Synthesis of the bicyclo-[4.3.0]thymidyl nucleoside via Pd(II)-mediated ring expansion chemistry

A variety of modified nucleosides to improve antisense oligodeoxynucleotide properties such as target affinity, nuclease resistance, and pharmacokinetics were developed in the last two decades. In the context of conformational restriction we present here the synthesis of the [4.3.0]-bicyclo-DNA thymine monomer via Pd(II)-mediated ring expansion of an intermediate of the tricyclo-DNA synthesis.

LRH-1-mediated glucocorticoid synthesis in enterocytes protects against inflammatory bowel disease

Liver receptor homolog-1 (LRH-1) is a nuclear receptor involved in intestinal lipid homeostasis and cell proliferation. Here we show that haploinsufficiency of LRH-1 predisposes mice to the development of intestinal inflammation. Besides the increased inflammatory response, LRH-1 heterozygous mice exposed to 2,4,6-trinitrobenzene sulfonic acid show lower local corticosterone production as a result of an impaired intestinal expression of the enzymes CYP11A1 and CYP11B1, which control the local synthesis of corticosterone in the intestine. Local glucocorticoid production is strictly enterocyte-dependent because it is robustly reduced in epithelium-specific LRH-1-deficient mice. Consistent with these findings, colon biopsies of patients with Crohn's disease and ulcerative colitis show reduced expression of LRH-1 and genes involved in the production of glucocorticoids. Hence, LRH-1 regulates intestinal immunity in response to immunological stress by triggering local glucocorticoid production. These findings underscore the importance of LRH-1 in the control of intestinal inflammation and the pathogenesis of inflammatory bowel disease.

Design, synthesis and pharmacological characterization of analogs of 2-aminoethyl diphenylborinate (2-APB), a known store-operated calcium channel blocker, for inhibition of TRPV6-mediated calcium transport

2-Aminoethyl diphenylborinate (2-APB) is a known modulator of the IP3 receptor, the calcium ATPase SERCA, the calcium release-activated calcium channel Orai and TRP channels. More recently, it was shown that 2-APB is an efficient inhibitor of the epithelial calcium channel TRPV6 which is overexpressed in prostate cancer. We have conducted a structure-activity relationship study of 2-APB congeners to understand their inhibitory mode of action on TRPV6. Whereas modifying the aminoethyl moiety did not significantly change TRPV6 inhibition, substitution of the phenyl rings of 2-APB did. Our data show that the diaryl borinate moiety is required for biological activity and that the substitution pattern of the aryl rings can influence TRPV6 versus SOCE inhibition. We have also discovered that 2-APB is hydrolyzed and transesterified within minutes in solution.

Effect of electroporation-mediated diphtheria toxin A expression on PSA positive human prostate xenograft tumors in SCID mice

Current therapies to treat prostate cancer are often limited. Since it has been shown that very low concentrations of diphtheria toxin A (DT-A) result in abrogation of protein synthesis and apoptosis of cells, DT-A might serve as an efficient killer in cancer gene therapy. For this purpose we investigated in a quantitative manner using a stereological approach the apoptotic effect of DT-A in androgen receptor (AR) and prostate specific antigen (PSA) expressing cells after tumor formation in both flanks of SCID mice.

Synthesis and biological activity of new functionalized epothilones for prodrug design and tumor targeting

Epothilones are potent antiproliferative agents, which have served as successful lead structures for anticancer drug discovery. However, their therapeutic efficacy would benefit greatly from an increase in their selectivity for tumor cells, which may be achieved through conjugation with a tumor-targeting moiety. Three novel epothilone analogs bearing variously functionalized benzimidazole side chains were synthesized using a strategy based on palladium-mediated coupling and macrolactonization. The synthesis of these compounds is described and their in vitro biological activity is discussed with respect to their interactions with the tubulin/microtubule system and the inhibition of human cancer cell proliferation. The additional functional groups may be used to synthesize conjugates of epothilone derivatives with a variety of tumor-targeting moieties.

Synthesis, maturation, and trafficking of human Na+-dicarboxylate cotransporter NaDC1 requires the chaperone activity of cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na(+)-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.

Evidence for dissociation of TLR mRNA expression and TLR agonist-mediated functions in bovine macrophages

Toll-like receptors are of key importance in the recognition of and response to infectious agents by cells of the innate immune system. TLR mRNA expression and TLR-mediated functions were determined in bovine macrophages (MPhi) infected with bovine viral diarrhea virus (BVDV) or stimulated with interferon-gamma (IFN-gamma) in order to see whether they are correlated under these conditions. As parameters quantitative real time RT-PCR (QRT-PCR) for TLR2, TLR3 and TLR4, NO and TNF production were measured. Triggering of bovine MPhi with bona fide TLR2 and TLR4 agonists (lipopolysaccharide, lipoteichoic acid, peptidoglycan, lipopetide) led to NO and TNF production but neither TLR3 nor TLR9 agonists (double-stranded RNA, CpG DNA) showed this effect. The mRNA expression of TLR2, TLR3 and TLR4 was neither influenced by MPhi costimulation with IFN-gamma nor by MPhi preinfection with BVDV nor by the ligands themselves. However, NO production induced by TLR2 or TLR4 agonists was strongly modulated either by IFN-gamma costimulation or BVDV preinfection. Thus costimulation of MPhi with IFN-gamma resulted in an increase of both NO synthesis and TNF expression by cells stimulated simultaneously by TLR2 or TLR4 agonists. Preinfection of bovine MPhi by BVDV resulted in upregulation of TLR2- and TLR4-mediated NO synthesis. Collectively, these data show that TLR-mediated functions may be modulated by viral infection or activation via IFN-gamma of MPhi whereas the mRNA concentrations of relevant TLR members were not significantly influenced. Thus, the amount of TLR2, TLR3 and TLR4 mRNA transcripts is stable at least under the conditions tested. More importantly, modulation of TLR-mediated responses was dissociated from mRNA expression of TLR members.

Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances

Concern over possible adverse effects of endocrine-disrupting compounds on fish has caused the development of appropriate testing methods. In vitro screening assays may provide initial information on endocrine activities of a test compound and thereby may direct and optimize subsequent testing. Induction of vitellogenin (VTG) is used as a biomarker of exposure of fish to estrogen-active substances. Since VTG induction can be measured not only in vivo but also in fish hepatocytes in vitro, the use of VTG induction response in isolated fish liver cells has been suggested as in vitro screen for identifying estrogenic-active substances. The main advantages of the hepatocyte VTG assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. In this article, we critically review the current knowledge on the VTG response of cultured fish hepatocytes to (anti)estrogenic substances. In particular, we discuss the sensitivity, specificity, and variability of the VTG hepatocyte assay. In addition, we review the available data on culture factors influencing basal and induced VTG production, the response to natural and synthetic estrogens as well as to xenoestrogens, the detection of indirect estrogens, and the sources of assay variability. The VTG induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature, etc.) and culture system (hepatocyte monolayers, aggregates, liver slices, etc.). The currently available database on estrogen-mediated VTG induction in cultured teleost hepatocytes is too small to support conclusive statements on whether there exist systematic differences of the VTG response between in vitro culture systems, VTG analytical methods or fish species. The VTG hepatocyte assay detects sensitively natural and synthetic estrogens, whereas the response to xenoestrogens appears to be more variable. The detection of weak estrogens can be critical due to the overshadow with cytotoxic concentrations. Moreover, the VTG hepatocyte assay is able to detect antiestrogens as well as indirect estrogens, i.e substances which require metabolic activation to induce an estrogenic response. Nevertheless, more chemicals need to be analysed to corroborate this statement. It will be necessary to establish standardized protocols to minimize assay variability, and to develop a set of pass-fail criteria as well as cut-offs for designating positive and negative responses.

Cell cycle-dependent regulation of extra-adrenal glucocorticoid synthesis in murine intestinal epithelial cells

Glucocorticoids are anti-inflammatory steroids with important applications in the treatment of inflammatory diseases. Endogenous glucocorticoids are mainly produced by the adrenal glands, although there is increasing evidence for extra-adrenal sources. Recent findings show that intestinal crypt cells produce glucocorticoids, which contribute to the maintenance of intestinal immune homeostasis. Intestinal glucocorticoid synthesis is critically regulated by the transcription factor liver receptor homologue-1 (LRH-1). As expression of steroidogenic enzymes and LRH-1 is restricted to the proliferating cells of the crypts, we aimed to investigate the role of the cell cycle in the regulation of LRH-1 activity and intestinal glucocorticoid synthesis. We here show that either pharmacological or molecular modulation of cell cycle progression significantly inhibited expression of steroidogenic enzymes and synthesis of glucocorticoids in intestinal epithelial cells. Synchronization of intestinal epithelial cells in the cell cycle revealed that expression of steroidogenic enzymes is preferentially induced at the G(1)/S stage. Differentiation of immature intestinal epithelial cells to mature nonproliferating cells also resulted in reduced expression of steroidogenic enzymes. This cell cycle-related effect on intestinal steroidogenesis was found to be mediated through the regulation of LRH-1 transcriptional activity. This mechanism may restrict intestinal glucocorticoid synthesis to the proliferating cells of the crypts.

TLR expression on neutrophils at the pulmonary site of infection: TLR1/TLR2-mediated up-regulation of TLR5 expression in cystic fibrosis lung disease

Cystic fibrosis (CF) lung disease is characterized by infection with Pseudomonas aeruginosa and a sustained accumulation of neutrophils. In this study, we analyzed 1) the expression of MyD88-dependent TLRs on circulating and airway neutrophils in P. aeruginosa-infected CF patients, P. aeruginosa-infected non-CF bronchiectasis patients, and noninfected healthy control subjects and 2) studied the regulation of TLR expression and functionality on neutrophils in vitro. TLR2, TLR4, TLR5, and TLR9 expression was increased on airway neutrophils compared with circulating neutrophils in CF and bronchiectasis patients. On airway neutrophils, TLR5 was the only TLR that was significantly higher expressed in CF patients compared with bronchiectasis patients and healthy controls. Studies using confocal microscopy and flow cytometry revealed that TLR5 was stored intracellularly in neutrophils and was mobilized to the cell surface in a protein synthesis-independent manner through protein kinase C activation or after stimulation with TLR ligands and cytokines characteristic of the CF airway microenvironment. The most potent stimulator of TLR5 expression was the bacterial lipoprotein Pam(3)CSK(4). Ab-blocking experiments revealed that the effect of Pam(3)CSK(4) was mediated through cooperation of TLR1 and TLR2 signaling. TLR5 activation enhanced the phagocytic capacity and the respiratory burst activity of neutrophils, which was mediated, at least partially, via a stimulation of IL-8 production and CXCR1 signaling. This study demonstrates a novel mechanism of TLR regulation in neutrophils and suggests a critical role for TLR5 in neutrophil-P. aeruginosa interactions in CF lung disease.