Ion adsorption at the rutile-water interface: linking molecular and macroscopic properties.

A comprehensive picture of the interface between aqueous solutions and the (110) surface of rutile (alpha-TiO2) is being developed by combining molecular-scale and macroscopic approaches, including experimental measurements, quantum calculations, molecular simulations, and Gouy-Chapman-Stern models. In situ X-ray reflectivity and X-ray standing-wave measurements are used to define the atomic arrangement of adsorbed ions, the coordination of interfacial water molecules, and substrate surface termination and structure. Ab initio calculations and molecular dynamics simulations, validated through direct comparison with the X-ray results, are used to predict ion distributions not measured experimentally. Potentiometric titration and ion adsorption results for rutile powders having predominant (110) surface expression provide macroscopic constraints of electrical double layer (EDL) properties (e.g., proton release) which are evaluated by comparison with a three-layer EDL model including surface oxygen proton affinities calculated using ab initio bond lengths and partial charges. These results allow a direct correlation of the three-dimensional, crystallographically controlled arrangements of various species (H2O, Na+, Rb+, Ca2+, Sr2+, Zn2+, Y3+, Nd3+) with macroscopic observables (H+ release, metal uptake, zeta potential) and thermodynamic/electrostatic constraints. All cations are found to be adsorbed as "inner sphere" species bonded directly to surface oxygen atoms, while the specific binding geometries and reaction stoichiometries are dependent on ionic radius. Ternary surface complexes of sorbed cations with electrolyte anions are not observed. Finally, surface oxygen proton affinities computed using the MUSIC model are improved by incorporation of ab initio bond lengths and hydrogen bonding information derived from MD simulations. This multitechnique and multiscale approach demonstrates the compatibility of bond-valence models of surface oxygen proton affinities and Stern-based models of the EDL structure, with the actual molecular interfacial distributions observed experimentally, revealing new insight into EDL properties including specific binding sites and hydration states of sorbed ions, interfacial solvent properties (structure, diffusivity, dielectric constant), surface protonation and hydrolysis, and the effect of solution ionic strength.

Dual analyte flow injection fluorescence immunoassays using thiophilic gel reactors and synchronous scanning detection.

Heterogeneous fluorescence immunoassays have been automated using flow injection manifolds incorporating thiophilic gel solid phase reactors to separate antibody-bound and unbound analyte molecules. Antibody elution is achieved by changes in ionic strength, thus allowing the use of pH sensitive fluorescent labels. This facilitates the development of dual analyte systems, in which two competitive immunoassays with separate labels are monitored in parallel. Detection of the fluorophores by high speed synchronous fluorescence scanning while the flow is briefly stopped utilises either one synchronous interval which detects both fluorophores, or two separate scans at different wavelength intervals, one for each fluorophore. Simultaneous analyses of serum albumin and transferrin exemplify these novel approaches. Spectroscopic interferences are very small, analyte recoveries are close to 100%, with a relative standard deviation of 5-6% and a sampling rate of 20 h-1.

Field evaluation of the Determine rapid human immunodeficiency virus diagnostic test in Honduras and the Dominican Republic.

Rapid detection of human immunodeficiency virus (HIV) infection can result in improved patient care and/or faster implementation of public health preventive measures. A new rapid test, Determine (Abbott, Abbott Park, Ill.), detects HIV type 1 (HIV-1) and HIV-2 antibodies within 15 min by using 50 microl of serum or plasma. No specialized equipment or ancillary supplies are required, and results are read visually. A positive result is noted by the appearance of a red line. An operational control (red line) indicates proper test performance. We evaluated the Determine rapid HIV detection test with a group of well-characterized serum samples (CD4 counts and viral loads were known) and serum samples from HIV-positive individuals at field sites in Honduras and the Dominican Republic. In the field evaluations, the results obtained by the Determine assay were compared to those obtained by local in-country HIV screening procedures. We evaluated serum from 100 HIV-positive patients and 66 HIV-negative patients. All samples gave the expected results. In a companion study, 42 HIV-positive samples from a Miami, Fla., serum bank were tested by the Determine assay. The samples had been characterized in terms of CD4 counts and viral loads. Fifteen patients had CD4 counts <200 cells/mm(3), while 27 patients had CD4 counts >200 cells/mm(3). Viral loads ranged from 630 to 873,746 log(10) copies/ml. All samples from the Miami serum bank were positive by the Determine test. Combined results from the multicenter studies indicated that the correct results were obtained by the Determine assay for 100% (142 of 142) of the HIV-positive serum samples and 100% (66 of 66) of the HIV-negative serum samples. The Determine test was simple to perform and the results were easy to interpret. The Determine test provides a valuable new method for the rapid identification of HIV-positive individuals, especially in developing countries with limited laboratory infrastructures.

