LpxA: A natural nanotube

UDP‐N‐acetylglucosamine 3‐O‐acyltransferase is a protein with a left‐handed parallel β‐helix, which is a natural nanotube. They are associated with unusual high stability. To identify the reason behind the structural stability of β‐helical nanotubular structure, we have performed a total of 4 μs molecular dynamics simulations of the protein in implicit solvent at four different temperatures and monitored the unfolding pathway. The correlation in movement between different regions of the nanotubular structure has been identified from the dynamical cross‐correlation map and contribution of some specific residues towards unfolding transition has been identified by principal component analysis. Difference in stability of the three loop regions has also been characterized. Construction of the unfolding conformational energy landscape identifies the probable intermediates that can appear in the unfolding pathway of the protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 845–853, 2010.     

Transport of liposomal and albumin loaded curcumin to living cells: an absorption and fluorescence spectroscopic study

Curcumin, a lipid soluble antioxidant, exhibits solvent and medium sensitive absorption and fluorescence properties. Using such changes, the average binding constants of curcumin to phosphatidylcholine (PC) liposomes and human serum albumin (HSA) were estimated to be 2.5 x 10(4) M(-1) and 6.1 x 10(4) M(-1) respectively. From the studies on temperature dependent fluorescence anisotropy of liposomal curcumin and its fluorescence quenching by acrylamide and iodide, it was concluded that curcumin is located in the gel phase of the liposomes. Similarly from the studies on quenching of tryptophan fluorescence in HSA by curcumin, it was found to be in the same domain as that of tryptophan. Both liposomal and HSA vehicles were examined for the transfer of curcumin to spleen lymphocyte cells, EL4 lymphoma cell line and compared with aqueous DMSO vehicles. From these studies it was found that liposomal vehicle is capable of loading more curcumin in to cells than HSA or aqueous-DMSO, and lymphoma cells show preferential uptake of curcumin to lymphocytes. The fluorescence of curcumin in EL4 lymphoma cells was found to be significantly higher as compared to the lymphocytes. The present study demonstrates a simple and quantitative method of estimation of curcumin delivered to cells by different vehicles using absorption and fluorescence spectroscopy.     

Ionic liquids as solvents for biopolymers: Acylation of starch and zein protein

Biopolymers such as starch and zein protein were found to be soluble at 80 °C in ionic liquids such as 1-butyl-3-methylimidazolium chloride (BMIMCl) and 1-butyl-3-methylimidazolium dicyanamide (BMIMdca) in concentrations up to 10% (w/w). Higher concentrations of biopolymers in these novel solvents resulted in solutions with too high viscosity to stir. Solutions of both starch and zein in BMIMCl were acylated with anhydrides in presence of pyridine to give acetyl starch and benzoyl zein with various degrees of substitution. Without pyridine the acylation reaction did not proceed. ¹H NMR and IR spectroscopies were used to determine the degree of substitution of starch. Viscosity studies indicated that the starch underwent slight reduction in molecular weight during the course of acylation. Starch was also soluble in other non-conventional solvents such as choline chloride/oxalic acid and choline chloride/ZnCl₂. However, zein was insoluble in these solvents.     

Mitochondrial Superoxide Radicals Differentially Affect Muscle Activity and Neural Function

