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PubMed2006

Microbial diversity in sediments of saline Qinghai Lake, China: linking geochemical controls to microbial ecology.

Saline lakes at high altitudes represent an important and extreme microbial ecosystem, yet little is known about microbial diversity in such environments. The objective of this study was to examine the change of microbial diversity from the bottom of the lake to sediments of 40 cm in depth in a core from Qinghai Lake. The lake is saline (12.5 g/L salinity) and alkaline (pH 9.4) and is located on the Qinghai-Tibetan Plateau at an altitude of 3196 m above sea level. Pore water chemistry of the core revealed low concentrations of sulfate and iron (<1 mM), but high concentrations of acetate (40-70 mM) and dissolved organic carbon (1596-5443 mg/L). Total organic carbon and total nitrogen contents in the sediments were approximately 2 and <0.5%, respectively. Acridine orange direct count data indicated that cell numbers decreased from 4 x 10(9) cells/g at the water-sediment interface to 6 x 10(7) cells/g wet sediment at the 40-cm depth. This change in biomass was positively correlated with acetate concentration in pore water. Phospholipid fatty acid (PLFA) community structure analyses determined decrease in the proportion of the Proteobacteria and increase in the Firmicutes with increased depth. Characterization of small subunit (SSU) rRNA genes amplified from the sediments indicated a shift in the bacterial community with depth. Whereas the alpha-, beta-, and gamma-Proteobacteria and the Cytophaga/Flavobacterium/Bacteroides (CFB) were dominant at the water-sediment interface, low G + C gram-positive bacteria (a subgroup of Firmicutes) became the predominant group in the anoxic sediments. Both PLFA and the sequence data showed similar trend. The Proteobacteria, CFB, and gram-positive bacteria are present in other saline lakes, but the presence of Actinobacteria and Acidobacteria/Holophaga in significant proportions in the Qinghai Lake sediments appears to be unique. The archaeal diversity was much lower, and clone sequences could be grouped in the Euryarchaeota and Crenarchaeota domains. The archaeal clones were not related to any known cultures but to sequences previously found in methane-rich sediments. Acetate-utilizing methanogens were isolated from sediment incubations, and alpha- and gamma-proteobacterial isolates were obtained from a water sample from the lake-bottom (23 m). Our data collectively showed that the observed diversity and shift in the community structure with depth was correlated with geochemical parameters (the redox state and availability of electron acceptor and donor). Heterotrophic methanogenesis is possibly adominant metabolic process in the Qinghai Lake sediments. These results reinforce the importance of geochemical controls on microbial ecology in saline and alkaline lake environments.

Acridine Orange↗

PubMed2005

Meprin metalloprotease expression and regulation in kidney, intestine, urinary tract infections and cancer.

Meprins are unique plasma membrane and secreted metalloproteinases that are highly regulated at the transcriptional and post-translational levels. Meprin alpha and beta subunits are abundantly expressed in kidney and intestinal epithelial cells, are secreted into the urinary tract and intestinal lumen, and are found in leukocytes and cancer cells under certain conditions. Their location and proteolytic activities indicate functions at the interface of the host and the external environment, and in trafficking of macrophages and metastases of cancer cells. These proteases can be detrimental when there is tissue damage or disruption, as in acute renal injury or intestinal inflammation, and there is evidence they are involved in movement of leukocytes and cancer cells to sites of infection or in metastasis, respectively.

Animals↗

PubMed2005

Site-directed fluorescence labeling reveals differences on the R-conformer of glucosamine 6-phosphate deaminase of Escherichia coli induced by active or allosteric site ligands at steady state.

Engineered glucosamine 6-phosphate deaminase of Escherichia coli with unique reactive cysteines at positions 164 or 206 was created by site-directed mutagenesis to monitor the allosteric transition in solution by the fluorescence emission of the bimane or dansyl-amidoethyl groups attached to the indicated residues. The selection of both positions was due to the differential interaction of these residues between T- and R-conformers at the interface of two trimers that form the hexameric structure; in the T-conformer, residue 164 or 206 presents only intrasubunit contacts, but in the R-conformer, new intersubunit contacts are established. As in the wild-type enzyme, fluorescent-labeled mutants show no modification on the allosteric activation of the K-system, only the kcat was reduced to a value of 72 s(-1) (approximately 50% of wild-type). With these preparations, conformational changes were detected by the fluorescence emission spectra at steady state when the active site or the allosteric site ligands were titrated. Despite the similar changes in the fluorescence spectra that were correlated with the induction of the R-state, differences were observed at the maximal change in the fluorescence spectra and in the relative solvent polarities at the positions labeled. These data suggested structural differences in the conformation of the R-state when it is induced from the active site or from the allosteric site, which is not consistent with the two-state structural model proposed by previous crystallographic studies of this enzyme.

