Enhancing the ratio of molecular ions to non-covalent compounds in the electrospray interface of LC-MS in quantitative analysis

A common problem encountered during the development of MS methods for the quantitation of small organic molecules by LGMS is the formation of non-covalently bound species or adducts in the electrospray interface. Often the population of the molecular ion is insignificant compared to those of all other forms of the analyte produced in the electrospray, making it difficult to obtain the sensitivity required for accurate quantitation. We have investigated the effects of the following variables: orifice potential, nebulizer gas flow, temperature, solvent composition and the sample pH on the relative distributions of ions of the types MH+, MNa+, MNH+, and 2MNa(+), where M represents a 4 small organic molecule: BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile). Orifice potential, solvent composition and the sample pH had the greatest influence on the relative distributions of these ions, making these parameters the most useful for optimizing methods for the quantitation of small molecules.

Lemon oil to beta-cyclodextrin ratio effect on the inclusion efficiency of beta-cyclodextrin and the retention of oil volatiles in the complex

Microencapsulation of lemon oil was undertaken with beta-cyclodextrin using a precipitation method at the five lemon oil to beta-cyclodextrin ratios of 3:97, 6:94, 9:91, 12:88, and 15:85 (w/w) in order to determine the effect of the ratio of lemon oil to beta-cyclodextrin on the inclusion efficiency of beta-cyclodextrin for encapsulating oil volatiles. The retention of lemon oil volatiles reached a maximum at the lemon oil to beta-cyclodextrin ratio of 6:94; however, the maximum inclusion capacity of beta-cyclodextrin and a maximum powder recovery were achieved at the ratio of 12:88, in which the beta-cyclodextrin complex contained 9.68% (w/w) lemon oil. The profile and proportion of selected flavor compounds in the beta-cyclodextrin complex and the starting lemon oil were not significantly different.

Sulfite : Cytochrome c oxidoreductase from Thiobacillus novellus - Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC 1.8.2.1), Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alpha beta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa monoheme cytochrome c(552) smbunit (midpoint redox potential, Em(8.0) = +280 mV), The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K-m values for sulfite and cytochrome c(550) were determined to be 27 and 4 mu M, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.

Epidemiological and molecular evidence supports the zoonotic transmission of Glardia among humans and dogs living in the same community

Giardia duodenalis isolates recovered from humans and clogs living in the same locality in a remote tea-growing community of northeast India were characterized at 3 different loci; the SSU-rDNA, elongation factor 1-alpha (ef1-alpha) and triose phosphate isomerase (tpi) gene. Phylogenetic analysis of the SSU-rDNA and ef1-alpha genes provided poor genetic resolution of the isolates within various assemblages, stressing the importance of using multiple loci when inferring genotypes to Giardia. Analysis of the tpi gene provided better genetic resolution and placed canine Giardia isolates within the genetic groupings of human isolates (Assemblages A and B). Further evidence for zoonotic transmission was supported by epidemiological data showing a highly significant association between the prevalence of Giardia in humans and presence of it Giardia-positive dog in the same household (odds ratio 3.01, 95%) CI, 1.11, 8.39, P = 0.0000).

Molecular Lego (R): non-centrosymmetric alignment within interdigitating layers

(E)-N-Hexadecyl-4-[2-(4-octadecyloxynaphthyl) ethenyl] quinolinium bromide, which has a wide-bodied chromophore and terminal n-alkyl groups, adopts a U-shape when spread at the air-water interface but a stretched conformation when compressed to ca. 35 mN m(-1). The high-pressure phase has a narrow stability range prior to collapse but may be extended from 40 to 60 mN m(-1) by co-spreading the dye in a 1 : 1 ratio with docosanoic acid. The mixed Langmuir-Blodgett (LB) film has a monolayer thickness of 4.6 +/- 0.2 nm which decreases to 2.5 +/- 0.1 nm layer(-1) in the bulk, the reduction arising from an interdigitating layer arrangement, both top and bottom. It is the first example of LB-Lego(R) and, in addition, represents the only fully interdigitating structure with non-centrosymmetrically aligned chromophores. They are tilted 38 degrees from the substrate normal. The second-harmonic intensity increases quadratically with the number of layers, i.e. as I-(N)(2 omega) = (I(1)N2)-N-2 omega, with a second-order susceptibility of chi ((2))(zzz) = 30 pm V-1 at 1064 nm for refractive indices of n(omega) = 1.55 and n(2 omega) = 1.73, d = 2.5 nm layer(-1) and phi = 38 degrees. Angle resolved X-ray photoelectron spectra (XPS) of these films provide no evidence of the bromide counterion, which suggests that it is replaced by OH 2 or HCO3-, which occur naturally in the aqueous subphase, or C21H43COO- from the co-deposited fatty acid. This probably applies to all cationic dyes deposited by the LB technique.

