Production of transgenic <Emphasis Type="Italic">Podophyllum peltatum via Agrobacterium tumefaciens</Emphasis>-mediated transformation

Transgenic Podophyllum peltatum plants were successfully produced by Agrobacterium tumefaciens-mediated transformation. Embryogenic callus was co-cultivated with Agrobacterium tumefaciens harboring a binary vector pBI 121 carrying β-glucuronidase (GUS) and neomycinphosphotransferase (NPT II) gene. GUS-histochemical analysis revealed that, 50 μM acetosyringone treatments during Agrobacterium infection and 3 d co-cultivation with Agrobacterium showed enhanced transformation efficiency. Percentage of GUS positive callus increased rapidly as the subculture time proceeded on selection medium containing 100 mg dm⁻³ kanamycin. Kanamycin resistant somatic embryos were formed from embryogenic callus after cultivation with 11.35 μM abscisic acid (ABA) for 3 weeks and then on hormone-free selection medium. Somatic embryos were germinated and converted into plantlets on medium containing 2.89 μM gibberellic acid (GA₃). The integration of GUS and NPT II gene into transgenic plants was confirmed by polymerase chain reaction and Southern analysis.     

In vitro organogenesis and plant formation in Acacia sinuata

In vitro morphogenesis via organogenesis was achieved from callus cultures derived from hypocotyl explants of Acacia sinuata on MS (Murashige and Skoog, 1962) medium. Calli were induced from hypocotyl explants excised from 7-day-old seedlings on MS medium containing 3% sucrose, 0.8% agar, 6.78 μM 2,4-dichlorophenoxyacetic acid and 2.22 μM 6-benzylaminopurine. Regeneration of adventitious buds from callus was achieved when they were cultured on MS medium supplemented with 10% coconut water, 13.2 μM 6-benzylaminopurine and 3.42 μM indoleacetic acid. Addition of gibberellic acid (1.73 μM) favored shoot elongation. Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 7.36 μM indolebutyric acid. Rooted plantlets, thus developed were hardened and successfully established in the soil. This protocol yielded an average of 20 plants per hypocotyl explant over a period of 4 months. This revised version was published online in June 2006 with corrections to the Cover Date.     

Candidate vaccine antigens and genes in Pasteurella multocida.

Pasteurella multocida is the causative agent of fowl cholera and other diseases of production animals. Isolates are classified into five groups based on capsular antigens and into 16 serotypes based on LPS antigens. Strains causing fowl cholera are most frequently designated A:1, A:3 or A:4. Whole cell bacterins can provide some degree of protection, but only against the homologous LPS serotype. There is good evidence that cross-protective antigens are expressed only under in vivo conditions. Empirically derived, live, attenuated vaccines can protect against heterologous serotypes, but because the basis for attenuation is undefined, reversion to virulence is not uncommon. Work in our laboratory is aimed at using a variety of approaches to identify potential protective antigens or virulence genes to be used as candidates for attenuating mutations or as the basis for vaccine antigen delivery systems. The gene encoding an outer membrane protein, Oma87, which is a homologue of the D15 protective antigen of Haemophilus influenzae, was cloned and sequenced. Rabbit antiserum prepared against recombinant Oma87 could passively protect mice against infection. Type 4 fimbriae form the basis of vaccines against ovine footrot and bovine keratoconjunctivitis. We have identified type 4 fimbriae on the surface of P. multocida, purified the fimbrial subunit protein, PtfA, and determined its N-terminal amino acid sequence. Subsequent cloning of the ptfA gene and its inactivation will now be used to assess the importance of type 4 fimbriae in virulence. There has long been anecdotal evidence for the importance of capsule in virulence, but unequivocal genetic evidence for such a role is lacking. We have cloned and characterised the capsule biosynthetic locus in P. multocida A:1 and identified four bex genes involved in capsule transport and genes encoding enzymes involved in the biosynthesis and transfer of the N-acetyl glucosamine and glucuronic acid components of the capsule. It has been suggested that the low concentration of available iron in vivo acts as an environmental cue for expression of cross-protective antigens. Accordingly, we have cloned and characterised the gene encoding transferrin binding protein, Tbpl, so that its role in immunity and virulence can be investigated. Although P. multocida is not normally considered haemolytic, we have observed haemolysis under anaerobic conditions. Standard library construction and screening resulted in the identification of the mesA gene which encodes an esterase enzyme resulting in a haemolytic phenotype under anaerobic conditions. Virulence studies with mesA- mutants were performed to assess its role in pathogenesis. Using a promoterless phoA gene vector system, the cloning of proteins homologous to known surface proteins of other species as well as proteins unique to P. multocida, allowing their potential as vaccine components to be assessed.     

