Interaction of EcoP1 modification methylase with S-adenosyl-L-methionine: a UV-crosslinking study

EcoP1 modification methylase was radioactively labeled when incubated with S-adenosyl-L-[methyl-3H]methionine in the presence of ultraviolet light. Crosslinking of the enzyme as detected by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel followed by fluorography and autoradiography, was shown to be specific by a number of criteria. More importantly, EcoP1 modification methylase was also radioactively labeled with S-adenosyl-L-[carboxyl-14C]methionine demonstrating that labeling involved binding of the entire AdoMet molecule rather than methylation of the protein. Further, c2 EcoP1 mutant DNA modification methylases which show negligible or very little methylation activity, correspondingly formed a weak or no adduct upon crosslinking. These results suggest that photolabeling of EcoP1 DNA modification methylase occurs at the AdoMet binding site.

Role of neutral metabolites in microbial conversion of 3beta-acetoxy-19-hydroxycholest-5-ene into estrone

Biotransformation of 3 beta-acetoxy-19-hydroxycholest-5-ene (19-HCA, 6 g) by Moraxella sp. was studied. Estrone (712 mg) was the major metabolite formed. Minor metabolites identified were 5 alpha-androst-1-en-19-ol-3,17-dione (33 mg), androst-4-en-19-ol-3,17-dione (58 mg), androst-4-en-9 alpha,19-diol-3,17-dione (12 mg), and androstan-19-ol-3,17-dione (1 mg). Acidic metabolites were not formed. Time course experiments on the fermentation of 19-HCA indicated that androst-4-en-19-ol-3,17-dione was the major metabolite formed during the early stages of incubation. However with continuing fermentation its level dropped, with a concomitant increase in estrone. Fermentation of 19-HCA in the presence of specific inhibitors or performing the fermentation for a shorter period (48 h) did not result in the formation of acidic metabolites. Resting-cell experiments carried out with 19-HCA (200 mg) in the presence of alpha,alpha'-bipyridyl led to the isolation of three additional metabolites, viz., cholestan-19-ol-3-one (2 mg), cholest-4-en-19-ol-3-one (10 mg), and cholest-5-en-3 beta,19-diol (12 mg). Similar results were also obtained when n-propanol was used instead of alpha,alpha'-bipyridyl. Resting cells grown on 19-HCA readily converted both 5 alpha-androst-1-en-19-ol-3,17-dione and androst-4-en-19-ol-3,17-dione into estrone. Partially purified 1,2-dehydrogenase from steroid-induced Moraxella cells transformed androst-4-en-19-ol-3,17-dione into estrone and formaldehyde in the presence of phenazine methosulfate, an artificial electron acceptor. These results suggest that the degradation of the hydrocarbon side chain of 19-HCA does not proceed via C-22 phenolic acid intermediates and complete removal of the C-17 side chain takes place prior to the aromatization of the A ring in estrone. The mode of degradation of the sterol side chain appears to be through the fission of the C-17-C-20 bond. On the basis of these observations, a new pathway for the formation of estrone from 19-HCA in Moraxella sp. has been proposed.

Purification and partial characterization of acyl carrier proteins from developing oil seeds of pisa (Actinodaphne hookeri) and ground nut (Arachis hypogaea)

Acyl carrier proteins (ACP) were purified to homogeneity in the active form from developing seeds of pisa (Actinodaphne hookeri) which synthesizes exclusively trilaurin and from ground nut (Arachis hypogaea) which synthesizes triacylglycerols containing long chain fatty acids. Two major isoforms of ACPs were purified from developing pisa seeds using DEAE-cellulose, Superose-6 FPLC and C-4 reversed phase HPLC chromatographic methods. In contrast, only a single form of ACP was present in ground nut seeds which was purified by anion-exchange and activated thiol-Sepharose 4B affinity chromatography. The two isoforms of ACPs from pisa showed nearly the same specific activity of 6,706 and 7,175 pmol per min per mg protein while ground nut ACP showed a specific activity of 3,893 pmol per min per mg protein when assayed using E. coli acyl-ACP synthetase and [1-C-14]palmitic acid. When compared with E. coli ACP, the purified ACPs from both the seeds showed considerable difference in their mobility in native PAGE, but showed similar mobility in SDS-PAGE under reducing conditions. In the absence of reducing agents formation of dimers was quite prominent. The ACPs from both the seed sources were acid- and heat-stable. The major isoform of pisa seed ACP and the ground nut ACP contain 91 amino acids with M(r) 11,616 and 1,228 respectively. However, there is significant variation in their amino acid composition. A comparision of the amino acid sequence in the N-terminal region of pisa and ground nut seed ACPs showed considerable homology between themselves and with other plant ACPs but not with E. coli ACP.

