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PubMed2002

Quantitation of trace components in liquid process streams by direct liquid sampling mass spectrometry.

This paper describes the development of a liquid sampling approach for trace analysis by electron impact ionization magnetic sector mass spectrometry, with no chromatographic separation. Development of a liquid sample introduction interface based on the principle of Programmable Temperature Vaporizing (PTV) GC injection is shown. A univariate procedure for the analysis of trace (mg kg(-1)) propanoic acid in acetic acid was developed. Results from the laboratory-based analysis of acetic acid are presented and compared with conventional GC analysis. The detection limit was 16 mg l(-1) and the speed of analysis was employed to acquire 30 scans per minute thus reducing the confidence intervals of the results and potentially allowing production plants to run much closer to sales specifications. For the analysis of more complex samples where the analytes contained no unique ions, multivariate analysis was employed and up to three scans per minute were acquired. Results from the analysis of an ester for six trace impurities are shown. Calibration was by partial least squares regression. The detection limits for these components were 20-30 mg kg(-1), well within the required product specifications. The system proved to be robust and easy to operate, with analyses being carried out over a period of several months requiring no maintenance of the spectrometer and only cleaning of the injection liner of the PTV injector on a monthly basis.

Journal Article↗

PubMed2000

The ionization of a buried glutamic acid is thermodynamically linked to the stability of Leishmania mexicana triose phosphate isomerase.

The amino acid sequence of Leishmania mexicana triose phosphate isomerase is unique in having at position 65 a glutamic acid instead of a glutamine. The stability properties of LmTIM and the E65Q mutant were investigated by pH and guanidinium chloride-induced unfolding. The crystal structure of E65Q was determined. Three important observations were made: (a) there are no structural rearrangements as the result of the substitution; (b) the mutant is more stable than the wild-type; and (c) the stability of the wild-type enzyme shows strong pH dependence, which can be attributed to the ionization of Glu65. Burying of the Glu65 side chain in the uncharged environment of the dimer interface results in a shift in pKa of more than 3 units. The pH-dependent decrease in overall stability is due to weakening of the monomer-monomer interactions (in the dimer). The E65Q substitution causes an increase in stability as the result of the formation of an additional hydrogen bond in each subunit (DeltaDeltaG degrees of 2 kcal.mol-1 per monomer) and the elimination of a charged group in the dimer interface (DeltaDeltaG degrees of at least 9 kcal.mol-1 per dimer). The computated shift in pKa and the stability of the dimer calculated from the charge distribution in the protein structure agree closely with the experimental results. The guanidinium chloride dependence of the unfolding constant was smaller than expected from studies involving monomeric model proteins. No intermediates could be identified in the unfolding equilibrium by combining fluorescence and CD measurements. Study of a stable monomeric triose phosphate isomerase variant confirmed that the phenomenon persists in the monomer.

Animals↗

PubMed2000

Two tricks in one bundle: helix-turn-helix gains enzymatic activity.

Many examples of enzymes that have lost their catalytic activity and perform other biological functions are known. The opposite situation is rare. A previously unnoticed structural similarity between the lambda integrase family (Int) proteins and the AraC family of transcriptional activators implies that the Int family evolved by duplication of an ancient DNA-binding homeodomain-like module, which acquired enzymatic activity. The two helix-turn-helix (HTH) motifs in Int proteins incorporate catalytic residues and participate in DNA binding. The active site of Int proteins, which include the type IB topoisomerases, is formed at the domain interface and the catalytic tyrosine residue is located in the second helix of the C-terminal HTH motif. Structural analysis of other 'tyrosine' DNA-breaking/rejoining enzymes with similar enzyme mechanisms, namely prokaryotic topoisomerase I, topoisomerase II and archaeal topoisomerase VI, reveals that the catalytic tyrosine is placed in a HTH domain as well. Surprisingly, the location of this tyrosine residue in the structure is not conserved, suggesting independent, parallel evolution leading to the same catalytic function by homologous HTH domains. The 'tyrosine' recombinases give a rare example of enzymes that evolved from ancient DNA-binding modules and present a unique case for homologous enzymatic domains with similar catalytic mechanisms but different locations of catalytic residues, which are placed at non-homologous sites.