Biosynthesis and regulation of coronatine, a non-host-specific phytotoxin produced by Pseudomonas syringae.

Many P. syringae pathovars are known to produce low-molecular-weight, diffusible toxins in infected host plants. These phytotoxins reproduce some of the symptoms of the relevant bacterial disease and are effective at very low concentrations. Phytotoxins generally enhance the virulence of the P. syringae pathovar which produces them, but are not required for pathogenesis. Genes encoding phytotoxin production have been identified and cloned from several P. syringae pathovars. With the exception of coronatine, toxin biosynthetic gene clusters are generally chromosomally encoded. In several pathovars, the toxin biosynthetic gene cluster also contains a resistance gene which functions to protect the producing strain from the biocidal effects of the toxin. In the case of phaseolotoxin, a resistance gene (argK) has been utilized to engineer phaseolotoxin-resistant tobacco plants. Although P. syringae phytotoxins can induce very similar effects in plants (chlorosis and necrosis), their biosynthesis and mode of action can be quite different. Knowledge of the biosynthetic pathways to these toxins and the cloning of the structural genes for their biosynthesis has relevance to the development of new bioactive compounds with altered specificity. For example, polyketides constitute a huge family of structurally diverse natural products including antibiotics, chemotherapeutic compounds, and antiparasitics. Most of the research on polyketide synthesis in bacteria has focused on compounds synthesized by Streptomyces or other actinomycetes. It is also important to note that it is now possible to utilize a genetic rather than synthetic approach to biosynthesize novel polyketides with altered biological properties (Hutchinson and Fujii, 1995; Kao et al., 1994; Donadio et al., 1993; Katz and Donadio, 1993). Most of the reprogramming or engineering of novel polyketides has been done using actinomycete PKSs, but much of this technology could also be applied to polyketides synthesized by Pseudomonas when sufficient sequence information is available. It is important to note that Pseudomonas produces a variety of antimicrobial compounds from the polyketide pathway, including mupirocin (pseudomonic acid) (Feline et al., 1977), pyoluteorin (Cuppels et al., 1986), and 2-4 diacetylphloroglucinol (Phl) (Bangera and Thomashow, 1996). Pseudomonic acid is valued for its pharmaceutical properties as an antibiotic (Aldridge, 1992), whereas pyoluteorin and Phl have antifungal properties (Howell and Stipanovic, 1980; Keel et al., 1992). A thorough understanding of the biosynthetic pathway to polyketide phytotoxins such as coronatine may ultimately lead to the development of novel compounds with altered biological properties. Thus, specific genes in the biosynthetic pathways of P. syringae phytotoxins could be deployed in other systems to develop new compounds with a wide range of activities.

The use of polymeric liquid enteral diets for nutritional support in seriously ill or injured small animals: clinical results in 200 patients.

This prospective, multicenter study evaluated the use of four polymeric liquid enteral (PLE) diets manufactured for dogs and cats in 200 ill or injured patients. Polymeric liquid enteral diets were administered by free-choice feeding, syringe, or feeding tube for up to 208 days. Overall results indicated a 4.9% incidence of vomiting in dogs and a 7.9% incidence in cats; an 8.9% incidence of diarrhea in dogs and an 18.4% incidence in cats. Patients fed the PLE diets seven days or longer had an average increase in body weight of 1.4% in dogs, an average decrease in body weight of 3.8% in cats, increases in lymphocyte counts, and mild decreases in serum albumin.

