Synthesis and biological activities of phenyl piperazine-based peptidomimetic growth hormone secretagogues

A new class of potent, orally active phenyl piperazine-based GH secretagogues have been discovered from attempts to mimic the arrangement of the phenyl substituent in the spiroindanyl piperidine and spiroindoline sulfonamide privileged structures of 4 and 1, respectively. The best of these compounds, 18 (EC50 = 2.8 nM) is nearly as potent as MK-0677 for releasing GH from rat pituitary cells.     

Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment

In a pilot study to establish fetal nucleated red blood cell (NRBC) detection in maternal blood, trisomy 13 was diagnosed by FISH analysis at 11 weeks' gestation. The NRBCs were detected after a single-step ficoll density gradient enrichment. In blood samples taken both before and after CVS, 52 and 80 NRBCs, respectively, were found to be positive for fetal haemoglobin. In 47 per cent of these cells, FISH analysis for X and Y chromosomes confirmed the fetal sex. Moreover, 48 per cent of these NRBCs showed three fluorescent signals for a chromosome 13 probe, which confirmed the diagnosis of trisomy 13, previously detected at CVS karyotyping. This is the first report of non-invasive prenatal diagnosis of trisomy 13, i.e., pre-CVS, in the first trimester. The high number of fetal NRBCs detected indicates a connection with aneuploidy, probably due to early impairment of the feto-maternal barrier.     

Insulin-like growth factor binding protein-1 at mouse neuromuscular synapses

The insulin-like growth factor (IGF) signaling system includes the growth factors and their cell surface receptors, along with circulating IGF binding proteins (IGFBPs) that may alter and modulate the action of these neurotrophic hormones. These IGFBPs, along with IGFs and receptors, have been detected in various tissues including the brain. In this study, using polyclonal antibody to human IGFBP-1 or bovine IGFBP-2, we found that mouse muscle extracts contain similar-sized proteins that cross-react with these antibodies on Western immunoblots. After establishing that these antibodies reacted with the homologous murine IGFBPs, we performed immunocytochemistry to demonstrate the localization of IGFBP-1 at the neuromuscular junction, a model nicotinic, cholinergic synapse, as well as within intramuscular nerves. IGFBP-2, a distinct macromolecule, is present on the surface of muscle fibers and is not present within synapses or nerves.     

Antimetabolite-induced increases in the invasive capacity of murine leukaemia L1210 cells

Pretreatment of murine leukaemia L1210 cells with non-lethal concentrations of various antimetabolites increased the in vitro invasive capacity of these cells into monolayers of rat embryo fibroblasts. The increase in invasive capacity was partly correlated with the induced cell cycle arrest. The concomitant increase in cell surface fucosylation and inhibition of invasion with sulphate indicate a role for glycoproteins in this process. Our results suggest that treatment with antimetabolites may lead to a more aggressive phenotype by altering cell surface properties.     

Ecosystem Service Impacts of Urban Water Supply and Demand Management

Utilities face the challenge of enhancing long-term water security while minimising undesirable economic, social and environmental impacts of supply and demand management options. This paper provides an example of how the ecosystem services concept can be used to enumerate and organise broad impacts of water supply options. A case study of Adelaide, South Australia, is used to examine costs and benefits associated with different sources of water and source-water mix scenarios. Ecosystem service impacts are estimated using estimates from the literature. Seven water supply and demand management options are considered for Adelaide: 1) the River Murray, 2) Mt. Lofty Ranges catchments, 3) wastewater reuse, 4) desalination, 5) stormwater harvesting, 6) groundwater and 7) water conservation. The largest costs are associated with sourcing water from conservation measures such as water restrictions on outdoor watering estimated at $1.87/kL. Salinity damage costs associated with residential uses are estimated at up to $1.54/kL. Salinity damage costs of wastewater reuse were estimated at $1.16/kL. The largest benefit is coastal amenity services associated with stormwater harvesting and treatment estimated at $1.03/kL. Results show that there is a trade-off between financial costs and ecosystem services impacts with source-water mix scenarios with the highest ecosystem services cost having the lowest financial O&M cost and vice versa. This highlights the importance of taking ecosystem services into account when evaluating water supply options.     