Amino Acid Sequence↗

PubMed2002

A novel heterodimerization domain, CRM1, and 14-3-3 control subcellular localization of the MondoA-Mlx heterocomplex.

Among members of the bHLHZip family of transcriptional regulators, MondoA and Mlx have the unique property of cytoplasmic localization. We have proposed that MondoA-Mlx heterodimers accumulate in the nucleus in response to extracellular cues. Our previous work implicated heterodimerization between MondoA and Mlx and a conserved domain in the N terminus of MondoA as important determinants of MondoA-Mlx subcellular localization. MondoA and Mlx share sequence similarity in their bHLHZip domains and C termini. Here we show that for both MondoA and Mlx, this C-terminal domain has cytoplasmic localization activity that is required by the protein monomers to accumulate in the cytoplasm. This C-terminal domain is also a novel dimerization interface that functions independently of the leucine zipper to mediate heterotypic interactions between MondoA and Mlx. Dimerization between MondoA and Mlx inactivates the cytoplasmic localization activity of their C termini and is necessary for the heterocomplex to accumulate in the nucleus. MondoA-Mlx heterodimers, while poised for nuclear entry, are retained in the cytoplasm by conserved domains in the N terminus of MondoA. Mondo conserved regions (MCRs) II and III contribute to cytoplasmic localization of MondoA-Mlx by functioning as a CRM1-dependent nuclear export signal and as a novel binding site for 14-3-3 family members, respectively. We propose that the nuclear accumulation of MondoA and Mlx is a two-step process. First, heterodimerization abolishes the cytoplasmic localization activity of their C termini. Second, an extracellular signal(s) must overcome the cytoplasmic localization function imparted by CRM1 and 14-3-3 binding to the N terminus of MondoA.

14-3-3 Proteins↗

PubMed1993

Otolaryngic allergy.

The treatment of chronic sinusitis and nasal polyposis requires an interface between allergy and otolaryngology. The tendency of allergy to produce chronic symptoms from repeated exposure to inhalant or food antigens plays a role in both the production and chronicity of sinus disease. Mild, seasonal, or easily avoidable allergens may be well treated by proper selection of antihistamines, cromolyn sodium, topical steroids, or the judicious use of systemic steroids, as well as environmental control. More severe or perennial symptoms require identification by in vivo or in vitro testing techniques, with desensitization for offending inhalant allergens and dietary restrictions or injection therapy for food antigens. The otolaryngologist dealing with sinus disease is in a unique position to offer optimal patient care by combining surgical skills with specific medical treatment of allergy.

Adrenal Cortex Hormones↗

PubMed1995

Cat 6 mo increases symptoms: online physician charting and more.

The physicians of the Children's Asthma Center and the computer scientists of Information Services at Texas Children's Hospital set out to design a system that is comfortable to use, structured enough to effectively measure outcomes, yet flexible enough to conserve the individuality of the patient. To achieve these goals, we examined how the differential diagnosis process is applied to clinical decision making and implemented it in a clinical workstation. Unique patterns representing the state of the patient's disease are formed by dynamically selecting pertinent sets of observations, assigning attributes to these observations, and describing relationships between observations and/or sets of observations.

Asthma↗

PubMed2004

Novel structure and nucleotide binding properties of HI1480 from Haemophilus influenzae: a protein with no known sequence homologues.

