Titration calorimetric studies to elucidate the specificity of the interactions of polymyxin B with lipopolysaccharides and lipid A

Lipopolysaccharide (LPS), the major cell wall constituent of Gram-negative bacteria, evokes a multitude of biological effects in mammals including pyrogenicity and toxic shock syndrome. Polymyxin B (PmB), a polycationic cyclic peptide, is known to neutralize most of its activities. The nature of the interaction of PmB with LPS and lipid A was investigated by isothermal titration calorimetry. PmB binds to LPS as well as lipid A stoichiometrically and non-co-operatively with micromolar affinity. These interactions are driven primarily by a favourable change in entropy (delta S) and are endothermic in nature. These positive changes in enthalpies decrease with increasing temperature, yielding a heat capacity change, delta Cp, of -2385 J.mol-1.degree-1 for PmB-LPS interactions while the binding of PmB to lipid A displays a delta Cp of -2259 J.mol-1.degree-1. The negative heat capacity changes provide strong evidence for the role of hydrophobic interactions as the driving force for the association of PmB with LPS and lipid A. A correlation of the energetics of these interactions with analyses of the molecular models of PmB suggests that a cluster of solvent-exposed non-polar amino acid side-chains that line one surface of the molecule, together with a ring of positively charged residues on its other surface, are responsible for its strong and stoichiometric binding to LPS.

Phenotypic and Functional Characterization of Human Mammary Stem/Progenitor Cells in Long Term Culture

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. Methodology: Single cell suspensions derived from human breast `organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. Principal Findings: We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. Conclusions: Thus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance.

A novel DNA intercalator, butylamino-pyrimido[4',5':4,5]selenolo(2,3-b)quinoline, induces cell cycle arrest and apoptosis in leukemic cells

DNA intercalators are one of the most commonly used chemotherapeutic agents. Novel intercalating compounds of pyrimido[4',5':4,5]selenolo(2,3-b)quinoline series having a butylamino or piperazino group at fourth position (BPSQ and PPSQ, respectively) are studied. Our results showed that BPSQ induced cytotoxicity whereas PPSQ was cytostatic. The cytotoxicity induced by BPSQ was concentration- and time-dependent. Cell cycle analysis and tritiated thymidine assay revealed that BPSQ affects the cell cycle progression by arresting at S phase. The absence of p-histone H3 and reduction in the levels of PCNA in the cells treated with BPSQ further confirmed the cell cycle arrest. Further, annexin V staining, DNA fragmentation, nuclear condensation and changes in the expression levels of BCL2/BAD confirmed the activation of apoptosis. Activation of caspase 8 and lack of cleavage of caspase 9, caspase 3 and PARP suggest the possibility of BPSQ triggering extrinsic pathway for induction of apoptosis, which is discussed. Hence, we have identified a novel compound which would have clinical relevance in cancer chemotherapeutics.

Isolation and visualization of active presynaptic filaments of recA protein and single-stranded DNA

In the presence of ATP, recA protein forms a presynaptic complex with single-stranded DNA that is an obligatory intermediate in homologous pairing. Presynaptic complexes of recA protein and circular single strands that are active in forming joint molecules can be isolated by gel filtration. These isolated active complexes are nucleoprotein filaments with the following characteristics: (i) a contour length that is at least 1.5 times that of the corresponding duplex DNA molecule, (ii) an ordered structure visualized by negative staining as a striated filament with a repeat distance of 9.0 nm and a width of 9.3 nm, (iii) approximately 8 molecules of recA protein and 20 nucleotide residues per striation. The widened spacing between bases in the nucleoprotein filament means that the initial matching of complementary sequences must involve intertwining of the filament and duplex DNA, unwinding of the latter, or some combination of both to equalize the spacing between nascent base pairs. These experiments support the concept that recA protein first forms a filament with single-stranded DNA, which in turn binds to duplex DNA to mediate both homologous pairing and subsequent strand exchange.

Exfoliated graphite–ruthenium oxide composite electrodes for electrochemical supercapacitors

The performance of exfoliated graphite (EG)–ruthenium oxide (RuOx) composites as binderless electrodes is evaluated for electrochemical capacitors (ECs). A composite of EG–RuOx is prepared by a modified sol–gel process. The material is characterized using X-ray diffraction and microscopy. Electrochemical capacitors with the composite electrodes in the presence of aqueous sulfuric acid (H2SO4) electrolyte are evaluated using voltammetry, impedance and charge–discharge studies. Cyclic voltammetry reveals very stable current–voltage behaviour up to several thousands of cycles, as well as high specific capacitances, e.g., a few hundreds of farads per gram for the composite that contains 16.5 wt.% RuOx.

