Guanidinium chloride and urea denaturations of beta-lactoglobulin A at pH 2.0 and 25 degrees C: the equilibrium intermediate contains non-native structures (helix, tryptophan and hydrophobic patches)

We have carried out guanidinium chloride (GdmCl) and urea denaturations of bovine beta-lactoglobulin A (beta-lgA) at pH 2.0 and 25 degrees C, using far-UV and near-UV circular dichroism, near-UV absorption and tryptophan fluorescence spectroscopies. The stable intermediate state that occurs during GdmCl denaturation has been characterized by the far- and near-UV circular dichroism, tryptophan difference absorption, tryptophan fluorescence and 8-anilino-1-naphthalene sulphonic acid binding measurements. Following conclusions have been reached. (a) Urea-induced denaturation is not a two-state process. (b) GdmCl-induced denaturation is composed of two distinct two-state processes. (c) alpha-Helical content, burial of tryptophan residues and burial of hydrophobic surface area are more in the GdmCl-induced stable intermediate than those originally present in the native protein.     

Voltage-dependent Inwardly Rectifying Potassium Conductance in the Outer Membrane of Neuronal Mitochondria

Potassium fluxes integrate mitochondria into cellular activities, controlling their volume homeostasis and structural integrity in many pathophysiological mechanisms. The outer mitochondrial membrane (OMM) is thought to play a passive role in this process because K⁺ is believed to equilibrate freely between the cytosol and mitochondrial intermembrane space. By patch clamping mitochondria isolated from the central nervous systems of adult mitoCFP transgenic mice, we discovered the existence of I_OMMKi, a novel voltage-dependent inwardly rectifying K⁺ conductance located in the OMM. I_OMMKi is regulated by osmolarity, potentiated by cAMP, and activated at physiological negative potentials, allowing K⁺ to enter the mitochondrial intermembrane space in a controlled regulated fashion. The identification of I_OMMKi in the OMM supports the notion that a membrane potential could exist across this membrane in vivo and suggests that the OMM possesses regulated pathways for K⁺ uptake.     

D224V and S128Y mutation in FSHRED influence thumb movement differentially: An intricate insight gained by short-term molecular dynamics simulation

Follicle-stimulating hormone-follicle-stimulating hormone receptor (FSH-FSHR) interaction is one of the most thoroughly studied signaling pathways primarily because of being implicated in sexual reproduction in mammals by way of maintaining gonadal function and sexual fertility. Despite material advances in understanding the role of point mutations, their mechanistic basis in FSH-FSHR signaling is still confined to mystically altered behavior of sTYS335 (sulfated tyrosine) yet lacking a substantial theory. To understand the structural basis of receptor modulation, we choose two behaviorally contradicting mutations, namely S128Y (activating) and D224Y (inactivating), found in FSH receptor responsible for ovarian hyperstimulation syndrome and ovarian dysgenesis, respectively. Using short-term molecular dynamics simulations, the atomic scale investigations reveal that the binding pattern of sTYS with FSH and movement of the thumb region of FSHR show distinct contrasting patterns in the two mutants, which supposedly could be a critical factor for differential FSHR behavior in activating and inactivating mutations.     

Antitumor activity of curcumin is mediated through the induction of apoptosis in AK-5 tumor cells

Curcumin, the yellow pigment of turmeric (Curcuma longa), used commonly as a spice, has been shown to possess anticarcinogenic activity. Curcumin inhibited AK-5 tumor growth and induced apoptosis in AK-5 cells. Curcumin induced apoptosis is mediated through the activation of caspase-3, which is specifically inhibited by the tetrapeptide Ac-DEVD-CHO. In addition, curcumin induced tumor cell death is caused through the generation of reactive oxygen intermediates which is inhibited by N-acetyl-L-cysteine. Our studies suggest that the apoptotic process induced by curcumin is the mechanism mediating AK-5 tumor cell death.     

Spectroscopic studies of structural changes in two beta-sheet-forming peptides show an ensemble of structures that unfold noncooperatively