The crystal structure of the Haemophilus influenzae protein HI1480 was determined at 2.1-A resolution. The amino acid sequence of HI1480 is unique, having no homology with other known protein sequences. The protein adopts a novel alpha+beta fold, and associates into a dimer of tightly associated dimers. The tight dimers are formed by intermolecular interactions that are mediated by an antiparallel beta-barrel involving both monomers. Helical regions of two dimers mediate the tetramer formation. The helical region contains a four-helix bundle that has been seen only in the anticodon binding domains of class I tRNA synthetases. A cluster of four residues, Tyr18, Arg134, Glu26, and Lys12 is located in a depression formed at the four-helix bundle/ beta-barrel interface. The arrangement is suggestive of an active center, possibly a catalytic site. The HI1480 gene is located within the Mu-like prophage region of H. influenzae, has no homology to bacteriophage genes, and is flanked by transposases. Hence, this is an example of horizontal transfer from an unknown organism. Gel mobility shift assays revealed that HI1480 binds DNA and RNA molecules. Double-stranded DNA is favored over single-stranded DNA, and longer DNA molecules are bound better than shorter ones.

Amino Acid Sequence↗

PubMed1999

The structure of pyruvate kinase from Leishmania mexicana reveals details of the allosteric transition and unusual effector specificity.

Glycolysis occupies a central role in cellular metabolism, and is of particular importance for the catabolic production of ATP in protozoan parasites such as Leishmania and Trypanosoma. In these organisms pyruvate kinase plays a key regulatory role, and is unique in responding to fructose 2,6-bisphosphate as allosteric activator. The determination of the first eukaryotic pyruvate kinase crystal structure in the T-state is reported. A comparison of the leishmania and yeast R-state enzymes reveals fewer differences than the previous comparison of Escherichia coli T-state and rabbit muscle non-allosteric enzymes. Structural changes related to the allosteric transition can therefore be distinguished from those that are a consequence of the inherent wide structural divergence between bacterial and mammalian proteins. The allosteric transition involves significant changes in a tightly packed array of eight alpha helices at the interface near the catalytic site. At the other interface the allosteric transition appears to be accompanied by the bending of a ten-stranded intersubunit beta sheet adjacent to the effector site. Helix Calpha1 makes contacts to the N-terminal helical domain and bridges both interfaces. A comparison of the effector sites of the leishmania and yeast enzymes reveals the structural basis for the different effector specificity. Two loops comprising residues 443-453 and 480-489 adopt very different conformations in the two enzymes, and Lys453 and His480 that are a feature of trypanosomatid enzymes provide probable ligands for the 2-phospho group of the effector molecule. These differences offer an opportunity for the design of drugs that would bind to the trypanosomatid enzymes but not to those of the mammalian host.

Allosteric Regulation↗

PubMed2006

Facilitated transfer of alkali-metal cations by dibenzo-18-crown-6 across the electrochemically polarized interface between an aqueous solution and a hydrophobic room-temperature ionic liquid.

The facilitated transfer of alkali metal cations (Li+, Na+, K+, Rb+, Cs+) by dibenzo-18-crown-6 (DB18C6) across the electrochemically polarizable interface between an aqueous solution (W) and a hydrophobic ionic liquid, N-octadecylisoquinolinium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate ([C18Iq][TFPB]), has been studied using cyclic voltammetry at the interface formed at the tip of a micropipet. In cyclic voltammograms (CVs), the current due to the facilitated transfer of the cations by DB18C6 from W to [C18Iq][TFPB] can be measured within the polarized potential window of the [C(18)Iq][TFPB]|W interface. The stoichiometry of the complexes in [C18Iq][TFPB] for Li+, Na+, K+, and Rb+ are found to be 1:1 while for the Cs+ transfer both 1:1 and 1:2 complexes are likely to be formed. The formation constants of the 1:1 complexes for Li+, Na+, K+, and Rb+ in [C18Iq][TFPB], , evaluated from CVs are log = 5.0, 7.0, 8.2, and 7.3, respectively. The value for K+ is 1 order of magnitude greater than that for Na+. This higher selectivity of DB18C6 to K+ over Na+ in [C18Iq][TFPB] compared with that in molecular solvents suggests that the RTIL provides a unique solvation environment for the complexations of DB18C6 with the ions.

