Vigorous Washing Research Articles

There Aren't Any Kitchens in Heaven

There Aren't Any Kitchens in Heaven

There Aren't Any Kitchens in Heaven

There Aren't Any Kitchens in Heaven

DNA hybridization on silica microbeads that are physically adsorbed as arrays on glass surfaces

Detection of DNA hybridization was done on silica microbeads that were physically adsorbed onto glass surfaces. DNA oligonucleotide probes of approximately 20 mer size were immobilized on beads of 5 μm diameter after the microbeads were first silanized with 3-glycidoxypropyltrimethoxysilane. The suspension of silica microbeads in aqueous solution was spotted on the glass slides. After drying, the glass surface was washed with water and 1 × SSC buffer. Significant numbers of microbeads remained physically adsorbed onto the glass surfaces even after vigorous washing with buffer solution. After hybridization using approximately 200 mer PCR targets strands, the glass slides were scanned using a standard laser confocal fluorescence microscope microarray reader. The total time for completion of hybridization assays was less than 20 min for 100 nM samples of target oligonucleotide. The clinical utility of the method was demonstrated by detection of single base pair mutations in the survival motor neuron gene that is associated with the childhood disease Spinal Muscular Atrophy. The method proved to provide a readily adaptable strategy for immobilization of different probes in an array format, and provided for SNP detection on a disposable slide without cross contamination.

Lipopolysaccharide stimulation converts vigorously washed dendritic cells (DCs) to nonexhausted DCs expressing CD70 and evoking long-lasting type 1 T cell responses

A great variety of in vitro culture protocols for human monocyte-derived dendritic cells (mo-DCs) has been used to generate DCs suitable for use in immunotherapy. It is thought that activated DCs undergo one-way differentiation into "exhausted" DCs. In the present study, we contrived an in vitro method for facilitating expression of CD70 by mature DCs. This was achieved by vigorous washing of mo-DCs before exposure to lipopolysaccharide (LPS). Unexpectedly, these mature DCs retain expression of some interleukin (IL)-12 family members after extended periods and maintain their ability to stimulate type 1 T cell responses. In contrast, DCs exposed to IL-4 before LPS stimulation or LPS-stimulated DCs not exposed to washing stress before activation failed to express CD70 and did differentiate into exhausted DCs. It is interesting that DCs expressing CD70 (CD70+ DCs) induced interferon-gamma production from purified, allogeneic CD8+ T cells through a direct CD27-CD70 interaction. This is evidence for a pathway resulting in generation of CD8 T effectors by B7-independent mechanisms. These data suggest that exposure of immature DCs to LPS stimulation contributes to their terminal differentiation into CD70+ DCs, which have potent ability to prolong type 1 T cell responses through alternative pathways.

Have We Been Reprocessing the Endoscopic Biopsy Forceps Adequately?