A characterization of the conformation and stability of model peptide systems that form beta-sheets in aqueous solutions is considerably important in gaining insights into the mechanism of beta-sheet formation in proteins. We have characterized the conformation and equilibrium folding and unfolding of two 20-residue peptides whose NMR spectra suggest a three-stranded beta-sheet topology in aqueous solution: Betanova [Kortemme, T., Ramirez-Alvarado, M., and Serrano, L. (1998) Science 281, 253-256] and (D)P(D)P with d-Pro-Gly segments at the turns [Schenck, H. L., and Gellman, S. H. (1998) J. Am. Chem. Soc. 120, 4869-4870]. Both circular dichroism (CD) and infrared measurements indicate only 20-26% beta-sheet-like structure at 5 degrees C for Betanova and 42-59% beta-sheet for (D)P(D)P. For both peptides, the CD and infrared spectra change nearly linearly with increasing temperatures (or urea concentrations) and lack a sigmoidal signature characteristic of cooperative unfolding. Fluorescence resonance energy transfer (FRET) measurements between donor and acceptor molecules attached to the two ends confirm that Betanova is largely unstructured even at 10 degrees C; the average end-to-end distance estimated from FRET is closer to that of a random coil than a structured beta-sheet. In (D)P(D)P, the FRET results indicate a more compact structure that remains compact even at high temperatures (approximately 80 degrees C) or high urea concentrations (approximately 8 M). These results indicate that both these peptides access an ensemble of conformations at all temperatures or denaturant concentrations, with no significant free energy barrier separating the "folded" and "unfolded" conformations.     

Response of the NAD(P)H-oxidising flavohaemoglobin (Hmp) to prolonged oxidative stress and implications for its physiological role in Escherichia coli

The Escherichia coli flavohaemoglobin (Hmp) has a globin-like N-terminal domain and a ferredoxin-NADP-reductase-like C-terminal domain. We show here that purified Hmp oxidises both NADH and NADPH with K _m values of 1.8 and 19.6 μM, respectively. Prolonged incubation of a hmp-lacZ fusion strain with the redox cycling agent paraquat resulted in a 28-fold induction of hmp gene expression, nearly 3-fold higher than after short periods of exposure. A strain overproducing Hmp was significantly more sensitive to paraquat than was the wild-type strain but, in vitro, purified Hmp was not an effective NADPH-paraquat diaphorase. Prolonged incubation of a wild-type strain with paraquat increased intracellular Hmp to spectrally detectable levels.     

Molecular characterization of secretory proteins Rv3619c and Rv3620c from Mycobacterium tuberculosis H37Rv

Rv3619c and Rv3620c are the secretory, antigenic proteins of the ESAT-6/CFP-10 family of Mycobacterium tuberculosis H37Rv. In this article, we show that Rv3619c interacts with Rv3620c to form a 1 : 1 heterodimeric complex with a dissociation constant (K(d)) of 4.8 × 10(-7) M. The thermal unfolding of the heterodimer was completely reversible, with a T(m) of 48 °C. The comparative thermodynamics and thermal unfolding analysis of the Rv3619c-Rv3620c dimer, the ESAT-6-CFP-10 dimer and another ESAT family heterodimer, Rv0287-Rv0288, revealed that the binding strength and stability of Rv3619c-Rv3620c are relatively lower than those of the other two pairs. Molecular modeling and docking studies predict the structure of Rv3619c-Rv3620c to be similar to that of ESAT-6-CFP-10. Spectroscopic studies revealed that, in an acidic environment, Rv3619c and Rv3620c lose their secondary structure and interact weakly to form a complex with a lower helical content, indicating that Rv3619c-Rv3620c is destabilized at low pH. These results, combined with those of previous studies, suggest that unfolding of the proteins is required for dissociation of the complex and membrane binding. In the presence of membrane mimetics, the α-helical contents of Rv3619c and Rv3620 increased by 42% and 35%, respectively. In mice, the immune response against Rv3619c protein is characterized by increased levels of interferon-γ, interleukin-12 and IgG(2a) , indicating a dominant Th1 response, which is mandatory for protection against mycobacterial infection. This study therefore emphasizes the potential of Rv3619c as a subunit vaccine candidate.     

India and the Muslim Punjab: a unified approach to South Asian kinship

The comparison of kinship of different South Asian regions with the Muslim Punjab reveals basic invariants, thus denying a fundamental division between the Dravidian South and the Indo-Aryan North, though concepts of kinship reveal themselves in different media: Muslim Punjabi kinship is expressed primarily in gift exchange, but exhibits characteristics commonly associated with the South Indian terminology. The present article argues for an approach which takes meaning embedded in the 'act' or 'person' as non-separable from 'culture', and grasps kinship as structuring the dynamic relation between self and other, regarded as ontological categories.  

Résumé

La comparaison de la parenté entre les différentes régions du sud de l'Asie et le Pendjab musulman révèle des constantes fondamentales et remet ainsi en question la division de principe entre le Sud dravidien et le Nord indo-aryen, bien que les concepts de parenté trouvent des expressions différentes : dans le Pendjab musulman, la parenté s'exprime principalement à travers l'échange de dons mais présente des caractéristiques habituellement associées à la terminologie du sud de l'Inde. Le présent article plaide en faveur d'une approche considérant la signification de « l'acte >> ou de la « personne >> en tant qu'entité indissociable de la « culture >>, et aborde la parenté comme un moyen de structurer la relation dynamique entre soi-même et l'autre, considérés comme des catégories ontologiques.