Molecules: Molecular Diversity

Molecules, Vol. 17, Pages 796-808: Quantum Dot- Conjugated Anti-GRP78 scFv Inhibits Cancer Growth in Mice

Weiming Xu — Mon, 16 Jan 2012 00:00:00 CET

Semiconductor quantum dots (Qdots) have recently been shown to offer significant advantages over conventional fluorescent probes to image and study biological processes. The stability and low toxicity of QDs are well suited for biological applications. Despite this, the potential of Qdots remains limited owing to the inefficiency of existing delivery methods. By conjugating Qdots with small antibody fragments targeting membrane-bound proteins, such as GRP78, we demonstrate here that the Quantum dot- Anti-GRP78 scFv (Qdot-GRP78) retains its immunospecificity and its distribution can be monitored by visualization of multi-color fluorescence imaging both in vitro and in vivo. Moreover we demonstrate here for the first time that Qdot-GRP78 scFv bioconjugates can be efficiently internalized by cancer cells, thus upregulate phophosphate-AKT-ser473 and possess biological anti-tumour activity as shown by inhibition of breast cancer growth in a xenograft model. This suggests that nanocarrier-conjugated scFvs can be used as a therapeutic antibody for cancer treatment.

Molecules, Vol. 17, Pages 145-150: The Biological Response of Spermidine Induced by Ionization Radiation

Changhyun Roh — Fri, 23 Dec 2011 00:00:00 CET

Globally, there are concerns over the risks associated with radiation exposure, so it is important to understand the biological effects of radiation exposure. Driven by the need to detect the presence of radiation exposure, biomarkers to monitor potential exposure after radiological accidents can be developed and would be extremely valuable for biological response. In this study, the behavior of spermidine as a biomarker was investigated in a C57BL/6 mouse model exposed to an acute whole-body sublethal dose of 6 Gy. The spermidine content values in serum increased for up to two days after 6 Gy irradiation. However, the enhanced spermidine content observed on day +3 in irradiated mice returned to normal levels on the subsequent five days. The result indicates that spermidine can be used as a biomarker of biological response to radiation exposure.

Molecules, Vol. 16, Pages 10570-10591: Comparison of A and B Starch Granules from Three Wheat Varieties

Jie Zeng — Mon, 19 Dec 2011 00:00:00 CET

Three starches from the wheat varieties AK58, ZM18 and YZ4110 were separated into large (A) and small (B) granules, which were characterized structurally and evaluated for their functional properties. SEM results showed that the size of A-granules from ZM18 and YZ4110 were about the same, but the sizes of A-granules and B-granules from AK58 were larger than those of ZM18 and YZ4110. FTIR spectra showed that all the samples exhibited a similar pattern, with seven main modes with maximum absorbance peaks near 3,500, 3,000, 1,600, 1,400, 1,000, 800, 500 cm−1. The B-granules of ZM18 and YZ4110 had less amylose content, although the difference among the total amylose contents of the three unfractionated starches was not significant. X-ray diffraction (XRD) patterns showed predominantly A-type crystallinity for all the starches. The A-granules showed sharper XRD patterns than the other starches. DSC analysis showed that the A-granules had broader ranges of gelatinization temperatures than the B-granules from the same wheat variety. The gelatinization enthalpy (ΔH) of A-granules was higher than that of B-granules. AK58 exhibited the smallest enthalpy, while ZM18 showed the largest enthalpy. In pasting tests, the A-granule starch of AK58 had higher peak, final and setback viscosity, lower breakdown and pasting temperature, and the B-granule starch and unfractionated starch of AK58 had lower peak, breakdown, final and setback viscosity and higher pasting temperature than ZM18 and YZ4110.

Molecules, Vol. 16, Pages 10078-10092: Genetic Diversity and Identification of Chinese-Grown Pecan Using ISSR and SSR Markers

Xiao-Dong Jia — Tue, 06 Dec 2011 00:00:00 CET

Pecan is an important horticultural nut crop originally from North America and now widely cultivated in China for its high ecological, ornamental and economic value. Currently, there are over one hundred cultivars grown in China, including introduced American cultivars and Chinese seedling breeding cultivars. Molecular markers were used to assess the genetic diversity of these cultivars and to identify the pedigrees of fine pecan plants with good characteristics and no cultivar-related data. A total of 77 samples grown in China were studied, including 14 introduced cultivars, 12 domestic seedling breeding cultivars, and 49 fine pecan plants with no cultivar data, together with Carya cathayensis and Juglans nigra. A total of 77 ISSR and 19 SSR primers were prescreened; 10 ISSR and eight SSR primers were selected, yielding a total of 94 amplified bands (100% polymorphic) in the range of 140–1,950 bp for the ISSR and 70 amplified bands (100% polymorphic) in the range of 50–350 bp for SSR markers. Genetic diversity analyses indicated Chinese-grown pecan cultivars and fine plants had significant diversity at the DNA level. The dengrograms constructed with ISSR, SSR or combined data were very similar, but showed very weak grouping association with morphological characters. However, the progeny were always grouped with the parents. The great diversity found among the Chinese cultivars and the interesting germplasm of the fine pecan plants analyzed in this study are very useful for increasing the diversity of the pecan gene pool. All 77 accessions in this study could be separated based on the ISSR and SSR fingerprints produced by one or more primers. The results of our study also showed that ISSR and SSR techniques were both suitable for genetic diversity analyses and the identification of pecan resources.

Molecules, Vol. 16, Pages 9838-9849: The Effect of Recycling Flux on the Performance and Microbial Community Composition of a Biofilm Hydrolytic-Aerobic Recycling Process Treating Anthraquinone Reactive Dyes

Yuanpeng Wang — Fri, 25 Nov 2011 00:00:00 CET

Synthetic dyes are extensively used and rarely degraded. Microbial decomposition is a cost-effective alternative to chemical and physical degradation processes. In this study, the decomposition of simulated anthraquinone reactive dye (Reactive Blue 19; RB19) at a concentration of 400-mg/L in wastewater by a biofilm hydrolytic-aerobic recycling system was investigated over a range of recycling fluxes. The 16S rDNA-based fingerprint technique was also used to investigate the microbial community composition. Results indicated that the recycling flux was a key factor that influenced RB19 degradation. The RB19 and COD removal efficiency could reach values as high as 82.1% and 95.4%, respectively, with a recycling flux of 10 mL/min. Molecular analysis indicated that some strains were similar to Aeromonadales, Tolumonas, and some uncultured clones were assumed to be potential decolorization bacteria. However, the microbial community composition in the reactors remained relatively stable at different recycling fluxes. This study provided insights on the decolorization capability and the population dynamics during the decolorization process of anthraquinone dye wastewater.

