Metabolism of 5β-pregnane-3,20-dione and 3β-hydroxy-5β-pregnan-20-one by Digitalis suspension cultures

Digitalis purpurea normal callus suspension culture is capable of metabolizing 5β-pregnane-3,20-dione (1) to 3β-hydroxy-5β-pregnan-20-one (2), 3α-hydroxy-5β-pregnan-20-one (3), 3β-hydroxy-5β-pregnan-20-one glucoside (7) and 3α-hydroxy-5β-pregnan-20-one glucoside (8). Digitalis purpurea habituated callus suspension culture is also capable of metabolizing 1 to 2, 3, 5β-pregnane-3β,20β-diol (5), (7), (8), 5β-pregnane-3β,20α-diol monoglucoside (9) and 5β-pregnane-3α,20α-diol monoglucoside (11). Furthermore, it was observed that 3β-hydroxy-5β-pregnan-20-one (2) is converted to 7, 9 and 11 by both suspension cultures. At the same time, 1, 3, 5 and 8 were detected in normal callus, while 5β-pregnane-3β,20α-diol (4) and 5β-pregnane-3β,20β-diol monoglucoside (10) were present in the habituated callus culture.     

Selectivity and potency of the retroprogesterone dydrogesterone in vitro

Dydrogesterone is widely used for menstrual disorders, endometriosis, threatened and habitual abortion and postmenopausal hormone replacement therapy. Although progestins have a promiscuous nature, dydrogesterone does not have clinically relevant androgenic, estrogenic, glucocorticoid or mineralocorticoid activities. To date, systematic biochemical characterization of this progestin and its active main metabolite, 20α-dihydrodydrogesterone, has not been performed in comparison to progesterone. The objective of this study was to evaluate the selectivity and potential androgenic/antiandrogenic effects of dydrogesterone and its metabolite in comparison to progesterone and medroxyprogesterone acetate by analyzing their interference with AR signaling in vitro. We characterized dydrogesterone and its metabolite for their binding and transactivation of androgen and other steroid hormone receptors and for their potential inhibitory effects against androgen biosynthetic enzymes, 17β-hydroxysteroid dehydrogenase types 3 and 5 and 5α-reductase types 1 and 2. We found that dydrogesterone resembled progesterone mainly in its progestogenic effects and less in its androgenic, anti-androgenic, glucocorticoid and antiglucocorticoid effects; whereas, 20α-dihydrodydrogesterone showed reduced progestogenic potency with no androgenic, glucocorticoid and mineralocorticoid effects. Effects on the androgen and glucocorticoid receptor differed depending on the technology used to investigate transactivation. Progesterone, but not dydrogesterone and 20α-dihydrodydrogesterone, exerted anti-androgenic effects at the pre-receptor level by inhibiting 5α-reductase type 2. Dydrogesterone, 20α-dihydrodydrogesterone and progesterone inhibited the biosynthesis of testosterone catalyzed by 17β-hydroxysteroid dehydrogenase types 3 and 5; however, due to their micromolar K_i values, these activities appeared to be not of relevance at therapeutic levels. Overall, our data show that the anti-androgenic potential of dydrogesterone and 20α-dihydrodydrogesterone is less pronounced compared to progesterone.     

Genetic diversity and prevalence of CCR2-CCR5 gene polymorphisms in the Omani population

Polymorphisms in the regulatory region of the CCR5 gene affect protein expression and modulate the progress of HIV-1 disease. Because of this prominent role, variations in this gene have been under differential pressure and their frequencies vary among human populations. The CCR2V64I mutation is tightly linked to certain polymorphisms in the CCR5 gene. The current Omani population is genetically diverse, a reflection of their history as traders who ruled extensive regions around the Indian Ocean. In this study, we examined the CCR2-CCR5 haplotypes in Omanis and compared the patterns of genetic diversity with those of other populations. Blood samples were collected from 115 Omani adults and genomic DNA was screened to identify the polymorphic sites in the CCR5 gene and the CCR2V64I mutation. Four minor alleles were common: CCR5-2554T and CCR5-2086G showed frequencies of 49% and 46%, respectively, whereas CCR5-2459A and CCR5-2135C both had a frequency of 36%. These alleles showed moderate levels of heterozygosity, indicating that they were under balancing selection. However, the well-known allele CCR5Δ32 was relatively rare. Eleven haplotypes were identified, four of which were common: HHC (46%), HHE (20%), HHA (14%) and HHF*2 (12%).     

STE20 phosphorylation of AMPK-related kinases revealed by biochemical purifications combined with genetics

We have recently purified mammalian sterile 20 (STE20)–like kinase 3 (MST3) as a kinase for the multifunctional kinases, AMP-activated protein kinase–related kinases (ARKs). However, unresolved questions from this study, such as remaining phosphorylation activities following deletion of the Mst3 gene from human embryonic kidney cells and mice, led us to conclude that there were additional kinases for ARKs. Further purification recovered Ca²⁺/calmodulin-dependent protein kinase kinases 1 and 2 (CaMKK1 and 2), and a third round of purification revealed mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5) as potential kinases of ARKs. We then demonstrated that MST3 and MAP4K5, both belonging to the STE20-like kinase family, could phosphorylate all 14 ARKs both in vivo and in vitro. Further examination of all 28 STE20 kinases detected variable phosphorylation activity on AMP-activated protein kinase (AMPK) and the salt-inducible kinase 3 (SIK3). Taken together, our results have revealed novel relationships between STE20 kinases and ARKs, with potential physiological and pathological implications.     

Characterization of human phosphodiesterase 12 and identification of a novel 2'-5' oligoadenylate nuclease - The ectonucleotide pyrophosphatase/phosphodiesterase 1

The vertebrate 2-5A system is part of the innate immune response and central to cellular antiviral activities. Upon activation by viral double-stranded RNA, 5′-triphosphorylated, 2′-5′-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2′-5′ oligoadenylate synthetases. The 2-5As bind and activate RNase L, an unspecific endoribonuclease, resulting in viral and cellular RNA decay. Given that most endogenous RNAs are degraded by RNase L, continued enzyme activity will eventually lead to cell growth arrest and cell death. This is averted, when 2-5As and their 5′-dephosphorylated forms, the so-called 2-5A core molecules, are cleaved and thus inactivated by 2′-5′-specific nuclease(s), e.g. phosphodiesterase 12, thereby turning RNase L into its latent form. In this study, we have characterized the human phosphodiesterase 12 in vitro focusing on its ability to degrade 2-5As and 2-5A core molecules. We have found that the enzyme activity is distributive and is influenced by temperature, pH and divalent cations. This allowed us to determine V_max and K_m kinetic parameters for the enzyme. We have also identified a novel 2′-5′-oligoadenylate nuclease; the human plasma membrane-bound ectonucleotide pyrophosphatase/phosphodiesterase 1, suggesting that 2-5A catabolism may be a multienzyme-regulated process.     

Design,synthesis and biological evaluation of novel pyrimidine, 3-cyanopyridine and m-amino-N-phenylbenzamide based monocyclic EGFR tyrosine kinase inhibitors

36 new compounds with the typical skeleton of 4-anilino-5-vinyl/ethynyl pyrimidine, 4-anilino-3-cyano-5-vinyl/ethynyl/phenyl pyridine, and m-amino-N-phenylbenzamide, are designed, synthesized and selectively tested on EGFR, ErbB-2 kinases, and A-549, HL60 cells growth inhibition. Results from the bioactivity and chemical structures yield preliminary structure–activity relationships (SARs). The most potent 5-ethynylpyrimidine derivative 20a has an IC₅₀ value of 45 nM to EGFR kinase. Several compounds of other series also show IC₅₀ values <1 μM for EGFR and <5 μM for A-549 and HL60 cells growth inhibition.     

C20-trifluoro-5-oxo-ETE: a metabolically stable 5-oxo-ETE derivative

The total synthesis of C₂₀-trifluoro-6(E),8(Z),11(Z),14(Z) 5-oxo-ETE is reported. This compound was designed as an ω-oxidation-resistant analog of 5-oxo-ETE that would be resistant to metabolism. The trifluoro derivative of 5-oxo-ETE stimulated calcium mobilization in neutrophils and desensitized these cells to subsequent exposure to 5-oxo-ETE.     

PRMT5 acts as a tumor suppressor by inhibiting Wnt/β-catenin signaling in murine gastric tumorigenesis

Previous studies have demonstrated the in vitro oncogenic role of protein arginine methyltransferase 5 (PRMT5) in gastric cancer cell lines. The in vivo function of PRMT5 in gastric tumorigenesis, however, is still unexplored. Here, we showed that Prmt5 deletion in mouse gastric epithelium resulted in spontaneous tumorigenesis in gastric antrum. All Prmt5-deficient mice displayed intestinal-type gastric cancer within 4 months of age. Of note, 20% (2/10) of Prmt5 mutants finally developed into invasive gastric cancer by 8 months of age. Gastric cancer caused by PRMT5 loss exhibited the increase in Lgr5⁺ stem cells, which are proposed to contribute to both the gastric tumorigenesis and progression in mouse models. Consistent with the notion that Lgr5 is the target of Wnt/β-catenin signaling, whose activation is the most predominant driver for gastric tumorigenesis, Prmt5 mutant gastric cancer showed the activation of Wnt/β-Catenin signaling. Furthermore, in human gastric cancer samples, PRMT5 deletion and downregulation were frequently observed and associated with the poor prognosis. We propose that as opposed to the tumor-promoting role of PRMT5 well-established in the progression of various cancer types, PRMT5 functions as a tumor suppressor in vivo, at least during gastric tumor formation.     

5-Hydroxytryptamine stimulates 86Rb+ efflux from pancreatic β-cells

The effects of 5-hydroxytryptamine and 5-hydroxytryptophan on ⁸⁶Rb⁺ efflux from prelabelled ob/ob-mouse islets were studied to better understand the cellular mechanisms underlying the effects of 5-hydroxytryptamine and 5-hydroxytryptophan on insulin release. 5-Hydroxytryptophan (4 mM) had no effect on ⁸⁶Rb⁺ efflux either at a low (3 mM) or at a high (20 mM) d-glucose concentration, whereas 5-hydroxytryptamine (4 mM) stimulated ⁸⁶Rb⁺ efflux at both glucose concentrations. These results indicate that 5-hydroxytryptamine may reduce glucose-induced insulin release by inhibiting early steps in the β-cell stimulus-secretion coupling.     

Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger

In early 2013, a Bengal tiger (Panthera tigris) in a zoo died of respiratory distress. All specimens from the tiger were positive for HPAI H5N1, which were detected by real-time PCR, including nose swab, throat swab, tracheal swab, heart, liver, spleen, lung, kidney, aquae pericardii and cerebrospinal fluid. One stain of virus, A/Tiger/JS/1/2013, was isolated from the lung sample. Pathogenicity experiments showed that the isolate was able to replicate and cause death in mice. Phylogenetic analysis indicated that HA and NA of A/Tiger/JS/1/2013 clustered with A/duck/Vietnam/OIE-2202/2012 (H5N1), which belongs to clade 2.3.2.1. Interestingly, the gene segment PB2 shared 98% homology with A/wild duck/Korea/CSM-28/20/2010 (H4N6), which suggested that A/Tiger/JS/1/2013 is a novel reassortant H5N1 subtype virus. Immunohistochemical analysis also confirmed that the tiger was infected by this new reassortant HPAI H5N1 virus. Overall, our results showed that this Bengal tiger was infected by a novel reassortant H5N1, suggesting that the H5N1 virus can successfully cross species barriers from avian to mammal through reassortment.     

Stereospecific reduction of progesterone by Pisum sativum

[4 -¹⁴C]-Progesterone was applied to the leaves of growing pea plants, Pisum sativum. After 3 weeks, about 50% of the administered steroid was reduced, about 20% being reduced to 5α-pregnane-3α,20β-diol as the major metabolite. The radioactivities of 5α-pregnane-3α,20α-diol and 5α-pregnane-3α,20β-diol after 3 weeks were more than twice those after one week. The following radioactive metabolises were also isolated: 5α-pregnane-3,20-dione; 20α-hydroxy-4- pregnen-3-one; 20β-hydroxy-4-pregnen-3-one; 3α-hydroxy-5α-pregnan-20-one; 3α-hydroxy-5β-pregnan-20-one; 3β-hydroxy- 5α-pregnan-20-one; 20β-hydroxy-5α-pregnan-3-one; 5α-pregnane-3β,20β-diol; and 5β-pregnane-3α,20β-diol. The radioactivities of the 5α-pregnane derivatives were considerably higher than those of the corresponding 5β-pregnane derivatives.     

