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PubMed2004

Nanoscale lead and noble gas inclusions in aluminum: structures and properties.

Transmission electron microscopy has been used for structural and physical characterization of nanoscale inclusions of lead and noble gases in aluminum. When the inclusion sizes approach nanoscale dimensions, many of their properties are seen to deviate from similar properties in bulk and in most cases the deviations will increase as the inclusion sizes decrease. Binary alloys of lead and noble gases with aluminum are characterized by extremely low mutual solubilities and inclusions will, therefore, exist as practically pure components embedded in the aluminum matrix. Furthermore, the thermal vacancy mobility in aluminum at and above room temperature is sufficiently high to accommodate volume strains associated with the inclusions thus leading to virtually strain free crystals. The inclusions grow in parallel cube alignment with the aluminum matrix and have a cuboctahedral shape, which reflects directly the anisotropy of the interfacial energies. Inclusions in grain boundaries can have single crystalline or bicrystalline morphology that can be explained from a generalized Wulff analysis such as the xi-vector construction. The inclusions have been found to display a variety of nanoscale features such as high Laplace pressure, size-dependent superheating during melting, deviations from the Wulff shape displaying magic size effects, a shape dependence of edge energy, and so on. All these effects have been observed and monitored by TEM using conventional imaging conditions and high-resolution conditions in combination with in-situ analysis at elevated temperatures.

Algorithms↗

PubMed1994

Phospholipid headgroup-headgroup electrostatic interactions in mixed bilayers of cardiolipin with phosphatidylcholines studied by 2H NMR.

The headgroup-headgroup interactions in binary mixed bilayers of diacylphosphatidylcholines (PC) and cardiolipin were analyzed by 2H NMR. Specific changes in the quadrupole splittings of the choline headgroup deuterated PC at alpha,beta-methylenes, and gamma-methyls are observed upon the insertion of the negatively charged tetraacylphospholipid, cardiolipin. The effects are consistent with an electrostatic interaction between PC and cardiolipin headgroups, in which a concerted conformational reorientation of the entire phosphocholine moiety toward the membrane surface is involved. On the basis of the "choline-tilt" model by Macdonald and co-workers (1991) the variations in the quadrupole splittings are consistent with a change in orientation of the choline P-N vector up to 23 degrees for the highest cardiolipin concentrations. Additional information on headgroup conformational changes was obtained through the analysis of the dependence on temperature of the quadrupole splittings for the various deuterium-labeled segments. Evaluation of the deuterium spin-lattice (T1) relaxation times for the deuterons in the various positions of the choline headgroup in mixed bilayers of PC and cardiolipin showed that the internal fast segmental motions were not affected on addition of cardiolipin to PC membranes.

Cardiolipins↗

PubMed2002

Carcinogenic chromium(VI) induces cross-linking of vitamin C to DNA in vitro and in human lung A549 cells.

Reductive activation of carcinogenic Cr(VI) is required for the induction of DNA damage and mutations. Here, we examined the formation of Cr-DNA adducts in the reactions of Cr(VI) with its dominant biological reducer, vitamin C (ascorbate). Reductive conversion of Cr(VI) to Cr(III) by ascorbate produced stable Cr-DNA adducts, of which approximately 25% constituted ascorbate-Cr(III)-DNA cross-links. No evidence was found for the involvement of Cr(V) or Cr(IV) intermediates in the formation of either binary or ternary adducts. The cross-linking reaction was consistent with the attack of DNA by transient Cr(III)-ascorbate complexes. The yield of Cr(III)-DNA adducts was similar on dsDNA and AGT, ACT, or CT oligonucleotides and was strongly inhibited by Mg(2+), suggesting predominant coordination of Cr(III) to DNA phosphate oxygens. We also detected cross-linking of ascorbate to DNA in Cr(VI)-exposed human lung A549 cells that were preincubated with dehydroascorbic acid to create normal levels of intracellular ascorbate. Ascorbate-Cr-DNA cross-links accounted for approximately 6% of the total Cr-DNA adducts in A549 cells. Shuttle-vector experiments showed that ascorbate-Cr-DNA cross-links were mutagenic in human cells. Our results demonstrate that in addition to reduction of Cr(VI) to DNA-reactive Cr(III), vitamin C contributes to the genotoxicity of Cr(VI) via a direct chemical modification of DNA. The absence of Asc in A549 and other human cultured cells indicates that cells maintained under the usual in vitro conditions lack the most important reducing agent for Cr(VI) and would primarily display slow thiol-dependent activation of Cr(VI).