Molecules, Vol. 16, Pages 9600-9610: Interaction of the Main Components from the Traditional Chinese Drug Pair Chaihu-Shaoyao Based on Rat Intestinal Absorption

Yan Chen — Thu, 17 Nov 2011 00:00:00 CET

The Chaihu-Shaoyao drug pair (Bupleuri Radix and Paeoniae Radix Alba) which is a traditional Chinese drug pair, has been widely used for anti-inflammatory purposes. Saikosaponin a (SSA), saikosaponin d (SSD) and paeoniflorin are identified as the main components in the pair. The present study focused on the interaction of the main components based on investigating their intestinal absorption using a four-site perfused rat intestinal model in order to clarify the mechanism of the compatibility of Chaihu-Shaoyao. The concentrations of SSA, SSD and paeoniflorin in the intestinal perfusate were determined by LC/MS or UPLC (Ultra Performance Liquid Chromatography) methods, followed by P*eff (effective permeability) and 10% ABS (the percent absorption of 10 cm of intestine) calculations. The results showed that all of the three main components displayed very low permeabilities（P*eff < 0.4), which implied their poor absorption in the rat intestine. The absorption levels of SSA and SSD were similar in intestine and higher in ileum than those in other intestinal regions in the decreasing order: colon, jejunum and duodenum. However, there is no significant difference in the absorption of paeoniflorin in the four segments (P < 0.05). The P*eff values of paeoniflorin exhibited an almost 2.11-fold or 1.90-fold increase in ileum when it was co-administrated with SSA and SSD, as well as 2.42-, 2.18-fold increase in colon, respectively, whereas the absorptions of SSA and SSD were not influenced by paeoniflorin. In conclusion, SSA and SSD could promote the absorption of paeoniflorin. To some extent this might explain the nature of the compatibility mechanisms of composite formulae in TCMs.

Molecules, Vol. 16, Pages 9534-9552: The Effects of Combined Adiponectin-Metformin on Glucose and Lipids Levels in Mice and Acute Toxicity and Anti-Ulcerogenic Activity of Adiponectin Against Ethanol-Induced Gastric Mucosal Injuries in Rat

Atieh A. Fard — Tue, 15 Nov 2011 00:00:00 CET

Adiponectin is a protein hormone secreted entirely by abdominal fat tissue. It exhibits various biological activities. The present study was performed to evaluate the effects of metformin alone or in combination with adiponectin on blood glucose, TG (triglyceride), CHOL (Total cholesterol), LDL (Low density lipoprotein) and HDL (High density lipoprotein) levels in mice and also to evaluate the anti-ulcerogenic activity of adiponectin against ethanol induced gastric mucosal injury in rats. Three groups of mice were gavaged with 1% volume/body weight high fat-sucrose. Metformin at a dosage of 250 mg/kg was added to the feed and a dosage of 2.5 mg/kg adiponectin was injected intraperitoneally (i.p). Blood glucose was measured at one hour intervals for five hours. Blood concentrations of TG, CHOL, LDL and HDL were also measured at the end of the fifth hour of the experiment. On the other hand, four groups of adult healthy rats were i.p. injected with distilled water, omeprazole 20 mg/kg, 2.5 mg/kg and 5 mg/kg adiponectin one hour before oral administration of absolute ethanol to generate gastric mucosal injury. After an additional hour the rats were sacrificed and the ulcer areas of the gastric walls were determined. Furthermore, an acute toxicity study has indicated no mortality with 5 mg/kg dose of adiponectin injected i.p in rats and no major clinical signs of toxicity were observed. The results indicate that the effect of a combination of metformin and adiponectin on blood glucose and HDL is quite effective. Histology of the gastric wall of negative control rats revealed severe damage of gastric mucosa, along with edema and leucocyte infiltration of the submucosal layer compared to rats pre-treated with either omeprazole or adiponectin extract where there was marked gastric protection along with reduction or inhibition of edema and leucocytes infiltration. The results suggest that combination of metfomin and adiponectin give a promising antidiabetic effect and also, adiponectin promotes ulcer protection as ascertained by the comparative decrease of ulcer areas, reduction of edema and leucocytes infiltration of the submucosal layer.

Molecules, Vol. 16, Pages 9077-9089: Molecular Characterization and Tandem Mass Spectrometry of the Lectin Extracted from the Seeds of Dioclea sclerocarpa Ducke

Jorge Luis Almeida Correia — Fri, 28 Oct 2011 00:00:00 CEST

Lectin from the seeds of Dioclea sclerocarpa (DSL) was purified in a single step by affinity chromatography on a Sephadex G-50 column. The primary sequence, as determined by tandem mass spectrometry, revealed a protein with 237 amino acids and 81% of identity with ConA. DSL has a molecular mass of 25,606 Da. The β and γ chains weigh 12,873 Da and 12,752 Da, respectively. DSL hemagglutinated rabbit erythrocytes (both native and treated with proteolytic enzymes), showing stability even after one hour of exposure to a specific pH range. The hemagglutinating activity of DSL was optimal between pH 6.0 and 8.0, but was inhibited after incubation with D-galactose and D-glucose. The pure protein possesses a molecular mass of 25 kDa by SDS-PAGE and 25,606 Da by mass spectrometry. The secondary structure content was estimated using the software SELCON3. The results indicate that b-sheet secondary structures are predominant in DSL (approximately 42.3% antiparallel b-sheet and 6.7% parallel b-sheet). In addition to the b-sheet, the predicted secondary structure of DSL features 4.1% a-helices, 15.8% turns and 31.3% other contributions. Upon thermal denaturation, evaluated by measuring changes in ellipticity at 218 nm induced by a temperature increase from 20 °C to 98 °C, DSL displayed cooperative sigmoidal behavior with transition midpoint at 84 °C and permitted the observation of two-state model (native and denatured).