Journal Article↗

PubMed1997

One residency's experience with the electronic residency application service.

For medical students and residency programs alike, the residency application process is time-consuming. This paper examines one program's experience with a computerized system designed by the Association of American Medical Colleges (AAMC) to simplify and standardize the filing and receipt of applications over the Internet. A large-scale pilot test of the Electronic Residency Application Service (ERAS) was implemented in 1995-1996 for applicants to first-year residency positions in OB-GYN. Each student completed the computerized application, which was transmitted, along with other documents, to student-specified programs by the dean's office via the AAMC "electronic post office." ERAS will be extended to family practice residencies in 1997-1998. A major advantage of ERAS to residency programs is that materials were received in a well-organized, complete, and consistent format. Built-in filters allow grouping of applicants according to various criteria. Opening envelopes and filing documents is no longer necessary. Each student completes one application, and faculty write one letter of recommendation per student. Disadvantages of the 1995-1996 system related to the software, which had an inflexible interface and did not allow a spreadsheet view of the database. Personal statements and recommendation letters were often sent as unformatted ASCII text and were difficult to read. Dean's offices reported problems scanning documents such as transcripts and photographs. These problems led to resubmission of materials and receipt of duplicate copies. With the standard application format, ASCII-style personal statements and "generic" recommendation letters caused applicants to lose individuality. Specific recommendations to the AAMC for improving ERAS include providing a spreadsheet view, allowing students and faculty to write personal statements and letters in standard word processing formats, and allowing faculty to send unique letters to specific residency programs.

Computer Communication Networks↗

PubMed1986

Unique characteristics of local responses in host resistance to mucosal parasitic infections.

Because of the tremendous impact that parasitic infections have on the health and productivity of humans and domestic animals, considerable research effort has been focused upon understanding the mechanisms of host-parasite coexistence, host resistance and immunopathology. Studies have employed a range of approaches including: kinetic analysis of parasite establishment, development, fecundity and survival in naive and previously-infected hosts; correlation between parasite survival and histopathologic responses at the site of infection; vaccination with attenuated parasites or their products; cellular and serum transfer of immunity to naive or immunocompromised hosts; pharmacologic manipulation of potential mediators of host defense using agonistic and antagonistic drugs. However, it is becoming increasingly clear that to understand the mechanisms associated with host resistance and parasite survival, one must define the characteristics of the local microenvironment at the host-parasite interface. One of the approaches by which such studies can be made involves the isolation and characterization of cells derived from the local infection site. This manuscript reviews some of these studies on local aspects of mucosal immune responses in parasitic infections. Examples that will be discussed include IgA antibody, intraepithelial leukocytes from the intestine, intestinal mast cell populations, macrophages derived from bronchoalveolar lavage, and local immunoregulatory responses during respiratory and intestinal parasitic infection. These studies have established unequivocally that local responses to mucosal parasitic infection can only be appropriately investigated using cells derived from the specific microenvironment. This conclusion should encourage others to further study these local responses and to be innovative in investigating unexplored aspects of the host-parasite interface.

Animals↗

PubMed1994

The thermodynamic and hydrodynamic properties of macromolecules that influence the hydrodynamics of porous systems.

The water flow across porous, semipermeable membranes associated with osmosis and filtration under a variety of conditions is analysed and compared to macromolecular diffusion across free-liquid boundaries, diffusion and sedimentation in the ultracentrifuge, and tracer diffusion of water. This study establishes that osmosis can be explained in terms of the irreversible thermodynamics of diffusion. For macromolecular osmotically active solutes in the semidilute concentration regime the water flows across semipermeable porous membranes are interpreted in terms of a rate-limiting solute-solvent exchange layer that exists on the solution side of the membrane adjacent to the membrane pore; both osmosis and filtration will be governed by these exchange layers. These exchange layers also yield unique properties of their constituent molecules in systems where there is osmotic equilibration between solutions of different solutes. This study also establishes the need to consider the internal osmotic pressure of membranes in the pressure balance associated with the flow across the membrane. The complex situation of partially permeable membranes is analysed for the simple case where there are no mechanical gradients and there is only one osmotically active solution that creates a rate-limiting exchange layer. This treatment predicts that the flow will be governed primarily by the osmotic pressure difference associated with the partitioning of the solute at the membrane-solution interface.

Animals↗

PubMed2000

A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL.

Apoptosis-inducing ligand 2 (Apo2L, also called TRAIL), a member of the tumor necrosis factor (TNF) family, induces apoptosis in a variety of human tumor cell lines but not in normal cells [Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C.-P., Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995) Immunity 3, 673-682; Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Ashkenazi, A. (1996) J. Biol. Chem. 271, 12687-12690]. Here we describe the structure of Apo2L at 1.3 A resolution and use alanine-scanning mutagenesis to map the receptor contact regions. The structure reveals a homotrimeric protein that resembles TNF with receptor-binding epitopes at the interface between monomers. A zinc ion is buried at the trimer interface, coordinated by the single cysteine residue of each monomer. The zinc ion is required for maintaining the native structure and stability and, hence, the biological activity of Apo2L. This is the first example of metal-dependent oligomerization and function of a cytokine.

Alanine↗

PubMed1999

Crystal structure of the CD2-binding domain of CD58 (lymphocyte function-associated antigen 3) at 1.8-A resolution.

The binding of the cell surface molecule CD58 (formerly lymphocyte function-associated antigen 3) to its ligand, CD2, significantly increases the sensitivity of antigen recognition by T cells. This was the first heterophilic cell adhesion interaction to be discovered and is now an important paradigm for analyzing the structural basis of cell-cell recognition. The crystal structure of a CD2-binding chimeric form of CD58, solved to 1.8-A resolution, reveals that the ligand binding domain of CD58 has the expected Ig superfamily V-set topology and shares several of the hitherto unique structural features of CD2, consistent with previous speculation that the genes encoding these molecules arose via duplication of a common precursor. Nevertheless, evidence for considerable divergence of CD2 and CD58 is also implicit in the structures. Mutations that disrupt CD2 binding map to the highly acidic surface of the AGFCC'C" beta-sheet of CD58, which, unexpectedly, lacks marked shape complementarity to the equivalent, rather more basic CD58-binding face of human CD2. The specificity of the very weak interactions of proteins mediating cell-cell recognition may often derive largely from electrostatic complementarity, with shape matching at the protein-protein interface being less exact than for interactions that combine specificity with high affinity, such as those involving antibodies.

Amino Acid Sequence↗

PubMed1986

Isolation and Characterization of Tonoplast from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L.

Tonoplasts were isolated in a high purity from etiolated young seedlings of Vigna radiata L. (mung bean) utilizing a sucrose density gradient system. The excised hypocotyls were homogenized in a sorbitol-buffer system and the 3,600 to 156,000g pellets obtained after the differential centrifugations were suspended in a sorbitol medium and loaded on a linear sucrose density gradient. After centrifugation at 89,000g for 2 hours, tonoplasts were banded at the sample load/sucrose interface. Assessed by electron microscopy and marker enzymes, the purity and the quantity were found to be sufficient for biochemical and biophysical analyses. The tonoplasts were associated with NO(3) (-)-sensitive and vana-date-insensitive ATPase. The tonoplast ATPase was stimulated by proton ionophores such as carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone and gramicidin D, suggesting a proton-pumping enzyme. In the presence of ATP and Mg(2+), a proton gradient was formed in the isolated tonoplast vesicles as assessed by fluorescence quenching of quinacrine. The tonoplasts contained several kinds of mannosylated or glycosylated glycoproteins and a major protein (65 kilodaltons) which was unique to the membranes.

