Molecular weight characterization of poly[(R)-3-hydroxybutyrate] synthesized by genetically engineered strains of Escherichia coli

This study investigated the relationship of growth conditions, host strains and molecular weights of poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesized by genetically engineered Escherichia coli. Various PHA synthases belonging to types I-IV enzymes were expressed in E. coli JM109 under the same experimental conditions, and the molecular weights of the polymers were characterized by gel permeation chromatography. The results demonstrate that P(3HB) polymers have varied molecular weights and polydispersities dependent on the characteristics of the individual PHA synthase employed. P(3HB) with high number-average molecular weights (M_n) [(1.5-4.0) × 10⁶] and narrow polydispersities (1.6-1.8) were synthesized by PHA synthases from Ralstonia eutropha (type I), Delftia acidovorans (type I) and Allochromatium vinosum (type III). Contrary to these, P(3HB) with relatively low M_n [(0.17-0.79) × 10⁶] and broad polydispersities (2.2-9.0) were synthesized by PHA synthases from Aeromonas caviae (type I), Pseudomonas sp. 61-3 (type II) and Bacillus sp. INT005 (type IV). Furthermore, the molecular weights of P(3HB) synthesized under various culture conditions, in various hosts of E. coli and by mutants of PHA synthase were characterized. It was found that, in addition to culture pH [Kusaka et al. Appl Microbiol Biotechnol 1997;47:140], other variances such as culture temperature, host strain and use of mutants are effective in changing polymer molecular weight.     

Improved synthesis of P(3HB-co-3HV-co-3HHx) terpolymers by mutant Cupriavidus necator using the PHA synthase gene of Chromobacterium sp. USM2 with high affinity towards 3HV

Unlike polyhydroxyalkanoates (PHAs) copolymers, the controlled and efficient synthesis of PHA terpolymers from triglycerides and fatty acids are yet to be established. This study demonstrates the production of P(3HB-co-3HV-co-3HHx) terpolymer with a wide range of 3HV monomer compositions from mixtures of crude palm kernel oil and 3HV precursors using a mutant Cupriavidus necator PHB⁻4 transformant harboring the PHA synthase gene (phaC) of a locally isolated Chromobacterium sp. USM2. The PHA synthase of Chromobacterium has an unusually high affinity towards 3HV monomer. P(3HB-co-3HV-co-3HHx) terpolymers with 3HV monomer composition ranging from 2 to 91 mol% were produced. Generation of 3HHx monomers was affected by the concentration and feeding time of 3HV precursor. P(3HB-co-24 mol% 3HV-co-7 mol% 3HHx) exhibited mechanical properties similar to that of common low-density polyethylene. P(3HB-co-3HV-co-3HHx) terpolymers with a wide range of 3HV molar fraction had been successfully synthesized by adding lower concentrations of 3HV precursors and using a PHA synthase with high affinity towards 3HV monomer.     

The synthesis of tetrahydropyridopyrimidones as a new scaffold for HIV-1 integrase inhibitors

An efficient synthesis of methyl 9-amino-3-hydroxy-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-2-carboxylate as a late stage intermediate for a new class of HIV-1 integrase inhibitors is described. After construction of the bicyclic core scaffold, a stereoselective introduction of the chiral amino group in the 9-position is achieved.     

De novo design based identification of potential HIV-1 integrase inhibitors: A pharmacoinformatics study

In the present study, pharmacoinformatics paradigms include receptor-based de novo design, virtual screening through molecular docking and molecular dynamics (MD) simulation are implemented to identify novel and promising HIV-1 integrase inhibitors. The de novodrug/ligand/molecule design is a powerful and effective approach to design a large number of novel and structurally diverse compounds with the required pharmacological profiles. A crystal structure of HIV-1 integrase bound with standard inhibitor BI-224436 is used and a set of 80,000 compounds through the de novo approach in LigBuilder is designed. Initially, a number of criteria including molecular docking, in-silico toxicity and pharmacokinetics profile assessments are implied to reduce the chemical space. Finally, four de novo designed molecules are proposed as potential HIV-1 integrase inhibitors based on comparative analyses. Notably, strong binding interactions have been identified between a few newly identified catalytic amino acid residues and proposed HIV-1 integrase inhibitors. For evaluation of the dynamic stability of the protein-ligand complexes, a number of parameters are explored from the 100 ns MD simulation study. The MD simulation study suggested that proposed molecules efficiently retained their molecular interaction and structural integrity inside the HIV-1 integrase. The binding free energy is calculated through the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) approach for all complexes and it also explains their thermodynamic stability. Hence, proposed molecules through de novo design might be critical to inhibiting the HIV-1 integrase.     

