Synthesis and antimicrobial evaluation of water-soluble, dendritic derivatives of epimeric 5alpha-cholestan-3-amines and 5alpha-cholestan-3-yl aminoethanoates

To examine the effect of negatively charged steroidal amphiphiles on antimicrobial activity, two pairs of epimeric, dendritic tricarboxylato amphiphiles--4-(2-carboxyethyl)-4-[3-(5alpha-cholestan-3-yl)ureido]heptanedioic acid (1) and 4-(2-carboxyethyl)-4-[3-(5alpha-cholestan-3-yloxycarbonylmethyl)ureido]heptanedioic acid (2)--were synthesized. A broad antimicrobial screen of 11 microbes revealed that these amphiphiles only showed good activity against a methicillin-resistant isolate of Staphylococcus aureus (MRSA) and modest activity against an unrelated strain of S. aureus. The best activity, a minimal inhibitory concentration (MIC) of 27 microM, was found for the 3beta epimer of 1 against MRSA.     

A Genome-wide Approach to Identify the Genes Involved in Biofilm Formation in E. coli

Biofilm forming cells are distinctive from the well-investigatedplanktonic cells and exhibit a different type of gene expression.Several new Escherichia coli genes related to biofilm formationhave recently been identified through genomic approaches suchas DNA microarray analysis. However, many others involved inthis process might have escaped detection due to poor expression,regulatory mechanism, or genetic backgrounds. Here, we screeneda collection of single-gene deletion mutants of E. coli named‘Keio collection’ to identify genes required forbiofilm formation. Of the 3985 mutants of non-essential genesin the collection thus examined, 110 showed a reduction in biofilmformation nine of which have not been well characterized yet.Systematic and quantitative analysis revealed the involvementof genes of various functions and reinforced the importancein biofilm formation of the genes for cell surface structuresand cell membrane. Characterization of the nine mutants of function-unknowngenes indicated that some of them, such as yfgA that geneticallyinteracts with a periplasmic chaperone gene surA together withyciB and yciM, might be required for the integrity of outermembrane.     

A recombinant multivalent combination vaccine protects against Chlamydia and genital herpes

Chlamydia trachomatis and Herpes simplex virus type 2 (HSV-2) genital infections pose a considerable public health challenge worldwide. Considering the high incidence of coinfections by the two pathogens, a combination vaccine that can be administered as a single regimen would be highly desirable. Recombinant Vibrio cholerae ghosts (rVCG) offer an attractive approach for the induction of humoral and cellular immune responses against human and animal pathogens. In this study, we evaluated a bivalent combination vaccine formulation comprising rVCG expressing chlamydial MOMP and HSV-2 glycoprotein D in mice for immunogenicity and protective efficacy against genital challenge with either pathogen. Mice immunized with the combination vaccine elicited secretory IgA and IgG2a antibodies to both chlamydial and HSV-2 antigens in serum and vaginal secretions. Robust antigen-specific mucosal and systemic T helper type 1 responses were induced in mice as measured by increased interferon-gamma levels produced by immune T cells in response to restimulation with target antigen in vitro. In addition, mice immunized with the combination vaccine were prophylactically protected from genital challenge with high doses of live Chlamydia and HSV-2. Thus, the combination vaccine regimen delivered by rVCG elicited adequate immune effectors that simultaneously protected against the individual pathogens.     

Comparing micellar, hemolytic, and antibacterial properties of di- and tricarboxyl dendritic amphiphiles

Homologous dicarboxyl dendritic amphiphiles-RCONHC(CH(3))(CH(2)CH(2)COOH)(2), 4(n); and ROCONHC(CH(3))(CH(2)CH(2)COOH)(2), 5(n), where R=n-C(n)H(2)(n)(+1) and n=13-22 carbon atoms-were synthesized. Critical micelle concentrations (CMCs) in aqueous triethanolamine solutions and at pH 7.4 were measured along with hemolytic activity (effective concentrations, EC(10)) in phosphate-buffered saline (PBS). LogCMC showed a linear dependence on chain length (n); the longest chain in each series had the lowest CMC-in triethanolamine: 4(21), 180μM and 5(22), 74μM and at pH 7.4: 4(21), 78μM and 5(22), 33μM. These two series, 4(n) and 5(n), and three series of homologous tricarboxyl dendritic amphiphiles-RCONHC(CH(2)CH(2)COOH)(3), 1(n); ROCONHC(CH(2)CH(2)COOH)(3), 2(n); RNHCONHC(CH(2)CH(2)COOH)(3), 3(n), where R=n-C(n)H(2)(n)(+1) and n=13-22 carbon atoms-were tested for growth inhibition of Staphylococcus aureus strain ATCC 6358 and methicillin-resistant S. aureus (MRSA) strain ATCC 43330 by microdilution in 0.1-strength brain heart infusion broth (BHIB). Amphiphiles 4(19), 4(21), 5(18), and 5(20) showed the strongest antibacterial activity (2.2-3.4μg/mL) against S. aureus (vancomycin, MIC=0.25μg/mL). These four plus 1(21), 2(20), 2(22), and 3(20) exhibited the strongest antibacterial activity (1.7-6.8μg/mL) against MRSA (vancomycin, MIC=0.25μg/mL). The MICs of these amphiphiles against six clinical MRSA were similar to those against the ATCC strain. In PBS, EC(10)s of the most active homologues ranged from 7 to 18μg/mL and 18 to 220μg/mL for di- and tricarboxyl dendritic amphiphiles, respectively. To assess the potential safety of using dendritic amphiphiles as drugs, measurements of micellar and hemolytic properties were conducted in the same medium (full-strength BHIB) that was used for antibacterial activity. The CMCs (9-36μg/mL, ～18-72μM) of ten amphiphiles were measured by microdilution (log2 progression) with dye-covered beads. The EC(10)s were similar to those in PBS. The MICs of most amphiphiles (14-72μg/mL) and vancomycin (1.1-2.2μg/mL) against both S. aureus and MRSA increased significantly compared to the MICs measured in 0.1-strength BHIB. The one exception, 5(18), had an MIC against S. aureus of 1.1μg/mL compared to vancomycin (2.2μg/mL). With CMC (9-18μg/mL) and EC(10) (16μg/mL) values higher than the MIC, 5(18) was discovered as a lead for further development.     