The cleaning of endoscopic biopsy forceps is the most important phase of the disinfection method, since it consists on the removal of all organic material. However, the typical spiral shape and their extremity in cup make difficult the adequate decontamination and cleaning the forceps. Aims: 1) To verify the removal efficacy of the remaining material on the forceps through three different methods; 2) To verify the cost of each method. Methods: We randomized the study including 60 biopsy forceps used routinely for the urease test, at the Endoscopic Unit of the University of Sao Paulo. We followed the methodology: forceps removal and cleaning with gauze; immersion in 0.5%-enzymatic solution during 20 minutes; vigorous mechanical washing and rinsing with water. After that, the forceps were separated in three groups, such as: group a. 20 forceps - without any additional cleaning method; group b. 20 forceps - immersion in 3% hydrogen peroxide solution (20 minutes); group c. 20 forceps - ultrasound washing with enzymatic detergent. At the second phase, the forceps were submitted to observation by using magnifying lens (20 times), by just one observer, who did not know the cleaning method used, classifying them in: appropriate cleaning method: absence of organic material; inappropriate method: presence of organic material (secretion or blood/tissue). After that, all forceps were reprocessed again with enzymatic solution and sterilized under ethylene oxide. Results: We could identify two distinct types: 1. blood or tissue (adhered to the cup); 2. whitish secretion (from the body of the forceps). We could also observe that the forceps submitted to the washing with peroxidase presented a brighter and lighter color, comparing to the ones treated only with enzymatic detergent. Cleaning method was considered appropriate in 20% (4/20) in group a; 70% (14/20) in group b, and 55% (11/20) in group c. So, the peroxidase and the ultrasound washing have presented a superior efficacy when comparing to the enzymatic detergent only; p < 0.01 and p = 0.014, respectively. The cost of desinfection for group a was U$14.00, group b U$64.50 and group c U$46.20. Conclusions: 1. The peroxidase adds better efficacy of desinfection method; 2. In spite of presenting a superior cost when comparing to the ultrasound washing, it is a safe and efficient method, because it also has bactericidal, fungicidal, viruscidal and sporicidal qualities. The cleaning of endoscopic biopsy forceps is the most important phase of the disinfection method, since it consists on the removal of all organic material. However, the typical spiral shape and their extremity in cup make difficult the adequate decontamination and cleaning the forceps. Aims: 1) To verify the removal efficacy of the remaining material on the forceps through three different methods; 2) To verify the cost of each method. Methods: We randomized the study including 60 biopsy forceps used routinely for the urease test, at the Endoscopic Unit of the University of Sao Paulo. We followed the methodology: forceps removal and cleaning with gauze; immersion in 0.5%-enzymatic solution during 20 minutes; vigorous mechanical washing and rinsing with water. After that, the forceps were separated in three groups, such as: group a. 20 forceps - without any additional cleaning method; group b. 20 forceps - immersion in 3% hydrogen peroxide solution (20 minutes); group c. 20 forceps - ultrasound washing with enzymatic detergent. At the second phase, the forceps were submitted to observation by using magnifying lens (20 times), by just one observer, who did not know the cleaning method used, classifying them in: appropriate cleaning method: absence of organic material; inappropriate method: presence of organic material (secretion or blood/tissue). After that, all forceps were reprocessed again with enzymatic solution and sterilized under ethylene oxide. Results: We could identify two distinct types: 1. blood or tissue (adhered to the cup); 2. whitish secretion (from the body of the forceps). We could also observe that the forceps submitted to the washing with peroxidase presented a brighter and lighter color, comparing to the ones treated only with enzymatic detergent. Cleaning method was considered appropriate in 20% (4/20) in group a; 70% (14/20) in group b, and 55% (11/20) in group c. So, the peroxidase and the ultrasound washing have presented a superior efficacy when comparing to the enzymatic detergent only; p < 0.01 and p = 0.014, respectively. The cost of desinfection for group a was U$14.00, group b U$64.50 and group c U$46.20. Conclusions: 1. The peroxidase adds better efficacy of desinfection method; 2. In spite of presenting a superior cost when comparing to the ultrasound washing, it is a safe and efficient method, because it also has bactericidal, fungicidal, viruscidal and sporicidal qualities.

Safety and Efficacy of Endoscopic Sphincterotomy and Bile Duct Clearance in Patients with Acute Cholangitis Due to the Common Bile Duct Stones