Cellular superoxide radicals (O₂⁻) are mostly generated during mitochondrial oxygen metabolism. O₂⁻ serves as the raw material for many reactive oxygen species (ROS) members like H₂O₂ and OH^.− radicals following its catalysis by superoxide dismutase (SOD) enzymes and also by autocatalysis (autodismutation) reactions. Mitochondrial ROS generation could have serious implications on degenerative diseases. In model systems overproduction of mitochondrial O₂⁻ resulting from the loss of SOD2 function leads to movement disorders and drastic reduction in life span in vertebrates and invertebrates alike. With the help of a mitochondrial SOD2 loss-of-function mutant, Sod2ⁿ²⁸³, we measured the sensitivity of muscles and neurons to ROS attack. Neural outputs from flight motor neurons and sensory neurons were unchanged in Sod2ⁿ²⁸³ and the entire neural circuitry between the giant fiber (GF) and the dorsal longitudinal muscles (DLM) showed no overt defect due to elevated ROS. Such insensitivity of neurons to mitochondrial superoxides was further established through neuronal expression of SOD2, which failed to improve survival or locomotive ability of Sod2ⁿ²⁸³. On the other hand, ultrastructural analysis of Sod2ⁿ²⁸³ muscles revealed fewer mitochondria and reduced muscle ATP production. By targeting the SOD2 expression to the muscle we demonstrate that the early mortality phenotype of Sod2ⁿ²⁸³ can be ameliorated along with signs of improved mobility. In summary, muscles appear to be more sensitive to superoxide attack relative to the neurons and such overt phenotypes observed in SOD2-deficient animals can be directly attributed to the muscle.BETWEEN Drosophila, mouse, and human, the enzymatic antioxidant defense system shares similar organization both structurally (Landis and Tower 2005) and functionally. Besides having a good degree of homology (Duttaroy et al. 1994; Landis and Tower 2005), other significant similarities include the presence of a single copy of Sod1 and Sod2 genes in each with no degree of functional complementation between these enzymes (Copin et al. 2000). While vertebrates have developed additional antioxidant defense enzymes such as glutathione peroxidase (Gpx) and extracellular superoxide dismutase (EcSOD or Sod3), neither Gpx nor an active SOD3 has been demonstrated in Drosophila, although a Sod3-like sequence has been identified (Landis and Tower 2005). Complete loss of SOD2 function is fatally injurious for both mice and Drosophila (Li et al. 1995; Lebovitz et al. 1996; Kirby et al. 2002; Duttaroy et al. 2003). The severe phenotypic effects of SOD2 loss of function have been attributed to elevated DNA damage and protein carbonylation (Golden and Melov 2001). SOD2 loss of function has also been attributed to “free radical attack” or “oxidative insult” on mitochondria where obvious mitochondrial damage was apparent from the inactivation of mitochondrial Fe-S cluster enzymes aconitase and succinate dehydrogenase (Melov et al. 1999; Kirby et al. 2002; Paul et al. 2007). Furthermore, impairment of cellular signaling, specifically those induced by reactive oxygen species (ROS) (Klotz 2005), might also play a very significant role in the early mortality effects of SOD2-deficient flies as indicated recently (Wicks et al. 2009).Sod2 null mice with damaged mitochondria display a number of pathologies including cardiomyopathy (Li et al. 1995), neurodegeneration, and seizures (Melov et al. 1998). Drosophila mutants of mitochondrial dysfunction are also claimed to be associated with neurodegeneration (Kretzschmar et al. 1997; Min and Benzer 1997, 1999; Rogina et al. 1997; Palladino et al. 2002, 2003; Celotto et al. 2006). In addition to the neurons, muscles are important targets for oxidative modification (Choksi and Papaconstantinou 2008; Choksi et al. 2008). Aerobic muscles with high mitochondrial content and high myoglobin levels, for example, show a significant increase in oxidative modification of all electron transport chain proteins compared to muscles with fewer mitochondria and less myoglobin (anaerobic muscle) (Choksi and Papaconstantinou 2008; Choksi et al. 2008). Mice lacking the Cu-ZnSOD enzyme suffer from a rapid loss of skeletal muscle mass, resembling an accelerated sarcopenia (Jackson 2006; Muller et al. 2006). We therefore set out to measure the impact of heightened superoxide concentration on neurons and muscles of Sod2ⁿ²⁸³ flies that are devoid of SOD2, the principal scavenger of superoxide radicals in mitochondria (Duttaroy et al. 2003; Belton et al. 2006).     

