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Biomedical subjects

R P Schneider

Publications and source records attributed to R P Schneider.

At least 19 recordsLinked to original sources
PubMed1999

Effect of nutrient limitation on adhesion characteristics of Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes a variety of diseases in humans including lung and ocular infections. Infections of the cornea are usually associated with wearing contact lenses and can result in loss of vision. This study aimed to determine the effect of carbon or nitrogen limitation on the adhesion to contact lenses of a strain of Ps. aeruginosa isolated from contact lens-related corneal inflammation. Cells were grown in a continuous culture apparatus in varying levels of glucose or ammonia to effect nutrient limitation. Adhesion to contact lenses was measured as total counts and viable counts. The cell surface hydrophobicity and charge were measured using adhesion to surface-modified Sepharose. Changes in lipopolysaccharide were determined using 1D SDS-PAGE and changes in cell-surface proteins were measured using 2D gel electrophoresis. The more the cultures were nitrogen limited, the greater the increase in adhesion to unworn hydrogel contact lenses 0.3 x 10(3) - 2.2 x 10(3) cells/mm2 on Etafilon A lenses. Cells that were carbon limited showed a greater increase in adhesion to contact lenses when the lenses had been coated in artificial tears. It appeared that lipopolysaccharide may have been involved in the constitutive adhesion to unworn lenses that occurred during C-limitation, whereas changes in the outer membrane proteins contributed to the increased adhesion under nitrogen limitation, or the change in adhesion that occurred to carbon-limited cells using contact lenses coated in artificial tears. Nine cell-surface proteins appeared during nitrogen limitation with kDa/pI of 75/4.8, 4.9, 5.0; 62/5.6; 89/6.5; 38/6.4; 28/1.5; 18/6.4; 12/4.5. Any or all of these may have been involved in the increased adhesion and further experiments are underway to examine this possibility.

Bacterial Adhesion↗
PubMed1999

Cell surface analysis techniques: What do cell preparation protocols do to cell surface properties?

Cell surface analysis often requires manipulation of cells prior to examination. The most commonly employed procedures are centrifugation at different speeds, changes of media during washing or final resuspension, desiccation (either air drying for contact angle measurements or freeze-drying for sensitive spectroscopic analysis, such as X-ray photoelectron spectroscopy), and contact with hydrocarbon (hydrophobicity assays). The effects of these procedures on electrophoretic mobility, adhesion to solid substrata, affinity to a number of Sepharose columns, structural integrity, and cell viability were systematically investigated for a range of model organisms, including carbon- and nitrogen-limited Psychrobacter sp. strain SW8 (glycocalyx-bearing cells), Escherichia coli (gram-negative cells without a glycocalyx), and Staphylococcus epidermidis (gram-positive cells without a glycocalyx). All of the cell manipulation procedures severely modified the physicochemical properties of cells, but with each procedure some organisms were more susceptible than others. Considerable disruption of cell surfaces occurred when organisms were placed in contact with a hydrocarbon (hexadecane). The majority of cells became nonculturable after air drying and freeze-drying. Centrifugation at a high speed (15,000 x g) modified many cell surface parameters significantly, although cell viability was considerably affected only in E. coli. The type of washing or resuspension medium had a strong influence on the values of cell surface parameters, particularly when high-salt solutions were compared with low-salt buffers. The values for parameters obtained with different methods that allegedly measure similar cell surface properties did not correlate for most cells. These results demonstrate that the methods used to prepare cells for cell surface analysis need to be critically investigated for each microorganism so that the final results obtained reflect the nature of the in situ microbial cell surface as closely as possible. There is an urgent need for new, reliable, nondestructive, minimally manipulative cell surface analysis techniques that can be used in situ.

Bacterial Adhesion↗
PubMed1998

An ocular strain of Pseudomonas aeruginosa is inflammatory but not virulent in the scarified mouse model.