Parents' and physicians' views on antibiotics.

OBJECTIVE: To describe parents' opinions and concerns about antibiotics and to contrast these opinions with those of pediatricians. DESIGN: Parents were surveyed using an interviewer-administered questionnaire and pediatricians were mailed a self-administered questionnaire. RESULTS: Parents from two private practices (N = 300) were largely white (84%) and had completed college (81%). The parents from a community health center (N = 100) were mostly black (80%) and had not completed college (91%). Twenty-nine percent of parents were worried that their children were receiving too many antibiotics. Eighty-five percent believed there were problems with receiving too many antibiotics, with 55% mentioning resistance or immunity as concerns. Eighteen percent of parents had given their child an antibiotic at home before consulting a physician. Parents believed that antibiotics were always or sometimes required for ear infections (93%), throat infections (83%), colds (32%), cough (58%), and fever (58%). Fourteen percent of parents believed that their child had required an antibiotic when the doctor did not prescribe one, with clinic parents significantly more likely to report this issue (22%) than private practice parents (12%). Nine percent believed that their doctor had prescribed an antibiotic unnecessarily (private practice = 12%, community health center = 3%). Parents from the private practices were also more likely to report requesting a specific antibiotic (34%) in comparison with 19% of clinic parents. Sixty-one percent of the physician surveys were returned after two mailings and a follow-up phone call. The pediatricians had been in practice for a median of 12 years, seeing a median of 110 patients per week. Fifty-eight percent of pediatricians reported that some, many, or most of the parents in their practices were worried that their children were receiving too many antibiotics. Seventy-one percent indicated that four or more times during the previous month, a parent had requested an antibiotic when the physician believed it was unnecessary, and 35% said that at least occasionally they went along with these requests. Sixty-one percent reported that parents requested a different antibiotic from the one they were going to prescribe at least four times in the previous month, and 30% of pediatricians said that they agreed to parents' requests often or most of the time. CONCLUSIONS: Both the parent and the physician surveys suggest that parents are concerned about the overuse of antibiotics, but often request them when their physicians believe they are unnecessary. Parents often administer antibiotics without physician knowledge, and many parents have misconceptions about which illnesses warrant antibiotic therapy. Understanding parents' concerns and beliefs about antibiotics and the range of physician practice styles with respect to antibiotics may direct the development of intervention strategies to reduce the inappropriate use of oral antibiotics.

Use of Tn5-gusA5 to investigate environmental and nutritional effects on gene expression in the coronatine biosynthetic gene cluster of Pseudomonas syringae pv. glycinea.

Pseudomonas syringae pv. glycinea PG4180 produces coronatine (COR), a chlorosis-inducing phytotoxin that consists of the polyketide coronafacic acid (CFA) coupled via an amide bond to the ethylcyclopropyl amino acid coronamic acid (CMA). Both CFA and CMA function as intermediates in the pathway to coronatine, and genes encoding their synthesis have been localized: however, the precise factors that regulate the production of COR and its precursors remain unclear. In the present study, a lambda delivery system for Tn5-gusA5 was developed and used to obtain transcriptional fusions in the COR gene cluster. Selected carbon (fructose and xylose) and amino acid (isoleucine and valine) sources significantly decreased COR biosynthesis at the transcriptional level. Transcriptional activity in the COR gene cluster was temperature dependent with maximal expression at 18-24 degrees C and significantly less expression at 14 and 30 degrees C. Interestingly, changes in osmolarity and the addition of complex carbon and nitrogen sources to the growth medium did not significantly affect COR gene expression, although both factors significantly impacted the quantity of COR produced. These results indicate that multiple factors impact COR production and only some of these directly affect transcription in the COR gene cluster.

Neoplastic transformation of mouse C3H10T1/2 cells following exposure to neutrons does not involve mutation of ras gene as analyzed by SSCP and cycle sequencing.