Ascorbic Acid↗

PubMed1995

Cloning, expression, purification, and characterization of 2'-deoxyuridylate hydroxymethylase from phage SPO1.

2'-Deoxyuridylate hydroxymethylase (dUMP-hmase) from phage SPO1 has been cloned and expressed in Escherichia coli. In crude extracts, the enzyme represents about 25% of the soluble protein and has a higher specific activity than the most purified preparation yet reported. The enzyme was purified to homogeneity by ion-exchange and hydrophobic chromatography. The subunits of dUMP-hmase are 45 kDa by SDS-PAGE and form dimers with a molecular mass of 89.2 kDa by analytical centrifugation. In addition to the normal reaction, dUMP-hmase catalyzes the 5,10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate)-independent tritium exchange of [5-3H]dUMP for protons of water and dehalogenation of 5-bromo-2'-deoxy-uridine-5'-monophosphate; the enzyme also forms a covalent binary adduct with pyridoxal 5'-monophosphate and a covalent ternary complex with 5-fluoro-2'-deoxyuridine-5'-monophosphate and CH2H4folate. Folic acid inhibits the tritium release catalyzed by dUMP-hmase in the presence of cofactor but has no effect on the catalysis of cofactor-independent tritium exchange.

Bacillus Phages↗

PubMed1989

Dependence on pH of substrate binding to a mutant lactose carrier, lacYun, in Escherichia coli. A model for H+/lactose symport.

The lacYun gene, which encodes a lactose carrier showing the uncoupled phenotype of substrate transport in Escherichia coli [Wilson, Kusch & Kashket (1970) Biochem. Biophys. Res. Commun. 40, 1409-1414], was cloned on a plasmid vector, pBR322. The binding of a substrate, p-nitrophenyl alpha-galactoside, to the lacYun carrier in membranes from the strain harbouring the lacYun clone showed a pH-dependence different from its binding to the wild-type lactose carrier. This finding indicated that the lacYun mutation confers higher affinity for H+ on the carrier, exerting its effect on the less efficient dissociation of substrate inside cells. The result coincides with the proposal [Yamato & Rosenbusch (1983) FEBS Lett. 151, 102-104] that the proton affecting the substrate binding is the coupling proton of the proton/lactose symport reaction, which allows only the ordered mechanism of binding of substrate to an H+-carrier binary complex. From the simplest model of the symport reaction, constructed on the basis of these results, the coupling site of energy in the carrier cycle of the transport reaction can be identified at the substrate-dissociation step inside cells.

Binding Sites↗

PubMed2000

Influence of the free-energy functional form on simulated morphology of spinodally decomposing blends

The spinodal decomposition of a binary mixture has been studied within several mesoscopic models. It has been found that the form of the equilibrium free energy has a crucial effect on the morphological development in asymmetric blends. We have shown that the principal quantity that determines the topology of the interface (and type of morphology) is the equilibrium minority phase volume fraction, while the transition from bicontinuous to droplet morphology can be treated as a percolation. The concentration dependence of the square gradient coefficient attributed for the Flory-Huggins-de Gennes free energy has no significant influences on the average domain growth, but can be distinguished experimentally from its constant-coefficient alternative by measuring the maximum wave vector of the scattering intensity as a function of the minority phase volume fraction for spinodally decomposing asymmetric blends. The concentration dependence of the Onsager coefficient has the weak, systematic effect of slowing down the morphological development. The local shape of the interface is not affected considerably by the concentration dependence of the square gradient and Onsager coefficient.