Safety and Efficacy of Endoscopic Sphincterotomy and Bile Duct Clearance in Patients with Acute Cholangitis Due to the Common Bile Duct Stones Neven Ljubicic, Luka Rotkvic Background: It has been shown that urgent endoscopic drainage is an effective treatment for acute cholangitis and is superior to surgical drainage with a better clinical outcome Because of the risk of post-sphincterotomy bleeding in these patients many endoscopists have advocated endoscopic drainage by using nasobiliary catheter or an indwelling stent without sphincterotomy. The aim of this study was to analyze safety and efficacy of endoscopic sphincterotomy (ES) and urgent stone extraction in patients with acute cholangitis due to the common bile duct stones.Methods: Between January andDecember 2002, one hundred and eleven patients with clinical jaundice and common bile duct stones were enrolled to an ES and urgent stone extraction. ES has been performed in a standard manner, always using the guide-wire. In patients with cholangitis vigorous washing of the common bile duct has been performed after removal of the stones. Forty-six patients fulfilled criteria for acute cholangitis. Prior to the ES all patients received intravenously ciprofloxacin for at least 24 hours. Results: ES has been performed successfully in 102 (92%) patients. Complete duct clearance has been achieved in 90% of patients without cholangitis and in 93% of patients with cholangitis (not significant). Complications occurred in 12% of patients without and in 16% of those with acute cholangitis (Table). In patients with post-sphincterotomy bleeding blood transfusion was not required. The median lengths of time for resolution of jaundice in patients with and without cholangitis were similar. Significant coagulopathy has been observed in two (4.5%) patients with acute cholangitis. In those patients there were no bleeding occurred after ES. Conclusions: ES and bile duct clearance is very effective and safe method even in the patients with acute cholangitis. Mild bleeding was the most common postsphincterotomy complication not required blood transfusion even in the presence of coagulopathy. *T1500 MRCP Vs ERCP: A Comparative Study in Diagnosis of Common Bile Duct Stones Luigi T. Familiari Background: Because it is a noninvasive method, without complications, the magnetic resonance cholangiography (MRCP) is going to reliably replace ERCP in the diagnosis of pancreato-biliary diseases. Althoughmany studies documented a high sensitivity and specificity of MRCP, its ability in detecting microlithiasis ( 270; I-GT > 50 UI/l). Patients underwent upper abdominal ultrasonography (US), thenMRCP and, within 72 h, diagnostic and/or operative ERCP. All patients signed an informed consent. The statistical analysis was based on the report of true positives and negatives, false positives and negatives. Results:106/123 patients completed the study. MRCP diagnosed lithiasis of CBD in 73; did not document stones in 33. ERCP confirmed the presence of lithiasis in 66/73 pts who were submitted to ES. Seven were not found to have stones after ES.Among 33 negative pts, ERCPdocumented stones in 9: in 4 only after ES (it was performed according to clinico-biochemical data and to CBD dilation, though ERCP did not identify stones). The stones were 2-3 mm. In 24/33 patients negative at MRCP, ERCP confirmed this response:10/24 were submitted to ES. ES was not performed in 14/24 subjects, without CBD dilation and/or cholestasis. After a 27.8 months follow-up (range: 4-54), 8/14 remained asymptomatic, 6 underwent laparoscopic cholecystectomy without finding of CBD stones at the intra-operative cholangiography. The sensitivity, specificity, diagnostic accuracy, PPV and NPV of MRCP were: 88%, 77%, 85%, 90%, 73%. Conclusions: asMRCPhas still a limitation in the diagnosis of small stones,missed at US, it leaves unrisolved the question of which patient is candidate to ERCP/ES. The recognition of microlithiasis should refer to other imaging techniques preliminar to ERCP (e.g. EUS) though their limited availability makes until now MRCP a method of choice in the diagnosis of biliary disease.

Xenogeneic cells and superantigen induce human T-cell activation in the absence of T-cell recognition of xenoantigen

