Bufadienolides. 8. 12(13→14)abeo skeletal rearrangements

Several methods were developed for converting isodigitoxigenin (2a) into methyl acetals 4b and 4c. Of these, methanolysis (followed by acetylation) of isodigitoxigenin in the presence of p-toluenesulfonic acid proved most useful. Each isomer reached an equilibrium corresponding to ca. 3:1 acetal 4c to 4b within 15 min in benzene containing p-toluenesulfonic acid. Addition of dihydropyran to the equilibrium mixture resulted in excellent conversion into vinyl ether 5a. Heating either acetal 4b or 4c in benzene containing p-toluenesulfonic acid led to a skeletal rearrangement culminating in formation of C-norcardenolide 6. In addition to results of physical measurements, the structure of spiran 6 was confirmed by degradation to methyl ketone 8. Similar rearrangement of isodigitoxigenin gave spiran 9 accompanied by C-norcardenolide 6. Treating lactone 9 with p-toluenesulfonic acid in methanol-water provided acetals 10a and 10b, which on further contact with p-toluenesulfonic acid in refluxing benzene gave lactone 9 and cardenolide 6. Evidence underlying the stereochemical assignments noted for structures 4, 9, and 10 was also discussed.

Synthetic investigations on testerone and its analogues—I *1: The total synthesis of 8-isotestosterone and its anthracene analogue

The total synthesis of 8-isotestosterone (II) and the corresponding anthracene analogue (III) following the benzohydrindane route is reported. Catalytic hydrogenation of trans-1β-acetoxy-8-methyl-4,5-(3′-methyl-4′-hydroxybenzo)-hydrindane (V) followed by oxidation has furnished two isomeric tricyclic keto acetates, viz. 1β,2α-(3′-acetoxycyclopentano)-2,5-dimethyl-6-keto-1α,2,3,4,4aα,-5α,6,7,8,8aα-decahydronaphthalene (VII) and 1β,2α-(3′-acetoxycyclopentano)-2,5-dimethyl-6-keto-1α,2,3,4,4aβ,5,6,7,8,8aβ-decahydronaphthalene (IX) which are cis-non-steroid and cis-steroid configurations of the same cyclopentano-cis-decalins. A difference in the direction of enolization of the keto acetate (VII) in alkylation reaction and enol acetylation towards the methine and the methylene carbon atoms respectively has been observed.

Steroids and related natural products. VII. Boron trifluoride etherate-lithium aluminum hydride reduction of smilagenin acetate

Reduction of smilagenin acetate (Va) using a boron trifluoride etherate-lithium aluminum hydride reagent, followed by hydrogen peroxide oxidation and acetylation, was found to yield: 3β-ethoxysmilagenin (Vb), 3β-ethoxydihydrosmilagenin acetate (VIa), dihydrosmilagenin diacetate (VIb), and a complex mixture of partially acetylated products. Similar reaction conditions were employed to convert dihydrodiosgenin (II) to dihydrochlorogenin (III). Boron trifluoride etherate-lithium aluminum hydride reduction of 3β-acetoxy-5α-cholestane and 3β-acetoxy-5α-lanostane (VIIIa) was shown to yield the corresponding 3β-ethoxy (e.g., VIIIb) derivatives.

Characterization of poly(ADP-ribosyl)ated domains of rat pachytene chromatin

Poly(ADP-ribosyl)ation of nuclear proteins was several-fold higher in the pachytene spermatocytes than in the premeiotic germ cells of the rat. Among the histones of the pachytene nucleus, histone subtypes H2A, H1 and H3 were poly(ADP-ribosyl)ated. Based on the immunoaffinity fractionation procedure of Malik, Miwa, Sugimara & Smulson [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] we have fractionated DNAase-II-solubilized chromatin into poly(ADP-ribosyl)ated chromatin (PAC) and non-poly(ADP-ribosyl)ated chromatin (non-PAC) domains on an anti-[poly(ADP-ribose)] IgG affinity matrix. Approx. 2.5% of the pachytene chromatin represented the PAC domains. A significant amount of [alpha-32P]dATP-labelled pachytene chromatin (labelled in vitro) was bound to the affinity matrix. The DNA of pachytene PAC domains had internal strand breaks, significant length of gaps and ligatable ends, namely 5'-phosphoryl and 3'-hydroxyl termini. On the other hand, the PAC domains from 18 h regenerating liver had very few gaps, if any. The presence of gaps in the pachytene PAC DNA was also evident from thermal denaturation studies. Although many of the polypeptides were common to the PAC domains of both pachytene and regenerating liver, the DNA sequences associated with these domains were quite different. A 20 kDa protein and the testis-specific histone H1t were selectively enriched in the pachytene PAC domains. The pachytene PAC domains also contained approx. 10% of the messenger coding sequences present in the DNAase-II-solubilized chromatin. The pachytene PAC domains, therefore, may represent highly enriched DNA-repair domains of the pachytene nucleus.

cAMP-regulated Protein Lysine Acetylases in Mycobacteria

Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellularcAMP found in both pathogenic and nonpathogenic mycobacteria suggest that additional and important biological processes are regulated by characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase.