About 25% of human tumors contain a mutated member of the ras gene family. Neutron exposure is an occupational risk in several work places and while we know that cells exposed to neutrons can become transformed, the molecular basis of this process is not understood. To determine whether neutron-induced cellular transformation involves ras mutation, C3H10T1/2 cells were exposed to a single dose of 5.9 MeV neutrons. Type II and type III foci were isolated and established as cell lines. A total of 34 foci were selected and expanded for analysis of tumorigenicity, chromosomal aberrations and mutations in members of the ras gene family. The presence of mutations in genomic DNA in N-ras or K-ras of each focus was examined by either single-strand conformational polymorphism (SSCP) analysis or by asymmetric PCR coupled cell cycle sequence analysis. Although chromosomal aberrations were detected at metaphase, no alterations in either ras gene were detected. We conclude that in vitro neutron-induced transformation must occur through a mechanism other than ras mutation.

Sixth and eighth graders and acquired immunodeficiency syndrome: the results of focus group analysis.

OBJECTIVE: To determine young adolescents' range of factual knowledge and beliefs about human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) among those who have received AIDS education. DESIGN: Focus group analysis. SETTING: Urban middle school. PARTICIPANTS: Two male and two female groups consisting of 4-7 students each, ages 11-15 years. RESULTS: The predominant responses in all four groups relating to factual knowledge of HIV transmission and mechanisms of prevention were correct. However, responses indicated that factual information had not been integrated into students' plans for situations involving relationships and sexual activity. All four groups shared the image of AIDS as a disease of adults. The girls frequently gave responses which included discussion and use of condoms, whereas only a few boys could realistically visualize using condoms. Many students, predominantly boys, identified with media figures such as Magic Johnson and saw themselves to be at risk for HIV, but the girls almost exclusively saw Magic Johnson as a rich, famous person and did not identify with him. Students gave suggestions about ways to improve AIDS education and recommended that AIDS education begin in the early grades. CONCLUSION: Standard AIDS education may be effective in teaching factual information about AIDS, but it may have little effect on students' future behavior. The format of AIDS education may need to be modified to better address adolescents' beliefs and behaviors regarding HIV and AIDS.

Characterization and transcriptional analysis of the gene cluster for coronafacic acid, the polyketide component of the phytotoxin coronatine.

Coronafacic acid (CFA), the polyketide component of the phytotoxin coronatine (COR), is activated and coupled to coronamic acid via amide bond formation, a biosynthetic step presumably catalyzed by the CFA ligase (cfl) gene product. The COR biosynthetic gene cluster in Pseudomonas syringae pv. glycinea PG4180 is located within a 32-kb region of a 90-kb plasmid designated p4180A. In the present study, a cloned region of p4180A complemented all CFA- mutants spanning an 18.8-kb region of the COR biosynthetic cluster. The genetic evidence presented in this study indicates that cfl and the CFA biosynthetic gene cluster are encoded by a single transcript and that transcription of all of the genes in this operon is directed by the cfl promoter. The cfl promoter was localized to a 0.37-kb region upstream of the transcriptional start site by progressive subcloning in pRG960sd, a vector containing a promoterless glucuronidase gene. Transcription of the cfl/CFA operon was temperature sensitive and showed maximal glucuronidase activity at 18 degrees C. Furthermore, transcription of the cfl/CFA operon was dependent on the functional activity of a modified two-component regulatory system located within the COR biosynthetic gene cluster. Thermoregulation of the cfl/CFA operon and the coronamic acid biosynthetic gene cluster via the modified two-component regulatory system is discussed.

Copper as a signal for alginate synthesis in Pseudomonas syringae pv. syringae.

