PhBPs show selectivity for tumour and microbial cells, which appears to be based on electrostatic interactions between the positive charge generally carried by these molecules and the negative charge found on the outer surface of these target cells. In some cases, a site of action for photoactivated PhBPs is the outer membrane/envelope of the target cell. Such action can involve the modification of membrane lipid and/or lipopolysaccharide, and the inactivation of essential proteins and enzymes, with these effects usually leading to cell lysis and death. However, more often, PhBPs are internalised by target cells, promoted by a variety of factors, including low pH and enzymatic reduction, and upon photoactivation, internalised, PhBPs are able to inflict damage on a number of intracellular targets. In tumour cells, PhBPs can photodamage DNA and the membranes of organelles, thereby inducing necrosis and/or apoptosis. In bacterial cells, whilst DNA is generally a primary target of PhBPs, these compounds can exhibit multiple sites of action within a given cell and show different sites of action between different bacterial species. This variable targeting makes PhBPs attractive propositions as alternatives to conventional antibiotics in that the emergence of bacterial strains with acquired resistance to these compounds appears to be highly unlikely.

Activated ras is known to dysregulate TGF-b signaling by altering the expression of TGF-b type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI,832) may directly restore TGF-b signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 mM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 mM concentrations (P40.004). The TGF-b effector gene p21 waf1/cip1 was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-b responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-b signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT -PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-b signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.

Structural genes for catechol 2,3-oxygenase (C230) were cloned from the TOL plasmids pWW5, pWW14, pWW74, pWW84, and pWW88 isolated fro*n Pseudomonas strains of diverse geographical origins. Each pKT230-based C230+ recombinant plasmid carried a 2.05-kilobase XhoI insert which showed strong homology in Southern hybridizations with the xyLE gene from the archetype TOL plasmid pWWO. Fragments were mapped for restriction endonuclease sites and were classified into two closely related groups on the basis of restriction maps. C230 structural genes were located on cloned fragments by a combination of subcloning and site-specific mutagenesis. All five TOL plasmids examined yielded clones whose maps differed from that of xyLE of pWWO by only a single XbaI site, but in addition plasmids pWW5, pWW74, and pWW88 carried a second, homologous C230 gene with seven further restriction site differences. The remaining plasmids, pWW14 and pWW84, carried a second nonhomologous C230 gene related to the second C230 gene (C2301) of TOL plasmid pWW15 described previously (H. Keil, M. R. Lebens, and P. A. Williams, J. Bacteriol. 163:248&255, 1985). Thus, each naturally occurring TOL plasmid in this study appears to carry genes for two meta cleavage dioxygenases.The degradation of toluene and some substituted toluenes via the meta pathway by Pseudomonas spp. is often plasmid mediated (7,15,19,26,34,35,39). TOL plasmids from different strains are diverse with regard to molecular sizes, fragmentation patterns by restriction endonucleases, selftransmissibility, rate of dissimilation ofp-methyl-substituted substrates, and formation of different classes of deletion mutants after growth on benzoate (7,17,27,35,37,38).Although the archetype TOL plasmid pWWO (34) has a single xylE gene coding for the ring cleavage dioxygenase catechol 2,3-oxygenase (C230), it was recently demonstrated in this laboratory that the 250-kilobase (kb) plasmid pWW15 carries two nonhomologous genes which specify the apparently unrelated enzymes C2301 and C2301I (16). To investigate the evolutionary relationship between the catabolic genes on different TOL plasmids, we cloned xylE genes from five TOL plasmids found in Pseudomonas strains isolated from soil samples of diverse geographical origins. Like pWW15, two of the plasmids reported here also carried two nonhomologous C230 genes. Unexpectedly, the other plasmids in this study each possessed two closely related C230 genes which exhibited strong homology with each other and with the xylE gene of pWWO. MATERIALS AND METHODSBacterial strains and plasmids. Pseudomonas strains and plasmids used in this study are described in Table 1. Strains MT74, MT84, and MT88 were isolated by selective enrichment on m-toluate minimal medium during this study; MT5 and MT14-26 have been described previously (7,26,35).

Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress conditions, such as cancer and hypoxia, with production also being linked to tumourigenesis. HSPA mRNA transcripts and proteins were examined in three human glioma cell lines, representing astrocytoma, oligodendroglioma and glioblastoma, plus 18 clinical brain tissue samples. GAPDH was used as a control gene throughout these studies and exhibited a consistent level of expression in a normal astrocyte cell line, tumourous cell lines and tissue samples. In contrast, the average HSPA mRNA copy numbers detected in glioblastoma tissue were between 1.8- and 8.8-fold higher than in lower grade glioma and control tissue, respectively, which is suggestive of a grade-related transcription profile. Similar patterns of grade-related expression were also observed in glioma cell lines. This study indicates for the first time that HSPA expression in glioma cells may possibly be grade related, and hence could have potential as a prognostic marker.

Type 2 diabetes mellitus (T2DM) is characterized by defects in haepatic glucose production, insulin action and insulin secretion, which can also lead to a variety of secondary disorders. The disease can lead to death without treatment and it has been predicted that T2DM will affect 215 million people world-wide by 2010. T2DM is a multifactorial condition whose precise genetic causes and biochemical defects have not been fully elucidated but at both levels, calpains appear to play a role. Positional cloning studies mapped T2DM susceptibility to CAPN10, the gene encoding the intracellular cysteine protease, calpain 10. Further studies have shown a number of non-coding polymorphisms in CAPN10 to be functionally associated with T2DM whilst the identification of coding polymorphisms, suggested that mutant calpain 10 proteins may also contribute to the disease. The presence of both calpain 10 and its mRNA have been demonstrated in tissues from several mammalian species whilst calpain 10 appears to be associated with pathways involved in glucose metabolism, insulin secretion and insulin action. It appears that other calpains may also participate in these pathways and here we present an overview of recent studies on calpains and their putative role in T2DM.