Aldose-Ketose Isomerases↗

PubMed2004

Human DNA ligase I completely encircles and partially unwinds nicked DNA.

The end-joining reaction catalysed by DNA ligases is required by all organisms and serves as the ultimate step of DNA replication, repair and recombination processes. One of three well characterized mammalian DNA ligases, DNA ligase I, joins Okazaki fragments during DNA replication. Here we report the crystal structure of human DNA ligase I (residues 233 to 919) in complex with a nicked, 5' adenylated DNA intermediate. The structure shows that the enzyme redirects the path of the double helix to expose the nick termini for the strand-joining reaction. It also reveals a unique feature of mammalian ligases: a DNA-binding domain that allows ligase I to encircle its DNA substrate, stabilizes the DNA in a distorted structure, and positions the catalytic core on the nick. Similarities in the toroidal shape and dimensions of DNA ligase I and the proliferating cell nuclear antigen sliding clamp are suggestive of an extensive protein-protein interface that may coordinate the joining of Okazaki fragments.

Binding Sites↗

PubMed1991

The leadership and executive potential of physicians in an era of managed care systems.

The emergence of managed care signals a need for an expanded role for physicians. Physician executives, trained in management, have the potential to become champions of clinical integrity, negotiators with patient agent organizations, public interpreters of hospital performance data, consultants to the profession, and designers of health care systems. Nonclinically trained administrators have been slow to recognize the unique skills and perspective that physicians bring to the managerial suite. The clash of professional and managerial cultures has often been perceived as precluding a strong working alliance between physician and nonphysician executives. As hospital margins are threatened, decision protocols must not be designed to incorporate teams of leaders who are able to overcome the managerial/clinical divide. The management of this interface will demand the attention of both administration and boards and may require the establishment of new structures in health care organizations.

Leadership↗

PubMedDate not supplied

Data mining and healthcare informatics.

OBJECTIVE: To acquaint members of the Academy with a relatively recent development in the area of data exploration and statistical analysis. METHODS: A review of the concepts and methods inherent in data-mining with a special emphasis on the those methods applicable to predictive modeling. RESULTS: Data-mining is demonstrated to be a useful tool for researchers in those circumstances where large amounts of information are available. CONCLUSIONS: With the advent and proliferation of on-line data collection, truly massive databases are now available to health care researchers. In that situation, data-mining methods yield some unique opportunities to researchers who wish to develop prediction models and to establish associations.

Database Management Systems↗

PubMed1993

Characterization of an intermediate in the folding pathway of phosphoglycerate kinase: chemical reactivity of genetically introduced cysteinyl residues during the folding process.

The unfolding-refolding kinetics of yeast phosphoglycerate kinase were studied using the chemical reactivity of genetically introduced cysteinyl residues as conformational probes and far-ultraviolet circular dichroism. A unique internal cysteinyl residue was introduced in several mutants at selected positions in the N- and C-domains. The cysteinyl residues were at positions 97 (the unique cysteinyl residue of the wild-type enzyme), 183 in the N-domain, 285 and 324 in the C-domain. A similar strategy has been used to study the unfolding-refolding transition under equilibrium conditions [Ballery et al. (1990) Protein Eng. 3, 199-204]. Except for the mutant C97A,A183C, whose cysteinyl residue is located at the domain interface, three labeling phases were observed during the refolding process, indicating the presence of three species, the unfolded, intermediate, and folded proteins. The comparison of the data obtained following the accessibility of the thiol group to 5,5'-dithiobis(2-nitrobenzoate) and ellipticity at 218 nm indicated that all mutants have the same folding pathway and allowed us to characterize the intermediate. In this species, each domain appeared to have a high content of secondary structure but a flexible tertiary structure; this intermediate, which had the characteristics of a molten globule, remained in fluctuating equilibrium with a widely unfolded form. The same folding intermediate was detected for mutant C97A,A183C; however, the cysteinyl residue being totally accessible to the reagent, it is likely that in this intermediate the interdomain interactions are not established. Domain pairing and formation of the native tertiary structure occur simultaneously in the slow phase of refolding. The validity and limitations of the methodology are discussed.

Circular Dichroism↗

PubMed2006

Lectin-recognizable colloidal dispersions stabilized by n-dodecyl beta-D-maltoside: particle-particle and particle-surface interactions.

