<Emphasis Type="Italic">N</Emphasis>-Acyl derivatives of Asn,new bacterial <Emphasis Type="Italic">N</Emphasis>-acyl <Emphasis Type="Italic">D</Emphasis>-amino acids with surfactant activity

Summary. New N-acyl D-amino acids were isolated from Bacillus pumilus IM 1801. Their structures were determined by chemical analysis and mass spectrometry. The lipid part was identified as a mixture of fatty acids with 11, 12, 13, 15, and 16 carbon atoms in the iso, anteiso or n configuration linked by an amide bond with a D-asparagine. They exhibited surfactant properties.     

Antibacterial activity of <Emphasis Type="Italic">N</Emphasis>-benzylsalicylthioamides

The in-vitro biological activity of N-benzylsalicylthioamides against 8 bacterial strains was determined by broth microdilution method; results were compared with those obtained with neomycin, penicillin G, ciprofloxacin and penicillin V. The compounds showed moderate to high activity against G⁺ bacteria; especially compounds 4, 6, 13, 16–21 and 24 exhibited comparable or higher activity than reference drugs. The antibacterial activity was analyzed by quantitative structure-activity relationship (QSAR). The antibacterial activity increased with lipophilicity, with the presence of halogens and with increasing value of Hammet substituent constant σ.     

Antibacterial activity of three South Indian seagrasses, <Emphasis Type="Italic">Cymodocea serrulata</Emphasis>, <Emphasis Type="Italic">Halophila ovalis</Emphasis> and <Emphasis Type="Italic">Zostera capensis</Emphasis>

The antibacterial properties of the three seagrasses namely Cymodocea serrulata, Halophila ovalis and Zostera capensis were tested against the human pathogens Staphylococcus aureus, Bacillus cereus, B. subtilis, Escherichia coli, Salmonella paratyphi, Salmonella typhimurium and Micrococcus luteus, using six different solvents namely, petroleum ether, chloroform, ethyl acetate, acetone, methanol and water. Ethyl acetate and methanol extracts showed maximum activity against most of the pathogens when compared to other solvents. Experiments are underway to isolate active compound(s) implicated in controlling the growth of the pathogens in vitro.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

Characterization of the <Emphasis Type="Italic">codY</Emphasis> gene and its influence on biofilm formation in <Emphasis Type="Italic">Bacillus </Emphasis><Emphasis Type="Italic">cereus</Emphasis>

The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant (M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation. Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type. Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels CodY represses production of an unknown protease and is involved in biofilm formation.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Invasion of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> into host cells is impaired by <Emphasis Type="Italic">N</Emphasis>-propionylmannosamine and other <Emphasis Type="Italic">N</Emphasis>-acylmannosamines

The etiologic agent of Chagas’ disease, Trypanosoma cruzi, is widely distributed in South America, affecting millions of people with thousands of deaths every year. Adherence of the infectious trypomastigote to host cells is mediated by sialic acid. T. cruzi cannot synthesize sialic acids on their own but cleave them from the host cells and link them to glycans on the surface of the parasites using the trans-sialidase, a GPI-anchored enzyme. The infectivity of the protozoan parasites strongly depends on the activity of this enzyme. In this report, we investigated whether the transfer of sialic acids from the host to the parasites can be attenuated using novel sialic acid precursors. The cell line 86-HG-39 was infected with T. cruzi and treated with defined N-acylmannosamine analogues bearing an elongated N-acyl side-chain. By treatment of these cells the number of T.cruzi infected cell was reduced up to 60%. We also showed that the activity of the bacterial sialidase C was reduced with N-glycan substrates with elongated N-acyl side chains of the terminal sialic acids. The affinity of this sialidase decreased with the length of the N-acyl side-chain. The data presented suggest that N-acyl modified sialic acid precursors can change the transfer of sialic acids leading to modification of infection. Since the chemotherapy of this disease is inefficient and afflicted by side effects, the need of effective drugs is lasting. These findings propose a new path to prevent the dissemination of T. cruzi in the human hosts. These compounds or further modified analogues might be a basis for the search of new agents against Chagas’ disease.     