The ratio of messenger RNA levels of receptor activator of nuclear factor kappa B ligand to osteoprotegerin correlates with bone remodeling indices in normal human cancellous bone but not in osteoarthritis

skeletal disease. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Receptor activator of nuclear factor KB ligand (RANKL) is a newly described regulator of osteoclast formation and function, the activity of which appears to be a balance between interaction with its receptor RANK and with an antagonist binding protein osteoprotegerin (OPG). Therefore, we have examined the relationship between the expression of RANKL, RANK, and OPG and indices of bone structure and turnover in human cancellous bone from the proximal femur. Bone samples were obtained from individuals with osteoarthritis (OA) at joint replacement surgery and from autopsy controls. Histomorphometric analysis of these samples showed that eroded surface (ES/BS) and osteoid surface (OS/BS) were positively associated in both control (p < 0.001) and OA (p < 0.02), indicating that the processes of bone resorption and bone formation remain coupled in OA, as they are in controls. RANKL, OPG, and RANK messenger RNA, (mRNA) were abundant in human cancellous bone, with significant differences between control and OA individuals. In coplotting the molecular and histomorphometric data, strong associations were found between the ratio of RANKL/OPG mRNA and the indices of bone turnover (RANKL/OPG vs. ES/BS: r = 0.93, p < 0.001; RANKL/OPG vs. OS/BS: r = 0.80, p < 0.001). These relationships were not evident in trabecular bone from severe OA, suggesting that bone turnover may be regulated differently in this disease. We propose that the effective concentration of RANKL is related causally to bone turnover.

Characterisation of templated xerogels for molecular sieve application

This paper presents the results of the characterisation of templated silica xerogels as precursor material for molecular sieve silica membranes for gas separation. The template agent integrated in the xerogel matrix is a methyl ligand covalently bended to the siloxane network in the form of methyltriethoxysilane (MTES). Several surface and microstructural characterisation techniques such as TGA, FTIR, NMR, and nitrogen adsorption have been employed to obtain information on the reaction mechanisms involved in the sol-gel processing of such molecular sieves. The characterisation results show the effects of processing parameters such as heat treatment temperature, and the concentration of the covalently bonded template on the development of the pore structure. It was found that calcination temperature significantly enhanced the condensation reactions thus resulted in more Si-O-Si groups being formed. This was also confirmed with the data of FTIR characterisation showing enhanced silicon bands at higher heat treatment temperatures. As a result of the promoted densification and shrinkable pore network the micropore volume also reduced with increasing methyl ligand molar ratio. However, the mean pore diameter does not change significantly with calcination temperature. While the contribution of the templates towards controlling pore size is less precise, increasing the methyl ligand molar ratio results in the broadening of the pore size distribution and lower pore volume. Higher template concentration induces the collapse of the xerogel matrix due to capillary stress promoting dense xerogels with low pore volume (C) 2001 Elsevier Science B.V. All rights reserved.

