Synthesis and evaluation of chiral bicyclic proline FKBP12 ligands

As part of our ongoing program to explore novel structural classes of FKBP12 ligands, we herein wish to report a new class of FKBP12 ligands containing chiral bicyclic proline analogues. Details of the synthetic routes, together with preliminary biological activity, will be presented.     

Enzymatic liberation of lycotetraose from the Solanum glycoalkaloid alpha-tomatine

The branched tetrasaccharide, O-β-d-glucopyranosyl-(1 → 2)-O-[β-d-xylopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-d-galactose (lycotetraose) is a key constituent of many biologically interesting natural products. Described herein is a convenient enzymatic preparation of lycotetraose from the readily available Solanum glycoalkaloid α-tomatine. The preparation makes use of the recombinant endo-glycosidase, tomatinase, from the plant pathogen Fusarium oxysporum f. sp. lycopersici.     

Expression of NPY Y1 and Y5 receptors in the hypothalamic paraventricular nucleus of aged Fischer 344 rats

Many mammals, nearing the end of life, spontaneously decrease their food intake and body weight, a stage we refer to as senescence. The spontaneous decrease in food intake and body weight is associated with attenuated responses to intracerebroventricular injections of neuropeptide Y (NPY) compared with old presenescent or with young adult rats. In the present study, we tested the hypothesis that this blunted responsiveness involves the number and expression of hypothalamic paraventricular nucleus (PVN) Y(1) and/or Y(5) NPY receptors, both of which are thought to mediate NPY-induced food intake. We found no significant difference in mRNA levels, via quantitative PCR, for Y(1) and Y(5) receptors in the PVN of senescent vs. presenescent rats. In contrast, immunohistochemistry indicated that the number of PVN neurons staining for Y(1) receptor protein was greater in presenescent compared with senescent rats. We conclude that a decreased expression and number of Y(1) or Y(5) receptors in the PVN cannot explain the attenuated responsiveness of the senescent rats to exogenous NPY.     

The impact of microsatellite electromorph size homoplasy on multilocus population structure estimates in a tropical tree (Corythophora alta) and an anadromous fish (Morone saxatilis)

Microsatellite allelic states are determined by electrophoretic sizing of polymerase chain reaction fragments to define electromorphs. Numerous studies have documented that identical microsatellite electromorphs are potentially heterogeneous at the DNA sequence level, a phenomenon called electromorph size homoplasy. Few studies have examined the impact of electromorph size homoplasy on estimates of population genetic parameters. We investigated the frequency of microsatellite electromorph size homoplasy for 12 loci in the tropical tree Corythophora alta and 11 loci in the anadromous fish Morone saxatilis by sequencing 14-23 homozygotes per locus sampled from multiple populations for a total of 453 sequences. Sequencing revealed no homoplasy for M. saxatilis loci. Seven C. alta loci exhibited homoplasy, including single and compound repeat motifs both with and without interruptions. Between 12.5 and 42.9% of electromorphs sampled per locus showed size homoplasy. Two methods of correction for homoplasy in C. alta generally produced little or no change in single-locus estimates of RST, except for two loci in which some additional differentiation among populations was revealed. Twelve-locus estimates of RST (including the seven loci corrected for homoplasy) were slightly greater than estimates from uncorrected data, although the 95% confidence intervals overlapped. The frequency of methodological errors such as clerical mistakes or sample mislabelling per genotype scored was estimated at 5.4 and 7.3% for C. alta and M. saxatilis, respectively. Simulations showed that the increase in RST produced by homoplasy correction was only slightly larger than variation in RST estimates expected to be caused by methodological errors.     

Statistical quantification of detachment rates and size distributions of cell clumps from wild-type (PAO1) and cell signaling mutant (JP1) Pseudomonas aeruginosa biofilms

The detachment of cells from bacterial biofilms is an important, yet poorly understood and largely unquantified phenomenon. Detached cell clumps from medical devices may form microemboli and lead to metastasis, especially if they are resistant to host defenses and antibiotics. In manufacturing plants detached clumps entering a process stream decrease product quality. Two strains of Pseudomonas aeruginosa, a wild type (PAO1) and a cell signaling mutant (JP1), were studied to (i) quantify and model detachment patterns and (ii) determine the influence of cell signaling on detachment. We collected effluent from a biofilm flowthrough reactor and determined the size distribution for cell detachment events by microscopic examination and image analysis. The two strains were similar in terms of both biofilm structure and detachment patterns. Most of the detachment events were single-cell events; however, multiple-cell detachment events contributed a large fraction of the total detached cells. The rates at which events containing multiple cells detached from the biofilm were estimated by fitting a statistical model to the size distribution data. For events consisting of at least 1,000 cells, the estimated rates were 4.5 events mm(-2) min(-1) for PAO1 and 4.3 events mm(-2) min(-1) for JP1. These rates may be significant when they are scaled up to the total area of a real biofilm-contaminated medical device surface and to the hours or days of patient exposure.     

