Syntheses of (<Emphasis Type="Italic">E</Emphasis>)- and (<Emphasis Type="Italic">Z</Emphasis>)-3-styrylchromones

Several (E)- and (Z)-3-styrylchromones were prepared by two different methodologies, the Wittig reaction of chromone-3-carboxaldehyde with benzylic ylides and the Knoevenagel condensation of chromone-3-carboxaldehyde with phenylacetic acids in the presence of potassium tert-butoxide under microwave irradiation. The Knoevenagel reaction followed by a decarboxylation offered an efficient and diastereoselective method for preparing (E)-3-styrylchromones in a shorter reaction time. It was also demonstrated that phenylacetic acid can also be substituted with success by phenylmalonic acid. The stereochemistry of all products was assigned by NMR experiments. Correspondence: Artur M. S. Silva, Chemistry Department, University of Aveiro, 3810-193 Aveiro, Portugal.     

Synthesis of 6-methyl-8<Emphasis Type="Italic">H</Emphasis>-dibenzo[<Emphasis Type="Italic">a</Emphasis>,<Emphasis Type="Italic">g</Emphasis>]quinolizin-8-imines <Emphasis Type="Italic">via Reissert</Emphasis> compounds

Alkylation of Reissert compounds derived from 3-methylisoquinolines with several 2-cyanobenzylbromides followed by hydrolytic cleavage provided the corresponding 1-benzyl-3-methylisoquinolines. Treatment of the latter with methylmagnesiumiodide caused cyclization to the title compounds rather than formation of 2-acetylbenzylisoquinolines.     

Lipids from <Emphasis Type="Italic">Halostachys caspica</Emphasis> and <Emphasis Type="Italic">Halocharis hispida</Emphasis>

The composition of lipids from the aerial parts of two species of halophytes from the family Chenopodiaceae, Halostachys caspica C. A. Mey. and Halocharis hispida Bge. was determined. Neutral lipids (NL, 62.1 and 54.2%, respectively) dominated the total lipids (TL) of these plants. More than a third of the NL were esters of aliphatic alcohols and phytosterols (FAE). Fatty acids 16:0, 18:1, and 18:2 dominated the acids of FAE; 16:0, 18:1, and 18:3, the phospholipids. The principal fatty acids of glycolipids were unsaturated acids (68.3 and 75.1%) with linolenic acid dominating (44.9 and 43.5%). Presented at the 7th International Symposium on the Chemistry of Natural Compounds, Tashkent, October 16–18, 2007. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 276–278, May–June, 2009.     

Stress Response Kinetics of Two Nisin Producer Strains of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>

The purpose of this study is to determine the survival and nisin production behaviors of two strains of Lactococcus lactis under different stress conditions that represent the food ecosystem. In this respect, the survival ratios of two nisin producers were determined under different pH, temperature, NaCl, and bile salt concentrations. Then, nisin production levels of the strains were determined at each stress conditions. Both strains had similar growth or inactivation patterns under the same stress conditions. NaCl and bile salt stresses on the survival ratio of the strains could be successfully described by the exponential decay function, whereas Gaussian function produced good fits for temperature and pH stresses. The nisin activity of two nisin producers (in their mid-exponential and/or early stationary phase) decreased dramatically under all stress conditions, except osmotic (NaCl) and low temperature applications. The results of this study showed that two nisin producers had similar adaptive responses under severe stress conditions, which could be described by appropriate mathematical equations. Moreover, the effect of harsh environment on the nisin activity of L. lactis strains depends on the stress factors applied.     

Antioxidant flavonoids from <Emphasis Type="Italic">Tamus communis</Emphasis> ssp. <Emphasis Type="Italic">cretica</Emphasis>

A new flavonoid, kaempferol-3,4′-di-O-α-L-rhamnopyranoside (1), and three known flavonoids (2–4) were isolated from the aerial parts of T. communis L. The structure of the new compound was elucidated on the basis of spectroscopic data. Compounds 1 and 2 showed significant antioxidant activity (IC₅₀ 187.151 ± 0.821 μM, and 92.079±0.513 μM, respectively), whereas compounds 3 and 4 showed moderate activity in DPPH free radical scavenging assays. Published in Khimiya Prirodnykh Soedinenii, No. 3, pp. 295–297, May–June, 2009.     