Pseudomonas aeruginosa is the most common pathogen among contact lens-associated infections. This study investigated the response of the murine cornea to infection with an ocular strain of P. aeruginosa isolated from a subject with an inflammatory adverse response to contact lens wear termed CLARE. Although this bacterium was isolated in confluency (greater than 2000 cfu lens-1) from the lens at the time of the inflammatory episode, no infection of the cornea subsequently developed. Male C57BL/6J mice (20 per strain) had their corneas scratched with a 26 gauge needle (3 parallel 1.0 mm wounds in the left eye only). The incisions were centered over the pupillary axis and penetrated the epithelial cell basal lamina and into the superficial stroma. The CLARE strain was found to persist (viable bacteria could be cultured from corneal homogenates) up to 8 hr, as did the virulent control strain ATCC 19660. At 24 hr, only ATCC 19660 could be cultured, indicating an inability of the strain isolated from CLARE, Paer1, to persist in the eye consistent with the human inflammatory episode. Histological examination of the mouse tissue showed further differences between infection by the two strains. Infection with ATCC 19660 resulted in tissue necrosis and a large population of polymorphonuclear leukocytes (PMNs) recruited to the wound site. In contrast, during infection with the CLARE strain, PMN recruitment was reduced and temporally delayed. The CLARE strain grew as well as ATCC 19660 in vitro but produced less protease activity, in particular less elastase. The decreased PMN response and decreased protease production by the CLARE strain may have been responsible for the lack of ocular damage and apparent healing of the wound. P. aeruginosa strains are considered to be invasive or cytotoxic to corneal tissue, however this strain may represent a third inflammatory type consistent with its differing pathology.

Animals↗
PubMed1998

A relatively small change in sodium chloride concentration has a strong effect on adhesion of ocular bacteria to contact lenses.

Adhesion of bacteria to hydrogel lenses is thought to be an initial step of ocular colonization allowing evasion of normal host defences. The salt concentration of media is an important parameter controlling microbial adhesion. Salinity varies from 0.97% NaCl equivalents in the open eye to 0.89% in the closed eye state. In this study, the effect of sodium chloride in the concentration range of 0.8-1.0% (w/v) NaCl on adhesion of ocular bacteria to soft contact lenses was investigated using a static adhesion assay. Pseudomonas aeruginosa was found to adhere to lenses in significantly greater amounts than Serratia marcescens, Flavobacterium meningosepticum, Stenotrophomonas maltophilia and Staphylococcus intermedius. Increasing NaCl from 0.8% to 1.0% (w/v) increased adhesion of all bacteria tested. This adhesion was strong since the organisms could not be removed by washing in low ionic buffer. Adhesion of these organisms did not correlate with their cell surface properties as determined by bacterial adhesion to hydrocarbons (BATH) and retention on sepharose columns.

Bacterial Adhesion↗
PubMed1998

Interactions of bacteria with contact lenses: the effect of soluble protein and carbohydrate on bacterial adhesion to contact lenses.

PURPOSE: During contact lens wear, components in the tear film adsorb to the contact lens surface to form a protein-rich coating. In this study, we investigated the importance of these components in bacterial adhesion to contact lenses. METHODS: Five strains of bacteria were tested for their ability to adhere to contact lenses (patient-worn; coated with lactoferrin, lysozyme, or an artificial tear formulation; or left uncoated) in the presence of various solutes (protein, carbohydrate). Bacterial adhesion was quantified by both a total count (light microscopy) and viable count (culturing on agar). RESULTS: None of the solutes were found to competitively inhibit bacterial adhesion to worn contact lenses. However, lactoferrin was observed to kill bacteria attached to worn contact lenses. CONCLUSIONS: We could find no evidence to indicate that bacterial adhesion to contact lenses is mediated by specific interactions between bacteria and contact lens-bound tear components. The interaction between attached bacteria and lactoferrin may be important in initiating inflammatory responses to contact lens wear.

Aeromonas hydrophila↗
PubMed1998

Tissue distribution and biotransformation of zopolrestat, an aldose reductase inhibitor, in rats.

Zopolrestat (Alond) is a new drug that is being evaluated as an aldose reductase inhibitor for the treatment of diabetic complications. 14C-labeled zopolrestat was orally administered to rats for a tissue distribution study and a bile duct cannulation metabolism study. Tissue samples from the distribution study were analyzed by complete oxidation and liquid scintillation counting. Urine and bile samples from the bile duct cannulation study were analyzed by microbore HPLC, with simultaneous radioactivity monitoring and atmospheric pressure ionization tandem mass spectrometry. The mass balance in the distribution study demonstrated that the greatest exposure (AUC0-infinity) occurred in the liver, followed by the ileum and large intestine. The time of maximal plasma concentrations for nearly all tissues was 4 hr after the dose, and the half-life of radioactivity in most tissues (8-10 hr) was similar to the half-life in plasma. For the bile duct-cannulated rat study, most of the radioactivity was recovered in the bile, indicating that biliary excretion is a major route of elimination of zopolrestat and its metabolites in rats. Numerous oxidative metabolites, as well as phase II conjugates, were identified in the bile and urine samples. Acyl glucuronides of zopolrestat and unchanged drug accounted for >85% of biliary radioactivity, whereas unchanged drug and degradation products of glutathione conjugates were identified as the major urinary metabolites.