DNA Clasping by Mycobacterial HU: The C-Terminal Region of HupB Mediates Increased Specificity of DNA Binding

Background: HU a small, basic, histone like protein is a major component of the bacterial nucleoid. E. coli has two subunits of HU coded by hupA and hupB genes whereas Mycobacterium tuberculosis (Mtb) has only one subunit of HU coded by ORF Rv2986c (hupB gene). One noticeable feature regarding Mtb HupB, based on sequence alignment of HU orthologs from different bacteria, was that HupB(Mtb) bears at its C-terminal end, a highly basic extension and this prompted an examination of its role in Mtb HupB function. Methodology/Principal Findings: With this objective two clones of Mtb HupB were generated; one expressing full length HupB protein (HupB(Mtb)) and another which expresses only the N terminal region (first 95 amino acid) of hupB (HupB(MtbN)). Gel retardation assays revealed that HupBMtbN is almost like E. coli HU (heat stable nucleoid protein) in terms of its DNA binding, with a binding constant (K-d) for linear dsDNA greater than 1000 nM, a value comparable to that obtained for the HU alpha alpha and HU alpha beta forms. However CTR (C-terminal Region) of HupB(Mtb) imparts greater specificity in DNA binding. HupB(Mtb) protein binds more strongly to supercoiled plasmid DNA than to linear DNA, also this binding is very stable as it provides DNase I protection even up to 5 minutes. Similar results were obtained when the abilities of both proteins to mediate protection against DNA strand cleavage by hydroxyl radicals generated by the Fenton's reaction, were compared. It was also observed that both the proteins have DNA binding preference for A: T rich DNA which may occur at the regulatory regions of ORFs and the oriC region of Mtb. Conclusions/Significance: These data thus point that HupB(Mtb) may participate in chromosome organization in-vivo, it may also play a passive, possibly an architectural role.

Mode of action of antitumour antibiotics-III: Modulation of permeability of nuclear membrane in the presence of the antibiotics

The binding characteristics of the antibiotics to nuclei and their effect on the permeability of nuclear membrane with respect to histones and ribonucleic acids have been investigated. The binding constant for chromomycin A3 was found to be 1.4 × 104M?1 and number of binding sites was equal to 3.48 ± 1.08 × 1012 molecules/nuclei. The antibiotic chromomycin A3 enhanced the uptake of lysine-rich histone, actinomycin D decreased the uptake and ethidium bromide had no effect. Chromomycin A3 also enhanced the release of acid insoluble fraction containing RNA from the nuclei, actinomycin D and ethidium bromide inhibited the release of acid insoluble fraction containing RNA. The relevance of this finding to the role of nuclear envelope in understanding the mechanism of action of the antibiotic has been discussed.

Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein

To understand the molecular basis of gene targeting, we have studied interactions of nucleoprotein filaments comprised of single-stranded DNA and RecA protein with chromatin templates reconstituted from linear duplex DNA and histones. We observed that for the chromatin templates with histone/DNA mass ratios of 0.8 and 1.6, the efficiency of homologous pairing was indistinguishable from that of naked duplex DNA but strand exchange was repressed. In contrast, the chromatin templates with a histone/DNA mass ratio of 9.0 supported neither homologous pairing nor strand exchange. The addition of histone H1, in stoichiometric amounts, to chromatin templates quells homologous pairing. The pairing of chromatin templates with nucleoprotein filaments of RecA protein-single-stranded DNA proceeded without the production of detectable networks of DNA, suggesting that coaggregates are unlikely to be the intermediates in homologous pairing. The application of these observations to strategies for gene targeting and their implications for models of genetic recombination are discussed.

DNA damage recognition in the normal epithelium of human prostate and seminal vesicles