The extent of interaction between human T-lymphocytes and xenogeneic antigen-presenting cells (APCs) is species-dependent. A successful interaction requires high-affinity receptor-ligand pairing across species and recognition of antigens presented by foreign major histocompatibility complex (MHC). A deficient human T-lymphocyte response to xenogeneic cells is likely the result of a defect in these interactions. However, the requirements for a T-cell response to superantigen (SAg) may differ from those of other T-cell responses. Using irradiated murine splenocytes, which are believed to be incapable of eliciting human T-cell responses, and porcine aortic endothelial cells (PAECs) as the APC populations, we studied the human T-lymphocyte response to antigens presented by these cells. Direct proliferation of human T-lymphocytes to SAg presented by murine APCs was demonstrated; it was blocked by anti–human LFA-1 and anti–murine MHC class II but not by anti–human MHC class II. PAECs also presented SAg to human T-cells, generating a proliferative response greater than the primary response to porcine xenoantigen. Culture of human T-cells with murine splenocytes or PAECs and SAg Staphylococcus enterotoxin A (SEA) for 7 days primed human T-cells to proliferate in a secondary culture in response to autologous APCs. This autologous secondary response was human MHC class II–dependent and was inhibited by anti–human LFA-1, anti–human CD2, and anti–human CD98. Surprisingly, both of these responses were also blocked by anti-SEA, suggesting that despite vigorous washing, a small amount of functionally important SAg was carried over from primary to secondary culture, probably bound to the surface of T-cells. Xenogeneic APCs, even those that fail to stimulate human T-cells directly, can serve as APCs for primary human T-cell responses. After such interactions T-cells can develop secondary responses in autologous interactions based on retention of minute amounts of SAg. Such interactions may have important implications for xenotransplantation.

The disintegrin-like metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell-cell adhesion

AbstractThe CX3C chemokine fractalkine (CX3CL1) exists as a membrane-expressed protein promoting cell-cell adhesion and as a soluble molecule inducing chemotaxis. Transmembrane CX3CL1 is converted into its soluble form by defined proteolytic cleavage (shedding), which can be enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA). PMA-induced CX3CL1 shedding has been shown to involve the tumor necrosis factor-α–converting enzyme (TACE), whereas the constitutive cleavage in unstimulated cells remains elusive. Here we demonstrate a role of the closely related disintegrin-like metalloproteinase 10 (ADAM10) in the constitutive CX3CL1 cleavage. The hydroxamate GW280264X, capable of blocking TACE as well as ADAM10, proved to be an effective inhibitor of the constitutive and the PMA-inducible CX3CL1 cleavage in CX3CL1-expressing ECV-304 cells (CX3CL1–ECV-304), whereas GI254023X, preferentially blocking ADAM10 but not TACE, reduced the constitutive cleavage only. Overexpression of ADAM10 in COS-7 cells enhanced constitutive cleavage of CX3CL1 and, more importantly, in murine fibroblasts deficient of ADAM10 constitutive CX3CL1 cleavage was markedly reduced. Thus, ADAM10 contributes to the constitutive shedding of CX3CL1 in unstimulated cells. Addressing the functional role of CX3CL1 shedding for the adhesion of monocytic cells via membrane-expressed CX3CL1, we found that THP-1 cells adhere to CX3CL1–ECV-304 cells but detach in the course of vigorous washing. Inhibition of ADAM10-mediated CX3CL1 shedding not only increased adhesive properties of CX3CL1–ECV-304 cells but also prevented de-adhesion of bound THP-1 cells. Our data demonstrate that ADAM10 is involved in the constitutive cleavage of CX3CL1 and thereby may regulate the recruitment of monocytic cells to CX3CL1-expressing cell layers.

Peptidylglycine-α-amidating monooxygenase and pro-atrial natriuretic peptide constitute the major membrane-associated proteins of rat atrial secretory granules

Peptidylglycine-α-amidating monooxygenase (PAM) is a bi-functional enzyme known to catalyze the post-translational bioactivation of signaling peptides. Although PAM is highly concentrated within the cardiac atrium, this tissue does not produce appreciable amounts of α-amidated peptides and thus, the function of PAM in atrium remains largely unknown. In this study, we demonstrate that PAM co-localizes in atrial secretory granules with the storage form of atrial natriuretic peptide (pro-ANP, amino acids 1–126), a hormone involved in the maintenance of blood pressure and fluid homeostasis. ANP is not amidated by PAM, but rather is processed to its active form (amino acids 99–126) by the proteolytic cleavage of pro-ANP. We demonstrate here by subcellular fractionation and biochemical analyses that PAM co-localizes with pro-ANP in secretory granules, where together they constitute the two most abundant membrane-associated proteins, accounting for approximately 95% of the total granular membrane protein. Respectively, light and electron microscopic immunohistochemistry show intense staining for PAM in atrial cardiomyocyctes and subcellular localization of PAM to secretory granules. Additionally, we demonstrate that while pro-ANP is readily found in the soluble contents of the granule lumen, significant amounts remain tightly associated with the membranes even after vigorous washing and estimate the molar ratio of pro-ANP to PAM to be approximately 30:1 in the membrane fraction. We postulate here that the primary function of PAM in the atrium is structural rather than enzymatic. In this regard, PAM may contribute to the packaging of pro-ANP within the secretory granule and possibly function in the presentation of pro-ANP for proteolytic processing.