Journal Article↗

PubMed2000

Stoichiometry of cyclin A-cyclin-dependent kinase 2 inhibition by p21Cip1/Waf1.

Progression through the eukaryotic cell cycle is regulated by phosphorylation, which is catalyzed by cyclin-dependent kinases. Cyclin-dependent kinases are regulated through several mechanisms, including negative regulation by p21 (variously called CAP20, Cip1, Sdi1, and WAF1). It has been proposed that multiple p21 molecules are required to inhibit cyclin-dependent kinases, such that p21 acts as a sensitive buffer of cyclin-dependent kinase activity or as an assembly factor for the complexes formed by the cyclins and cyclin-dependent kinases. Using purified, full-length proteins of known concentration (determined by absorbance) and cyclin A-Cdk2 of known activity (calibrated with staurosporine), we find that a 1:1 molar ratio of p21 to cyclin A-Cdk2 is able to inhibit Cdk2 activity both in the binary cyclin A-Cdk2 complex and in the presence of proliferating cell nuclear antigen (PCNA). Our results indicate that the mechanism of p21 inhibition of cyclin A-Cdk2 does not involve multiple molecules of bound p21.

Amino Acid Sequence↗

PubMed2006

A novel approach for accurate prediction of spontaneous passage of ureteral stones: support vector machines.

The objective of this study was to optimally predict the spontaneous passage of ureteral stones in patients with renal colic by applying for the first time support vector machines (SVM), an instance of kernel methods, for classification. After reviewing the results found in the literature, we compared the performances obtained with logistic regression (LR) and accurately trained artificial neural networks (ANN) to those obtained with SVM, that is, the standard SVM, and the linear programming SVM (LP-SVM); the latter techniques show an improved performance. Moreover, we rank the prediction factors according to their importance using Fisher scores and the LP-SVM feature weights. A data set of 1163 patients affected by renal colic has been analyzed and restricted to single out a statistically coherent subset of 402 patients. Nine clinical factors are used as inputs for the classification algorithms, to predict one binary output. The algorithms are cross-validated by training and testing on randomly selected train- and test-set partitions of the data and reporting the average performance on the test sets. The SVM-based approaches obtained a sensitivity of 84.5% and a specificity of 86.9%. The feature ranking based on LP-SVM gives the highest importance to stone size, stone position and symptom duration before check-up. We propose a statistically correct way of employing LR, ANN and SVM for the prediction of spontaneous passage of ureteral stones in patients with renal colic. SVM outperformed ANN, as well as LR. This study will soon be translated into a practical software toolbox for actual clinical usage.

Algorithms↗

PubMed2002

Bcc and Fcc transition metals and alloys: a central role for the Jahn-Teller effect in explaining their ideal and distorted structures.

Transition metal elements, alloys, and intermetallic compounds often adopt the body centered cubic (bcc) and face centered cubic (fcc) structures. By comparing quantitative density functional with qualitative tight-binding calculations, we analyze the electronic factors which make the bcc and fcc structures energetically favorable. To do so, we develop a tight-binding function, DeltaE(star), a function that measures the energetic effects of transferring electrons within wave vector stars. This function allows one to connect distortions in solids to the Jahn-Teller effect in molecules and to provide an orbital perspective on structure determining deformations in alloys. We illustrate its use by considering first a two-dimensional square net. We then turn to three-dimensional fcc and bcc structures, and distortions of these. Using DeltaE(star), we rationalize the differences in energy of these structures. We are able to deduce which orbitals are responsible for instabilities in seven to nine valence electron per atom (e(-)/a) bcc systems and five and six e(-)/a fcc structures. Finally we demonstrate that these results account for the bcc and fcc type structures found in both the elements and binary intermetallic compounds of group 4 through 9 transition metal atoms. The outline of a theory of metal structure deformations based on loss of point group operation rather than translational symmetry is presented.

Journal Article↗

PubMed2001

Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation.