Unusual complexation of Cu(I) by pyrimidine/pyridine-pyrazole derived ligands exploiting the molecular function of 2-mercapto-4,6-dimethylpyrimidine - Syntheses, crystal structures and electrochemistry

Reaction of 2 equiv. amount of copper(II) chloride dihydrate with 2 equiv. of methyl-5-methyl-1-(4,6-dimethyl-2-pyrimidyl)pyrazole-3-carboxylate (DpymPzC) in presence of 1 equiv. of 2-mercapto-4,6-dimethylpyrimidine (DpymtH) at pH ∼ 6 afforded the tricoordinated copper(I) complex [Cu(DpymPzC)Cl] (1). The same reaction with copper(II) perchlorate hexahydrate, as the metal salt under the same equivalent ratio at pH ∼ 6 formed the tetracoordinated copper(I) complex [Cu(DpymPzC)₂]ClO₄ (2). In both the cases, the role of DpymtH is nothing but only to reduce the copper(II) salt in situ finally forming the copper(I) complex. On the other hand, the direct reaction between the copper(I) thiocyanate and DpymPzC in 2:2 equiv. ratio produced a tricoordinated copper(I) complex [Cu(DpymPzC)SCN] (3). In a similar reaction of 2 equiv. amount of copper(II) chloride dihydrate with 2 equiv. amount of ethyl-5-methyl-1-(2-pyridyl)pyrazole-3-carboxylate (PyPzC) in presence of 1 equiv. of DpymtH at pH ∼ 6, an intense red coloured microcrystalline compound (4) was obtained. In contrast, 1 equiv. of PyPzC and 2 equiv. of DpymtH on reaction with 1 equiv. of copper(II) chloride dihydrate at pH ∼ 6 produced a novel tetranuclear mixed coordinated [Cu₄(DpymtH)₄Cl₄] complex (5). Here DpymtH plays dual role - a reducing agent for the copper(II) salt followed by a chelating ligand towards copper(I) so formed in situ. Among the above species, 1, 2 and 5 are crystallographically characterized. In 1, the central copper atom is in distorted triangular planar geometry with N₂Cl chromophore whereas in 2, the same is in distorted tetrahedral geometry with N₄ chromophore. Notably, the extent of distortion from the ideal geometry is more in 2. In 5, which is in chair conformation, out of four copper atoms, two being in S₂Cl chromophore are tricoordinated and the remaining two are tetracoordinated with NS₂Cl chromophore. The metal centers are bridged through DpymtH in its ‘thione’ form. Interestingly, the chelation (in part) results in formation of the highly stable four-membered two chelate rings around the two tetracoordinated copper atoms in 5. The two copper centers along the long arm of the chair are separated through a distance of 5.190 Å while those in the short arm are at a length of 3.629 Å. The electronic, IR spectra and electrochemistry of the complexes 1, 2 and 5 have also been investigated.     

Interaction of aromatic imino glycoconjugates with jacalin: experimental and computational docking studies

Altering the lectin properties by chemically modified glycoconjugates can have profound effect on their biological applications. In the present case, jacalin has been chosen to study the binding aspects toward glycoconjugates modified by connecting aromatic moieties through imine conjugation at their C-1- or C-2-positions. Out of 10 glycoconjugates, the galactosyl-naphthyl imine (1c) was found to be most effective toward agglutination inhibition (260 times better than galactose), quenching fluorescence intensity, and exhibiting greater binding (K_a, 1.3 × 10⁴ M⁻¹) with jacalin. The specific binding of galactose conjugates and the nonspecific binding of other conjugates have been demonstrated based on ITC. Changes in the secondary structures have been addressed by far- and near-UV CD spectroscopy. The present studies demonstrated that galactose-based conjugates bind at carbohydrate recognition domain (CRD) mainly through polar interactions in addition to exhibiting some nonpolar/hydrophobic interactions, whereas the conjugates other than galactose primarily interact through hydrophobic interactions. Binding of galactosyl conjugates at CRD has been further demonstrated by rigid docking.     