Molecules, Vol. 16, Pages 8463-8474: An Unexpected Reaction between 5-Hydroxymethylfurfural and Imidazolium-Based Ionic Liquids at High Temperatures

Zehui Zhang — Tue, 11 Oct 2011 00:00:00 CEST

A new compound was detected during the production of 5-hydroxymethylfurfural (HMF) from glucose and cellulose in the ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) at high temperatures. Further experiments found that it was derived from the reaction of HMF with [Bmim]Cl. The structure of new compound was established as 1-butyl-2-(5’-methyl-2’-furoyl)imidazole (BMI) based on nuclear magnetic resonance and mass spectrometry analysis, and a possible mechanism for its formation was proposed. Reactions of HMF with other imidazolium-based ionic liquids were performed to check the formation of BMI. Our results provided new insights in terms of side reactions between HMF and imidazolium-based ionic liquids, which should be valuable for designing better processes for the production of furans using biomass and related materials.

Molecules, Vol. 16, Pages 8319-8331: Role of Kupffer Cells in Thioacetamide-Induced Cell Cycle Dysfunction

Mirandeli Bautista — Thu, 29 Sep 2011 00:00:00 CEST

It is well known that gadolinium chloride (GD) attenuates drug-induced hepatotoxicity by selectively inactivating Kupffer cells. In the present study the effect of GD in reference to cell cycle and postnecrotic liver regeneration induced by thioacetamide (TA) in rats was studied. Two months male rats, intraveously pretreated with a single dose of GD (0.1 mmol/Kg), were intraperitoneally injected with TA (6.6 mmol/Kg). Samples of blood and liver were obtained from rats at 0, 12, 24, 48, 72 and 96 h following TA intoxication. Parameters related to liver damage were determined in blood. In order to evaluate the mechanisms involved in the post-necrotic regenerative state, the levels of cyclin D and cyclin E as well as protein p27 and Proliferating Cell Nuclear Antigen (PCNA) were determined in liver extracts because of their roles in the control of cell cycle check-points. The results showed that GD significantly reduced the extent of necrosis. Noticeable changes were detected in the levels of cyclin D1, cyclin E, p27 and PCNA when compared to those induced by thioacetamide. Thus GD pre-treatment reduced TA-induced liver injury and accelerated the postnecrotic liver regeneration. These results demonstrate that Kupffer cells are involved in TA-induced liver and also in the postnecrotic proliferative liver states.

Molecules, Vol. 16, Pages 7691-7705: Synthesis and Transformations of di-endo-3-Aminobicyclo-[2.2.2]oct-5-ene-2-carboxylic Acid Derivatives

Márta Palkó — Wed, 07 Sep 2011 00:00:00 CEST

all-endo-3-amino-5-hydroxybicyclo[2.2.2]octane-2-carboxylic acid (13) and all-endo-5-amino-6-(hydroxymethyl)bicyclo[2.2.2]octan-2-ol (10) were prepared via dihydro-1,3-oxazine or g-lactone intermediates by the stereoselective functionalization of an N-protected derivative of endo-3-aminobicyclo[2.2.2]oct-5-ene-2-carboxylic acid (2). Ring closure of b-amino ester 4 resulted in tricyclic pyrimidinones 15 and 16. The structures, stereochemistry and relative configurations of the synthesized compounds were determined by IR and NMR.

Molecules, Vol. 16, Pages 5916-5927: Industrial Aplication of Catalytic Systems for n-Heptane Isomerization

Laura Olivia Alemán-Vázquez — Thu, 14 Jul 2011 00:00:00 CEST

The ideal gasoline must have a high pump octane number, in the 86 to 94 range, and a low environmental impact. Alkanes, as a family, have much lower photochemical reactivities than aromatics or olefins, but only the highly branched alkanes have adequate octane numbers. The purpose of this work is to examine the possibilities of extending the technological alternative of paraffin isomerization to heavier feedstocks (i.e., n-heptane) using non-conventional catalytic systems which have been previously proposed in the literature: a Pt/sulfated zirconia catalyst and a molybdenum sub-oxide catalyst. Under the experimental conditions at which these catalysts have been evaluated, the molybdenum sub-oxide catalyst maintains a good activity and selectivity to isomerization after 24 h, while the Pt/sulfated zirconia catalyst shows a higher dimethylpentanes/methylhexanes ratio, probably due to a lower operating temperature, but also a high formation of cracking products, and presents signs of deactivation after 8 h. Though much remains to be done, the performance of these catalysts indicates that there are good perspectives for their industrial application in the isomerization of n-heptane and heavier alkanes.

Molecules, Vol. 16, Pages 5402-5421: Keeping an Eye on Myocilin: A Complex Molecule Associated with Primary Open-Angle Glaucoma Susceptibility

Farid Menaa — Mon, 27 Jun 2011 00:00:00 CEST

MYOC encodes a secretary glycoprotein of 504 amino acids named myocilin. MYOC is the first gene to be linked to juvenile open-angle glaucoma (JOAG) and some forms of adult-onset primary open-angle glaucoma (POAG). The gene was identified as an up-regulated molecule in cultured trabecular meshwork (TM) cells after treatment with dexamethasone and was originally referred to as trabecular meshwork-inducible glucocorticoid response (TIGR). Elevated intraocular pressure (IOP), due to decreased aqueous outflow, is the strongest known risk factor for POAG. Increasing evidence showed that the modulation of the wild-type (wt) myocilin protein expression is not causative of glaucoma while some misfolded and self-assembly aggregates of mutated myocilin may be associated with POAG in related or unrelated populations. The etiology of the disease remains unclear. Consequently, a better understanding of the molecular mechanisms underlyingPOAG is required to obtain early diagnosis, avoid potential disease progression, and develop new therapeutic strategies. In the present study, we review and discuss the most relevant studies regarding structural characterizations, expressions, molecular interactions, putative functions of MYOC gene and/or its corresponding protein in POAG etiology.