Complete 'melting' of charge order in hydrothermally grown Pr0.57Ca0.41Ba0.02MnO3 nanowires

Nanowires of Pr0.57Ca0.41Ba0.02MnO3 (PCBM) (diameter similar to 80-90 nm and length similar to 3.5 mu m) were synthesized by a low reaction temperature hydrothermal method. Single-phase nature of the sample was confirmed by XRD experiments. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the morphology and microstructures of the nanowires. While the bulk PCBM is known to exhibit charge order (CO) below 230 K along with a ferromagnetic transition at 110 K, SQUID measurements on the nanowires of PCBM show that the charge order is completely absent and a ferromagnetic transition occurs at 115 K. However, the magnetization in the nanowires is observed to be less compared to that in the bulk. This observation of the complete 'melting' of the charge order in the PCBM nanowires is particularly significant in view of the observation of only a weakening of the CO in the nanowires of Pr0.5Ca0.5MnO3. Electron paramagnetic resonance experiments were also carried out on the PCBM nanowires using an X-band EPR spectrometer. Characteristic differences were observed in the line width of nanowires when compared with that of the bulk.

Roles of residues in the interface of transient protein-protein complexes before complexation

Transient protein-protein interactions play crucial roles in all facets of cellular physiology. Here, using an analysis on known 3-D structures of transient protein-protein complexes, their corresponding uncomplexed forms and energy calculations we seek to understand the roles of protein-protein interfacial residues in the unbound forms. We show that there are conformationally near invariant and evolutionarily conserved interfacial residues which are rigid and they account for similar to 65% of the core interface. Interestingly, some of these residues contribute significantly to the stabilization of the interface structure in the uncomplexed form. Such residues have strong energetic basis to perform dual roles of stabilizing the structure of the uncomplexed form as well as the complex once formed while they maintain their rigid nature throughout. This feature is evolutionarily well conserved at both the structural and sequence levels. We believe this analysis has general bearing in the prediction of interfaces and understanding molecular recognition.

Helix and hairpin nucleation in short peptides using centrally positioned conformationally constrained dipeptide segments

The effect of incorporation of a centrally positioned Ac(6)c-Xxx segment where Xxx = (L)Val/(D)Val into a host oligopeptide composed of L-amino acid residues has been investigated. Studies of four designed octapeptides Boc-Leu-Phe-Val-Ac(6)c-Xxx-Leu-Phe-Val-OMe (Xxx = (D)Val 1, (L)Val 2) Boc-Leu-Val-Val-Ac(6)c-Xxx-Leu-Val-Val-OMe (Xxx = (D)Val 3, (L)Val 4) are reported. Diagnostic nuclear Overhouse effects characteristic of hairpin conformations are observed for Xxx = (D)Val peptides (1 and 3) while continuous helical conformation characterized by sequential NiH <-> Ni+1H NOEs are favored for Xxx = (L)Val peptides (2 and 4) in methanol solutions. Temperature co-efficient of NH chemical shifts are in agreement with distinctly different conformational preferences upon changing the configuration of the residue at position 5. Crystal structures of peptides 2 and 4 (Xxx = (L)Val) establish helical conformations in the solid state, in agreement with the structures deduced from NMR data. The results support the design principle that centrally positioned type I beta-turns may be used to nucleate helices in short peptides, while type I' beta-turns can facilitate folding into beta-hairpins.

Detection of glycated hemoglobin using 3-Aminophenylboronic acid modified graphene oxide

This paper presents the chemical synthesis of 3-Aminophenylboronic acid (APBA) modified graphene oxide (GO) and its application to the electrochemical detection of glycated hemoglobin (GHb). The compound (GO-APBA) was synthesized by forming an amide linkage between the amino group (-NH2) of APBA and the carboxylic group (-COOH) of GO. The compound was characterized using IR spectroscopy. Detection of GHb was carried out using Electrochemical Impedance Spectroscopic (EIS) measurements with GO-APBA modified glassy carbon electrode as the working electrode.

Synthesis, antimicrobial, DNA-binding and photonuclease studies of Cobalt(III) and Nickel(II) Schiff base complexes

New metal complexes of the type M(nih)(L)](PF6)(n)center dot xAH(2)O and M(nih)(2)](PF6)center dot xH(2)O (where M = Co(III) or Ni(II), L = 1,10-phenanthroline (phen)/or 2,2' bipyridine (bpy), nih = 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone, n = 2 or 1 and x = 3 or 2) have been synthesized and characterized by elemental analysis, magnetic, IR and H-1 NMR spectral data. The electronic and magnetic moment 2.97-3.07 B.M. data infers octahedral geometry for all the complexes. The IR data reveals that Schiff base (nih) form coordination bond with the metal ion through azomethine-nitrogen, phenolic-oxygen and carbonyl-oxygen in a tridentate fashion. In addition, DNA-binding properties of these six metal complexes were investigated using absorption spectroscopy, viscosity measurements and thermal denaturation methods. The results indicated that the nickel(II) complex strongly bind with calf-thymus DNA with intrinsic DNA binding constant K-b value of 4.9 x 10(4) M-1 for (3), 4.2 x 10(4) M-1 for (4), presumably via an intercalation mechanism compared to cobalt(III) complex with K-b value of 4.6 x 10(4) M-1 (1) and 4.1 x 10(4) M-1 (2). The DNA Photoclevage experiment shows that, the complexes act as effective DNA cleavage agent. (C) 2012 Elsevier B.V. All rights reserved.