Local hemostasis during laparoscopic partial nephrectomy using biodegradable hydrogels: initial porcine results.

Despite the advance of laparoscopic partial nephrectomy, significant technical limitations remain with regard to control of bleeding and closure of the collecting system. An attractive approach on the horizon for local hemostatic and wound control is the use of local tissue sealants. To date, sealants remain largely derived from natural biologic products and are difficult to apply laparoscopically with precise local control. In this study, we examined the novel strategy of forming occlusive tissue-adherent hydrogels utilizing a synthetic biodegradable polyethylene glycol-lactide copolymer (PEG-lactide) as an in situ occlusive barrier for hemostasis and wound control. Specifically, the objects of this study were to determine if PEG-lactide hydrogels could be formed intraperitoneally on renal tissue, to test the adhesiveness of the hydrogels to injured renal parenchyma, and to evaluate the ability of adherent hydrogel barriers to limit renal parenchymal bleeding and collecting system leakage following renal pole amputation or wedge excision. Five kidneys from three female pigs were used in a nonsurvival study. A standardized model for laparoscopic partial nephrectomy was created by performing wedge excision or polar amputation under vascular control using a laparoscopic Satinsky clamp. Bleeding briskness following injury was assessed utilizing a scoring system and free blood quantitated comparing a conventional "clamp and wait" strategy with an adherent hydrogel strategy. For the hydrogel group, PEG-lactide hydrogel primer and macromer were applied through laparoscopic ports. The hydrogel was polymerized using a xenon light source, and the pedicle clamp was released to observe for bleeding. A subsequent opposite polar injury was created to confirm renal perfusion and the sites were compared. The kidneys were removed, and the adhesion of the hydrogel to the renal parenchyma was examined. The PEG-lactide macromer was effectively applied to five kidneys following partial nephrectomy. In all cases, successful intraperitoneal in situ polymerization was achieved, with resultant hydrogel formation. Polymeric hydrogel adhesion to the cut renal parenchyma was assessed semiquantitatively following vigorous cyclic washing. In all cases, polymer gels remained adherent without any evidence of peeling, delamination, or separation from the underlying tissue surface. In the control group, the mean bleeding score was 2.63 +/- 0.48 v 0.00 +/- 0.00 in the gel-treated group (P < 0.001). Blood loss in the control group was 56 +/- 5 ml v 0.00 +/- 0.00 in the gel-treated group (P < 0.001). In an ex vivo retrograde ureteral perfusion, no leakage was observed at pressure as high as 100 mm Hg. In this feasibility study, a biodegradable PEG-lactide polymer system photopolymerized rapidly in situ on exposed renal parenchymal surfaces, forming adherent hydrogel barriers. When applied during vascular clamping, an adequate physical bond and patch-like cap was created to prevent bleeding at physiologic renal perfusion pressures. Use of locally applied occlusive hydrogels holds promise for hemostasis and local wound control during laparoscopic urologic procedures.