Molecules, Vol. 16, Pages 4923-4936: Derivatives of 10,16-Dihydroxyhexadecanoic Acid Isolated from Tomato (Solanum lycopersicum) as Potential Material for Aliphatic Polyesters

Daniel Arrieta-Baez — Wed, 15 Jun 2011 00:00:00 CEST

The main monomer of tomato cuticle, 10,16-dihydroxyhexadecanoic acid (or 10,16-dihydroxypalmitic acid; 10,16-DHPA), was isolated and used to efficiently synthesize two different monomers (16-hydroxy-10-oxo-hexadecanoic and 7-oxohexa-decanedioic acids) in addition to a dimer and linear and branched trimers. These compounds were fully characterized using NMR and MS techniques and could be used as starting materials for the synthesis of a wide range of chemicals and bio-polyesters, particularly the latter due to their physical properties, non-toxicity, and relative abundance among raw materials.

Molecules, Vol. 16, Pages 2599-2608: Studies on the Genetic Variation of the Green Unicellular Alga Haematococcus pluvialis (Chlorophyceae) Obtained from Different Geographical Locations Using ISSR and RAPD Molecular Marker

Noroozi Mostafa — Tue, 22 Mar 2011 00:00:00 CET

Haematococcus pluvialis (Flotow) is a unicellular green alga, which is considered to be the best astaxanthin-producing organism. Molecular markers are suitable tools for the purpose of finding out genetic variations in organisms; however there have been no studies conducted on ISSR or RAPD molecular markers for this organism. The DNA of 10 different strains of H. pluvialis (four strains from Iran, two strains from Finland, one strain from Switzerland and three strains from the USA) was extracted. A genetic similarity study was carried out using 14 ISSR and 12 RAPD primers. Moreover, the molecular weights of the bands produced ranged from 0.14 to 3.4 Kb. The PCA and dendrogram clustered the H. pluvialis strains into various groups according to their geographical origin. The lowest genetic similarity was between the Iran2 and USA2 strains (0.08) and the highest genetic similarity was between Finland1 and Finland2 (0.64). The maximum numbers of bands produced by the ISSR and RAPD primers were 35 and 6 bands, respectively. The results showed that ISSR and RAPD markers are useful for genetic diversity studies of Haematococcus as they showed geographical discrimination.

Molecules, Vol. 16, Pages 2561-2582: Purification, Biochemical Characterization, and Bioactive Properties of a Lectin Purified from the Seeds of White Tepary Bean (Phaseolus Acutifolius Variety Latifolius)

Carmen Valadez-Vega — Mon, 21 Mar 2011 00:00:00 CET

The present work shows the characterization of Phaseolus acutifolius variety latifolius, on which little research has been published, and provides detailed information on the corresponding lectin. This protein was purified from a semi-domesticated line of white tepary beans from Sonora, Mexico, by precipitation of the aqueous extract with ammonium sulfate, followed by affinity chromatography on an immobilized fetuin matrix. MALDI TOF analysis of Phaseolus acutifolius agglutinin (PAA) showed that this lectin is composed of monomers with molecular weights ranging between 28 and 31 kDa. At high salt concentrations, PAA forms a dimer of 63 kDa, but at low salt concentrations, the subunits form a tetramer. Analysis of PAA on 2D-PAGE showed that there are mainly three types of subunits with isoelectric points of 4.2, 4.4, and 4.5. The partial sequence obtained by LC/MS/MS of tryptic fragments from the PAA subunits showed 90–100% identity with subunits from genus Phaseolus lectins in previous reports. The tepary bean lectin showed lower hemagglutination activity than Phaseolus vulgaris hemagglutinin (PHA-E) toward trypsinized human A and O type erythrocytes. The hemagglutination activity was inhibited by N-glycans from glycoproteins. Affinity chromatography with the immobilized PAA showed a high affinity to glycopeptides from thyroglobulin, which also has N-glycans with a high content of N-acetylglucosamine. PAA showed less mitogenic activity toward human lymphocytes than PHA-L and Con A. The cytotoxicity of PAA was determined by employing three clones of the 3T3 cell line, demonstrating variability among the clones as follows: T4 (DI50 51.5 µg/mL); J20 (DI50 275 µg/mL), and N5 (DI50 72.5 µg/mL).

Molecules, Vol. 16, Pages 2107-2118: Detection of Cytotoxic Activity of Lectin on Human Colon Adenocarcinoma (Sw480) and Epithelial Cervical Carcinoma (C33-A)

Carmen Valadez-Vega — Wed, 02 Mar 2011 00:00:00 CET

Lectins comprise a heterogeneous class of proteins that recognize the carbohydrate moieties of glycoconjugates with high specificity. Numerous studies have shown that lectins are capable of recognizing specific carbohydrate moieties displayed by malignant cells or tissues. The present work was performed to investigate the effects of tepary bean (Phaseolus acutifolius) lectins on proliferation, colony formation, and alteration of DNA synthesis of human malignant cells. Tepary bean lectin showed dose dependent effects on the inhibition of viability as well as on colony formation in two human malignant cells lines (C33-A, Sw480); By contrast, tepary bean lectin only showed significant effects on DNA synthesis on Sw480 cells. Our results provide evidence of the anti- proliferative and cytotoxic effects of the tepary bean lectins on C33-A and Sw480 cells lines.

Molecules, Vol. 16, Pages 1378-1388: Effects of Rutin and Hesperidin and Their Al(III) and Cu(II) Complexes on in Vitro Plasma Coagulation Assays

Vesna Kuntić — Mon, 07 Feb 2011 00:00:00 CET

Two flavonoids, rutin and hesperidin, were investigated in vitro for anticoagulant activity through coagulation tests: activated partial thromboplastin time (aPTT), prothrombin time (PT) and thrombin time (TT). Only an ethanolic solution of rutin at the concentration of 830 µM prolonged aPTT, while TT and PT were unaffected. In order to evaluate whether the prolongation of aPTT was due to the decrease of coagulation factors, the experiment with deficient plasma was performed, showing the effects on factors VIII and IX. Since pharmacological activity of flavonoids is believed to increase when they are coordinated with metal ions, complexes of these flavonoids with Al(III) and Cu(II) ions were also tested. The results showed that complexes significantly prolonged aPTT and had no effects on PT and TT. Assay with deficient plasma (plasma having the investigated factor at less then 1%) revealed that complexes could bind to the coagulation factors, what may lead to a non-specific inhibition and aPTT prolongation. An effort was made to correlate stability of complexes with their anticoagulant properties.