Biosynthesis and characterization of novel polyhydroxyalkanoate polymers with high elastic property by Cupriavidus necator PHB4 transformant

We attempted to synthesize novel polyhydroxyalkanoate (PHA) containing new 3-hydroxy-4-methylvalerate (3H4MV) monomer from the transformed strain of Cupriavidus necator PHB⁻4 harboring the PHA synthase gene of Chromobacterium sp. USM2 (phaC_Cs). Novel PHA containing SCL and MCL monomers were successfully synthesized from crude palm kernel oil (CPKO) and isocaproic acid. Results showed that P(3HB-co-1 mol% 3HV-co-3 mol% 3H4MV-co-18 mol% 3HHx) possessed higher thermal stability, higher elastomeric behavior at room temperature and higher ductility than the P(3HB-co-5 mol% 3HHx). The novel PHA film was found to possess an interesting rubber-like elasticity and flexibility property which has not been reported. The soil surface degradation study showed that the novel PHA film was degraded faster than the 3HB homopolymer and copolymer with 5 mol% of 3HHx.     

Phage Langmuir monolayers and Langmuir–Blodgett films

Stable, insoluble Langmuir monolayer films composed of Staphylococcus aureus-specific lytic bacteriophage were formed at an air–water interface and characterized. The phage monolayer was very strong, withstanding a surface pressure of ～40 mN/m at 20 °C. The surface pressure–area (Π–A) isotherm possessed a shoulder at ～7 × 10⁴ nm²/phage particle, attributed to a change in phage orientation at the air–water interface from horizontal to vertical capsid-down/tail-up orientation as surface pressure was increased. The Π–A-dependence was accurately described using the Volmer equation of state, assuming horizontal orientation to an air–water interface at low surface pressures with an excluded area per phage particle of 4.6 × 10⁴ nm². At high pressures phage particles followed the space-filling densely packed disks model with a specific area of 8.5 × 10³ nm²/phage particle. Lytic phage monolayers were transferred onto gold-coated silica substrates from the air–water interface at a constant surface pressure of 18 mN/m by Langmuir–Blodgett method, then dried and analyzed by scanning electron microscopy (SEM) and ellipsometry. Phage specific adsorption (Γ) in Langmuir–Blodgett (LB) films measured by SEM was consistent with that calculated independently from Π–A isotherms at the transfer surface pressure of 18 mN/m (Γ = 23 phage particles/μm²). The 50 nm-thickness of phage monolayer measured by ellipsometer agreed well with the horizontal phage average size estimated by SEM. Surface properties of phage Langmuir monolayer compare well with other monolayers formed from nano- and micro-particles at the air–water interface and similar to that of classic amphiphiles 1,2-diphytanoyl-sn-glycero-3-phosphocholine (phospholipid) and stearic acid.     

Design, Practical Synthesis, and Biological Evaluation of Novel 6-(Pyrazolylmethyl)-4-quinoline-3-carboxylic Acid Derivatives as HIV-1 Integrase Inhibitors

A series of novel 6-(pyrazolylmethyl)-4-oxo-4H-quinoline-3-carboxylic acid derivatives bearing different substituents on the N-position of quinoline ring were designed and synthesized as potential HIV-1 integrase (IN) inhibitors, based on the structurally related GS-9137 scaffold. The structures of all new compounds were confirmed by ¹H-NMR, ¹³C-NMR and ESI (or HRMS) spectra. Detailed synthetic protocols and the anti-IN activity studies are also presented.     

