Design, synthesis, and biological evaluation of 4-phenylpyrrole derivatives as novel androgen receptor antagonists

A series of 4-phenylpyrrole derivatives D were designed, synthesized, and evaluated for their potential as novel orally available androgen receptor antagonists therapeutically effective against castration-resistant prostate cancers. 4-Phenylpyrrole compound 1 exhibited androgen receptor (AR) antagonistic activity against T877A and W741C mutant-type ARs as well as wild-type AR. An arylmethyl group incorporated into compound 1 contributed to enhancement of antagonistic activity. Compound 4n, 1-{[6-chloro-5-(hydroxymethyl)pyridin-3-yl]methyl}-4-(4-cyanophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonitrile exhibited inhibitory effects on tumor cell growth against the bicalutamide-resistant LNCaP-cxD2 cell line as well as the androgen receptor-dependent JDCaP cell line in a mouse xenograft model. These results demonstrate that this series of pyrrole compounds are novel androgen receptor antagonists with efficacy against prostate cancer cells, including castration-resistant prostate cancers such as bicalutamide-resistant prostate cancer.     

In vivo VL-targeted activation-induced apoptotic supraclonal deletion by a microbial B cell toxin

To interfere with host immune responses, some microbial pathogens produce proteins with the properties of superantigens, which can interact via conserved V region framework subdomains of the Ag receptors of lymphocytes rather than the complementarity-determining region involved in the binding of conventional Ags. In recent studies, we have elucidated how a model B cell superantigen affects the host immune system by targeting a conserved V(H) site on the Ag receptors of B lymphocytes. To determine whether these findings represent a general paradigm, we investigated the in vivo immunobiologic properties of protein L of Peptostreptococcus magnus (PpL), a microbial Ig-binding protein specific for a V region site on Ig L chains. Our studies confirmed that PpL binding is restricted to a subset of murine Vkappa-expressing B cells, and found that B cells with stronger PpL-binding activity are associated with certain B cell subsets: splenic marginal zone (CD21(high) CD23(low)), splenic CD1(+), peritoneal B-1a (IgD(low) CD5(+)), and CD21(high) CD24(high) B cells in peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches. Infusion of PpL triggered a sequence of events in B cell receptor (BCR)-targeted B cells, with rapid down-regulation of BCR, the induction of an activation phenotype, and limited rounds of proliferation. Apoptosis followed through a process heralded by the dissipation of mitochondrial membrane potential, the induction of the caspase pathway, DNA fragmentation, and the deposition of B cell apoptotic bodies. These studies define a common pathway by which microbial toxins that target V region-associated BCR sites induce programmed cell death.     

Polysaccharide/polynucleotide complexes. Part 6: complementary-strand-induced release of single-stranded DNA bound in the schizophyllan complex

Spectroscopic properties of single-stranded DNA/schizophyllan ternary complexes (ss-DNA2s-SPG), induced by addition of either complementary or noncomplementary strands, have been investigated. The addition of the complementary strands to ss-DNA2s-SPG induced the quick release of the bound ss-DNA to the complementary strands (both DNA and RNA), whereas the ternary complex was unaffected upon addition of noncomplementary strands. Our experiments imply that SPG has complexation properties indispensable to the gene carriers. As far as we know, there is no report on exploitation of such nonviral gene carriers that can accomplish an intelligent release of the bound ss-DNA toward the complementary strands. We believe, therefore, that SPG, a natural and neutral polysaccharide, has a great potential to become a new ss-DNA carrier.     

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.     

On the reflex coactivation of ankle flexor and extensor muscles induced by a sudden drop of support surface during walking in humans.

Recent studies have revealed that the stretch reflex responses of both ankle flexor and extensor muscles are coaugmented in the early stance phase of human walking, suggesting that these coaugmented reflex responses contribute to secure foot stabilization around the heel strike. To test whether the reflex responses mediated by the stretch reflex pathway are actually induced in both the ankle flexor and extensor muscles when the supportive surface is suddenly destabilized, we investigated the electromyographic (EMG) responses induced after a sudden drop of the supportive surface at the early stance phase of human walking. While subjects walked on a walkway, the specially designed movable supportive surface was unexpectedly dropped 10 mm during the early stance phase. The results showed that short-latency reflex EMG responses after the impact of the drop (<50 ms) were consistently observed in both the ankle flexor and extensor muscles in the perturbed leg. Of particular interest was that a distinct response appeared in the tibialis anterior muscle, although this muscle showed little background EMG activity during the stance phase. These results indicated that the reflex activities in the ankle muscles certainly acted when the supportive surface was unexpectedly destabilized just after the heel strike during walking. These reflex responses were most probably mediated by the facilitated stretch reflex pathways of the ankle muscles at the early stance phase and were suggested to be relevant to secure stabilization around the ankle joint during human walking.     

Effect of anti-regucalcin antibody on neutral phosphatase activity in rat liver cytosol: Involvement of endogenous regucalcin

The effect of anti-regucalcin monoclonal antibody on neutral phoshatase activity in rat liver cytosol was investigated. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate toward phosphatase asssy. Liver cytosolic phosphatase activity with three phosphoaminoacids was significantly increased in the presence of anti-regucalcin antibody (100 and 200 ng/ml) in the enzyme reaction mixture with calcium chloride (0.1 mM) or EGTA (1.0 mM). The effect of anti-regucalcin antibody was completely abolished in the presence of exogenous regucalcin (1.0 M), indicating the involvement of endogenous regucalcin. The anti-regucalcin anti body- increased phosphatase activity was not significantly altered in the presence of trifluoperazine (20 M), an antagonist of calmodulin, or akadaic acid (10 M), an inhibitor of protein phosphatase, although these inihibitors caused a slight decrease in liver cytosolic phosphatase activity. The effect of endogenous regucalcin might be not related to calmodulin, and it was insensitive to okadaic acid. The present findings suggest that endogenous regucalcin is involved in the regulation of protein phasphatase in rat liver cytoplasm.     

In vivo visualization of subendocardial arteriolar response in renovascular hypertensive hearts

Time-sequential responses to endothelium-dependent and -independent vasodilators and angiotensin-converting enzyme (ACE) inhibitors were studied in the subendocardial arterioles (Endo) of canine renovascular hypertension (HT) compared with subepicardial arterioles (Epi; both <120 microm) by charge-coupled device intravital microscope. Vascular responses to acetylcholine, papaverine, and cilazaprilat were compared between normotensive (NT) and HT dogs [4 wk and 12 wk of HT (4wHT and 12wHT)]. The acetylcholine-induced vasodilation of Endo in both 4wHT and 12wHT was smaller than that of NT (both P < 0.01 vs. 4wHT and 12wHT), and that of Epi was smaller than that of NT only in 12wHT (P < 0.05). The papaverine-induced vasodilation of Endo, but not Epi, was impaired only in 12wHT (both P < 0.01 vs. NT and 4wHT). Vasodilation by cilazaprilat remained unchanged at 4wHT and 12wHT in both Epi and Endo. In conclusion, at the early stage, the endothelium-dependent response of Endo was impaired, whereas at the later stage, the endothelium-dependent and -independent responses of Endo and the endothelium-dependent response of Epi were impaired. However, the vasodilatory responses to the ACE inhibitor were maintained in both Endo and Epi of HT.