Immobilization of homooligonucleotide probe layers onto Si/SiO 2 substrates: characterization by electrochemical impedance measurements and radiolabelling

Radiolabelling and electrochemical impedance measurements were used to characterize the immobilization of single stranded homooligonucleotides onto silica surfaces and their subsequent hybridization with complementary strands. The immobilization procedure consists of grafting an epoxysilane onto microelectronic grade Si/SiO 2 substrates, and coupling oligonucleotides bearing a hexylamine linker onto the epoxy moiety. Radiolabelling was used as a reference method to quantify the amount of immobilized and hybridized oligonucleotides. These results show that the Si/SiO 2 substrates modified with an epoxysilane yield a surface concentration of ∼10 11 strands/cm 2 for the immobilized oligonucleotides, after vigorous washings, and that ∼36% of these undergo hybridization with complementary strands. The impedance measurements, which provide a direct means of detecting variations in electrical charge accumulation across the semiconductor/oxide/electrolyte structure when the oxide surface is chemically modified, show that the semiconductor's flat band potential undergoes reproducible shifts of −150 and −100 mV following the immobilization and the hybridization step, respectively. These results demonstrate that electrochemical impedance measurements using chemically modified semiconductor/oxide/electrolyte structures of this type offer a viable alternative for the direct detection of complementary DNA strands upon hybridization.

Diagnostic potential of PhotoSELEX-evolved ssDNA aptamers

High sensitivity and specificity of two modified ssDNA aptamers capable of photocross-linking recombinant human basic fibroblast growth factor (bFGF(155)) were demonstrated. The aptamers were identified through a novel, covalent, in vitro selection methodology called photochemical systematic evolution of ligands by exponential enrichment (PhotoSELEX). The aptamers exhibited high sensitivity for bFGF(155) comparable with commercially available ELISA monoclonal antibodies with an absolute sensitivity of at least 0.058 ppt bFGF(155) under prevailing test conditions. The aptamers exquisitely distinguished bFGF(155) from consanguine proteins, vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). A commercially viable diagnostic system incorporating PhotoSELEX-evolved aptamers capable of simultaneous quantification of a large number of analyte molecules is also described. Such a system benefits from covalent bonding of aptamer to target protein allowing vigorous washing with denaturants to improve signal to noise.

Modification of indium tin oxide electrodes with nucleic acids: detection of attomole quantities of immobilized DNA by electrocatalysis

Indium tin oxide electrodes were modified with DNA, and the guanines in the immobilized nucleic acid were used as a substrate for electrocatalytic oxidation by Ru(bpy)3(3+) (bpy = 2,2'-bipyridine). Nucleic acids were deposited onto 12.6-mm2 electrodes from 9:1 DMF/water mixtures buffered with sodium acetate. The DNA appeared to denature in the presence of DMF, leading to adsorption of single-stranded DNA. The nucleic acid was not removed by vigorous washing or heating the electrodes in water, although incubation in phosphate buffer overnight liberated the adsorbed biomolecule. Acquisition of cyclic voltammograms or chronoamperomograms of Ru(bpy)3(2+) at the modified electrodes produced catalytic signals indicative of oxidation of the immobilized guanine by Ru(III). The electrocatalytic current was a linear function of the extent of modification with a slope of 0.5 microA/pmol of adsorbed guanine; integration of the current-time traces gave 2.2+/-0.4 electrons/guanine molecule. Use of long DNA strands therefore gave steep responses in terms of the quantity of adsorbed DNA strand. For example, electrodes modified with a 1497-bp PCR product from the HER-2 gene produced detectable catalytic currents when as little as 550 amol of strand was adsorbed, giving a sensitivity of 44 amol/mm2.

Composition and enzymatic activity of the extracellular matrix secreted by germlings of botrytis cinerea.

Germlings of Botrytis cinerea, an important fungal pathogen of plants, produce an extracellular matrix (ECM), or ensheathing film, that serves, in part, in their attachment (R. P. Doss, et al., Appl. Environ. Microbiol. 61:260-265, 1995). The composition of this film has been ascertained by using samples obtained by growing germlings on a glass surface, removing the fungal mycelium by vigorous washing, and collecting the tightly attached film by scraping the substratum with a razor blade. Slightly over half of the dry weight of the ECM was found to be carbohydrates (about 20%), proteins (about 28%), and lipids (about 6%). Hydrolysis of the carbohydrate portion of the ECM revealed that glucose was the most prominent monosaccharide present, comprising about 60% of the total monosaccharides. Also present were mannose (about 35%) and myo-inositol (about 5%). The proteinaceous fraction of the ECM was made up of a number of polypeptides separable by polyacrylamide gel electrophoresis. The lipid fraction of the ECM, analyzed by thin-layer chromatography, was made up of several simple lipid components, including free fatty acid, mono- and triacylglycerol, wax ester, fatty alcohol, and several unidentified components. No complex lipids were detected. Isolated ECM exhibited polygalacturonase and laccase activity and was able to catalyze the hydrolysis of p-nitrophenyl butyrate, a model substrate for assessing cutinase activity. Cellulase, pectin lyase, and pectin methyl esterase activities were noted with both heated and unheated ECM preparations. Proteinase activity was not detected.