C12-Helix development in ()n sequences - spectroscopic characterization of Boc-Aib-4(R)Val]-OMe oligomers

The solution conformations of the -hybrid oligopeptides Boc-Aib-4(R)Val]n-OMe (n = 1-8) in organic solvents have been probed by NMR, IR, and CD spectroscopic methods. In the solid state, this peptide series favors C12-helical conformations, which are backbone-expanded analogues of 310 helices in -peptide sequences. NMR studies of the six- (n = 3) and 16-residue (n = 8) peptides reveal that only two NH protons attached the N-terminus residues Aib(1) and 4(R)Val(2) are solvent-exposed. Sequential NiH-Ni+1H NOEs characteristic of local helical conformations are also observed at the residues. IR studies establish that chain extension leads to a large enhancement in the intensities of the hydrogen-bonded NH stretching bands (3343-3280 cm-1), which suggest elongation of intramolecularly hydrogen-bonded structures. The development of C12-helical structures upon lengthening of the sequence is supported by the NMR and IR observations. The CD spectra of the ()n peptides reveal a negative maximum at ca. 206 nm and a positive maximum at ca. 192 nm, spectral feature that are distinct from those of 310 helices in -peptides.

Whole exome sequencing identifies a novel splice-site mutation in ADAMTS17 in an Indian family with Weill-Marchesani syndrome

Purpose: Weill-Marchesani syndrome (WMS) is a rare connective tissue disorder, characterized by short stature, micro-spherophakic lens, and stubby hands and feet (brachydactyly). WMS is caused by mutations in the FBN1, ADAMTS10, and LTBP2 genes. Mutations in the LTBP2 and ADAMTS17 genes cause a WMS-like syndrome, in which the affected individuals show major features of WMS but do not display brachydactyly and joint stiffness. The main purpose of our study was to determine the genetic cause of WMS in an Indian family. Methods: Whole exome sequencing (WES) was used to identify the genetic cause of WMS in the family. The cosegregation of the mutation was determined with Sanger sequencing. Reverse transcription (RT)-PCR analysis was used to assess the effect of a splice-site mutation on splicing of the ADAMTS17 transcript. Results: The WES analysis identified a homozygous novel splice-site mutation c.873+1G>T in a known WMS-like syndrome gene, ADAMTS17, in the family. RT-PCR analysis in the patient showed that exon 5 was skipped, which resulted in the deletion of 28 amino acids in the ADAMTS17 protein. Conclusions: The mutation in the WMS-like syndrome gene ADAMTS17 also causes WMS in an Indian family. The present study will be helpful in genetic diagnosis of this family and increases the number of mutations of this gene to six.

Anticancer Effects of Brominated Indole Alkaloid Eudistomin H from Marine Ascidian Eudistoma viride Against Cervical Cancer Cells (HeLa)

Marine invertebrates called ascidians are prolific producers of bioactive substances. The ascidian Eudistoma viride, distributed along the Southeast coast of India, was investigated for its in vitro cytotoxic activity against human cervical carcinoma (HeLa) cells by the MTT assay. The crude methanolic extract of E. viride, with an IC50 of 53 mu g/ml, was dose-dependently cytotoxic. It was more potent at 100 mu g/ml than cyclohexamide (1 mu g/ml), reducing cell viability to 9.2%. Among nine fractions separated by chromatography, ECF-8 exhibited prominent cytoxic activity at 10 mu g/ml. The HPLC fraction EHF-21 of ECF-8 was remarkably dose- and time-dependently cytotoxic, with 39.8% viable cells at 1 mu g/ml compared to 51% in cyclohexamide-treated cells at the same concentration; the IC50 was 0.49,mu g/ml. Hoechst staining of HeLa cells treated with EHF-2I at 0.5 mu g/ml revealed apoptotic events such an cell shrinkage, membrane blebbing, chromatin condensation and formation of apoptotic bodies. Cell size and granularity study showed changes in light scatter, indicating the characteristic feature of cells dying by apoptosis. The cell-cycle analysis of HeLa cells treated with fraction EHF-21 at 1 mu g/ml showed the marked arrest of cells in G(0)/G(1), S and G(2)/M phases and an increase in the sub G(0)/G(1) population indicated an increase in the apoptotic cell population. The statistical analysis of the sub-G(1) region showed a dose-dependent induction of apoptosis. DNA fragmentation was also observed in HeLa cells treated with EHF-21. The active EHF-2I fraction, a brominated indole alkaloid Eudistomin H, led to apoptotic death of HeLa cells.

Clean localization super-resolution microscopy for 3D biological imaging

We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3D image. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells. (C) 2016 Author(s).