The co-transformation of a single plant genome with two independent T-DNA regions provides opportunities for genetic separation in subsequent generations. In an effective strategy, co-delivery events must form a high proportion of the total transformed population. In this study, using the model plant species tobacco (Nicotiana tabacum), it was shown that the frequency of co-transformation within a given To population could be as high as 100% and this was found to be dependent, at least in part, on designing the plasmid vectors so that the kbp size of the first selected T-DNA region was >2-fold that of the designated T-DNA region for co-transfer. Overall, 40-50% of To lines demonstrated the capacity for segregational separation of co-transformed T-DNA regions. Hence, the estimate of the required number of total transformants for such an independent strategy may seem to be as little as 2-fold that for a conventional, single T-DNA strategy, but we strongly temper such estimates with indications that high co-transformation frequencies may be associated with a higher incidence of linkage. In this co-transformation study we used a single (Agrobacterium) strain system in which a single binary plasmid contained either two or three T-DNA regions, each with a selectable marker. This arrangement could reveal that 'read-through' events within the Agrobacterium cells, resulting in the co-transfer of adjacent T-DNA regions as a single linked unit, accounted for up to 20% of co-transformed plant lines. Such read-through co-delivery appeared to be more frequent from the 'supervirulent' EHA101 A. tumefaciens strain, compared to the 'ordinary' LBA4404 strain. By using the binary plasmid with three selectable T-DNA regions, we have been able to consider the frequency of co-integration of a third independent T-DNA within a T0 subpopulation of co-transformants. This was found to be higher than expected. These observations were applied to the co-transfer of (unwanted) plasmid backbone sequences and showed that screening against such sequences may add a significant factor in achieving the desired, final genotype.

DNA, Bacterial↗

PubMed1997

Human babesiosis in Taiwan: asymptomatic infection with a Babesia microti-like organism in a Taiwanese woman.

An asymptomatic Babesia infection was confirmed by laboratory diagnoses. The intraerythrocytic protozoan (designed TW1) isolated from a 51-year-old Taiwanese woman appeared to be morphologically consistent with small-form piroplasm, and measurements indicated that it had a body size of 1.5 to 2.5 microm in diameter. The typical features of ring, binary, and tetrad forms were observed in Giemsa-stained thin blood smears. A persistent and low-grade parasitemia was established after hamster inoculation. Indirect immunofluorescent-antibody reactivities indicate that this strain (TW1) of Babesia was serologically related to, but not identical to, the Babesia species (B. microti) that infects rodents. Antibody titers in the patient's sera combined with the clinical symptoms suggested that the present case was a chronic and subclinical babesial infection. A neighborhood human serologic survey indicated that the infection may have been acquired accidentally from an infected rodent and localized within the same family. Indeed, rodents from areas around the neighborhood were trapped, and a high prevalence (83%) of babesial infection was observed. The possible vector responsible for the transmission remains to be identified.

Animals↗

PubMed1997

Multivariate methods for clustered ordinal data with applications to survival analysis.

Clustered data are the rule in many clinical specialties such as ophthalmology. Methods have been developed for the treatment of clustered continuous or binary outcome data. Less attention has been given to ordinal outcomes which occur frequently in ophthalmology. For example, grading systems of cataract and diabetic retinopathy are commonly used where a photograph is graded by comparison with a series of reference photographs of increasing severity. Some commonly used methods for the analysis of ordered categorical data include the proportional odds and continuation ratio models. It is difficult, however, to incorporate clustering effects into these models. Instead, for clusters of size two, we propose a generalization of the adjacent category model given by log[Pr(i + 1,j)/Pr(i,j)] = ui + (j - 1) lambda + beta' x, where Pr(i,j) denotes the probability that the right (left) eye has grade i(j), x is a vector of (person or eye-specific) covariates for the right eye, u and beta are vectors of location and covariate parameters and lambda is a clustering parameter. Based on this model, and a similar model interchanging the role of i and j, we derived a closed-form expression for Pr(i,j) as a function of u, lambda and beta and use Newton-Raphson method to maximize the likelihood. An extension of the method allows for extra agreement along the diagonal and is then a generalization of the agreement plus linear-by-linear association model proposed by Agresti in the setting of no covariates. We apply these methods to a data set of 43 diabetic subjects from the Harvard Clinical Cataract Research Center, where cortical cataract grade was the outcome. We also extend this methodology to a survival setting, where both censored and uncensored outcomes are available for individual cluster members, and one wishes to take clustering into account. We apply the survival analysis model to a data set of 1807 children (two ears per child) in the greater Boston area, who were followed for the development of otitis media over the first year of life.