Applications of accurate, high-precision Pb isotope ratio measurement by multi-collector ICP-MS

The isotope composition of Ph is difficult to determine accurately due to the lack of a stable normalisation ratio. Double and triple-spike addition techniques provide one solution and presently yield the most accurate measurements. A number of recent studies have claimed that improved accuracy and precision could also be achieved by multi-collector ICP-MS (MC-ICP-MS) Pb-isotope analysis using the addition of Tl of known isotope composition to Pb samples. In this paper, we verify whether the known isotope composition of Tl can be used for correction of mass discrimination of Pb with an extensive dataset for the NIST standard SRM 981, comparison of MC-ICP-MS with TIMS data, and comparison with three isochrons from different geological environments. When all our NIST SRM 981 data are normalised with one constant Tl-205/Tl-203 of 2.38869, the following averages and reproducibilities were obtained: Pb-207/Pb-206=0.91461+/-18; Pb-208/Ph-206 = 2.1674+/-7; and (PbPh)-Pb-206-Ph-204 = 16.941+/-6. These two sigma standard deviations of the mean correspond to 149, 330, and 374 ppm, respectively. Accuracies relative to triple-spike values are 149, 157, and 52 ppm, respectively, and thus well within uncertainties. The largest component of the uncertainties stems from the Ph data alone and is not caused by differential mass discrimination behaviour of Ph and Tl. In routine operation, variation of sample introduction memory and production of isobaric molecular interferences in the spectrometer's collision cell currently appear to be the ultimate limitation to better reproducibility. Comparative study of five different datasets from actual samples (bullets, international rock standards, carbonates, metamorphic minerals, and sulphide minerals) demonstrates that in most cases geological scatter of the sample exceeds the achieved analytical reproducibility. We observe good agreement between TIMS and MC-ICP-MS data for international rock standards but find that such comparison does not constitute the ultimate. test for the validity of the MC-ICP-MS technique. Two attempted isochrons resulted in geological scatter (in one case small) in excess of analytical reproducibility. However, in one case (leached Great Dyke sulphides) we obtained a true isochron (MSWD = 0.63) age of 2578.3 +/- 0.9 Ma, which is identical to and more precise than a recently published U-Pb zircon age (2579 3 Ma) for a Great Dyke websterite [Earth Planet. Sci. Lett. 180 (2000) 1-12]. Reproducibility of this age by means of an isochron we regard as a robust test of accuracy over a wide dynamic range. We show that reliable and accurate Pb-isotope data can be obtained by careful operation of second-generation MC-ICP magnetic sector mass spectrometers. (C) 2002 Elsevier Science B.V. All rights reserved.

Dynamical properties of a strongly correlated model for quarter-filled layered organic molecular crystals

The dynamical properties of an extended Hubbard model, which is relevant to quarter-filled layered organic molecular crystals, are analyzed. We have computed the dynamical charge correlation function, spectral density, and optical conductivity using Lanczos diagonalization and large-N techniques. As the ratio of the nearest-neighbor Coulomb repulsion, V, to the hopping integral, t, increases there is a transition from a metallic phase to a charge-ordered phase. Dynamical properties close to the ordering transition are found to differ from the ones expected in a conventional metal. Large-N calculations display an enhancement of spectral weight at low frequencies as the system is driven closer to the charge-ordering transition in agreement with Lanczos calculations. As V is increased the charge correlation function displays a collective mode which, for wave vectors close to (pi,pi), increases in amplitude and softens as the charge-ordering transition is approached. We propose that inelastic x-ray scattering be used to detect this mode. Large-N calculations predict superconductivity with d(xy) symmetry close to the ordering transition. We find that this is consistent with Lanczos diagonalization calculations, on lattices of 20 sites, which find that the binding energy of two holes becomes negative close to the charge-ordering transition.

The molecular basis for the lack of immunostimulatory activity of vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at similar to3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 mug/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.

Limited effect of anthropogenic habitat fragmentation on molecular diversity in a rain forest skink, Gnypetoscincus queenslandiae

