Proteomic analysis of the molecular response of Raji cells to maslinic acid treatment.

Maslinic acid, a natural pentacyclic triterpene has been shown to inhibit growth and induce apoptosis in some tumour cell lines. We studied the molecular response of Raji cells towards maslinic acid treatment. A proteomics approach was employed to identify the target proteins. Seventeen differentially expressed proteins including those involved in DNA replication, microtubule filament assembly, nucleo-cytoplasmic trafficking, cell signaling, energy metabolism and cytoskeletal organization were identified by MALDI TOF-TOF MS. The down-regulation of stathmin, Ran GTPase activating protein-1 (RanBP1), and microtubule associated protein RP/EB family member 1 (EB1) were confirmed by Western blotting. The study of the effect of maslinic acid on Raji cell cycle regulation showed that it induced a G1 cell cycle arrest. The differential proteomic changes in maslinic acid-treated Raji cells demonstrated that it also inhibited expression of dUTPase and stathmin which are known to induce early S and G2 cell cycle arrests. The mechanism of maslinic acid-induced cell cycle arrest may be mediated by inhibiting cyclin D1 expression and enhancing the levels of cell cycle-dependent kinase (CDK) inhibitor p21 protein. Maslinic acid suppressed nuclear factor-kappa B (NF-κB) activity which is known to stimulate expression of anti-apoptotic and cell cycle regulatory gene products. These results suggest that maslinic acid affects multiple signaling molecules and inhibits fundamental pathways regulating cell growth and survival in Raji cells.

Distinct types of rRNA operons exist in the genome of the actinomycete Thermomonospora chromogena and evidence for horizontal transfer of an entire rRNA operon.

We describe here the presence of two distinct types of rRNA operons in the genome of a thermophilic actinomycete Thermomonospora chromogena. The genome of T. chromogena contains six rRNA operons (rrn), of which four complete and two incomplete ones were cloned and sequenced. Comparative analysis revealed that the operon rrnB exhibits high levels of sequence variations to the other five nearly identical ones throughout the entire length of the operon. The coding sequences for the 16S and 23S rRNA genes differ by approximately 6 and 10%, respectively, between the two types of operons. Normal functionality of rrnB is concluded on the basis of the nonrandom distribution of nucleotide substitutions, the presence of compensating nucleotide covariations, the preservation of secondary and tertiary rRNA structures, and the detection of correctly processed rRNAs in the cell. Comparative sequence analysis also revealed a close evolutionary relationship between rrnB operon of T. chromogena and rrnA operon of another thermophilic actinomycete Thermobispora bispora. We propose that T. chromogena acquired rrnB operon from T. bispora or a related organism via horizontal gene transfer.

Molecular cloning and comparative sequence analyses of rRNA operons in Streptomyces nodosus ATCC 14899.

The genome of Streptomyces nodosus contains six ribosomal RNA (rRNA) operons. Four of the rRNA operons; rrnB, rrnD, rrnE and rrnF were cloned. We have completely sequenced all four operons, including a region 750 base pairs (bp) upstream of the 16S rRNA gene. The three rRNA genes present in each operon were closely linked in the order 16S-23S-5S. A sequence comparison of the four operons showed more than 99% sequence similarity between the corresponding 16S and 23S rRNA genes, and more than 97% similarity between 5S rRNA genes. The sequence differences observed between 23S rRNA genes appeared to be localized in two specific regions. Substantial sequence differences were found in the region upstream of the 16S rRNA gene as well as in the internal transcribed spacers. No tRNA gene was found in the 16S-23S spacer regions.

Efficient synthesis of mosquitocidal toxins in Asticcacaulis excentricus demonstrates potential of gram-negative bacteria in mosquito control.

The control of mosquitoes with chemical insecticides pollutes the environment and leads to resistance in mosquito populations. Bacterial control of mosquito larvae with Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis, which produce protein toxins, has proved useful, safe, and nonpolluting. These bacteria do, however, suffer from disadvantages, including rapid setting, UV sensitivity, and lack of persistance of spores, proteolysis of toxins, narrow host range, and high production costs. Here we show that the Gram-negative bacterium Asticcacaulis excentricus is a promising host for delivering toxins to mosquito larvae. Plasmid-transformed A. excentricus cells expressing the binary toxin of B. sphaericus exhibited toxicity to Culex and Anopheles mosquito larvae similar to that of the high-toxicity strains of B. sphaericus which produce several toxins. A. excentricus has potential advantages as a larvicide compared with the bacilli, especially persistance in the larval feeding zone, resistance to UV light, lack of toxin-degrading proteases, and low production costs.

Expression of mosquitocidal toxin genes in a gas-vacuolated strain of Ancylobacter aquaticus.

A series of plasmids bearing the binary toxin genes of Bacillus sphaericus 2297 or 2317.3, the 100-kDa toxin gene of B. sphaericus SSII-1, or the 130-kDa (cryIVB) toxin gene of Bacillus thuringiensis subsp. israelensis were constructed and introduced into Ancylobacter aquaticus by electroporation. The transformed A. aquaticus cells exhibited significant toxicity towards mosquito larvae, demonstrating a potential use of recombinant A. aquaticus for biological control of mosquitoes.