Journal Article↗

PubMedDate not supplied

Advances in nutrition and gastroenterology: summary of the 1997 A.S.P.E.N. Research Workshop.

BACKGROUND: The 1997 A.S.P.E.N. Research Workshop was held at the annual meeting in San Francisco, on January 26, 1997. The workshop focused on advances in clinical and basic research involving the interface between nutrient and luminal gastroenterology. METHODS: Presentations on the genetic regulation of gastrointestinal development, the molecular biology of small intestinal adaptation, the effect of nutrition support on intestinal mucosal mass, the relationship between nutrition and gastrointestinal motility, nutrient absorption, and gastrointestinal tract substrate metabolism were made by the preeminent leaders in the field. RESULTS: The investigators presented an insightful analysis of each topic by reviewing data from their own laboratories and the published literature. CONCLUSIONS: This workshop underscored the important interactions between nutrition and luminal gastroenterology at the basic science, metabolic/physiologic, and clinical levels. The integration of presentations from the different disciplines provided a unique interaction of information and ideas to advance our understanding of nutrition and gastrointestinal tract.

Digestive System Physiological Phenomena↗

PubMed2005

Computational identification of strain-, species- and genus-specific proteins.

BACKGROUND: The identification of unique proteins at different taxonomic levels has both scientific and practical value. Strain-, species- and genus-specific proteins can provide insight into the criteria that define an organism and its relationship with close relatives. Such proteins can also serve as taxon-specific diagnostic targets. DESCRIPTION: A pipeline using a combination of computational and manual analyses of BLAST results was developed to identify strain-, species-, and genus-specific proteins and to catalog the closest sequenced relative for each protein in a proteome. Proteins encoded by a given strain are preliminarily considered to be unique if BLAST, using a comprehensive protein database, fails to retrieve (with an e-value better than 0.001) any protein not encoded by the query strain, species or genus (for strain-, species- and genus-specific proteins respectively), or if BLAST, using the best hit as the query (reverse BLAST), does not retrieve the initial query protein. Results are manually inspected for homology if the initial query is retrieved in the reverse BLAST but is not the best hit. Sequences unlikely to retrieve homologs using the default BLOSUM62 matrix (usually short sequences) are re-tested using the PAM30 matrix, thereby increasing the number of retrieved homologs and increasing the stringency of the search for unique proteins. The above protocol was used to examine several food- and water-borne pathogens. We find that the reverse BLAST step filters out about 22% of proteins with homologs that would otherwise be considered unique at the genus and species levels. Analysis of the annotations of unique proteins reveals that many are remnants of prophage proteins, or may be involved in virulence. The data generated from this study can be accessed and further evaluated from the CUPID (Core and Unique Protein Identification) system web site (updated semi-annually) at http://pir.georgetown.edu/cupid. CONCLUSION: CUPID provides a set of proteins specific to a genus, species or a strain, and identifies the most closely related organism.

Algorithms↗

PubMed2001

Update for nurse anesthetists. Part 6. Full-body patient simulation technology: gaining experience using a malignant hyperthermia model.

Simulation technology is extremely useful for training workers in a variety of industries. Simulation concepts have been developed and refined in full-body patient simulators. Contemporary full-body patient simulators can be used to teach the essential concepts of many healthcare professions, and they offer practice dealing with critical events, concentrating on the thought and decision-making processes related to favorable patient outcomes. Full-body patient simulators provide practitioners the opportunity to manage realistic and advanced physical findings in a variety of areas and are capable of responding to a large number of pharmacologic agents and a host of clinical interventions. The ability to combine high-fidelity educational tools with both classroom lecture and clinical experience provides nurse anesthetists the opportunity for a unique experience in a highly controlled and safe environment. Numerous technological and educational advantages and the chance for hands-on assessment and treatment of some of anesthesia's rarest and most life-threatening events provide a one-of-a-kind educational session for CRNAs.

Computer-Assisted Instruction↗