First-principles relativistic calculation for 4f-5d transition energy of Ce in various fluoride hosts

In this paper, we investigated the 4f-5d transition energy of Ce³⁺ in various fluoride hosts based on the first-principles discrete-variational Dirac-Slater (DV-DS) calculations using Slater's transition-state theory. Especially, we focused on the lowest energy peak (1st peak) of 4f-5d transition for Ce³⁺ impurities. As the host crystals, we adopted the 15 fluorides, for which the experimental data of the lowest energy peak (1st peak) in 4f-5d transitions were available from literature except for NaMgF₃ and BaMgF₄. A high correlation between the experimental 1st peak energies and the theoretical ones was obtained which suggests a possibility to predict the 4f-5d transition energy of Ce³⁺ in various fluoride hosts using the first-principles calculation.     

Unusual change in molecular weight of polyhydroxyalkanoate (PHA) during cultivation of PHA-accumulating Escherichia coli

This study aimed to investigate the factors affecting molecular weight of poly[(R)-3-hydroxybutyrate] [P(3HB)] when polyhydroxyalkanoate (PHA) synthase (PhaRC_Bsp) from Bacillus sp. INT005 was used for P(3HB) synthesis in Escherichia coli JM109. It was found that the molecular weight of P(3HB) decreased with time in mid- and late-phase of culture and was strongly affected by culture temperature. At 37 °C culture temperature, the molecular weight of P(3HB) rapidly decreased from 4.4 × 10⁵ to 4.8 × 10⁴ with culture time, whereas it was almost unchanged at 25 °C. Kinetic analysis suggested that the decrease in molecular weight of P(3HB) was due to random scission of the polymer chain. The decrease in molecular weight of P(3HB) was not observed when PHA synthases other than PhaRC_Bsp were expressed. This study sheds light on the unique behaviour in molecular weight change of P(3HB) that is synthesized by E. coli expressing PhaRC_Bsp.     

Assembly of luminescent hybrids from co-polymers bearing functional 4-vinyl pyridine and europium aromatic carboxylate

Three binary europium complexes with benzoic acid and ortho (meta) methylbenzoic acids in the first stage. Then, these complexes were introduced into 4-vinyl pyridine-ethyl methacrylate co-polymer hosts using the former component as the second ligand. The composition of the hybrids was characterized by FT-IR and the photophysical property of them was investigated by UV spectra, luminescent spectra and luminescent lifetimes. The experiment results substantiate that 4-vinyl pyridine could co-ordinate to europium ions effectively with nitrogen atoms of the pyridine ring. Therefore, the intramolecular energy transfer process accomplished within polymer hybrids and red emissions of Eu³⁺ have been obtained.     

Bulk and interfacial behaviour of caseinoglycomacropeptide (GMP)

Caseinoglycomacropeptide (GMP) is a hydrophilic glycopeptide released from milk κ-casein by chymosin hydrolysis during cheese making. GMP is thought to be a potential ingredient for specific dietary applications with several health benefits. In this study GMP was characterized at the air–water interface and its behaviour was related with the self-assembly of GMP in solution as affected by pH. This GMP self-assembly was investigated by dynamic light scattering and the interfacial properties were determined by tensiometry and surface dilatational measurements at pH 4, 5 and 7. At pH 5 GMP exhibited higher surface pressure at equilibrium than at pH 7. At pH 4 the behaviour was more complex due to self-assembly close to GMP pI. Dynamic measurement showed that the adsorption/penetration rate constant (K_ads) is facilitated at higher GMP bulk concentrations, while the rate constant of rearrangement (K_r) decreased at higher GMP concentrations which could be attributed to the existence of a steric restriction due to the higher GMP load at the interface. K_r was higher at pH 5 because of lower electrostatic interactions close to the pI. The viscoelastic properties showed a complex behaviour due to the existence of protein–protein interactions depending on the GMP concentration, on the pH of the bulk and on the rates of diffusion, adsorption and rearrangement of GMP at the air–water interface.     

Tuning the anticancer activity of maltol-derived ruthenium complexes by derivatization of the 3-hydroxy-4-pyrone moiety

Organometallic ruthenium(II)-arene complexes coordinated to maltol-derived ligands were prepared and their anticancer activity against human tumor cell lines was studied. In addition, their hydrolysis behavior and reaction with 5′-GMP was tested and compared to the parent compound chlorido[2-methyl-3-(oxo-κO)-pyran-4(1H)-onato-κO4](η⁶-p-cymene)ruthenium(II) (Ru-maltol). Improved stability and in vitro anticancer activity at maintained GMP binding capability were observed, in comparison to the Ru-maltol complex.     