Plant-associated pseudomonads are commonly exposed to copper bactericides, which are applied to reduce the disease incidence caused by these bacteria. Consequently, many of these bacteria have acquired resistance or tolerance to copper salts. We recently conducted a survey of 37 copper-resistant (Cur) Pseudomonas spp., including P. cepacia, P. fluorescens, P. syringae, and P. viridiflava, and found that a subset of the P. syringae strains showed a dramatic increase in exopolysaccharide (EPS) production on mannitol-glutamate medium containing CuSO4 at 250 micrograms/ml. A modified carbazole assay indicated that the EPS produced on copper-amended media contained high levels of uronic acids, suggesting that the EPS was primarily alginic acid. Uronic acids extracted from selected strains were further confirmed to be alginate by demonstrating their sensitivity to alginate lyase and by descending paper chromatography following acid hydrolysis. Subinhibitory levels of arsenate, cobalt, lithium, rubidium, molybdenum, and mercury did not induce EPS production, indicating that alginate biosynthesis is not induced in P. syringae cells exposed to these heavy metals. A 200-kb plasmid designated pPSR12 conferred a stably mucoid phenotype to several P. syringae recipients and also increased their resistance to cobalt and arsenate. A cosmid clone constructed from pPSR12 which conferred a stably mucoid phenotype to several P. syringae strains but not to Pseudomonas aeruginosa was obtained. Results obtained in this study indicate that some of the signals and regulatory genes for alginate production in P. syringae differ from those described for alginate production in P. aeruginosa.

Construction of a yeast artificial chromosome contig spanning the spinal muscular atrophy disease gene region.

The childhood spinal muscular atrophies (SMAs) are the most common, serious neuromuscular disorders of childhood second to Duchenne muscular dystrophy. A single locus for these disorders has been mapped by recombination events to a region of 0.7 centimorgan (range, 0.1-2.1 centimorgans) between loci D5S435 and MAP1B on chromosome 5q11.2-13.3. By using PCR amplification to screen yeast artificial chromosome (YAC) DNA pools and the PCR-vectorette method to amplify YAC ends, a YAC contig was constructed across the disease gene region. Nine walk steps identified 32 YACs, including a minimum of seven overlapping YAC clones (average size, 460 kb) that span the SMA region. The contig is characterized by a collection of 30 YAC-end sequence tag sites together with seven genetic markers. The entire YAC contig spans a minimum of 3.2 Mb; the SMA locus is confined to roughly half of this region. Microsatellite markers generated along the YAC contig segregate with the SMA locus in all families where the flanking markers (D5S435 and MAP1B) recombine. Construction of a YAC contig across the disease gene region is an essential step in isolation of the SMA-encoding gene.

Effects of Environmental and Nutritional Factors on Production of the Polyketide Phytotoxin Coronatine by Pseudomonas syringae pv. Glycinea.

Pseudomonas syringae pv. glycinea PG4180 produces the polyketide phytotoxin coronatine. The effects of environmental, nutritional, and host factors on growth and coronatine production by PG4180 were examined by varying the components of a defined basal medium which contained the following nutrients per liter: glucose (10 g), NH(4)Cl (1 g), MgSO(4) . 7H(2)O (0.2 g), KH(2)PO(4) (4.1 g), K(2)HPO(4) . 3H(2)O (3.6 g), and FeCl(3) (2 muM). Bacterial growth was recorded as dry weight, and coronatine production was measured by high-performance liquid chromatography. Both growth and the quantity of coronatine synthesized were significantly affected by carbon source, nutrient levels (glucose, NH(4)Cl, phosphate, Mg, and SO(4)), amino acid supplements, and the presence of complex carbon and nitrogen sources. The yield of coronatine generally declined when conditions were varied from those in the basal medium. Coronatine production and growth were not affected when the pH was adjusted from 6.5 to 7.8. Increases in the osmolarity of the basal medium significantly decreased coronatine production without affecting growth. The addition of plant extracts, plant-derived secondary metabolites, or zinc did not affect growth or coronatine production, while the addition of millimolar levels of KNO(3) or micromolar levels of FeCl(3) significantly enhanced coronatine production. The yield of coronatine was maximized after a 7-day incubation at 18 degrees C and 280 rpm. The results of the present study were used to formulate a medium which allowed for enhanced coronatine production in nearly all strains of P. syringae tested. A rapid method for extracting coronatine from small volumes of culture supernatant was also developed.

Refinement of the spinal muscular atrophy locus to the interval between D5S435 and MAP1B.