Enzymatic synthesis of lignin-siloxane hybrid functional polymers

This study combines the properties of siloxanes and lignin polymers to produce hybrid functional polymers that can be used as adhesives, coating materials, and/or multifunctionalized thin-coating films. Lignin-silica hybrid copolymers were synthesized by using a sol-gel process. Laccases from Trametes hirsuta were used to oxidize lignosulphonates to enhance their reactivity towards siloxanes and then were incorporated into siloxane precursors undergoing a sol-gel process. In vitro copolymerization studies using pure lignin monomers with aminosilanes or ethoxytrimethylsilane and analysis by 2?Si NMR spectroscopy revealed hybrid products. Except for kraft lignin, an increase in lignin concentration positively affected the tensile strength in all samples. Similarly, the viscosity generally increased in all samples with increasing lignin concentration and also affected the curing time.     

Secondary metabolism in tobacco

Tobacco has been quite well studied phytochemically, more than 2500 compounds have been identified. Here, the secondary metabolism in tobacco will be reviewed in a biosynthetic perspective. Major groups of compounds which have extensively been studied are the isoprenoids, alkaloids, cinnamoylputrescines, flavonoids, and anthocyanins. Their biosynthetic pathways and its regulation, and their occurrence in cell cultures and in intact plants will be discussed.     

A Novel Variant of Narrow-Spectrum Antifungal Bacterial Lipopeptides That Strongly Inhibit <Emphasis Type="Italic">Ganoderma boninense</Emphasis>

Bacterial antifungal cyclic lipopeptides (ACLs) have become a promising alternative to synthetic fungicide to control pathogenic fungi. Bacillus sp. is known to produce three families of ACL, namely iturin, surfactin, and fengycin. In this paper, we characterized the ACLs produced by B. methylotrophicus HC51 (referred as HC51) mainly regarding its composition and effectivity against fungal plant pathogen. HC51 culture was tested against various pathogenic fungi and the ACLs were extracted and analyzed using liquid chromatography–electrospray ionization mass spectrometry. HC51 showed strong antifungal activity against the plant pathogens Ganoderma sp. and Fusarium sp. Cell-free methanol extract of HC51 contains iturin A and various variants of fengycin. C16 fengycin A was present in four fractions which indicates it as a major component of ACL from HC51. Five variants of fengycin were detected, four of which had been previously reported. We found a novel C17 fengycin F that is characterized by a substitution of l-ornithine into lysine. Considering that l-ornithine is an important building block of fengycin, this substitution suggests the possibility of an alternative pathway for fengycin biosynthesis.     

Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase,a potential drug target

Plasmodium falciparum is an apicomplexan parasite that causes the most severe malaria in humans. Due to a lack of effective vaccines and emerging of drug resistance parasites, development of drugs with novel mechanisms of action and few side effects are imperative. To this end, ideal drug targets are those essential to parasite viability as well as absent in their mammalian hosts. The mitochondrial electron transport chain (ETC) of P. falciparum is one source of such potential targets because enzymes, such as L-malate:quinone oxidoreductase (PfMQO), in this pathway are absent humans. PfMQO catalyzes the oxidation of L-malate to oxaloacetate and the simultaneous reduction of ubiquinone to ubiquinol. It is a membrane protein, involved in three pathways (ETC, the tricarboxylic acid cycle and the fumarate cycle) and has been shown to be essential for parasite survival, at least, in the intra-erythrocytic asexual stage. These findings indicate that PfMQO would be a valuable drug target for development of antimalarial with novel mechanism of action. Up to this point in time, difficulty in producing active recombinant mitochondrial MQO has hampered biochemical characterization and targeted drug discovery with MQO. Here we report for the first time recombinant PfMQO overexpressed in bacterial membrane and the first biochemical study. Furthermore, about 113 compounds, consisting of ubiquinone binding site inhibitors and antiparasitic agents, were screened resulting in the discovery of ferulenol as a potent PfMQO inhibitor. Finally, ferulenol was shown to inhibit parasite growth and showed strong synergism in combination with atovaquone, a well-described anti-malarial and bc₁ complex inhibitor.