An optimized method for cell-based phage display panning

Background: Traditional methods of phage display panning, bind purified antigen to plates or other solid phases to which libraries are then applied, followed by vigorous washings in detergent-supplemented buffers to select for specific phage Fab. These methods are not directly applicable to antigens in their native environment on cell surfaces or in settings where the target antigen is unknown. Objectives: To develop a model antigen system employing whole cells rather than purified protein immobilized on a substrate; to optimize methods for phage display panning using a cell-based system. Results: Specificity of binding of phage Fab to antigen on cells was demonstrated by output titer and by flow cytometry. Output titers showed a plateau and binding advantage, after four washes, corresponding to removal of most non-specifically bound phage. Enrichment advantage was independent of input phage number. Longer incubation times, to cell tolerance, improved specific binding. Temperature had modest impact as a variable in the panning and washing. An increase in output titers paralleled enrichment for specific phage Fab. Conclusion: An optimized method applied to whole cells can productively enrich specific phage Fab in mixtures with large excesses of non-specific phage Fab over several rounds of panning.

Distribution and establishment of the biocontrol fungus Talaromyces flavus in soil and on roots of solanaceous crops

Ascospores of Talaromyces flavus were applied to eggplant, tomato and potato as either a drench or in spherical granules (alginate prill). In subsequent samplings, the percentage colonization of roots was significantly greater than that of root tips. However, when populations of T. flavus were expressed as colony forming units g −1 fresh weight, recovery of T. flavus was significantly greater in root tips. Populations were lower in rhizosphere soil than from roots and root tips and populations in non-rhizosphere soil were the lowest of any location. Vigorous washing of the roots removed most of the T. flavus, indicating that T. flavus was superficially associated with the roots. Populations of T. flavus were greater when it was applied as a drench rather than in prill.

Prevention of community-acquired and nosocomial pneumonia.

Pneumonia is an important cause of morbidity and mortality in the United States. The provision of effective prophylaxis for pneumonia has become a major goal for both public health officials and individual physicians. Prophylaxis for community-acquired pneumonia is pathogen-specific and is directed toward the most common microorganisms that cause it. The 23-valent pneumococcal polysaccharide vaccine; the trivalent influenza vaccine; the Haemophilus b conjugate vaccine; and either trimethoprim-sulfamethoxazole, dapsone, or aerosolized pentamidine are recommended to prevent Streptococcus pneumoniae, influenza viruses, H. influenzae type b, and Pneumocystis carinii respectively. Except for the microorganisms listed above, the prevention of nosocomial pneumonia is not pathogen-specific. Rather, prevention of nosocomial pneumonia requires the use of infection control procedures, including patient and staff education; isolation of patients with highly contagious respiratory pathogens; vigorous hand washing; cleaning and sterilizaton of respiratory equipment; and use of sterile water in nebulizers and humidifiers. It also requires procedures to limit pooling and aspiration of secretions, such as positioning and rotation of the bed-bound patient; frequent suctioning of respiratory secretions using gloves and sterile suction catheters; and limiting enteral alimentation. Finally, selective decontamination of the digestive tract may be considered for intubated patients.