Boston↗

PubMed1994

Expression of mosquitocidal toxin genes in a gas-vacuolated strain of Ancylobacter aquaticus.

A series of plasmids bearing the binary toxin genes of Bacillus sphaericus 2297 or 2317.3, the 100-kDa toxin gene of B. sphaericus SSII-1, or the 130-kDa (cryIVB) toxin gene of Bacillus thuringiensis subsp. israelensis were constructed and introduced into Ancylobacter aquaticus by electroporation. The transformed A. aquaticus cells exhibited significant toxicity towards mosquito larvae, demonstrating a potential use of recombinant A. aquaticus for biological control of mosquitoes.

Animals↗

PubMed1995

[Excretion of promastigotes of Leishmania pifanoi by experimentally infected Lutzomyia youngi].

The increase in the promastigotes population of Leishmania pifanoi in Lutzomyia youngi experimentally infected and kept on 50% sacarose under constant conditions of temperature and humidity is described. Two stages in the differentiation and growth of the parasites are recognised between two and twenty-four hours after meals. The pleomorphic differentiation of the amastigotes in short promastigotes which multiply by binary division for 60 hours, when the rupture of the peritrophic membrane occurs, takes place within 48 hours. The second stage occurs between 72 and 96 hours when some of the parasites migrate to the esophagic valve and the rest of the free parasites are excreted in fecal drops as large, active promastigotes. The first drops excreted react positively to glucose or contain crystals of urate. The excess promastigotes of the second stage of development are eliminated in the last excretions and react positively to the Hemoscreen and Biuret tests for total proteins as also for glucose and account for 82% of the of drops excreted. The excretion of parasites by Lu. youngi is a normal stage in the growth of L. pifanoi into a vector.

Animals↗

PubMed2000

Characterisation of three novel cationic lipids as liposomal complexes with DNA.

Cationic lipids (CLs) are being increasingly exploited as transfection vectors for the delivery of DNA into eukaryotic cells. To obtain further insight to the complex formation and interactions between cationic liposomes and DNA, we characterised three novel cationic lipids, viz. bis[2-(11-phenoxyundecanoate)ethyl]-dimethylammonium bromide, N-hexadecyl-N-¿10-[O-(4-acetoxy)-phenylundecanoate]ethyl¿- dimethylammonium bromide, and bis[2-(11-butyloxyundecanoate)ethyl]dimethylammonium bromide. These lipids bear the same charged headgroup yet have different hydrophobic parts. Accordingly, we may anticipate their electrostatic interactions with DNA to be similar while differing in both thermal phase behaviour and physicochemical properties of their complexes with DNA. In keeping with the above all three lipids formed complexes with DNA as evidenced by light scattering, fluorescence spectroscopy and Langmuir film balance. Differential scanning calorimetry revealed very different phase behaviours for the binary mixtures of the three CLs with dimyristoylphosphatidylcholine and also provided evidence for DNA-induced lipid phase separation. These data were confirmed by compression isotherms and fluorescence microscopy of monolayers residing on an aqueous buffer, recorded both in the presence and absence of DNA. Importantly, binding to cationic liposomes appears to prevent thermal denaturation of DNA upon heating of the complexes. Likewise, renaturation of heat-treated DNA complexed with the cationic liposomes appears to be abolished as well.

Cations↗

PubMed1999

Introduction of the IBD information into the weighted pairwise correlation method for linkage analysis.

