Structure and activity cyclase activated of OK-GC: A kidney receptor-guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4�75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Structure and activity of OK-GC: a kidney receptor guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

The repertoire of protein kinases encoded in the draft version of the human genome: atypical variations and uncommon domain combinations

Background: Phosphorylation by protein kinases is central to cellular signal transduction. Abnormal functioning of kinases has been implicated in developmental disorders and malignancies. Their activity is regulated by second messengers and by the binding of associated domains, which are also influential in translocating the catalytic component to their substrate sites, in mediating interaction with other proteins and carrying out their biological roles. Results: Using sensitive profile-search methods and manual analysis, the human genome has been surveyed for protein kinases. A set of 448 sequences, which show significant similarity to protein kinases and contain the critical residues essential for kinase function, have been selected for an analysis of domain combinations after classifying the kinase domains into subfamilies. The unusual domain combinations in particular kinases suggest their involvement in ubiquitination pathways and alternative modes of regulation for mitogen-activated protein kinase kinases (MAPKKs) and cyclin-dependent kinase (CDK)-like kinases. Previously unexplored kinases have been implicated in osteoblast differentiation and embryonic development on the basis of homology with kinases of known functions from other organisms. Kinases potentially unique to vertebrates are involved in highly evolved processes such as apoptosis, protein translation and tyrosine kinase signaling. In addition to coevolution with the kinase domain, duplication and recruitment of non-catalytic domains is apparent in signaling domains such as the PH, DAG-PE, SH2 and SH3 domains. Conclusions: Expansion of the functional repertoire and possible existence of alternative modes of regulation of certain kinases is suggested by their uncommon domain combinations. Experimental verification of the predicted implications of these kinases could enhance our understanding of their biological roles.

The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy

Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. In this paper, experimentally derived observations are reported that help in explaining why a proline→leucine mutation at position 50 of human GCAP1 results in cone–rod dystrophy in a family carrying this mutation. The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at position 50. In addition, serine replaced a glutamic acid residue at position 6 to promote N‐terminal myristoylation, yielding the construct GCAP1 E6S/P50L. The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1. In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra. However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter [Ca2+] and result in death of cells.

Identification of a core promoter and a novel isoform of the human TSCI gene transcript and structural comparison with mouse homolog

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with loci on chromosome 9q34.12 (TSC1) and chromosome 16p13.3 (TSC2). Genes for both loci have been isolated and characterized. The promoters of both genes have not been characterized so far and little is known about the regulation of these genes. This study reports the characterization of the human TSC1 promoter region for the first time. We have identified a novel alternative isoform in the 5' untranslated region (UTR) of the TSC1 gene transcript involving exon 1. Alternative isoforms in the 5' UTR of the mouse Tsc1 gene transcript involving exon I and exon 2 have also been identified. We have identified three upstream open reading frames (uORFs) in the 5' UTR of the TSC1/Tsc1 gene. A comparative study of the 5' UTR of TSC1/Tsc1 gene has revealed that there is a high degree of similarity not only in the sequence but also in the splicing pattern of both human and mouse TSC1 genes. We have used PCR methodology to isolate approximately 1.6 kb genomic DNA 5' to the TSC1 cDNA. This sequence has directed a high level of expression of luciferase activity in both HeLa and HepG2 cells. Successive 5' and 3' deletion analysis has suggested that a -587 bp region, from position +77 to -510 from the transcription start site (TSS), contains the promoter activity. Interestingly, this region contains no consensus TATA box or CAAT box. However, a 521-bp fragment surrounding the TSS exhibits the characteristics of a CpG island which overlaps with the promoter region. The identification of the TSC1 promoter region will help in designing a suitable strategy to identify mutations in this region in patients who do not show any mutations in the coding regions. It will also help to study the regulation of the TSC1 gene and its role in tumorigenesis. (C) 2003 Elsevier B.V. All rights reserved.

Synthesis and Antileukemic Activity of Novel 4-(3-(Piperidin-4-yl) Propyl)Piperidine Derivatives

To explore the anticancer effect associated with the piperidine framework, several (substituted phenyl) {4-[3-(piperidin-4-yl)propyl]piperidin-1-yl} methanone derivatives 3(a-i) were synthesized. Variation in the functional group at N-terminal of the piperidine led to a set of compounds bearing amide moiety. Their chemical structures were confirmed by (1)H NMR, IR and mass spectra analysis. Among these, compounds 3a, 3d and 3e were endowed with antiproliferative activity. The most active compound among this series was 3a with nitro and fluoro substitution on the phenyl ring of aryl carboxamide moiety, which inhibited the growth of human leukemia cells (K562 and Reh) at low concentration. Comparison with other derivative (3h) results shown by LDH assay, cell cycle analysis and DNA fragmentation suggested that 3a is more potent to induce apoptosis.