Microarray data assembler

SUMMARY: Large volumes of microarray data are generated and deposited in public databases. Most of this data is in the form of tab-delimited text files or Excel spreadsheets. Combining data from several of these files to reanalyze these data sets is time consuming. Microarray Data Assembler is specifically designed to simplify this task. The program can list files and data sources, convert selected text files into Excel files and assemble data across multiple Excel worksheets and workbooks. This program thus makes data assembling easy, saves time and helps avoid manual error. AVAILABILITY: The program is freely available for non-profit use, via email request from the author, after signing a Material Transfer Agreement with Johns Hopkins University.     

Isolation and Characterization of A Metal-resistant Pseudomonas Aeruginosa Strain

Summary The use of microorganisms to remove heavy metals from industrial effluent is an area of extensive research and development. Attempts have been made to isolate and characterize metal-resistant microorganisms from treated oil mill industry effluent wastewater samples. The metal-resistant organisms that showed values of minimum inhibitory concentration towards metals (Cd, Cr, Ni and Pb) ranging from 100 to 800 ppm level were screened. A potent metal-resistant organism, isolate BC15 from the wastewater samples was tentatively identified as Pseudomonas sp. Detailed analysis of morphological, biochemical and 16S rDNA sequence of the isolate revealed that it is closely related to Pseudomonas aeruginosa (94%). Pseudomonas BC15 was capable of absorbing 93% Ni, 65% Pb, 50% Cd and 30% Cr within 48 h from the medium containing 100 mg of each heavy metal per liter. The multiple metal tolerance of this strain was also associated with resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, erythromycin, kanamycin and streptomycin.     

Synthesis of some novel imidazole-based dicationic carbazolophanes as potential antibacterials

Synthesis of fluorescent imidazole-based dicationic carbazolophanes incorporating various spacer units is described. Interestingly, the cyclophanes 2a and 5a incorporating a pyridine moiety exhibited superior antibacterial activity against most of the pathogenic bacteria in the tested concentrations as compared to the other cyclophanes as well as the test control, benzalkonium chloride (BAC), cetylpyridinium chloride (CPC) and tetracycline.     

Thermodynamics of phosphorylcholine and lysophosphatidylcholine binding to the major protein of bovine seminal plasma, PDC-109

PDC-109 binds to sperm plasma membranes by specific interaction with choline phospholipids and induces cholesterol efflux, a necessary event before capacitation - and subsequent fertilization - can occur. The binding of phosphorylcholine (PrC) and lysophosphatidylcholine (Lyso-PC) with PDC-109 was investigated by monitoring the ligand-induced changes in the absorption spectrum of PDC-109. At 20 degrees C, the association constants (K(a)), for PrC and Lyso-PC were obtained as 81.4M(-1) and 2.02 x 10(4) M(-1), respectively, indicating that the binding of Lyso-PC to PDC-109 is 250-fold stronger than that of PrC. From the temperature dependence of the K(a) values, enthalpy of binding (DeltaH(0)) and entropy of binding (DeltaS(0)), were obtained as -79.7 and -237.1 J mol(-1)K(-1) for PrC and -73.0 kJ mol(-1) and -167.3 J mol(-1)K(-1) for Lyso-PC, respectively. These results demonstrate that although the binding of these two ligands is driven by enthalpic forces, smaller negative entropy of binding associated with Lyso-PC results in its significantly stronger binding.     

Incorporation of outer membrane protein OmpG in lipid membranes: protein-lipid interactions and beta-barrel orientation

OmpG is an intermediate size, monomeric, outer membrane protein from Escherichia coli, with n beta = 14 beta-strands. It has a large pore that is amenable to modification by protein engineering. The stoichiometry ( N b = 20) and selectivity ( K r = 0.7-1.2) of lipid-protein interaction with OmpG incorporated in dimyristoyl phosphatidylcholine bilayer membranes was determined with various 14-position spin-labeled lipids by using EPR spectroscopy. The limited selectivity for different lipid species is consistent with the disposition of charged residues in the protein. The conformation and orientation (beta-strand tilt and beta-barrel order parameters) of OmpG in disaturated phosphatidylcholines of odd and even chain lengths from C(12:0) to C(17:0) was determined from polarized infrared spectroscopy of the amide I and amide II bands. A discontinuity in the protein orientation (deduced from the beta-barrel order parameters) is observed at the point of hydrophobic matching of the protein with lipid chain length. Compared with smaller (OmpA; n beta = 8) and larger (FhuA; n beta = 22) monomeric E. coli outer membrane proteins, the stoichiometry of motionally restricted lipids increases linearly with the number of beta-strands, the tilt (beta approximately 44 degrees ) of the beta-strands is comparable for the three proteins, and the order parameter of the beta-barrel increases regularly with n beta. These systematic features of the integration of monomeric beta-barrel proteins in lipid membranes could be useful for characterizing outer membrane proteins of unknown structure.