Polyploid Formation Between <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Trichoderma viride</Emphasis> For Enhanced Citric Acid Production From Cellulose

The first-stage heterokaryons, obtaining from intergeneric protoplast fusion between Aspergillus niger (Y-b) and Trichoderma viride (M5S51), showed slow growth and mixed morphologies on minimal medium. The fusants were classified into heterokaryon and prototrophic haploid, showing the morphology as that of A. niger. The heterokaryon strains formed conidia with the same nutritional requirements as those of the original auxotrophic mutant strains. After several subcultivations on minimal medium containing d-camphor, some heterokaryon strains formed larger two to seven nuclei/conidium as compared to one nucleus/conidium of the auxotrophic mutant and prototrophic strains, indicating that the new hybrids were generated. Interestingly, three fusant strains AT 11-2-3, AT 11-2-10, and AT 11-2-14 produce 19.2, 6.1, and 10.5 g/l citric acid, respectively, in semisolid culture containing cellulose, whereas A. niger Yang no. 2 could not use carboxymethyl cellulose as the sole carbon source for citric acid production. In addition, the average maximum beta-glucosidase and carboxymethylcellulase productions from AT 11-2-3, AT 11-2-10, and AT 11-2-14 were about 16- and 4-folds higher than those of A. niger, respectively.     

Rapid molecular typing of <Emphasis Type="Italic">Tuber melanosporum</Emphasis>, <Emphasis Type="Italic">T. brumale</Emphasis> and <Emphasis Type="Italic">T. indicum</Emphasis> from tree seedlings and canned truffles

We have developed new DNA extraction and purification procedures for investigation of mycorrhized seedlings and canned truffles. Use of these procedures on approximately 100 mg initial material enabled good sample representation. For mycorrhized seedlings, Taq polymerase inhibitors were discarded irrespective of tree species. In routine analysis we systematically used consensus primers ITS1/ITS4 to check the absence of Taq polymerase inhibitors and the presence of fungus DNA. Positive response with ITS validates other positive or negative PCR results. Absence of amplification with ITS prevents validation of other results. For canned truffles, DNA harvested from ascocarps sterilized for one and a half hours at 115°C was amplified with specific primers. We have developed consensus primers, named R12/F12, to check for the presence of amplifiable fungus DNA and the absence of Taq polymerase inhibitors. Here also, positive response with consensus R12/F12 validates other positive or negative PCR results. We have developed one primer pair specific for T. brumale and another specific for T. melanosporum. We can then characterize these two taxa, which enables the use of truffle or truffled French designations. We can also characterize T. indicum, the Asiatic black truffle that might fraudulently be sold as T. melanosporum and T. brumale. These three specific primer pairs were used independently of DNA extraction from tree seedlings or canned truffles. Our process is specific, sensitive, convenient, and quick.J.P. Douet and D. Mabru have contributed equally to this work     

Splitting of the <Emphasis Type="Italic">d</Emphasis><Superscript><Emphasis Type="Italic">N</Emphasis></Superscript> atomic states in icosahedral 3<Emphasis Type="Italic">d</Emphasis> metal endofullerenes M@C<Subscript>60</Subscript>

Group-theoretical and quantum-chemical investigations of the spectrum of low-lying excited states have been performed by the ROHF and FCI-RAS (Full CI in Restricted Active Space) methods for 3d metal endofullerenes (MEFs) M@C₆₀ (M =Mn, Cr, and Fe) in different charged states. The major purpose of this study is quantum-chemical verification of the anomalous (“non-Bethe’s”) character of splitting of the d ^N atomic states in an electrostatic field with icosahedral symmetry, predicted previously within the theory of integral invariants theory. The interrelation between the integral invariants theory and the quantumchemical methods applied in this work is considered in detail. Our calculations suggest that the d ^N atomic states in the icosahedral field generated by fullerene C60 (I _h ) on a metal atom (ion) remain non-split for different charged states of the metal and C₆₀. Reasons for this phenomenon and other possible approaches to verification of the prediction are discussed. It is demonstrated that the d ^N states of the encapsulated metal are split in icosahedral 3d MEFs only under very strong compression of these structures.     

Screening and Immobilization <Emphasis Type="Italic">Burkholderia</Emphasis> sp. GXU56 Lipase for Enantioselective Resolution of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-Methyl Mandelate

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.     

Crystal and molecular structure of <Emphasis Type="Italic">tris</Emphasis>(<Emphasis Type="Italic">m</Emphasis>-chlorophenyl)phosphine selenide

The crystal and molecular structure of tris(m-chlorophenyl)phosphine selenide, C₁₈H₁₂Cl₃PSe (I), was investigated by X-ray diffraction (XRD) analysis. The trigonal rhombohedral structure of I (space group \(R\overline 3 c\), a = 14.110(2) Å, c = 32.360(4) Å, Z = 12) was solved by direct methods and refined by least squares in an anisotropic approximation (R = 0.029) for 1319 averaged measured reflections (CAD-4 automatic diffractometer, λCuK_α).     

Investigation of kernel oils of <Emphasis Type="Italic">Quercus robur</Emphasis> and <Emphasis Type="Italic">Quercus cerris</Emphasis>

The kernel oils of Quercus robur and Quercus cerris were obtained by Soxhlet extraction using petroleum ether. Oil yields were found to be 5.2–5.6% and 4.3–4.8% for Q. robur and Q. cerris kernel, respectively (expressed in g per 100 g of dried plant material). The physical and chemical constants, unsaponifiable matter and total fatty acids were determined. The total fatty acid composition of oils was determined by GC in the methyl ester form. Considering the composition and content of fatty acids, the examined kernel oils were very similar. Seven fatty acid components were identified in both oils: palmitic, stearic, arachidic, palmitoleic, oleic, linoleic, and -linolenic. In Q. robur and Q. cerris kernel oils the principal acids were oleic (44.3% and 43.0%, respectively) and linoleic (37.2% and 32.6%, respectively), followed by a significant amount of palmitic acid.Published in Khimiya Prirodnykh Soedinenii, No. 5, pp. 347–348, September–October, 2004.     

The smallest Hosoya index in (<Emphasis Type="Italic">n</Emphasis>, <Emphasis Type="Italic">n</Emphasis> + 1)-graphs

A (n, n + 1)-graph G is a connected simple graph with n vertices and n + 1 edges. In this paper, we determine the lower bound for the Hosoya index in (n, n + 1)-graphs in terms of the order n, and characterize the (n, n + 1)-graph with the smallest Hosoya index.     

New flavonoid-<Emphasis Type="Italic">C</Emphasis>-glycosides from <Emphasis Type="Italic">Triticum aestivum</Emphasis>

Two new flavonoid-C-glycosides named triticuside A (1a) and triticuside B (1b) were isolated from bran of Triticum aestivum L. The structures of the two new compounds were elucidated by spectral techniques including ¹H NMR, ¹³C NMR as well as HSQC, HMBC, and COSY. Published in Khimiya Prirodnykh Soedinenii, No. 2, pp. 135–137, March–April, 2008.     

Optimization of Spore and Antifungal Lipopeptide Production During the Solid-state Fermentation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis strain TrigoCor 1448 was grown on wheat middlings in 0.5-l solid-state fermentation (SSF) bioreactors for the production of an antifungal biological control agent. Total antifungal activity was quantified using a 96-well microplate bioassay against the plant pathogen Fusarium oxysporum f. sp. melonis. The experimental design for process optimization consisted of a 2⁶⁻¹ fractional factorial design followed by a central composite face-centered design. Initial SSF parameters included in the optimization were aeration, fermentation length, pH buffering, peptone addition, nitrate addition, and incubator temperature. Central composite face-centered design parameters included incubator temperature, aeration rate, and initial moisture content (MC). Optimized fermentation conditions were determined with response surface models fitted for both spore concentration and activity of biological control product extracts. Models showed that activity measurements and spore production were most sensitive to substrate MC with highest levels of each response variable occurring at maximum moisture levels. Whereas maximum antifungal activity was seen in a limited area of the design space, spore production was fairly robust with near maximum levels occurring over a wider range of fermentation conditions. Optimization resulted in a 55% increase in inhibition and a 40% increase in spore production over nonoptimized conditions.     

An efficient <Emphasis Type="Italic">Mannich</Emphasis>-type synthesis of <Emphasis Type="Italic">bis</Emphasis>(pyrido[2,1-<Emphasis Type="Italic">b</Emphasis>][1,3,5]thiadiazin-7-yl)-methanes

Morpholinium 3-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-thiolate upon treatment with primary amines and a formaldehyde excess under mild conditions produces bis(pyrido[2,1-b][1,3,5]thiadiazin-7-yl)methane derivatives in good yields (67–87%). Correspondence: Victor V. Dotsenko, State Enterprise “Luganskstandartmetrology”, 91021 Lugansk, Ukraine.     

Expression of Recombinant Proteins in <Emphasis Type="Italic">Pichia Pastoris</Emphasis>

Pichia pastoris has been used extensively and successfully to express recombinant proteins. In this review, we summarize the elements required for expressing heterologous proteins, and discuss various factors in applying this system for protein expression. These elements include vectors, host strains, heterologous gene integration into the genome, secretion factors, and the glycosylation profile. In particular, we discuss and evaluate the recent progress in optimizing the fermentation process to improve the yield and stability of expressed proteins. Optimization can be achieved by controlling the medium composition, pH, temperature, and dissolved oxygen, as well as by methanol induction and feed mode.     