To examine the effects of recent habitat fragmentation, we assayed genetic diversity in a rain forest endemic lizard, the prickly forest skink (Gnypetoscincus queenslandiae), from seven forest fragments and five sites in continuous forest on the Atherton tableland of northeastern Queensland, Australia. The rain forest in this region was fragmented by logging and clearing for dairy farms in the early 1900s and most forest fragments studied have been isolated for 50-80 years or nine to 12 skink generations. We genotyped 411 individuals at nine microsatellite DNA loci and found fewer alleles per locus in prickly forest skinks from small rain forest fragments and a lower ratio of allele number to allele size range in forest fragments than in continuous forest, indicative of a decrease in effective population size. In contrast, and as expected for populations with small neighbourhood sizes, neither heterozygosity nor variance in allele size differed between fragments and sites in continuous forests. Considering measures of among population differentiation, there was no increase in F-ST among fragments and a significant isolation by distance pattern was identified across all 12 sites. However, the relationship between genetic (F-ST) and geographical distance was significantly stronger for continuous forest sites than for fragments, consistent with disruption of gene flow among the latter. The observed changes in genetic diversity within and among populations are small, but in the direction predicted by the theory of genetic erosion in recently fragmented populations. The results also illustrate the inherent difficulty in detecting genetic consequences of recent habitat fragmentation, even in genetically variable species, and especially when effective population size and dispersal rates are low.

On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (he) to light chain (lc) genes encoded on either one or two plasmids. However, there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab. In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG(4) Mab, cB72.3, by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches. Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data. On the basis of this information, we hypothesized that utilization of he genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3 Mab was transiently produced by CHO cells at constant he and varying lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc gene dose. To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab, we compared the intra- and extracellular ratios of HC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab, cB72.3. Intra- and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5, less than that observed on transient expression of the same Mab in parental CHO cells using the same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ. In the case of the latter, we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.

The interlayer swelling and molecular packing in organoclays

Understanding the interlayer swelling and molecular packing in organoclays is important to the formation and design of polymer nanocomposites. This paper presents recent experimental and molecular simulation studies on a variety of organoclays that show a linear relationship between the increase of d-spacing and the mass ratio between organic and clay. A denser molecular packing is observed in organoclays containing surfactants with hydroxyl-ethyl units. Moreover, our simulation results show that the head (nitrogen) groups are essentially tethered to the clay surface while the long hydrocarbon chains tend to adopt a layering structure with disordered conformation, which contrasts with the previous assumptions of either the chains lying parallel to the clay surface or being tilted at rather precise angles. (c) 2005 Elsevier Inc. All rights reserved.

Novel approach to tailoring molecular weight distribution and structure with a difunctional RAFT agent

This work has demonstrated that for the first time a single RAFT agent (i. e., difunctional) can be used in conjunction with a radical initiator to obtain a desired M-n and PDI with controlled rates of polymerization. Simulations were used not only to verify the model but also to provide us with a predictive tool to generate other MWDs. It was also shown that all the MWDs prepared in this work could be translated to higher molecular weights through chain extension experiments with little or no compromise in the control of end group functionality. The ratio of monofunctional to difunctional SdC(CH2Ph)S- end groups, XPX and XP (where X) S=C(CH2Ph) S-), can be controlled by simply changing the concentration of initiator, AIBN. Importantly, the amount of dead polymer is extremely low and fulfils the criterion as suggested by Szwarc (Nature 1956) that to meet living requirements nonfunctional polymeric species formed by side reactions in the process should be undetectable by analytical techniques. In addition, this novel methodology will allow the synthesis of AB, ABA, and statistical multiblock copolymers with predetermined ratios to be produced in a one-pot reaction.

Molecular sieve silica membranes for H2/CO separation

Efficient separation of fuel gas (H2) from other gases in reformed gas mixtures is becoming increasingly important in the development of alternative energy systems. A highly efficient and new technology available for these separations is molecular sieve silica (MSS) membranes derived from tetraethyl-orthosilicate (TEOS). A permeation model is developed from an analogous electronic system and compared to transport theory to determine permeation, selectivity and apparent activation of energy based on experimental values. Experimental results for high quality membranes show single gas permselectivity peaking at 57 for H2/CO at 150°C with a H2 permeation of 5.14 x 10^-8 mol.m^-2.s^-1.Pa^-1. Higher permeance was also achieved, but at the expense of selectivity. This is the case for low quality membranes with peak H2 permeation at 1.78 x 10-7 mol.m-2.s-1.Pa-1 at 22°C and H2/CO permselectivity of 4.5. High quality membranes are characterised with positive apparent activation energy while the low quality membranes have negative values. The model had a good fit of r-squared of 0.99-1.00 using the experimental data.