An Oxidized Ergosterol from <Emphasis Type="Italic">Pleurotus cystidiosus</Emphasis> Active Against Anthracnose Causing <Emphasis Type="Italic">Colletotrichum</Emphasis><Emphasis Type="Italic">gloeosporioides</Emphasis>

This study was undertaken to study the antifungal activity of Pleurotus cystidiosus against Colletotrichum gloeosporioides. This was achieved by fractionating the mushroom, P. cystidiosus initially to acetone (A), dichloromethane (D), and hexane (H) and studying the antifungal activity using the standard poisoned food technique. All the test solutions used were in the concentration of 20,000 ppm. The percentage inhibition of extracts A, D, and H was 12, 7, and 0.4%, respectively. Antifungal assay guided fractionation of the most active extract A resulted in four fractions; A1, A2, A3, and A4 having 12, 22, 0, and 17% percentage inhibitions, respectively. Fractions A2 and A4 were selected for further purifications. Normal phase column chromatography of A2 gave A2-1, A2-2, A2-3, and A2-4, with percentage inhibitions 7, 5, 26, and 13%, respectively. The fraction with the highest inhibitory activity (A2-3) was further separated using the Chromatotron and a single compound (A2-3-13) with 41% inhibition was isolated. Structure elucidation of this compound using 1D and 2D NMR spectroscopy proved this compound to be 3beta, 5alpha, 6beta-trihydroxyergosta-7,22-diene.     

Decrease of <Emphasis Type="Italic">N</Emphasis>-nitrosodimethylamine and <Emphasis Type="Italic">N</Emphasis>-nitrosodiethylamine by <Emphasis Type="Italic">Lactobacillus pentosus</Emphasis> R3 is associated with surface-layer proteins

The objective of this study was to evaluate the ability of five strains of meat-borne bacteria to decrease N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) and to elucidate the mechanism in Mann-Rogosa-Sharp (MRS) broth. Lactobacillus pentosus R3 was found to be the most effective in decreasing the concentration of the two N-nitrosamines (NAs) in MRS broth, with a rate of 22.05% for NDMA and 23.31% for NDEA. The concentration of the two NAs could not be reduced by either extracellular metabolites or intracellular extracts of Lb. pentosus R3 (P?>?0.05), and proteinaceous substances in the cell debris were found to be responsible for the decrease. These were surface-layer proteins (SLPs) located on the cell wall. Therefore, the decrease in NDMA and NDEA by Lb. pentosus R3 is associated with its SLPs. Lb. pentosus R3 may be developed as a starter culture in the production of fermented foods with lower NAs.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Methods for syntheses of <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="Italic">DL</Emphasis>-aspartic acid derivatives

Summary. A novel practical method for the synthesis of N-methyl-DL-aspartic acid 1 (NMA) and new syntheses for N-methyl-aspartic acid derivatives are described. NMA 1, the natural amino acid was synthesized by Michael addition of methylamine to dimethyl fumarate 5. Fumaric or maleic acid mono-ester and -amide were regioselectively transformed into beta-substituted aspartic acid derivatives. In the cases of maleamic 11a or fumaramic esters 11b, the α-amide derivative 13 was formed, but hydrolysis of the product provided N-methyl-DL-asparagine 9 via base catalyzed ring closure to DL-α-methylamino-succinimide 4, followed by selective ring opening. Efficient methods were developed for the preparation of NMA-α-amide 13 from unprotected NMA via sulphinamide anhydride 15 and aspartic anhydride 3 intermediate products. NMA diamide 16 was prepared from NMA dimethyl ester 6 and methylamino-succinimide 4 by ammonolysis. Temperature-dependent side reactions of methylamino-succinimide 4 led to diazocinone 18, resulted from self-condensation of methylamino-succinimide via nucleophyl ring opening and the subsequent ring-transformation.     

1-(<Emphasis Type="Italic">N</Emphasis>-Trifluoroacetylamino)alkylphosphonic acids: synthesis and properties

Summary. The 1-(N-trifluoroacetylamino)alkylphosphonic acids (TFA-AA^P) – sub-products in the synthesis of O,O-dialkyl 1-(N-trifluoroacetylamino)alkylphosphonates and O,O-diethyl 1-aminoalkylphosphonates, were synthesized in two-stage transformations of 1-aminoalkylphosphonic acids including: trifluoroacetylation of 1-aminoalkylphosphonic acids (AA^P) using a trifluoroacetic anhydride/trifluoroacetic acid reagent (AA^P + TFAA/TFA→2) and subsequent hydrolysis of the intermediary compounds 2 into desired TFA-AA^P (2→TFA-AA^P). These intermediates 2 presented mixtures of the type of mixed anhydrides of TFAA and 1-(N-trifluoroacetylamino)alkylphosphonic, pyrophosphonic and polyphosphonic acids, which underwent rapid and quantitative conversion to corresponding TFA-AA^P during treatment with an excess of water. The title acids were isolated by direct evaporation of the corresponding post-reaction mixtures, and their physicochemical proprieties, including deacylation abilities, were determined. TFA-AA^P compounds can be re-converted into the starting amino acids AA^P under respectively mild conditions (AA^P→TFA-AA^P→AA^P).     

