Co) and copper (Cu) ferrihydrite nanoparticles (Fh-NPs, Cu-Fh-NPs, and Co-Fh-NPs) were studied by spectroscopic and molecular approaches. Fluorescence spectroscopy revealed a static quenching mechanism for all three types of Fh-NPs. All Fh-NPs interacted with HST with low affinity, and the binding was driven by hydrogen bonding and van der Waals forces for simple Fh-NPs and by hydrophobic interactions for Cu-Fh-NPs and Co-Fh-NPs binding, respectively. Of all samples, simple Fh-NPs bound the most to the HST binding site. Fluorescence resonance energy transfer (FRET) allowed the efficient determination of the energy transfer between HST and NPs and the distance at which the transfer takes place and confirmed the mechanism of quenching. The denaturation of the HST is an endothermic process, both in the case of apo HST and HST in the presence of the three types of Fh-NPs. Molecular docking studies revealed that Fh binds with a low affinity to HST (K_a = 9.17 × 10³ M⁻¹) in accord with the fluorescence results, where the interaction between simple Fh-NPs and HST was described by a binding constant of 9.54 × 10³ M⁻¹.     

Genetic Susceptibility to Periodontal Disease in Down Syndrome: A Case-Control Study

Severe periodontitis is prevalent in Down syndrome (DS). This study aimed to identify genetic variations associated with periodontitis in individuals with DS. The study group was distributed into DS patients with periodontitis (n = 50) and DS patients with healthy periodontium (n = 36). All samples were genotyped with the “Axiom Spanish Biobank” array, which contains 757,836 markers. An association analysis at the individual marker level using logistic regression, as well as at the gene level applying the sequence kernel association test (SKAT) was performed. The most significant genes were included in a pathway analysis using the free DAVID software. C12orf74 (rs4315121, p = 9.85 × 10⁻⁵, OR = 8.84), LOC101930064 (rs4814890, p = 9.61 × 10⁻⁵, OR = 0.13), KBTBD12 (rs1549874, p = 8.27 × 10⁻⁵, OR = 0.08), PIWIL1 (rs11060842, p = 7.82 × 10⁻⁵, OR = 9.05) and C16orf82 (rs62030877, p = 8.92 × 10⁻⁵, OR = 0.14) showed a higher probability in the individual analysis. The analysis at the gene level highlighted PIWIL, MIR9-2, LHCGR, TPR and BCR. At the signaling pathway level, PI3K-Akt, long-term depression and FoxO achieved nominal significance (p = 1.3 × 10⁻², p = 5.1 × 10⁻³, p = 1.2 × 10⁻², respectively). In summary, various metabolic pathways are involved in the pathogenesis of periodontitis in DS, including PI3K-Akt, which regulates cell proliferation and inflammatory response.     

Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics

Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the V_H–V_L sequence with a (Gly₄Ser)₃ linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10⁻⁴ M⁻¹·s⁻¹ and 6.29 × 10⁻³ s⁻¹, respectively, with an affinity value exceeding 10⁷ M⁻¹ (k_on/k_off), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor.     

Melatonin as a Signaling Molecule for Metabolism Regulation in Response to Hypoxia in the Crab Neohelice granulata

Melatonin has been identified in a variety of crustacean species, but its function is not as well understood as in vertebrates. The present study investigates whether melatonin has an effect on crustacean hyperglycemic hormone (CHH) gene expression, oxygen consumption (VO₂) and circulating glucose and lactate levels, in response to different dissolved-oxygen concentrations, in the crab Neohelice granulata, as well as whether these possible effects are eyestalk- or receptor-dependent. Melatonin decreased CHH expression in crabs exposed for 45 min to 6 (2, 200 or 20,000 pmol·crab⁻¹) or 2 mgO₂·L⁻¹ (200 pmol·crab⁻¹). Since luzindole (200 nmol·crab⁻¹) did not significantly (p > 0.05) alter the melatonin effect, its action does not seem to be mediated by vertebrate-typical MT1 and MT2 receptors. Melatonin (200 pmol·crab⁻¹) increased the levels of glucose and lactate in crabs exposed to 6 mgO₂·L⁻¹, and luzindole (200 nmol·crab⁻¹) decreased this effect, indicating that melatonin receptors are involved in hyperglycemia and lactemia. Melatonin showed no effect on VO₂. Interestingly, in vitro incubation of eyestalk ganglia for 45 min at 0.7 mgO₂·L⁻¹ significantly (p < 0.05) increased melatonin production in this organ. In addition, injections of melatonin significantly increased the levels of circulating melatonin in crabs exposed for 45 min to 6 (200 or 20,000 pmol·crab⁻¹), 2 (200 and 20,000 pmol·crab⁻¹) and 0.7 (200 or 20,000 pmol·crab⁻¹) mgO₂·L⁻¹. Therefore, melatonin seems to have an effect on the metabolism of N. granulata. This molecule inhibited the gene expression of CHH and caused an eyestalk- and receptor-dependent hyperglycemia, which suggests that melatonin may have a signaling role in metabolic regulation in this crab.     

Host-Guest Complexation Studied by Fluorescence Correlation Spectroscopy: Adamantane�CCyclodextrin Inclusion

The host-guest complexation between an Alexa 488 labelled adamantane derivative and β-cyclodextrin is studied by Fluorescence Correlation Spectroscopy (FCS). A 1:1 complex stoichiometry and a high association equilibrium constant of K = 5.2 × 10⁴ M⁻¹ are obtained in aqueous solution at 25 °C and pH = 6. The necessary experimental conditions are discussed. FCS proves to be an excellent method for the determination of stoichiometry and association equilibrium constant of this type of complexes, where both host and guest are nonfluorescent and which are therefore not easily amenable to standard fluorescence spectroscopic methods.     

Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library

A mangrove soil metagenomic library was constructed and a β-agarase gene designated as AgaML was isolated by functional screening. The gene encoded for a 659-amino-acids polypeptide with an estimated molecular mass of 71.6 kDa. The deduced polypeptide sequences of AgaML showed the highest identity of 73% with the glycoside hydrolase family 16 β-agarase from Microbulbifer agarilyticus in the GenBank database. AgaML was cloned and highly expressed in Escherichia coli BL21(DE3). The purified recombinant protein, AgaML, showed optimal activity at 50 °C and pH 7.0. The kinetic parameters of K_m and V_max values toward agarose were 4.6 mg·mL⁻¹ and 967.5 μM·min⁻¹·mg⁻¹, respectively. AgaML hydrolyzed the β-1,4-glycosidic linkages of agar to generate neoagarotetraose (NA4) and neoagarohexaose (NA6) as the main products. These characteristics suggest that AgaML has potential application in cosmetic, pharmaceuticals and food industries.