Highly biocompatible and water-dispersible, amine functionalized magnetite nanoparticles, prepared by a low temperature, air-assisted polyol process: a new platform for bio-separation and diagnostics

A low temperature polyol process, based on glycolaldehyde mediated partial reduction of FeCl3 center dot 6H(2)O at 120 degrees C in the presence of sodium acetate as an alkali source and 2,2'-(ethylenedioxy)-bis-(ethylamine) as an electrostatic stabilizer has been used for the gram-scale preparation of biocompatible, water-dispersible, amine functionalized magnetite nanoparticles (MNPs) with an average diameter of 6 +/- 0.75 nm. With a reasonably high magnetization (37.8 e.m.u.) and amine groups on the outer surface of the nanoparticles, we demonstrated the magnetic separation and concentration implications of these ultrasmall particles in immunoassay. MRI studies indicated that these nanoparticles had the desired relaxivity for T-2 contrast enhancement in vivo. In vitro biocompatibility, cell uptake and MR imaging studies established that these nanoparticles were safe in clinical dosages and by virtue of their ultrasmall sizes and positively charged surfaces could be easily internalized by cancer cells. All these positive attributes make these functional nanoparticles a promising platform for further in vitro and in vivo evaluations.

Proteomic profiling of forskolin-induced differentiated BeWo cells: an in-vitro model of cytotrophoblast differentiation

Placental trophoblastic differentiation is characterized by the fusion of monolayer cytotrophoblasts into syncytiotrophoblasts. During this process of differentiation, several morphological and biochemical changes are known to occur, and this model has been employed to investigate the changes that occur at the gene and protein level during differentiation. Using the sensitive technique of proteomics [two-dimensional gel electrophoresis (2DGE)], changes in protein profile were evaluated in the control and forskolin-induced differentiated cells of trophoblastic choriocarcinoma BeWo cell line. Several proteins were differentially expressed in control and differentiated cells. Four major proteins were up-regulated as assessed by silver staining, and were further characterized as c-h-ras p 21 (phosphorylated), retinoblastoma susceptibility protein I and integrase interactor protein 1. These proteins are known to play an important role in growth arrest of cells, and thus may play a role in initiating the process of differentiation.

Detection,amplification and sequence homology of sodC in clinical isolates of Salmonella sp

Background & objectives: Periplasmic copper and zinc superoxide dismutase (Cu,Zn-SOD or SodC) is an important component of the antioxidant shield which protects bacteria from the phagocytic oxidative burst. Cu,Zn-SODs protect Gram-negative bacteria against oxygen damage which have also been shown to contribute to the pathogenicity of these bacterial species. We report the presence of SodC in drug resistant Salmonella sp. isolated from patients suffering from enteric fever. Further sodC was amplified, cloned into Escherichia coli and the nucleotide sequence and amino acid sequence homology were compared with the standard strain Salmonella Typhimurium 14028. Methods: Salmonella enterica serovar Typhi (S. Typhi) and Salmonellaenterica serovar Paratyphi (S. Paratyphi) were isolated and identified from blood samples of the patients. The isolates were screened for the presence of Cu, Zn-SOD by PAGE using KCN as inhibitor of Cu,Zn-SOD. The gene (sodC) was amplified by PCR, cloned and sequenced. The nucleotide and amino acid sequences of sodC were compared using CLUSTAL X.Results: SodC was detected in 35 per cent of the Salmonella isolates. Amplification of the genomic DNA of S. Typhi and S. Paratyphi with sodC specific primers resulted in 519 and 515 bp amplicons respectively. Single mutational difference at position 489 was observed between thesodC of S. Typhi and S. Paratyphi while they differed at 6 positions with the sodC of S. Typhimurium 14028. The SodC amino acid sequences of the two isolates were homologous but 3 amino acid difference was observed with that of standard strain S. Typhimurium 14028.Interpretation & conclusions: The presence of SodC in pathogenic bacteria could be a novel candidate as phylogenetic marker.

Optical, Field-Emission, and Antimicrobial Properties of ZnO Nanostructured Films Deposited at Room Temperature by Activated Reactive Evaporation

ZnO nanostructures were deposited on flexible polymer sheet and cotton fabrics at room temperature by activated reactive evaporation. Room-temperature photoluminescence spectrum of ZnO nanostructured film exhibited a week intrinsic UV emission and a strong broad yellow-orange visible emission. TEM and HRTEM studies show that the grown nanostructures are crystalline in nature and their growth direction was indentified to be along [002]. ZnO nanostructures grown on the copper-coated flexible polymer sheets exhibited stable field-emissio characteristics with a threshold voltage of 2.74 V/mu m (250 mu A) and a very large field enhancement factor (beta) of 23,213. Cotton fabric coated with ZnO nanostructures show an excellent antimicrobial activity against Staphylococcus aureus bacteria (Gram positive), and similar to 73% reduction in the bacterial population is achieved compared to uncoated fabrics after 4 h in viability. Using a shadow mask technique, we also selectively deposited the nanostructures at room temperature on polymer substrates.