Physiological characterisation of <Emphasis Type="Italic">acuB</Emphasis> deletion in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The acuB gene of Aspergillus niger is an ortholog of facB in Aspergillus nidulans. Under carbon-repression conditions, facB is repressed, thereby preventing acetate metabolism when the repressing carbon source is present. Even though facB is reported to be repressed directly by CreA, it is believed that a basal level of FacB activity exists under glucose-repressive conditions. In the present study, the effect of deletion of acuB on the physiology of A. niger was assessed. Differences in organic acid and acetate production, enzyme activities and extracellular amino and non-amino organic acid production were determined under glucose-repressing and -derepressing conditions. Furthermore, consumption of alternative carbon sources (e.g. xylose, citrate, lactate and succinate) was investigated. It was shown that AcuB has pleiotropic effects on the physiology of A. niger. The results indicate that metabolic pathways that are not directly involved in acetate metabolism are influenced by acuB deletion. Clear differences in organic acid consumption and production were detected between the ∆acuB and reference strain. However, the hypothesis that AcuB is responsible for basal AcuA activity necessary for activation of acetate metabolic pathways, even during growth on glucose, could not be confirmed. The experiments demonstrated that also when acuB was deleted, no acetate was formed. Therefore, AcuB cannot be the only activator of AcuA, and another control mechanism has to be available for activating AcuA.     

<Emphasis Type="Italic">N</Emphasis>-halamine biocidal coatings

Novel N-halamine siloxane and epoxide coatings are described. The coatings can be rendered biocidal by exposure to dilute bleach. Once the bound chlorine is lost from the coatings, it can be regenerated by further exposure to dilute bleach. Synthetic schemes and biocidal efficacy data are presented. The stabilities of the bound chlorine on the surfaces are also addressed. Substrates employed include sand, textiles, and paint. Potential uses for the technology are discussed.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Plant regeneration from <Emphasis Type="Italic">Gossypium davidsonii</Emphasis> protoplasts <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

Protoplasts isolated from wild cotton Gossypium davidsonii were cultured in KM₈P medium supplemented with different phytohormones. The most effective combination was 0.45 μM 2,4-dichlorophenoxyacetic acid, 2.68 μM α-naphthaleneacetic acid and 0.93 μM kinetin and the division percentage at the 8^th day was 30.78 ± 3.04 %. The density of protoplasts at 2–10 × 10⁵ cm⁻³ was suitable for protoplast division and calli formation, with a division percentage of 32.21 ± 3.64 % and a plating efficiency of 9.12 ± 2.61 % at the 40^th day. The optimal osmotic potential was achieved using 0.5 M glucose or 0.1 M glucose plus 0.5 M mannitol. Protoplasts were cultured in three ways, a double-layer culture system, with liquid over solid medium was proved to be the best way. Embryo induction was further increased by addition of 0.14 μM gibberellic acid.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot culture and antimicrobial activity of <Emphasis Type="Italic">Berberis buxifolia</Emphasis> Lam

Berberis buxifolia Lam., known as “Calafate”, is a plant native to Argentina that exhibits antimicrobial activity. This biological activity is attributed to the isoquinoline alkaloid berberine. The aim of this research was to test the antimicrobial properties of different extracts of this species, taking berberine as the reference molecule, and to examine if the expression of bacterial multidrug resistance (MDR) efflux pumps could be responsible for possible resistance mechanisms. To this end, a wild-type and a mutant strain of Staphylococcus aureus with a defective MDR efflux pump were used and the minimum inhibitory concentrations of the extracts were determined. The studies were carried out with infusions of in vivo shoots and “Calafate” commercial tea, as well as with the media derived from shoot cultures incubated with different plant growth regulators (thidiazuron, picloram, and jasmonic acid). As far as antimicrobial activity is concerned, all the extracts tested were significantly more effective than berberine standard. “Calafate” commercial tea and shoot tea had inhibitory concentrations similar to the one observed for ampicillin standard. The media from the shoot cultures, however, were significantly more effective than all the others, particularly the one derived from jasmonic acid, suggesting the presence of compounds that could be acting synergistically with berberine. There were no differences in antimicrobial activity against the wild-type and the mutant S. aureus; no definite conclusions could be drawn concerning the relationship between MDR pumps and possible pathogen resistance to extracts of B. buxifolia.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Synthesis of (<Emphasis Type="Italic">2S</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)-4-phenylamino-5-oxoproline derivatives

Summary. The paper describes the synthesis of (2S,4S)-4-(N-Ts)- and (2S,4S)-4-(N-Boc)-phenylamino-5-oxoprolines (pyroglutamic acid). These derivatives have been shown to be useful for synthesis of their amides and peptides in spite of steric hindrances caused by bulky groups adjacent to the reaction centre. Under the conditions applied no lactam ring opening and no loss of stereochemical integrity of any of the chiral centres were observed, which has been confirmed by NMR techniques. Received December 29, 2000 Accepted June 26, 2001