Administration, Oral↗
PubMed1998

Bioavailability, multiple-dose pharmacokinetics, and biotransformation of the aldose reductase inhibitor zopolrestat in dogs.

Zopolrestat (Alond) is a new drug that is being evaluated as an aldose reductase inhibitor for the treatment of diabetic complications. The bioavailability in dogs of a 2 mg/kg oral dose of zopolrestat was 97.2%. In a 1-year, multiple-dose, pharmacokinetic study, systemic exposure increased with increasing dose (50, 100, and 200 mg/kg/day), and there were no consistent changes in exposure with multiple dosing. Renal clearance at 1 year appeared to be higher in males. The magnitude of the potential gender difference in exposure was relatively small and was unlikely to have had a meaningful impact on the pharmacokinetics of zopolrestat in dogs. In studies with bile duct-cannulated dogs, radioactivity from [14C]zopolrestat was primarily eliminated as unchanged drug and acyl glucuronide in the bile and feces (77.3% of the dose) and in urine (18.3% of the dose). The concentrations of acyl glucuronide in urine and feces were approximately 50% of the zopolrestat concentrations. Minor metabolites (each accounting for <1% of the dose) included those resulting from hydroxylation of the phthalazinone ring and glutathione conjugation of the benzothiazole ring.

Administration, Oral↗
PubMed1997

Growth of gram-negative bacteria in a simulated ocular environment.

PURPOSE: Gram-negative bacteria are major pathogens in the ocular environment, especially when contact lenses are worn. The source of many of these organisms, including Pseudomonas aeruginosa, is thought to be environmental. METHODS: A defined medium is described here that resembles both tear fluid and the environment they are derived from. RESULTS: Gram-negative ocular bacteria were found to grow successfully in this medium. Bactericidal tear proteins were added to this medium and pathogenic strains were not only able to resist their effects but were able to use them for growth. CONCLUSION: Utilization of tear proteins for growth may be an important virulence factor of pathogenic bacteria in the ocular environment.

Eye Proteins↗
PubMed1997

Role of tear fluid in the growth of gram-negative bacteria on contact lenses.

Incidents of CLARE (contact lens induced acute red eye) are associated with high numbers of Gram-negative bacteria on the lens surface. This study found that most strains of bacteria implicated in CLARE adhere poorly to the lens surface regardless of whether the lens surface was clean or had been worn in the eye. The high numbers of bacteria on the lens surface result from bacterial growth on the lens surface after adhesion has occurred. Using an artificial tear formulation and real tears, this study demonstrated that the tear fluid provides the necessary nutrients required for bacterial growth.

Bacterial Adhesion↗
PubMed1996

Homology between genes for aromatic hydrocarbon degradation in surface and deep-subsurface Sphingomonas strains.

The cloned genes for aromatic hydrocarbon degradation from Sphingomonas yanoikuyae B1 were utilized in Southern hybridization experiments with Sphingomonas strains from the surface and deep-subsurface environments. One hybridization pattern was obtained with BamHI-digested genomic DNAs for two surface strains, while a differing pattern was seen for five deep-subsurface strains. The cross-hybridizing genes were located in the chromosomes of the surface strains and on plasmids in the deep-subsurface strains.

Bacteria↗
PubMed1995

Quantitative determination of the antibiotic azithromycin in human serum by high-performance liquid chromatography (HPLC)-atmospheric pressure chemical ionization mass spectrometry: correlation with a standard HPLC-electrochemical method.

A specific assay for the quantitative determination of the new antibiotic azithromycin in a low volume of human serum is described. The assay uses on-line high-performance liquid chromatography (HPLC) and atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI). Deuterium-labeled azithromycin was synthesized and used as the internal standard of the assay. The drug and the internal standard are extracted from 50 microliters of serum, and aliquots are injected onto a standard reverse-phase HPLC column. The effluent from the HPLC column at 1 ml/min is introduced into the atmospheric pressure source of a SCIEX API III mass spectrometer. Azithromycin concentrations in serum are determined by the selected monitoring of the protonated molecular ions of the drug and the internal standard. Our assay yields accurate and precise results over the range 10 ng/ml to 250 ng/ml. The correlation between the assay and a standard HPLC-electrochemical method, requiring a larger volume of serum, has been determined. The two methods showed excellent agreement. Because of its low volume requirement, our HPLC-APCI assay can be substituted for the standard assay for the investigation of azithromycin pharmacokinetics in children.