Interplay between NS3 protease and human La protein regulates translation-replication switch of Hepatitis C virus

HCV NS3 protein plays a central role in viral polyprotein processing and RNA replication. We demonstrate that the NS3 protease (NS3(pro)) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding. More importantly, NS3(pro) binding to the SLIV hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, resulting in inhibition of HCV-IRES activity. Although overexpression of both NS3(pro) as well as the full length NS3 protein decreased the level of HCV IRES mediated translation, replication of HCV replicon RNA was enhanced significantly. These observations suggest that the NS3(pro) binding to HCV IRES reduces translation in favor of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might regulate the molecular switch from translation to replication of HCV.

UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of Candida albicans

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFN gamma and TNF alpha levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens.

Synthesis and Antileukemic Activity of Novel 2-(4-(2,4-dimethoxybenzoyl)phenoxy)-1-(4-(3-(piperidin-4-yl)propyl)piper idin-1-yl)ethanone Derivatives

A series of novel 2-(4-(2,4-dimethoxybenzoyl)phenoxy)-1-(4-(3-(piperidin-4-yl)propyl) piperidin-1-yl)ethanone derivatives 9(ae) and 10(ag) were synthesized and characterized by 1H NMR, IR, mass spectral, and elemental analysis. These novel compounds were evaluated for their antileukemic activity against two human leukemic cell lines (K562 and CEM) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. Some of the tested compounds showed good antiproliferative activity with IC50 values ranging from 1.6 to 8.0 mu m. Compound 9c, 9e, and 10f with an electron-withdrawing halogen substituent at the para position on the phenyl ring showed excellent in vitro potency against tested human leukemia cells (K562 and CEM).

Ferrocene-Conjugated Copper(II) Complexes of L-Methionine and Phenanthroline Bases: Synthesis, Structure, and Photocytotoxic Activity

Ferrocene-conjugated reduced Schiff base (Fc-metH) copper(II) complexes of L-methionine and phenanthroline bases, namely, Cu(Fc-met)(B)](NO3), where B is 1,10-phenanthroline (phen in 1), dipyrido3,2-d:2',3'-f]quinoxaline (dpq in 2), dipyrido3,2-a:2',3'-c]phenazine (dppz in 3), and 2-(naphthalen-1-yl)-1H-imidazo4,5-f]1,10]phenanthroline (nip in 4), were prepared and characterized and their photocytotoxicity studied (Fc = ferrocenyl moiety). Complexes Cu(Ph-met)(B)](NO3) of the reduced Schiff base from benzaldehyde and L-methionine (Ph-metH) and B (phen in 5, dppz in 6) were prepared and used as control species. Complexes 1 and 5 were structurally characterized by X-ray crystallography. Complex 1 as a discrete monomer has a CuN3OS core with the thiomethyl group as the axial ligand. Complex 5 has a polymeric structure with a CuN3O2 core in the solid state. Complexes 5 and 6 are formulated as Cu(Ph-met)(B)(H2O)] (NO3) in an aqueous phase based on the mass spectral data. Complexes 1-4 showed the Cu(II)-Cu(I) and Fc(+)-Fc redox couples at similar to 0.0 and similar to 0.5 V vs SCE, respectively, in DMF-0.1 M (Bu4N)-N-n](ClO4). A Cu(II)-based weak d-d band near 600 nm and a relatively strong ferrocenyl band at similar to 450 nm were observed in DMF-Tris-HCl buffer (1:4 v/v). The complexes bind to calf thymus DNA, exhibit moderate chemical nuclease activity forming (OH)-O-center dot radical species, and are efficient photocleavers of pUC19 DNA in visible light of 454, 568, and 647 rim, forming (OH)-O-center dot radical as the reactive oxygen species. They are cytotoxic in HeLa (human cervical cancer) and MCF-7 (human breast cancer) cells, showing an enhancement of cytotoxicity upon visible light irradiation. Significant change in the nuclear morphology of the HeLa cells was observed with 3 in visible light compared to the nonirradiated sample. Confocal imaging using 4 showed its nuclear localization within the HeLa cells.

Structure and Mechanistic Insights into Novel Iron-mediated Moonlighting Functions of Human J-protein Cochaperone, Dph4

J-proteins are obligate cochaperones of Hsp70s and stimulate their ATPase activity via the J-domain. Although the functions of J-proteins have been well understood in the context of Hsp70s, their additional co-evolved ``physiological functions'' are still elusive. We report here the solution structure and mechanism of novel iron-mediated functional roles of human Dph4, a type III J-protein playing a vital role in diphthamide biosynthesis and normal development. The NMR structure of Dph4 reveals two domains: a conserved J-domain and a CSL-domain connected via a flexible linker-helix. The linker-helix modulates the conformational flexibility between the two domains, regulating thereby the protein function. Dph4 exhibits a unique ability to bind iron in tetrahedral coordination geometry through cysteines of its CSL-domain. The oxidized Fe-Dph4 shows characteristic UV-visible and electron paramagnetic resonance spectral properties similar to rubredoxins. Iron-bound Dph4 (Fe-Dph4) also undergoes oligomerization, thus potentially functioning as a transient ``iron storage protein,'' thereby regulating the intracellular iron homeostasis. Remarkably, Fe-Dph4 exhibits vital redox and electron carrier activity, which is critical for important metabolic reactions, including diphthamide biosynthesis. Further, we observed that Fe-Dph4 is conformationally better poised to perform Hsp70-dependent functions, thus underlining the significance of iron binding in Dph4. Yeast Jjj3, a functional ortholog of human Dph4 also shows a similar iron-binding property, indicating the conserved nature of iron sequestration across species. Taken together, our findings provide invaluable evidence in favor of additional co-evolved specialized functions of J-proteins, previously not well appreciated.