Cyclic voltammetry of monel 400 in lithium hydroxide solution at elevated temperatures

The electrochemistry of Monel 400 in 1 mole/kg^?1 LiOH solution at 25, 125 and 250°C has been investigated using the technique of cyclic voltammetry. The general electrochemical behaviour is found to most closely approximate to that of the major component, nickel, although expansion of the current scale reveals anodic and cathodic peaks which probably arise from redox processes involving copper. The general similarity to nickel can be rationalized in terms of either the d electron theory for cupronickel alloys or phase separation, the latter being favoured in the present study. At 25°C the majority of charge consumed on sweeping the potential in the positive direction is involved in the formation of an oxide film at potentials close to oxygen evolution. This process is no longer observed at 250°C, due to a sharp reduction in the oxygen evolution overpotential with temperature. The majority of charge consumed on cyclic sweeping at this temperature is attributed to active dissolution of the nickel component of the alloy to form HNiO^?₂ (or Ni(OH)^?₂ (or Ni(OH)^?₃) at potentials slightly positive to the hydrogen evolution region.     

The expanding small heat-shock protein family, and structure predictions of the conserved "alpha-crystallin domain"

The ever-increasing number of proteins identified as belonging to the family of small heat-shock proteins (shsps) and alpha-crystallins enables us to reassess the phylogeny of this ubiquitous protein family. While the prokaryotic and fungal representatives are not properly resolved, most of the plant and animal shsps and related proteins are clearly grouped in distinct clades, reflecting a history of repeated gene duplications. The members of the shsp family are characterized by the presence of a conserved homologous "alpha-crystallin domain," which sometimes is present in duplicate. Predictions are made of secondary structure and solvent accessibility of this domain, which together with hydropathy profiles and intron positions support the presence of two similar hydrophobic beta-sheet-rich motifs, connected by a hydrophilic alpha-helical region. Together with an overview of the newly characterized members of the shsp family, these data help to define this family as being involved as stable structural proteins and as molecular chaperones during normal development and induced under pathological and stressful conditions.     

DNA-Directed alkylating agents. 7. Synthesis, DNA interaction, and antitumor activity of bis(hydroxymethyl)- and bis(carbamate)-substituted pyrrolizines and imidazoles

A series of bis(hydroxymethyl)-substituted imidazoles, thioimidazoles, and pyrrolizines and related bis(carbamates), linked to either 9-anilinoacridine (intercalating) or 4-(4-quinolinylamino)benzamide (minor groove binding) carriers, were synthesized and evaluated for sequence-specific DNA alkylation and cytotoxicity. The imidazole and thioimidazole analogues were prepared by initial synthesis of [(4-aminophenyl)alkyl]imidazole-, thioimidazole-, or pyrrolizine dicarboxylates, coupling of these with the desired carrier, and reduction to give the required bis(hydroxymethyl) alkylating moiety. The pyrrolizines were the most reactive alkylators, followed by the thioimidazoles, while the imidazoles were unreactive. The pyrrolizines and some of the thioimidazoles cross-linked DNA, as measured by agarose gel electrophoresis. Strand cleavage assays showed that none of the compounds reacted at purine N7 or N3 sites in the gpt region of the plasmid gpt2Eco, but the polymerase stop assay showed patterns of G-alkylation in C-rich regions. The corresponding thioimidazole bis(carbamates) were more selective than the bis(hydroxymethyl) pyrrolizines, with high-intensity bands at 5'-NCCN, 5'-NGCN and 5'-NCGN sequences in the PCR stopping assay ( indicates block sites). The data suggest that these targeted compounds, like the known thioimidazole bis(carbamate) carmethizole, alkylate exclusively at guanine residues via the 2-amino group, with little or no alkylation at N3 and N7 guanine or adenine sites. The cytotoxicities of the compounds correlated broadly with their reactivities, with the bis(hydroxymethyl)imidazoles being the least cytotoxic (IC50s >1 microM; P388 leukemia) and with the intercalator-linked analogues being more cytotoxic than the corresponding minor-groove-targeted ones. This was true also for the more reactive thioimidazole bis(carbamates) (IC50s 0.8 and 11 microM, respectively), but both were more active than the analogous "untargeted" carmethizole (IC50 20 microM). The bis(hydroxymethyl)pyrrolizine analogues were the most cytotoxic, with IC50s as low as 0.03 microM.     

'98 Escherichia coli SWISS-2DPAGE database update

The combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS-2DPAGE database to be established. This is part of the ExPASy molecular biology server accessible through the WWW at the URL address http://www.expasy.ch/ch2d/ch2d-top.html . Here we report recent progress in the development of the E. coli SWISS-2DPAGE database. Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining. Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman "sequence tag" approach. Some of the above identification techniques were coupled with database searching tools. Currently 231 polypeptides are identified on the E. coli SWISS-2DPAGE map: 64 have been identified by N-terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag. Of 153 proteins putatively identified by gel comparison, 65 have been confirmed. Many proteins have been identified using more than one technique. Faster progress in the E. coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E. coli genome.