Azithromycin↗
PubMed1995

Physical mapping and characterization of a catabolic plasmid from the deep-subsurface bacterium Sphingomonas sp. strain F199.

A supercoiled 180-kb plasmid, pNL1, has been isolated from the deep-subsurface, chemoheterotrophic Sphingomonas sp. strain F199, and a physical map was generated. Analysis of a pNL1-derived cosmid library indicated that catechol 2,3-dioxygenase activity was linked to two distinct regions of the plasmid. Thus, the genes for aromatic catabolism in this Sphingomonas strain are, at least in part, plasmid encoded.

Catechol 2,3-Dioxygenase↗
PubMed1994

Ductal adenocarcinoma arising in a heterotopic pancreas situated in a hiatal hernia.

We present a case of ductal adenocarcinoma originating in a heterotopic pancreas in a 60-year-old patient. The tumor developed at the esophagogastric junction in a hiatal hernia. The nontumoral pancreatic tissue showed ductal cystic dystrophy with enclosed stones, as well as lesions of chronic pancreatitis with metaplastic changes. The perigastric lymph nodes and the liver contained metastatic deposits. Malignant transformation in an ectopic pancreas is exceptional. To our knowledge, this would be the first case occurring in such a location. We review the literature on the subject and discuss the theory of the tumors arising in an aberrant pancreas.

Carcinoma, Ductal, Breast↗
PubMed1993

Structure of the rat p53 tumor suppressor gene.

Aberration within the p53 tumor suppressor gene is the most frequently identified genetic damage in human cancer. Regulatory functions proposed for the p53 protein include modulation of the cell cycle, cellular differentiation, signal transduction, and gene expression. Additionally, the p53 gene product may guard the genome against incorporation of damaged DNA. To facilitate study of its role in carcinogenesis using a common animal model, we determined the structure of the rat p53 gene. We identified 18 splice sites and defined 25 bases of the intervening sequences adjacent to these sites. We also discovered an allelic polymorphism that occurs within intron 5 of the gene. The rat gene approximates the mouse ortholog. It is 12 kb in length with the non-coding exon 1 separated from exon 2 by 6.2 kb of intervening sequence. The location and size of all rat gene introns approximate those of the mouse. Whereas the mouse and human genes each contain 11 exons, the rat p53 gene is composed of only 10. No intervening sequence occurs between the region of the rat gene corresponding to exons 6 and 7 of the mouse and human p53 genes. This implies intron 6 may be functionally insignificant for species in which it is retained. To extrapolate to p53 involvement in human tumorigenesis, we suggest that mutational events within intron 6 may not be of pathological significance unless splicing is hindered.

Animals↗
PubMed1993

Confirmation of danofloxacin residues in chicken and cattle liver by microbore high-performance liquid chromatography electrospray ionization tandem mass spectrometry.

A specific assay is described for the confirmatory identification of danofloxacin residues in edible tissues of cattle and chicken. The assay utilizes on-line microbore high-performance liquid chromatography and pneumatically assisted electrospray tandem mass spectrometry (MS/MS). Collision-induced dissociation of the danofloxacin protonated molecule results in two significant daughter ions. Monitoring both ions provides the specificity required for this confirmatory assay. Optimum electrospray and MS/MS operating conditions permitted the specific monitoring of danofloxacin and the confirmation of its residues in chicken and cattle liver extracts down to 50 ppb. The analysis of control liver or the commercially available antibacterial quinolones enrofloxacin and its metabolite ciprofloxacin gave no response under the assay conditions. The ratios of the two daughter ions were similar for danofloxacin standard solutions, fortified tissues and incurred tissues.

Animals↗
PubMed1992

Abnormal in vitro immunoglobulin synthesis in surgical patients.

Blood mononuclear cells from surgical patients produce large amounts of IgG in vitro. This synthesis is not increased by stimulation with pokeweed mitogen. To determine if this abnormal pattern of IgG synthesis extended to other immunoglobulin classes, surgical patients were stratified according to delayed-type hypersensitivity responses as reactive or anergic. Healthy personnel were studied as controls. Mononuclear cells were cultured without or with pokeweed mitogen, and IgG, IgM, and IgA were measured in supernatants. Unstimulated IgG and IgA synthesis was increased in surgical patients, especially in those with reduced delayed-type hypersensitivity responses. Synthesis of IgM was normal or low. With mitogen stimulation, IgG synthesis was increased in control and reactive subjects, but not in anergic subjects. For IgM, mitogen stimulation increased synthesis to a less than normal level in all patients. For IgA, synthesis was increased in all groups.

Adult↗