Substrate specificity of <Emphasis Type="Italic">Streptomyces</Emphasis> transglutaminases

Transglutaminase (TGase) is a multifunctional enzyme vital for many physiologic processes, such as cell differentiation, tissue regeneration, and plant pathogenicity. The acyl transfer function of the enzyme can activate primary amines and, consequently, attach them onto a peptidyl glutamine, a reaction important for various in vivo and in vitro protein crosslinking and modification processes. To understand better the structure-function relationship of the enzyme and to develop it further as an industrial biocatalyst, we studied TGase secreted by several Streptomyces species and Phytophthora cactorum. We purified the enzyme from S. lydicus, S. platensis, S. nigrescens, S. cinnamoneus, and S. hachijoensis. The pH and temperature profiles of S. lydicus, S. platensis, and S. nigrescens TGases were determined. The specificity of S. lydicus TGase toward its acyl-accepting amine substrates was characterized. Correlation of the electronic and steric features of the substrates with their reactivity supported the mechanism previously proposed for Streptomyces mobaraensis TGase.     

Glutaraldehyde-modified electrode for nonlabeling voltammetric detection of <Emphasis Type="Italic">p16</Emphasis><Superscript><Emphasis Type="Italic">INK4A</Emphasis></Superscript> gene

A nonlabeling electrochemical detection method for analyzing the polymerase-chain-reaction-amplified sequence-specific p16 ^INK4A gene, in which the basis for the covalent immobilization of deoxyribonucleic acid (DNA) probe is described, has been developed. The self-assembly process was based on the covalent coupling of glutaraldehyde (GA) as an arm molecule onto an amino-functional surface. The p16 ^INK4A gene was used as the model target for the methylation detection of early cancer diagnosis. An amino-modified DNA probe was successfully assembled on the GA-coupling surface through the formation of Schiff base under potential control. The hybridization of amino-modified DNA probes with the target was investigated by means of electrochemical measurements, including cyclic voltammetry and square wave voltammetry. Furthermore, the functions of GA coupling for sequence-specific detection were compared with those obtained based on mercaptopropionic acid. Hybridization experiments indicated that the covalent coupling of GA was suitable for the immobilization of DNA probe and was sensitive to the electrochemical detection of single-base mismatches of label-free DNA targets in hybridization. Moreover, reported probe-modified surfaces exhibited excellent stability, and the hybridization reactions were found to be completely reversible and highly specific for recognition in subsequent hybridization processes. The strategy provided the potential for taking full advantage of existing modified electrode technologies and was verified in microarray technology, which could be applied as a useful and powerful tool in electrochemical biosensor and microarray technology.     

A new stilbene glycoside from the <Emphasis Type="Italic">n</Emphasis>-butanol fraction of <Emphasis Type="Italic">Veratrum dahuricum</Emphasis>

A new stilbene glycoside, 5-methylresveratrol-3,4′-O-β-D-diglucopyranoside (1), was isolated from the n-butanol fraction of the rhizomes of Veratrum dahuricum, together with five known stilbenoids: resveratrol-3-O-β-D-glycoside (2), 4′-methylresveratrol-3-O-β-D-glycoside (3), oxyresveratrol-4′-O-β-D-glycoside (4), oxyresveratrol-3-O-β-D-glycoside (5), and oxyresveratrol-3,4′-O-β-D-diglycoside (6), and found for the first time in the investigated plant. The structures of six isolates were identified on the basis of 1D and 2D NMR data. Compounds 1–6 showed platelet aggregation inhibition, and compound 1 had an IC₅₀ value of 383.6 μM against platelet aggregation induced by AA. Published in Khimiya Prirodnykh Soedinenii, No. 3, pp. 279–282, May–June, 2009.     

Functional Expression of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> Protective Antigen in <Emphasis Type="Italic">E. coli</Emphasis>

The protective antigen (PA) of Bacillus anthracis is a potent immunogen and an important candidate vaccine. In addition, it is used in monitoring systems like enzyme-linked immunosorbent assay to assess antibodies against PA in immunized subjects. The low level of PA production in B. anthracis and the difficulty of separating it from other bacterial components have made the researchers do different studies with the aim of producing recombinant PA (rPA). In this study, to produce rPA as a recombinant protein vaccine, the partial sequence of protective antigen of B. anthracis, amino acids 175–764, as a potent immunogenic target was inserted in pET21b(+). This is a prokaryotic plasmid that carries an N-terminal T7.tag sequence. The integrity of constructed plasmid was confirmed using restriction enzyme mapping. rPA was expressed after induction with isopropyl β-d-1-thiogalactopyranoside in Escherichia coli BL21. Purification of rPA was done with an affinity system using anti T7.tag antibody. Electrophoresis and Western blotting confirmed the specificity of the expressed protein. BALB/c mice were immunized with obtained PA protein and evaluation of specific immunoglobulin G antibodies against PA in sera using Western blotting method and showed that rPA is immunogenic. The challenge of immunized mice with virulent strain of B. anthracis showed that rPA is functional to protect against pathogenic strain.