Molecules, Vol. 16, Pages 915-932: X-Ray Supramolecular Structure, NMR Spectroscopy and Synthesis of 3-Methyl-1-phenyl-1H-chromeno[4,3-c]pyrazol-4-ones Formed by the Unexpected Cyclization of 3-[1-(Phenyl-hydrazono)ethyl]-chromen-2-ones

Itzia I. Padilla-Martinez — Fri, 21 Jan 2011 00:00:00 CET

The molecular structures of nine 3-methyl-1-phenyl-1H-chromeno[4,3-c]pyrazol-4-one isomers, obtained by the oxidative cyclization of the corresponding 1-phenylhydrazono chromen-2-ones with copper acetate as catalyst, are reported. The molecular and supramolecular structures of the 8-chloro, 8-bromo- and 8-nitro isomers 2b-d, were established by X-ray diffraction. The halogenated isomers 2b and 2c are isomorphs, they crystallize as a triclinic system, space group P-1 with two molecules in the asymmetric unit. Compound 2d crystallizes as a monoclinic system, space group P21/m with two molecules in the unit cell. The 1-phenyl ring [Cg(4)] is almost perpendicularly positioned to the chromene-pyrazole ring system. This conformation is in agreement with the anisotropic NMR shielding effect exerted by the phenyl ring over H-9 in solution. The supramolecular architecture is almost controlled by C―H···A (A = O, p) and face to face p-stacking interactions. The observed p-stacking trend between chromene and pyrazole rings is given by the overlapping between the best donor and acceptor rings in each compound.

Molecules, Vol. 15, Pages 5525-5546: Nanofabrication of Nonfouling Surfaces for Micropatterning of Cell and Microtissue

Hidenori Otsuka — Tue, 10 Aug 2010 00:00:00 CEST

Surface engineering techniques for cellular micropatterning are emerging as important tools to clarify the effects of the microenvironment on cellular behavior, as cells usually integrate and respond the microscale environment, such as chemical and mechanical properties of the surrounding fluid and extracellular matrix, soluble protein factors, small signal molecules, and contacts with neighboring cells. Furthermore, recent progress in cellular micropatterning has contributed to the development of cell-based biosensors for the functional characterization and detection of drugs, pathogens, toxicants, and odorants. In this regards, the ability to control shape and spreading of attached cells and cell-cell contacts through the form and dimension of the cell-adhesive patches with high precision is important. Commitment of stem cells to different specific lineages depends strongly on cell shape, implying that controlled microenvironments through engineered surfaces may not only be a valuable approach towards fundamental cell-biological studies, but also of great importance for the design of cell culture substrates for tissue engineering. To develop this kind of cellular microarray composed of a cell-resistant surface and cell attachment region, micropatterning a protein-repellent surface is important because cellular adhesion and proliferation are regulated by protein adsorption. The focus of this review is on the surface engineering aspects of biologically motivated micropatterning of two-dimensional surfaces with the aim to provide an introductory overview described in the literature. In particular, the importance of non-fouling surface chemistries is discussed.

Molecules, Vol. 15, Pages 3010-3037: Assay Format as a Critical Success Factor for Identification of Novel Inhibitor Chemotypes of Tissue-Nonspecific Alkaline Phosphatase from High-Throughput Screening

Chung — Tue, 27 Apr 2010 00:00:00 CEST

The tissue-nonspecific alkaline phosphatase (TNAP) isozyme is centrally involved in the control of normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleoside triphosphate pyrophosphohydrolase-1 (NPP1) and the Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PPi), a potent inhibitor of mineralization. In this review we describe the serial development of two miniaturized high-throughput screens (HTS) for TNAP inhibitors that differ in both signal generation and detection formats, but more critically in the concentrations of a terminal alcohol acceptor used. These assay improvements allowed the rescue of the initially unsuccessful screening campaign against a large small molecule chemical library, but moreover enabled the discovery of several unique classes of molecules with distinct mechanisms of action and selectivity against the related placental (PLAP) and intestinal (IAP) alkaline phosphatase isozymes. This illustrates the underappreciated impact of the underlying fundamental assay configuration on screening success, beyond mere signal generation and detection formats.

Molecules, Vol. 15, Pages 2730-2748: MMPBSA Decomposition of the Binding Energy throughout a Molecular Dynamics Simulation of Amyloid-Beta (Aß10−35) Aggregation

Campanera — Thu, 15 Apr 2010 00:00:00 CEST

Recent experiments with amyloid-beta (Aβ) peptides indicate that the formation of toxic oligomers may be an important contribution to the onset of Alzheimer’s disease. The toxicity of Aβ oligomers depend on their structure, which is governed by assembly dynamics. However, a detailed knowledge of the structure of at the atomic level has not been achieved yet due to limitations of current experimental techniques. In this study, replica exchange molecular dynamics simulations are used to identify the expected diversity of dimer conformations of Aβ10−35 monomers. The most representative dimer conformation has been used to track the dimer formation process between both monomers. The process has been characterized by means of the evolution of the decomposition of the binding free energy, which provides an energetic profile of the interaction. Dimers undergo a process of reorganization driven basically by inter-chain hydrophobic and hydrophilic interactions and also solvation/desolvation processes.