Exploration of the 5-bromopyrimidin-4(3H)-ones as potent inhibitors of PDE5

The substituents both at the 6-position of the 5-bromopyrimidinone ring and at the 5′-position of the phenyl ring of 5-bromopyrimidin-4(3H)-ones were explored. 5-Bromo-6-isopropyl-2-(2-propoxy-phenyl)pyrimidin-4(3H)-one was identified as a new scaffold for potent PDE5 inhibitors. The crystal structures of PDE5/2e and PDE5/10a complexes provided a structural basis for the inhibition of 5-bromopyrimidinones to PDE5. In addition, it was also found that there is a great tolerance for the substitution at the 5′-position of the phenyl ring of 5-bormopyrimidinones and the resulted compound 13a has the highest inhibition activity to PDE5 (IC₅₀, 1.7 nM).     

Baihui (DU20)-penetrating-Qubin (GB7) acupuncture regulates microglia polarization through miR-34a-5p/Klf4 signaling in intracerebral hemorrhage rats

Intracerebral hemorrhage (ICH) is the most devastating subtype of stroke with high morbidity and mortality. The previous study has confirmed the therapeutic effect of Baihui (DU20)-penetrating-Qubin (GB7) acupuncture on ICH, while the related mechanism is left to be revealed. The aim of this study was to investigate the relevant mechanisms. ICH rat models were established utilizing the autologous blood injection method and the beneficial effect was found after DU20-penetrating-GB7 acupuncture along with decreased miR-34a-5p levels in the perihemorrhagic penumbra. Inversely, upregulating miR-34a-5p expression inhibited microglia M2 polarization while accelerated M1 polarization through targeting Krüppel-like factor 4 (Klf4), and thereby diminished the protective effect of DU20-penetrating-GB7 acupuncture on ICH. The results suggested the therapeutic effect of DU20-penetrating-GB7 acupuncture on ICH might be attributed to its modulation on microglia polarization through miR-34a-5p/Klf4 signaling.     

Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K_m, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.     

Macrophage ABCA5 deficiency influences cellular cholesterol efflux and increases susceptibility to atherosclerosis in female LDLr knockout mice

Objectives

To determine the role of macrophage ATP-binding cassette transporter A5 (ABCA5) in cellular cholesterol homeostasis and atherosclerotic lesion development.

Methods and results

Chimeras with dysfunctional macrophage ABCA5 (ABCA5^−M/−M) were generated by transplantation of bone marrow from ABCA5 knockout (ABCA5^−/−) mice into irradiated LDLr^−/− mice. In vitro, bone marrow-derived macrophages from ABCA5^−M/−M chimeras exhibited a 29% (P < 0.001) decrease in cholesterol efflux to HDL, whereas a 21% (P = 0.07) increase in cholesterol efflux to apoA-I was observed. Interestingly, expression of ABCA1, but not ABCG1, was up-regulated in absence of functional ABCA5 in macrophages. To induce atherosclerosis, the transplanted LDLr^−/− mice were fed a high-cholesterol Western-type diet (WTD) for 6, 10, or 18 weeks, allowing analysis of effects on initial as well as advanced lesion development. Atherosclerosis development was not affected in male ABCA5^−M/−M chimeras after 6, 10, and 18 weeks WTD feeding. However, female ABCA5^−M/−M chimeras did develop significantly (P < 0.05) larger aortic root lesions as compared with female controls after 6 and 10 weeks WTD feeding.

Conclusions

ABCA5 influences macrophage cholesterol efflux, and selective disruption of ABCA5 in macrophages leads to increased atherosclerotic lesion development in female LDLr^−/− mice.     

Methylation of the DFNA5 increases risk of lymph node metastasis in human breast cancer

The pathogenesis of breast cancer involves multiple genetic and epigenetic events. In this study, we report an epigenetic alteration of DFNA5 in human breast cancer. DFNA5 gene was silenced in breast cancer cell lines that were methylated in the DFNA5 promoter, and restored by treatment with the demethylating agent, 5-aza-dC, and gene knock-down of DFNA5 increased cellular invasiveness in vitro. The mRNA expression of DFNA5 in breast cancer tissues was down-regulated as compared to normal tissues. Moreover, the DFNA5 promoter was found to be methylated in primary tumor tissues with high frequency (53%, 18/34). Quantitative methylation-specific PCR of DFNA5 clearly discriminated primary breast cancer tissues from normal breast tissues (15.3%, 2/13). Moreover, methylation status of DFNA5 was correlated with lymph node metastasis in breast cancer patients. Our data implicate DFNA5 promoter methylation as a novel molecular biomarker in human breast cancer.