Cytosolic Cysteine Synthase Switch Cysteine and Mimosine Production in <Emphasis Type="Italic">Leucaena leucocephala</Emphasis>

In higher plants, multiple copies of the cysteine synthase gene are present for cysteine biosynthesis. Some of these genes also have the potential to produce various kinds of β-substitute alanine. In the present study, we cloned a 1275-bp cDNA for cytosolic O-acetylserine(thiol)lyase (cysteine synthase) (Cy-OASTL) from Leucaena leucocephala. The purified protein product showed a dual function of cysteine and mimosine synthesis. Kinetics studies showed pH optima of 7.5 and 8.0, while temperature optima of 40 and 35 °C, respectively, for cysteine and mimosine synthesis. The kinetic parameters such as apparent K_m, k_cat were determined for both cysteine and mimosine synthesis with substrates O-acetylserine (OAS) and Na₂S or 3-hydroxy-4-pyridone (3H4P). From the in vitro results with the common substrate OAS, the apparent k_cat for Cys production is over sixfold higher than mimosine synthesis and the apparent K_m is 3.7 times lower, suggesting Cys synthesis is the favored pathway.     

Carbohydrate Hydrogels with Stabilized Phage Particles for Bacterial Biosensing: Bacterium Diffusion Studies

Bacteriophage particles have been reported as potentially useful in the development of diagnosis tools for pathogenic bacteria as they specifically recognize and lyse bacterial isolates thus confirming the presence of viable cells. One of the most representative microorganisms associated with health care services is the bacterium Pseudomonas aeruginosa, which alone is responsible for nearly 15 % of all nosocomial infections. In this context, structural and functional stabilization of phage particles within biopolymeric hydrogels, aiming at producing cheap (chromogenic) bacterial biosensing devices, has been the goal of a previous research effort. For this, a detailed knowledge of the bacterial diffusion profile into the hydrogel core, where the phage particles lie, is of utmost importance. In the present research effort, the bacterial diffusion process into the biopolymeric hydrogel core was mathematically described and the theoretical simulations duly compared with experimental results, allowing determination of the effective diffusion coefficients of P. aeruginosa in the agar and calcium alginate hydrogels tested.     

A molecular-clip-based approach to cofacial zinc-porphyrin complexes

We have synthesized molecular-clip-based cofacial zinc-porphyrin complexes. The complexes are shown to be new efficient receptors for the complexation of 1,4-diazabicyclo[2,2,2]octane, acridinium ions, 1,4,5,8-naphthalene tetracarboxylic dianhydride (NTDA) and 1,2,4,5-benzenetetracarboxylic dianhydride (BTDA). Large binding constants (K_asso) in the range of 2.0 × 10⁴-1.1 × 10⁸ M⁻¹ were obtained for the 1:1 complexes of molecular tweezers (3) and rectangle (8) by inserting the hosts between the two porphyrin rings. The values of K and the spectroscopic results suggest that 3 and 8 serve as effective building blocks to capture the hosts. Semi-empirical calculations show that the guests position themselves within the cleft of the bis-porphyrins.     

Briarane-Related Diterpenoids from Octocoral Briareum stechei

A known polyoxygenated briarane, briaexcavatolide P (1), was isolated from a Formosan octocoral Briareum stechei. Moreover, the same species B. stechei, collected from Okinawan waters, yielded three chlorine-containing briaranes, including two new compounds, briastecholides B (2) and C (3) as well as a known analogue, briarenol R (4). The structures of 1–4 were established using spectroscopic methods. In addition, briarane 1 demonstrated anti-inflammatory activity in lipo-polysaccharide-induced RAW 264.7 mouse macrophage cells by suppressing the expression of inducible nitric oxide synthase (iNOS) protein.     