Immobilization of Antibodies on a Photoactive Self-Assembled Monolayer on Gold

This paper presents a strategy for immobilizing biomolecules on a photoactivable surface. A self-assembled monolayer is prepared by adsorbing an ω-functionalized dialkyl disulfide on gold. Functional groups of this monolayer are converted in two steps into a benzophenone derivative with an overall yield of 50 ± 10%. Several independent techniques (ellipsometry, X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy, radiolabel assay, and autoradiography) characterize the reaction and photoimmobilization of antibodies on this surface. The photoimmobilized antibodies cover the surface as a homogeneous and dense monolayer that could not be disrupted by vigorous washing with the detergent Tween 20. Immunoassays demonstrated specific recognition of the immobilized immunoglobulins as measured by their complexation with alkaline phosphatase-linked antibodies. The method of photoimmobilization used here leads to a homogeneous single layer of IgGs, in which the proteins maximize their contact with the surface. Residual adsorption of IgG on the nonirradiated surface of benzophenone remains one limitation of this approach. Progressively higher coverages of IgGs on the surface did not lead to strictly proportional changes of the biological activity of these surfaces, probably because of interactions between the IgGs in the film. This method of photoimmobilization is nonetheless useful as an experimental system to immobilize other proteins because it is simple, flexible, and efficient.

Association with MDCK epithelial cells by Salmonella typhimurium is reduced during utilization of carbohydrates.

Association of Salmonella typhimurium with MDCK epithelial cells in monolayers, represented primarily by intracellular bacteria after 30 min of contact, with centrifugation followed by vigorous washing, was measured during aerobic and anaerobic growth of the bacteria in brain heart infusion broth. Cell association was greatest during a short period in the late log phase of growth under aerobic conditions. At this time, the pH of the growth medium was changing from acid to alkaline and glucose (0.2% initially) was exhausted. Addition of excess glucose (0.5%) to brain heart infusion broth, which was not exhausted before the bacteria entered the stationary phase of growth, in which cell association dropped sharply, resulted in repression of cell association by the bacteria. The repressive effect of glucose on cell association could not be reversed by exogenous cyclic AMP in the bacterial growth medium. Under anaerobic conditions, the effect of glucose on cell association by the bacteria was not as great and the glucose was not exhausted before the bacteria entered the stationary phase. When S. typhimurium was grown in a rich but carbohydrate-free medium, cell association by the bacteria increased earlier in the growth cycle under both aerobic and anaerobic conditions. The addition of glucose and certain other utilizable carbohydrates to this medium caused a repression of cell association by S. typhimurium that was greater under aerobic growth conditions. These results show that cell association by S. typhimurium, which is accompanied by rapid internalization (cell invasion), is the same under aerobic and anaerobic conditions if the bacteria are grown to the log phase in a carbohydrate-free medium. This suggests that prior reports of greater cell invasion by S. typhimurium during anaerobic growth may have arisen from the use of media containing carbohydrates which were found to be more repressive during aerobic growth of the bacteria.

Identification and localisation of a synaptosomal membrane prolyl endopeptidase from bovine brain.

Prolyl endopeptidase, which has long been recognised for its importance in the degradation of several neuropeptides such as thyroliberin, luteinising hormone releasing hormone, angiotensin, substance P and neurotensin, has been widely characterised as a cytosolic enzyme. However, in this paper, we report the presence of a prolyl endopeptidase activity in the particulate fractions of bovine brain, which is distinct from that in the cytoplasm. This previously uncharacterised activity was found to reside in the synaptosomal membranes, a location which is highly significant for the inactivation of neuropeptides in brain. Following vigorous salt washing and osmotic shock, the prolyl endopeptidase activity was released from the membranes by treatment with the detergent Triton X-100, and was partially purified by gel filtration on a Sephacryl S-200HR column. This prolyl endopeptidase activity was shown to have a molecular mass (87 kDa) higher than the cytosolic prolyl endopeptidase but, from initial investigation, appears to demonstrate a similarly broad substrate specificity towards proline-containing neuropeptides. The partially purified enzyme was inhibited by certain thiol-protease inhibitors and was also found to be sensitive to the metal chelator 1,10-phenanthroline.