Molecules, Vol. 15, Pages 2609-2622: High-Throughput Behavioral Screens: the First Step towards Finding Genes Involved in Vertebrate Brain Function Using Zebrafish

Gerlai — Mon, 12 Apr 2010 00:00:00 CEST

The zebrafish has been in the forefront of developmental biology for three decades and has become a favorite of geneticists. Due to the accumulated genetic knowledge and tools developed for the zebrafish it is gaining popularity in other disciplines, including neuroscience. The zebrafish offers a compromise between system complexity (it is a vertebrate similar in many ways to our own species) and practical simplicity (it is small, easy to keep, and prolific). Such features make zebrafish an excellent choice for high throughput mutation and drug screening. For the identification of mutation or drug induced alteration of brain function arguably the best methods are behavioral test paradigms. This review does not present experimental examples for the identification of particular genes or drugs. Instead it describes how behavioral screening methods may enable one to find functional alterations in the vertebrate brain. Furthermore, the review is not comprehensive. The behavioral test examples presented are biased according to the personal interests of the author. They will cover research areas including learning and memory, fear and anxiety, and social behavior. Nevertheless, the general principles will apply to other functional domains and should represent a snapshot of the rapidly evolving behavioral screening field with zebrafish.

Molecules, Vol. 15, Pages 2478-2490: Automation in the High-throughput Selection of Random Combinatorial Libraries—Different Approaches for Select Applications

Glökler — Thu, 08 Apr 2010 00:00:00 CEST

Automation in combination with high throughput screening methods has revolutionised molecular biology in the last two decades. Today, many combinatorial libraries as well as several systems for automation are available. Depending on scope, budget and time, a different combination of library and experimental handling might be most effective. In this review we will discuss several concepts of combinatorial libraries and provide information as what to expect from these depending on the given context.

Molecules, Vol. 15, Pages 1987-1999: QSAR Models for Reproductive Toxicity and Endocrine Disruption Activity

Novič — Mon, 22 Mar 2010 00:00:00 CET

Reproductive toxicity is an important regulatory endpoint, which is required in registration procedures of chemicals used for different purposes (for example pesticides). The in vivo tests are expensive, time consuming and require large numbers of animals, which must be sacrificed. Therefore an effort is ongoing to develop alternative In vitro and in silico methods to evaluate reproductive toxicity. In this review we describe some modeling approaches. In the first example we describe the CAESAR model for prediction of reproductive toxicity; the second example shows a classification model for endocrine disruption potential based on counter propagation artificial neural networks; the third example shows a modeling of relative binding affinity to rat estrogen receptor, and the fourth one shows a receptor dependent modeling experiment.

Molecules, Vol. 15, Pages 1690-1704: HTS-Driven Discovery of New Chemotypes with West Nile Virus Inhibitory Activity

Dong Hoon Chung — Fri, 12 Mar 2010 00:00:00 CET

West Nile virus (WNV) is a positive sense, single-stranded RNA virus that can cause illness in humans when transmitted via mosquito vectors. Unfortunately, no antivirals or vaccines are currently available, and therefore efficient and safe antivirals are urgently needed. We developed a high throughput screen to discover small molecule probes that inhibit virus infection of Vero E6 cells. A primary screen of a 13,001 compound library at a 10 µM final concentration was conducted using the 384-well format. Z′ values ranged from 0.54–0.83 with a median of 0.74. Average S/B was 17 and S/N for each plate ranged from 10.8 to 23.9. Twenty-six compounds showed a dose response in the HT screen and were further evaluated in a time of addition assay and in a titer reduction assay. Seven compounds showed potential as small molecule probes directed at WNV. The hit rate from the primary screen was 0.185% (24 compounds out of 13,001 compounds) and from the secondary screens was 0.053% (7 out of 13,001 compounds) respectively.

Molecules, Vol. 15, Pages 1645-1667: Hitherto Unrecognized Fluorescence Properties of Coniferyl Alcohol

Komandoor Elayavalli Achyuthan — Thu, 11 Mar 2010 00:00:00 CET

We instituted a quasi-quality assurance program for demonstrating coniferyl alcohol’s fluorescence and fluorescence diminishment following enzymatic oxidation. The magnitude of diminishment was a measure of catalysis. High throughput screening was performed in pseudo-kinetic and endpoint modes by measuring the fluorescence at 416 nm following excitation at 290, 310 or 340 nm. Dose-response tracings were linear between two and three orders of magnitude with average limits of detection and quantitation of 1.8 and 6.9 mM coniferyl alcohol, respectively. Oxidation was evident with 0.025 mg/mL laccase or 0.003 mg/mL peroxidase or inside 5 min using 0.5 mg/mL laccase or 5 mM substrate. Sodium chloride inhibited (IC50, 25 mM) laccase oxidation of coniferyl alcohol. Fluorescence from 10 concentrations (1 to 1000 mM) of coniferyl alcohol was stable for 24 hours over 14 excitation/emission cycles at 3 different combinations of excitation and emission wavelengths. In conclusion, coniferyl alcohol absorption and fluorescence assays should facilitate biomass lignin analyses and improve delignification.

Molecules, Vol. 15, Pages 1590-1631: Preparation of 16β-Estradiol Derivative Libraries as Bisubstrate Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 1 Using the Multidetachable Sulfamate Linker

Marie Bérubé — Wed, 10 Mar 2010 00:00:00 CET

Combinatorial chemistry is a powerful tool used to rapidly generate a large number of potentially biologically active compounds. In our goal to develop bisubstrate inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) that interact with both the substrate (estrone or estradiol) and the cofactor (NAD(P)H) binding sites, we used parallel solid-phase synthesis to prepare three libraries of 16β-estradiol derivatives with two or three levels of molecular diversity. From estrone, we first synthesized a sulfamate precursor that we loaded on trityl chloride resin using the efficient multidetachable sulfamate linker strategy recently developed in our laboratory. We then introduced molecular diversity [one or two amino acid(s) followed by a carboxylic acid] on steroid nucleus by Fmoc peptide chemistry. Finally, after a nucleophilic cleavage, libraries of 30, 63 and 25 estradiol derivatives were provided. A library of 30 sulfamoylated estradiol derivatives was also generated by acidic cleavage and its members were screened for inhibition of steroid sulfatase. Biological evaluation on homogenated HEK-293 cells overexpressing 17β-HSD1 of the estradiol derivatives carrying different oligoamide-type chains at C-16 first revealed that three levels of molecular diversity (a spacer of two amino acids) were necessary to interact with the adenosine part of the cofactor binding site. Second, the best inhibition was obtained when hydrophobic residues (phenylalanine) were used as building blocks.

Molecules, Vol. 15, Pages 215-225: Screening and Improvement of an Anti-VEGF DNA Aptamer

Yoshihiko Nonaka — Thu, 07 Jan 2010 00:00:00 CET

To obtain an aptamer with a high affinity for vascular endothelial growth factor (VEGF), we focused on the receptor-binding domain (RBD) of VEGF as a target epitope. Three rounds of screening gave Vap7, which bound to the VEGF isoforms VEGF121 and VEGF165 with KD values of 1.0 nM and 20 nM, respectively. Moreover, Vap7 showed specificity within the VEGF family. Secondary structure predictions and circular dicrhoism suggested that Vap7 folds into a G-quadruplex structure. We obtained a mutant aptamer that contains only this region of the aptamer sequence. This truncated mutant (V7t1) bound to both VEGF121 and VEGF165 with KD values of 1.1 nM and 1.4 nM, respectively. Its sequence was 5'-TGTGGGGGTGGACGGGCCGGGTAGA-3', and it appeared to form a G-quadruplex structure. We also produced an aptamer heterodimer consisting of our previously derived aptamer (del5-1), which binds to the heparin-binding domain of VEGF, linked to V7t1. The resulting heterodimer bound strongly to VEGF165 with a KD value of 4.7 × 102 pM.

Molecules, Vol. 15, Pages 1-11: Upgrading SELEX Technology by Using Lambda Exonuclease Digestion for Single-Stranded DNA Generation

Meltem Avci-Adali — Thu, 24 Dec 2009 00:00:00 CET

The generation of single-stranded DNA (ssDNA) molecules plays a key role in the SELEX (Systematic Evolution of Ligands by EXponential enrichment) combinatorial chemistry process and numerous molecular biology techniques and applications, such as DNA sequencing, single-nucleotide polymorphism (SNP) analysis, DNA chips, DNA single-strand conformation polymorphism (SSCP) analysis and many other techniques. The purity and yield of ssDNA can affect the success of each application. This study compares the two ssDNA production methods, the strand separation by streptavidin-coated magnetic beads and alkaline denaturation and the lambda exonuclease digestion, in regard to the purity of generated ssDNA and the efficiency. Here, we demonstrate the considerable benefits of ssDNA production by lambda exonuclease digestion for in vitro selection of DNA aptamers. We believe that the generation of ssDNA aptamers using this method will greatly improve the success rate of SELEX experiments concerning the recovery of target-specific aptamers.

Molecules, Vol. 14, Pages 5223-5234: Generation of 500-Member Library of 10-Alkyl-2-R1,3-R2-4,10-Dihydrobenzo[4,5]imidazo[1,2-α]pyrimidin-4-ones

Svetlana M. Sirko — Tue, 15 Dec 2009 00:00:00 CET

Representative benzimidazopyrimidinones were previously reported to be intercalating antitumor agents. In this work, we used 2-substituted 4,10-dihydrobenzo [4,5]imidazo[1,2-α]pyriminin-4-ones for their diversification by regioselective alkylation. Under the conditions established, the alkylation gave 10-alkyl derivatives which permitted the parallel generation of a 500-member library of the title compounds.

Molecules, Vol. 14, Pages 5179-5188: Pore Forming Properties of Cecropin-Melittin Hybrid Peptide in a Natural Membrane

Alberto Milani — Fri, 11 Dec 2009 00:00:00 CET

The pore forming properties of synthetic cecropin-melittin hybrid peptide (Acetyl-KWKLFKKIGAVLKVL-CONH2; CM15) were investigated by using photoreceptor rod outer segments (OS) isolated from frog retinae obtained by using the whole-cell configuration of the patch-clamp technique. CM15 was applied (and removed) to (from) the OS in ~50 ms with a computer-controlled microperfusion system. Once the main OS endogenous conductance was blocked with light, the OS membrane resistance was ≥1 GΩ, allowing high resolution, low-noise recordings. Different to alamethicines, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate a toroidal mechanism of pore formation by CM15.

Molecules, Vol. 14, Pages 4758-4778: Spectroscopic Analyses of the Biofuels-Critical Phytochemical Coniferyl Alcohol and Its Enzyme-Catalyzed Oxidation Products

Komandoor Elayavalli Achyuthan — Mon, 23 Nov 2009 00:00:00 CET

Lignin composition (monolignol types of coniferyl, sinapyl or p-coumaryl alcohol) is causally related to biomass recalcitrance. We describe multiwavelength (220, 228, 240, 250, 260, 290, 295, 300, 310 or 320 nm) absorption spectroscopy of coniferyl alcohol and its laccase- or peroxidase-catalyzed products during real time kinetic, pseudokinetic and endpoint analyses, in optical turn on or turn off modes, under acidic or basic conditions. Reactions in microwell plates and 100 μL volumes demonstrated assay miniaturization and high throughput screening capabilities. Bathochromic and hypsochromic shifts along with hyperchromicity or hypochromicity accompanied enzymatic oxidations by laccase or peroxidase. The limits of detection and quantitation of coniferyl alcohol averaged 2.4 and 7.1 μM respectively, with linear trend lines over 3 to 4 orders of magnitude. Coniferyl alcohol oxidation was evident within 10 minutes or with 0.01 μg/mL laccase and 2 minutes or 0.001 μg/mL peroxidase. Detection limit improved to 1.0 μM coniferyl alcohol with Km of 978.7 ± 150.7 μM when examined at 260 nm following 30 minutes oxidation with 1.0 μg/mL laccase. Our assays utilized the intrinsic spectroscopic properties of coniferyl alcohol or its oxidation products for enabling detection, without requiring chemical synthesis or modification of the substrate or product(s). These studies facilitate lignin compositional analyses and augment pretreatment strategies for reducing biomass recalcitrance.

Molecules, Vol. 14, Pages 4707-4715: Chemosystematic Value of the Essential Oil Composition of Thuja species Cultivated in Poland—Antimicrobial Activity

Dimitroula Tsiri — Thu, 19 Nov 2009 00:00:00 CET

In the framework of the correlation between chemotaxonomy and chemical analysis studies, the chemical composition of the essential oils of four varieties of Thuja species cultivated in Poland − T. occidentalis ‘globosa’, T. occidentalis ‘aurea’, T. plicata and T. plicata ‘gracialis’ − were investigated by GC and GC-MS. Thirty-one compounds were identified from T. occidentalis ‘globosa’, representing 96.92% of the total oil; twenty-seven from T. occidentalis ‘aurea’ (94.34%); thirty-one from T. plicata (94.75%); and thirty compounds from T. plicata ‘gracialis’ (96.36%). The main constituents in all samples were the monoterpene ketones α- and β-thujone, fenchone and sabinene, as well as the diterpenes beyerene and rimuene.The chemosystematic value of the total ketone content of all samples (which varied from 54.30–69.18%) has been discussed and investigated. The constituents, beyerene and the mixture of α- and β-thujone, were isolated from the oils and tested against six Gram-positive and -negative bacteria and three pathogenic fungi. The oils of the two T. plicata species exhibited significant antimicrobial activity, while the mixture of α- and β-thujone showed very strong activity as well.

Molecules, Vol. 14, Pages 4546-4569: Simple Sequence Repeat Polymorphisms (SSRPs) for Evaluation of Molecular Diversity and Germplasm Classification of Minor Crops

Yong-Jin Park — Tue, 10 Nov 2009 00:00:00 CET

Evaluation of the genetic diversity among populations is an essential prerequisite for the preservation of endangered species. Thousands of new accessions are introduced into germplasm institutes each year, thereby necessitating assessment of their molecular diversity before elimination of the redundant genotypes. Of the protocols that facilitate the assessment of molecular diversity, SSRPs (simple sequence repeat polymorphisms) or microsatellite variation is the preferred system since it detects a large number of DNA polymorphisms with relatively simple technical complexity. The paucity of information on DNA sequences has limited their widespread utilization in the assessment of genetic diversity of minor or neglected crop species. However, recent advancements in DNA sequencing and PCR technologies in conjunction with sophisticated computer software have facilitated the development of SSRP markers in minor crops. This review examines the development and molecular nature of SSR markers, and their utilization in many aspects of plant genetics and ecology.

Molecules, Vol. 14, Pages 3436-3445: The Use of MoStBioDat for Rapid Screening of Molecular Diversity

Andrzej Bak — Tue, 08 Sep 2009 00:00:00 CEST

MoStBioDat is a uniform data storage and extraction system with an extensive array of tools for structural similarity measures and pattern matching which is essential to facilitate the drug discovery process. Structure-based database screening has recently become a common and efficient technique in early stages of the drug development, shifting the emphasis from rational drug design into the probability domain of more or less random discovery. The virtual ligand screening (VLS), an approach based on high-throughput flexible docking, samples a virtually infinite molecular diversity of chemical libraries increasing the concentration of molecules with high binding affinity. The rapid process of subsequent examination of a large number of molecules in order to optimize the molecular diversity is an attractive alternative to the traditional methods of lead discovery. This paper presents the application of the MoStBioDat package not only as a data management platform but mainly in substructure searching. In particular, examples of the applications of MoStBioDat are discussed and analyzed.

Molecules, Vol. 14, Pages 2717-2728: Headspace, Volatile and Semi-Volatile Organic Compounds Diversity and Radical Scavenging Activity of Ultrasonic Solvent Extracts from Amorpha fruticosa Honey Samples

Igor Jerković — Mon, 27 Jul 2009 00:00:00 CEST

Volatile organic compounds of Amorpha fruticosa honey samples were isolated by headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE), followed by gas chromatography and mass spectrometry analyses (GC, GC-MS), in order to obtain complementary data for overall characterization of the honey aroma. The headspace of the honey was dominated by 2-phenylethanol (38.3–58.4%), while other major compounds were trans- and cis-linalool oxides, benzaldehyde and benzyl alcohol. 2-Phenylethanol (10.5–16.8%) and methyl syringate (5.8–8.2%) were the major compounds of ultrasonic solvent extracts, with an array of small percentages of linalool, benzene and benzoic acid derivatives, aliphatic hydrocarbons and alcohols, furan derivatives and others. The scavenging ability of the series of concentrations of the honey ultrasonic solvent extracts and the corresponding honey samples was tested by a DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. Approximately 25 times lower concentration ranges (up to 2 g/L) of the extracts exhibited significantly higher free radical scavenging potential with respect to the honey samples.

Molecules, Vol. 14, Pages 1660-1701: On Two Novel Parameters for Validation of Predictive QSAR Models

Partha Pratim Roy — Wed, 29 Apr 2009 00:00:00 CEST

Validation is a crucial aspect of quantitative structure–activity relationship (QSAR) modeling. The present paper shows that traditionally used validation parameters (leave-one-out Q2 for internal validation and predictive R2 for external validation) may be supplemented with two novel parameters rm2 and Rp2 for a stricter test of validation. The parameter rm2(overall) penalizes a model for large differences between observed and predicted values of the compounds of the whole set (considering both training and test sets) while the parameter Rp2 penalizes model R2 for large differences between determination coefficient of nonrandom model and square of mean correlation coefficient of random models in case of a randomization test. Two other variants of rm2 parameter, rm2(LOO) and rm2(test), penalize a model more strictly than Q2 and R2pred respectively. Three different data sets of moderate to large size have been used to develop multiple models in order to indicate the suitability of the novel parameters in QSAR studies. The results show that in many cases the developed models could satisfy the requirements of conventional parameters (Q2 and R2pred) but fail to achieve the required values for the novel parameters rm2 and Rp2. Moreover, these parameters also help in identifying the best models from among a set of comparable models. Thus, a test for these two parameters is suggested to be a more stringent requirement than the traditional validation parameters to decide acceptability of a predictive QSAR model, especially when a regulatory decision is involved.