Size-mediated cytotoxicity of nanocrystalline titanium dioxide, pure and zinc-doped hydroxyapatite nanoparticles in human hepatoma cells

Nanoparticles are highly used in biological applications including nanomedicine. In this present study, the interaction of HepG2 hepatocellular carcinoma cells (HCC) with hydroxyapatite (HAp), zinc-doped hydroxyapatite, and titanium dioxide (TiO2) nanoparticles were investigated. Hydroxyapatite, zinc-doped hydroxyapatite and titanium dioxide nanoparticles were prepared by wet precipitation method. They were subjected to isochronal annealing at different temperatures. Particle morphology and size distribution were characterized by X-ray diffraction and transmission electron microscope. The nanoparticles were co-cultured with HepG2 cells. MTT assay was employed to evaluate the proliferation of tumor cells. The DNA damaging effect of HAp, Zn-doped HAp, and TiO2 nanoparticles in human hepatoma cells (HepG2) were evaluated using DNA fragmentation studies. The results showed that in HepG2 cells, the anti-tumor activity strongly depend on the size of nanoparticles in HCC cells. Cell cycle arrest analysis for HAp, zinc-doped HAp, and TiO2 nanoparticles revealed the influence of HAp, zinc-doped HAp, and titanium dioxide nanoparticles on the apoptosis of HepG2 cells. The results imply that the novel nano nature effect plays an important role in the biomedicinal application of nanoparticles.

Anticancer property of Bryophyllum pinnata (Lam.) Oken. leaf on human cervical cancer cells

Background: Bryophyllum pinnata (B. pinnata) is a common medicinal plant used in traditional medicine of India and of other countries for curing various infections, bowel diseases, healing wounds and other ailments. However, its anticancer properties are poorly defined. In view of broad spectrum therapeutic potential of B. pinnata we designed a study to examine anti-cancer and anti-Human Papillomavirus (HPV) activities in its leaf extracts and tried to isolate its active principle. Methods: A chloroform extract derived from a bulk of botanically well-characterized pulverized B. pinnata leaves was separated using column chromatography with step-gradient of petroleum ether and ethyl acetate. Fractions were characterized for phyto-chemical compounds by TLC, HPTLC and NMR and Biological activity of the fractions were examined by MTT-based cell viability assay, Electrophoretic Mobility Shift Assay, Northern blotting and assay of apoptosis related proteins by immunoblotting in human cervical cancer cells. Results: Results showed presence of growth inhibitory activity in the crude leaf extracts with IC50 at 552 mu g/ml which resolved to fraction F4 (Petroleum Ether: Ethyl Acetate:: 50: 50) and showed IC50 at 91 mu g/ml. Investigations of anti-viral activity of the extract and its fraction revealed a specific anti-HPV activity on cervical cancer cells as evidenced by downregulation of constitutively active AP1 specific DNA binding activity and suppression of oncogenic c-Fos and c-Jun expression which was accompanied by inhibition of HPV18 transcription. In addition to inhibiting growth, fraction F4 strongly induced apoptosis as evidenced by an increased expression of the pro-apoptotic protein Bax, suppression of the anti-apoptotic molecules Bcl-2, and activation of caspase-3 and cleavage of PARP-1. Phytochemical analysis of fraction F4 by HPTLC and NMR indicated presence of activity that resembled Bryophyllin A. Conclusions: Our study therefore demonstrates presence of anticancer and anti-HPV an activity in B. pinnata leaves that can be further exploited as a potential anticancer, anti-HPV therapeutic for treatment of HPV infection and cervical cancer.

Ag@AgI, Core@Shell Structure in Agarose Matrix as Hybrid: Synthesis, Characterization, and Antimicrobial Activity

A novel in situ core@shell structure consisting of nanoparticles of Ag (Ag Nps) and AgI in agarose matrix (Ag@ AgI/agarose) has been synthesized as a hybrid, in order to have an efficient antibacterial agent for repetitive usage with no toxicity. The synthesized core@shell structure is very well characterized by XRD, UV-visible, photoluminescence, and TEM. A detailed antibacterial studies including repetitive cycles are carried out on Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria in saline water, both in dark and on exposure to visible light. The hybrid could be recycled for the antibacterial activity and is nontoxic toward human cervical cancer cells (HeLa cells). The water insoluble Ag@AgI in agarose matrix forms a good coating on quartz, having good mechanical strength. EPR and TEM studies are carried out on the Ag@AgI/agarose and the bacteria, respectively, to elucidate a possible mechanism for killing of the bacteria.

Binding of Gemini Bisbenzimidazole Drugs with Human Telomeric G-Quadruplex Dimers: Effect of the Spacer in the Design of Potent Telomerase Inhibitors

The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3'-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or nmers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3) 8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties.