Analysis of a conformation-specific epitope of the alpha subunit of human chorionic gonadotropin: Study using monoclonal antibody probes

Monoclonal antibodies (MAbs) have been used extensively for identification of sequence-specific epitopes using either the ELISA or/and IRMA methods, However, attempts to use MAbs for identification of conformation-specific epitopes have been very few as they are considered very labile. We have investigated the stability of conformation-specific epitopes of human chorionic gonadotropin (hCG) using a quantitative solid-phase radioimmnunoassay (SPRIA) technique. Several epitopes are stable to mild modification (chemical and proteolytic) conditions, and epitopes show differential stability for these modifications. Based on these observations, a monoclonal antibody (MAb 16) for an a-subunit-specific epitope of hCG has been used to monitor changes at the epitopic site (identified as epitope 16) on modification of hCG, using SPRIA with immobilized MAb 16. Modifications of amino groups, hydroxyl group of tyrosine as well as carboxyl group of Asp/Glu all bring about sufficient changes in the epitope integrity. Peptide bond hydrolysis at lysine residues damages the epitope, but not at arginine residues, Hydrolysis at tyrosine does not affect the epitope, though modification of the side-chain of tyrosine inactivates the epitope. Destruction of the epitope occurs on reduction of the disulphide bonds. Partial retention of the epitope activity is seen on modification of carboxyl or the epsilon-amino groups of lysine. Based on these results four to six amino acids have been identified to be at the epitopic site, and the data suggest that two peptide segments are brought together by the disulphide bond Cys10-Cys60 to form the epitope.

Vinyl Monomer Based Polyperoxides as Potential Initiators for Radical Polymerization: An Exploratory Investigation with Poly(.alpha.-methylstyrene peroxide)

We describe the use of poly(alpha-methylstyrene peroxide) (P alpha MSP), an alternating copolymer of alpha-methylstyrene and oxygen, as initiator for the radical polymerization of vinyl monomers. Thermal decomposition of P alpha MSP in 1,4-dioxane follows first-order kinetics with an activation energy (E(a)) of 34.6 kcal/mol. Polymerization of methyl methacrylate (MMA) and styrene using P alpha MSP as an initiator was carried out in the temperature range 60-90 degrees C. The kinetic order with respect to the initiator and the monomer was close to 0.5 and 1.0, respectively, for both monomers. The E(a) for the polymerization was 20.6 and 22.9 kcal/mol for MMA and styrene, respectively. The efficiency of P alpha MSP was found to be in the range 0.02-0.04. The low efficiency of P alpha MSP was explained in terms of the unimolecular decomposition of the alkoxy radicals which competes with primary radical initiation. The presence of peroxy segments in the main chain of PMMA and polystyrene was confirmed from spectroscopic and DSC studies. R(i)'/2I values for P alpha MSP compared to that of BPO at 80 degrees C indicate that P alpha MSP can be used as an effective high-temperature initiator.

Mycobacterium tuberculosis Expresses ftsE Gene Through Multiple Transcripts

Bacterial FtsE gene codes for the ATP-binding protein, FtsE, which in complex with the transmembrane protein, FtsX, participates in diverse cellular processes. Therefore, regulated expression of FtsE and FtsX might be critical to the human pathogen, Mycobacterium tuberculosis, under stress conditions. Although ftsX gene of M. tuberculosis (MtftsX) is known to be transcribed from a promoter inside the upstream gene, ftsE, the transcriptional status of ftsE gene of M. tuberculosis (MtftsE) remains unknown. Therefore, the authors initiated transcriptional analyses of MtftsE, using total RNA from M. tuberculosis cells that were grown under stress conditions, which the pathogen is exposed to, in granuloma in tuberculosis patients. Primer extension experiments showed the presence of putative transcripts, T1, T2, T3, and T4. T1 originated from the intergenic region between the upstream gene, MRA_3135, and MtftsE. T2 and T3 were found initiated from within MRA_3135. T4 was transcribed from a region upstream of MRA_3135. RT-PCR confirmed co-transcription of MRA_3135 and MtftsE. The cloned putative promoter regions for T1, T2, and T3 elicited transcriptional activity in Mycobacterium smegmatis transformants. T1, T2, and T3, but no new transcript, were present in the M. tuberculosis cells that were grown under the stress conditions, which the pathogen is exposed to in granuloma in tuberculosis patients. It showed lack of modulation of MtftsE transcripts under the stress conditions tested, indicating that ftsE may not have a stress response-specific function in M. tuberculosis.

A novel (main chain) (side chain) polymeric peroxide: Synthesis and initiating characteristics of poly(alpha-(tert-butylperoxymethyl)styrene peroxide)

A novel (main chain)-(side chain) vinyl polyperoxide, poly(alpha-(tert-butylperoxymethyl)styrene peroxide) (MCSCPP), an alternating copolymer of alpha-(tert-butylperoxymethyl)styrene (TPMS) and oxygen, has been synthesized by the oxidative polymerization of TPMS. The MCSCPP was characterized by H-1 NMR, C-13 NMR, IR, DSC, EI-MS, and GC-MS studies. The overall activation energy (E(a)) for the degradation of MCSCPP was found to be 27 kcal/mol. Formaldehyde and alpha-(tert-butylperoxy)acetophenone (TPAP) were identified as the primary degradation products of MCSCPP; TPAP was found to undergo further degradation. The side chain peroxy groups were found to be thermally more stable than those in the main chain. Polymerization of styrene in the presence of MCSCPP as initiator, at 80 degrees C, follows classical kinetics. The presence of peroxy segments in the polystyrene chain was confirmed by both H-1 NMR and thermal decomposition studies. Interestingly, unlike other vinyl polyperoxides, the MCSCPP initiator shows an increase in molecular weight with conversion.

Photoinitiating capabilities of vinyl polyperoxides and metamorphosis of block-into-block copolymer

This article describes the first comprehensive study on the use of a vinyl polyperoxide, namely poly(styrene peroxide) (PSP), an equimolar alternating copolymer of oxygen and styrene, as a photoinitiator for free radical polymerization of vinyl monomers like styrene. The molecular weight, yield, structure and thermal stability of polystyrene (PS) thus obtained are compared with PS made using a simple peroxide like di-t-butyl peroxide. Interestingly, the PS prepared using PSP contained PSP segments attached to its backbone preferably at the chain ends. This PSP-PS-PSP was further used as a thermal macroinitiator for the preparation of another block copolymer PS-b-PMMA by reacting PSP-PS-PSP with methyl methacrylate (MMA). The mechanism of block copolymerization has been discussed. (C) 1996 John Wiley & Sons, Inc.

Structural variants of hyperbranched polyesters

Hyperbranched polyesters based on 3,5-dihydroxybenzoic acid and its derivatives were prepared by self-condensation of the corresponding ester under standard trans-esterification conditions. The spacer segment length that connects the branching points was systematically varied by starting from the appropriate ethyl 3,5-bis(omega-hydroxyoligo(ethyleneoxy))benzoate. The thermal properties of the hyperbranched polyesters were studied using DSC, and they have been compared with those of the linear analogues prepared from the corresponding p-hydroxybenzoic acid derivatives and also with the molecularly ''kinked'' analogues prepared from the meta isomers. These hyperbranched polyesters were also terminally functionalized by using a potentially mesogenic 4-butoxybiphenylcarboxylic acid derivative in an attempt to prepare novel hyperbranched liquid crystalline polyesters. This was achieved by copolymerization of the AB(2) monomer with the mesogenic A-type capping unit. These polymers were found to be amorphous and did not exhibit any liquid crystalline phases, probably due to the random distribution of the mesogenic segments on the polymer framework, making it difficult to both crystallize and form mesophases.

Surfactant lipids containing aromatic units produce vesicular membranes with high thermal stability

Six new vesicle-forming, cationic surfactant lipids are synthesized. Four of them contain 'flat' aromatic units at different locations of hydrophobic segments. In order to estimate the influence of aromatic units in the lipid monomer two other surfactant lipids of related structure with n-butyloxy units in the places of aromatic groups were also prepared. Transmission electron microscopy confirmed the vesicular membrane formation from these newly synthesized lipids. DSC or temperature-dependent keto-enol tautomerism of benzoylacetanilide-doped vesicles reveal a remarkable increase in the thermal stability of the membranes formed from aromatic surfactant lipids in contradistinction to their counterparts that contain n-butyloxy units. The enhanced thermal stability originates presumably as a consequence of inter-monomer stacking.

Synthesis, Thermotropic Behavior, and Permeability Properties of Vesicular Membranes Composed of Cationic Mixed-Chain Surfactants

In order to elucidate the role of the linkage region that connects polar headgroups with hydrophobic segments in a lipid monomer, cationic mixed-chain amphiphiles containing acyl and alkyl hydrophobic segments connected at the level of Me(2)N(+) headgroups 2a-d were synthesized. Related dialkyldimethyl-ammonium ion surfactants 1a-e and diacyl systems 3a-c were also synthesized. Despite mismatch in the connector region, amphiphiles 2a-d form bilayer vesicles like their dialkyl and diacyl counterparts, as revealed by electron microscopy. Introduction of an ester connector function between the polar and hydrophobic parts raises the phase transition temperature (T-m), transition enthalpies, and resistance to ion permeation. Consideration of energy minimized conformations points toward the importance of differences in the depth of chain penetration into the putative bilayer.

RPS: Repeats in Protein Sequences

Repeats are two or more contiguous segments of amino acid residues that are believed to have arisen as a result of intragenic duplication, recombination and mutation events. These repeats can be utilized for protein structure prediction and can provide insights into the protein evolution and phylogenetic relationship. Therefore, to aid structural biologists and phylogeneticists in their research, a computing resource (a web server and a database), Repeats in Protein Sequences (RPS), has been created. Using RPS, users can obtain useful information regarding identical, similar and distant repeats (of varying lengths) in protein sequences. In addition, users can check the frequency of occurrence of the repeats in sequence databases such as the Genome Database, PIR and SWISS-PROT and among the protein sequences available in the Protein Data Bank archive. Furthermore, users can view the three-dimensional structure of the repeats using the Java visualization plug-in Jmol. The proposed computing resource can be accessed over the World Wide Web at http://bioserver1.physics.iisc.ernet.in/rps/.

A natural compound, methyl angolensate, induces mitochondrial pathway of apoptosis in Daudi cells

Natural products discovered from medicinal plants have played an important role in the treatment of cancer. In an effort to identify novel small molecules which can affect the proliferation of lymphoma cells, we tested methyl angolensate (MA), a plant derived tetranortriterpenoid, purified from the crude extract of the root callus of Soymida febrifuga commonly known as Indian red wood tree. We have tested MA for its cytotoxic properties on Burkitt's lymphoma cell lines, using various cellular assays. We observed that MA induces cytotoxicity in Daudi cells in a dose-dependent manner using trypan blue, MTT and LDH assays. We find that the treatment with MA led to activation of DNA double-strand break repair proteins including KU70 and KU80, suggesting the activation of nonhomologous DNA end joining pathway in surviving cells. Further, we find that methyl angolensate could induce apoptosis by cell cycle analysis, annexin V-FITC staining, DNA fragmentation and PARP cleavage. Besides, MA treatment led to reactive oxygen species generation and loss of mitochondrial transmembrane potential. These results suggest the activation of mitochondrial pathway of apoptosis. Hence, we identify MA as a potential chemotherapeutic agent against Daudi cells.

In vitro plant regeneration from different seedling explants of blackgram [Vigna mungo (L.) Hepper] via organogenesis

Callus induction and morphogenesis from different blackgram explants were tested on MS basal medium supplemented with B5 vitamins, IAA, NAA, IBA, KIN and BAP individually and in combinations. The explants were hypocotyl, epicotyl, axillary bud, cotyledonary node and immature leaf. The optimal levels of the frequency of callus induction was 22.8 mu M of IAA or 16.1 mu M NAA and in combination with 2.2 mu M of BAP. Among the seedling explants, hypocotyl was found to be more efficient in producing callus. Shoots mere induced from callus cultures of hypocotyls, epicotyls, axillary bud, cotyledonary node and immature leaf with varying frequencies in the medium containing KIN (2.3-9.3 mu M) or BAP (2.2-8.8 mu M) and in combination with IAA (2.8 mu M) or NAA (2.6 mu M). Multiple shoots were obtained using cotyledonary node segments. The regenerated shoots rooted best on MS basal medium containing 9.8 mu M IBA. Seventy three per cent of the shoots produced roots, and 80-85% of the plantlets survived under greenhouse condition.

In vitro regeneration from immature cotyledon explant and protein profile changes during organogenesis in groundnut (Arachis hypogaea L.)

Complete plants were regenerated from in vitro cultured immature cotyledon segments of groundnut (Arachis hypogaea L. cv. TMV-7) by organogenesis. Callus cultures were best Initiated from immature cotyledon segments on MS (Murashige and Skoog) salts containing B5 vitamins supplemented with indole-3-acetic acid (IAA) and alpha -naphthalene acetic acid (NAA; 4.0 mg L-1) and kinetin (KIN; 0.5 L-1). Calluses were transferred to a medium containing KIN (2.0 mg L-1) and IAA and NAA (0.5 mg L-1) for shoot Initiation. The regenerated shoots were transferred to a medium containing Indole-3-butyric acid (IBA; 2.0 mg L-1) and KIN (0.2 mg L-1) for developing roots. In vitro produced plantlets developed sucessfully, matured, and set seed. The protein profiles [sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE)] of callus, callus with shoot, and callus with shoot and root showed differences.

Revisiting the earthquake sources in the Himalaya: Perspectives on past seismicity

The similar to 2500 km-long Himalaya plate boundary experienced three great earthquakes during the past century, but none of them generated any surface rupture. The segments between the 1905-1934 and the 1897-1950 sources, known as the central and Assam seismic gaps respectively, have long been considered holding potential for future great earthquakes. This paper addresses two issues concerning earthquakes along the Himalaya plate boundary. One, the absence of surface rupture associated with the great earthquakes, vis-a-vis the purported large slip observed from paleoseismological investigations and two, the current understanding of the status of the seismic gaps in the Central Himalaya and Assam, in view of the paleoseismological and historical data being gathered. We suggest that the ruptures of earthquakes nucleating on the basal detachment are likely to be restricted by the crustal ramps and thus generate no surface ruptures, whereas those originating on the faults within the wedges promote upward propagation of rupture and displacement, as observed during the 2005 Kashmir earthquake, that showed a peak offset of 7 m. The occasional reactivation of these thrust systems within the duplex zone may also be responsible for the observed temporal and spatial clustering of earthquakes in the Himalaya. Observations presented in this paper suggest that the last major earthquake in the Central Himalaya occurred during AD 1119-1292, rather than in 1505, as suggested in some previous studies and thus the gap in the plate boundary events is real. As for the Northwestern Himalaya, seismically generated sedimentary features identified in the 1950 source region are generally younger than AD 1400 and evidence for older events is sketchy. The 1897 Shillong earthquake is not a decollement event and its predecessor is probably similar to 1000 years old. Compared to the Central Himalaya, the Assam Gap is a corridor of low seismicity between two tectonically independent seismogenic source zones that cannot be considered as a seismic gap in the conventional sense. (C) 2011 Elsevier B.V. All rights reserved.

Structural sensitivity as a measure of redundancy

The conventional definition of redundancy is applicable to skeletal structural systems only, whereas the concept of redundancy has never been discussed in the context of a continuum. Generally, structures in civil engineering constitute a combination of both skeletal and continuum segments. Hence, this gaper presents a generalized definition of redundancy that has been defined in terms of structural response sensitivity, which is applicable to both continuum and discrete structures. In contrast to the conventional definition of redundancy, which is assumed to be fixed for a given structure and is believed to be independent of loading and material properties, the new definition would depend on strength and response of the structure at a given stage of its service life. The redundancy measure proposed in this paper is linked to the structural response sensitivities. Thus, the structure can have different degrees of redundancy during its lifetime, depending on the response sensitivity under consideration It is believed that this new redundancy measure would be more relevant in structural evaluation, damage assessment, and reliability analysis of structures at large.

Preincubation of cut tobacco leaf explants promotes Agrobacterium-mediated transformation by increasing vir gene induction

The effects of preincubation of cut tobacco leaf explants on Agrobacterium transformation efficiency and induction of Agrobacterium virE-lacZ fusion were evaluated. Transformation efficiency was evaluated by histochemical and fluorometric analysis of beta-glucuronidase in leaf rings transformed with Agrobacterium tumefaciens strain LBA4404(pKIWI105). The transformation efficiency increased by 2-fold, 5-fold, and 4.3-fold upon preincubation for 24, 48, and 72 h, respectively. Preincubation for 24, 48, and 72 h increased the ability of tobacco leaf segments to induce Agrobacterium virE by 2.3-fold, 3.5-fold and 4.5-fold, respectively. The requirement of preincubation for increased transformation efficiency was obviated by the addition of 100 mu M acetosyringone to the freshly cut leaf rings cocultivated with Agrobacterium. The production of vii gene inducers by the leaf rings during the preincubation period is an important factor that contributes to increased transformation efficiency of Agrobacterium upon preincubation. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.