Prostate cancer is one of the most prevalent cancer types in men. The development of prostate tumors is known to require androgen exposure, and several pathways governing cell growth are deregulated in prostate tumorigenesis. Recent genetic studies have revealed that complex gene fusions and copy - number alterations are frequent in prostate cancer, a unique feature among solid tumors. These chromosomal aberrations are though to arise as a consequence of faulty repair of DNA double strand breaks (DSB). Most repair mechanisms have been studied in detail in cancer cell lines, but how DNA damage is detected and repaired in normal differentiated human cells has not been widely addressed. The events leading to the gene fusions in prostate cancer are under rigorous studies, as they not only shed light on the basic pathobiologic mechanisms but may also produce molecular targets for prostate cancer treatment and prevention. Prostate and seminal vesicles are part of the male reproductive system. They share similar structure and function but differ dramatically in their cancer incidence. Approximately fifty primary seminal vesicle carcinomas have been reported worldwide. Surprisingly, only little is known on why seminal vesicles are resistant to neoplastic changes. As both tissues are androgen dependent, it is a mystery that androgen signaling would only lead to tumors in prostate tissue. In this work, we set up novel ex vivo human tissue culture models of prostate and seminal vesicles, and used them to study how DNA damage is recognized in normal epithelium. One of the major DNA - damage inducible pathways, mediated by the ATM kinase, was robustly activated in all main cell types of both tissues. Interestingly, we discovered that secretory epithelial cells had less histone variant H2A.X and after DNA damage lower levels of H2AX were phosphorylated on serine 139 (γH2AX) than in basal or stromal cells. γH2AX has been considered essential for efficient DSB repair, but as there were no significant differences in the γH2AX levels between the two tissues, it seems more likely that the role of γH2AX is less important in postmitotic cells. We also gained insight into the regulation of p53, an important transcription factor that protects genomic integrity via multiple mechanisms, in human tissues. DSBs did not lead to a pronounced activation of p53, but treatments causing transcriptional stress, on the other hand, were able to launch a notable p53 response in both tissue types. In general, ex vivo culturing of human tissues provided unique means to study differentiated cells in their relevant tissue context, and is suited for testing novel therapeutic drugs before clinical trials. In order to study how prostate and seminal vesicle epithelial cells are able to activate DNA damage induced cell cycle checkpoints, we used primary cultures of prostate and seminal vesicle epithelial cells. To our knowledge, we are the first to report isolation of human primary seminal vesicle cells. Surprisingly, human prostate epithelial cells did not activate cell cycle checkpoints after DSBs in part due to low levels of Wee1A, a kinase regulating CDK activity, while primary seminal vesicle epithelial cells possessed proficient cell cycle checkpoints and expressed high levels of Wee1A. Similarly, seminal vesicle cells showed a distinct activation of the p53 - pathway after DSBs that did not occur in prostate epithelial cells. This indicates that p53 protein function is under different control mechanisms in the two cell types, which together with proficient cell cycle checkpoints may be crucial in protecting seminal vesicles from endogenous and exogenous DNA damaging factors and, as a consequence, from carcinogenesis. These data indicate that two very similar organs of male reproductive system do not respond to DNA damage similarly. The differentiated, non - replicating cells of both tissues were able to recognize DSBs, but under proliferation human prostate epithelial cells had deficient activation of the DNA damage response. This suggests that prostate epithelium is most vulnerable to accumulating genomic aberrations under conditions where it needs to proliferate, for example after inflammatory cellular damage.

Stereoselective total synthesis of (+/-)-tochuinyl acetate and (+/-)-dihydrotochuinyl acetates

Details of the first total synthesis of the marine natural product dihydrotochuinyl acetate is described. Cyclopentenone annulation of p-methylacetophenone via a Claisen rearrangement-Wacker oxidation based sequence generated the cyclopentenone 3, a known precursor for the sesquiterpenes cuparene, laurene, alpha-cuparenone and beta-cuparenones. Conversion of the ketone moiety into a carboxylate followed by stereoselective alkylation and reduction transformed the cyclopentenone 3 into the primary alcohol 19. Birch reduction of the alcohol 19 followed by acetylation furnished (+/-)-dihydrotochuinyl acetate, whereas direct acetylation of 19 furnished (+/-)-tochuinyl acetate. (C) 1998 Elsevier Science Ltd. All rights reserved.

Characterization of RNA polymerase III transcription factor TFIIIC from the mulberry silkworm, Bombyx mori

Fractionation of nuclear extracts from posterior silk glands of mulberry silkworm Bombyx mori. resolved the transcription factor TFIIIC into two components (designated here as TFIIIC and TFIIIC1) as in HeLa cell nuclear extracts. The reconstituted transcription of tRNA genes required the presence of both components. The affinity purified TFIIIC is a heteromeric complex comprising of five subunits ranging from 44 to 240 kDa. Of these, the 51-kDa subunit could be specifically crosslinked to the B box of tRNA(1)(Gly). Purified swTFIIIC binds to the B box sequences with an affinity in the same range as of yTFIIIC or hTFIIIC2. Although an histone acetyl transferase (HAT) activity was associated with the TFIIIC fractions during the initial stages of purification. the HAT activity, unlike the human TFIIIC preparations, was separated at the final DNA affinity step. The tRNA transcription from DNA template was independent of HAT activity but the repressed transcription from chromatin template could be partially restored by external supplementation of the dissociated HAT activity. This is the first report on the purification and characterization of TFIIIC from insect systems.