Fugacity coefficients. Enthalpy, and second virial coefficients of boron trifluoride

Fugacity coefficients and isothermal changes of enthalpy have been calculated and reported. The calculations cover a temperature range of 0° to 75°C. up to gas densities of 1.0 gram per cc. The generalized Benedict-Webb-Rubin constants evaluated from generalized PVT relations is found to predict the experimental data with an over-all absolute deviation of 3.1%. Second virial coefficients and potential energy parameters for Lennard-Jones (12-6) potential energy function are reported also.

Biosynthesis of sulfated mucopolysaccharides in relation to vitamins A and K

1.(1) Incorporation of Na235SO4 into acid mucopolysaccharides of intestine and colon tissue has been studied in normal, vitamin A-deficient and excess vitamin A-fed rats. 2. (2) Vitamin A deficiency resulted in a significant decrease in [35S]sulfate incorporation into mucopolysaccharides isolated from intestines of male rats. There was no significant change in the total mucopolysaccharides per gram of fresh tissue. 3. (3) When rats are fed excessive amounts of retinyl acetate, increased [35S]sulfate incorporation into mucopolysaccharides of rat intestine and colon is observed. 4. (4) Supplementation of vitamin K1 to rats fed excessive amounts of vitamin A restores the incorporation of [35S]sulfate into the acid mucopolysaccharides to the normal level. 5. (5) The implications of these findings with special reference to the role of vitamins A and K in the synthesis of sulfated mucopolysaccharides are discussed.

Biosynthesis of valine and isoleucine in plants II. Dihydroxyacid dehydratase from Phaseolus radiatus

1. 1. An enzyme catalysing the conversion of α,β-dihydroxyisovalerate and α,β-dihydroxy-β-methylvalerate to α-ketoisovalerate and α-keto-β-methylvalerate has been partially purified from green gram (Phaseolus radiatus), and its characteristics studied. 2. 2. A natural inhibitor, heat stable and inorganic in nature, was observed in the crude extracts. 3. 3. The observed Km values for α-β-dihydroxyisovalerate and α,β-dihydroxy-β-methylvalerate were 2.4 · 10-3 M and 9 · 10-4 M, respectively. 4. 4. The enzyme required the presence of a divalent metal ion (Mg2+, Mn2+ or Fe2+) for maximal activity. Heavy metals like Ag+ and Hg2+ were inhibitory. 5. 5. The optimal activity was around pH 8.0 and the optimum temperature at 52°. The activation energy is found to be 12 600 cal/mole. 6. 6. The enzyme was inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and sulphydryl compounds like cysteine, glutathione, 2-mercaptoethanol and 2,3-dimercaptopropanol. The inhibition by p-hydroxymercuribenzoate could not be reversed by any of the sulfhydryl compounds tested.

Biosynthesis of valine and isoleucine in plants I. Formation of α-acetolactate in Phaseolus radiatus

1. 1. The presence of an enzyme system in plants catalyzing the formation of α-acetolactate from pyruvate has been demonstrated; the system in green gram (Phaseolus radiatus) has been partially purified and its characteristics have been studied.2. Free acetaldehyde is formed as a product of the reaction and so the reaction is mainly diverted towards the formation of acetoin. 3. The system requires thiamine pyrophosphate and a divalent metal ion (Mn2+ or Mg2+) for maximum activity. The optimum pH is around 6.0 and the optimum temperature is 60°. 4. The system is very labile in absence of pyruvate, Mn2+ and DPT. 5. The Km values for pyruvate, Mn2+, Mg2+ and DPT are 3·10−2 M. 5·10−5 M, 2·10−5 M, and e·10−6 M respectively. The activation energy is 3540 cal/mole. 6. The enzyme is strongly inhibited by p-chloromercuribenzoate and the inhibition can be reversed partially by 2-mercaptoethanol, BAL or cysteine. Heavy metals, such as Hg2+ and Ag+, are inhibitory but l-valine does not inhibit the reaction.

Studies on the enzyme hydrolysing flavin mononucleotide in plants: II. Phosphotransferase activity of the partially purified enzyme from Phaseolus radiatus

The preparation of the enzyme hydrolysing FMN whose partial purification from green-gram extracts is described in the preceding paper, has been shown to possess phosphotransferase activity. The enzyme could transfer the phosphate group cleaved from FMN to acceptors like thiamine, pyridoxal, pyridoxamine and nucleosides resulting in the formation of their corresponding phosphate esters and nucleotides. The properties of the enzyme hydrolysing FMN and the phosphotransferase activity of the preparation are compared.

Tryptophan synthetase in plants

Infiltration experiments with the intact seeds of Bengal gram (Cicer arietinum) indicated that indole and serine are the immediate precursors of tryptophan in this legume. The enzyme, tryptophan synthetase, has been demonstrated in cell-free extracts of the resting seeds. The optimum pH of the reaction was 5.5, and the Km value for indole at a constant serine concentration of 10−4M was 0.57 × 10−4M. There was a specific requirement for pyridoxal phosphate. Heavy-metal ions were inhibitory.