Dynamics of predominant microbiota in the human gastrointestinal tract and change in luminal enzymes and immunoglobulin profile during high-altitude adaptation

High-altitude (HA) visitors like pilgrims, trackers, scientists and military personnel face a group of nonspecific gastrointestinal (GI) symptoms during acclimatization to hypobaric hypoxia. In order to investigate the alteration of indigenous gastrointestinal microbiota in the development of such GI symptoms, an experiment was conducted for the enumeration of dominant cultivable faecal microbiota of 15 soldiers at base level (Delhi) and during their 15-day acclimatization at 3,505 m HA (Leh). At HA, faecal microbiota analysis revealed that total aerobes decreased significantly with increase of total and facultative anaerobes. The strict anaerobes like Bifidobacterium sp., Bacteroidetes sp. and Lactobacillus sp. exhibited positive growth direction index (GDI) like other predominant obligate anaerobes Clostridium perfringens and Peptostreptococcus sp. Different enzymes like amylase, proteinase and polyphenol hydrolase produced by different bacterial populations showed positive GDI, whereas phosphatase producers exhibited negative GDI. The levels of microbe-originated enzymes like amylase, proteinase, alkaline phosphatase and β-glucuronidase were also elevated during HA acclimatization. In addition, in vitro gas production ability was enhanced with increase of faecal immunoglobulins IgA and IgG. We demonstrated that hypoxic environment at HA had the potential to alter the gut microbial composition and its activities that may cause GI dysfunctions.     

In vitro plant regeneration from cotyledonary nodes of Withania somnifera (L.) Dunal and assessment of clonal fidelity using RAPD and ISSR markers

An efficient large-scale clonal propagation protocol has been described for Withania somnifera (L.) Dunal, a valuable medicinal plant, using cotyledonary nodes derived from axenic seedlings. Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) (MS) medium supplemented with 1.0 mg l^?1 N ⁶-benzyladenine (BA) was found to be optimum for production of multiple shoots (100 % shoot proliferation frequency and 16.93 shoots per explant). Successive shoot cultures were established by repeatedly sub-culturing the original cotyledonary node on a fresh medium after each harvest of newly formed shoots. Multiple shoot proliferation was also achieved from nodal segments derived from in vitro raised shoots on MS medium augmented with 1.0 mg l^?1 BA. Regenerated shoots were best rooted (95.2 %, 38.7 roots per shoot) in half-strength MS medium supplemented with 1.0 mg l^?1 indole-3-butyric acid. The plantlets were successfully acclimated and established in soil. Random amplified polymorphic DNA and inter-simple sequence repeats analysis revealed a homogeneous amplification profile for all micropropagated plants analyzed validating the genetic fidelity of the in vitro regenerated plants.     

Preferential binding of fisetin to the native state of bovine serum albumin: spectroscopic and docking studies

We have investigated the binding of the biologically important flavonoid fisetin with the carrier protein bovine serum albumin using multi-spectroscopic and molecular docking methods. The binding constants were found to be in the order of 10⁴ M^?1 and the number of binding sites was determined as one. MALDI-TOF analyses showed that one fisetin molecule binds to a single bovine serum albumin (BSA) molecule which is also supported by fluorescence quenching studies. The negative Gibbs free energy change (?G°) values point to a spontaneous binding process which occurs through the presence of electrostatic forces with hydrophobic association that results in a positive entropy change (+51.69 ± 1.18 J mol^?1 K^?1). The unfolding and refolding of BSA in urea have been studied in absence and presence of fisetin using steady-state fluorescence and lifetime measurements. Urea denaturation studies indicate that fisetin is gradually released from its binding site on the protein. In the absence of urea, an increase in temperature that causes denaturation of the protein results in the release of fisetin from its bound state indicating that fisetin binds only to the native state of the protein. The circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopic studies showed an increase in % α-helix content of BSA after binding with fisetin. Site marker displacement studies in accordance with the molecular docking results suggested that fisetin binds in close proximity of the hydrophobic cavity in site 1 (subdomain IIA) of the protein. The PEARLS (Program of Energetic Analysis of Receptor Ligand System) has been used to estimate the interaction energy of fisetin with BSA and the results are in good correlation with the experimental findings.