Influence of transcriptional and translational control sequences on the expression of foreign genes in Caulobacter crescentus.

The influence of expression control sequences (ECSs; promoters and ribosome-binding sites [RBSs]), transcriptional terminators, and gene orientation on the expression of the Escherichia coli lacZ gene in the gram-negative microorganisms Caulobacter crescentus and E. coli was investigated. A series of broad-host-range expression vectors, based on the RK2 plasmid derivative pRK248, were constructed. The ECSs included the tac promoter, the promoter for the surface layer protein of C. crescentus, and promoters from a number of gram-positive bacteria together with their associated RBSs. In addition, synthetic ECSs were constructed by using different combinations of promoters and RBSs. lacZ expression was found to be dependent on the nature of the promoter and RBS and, to a lesser extent, on the presence of a transcriptional terminator and the orientation of the promoter-lacZ construct in pRK248. The relative efficiencies of the various ECSs in driving lacZ expression differed markedly in C. crescentus and E. coli. In C. crescentus, the ECS ptac1 (tac promoter and consensus RBS for C. crescentus mRNAs) appeared to be the most efficient, producing 12-fold-higher activity than did pSL (promoter for the surface layer protein of C. crescentus and its putative RBS). pSL was not transcribed in E. coli, whereas various promoters from gram-positive microorganisms were transcribed in both C. crescentus and E. coli. A number of ECSs were also used to drive mosquitocidal toxin gene expression in C. crescentus, and a correlation between toxin expression and lacZ expression was observed.

Arachidonic acid regulates the binding of human interferon in human skin fibroblasts.

The induction of the antiviral state in human fibroblasts by human interferon is inhibited by arachidonic acid, its analogues 5,8,11,14-eicosatetraynoic and 5,8,11-eicosatriynoic acids, as well as by sodium arachidonate. The fatty acids myristic or oleic acid and sodium palmitate do not inhibit the antiviral action of interferon. Experiments were conducted to investigate the mechanism by which arachidonic acid could inhibit the action of interferon. No correlation between cellular lipoxygenase activities and the inhibition of antiviral action of interferon was observed in the fatty acid treated cells. Likewise, the cyclooxygenase inhibitors indomethacin and oxyphenylbutazone do not inhibit the interferon-induced antiviral state. Taken together, the inhibition of interferon action by arachidonates is unlikely to be mediated by cyclooxygenase or lipoxygenase-generated intermediates, even though arachidonates are known to affect the activity of these enzymes in vitro. Measurement of interferon receptors in the fatty acid treated cells showed that arachidonic acid, sodium arachidonate and its analogues decreased the number of human type I interferon receptors available for binding, and inhibited the transcription of the interferon-induced 6-16 gene and the induction of cellular (2'-5')-oligoadenylate synthetase, suggesting the mechanism of inhibition is mediated at the level of the interferon receptor. The significance of the finding that arachidonic acid, a common fatty acid of cells and serum, can affect the antiviral action of interferon is discussed.

Rapid and transient rise in diacylglycerol concentration in Daudi cells exposed to interferon.

Human beta-interferon stimulates a 4-fold increase in the concentration of diacyglycerol and a 2-fold increase in the concentration of inositol monophosphate in Daudi (human B-lymphoblastoid) cells within 30 sec of exposure of the cells to interferon. The increase in diacylglycerol and in inositol monophosphate is transient and the concentrations of these compounds decrease to basal levels within 10 min. Preincubation of human beta-interferon with anti-interferon antibodies inhibits this effect as well as the binding of interferon to Daudi cells. Diacylglycerol concentrations were unaffected in mouse A9 cells (fibroblasts) incubated with human beta-interferon and in Daudi cells incubated with human gamma-interferon. Mouse A9 cells are insensitive to human interferon and Daudi cells are insensitive to human gamma-interferon. The magnitude of the increase in diacylglycerol concentration stimulated by interferon can be correlated to the interferon-induced inhibition of Daudi cell division in a dose-responsive manner. Phorbol 12-myristate 13-acetate also inhibits Daudi cell division in a dose-responsive manner. It is likely that the sharp and transient increase in diacylglycerol concentration represents one of the early biochemical changes in Daudi cells exposed to interferon.

An early event in the interferon-induced transmembrane signaling process.

Human interferon stimulates a transient two- to threefold increase in the concentration of diacylglycerol and inositol tris-phosphate within 15 to 30 seconds of cell exposure to interferon. Antibodies to interferon inhibit this effect. The stimulation was measurable in isolated cell membranes exposed to interferon. Human alpha and beta, but not gamma, interferon stimulate this increase in cells containing the appropriate interferon receptor. The effect was proportional to the number of interferon receptors. Both the diacylglycerol increase and antiviral effects induced by interferon could be correlated in terms of dose dependence. Thus, a transient diacylglycerol increase is an early event in the interferon-induced transmembrane signaling process.