Recently, we reported that it is possible to utilize sugars as stabilizing agents for colloidal particles. This study shows that when n-dodecyl beta-D-maltoside (DDM) is utilized as a dispersing and stabilizing agent in the synthesis and stabilization of poly[methyl methacrylate-co-(n-butyl acrylate)] (p-MMA/nBA) colloidal particles, stable colloidal dispersions can be formed. Since understanding of sugar-protein interactions have numerous practical and scientific implications, these studies examine DDM-stabilized p-MMA/nBA colloidal particles and their specific binding properties with concanavalin A (Con A). By use of spectroscopic analysis, unique binding characteristics that are a function of DDM concentration, time, and the concentration of Con A are detected. When DDM-stabilized p-MMA/nBA particles are allowed to coalesce, DDM is released from the particle surfaces and, under suitable conditions, selectively stratifies in the areas of the excess of interfacial energy near the film-air (F-A) interface, thus providing sites for attracting Con A via alpha-glucose-OH hydrogen bonding. Consequently, adsorption of Con A at the F-A interfaces occur and the degree of adsorption is controlled by the amount of DDM at the F-A interface.

Adsorption↗

PubMed1985

Thickness of ice on perennially frozen lakes.

The dry valleys of southern Victoria Land, constituting the largest ice-free expanse in the Antarctic, contain numerous lakes whose perennial ice cover is the cause of some unique physical and biological properties. Although the depth, temperature and salinity of the liquid water varies considerably from lake to lake, the thickness of the ice cover is remarkably consistent, ranging from 3.5 to 6 m, which is determined primarily by the balance between conduction of energy out of the ice and the release of latent heat at the ice-water interface and is also affected by the transmission and absorption of sunlight. In the steady state, the release of latent heat at the ice bottom is controlled by ablation from the ice surface. Here we present a simple energy-balance model, using the measured ablation rate of 30 cm yr-1, which can explain the observed ice thickness.

Antarctic Regions↗

PubMed2000

Imaging the vortex-lattice melting process in the presence of disorder

General arguments suggest that first-order phase transitions become less sharp in the presence of weak disorder, while extensive disorder can transform them into second-order transitions; but the atomic level details of this process are not clear. The vortex lattice in superconductors provides a unique system in which to study the first-order transition on an inter-particle scale, as well as over a wide range of particle densities. Here we use a differential magneto-optical technique to obtain direct experimental visualization of the melting process in a disordered superconductor. The images reveal complex behaviour in nucleation, pattern formation, and solid-liquid interface coarsening and pinning. Although the local melting is found to be first-order, a global rounding of the transition is observed; this results from a disorder-induced broad distribution of local melting temperatures, at scales down to the mesoscopic level. We also resolve local hysteretic supercooling of microscopic liquid domains, a non-equilibrium process that occurs only at selected sites where the disorder-modified melting temperature has a local maximum. By revealing the nucleation process, we are able to experimentally evaluate the solid-liquid surface tension, which we find to be extremely small.

Journal Article↗

PubMed2003

Automatic design of gene-specific sequence tags for genome-wide functional studies.

MOTIVATION: The availability of complete genome sequences allows the identification of short DNA segments that are specific to each annotated gene. Such unique gene sequence tags (GSTs) replace advantageously cDNAs in microarray transcript profiling experiments. In particular, probes corresponding to individual members of multigene families can be chosen carefully to avoid cross-hybridization events. RESULTS: The Specific Primer and Amplicon Design Software (SPADS) was constructed to delineate the more divergent regions in each gene by comparing them with a completely annotated genome sequence and to select optimal primer pairs for the polymerase chain reaction amplification of one divergent region per gene. SPADS is a unique integrated tool to design specific GSTs from any public or private genome sequences and allows the user to fine-tune GST size and specificity. SPADS has been used to obtain probes for whole genome and family-wide transcript profiling, as well as inserts for gene-specific knock-out experiments. AVAILABILITY: The GENOPLANTE SPADS source code and web interface are available upon request. The online version is accessible via http://genoplante-info.infobiogen.fr/spads and via http://oberon.fvms.ugent.be:8080/SPADS/

Algorithms↗

PubMed2001

Differential chemical labeling of the AlcR DNA-binding domain from Aspergillas nidulans versus its complex with a 16-mer DNA target: identification of an essential tryptophan involved in the recognition and the interaction with the nucleic acid.

DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown to involve a consensus basic region as in the other zinc cluster proteins. However, additional interactions between some residues and DNA were suspected, among which were a hypothetic hydrophobic interaction between Trp45 and the T residue of the consensus TGCGG sequence. In the present study, the differential chemical labeling of both the free protein and the protein/DNA complex showed significantly different behaviors of the three tryptophan residues comprised in the AlcR sequence toward the Koshland reagent. The spectacular decreased reaction rate for Trp45 within the complex confirmed the location of this residue at the protein/DNA interface. A similar result obtained with Trp53, an amino acid present at the C-terminal side of AlcR, also indicated its involvement in the DNA recognition. In contrast, the formation of the complex accompanied by an allosteric rearrangement allowed the Trp36 to be much more exposed to the solvent than in the free protein. These data provide additional evidence that the unique specificity of AlcR among the zinc binuclear cluster family results in new types of interactions between AlcR and its cognate targets. From a methodological point of view, the approach of differential chemical labeling combined with mass spectrometric analyses proved to be an interesting tool for the recognition of hydrophobic interactions between the tryptophan residues of a protein and its macromolecular target.

Amino Acid Sequence↗

PubMed1990

Reconstituted skin in culture: a simple method with optimal differentiation.

Human skin is a unique organ, which can be reconstituted in vitro and represents an interesting system for studying cell proliferation and differentiation. A simple technique for producing reconstituted skin with optimal epidermal differentiation is described and characterized. A 4-mm punch biopsy of normal human skin is deposited on the epidermal side of mortified de-epidermized human dermis maintained at the air-liquid interface with a metallic support. The culture medium contains insulin, epidermal growth factor (EGF), cholera toxin, hydrocortisone, penicillin/streptomycin and fungizone. A well-differentiated epidermis develops within 15 days. Morphological and ultrastructural studies show a neoepidermis resembling normal skin. Differentiation markers such as involucrin, filaggrin, and various cytokeratins detected with pancytokeratin antibody are present and confirm this resemblance. The keratin profile is comparable to that observed in other skin culture models. A basement-membrane-like structure is reconstituted with hemidesmosomes and anchoring-filament formation. Bullous pemphigoid (BP) antigen is observed at the dermo-epidermal junction after 21 days of culture. Moreover, both dermal substrates and punch biopsies can be kept frozen for long-term storage, with little or no loss of epidermal growth kinetics and morphology. This skin culture technique is rapid, simple, economical and reproducible. Characterization has here shown high-quality epidermal differentiation. Scientists interested in epidermal in vitro studies should take interest in all these advantages.

Antibodies, Monoclonal↗

PubMed2003

Flow phase diagrams for concentration-coupled shear banding.

After surveying the experimental evidence for concentration coupling in the shear banding of wormlike micellar surfactant systems, we present flow phase diagrams spanned by shear stress Sigma (or strain rate gamma) and concentration, calculated within the two-fluid, non-local Johnson-Segalman (d-JS-phi) model. We also give results for the macroscopic flow curves Sigma(gamma,phi) for a range of (average) concentrations phi. For any concentration that is high enough to give shear banding, the flow curve shows the usual non-analytic kink at the onset of banding, followed by a coexistence "plateau" that slopes upwards, dSigma/dgamma>0. As the concentration is reduced, the width of the coexistence regime diminishes and eventually terminates at a non-equilibrium critical point [Sigmac,phic,gammac]. We outline the way in which the flow phase diagram can be reconstructed from a family of such flow curves, Sigma(gamma,phi), measured for several different values of phi. This reconstruction could be used to check new measurements of concentration differences between the coexisting bands. Our d-JS-phi model contains two different spatial gradient terms that describe the interface between the shear bands. The first is in the viscoelastic constitutive equation, with a characteristic (mesh) length l. The second is in the (generalised) Cahn-Hilliard equation, with the characteristic length xi for equilibrium concentration-fluctuations. We show that the phase diagrams (and so also the flow curves) depend on the ratio r congruent with l/xi, with loss of unique state selection at r=0. We also give results for the full shear-banded profiles, and study the divergence of the interfacial width (relative to l and xi) at the critical point.

Journal Article↗

PubMed1986

Human cytomegalovirus: development and progression of nuclear inclusions by primary clinical isolates and laboratory-adapted strains.

The morphogenesis of cytomegalovirus (CMV) nuclear inclusions (NIs) was investigated using unadapted clinical isolates and adapted laboratory strains. Both adapted and unadapted strains of CMVs induced NIs whose morphologic appearance was similar in human fibroblastic cells. Early NIs appeared as ring-like structures composed of dense granular and fibrillar material, while late NIs appeared to consist of multiple electron-lucent areas containing coarse granules and bounded by electron-dense fibrillar material (cellulae). Capsids and nucleocapsids were associated primarily with the electron-dense fibrillar material; however, developing nucleocapsids were most often observed at the interface of the electron-dense and -lucent areas. Although there was some variation in the rate of development and maturation of the NIs with the intensity of infection, all CMVs examined produced late NIs with similar organizational patterns consisting of cellulae. Substitution of human fibroblastic cells derived from various tissues as cellular substrate did not appreciably affect the results. Thus, the unique organization of the CMV late NI, consisting of multiple cellulae, appears to be an intrinsic feature of CMV replication since it seems to be independent of the extent of laboratory adaptation, the virus strain, the intensity of infection, or the cell type.

