Targeting the alpha-folate receptor with cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase

The alpha-FR has been reported to be overexpressed in many carcinomas, in particular those of the ovary and uterus. The high expression of alpha-FR in some tumours compared with normal tissues has been exploited over the last decade for folate-mediated targeting of macromolecules, anticancer drugs, imaging agents and nucleic acids to cancer cells. CB300638, a cyclopenta[g]quinazoline-based inhibitor of thymidylate synthase (TS), has been reported to have high affinity for the receptor and selectivity for alpha-FR overexpressing tumour cell lines. In this study, the structural features of the molecule, in particular modifications at the 2-position, have been investigated with respect to TS inhibition, affinity for the alpha-FR and reduced folate carrier (RFC) and activity in A431-FBP cells (transfected with human alpha-FR) compared with neo-transfected A431 cells. Compounds 1a,b, 2a,b and 3a,b were synthesised utilising multistep sequences. It was found that the 2-substituent does not affect the affinity for the alpha-FR; however, it greatly affects selectivity for A431-FBP cells, and suggests that there are factors other than TS inhibition and alpha-FR affinity that are important for the activity of these compounds. Compound 2b (2-CH2OH derivative) displayed the highest selectivity for the A431-FBP cells compared with A431 cells.     

Adrenalectomy Blocks the Compensatory Increases in UT-A1 and AQP2 in Diabetic Rat Kidney

In normal rats we showed that glucocorticoids participate in the downregulation of UT-A1 protein abundance in the inner medullary tip and in lowering of basal and vasopressin-stimulated facilitated urea permeability in terminal IMCDs. To examine the relevance of this response to a rat model of human disease, we studied rats with uncontrolled diabetes mellitus (DM) induced by streptozotocin (STZ), since these rats have increased corticosterone production and urea excretion. We found that at 3 days of DM, UT-A1 protein abundance is downregulated in the inner medullary tip compared to pair-fed control rats, while DM for more than 7 days caused an increase in UT-A1. To test whether adrenal steroids could be a mechanism contributing to the latter increase, we studied adrenalectomized rats (ADX), ADX rats given STZ to induce diabetes (ADX + STZ), and ADX + STZ rats receiving exogenous aldosterone or dexamethasone. In contrast to control rats, UT-A1 protein abundance was not increased by prolonged DM in the ADX rats. Aquaporin 2 (AQP2) was not increased in the inner medullas of 10-day DM rats either. However, UT-A1 protein abundance was significantly reduced in the inner medullary tips from both diabetic aldosterone-treated (40 ± 2%) and dexamethasone-treated (43 ± 2%) ADX rats compared to diabetic ADX rats without steroid replacement. AQP2 was unaffected by steroid hormone treatments. Thus, both mineralocorticoids and glucocorticoids downregulate UT-A1 protein abundance in rats with uncontrolled diabetes mellitus for 10 days. These results suggest that: 1) the increase in UT-A1 observed in DM is dependent upon having adrenal steroids present; and 2) adrenal steroids are not sufficient to enable the compensatory rise in UT-A1 to a steroid-deficient diabetic animal.     

The type and yield of lipopolysaccharide from symbiotically deficient rhizobium lipopolysaccharide mutants vary depending on the extraction method

At least 18 lipopolysaccharide (LPS) extraction methods are available, and no single method is universally applicable. Here, the LPSs from four R.etli, one R.leguminosarum bv. trifolii mutant, 24AR, and the R.etli parent strain, CE3, were isolated by hot phenol/water (phi;/W), and phenol/EDTA/triethylamine (phi/EDTA/TEA) extraction. The LPS in various preparations was quantified, analyzed by deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE), and by immunoblotting. These rhizobia normally have two prominent LPS forms: LPS I, which has O-polysaccharide, and LPS II, which has none. The LPS forms obtained depend on the method of extraction and vary depending on the mutant that is extracted. Both methods extract LPS I and LPS II from CE3. The phi/EDTA/TEA, but not the phi/W, method extracts LPS I from mutants CE358 and CE359. Conversely, the phi;/W but not the phi;/EDTA/TEA method extracts CE359 LPS V, an LPS form with a truncated O-polysaccharide. phi/EDTA/TEA extraction of mutant CE406 gives good yields of LPS I and II, while phi/W extraction gives very small amounts of LPS I. The LPS yield from all the strains using phi/EDTA/TEA extraction is fairly consistent (3-fold range), while the yields from phi/W extraction are highly variable (850-fold range). The phi/EDTA/TEA method extracts LPS I and LPS II from mutant 24AR, but the phi/W method partitions LPS II exclusively into the phenol phase, making its recovery difficult. Overall, phi/EDTA/TEA extraction yields more forms of LPS from the mutants and provides a simpler, faster, and less hazardous alternative to phi/W extraction. Nevertheless, it is concluded that careful analysis of any LPS mutant requires the use of more than one extraction method.     

Double prenylation by RabGGTase can proceed without dissociation of the mono-prenylated intermediate

Rab geranylgeranyltransferase (RabGGTase) catalyzes the prenylation of Rab proteins. Despite possessing a single active site, RabGGTase is able to add geranylgeranyl moieties onto each of the two C-terminal cysteine residues of Rab. We have studied the kinetics of Rab double prenylation employing a combination of a novel high pressure liquid chromatography (HPLC)-based in vitro prenylation assay and fluorescence spectroscopy. Transfer of the first geranylgeranyl group proceeds with a k(1) = 0.16 s(-1), while the conversion from singly to double prenylated Rab is 4-fold slower (k(2) = 0.039 s(-1)). We found that following the first transfer reaction, the conjugated lipid is removed from the active site of RabGGTase but mono-prenylated Rab.REP complex remains bound to RabGGTase with a K(d) < 1 nm. In contrast to the doubly prenylated Rab7.REP dissociation of the mono-prenylated species from RabGGTase was only weakly stimulated by phosphoisoprenoid. Based on the obtained rate constants we calculated that at least 72% of mono-prenylated Rab molecules proceed to double prenylation without dissociating from RabGGTase. The obtained data provides an explanation of how RabGGTase discriminates between mono-prenylated intermediate and double prenylated reaction product. It also indicates that the phosphoisoprenoid acts both as a substrate and as a sensor governing the kinetics of protein.protein interactions in the double prenylation reaction.     

Chloroplast DNA inheritance and variation in <Emphasis Type="Italic">Leucadendron</Emphasis> species (Proteaceae) as revealed by PCR-RFLP

The inheritance of chloroplast DNA (cpDNA) in Leucadendron species was studied by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis. A total of 100 progeny from five interspecific crosses involving seven parental species were tested, and all progeny exhibited the cpDNA restriction fragment pattern of the female parent, indicating that cpDNA in Leucadendron is maternally inherited. PCR-RFLP was also employed to study cpDNA variation among 21 Leucadendron species. Parsimony analysis using a heuristic search resulted in a phylogenetic tree that showed limited agreement to the taxonomic classification of Leucadendron species, based on morphological characteristics. The incongruence between cpDNA phylogenetic and taxonomic groupings in Leucadendron may be due to reticulate evolution involving a combination of hybridization and introgression, convergent evolution and/or lineage sorting at the interspecific, intersubsectional and intersectional levels.