The childhood-onset SMA locus has been mapped to chromosome 5q13, in a region bounded by the proximal locus, D5S6, and the closely linked distal loci, D5S112 and MAP1B. We now describe a highly polymorphic, tightly linked microsatellite marker (D5S435) that is very likely the closest proximal marker to the SMA locus. Multipoint linkage analysis firmly establishes the following order of markers at 5q13: centromere-D5S76-D5S6-D5S435-MAP1B/D5S112- D5S39-telomere. The data indicate that SMA resides in an approximately 0.7-cM (range 0.1-2.1) region between D5S435 and MAP1B. This finding reduces by approximately fourfold the genetic region that most likely harbors the SMA locus and will facilitate the physical mapping and cloning of the disease gene region.

Flow injection electrochemical enzyme immunoassay for theophylline using a protein A immunoreactor and p-aminophenyl phosphate-p-aminophenol as the detection system.

A competitive electrochemical enzyme immunoassay has been developed for the antiasthmatic drug theophylline, utilizing a controlled-pore glass-protein A immunoreactor and flow injection techniques. p-Aminophenyl phosphate, a substrate for alkaline phosphatase, has been used in this assay, and its hydrolysis product p-aminophenol was determined at +0.2 V versus the saturated calomel electrode. For each sample the antibody-protein A reaction takes place at near-neutral pH, and the complexes are eluted at acid pH. Serum theophylline has been determined by this method, and good relative standard deviations and percentage recoveries have been achieved.

Flow injection immunoassay using a protein A immunoreactor.

Competitive immunoassays have been developed for the immunosuppressant cyclosporin A and the anti-asthmatic drug theophylline utilizing identical controlled pore glass-protein A microcolumns and flow injection techniques. For cyclosporin A the assay was based on a monoclonal antibody with fluorescence detection whilst for theophylline sheep antiserum and electrochemical detection was used.

Analysis of Brucella lipopolysaccharide with specific and cross-reacting monoclonal antibodies.

Monoclonal antibodies which bind Brucella A lipopolysaccharide (LPS)-specific, M LPS-specific, or cross-reactive epitopes were used as reagents in quantitative dot blot, Western blot (immunoblot), and immunoprecipitation analysis of Brucella whole cells, whole-cell extracts, and purified LPS preparations. This set of monoclonal antibodies detected four unique epitopes on Brucella LPS. The specificity of monoclonal antibodies reactive with Brucella unique (A and M) and common (C and C/Y) LPS epitopes was demonstrated by blot analysis. The serotype specificity of monoclonal antibodies for A LPS of B. abortus 1119.3 or M LPS of Brucella melitensis 16M was confirmed. Type C monoclonal antibodies recognized epitopes on Brucella A and M LPS and did not cross-react with Yersinia enterocolitica O:9. In Western blots, type C monoclonal antibodies were bound by epitopes on Brucella A and M LPSs ranging in Mrs from 30,000 to 70,000, relative to marker proteins. Type C/Y monoclonal antibodies were cross-reactive with Y. enterocolitica O:9 and recognized Brucella A LPS epitopes with a restricted Mr ranging only from 40,000 to 50,000, relative to marker proteins. Type C/Y monoclonal antibodies also displayed a more restricted pattern of binding to Brucella M LPS. The monoclonal antibodies were able to detect 5 to 50 pg of a purified A LPS preparation in dot blots. The limits of detection by the monoclonal antibodies of a purified M LPS preparation ranged from 0.05 to 50 pg. Monoclonal antibody analysis of whole-cell preparations also demonstrated quantitative differences in the presence of the respective epitopes. The binding profiles of the monoclonal antibodies to Brucella whole cells varied between acetone- and chloroform-killed organisms as well as between species and strains. The lower limit of detection of any whole-cell preparation by the dot blot technique was 10(5) CFU. Binding profiles in Western blots and endotoxin activity of immunoprecipitates obtained with these monoclonal antibodies further defined the Brucella LPS antigens. These monoclonal antibodies and the techniques described may be useful in monitoring the antigenic content of Brucella vaccines and diagnostics.