Cool temperature hinders flux from glucose to sucrose during cellulose synthesis in secondary wall stage cotton fibers

Current knowledge about the integration of cellulose synthesis into cellular carbon metabolism and the cool temperature sensitivity of cellulose synthesis is reviewed briefly. Roles for sucrose synthase (to channel UDP-glucose to the cellulose synthase) and sucrose phosphate synthase (to recycle the fructose released by sucrose synthase to more sucrose) in secondary wall cellulose synthesis are described. Data are presented that implicate sucrose synthesis within cotton fibers as a particularly cool temperature-sensitive step in the partitioning of carbon to cellulose. Sugar metabolism during fiber secondary wall deposition was analyzed in in vitro cultures of ovules from two cultivars of Gossypium hirsutum L. (cv. Acala SJ-1 and cv. Paymaster HS 200), which had different levels of cool temperature sensitivity. The sizes of the sucrose, glucose, and fructose pools within fibers at 4 and 7 h after a temperature shift to 15 or 34 °C did not change in either cultivar. Feeding exogenous U-¹⁴C-glucose in pulse and pulse/chase experiments showed that uptake of glucose and transport through the ovule into fibers occurred at the same rate at 34 and 15 °C. In contrast, the flux from glucose to sucrose within fibers was greatly hindered at 15 °C in both cultivars. Since sucrose is the preferred donor of UDP-Glc to the cellulose synthase during secondary wall deposition, this sensitivity in sucrose synthesis is likely to at least partially explain the cool temperature sensitivity of cotton fiber cellulose synthesis that is observed in the field.     

Molecular recognition of biotin, barbital and tolbutamide with new synthetic receptors

We have measured, by means of NMR titrations, the binding constants for the complexes between hosts N,N′-bis(6-methylpyridin-2-yl)-1,3-benzenedicarboxamide (7) and 4-chloro-N,N′-bis(6-methylpyridin-2-yl)-2,6-pyridinedicarboxamide (8, hydrated) with biotin methyl ester (1), N,N′-dimethylurea (2), 2-imidazolidone (3), N,N′-trimethylenurea (4), barbital (5) and tolbutamide (6) as guests. Molecular Mechanics calculations (Monte Carlo Conformational Search, AMBER and OPLS force fields, MacroModel v.8.1) on the complexes formed between the foregoing guests and hosts 7 and 8, comparatively with 4-oxo-N,N′-bis(6-methylpyridin-2-yl)-1,4-dihydro-2,6-pyridinedicarboxamide (9a) have been carried out in order to determine the correlation between experimental and theoretical results and to understand the behaviour of the designed new hosts. Finally we have performed single point DFT [B3LYP/6-31G(d,p)] calculations on the optimised Molecular Mechanics geometries for the complexes between hosts 7-9 and water.     

Identification and expression analysis of genes encoding putative cellulose synthases (CesA) in the hybrid aspen, Populus tremula (L.) × P. tremuloides (Michx.)

Cellulose is synthesized in plant cell walls by large membrane-bound protein complexes proposed to contain several copies of the catalytic subunit of the cellulose synthase, CesA. Here we report identification of 10 distinct CesA genes within a database of 100,000 ESTs of the hybrid aspen, Populus tremula (L.) × P. tremuloides (Michx.). Expression analyses in normal wood undergoing xylogenesis and in tension wood indicate xylem specific expression of four putative CesA isoenzymes, PttCesA1, PttCesA3-1, PttCesA3-2 and PttCesA9. Both the protein sequences and the expression profiles of PttCesA3-1 and PttCesA3-2 are very similar, and they may thus represent redundant copies of an enzyme with essentially the same function. Further, one of the generally more constitutively expressed CesA genes, PttCesA2, seems to be activated on the opposite side of a tension wood induced stem, while PttCesA6 appears to be more specific for leaf tissues. The rest of the hybrid aspen CesA genes were found to be relatively evenly expressed over the poplar tissues hereby studied.     

Study of Fluorescence System of Gmanylic Acid-Tb3+ and the Determination of Gmanylic Acid

Abstract

It was found that gmanylic acid (GMP) can be selectively completed with Tb³⁺ at pH 6.0-6.6, which then emits strong fluorescence characteristic of Tb³⁺. This reaction can be used for the determination of GMP in presence of adenylic acid (AMP), uridylic acid (UMP) and cylidylic acid (CMP). A linear relationship is obtained between the fluorescence intensity and GMP concentration in the range of 2.0×10^?7 - 1.0×10^?4M. The detection limit is 2.0×10^?8 M. The results showed that the composition ratio and apparent stability constant of GMP-Tb complex were 1:1 for GMP Tb³⁺- and 2.3×10^?5, respectively.