Synergy between the N-terminal and C-terminal domains of Mycobacterium tuberculosis HupB is essential for high-affinity binding, DNA supercoiling and inhibition of RecA-promoted strand exchange

The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN), HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair.

Biochemical characterization of a recombinant Swainsona canescens calcium-dependent protein kinase (ScCPK1)

Calcium-dependent protein kinases (CPKs) constitute a unique family of kinases involved in many physiological responses in plants. Biochemical and kinetic properties of a recombinant Swainsona canescens calcium-dependent protein kinase (ScCPK1) were examined in this study. The optimum pH and temperature for activity were pH 7.5 and 37 degrees C, respectively. Substrate phosphorylation activity of ScCPK1 was calmodulin (CaM) independent. Yet CaM antagonists, W7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and calmidazolium inhibited the activity with IC50 values of 750 nM and 350 pM, respectively. Both serine and threonine residues were found to be phosphorylated in auto-phosphorylated ScCPK1 and in histone III-S phosphorylated by ScCPK1. The Ca2+] for half maximal activity (K-0.5) was found to be 0.4 mu M for ScCPK1 with histone III-S as substrate. Kinetic analysis showed that Km of ScCPK1 for histone III-S was 4.8 mu M. These data suggest that ScCPK1 is a functional Ser/Thr kinase, regulated by calcium, and may have a role in Ca2+-mediated signaling in S. canescens. (C) 2012 Elsevier Masson SAS. All rights reserved.

MicroRNA-155 Is Required for Mycobacterium bovis BCG-Mediated Apoptosis of Macrophages

Pathogenic rnycobacteria, including Mycobacterium tuberculosis and Mycobacterium bovis, cause significant morbidity and mortality worldwide. However, the vaccine strain Mycobacterium bovis BCG, unlike virulent strains, triggers extensive apoptosis of infected macrophages, a step necessary for the elicitation of robust protective immunity. We here demonstrate that M. bovis BCG triggers Toll-like receptor 2 (TLR2)-dependent microRNA-155 (miR-155) expression, which involves signaling cross talk among phosphatidylinositol 3-kinase (PI3K), protein kinase C delta (PKC delta), and mitogen-activated protein kinases (MAPKs) and recruitment of NF-kappa B and c-ETS to miR-155 promoter. Genetic and signaling perturbations presented the evidence that miR-155 regulates PKA signaling by directly targeting a negative regulator of PKA, protein kinase inhibitor alpha (PKI-alpha). Enhanced activation of PKA signaling resulted in the generation of PKA C-alpha; phosphorylation of MSK1, cyclic AMP response element binding protein (CREB), and histone H3; and recruitment of phospho-CREB to the apoptotic gene promoters. The miR-155-triggered activation of caspase-3, BAK1, and cytochrome c translocation involved signaling integration of MAPKs and epigenetic or posttranslational modification of histones or CREB. Importantly, M. bovis BCG infection-induced apoptosis was severely compromised in macrophages derived from miR-155 knockout mice. Gain-of-function and loss-of-function studies validated the requirement of miR-155 for M. bovis BCG's ability to trigger apoptosis. Overall, M. bovis BCG-driven miR-155 dictates cell fate decisions of infected macrophages, strongly implicating a novel role for miR-155 in orchestrating cellular reprogramming during immune responses to mycobacterial infection.

Molecular cloning, overexpression, and characterization of autophosphorylation in calcium-dependent protein kinase 1 (CDPK1) from Cicer arietinum

In plants, calcium-dependent protein kinases (CDPKs) are key intermediates in calcium-mediated signaling that couple changes in Ca2+ levels to a specific response. In the present study, we report the high-level soluble expression of calcium-dependent protein kinase1 from Cicer arietinum (CaCDPK1) in Escherichia coli. The expression of soluble CaCDPK1 was temperature dependent with a yield of 3-4 mg/l of bacterial culture. CaCDPK1 expressed as histidine-tag fusion protein was purified using Ni-NTA affinity chromatography till homogeneity. The recombinant CaCDPK1 protein exhibited both calcium-dependent autophosphorylation and substrate phosphorylation activities with a V (max) and K (m) value of 13.2 nmol/min/mg and 34.3 mu M, respectively, for histone III-S as substrate. Maximum autophosphorylation was seen only in the presence of calcium. Optimum temperature for autophosphorylation was found to be 37 A degrees C. The recombinant protein showed optimum pH range of 6-9. The role of autophosphorylation in substrate phosphorylation was investigated using histone III-S as exogenous substrate. Our results show that autophosphorylation happens before substrate phosphorylation and it happens via intra-molecular mechanism as the activity linearly depends on enzyme concentrations. Autophosphorylation enhances the kinase activity and reduces the lag phase of activation, and CaCDPK1 can utilize both ATP and GTP as phosphodonor but ATP is preferred than GTP.