The Weighted Pairwise Correlation (WPC) approach is a non-parametric method of linkage analysis that allows analysis of any kind of phenotypes (quantitative, binary, binary with age of onset) and to consider all pairs of relatives in a pedigree. The principle of this method is to test whether two relatives having close phenotypes also resemble at the marker locus more than expected under the null hypothesis of no linkage. So far, this marker resemblance was estimated by the proportion of alleles shared Identical By State (IBS) by the two relatives. Here, we propose a method to incorporate the Identical By Descent (IBD) information into the WPC approach. For any kind of relative pairs, the computation of the proportion of alleles shared IBD is based on the identification of the closest couple of ancestors, denoted as the reference couple. The IBD information is obtained for pairs of relatives having the same reference couple using individual genotypic vectors derived from this couple. This reconstruction of the IBD information is performed rapidly even in large pedigrees. Simulation studies conducted under various genetic models demonstrate that the use of IBD instead of IBS information leads to a large increase of power, especially in the situation of poorly informative markers.

Genetic Linkage↗

PubMed2001

Motions in binary mixtures of hard colloidal spheres: melting of the glass.

Dynamic light-scattering experiments are performed on binary mixtures of hard-sphere-like colloidal suspensions with a size ratio of 0.6. The optical properties of the particles are such that the relative contrast of the two species is very sensitive to temperature, a feature that is exploited to obtain the three partial coherent intermediate scattering functions. The glass transition is identified by the onset of structural arrest, or arrest of the alpha process, on the time scale of the experiment. This is observed in a one-component suspension at a packing fraction of 0.575. The intermediate scattering functions measured on the mixtures quantify how, on introduction of the smaller spheres, the alpha process is released, i.e., how the glass melts. Increasing the fraction of smaller particles causes the alpha process to speed up but, at a given wave vector, also incurs a change to its amplitude in proportion to the change in the (partial) structure factor.

Journal Article↗

PubMed2001

Adenovirus-mediated Bax overexpression for the induction of therapeutic apoptosis in prostate cancer.

Using adenoviral technology, we overexpressed the proapoptotic molecules pro-caspase-3, pro-caspase-7, and Bax to induce therapeutic apoptosis of prostate cancer cell lines growing in vitro and in vivo. Because overexpressed pro-caspase-3 did not undergo autocatalytic activation in any of the five prostate cancer cell lines evaluated, this strategy was unable to engage any component of the apoptotic pathway. Overexpressed pro-caspase-7 was proteolytically cleaved in LNCaP and LnCaP-Bcl-2 cells but not in PC-3, DU-145, or TsuPr(1) cells. Cleavage was associated with engagement of many components of the apoptotic pathway, including DEVDase activity, cleavage of intracellular caspase targets such as the DNA fragmentation factor and the proapoptotic Bid, release of cytochrome c from the mitochondria to the cytoplasm, and terminal deoxynucleotidyl transferase-mediated nick end labeling. No apoptosis was observed in the cells where caspase-7 did not undergo autocatalytic activation. Searching for an approach that would more reliably induce therapeutic apoptosis of prostate cancer cell lines, we used a binary adenoviral system to overexpress the proapoptotic molecule Bax. Bax was dramatically overexpressed and caused apoptosis of every cell line infected by engaging the mitochondrial pathway, including proteolytic cleavage and catalytic activation of the caspases, cleavage of caspase substrates, release of cytochrome c from the mitochondria, and DNA fragmentation. Furthermore, three injections of the Bax overexpression system into PC-3 cell tumors in nude mice in vivo caused a 25% regression in tumor size corresponding to a 90% reduction relative to continued tumor growth in animals that received injections with the control binary system expressing Lac-Z. These experiments show that adenovirus-mediated Bax overexpression is capable of inducing therapeutic programmed cell death in vitro and in vivo by activating the mitochondrial pathway of apoptosis. On the basis of these studies, we conclude that manipulation of Bax expression is an attractive new gene therapy approach for the treatment of prostate cancer.

Adenoviridae↗