Fatty acid interactions with proteins: what X-ray crystal and NMR solution structures tell us

The interactions of fatty acids with proteins have been studied by a variety of conventional approaches for decades. However, only limited aspects of fatty acid-protein interactions have been elucidated, even with the integration of information gleaned from the many techniques. Judgments must be made about what information is most reliable, particularly when derivatives of fatty acids are substituted for natural fatty acids. In recent years, the application of techniques of structural biology has brought about dramatic advances in this important area of lipid research. High-resolution crystallographic and NMR structures of several proteins with bound fatty acids reveal the complete tertiary structure of the protein and molecular details of fatty acid-protein interactions. The examples presented include most of the known structures of (non-enzymatic) proteins that bind fatty acids. The proteins are found in very different compartments of cells and organisms: the plasma compartment (human serum albumin); the cytosolic compartment of mammalian cells (fatty acid- binding proteins); the cytosol of plant cells (nonspecific lipid-transfer protein); the nucleus of mammalian cells (peroxisome proliferator-activated receptor and hepatic nuclear factor 4); and a bacterial membrane (halorhodopsin). This review discusses the structural features of these proteins and their binding pocket(s) and compares the specific modes of their interactions with fatty acids.     

Proteomic analysis of changes in the extracellular matrix of Arabidopsis cell suspension cultures induced by fungal elicitors

A proteomic approach has been applied to investigate changes in the extracellular matrix of Arabidopsis thaliana cell suspension cultures following treatments with two fungal pathogen elicitors, chitosan and extracts of Fusarium moniliforme. The oxidative burst and induction of glutathione S-transferase were used as markers for induction of the pathogen defence response. Changes in the cell wall and culture filtrate proteome were profiled. Proteins whose abundance changed reproducibly were analysed via matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS). An increase in the level of two classical cell wall proteins (a putative endochitinase and a polygalacturonase inhibiting protein) and two novel proteins (a putative receptor-like protein kinase and a probable apospory-associated protein) were seen at 24 hours following elicitation. The level of an unknown protein and a hypothetical protein, which has some homology to serine carboxypeptidases, were decreased at 24 hours post-elicitation. In the culture filtrate extracts, we identified two pathogen elicitor responsive proteins, a xyloglucan endo-1,4-beta-D glucanases (XEG) and a peroxidase. Using a combination of two-dimensional polyacrylamide gel electrophoresis, immunoblotting with a phosphotyrosine-specific antibody, and MALDI-TOF MS we discovered that spots that represent putative lectin receptor-like kinase, a putative endochitinase and a XEG possess phosphorylated tyrosine residues. The identification of phosphorylated bona fide cell wall proteins and a putative extracellular receptor-like kinase with no transmembrane domain implicate the existence of an extracellular phosphorylation network which could be involved in intercellular communication.     

Benzofuranyl 3,5-bis-polyamine derivatives as time-dependent inhibitors of trypanothione reductase

The synthesis and evaluation of 3,5-disubstituted benzofuran derivatives as time-dependent inhibitors of the protozoan oxidoreductase trypanothione reductase are reported. These molecules were designed as simplified mimetics of the naturally occurring spermidine-bridged macrocyclic alkaloid lunarine 1, a known time-dependent inhibitor of trypanothione reductase. In this series of compounds the bis-polyaminoacrylamide derivatives 2-4 were all shown to be competitive inhibitors, but only the bis-4-methyl-piperazin-1-yl-propylacrylamide derivative 4 displayed time-dependent activity. The kinetics of time dependent inactivation of trypanothione reductase by 1 and 4 have been determined and are compared and discussed herein.     

Rapamycin potentiates transforming growth factor beta-induced growth arrest in nontransformed,oncogene-transformed,and human cancer cells

Transforming growth factor beta (TGF-beta) induces cell cycle arrest of most nontransformed epithelial cell lines. In contrast, many human carcinomas are refractory to the growth-inhibitory effect of TGF-beta. TGF-beta overexpression inhibits tumorigenesis, and abolition of TGF-beta signaling accelerates tumorigenesis, suggesting that TGF-beta acts as a tumor suppressor in mouse models of cancer. A screen to identify agents that potentiate TGF-beta-induced growth arrest demonstrated that the potential anticancer agent rapamycin cooperated with TGF-beta to induce growth arrest in multiple cell lines. Rapamycin also augmented the ability of TGF-beta to inhibit the proliferation of E2F1-, c-Myc-, and (V12)H-Ras-transformed cells, even though these cells were insensitive to TGF-beta-mediated growth arrest in the absence of rapamycin. Rapamycin potentiation of TGF-beta-induced growth arrest could not be explained by increases in TGF-beta receptor levels or rapamycin-induced dissociation of FKBP12 from the TGF-beta type I receptor. Significantly, TGF-beta and rapamycin cooperated to induce growth inhibition of human carcinoma cells that are resistant to TGF-beta-induced growth arrest, and arrest correlated with a suppression of Cdk2 kinase activity. Inhibition of Cdk2 activity was associated with increased binding of p21 and p27 to Cdk2 and decreased phosphorylation of Cdk2 on Thr(160). Increased p21 and p27 binding to Cdk2 was accompanied by decreased p130, p107, and E2F4 binding to Cdk2. Together, these results indicate that rapamycin and TGF-beta cooperate to inhibit the proliferation of nontransformed cells and cancer cells by acting in concert to inhibit Cdk2 activity.