Cell Nucleus↗

PubMed1998

Influence of non-P region domains on selectivity filter properties in voltage-gated K+ channels.

The selectivity filter in voltage-gated K+ channels is formed at the interface of the pore loops (S5-S6 loop) from four channel subunits. Whereas most K+ channels are essentially impermeable to Na+, the Kv2.1 K+ channel conducts Na+ relatively well in the absence of K+ and selects for K+ over Na+ at least partially by an affinity-based competition mechanism. To examine whether the ability of Kv2.1 to conduct Na+ reflected unique properties of either its S5-S6 loop or channel domains that held the S5-S6 loop in place (the scaffolding), we studied chimeras made from Kv1.3 (which is completely impermeable to Na+) and Kv2.1. Chimeras that contained either the S5-S6 loop from Kv1.3 inserted into the Kv2.1 scaffolding or vice versa both made highly selective K+ channels that conducted Na+ and displayed competition between Na+ and K+ for conduction through the pore: In channels that contained the S5-S6 loop from Kv2.1, concentration-dependent block of Na+ current by either external or internal K+ differed depending on whether Kv2.1 or Kv1.3 donated the scaffolding. These results indicate that neither the S5-S6 loop nor the scaffolding from Kv2.1 possess unique attributes that permit Na+ to conduct through the channel. Furthermore, these results indicate that the competitive interaction between K+ and Na+ at the selectivity filter is determined not only by the S5-S6 loop but also by the scaffolding that holds the S5-S6 loop.

Cations↗

PubMedDate not supplied

Refractive index matching and clear emulsions.

Refractive index (RI) matching is a unique way of making clear emulsions to meet market trends. However, RI matching has not been sufficiently investigated in terms of physical principles and methodologies. Snell's law (n2 sin r2= n1 sin r1) is applicable to cosmetic emulsions. When oil phase and water phase have equal RI (n2 = n1) values, light will not bend as it strikes obliquely at the emulsion interface. Instead, light is transmitted through the emulsion without refraction, which produces clarity. Theoretical RI values in solution can be calculated with summation of the product of the weight percentage and refractive index of each ingredient (RI(mix) = [W1 x n1 + W2 x n2 + W3 x n3 + + Wn x nn]Wtau). Oil-phase RI values are normally at 1.4 or higher. Glycols are used to adjust the water phase RI, since they typically have larger RI values than water. Noticeable deviations from calculated RI values are seen in experimentally prepared solutions. Three basic deviation types are observed: negative, positive, and slightly negative or positive, which can occur in glycol aqueous solutions at different concentrations. The deviations are attributed to changes in molecular interaction between molecules in solution, which can lead to changes in specific gravity. Negative RI deviation corresponds to a decrease in specific gravity, and positive RI deviation corresponds to an increase in specific gravity. RI values will deviate from calculated values since an increase or decrease in specific gravity leads to a change in optical density.

Cosmetics↗

PubMed2006

Optimizing the surface plasmon resonance/mass spectrometry interface for functional proteomics applications: how to avoid and utilize nonspecific adsorption.

A great challenge in functional or interaction proteomics is to map protein networks and establish a functional relationship between expressed proteins and their effects on cellular processes. These cellular processes can be studied by characterizing binding partners to a "bait" protein against a complex background of other molecules present in cells, tissues, or biological fluids. This so-called ligand fishing process can be performed by combining surface plasmon resonance biosensors with MS. This combination generates a unique and automated method to quantify and characterize biomolecular interactions, and identify the interaction partners. A general problem in chip-based affinity separation systems is the large surface-to-volume ratio of the fluidic system. Extreme care, therefore, is required to avoid nonspecific adsorption, resulting in losses of the target protein and carry-over during the affinity purification process, which may lead to unwanted signals in the final MS analysis and a reduction in sensitivity. In this study, carry-over of protein and low-molecular weight substances has been investigated systematically and cleaning strategies are presented. Furthermore, it is demonstrated that by the introduction of colloidal particles as a capturing and transporting agent, the recovery yield of the affinity-purified ligand could be improved nearly twofold.

Adsorption↗