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The preparation of nylon-tube-supported hexokinase and glucose 6-phosphate dehydrogenase and the use of the co-immobilized enzymes in the automated determination of glucose.

Triethyloxonium tetrafluoroborate was used to O-alkylate nylon-tube thus producing the imidate salt of the nylon which was further made to react with 1,6-diaminohexane. 2. Hexokinase (EC 2.7.1.1) and glucose 6-phosphate dehydrogenase (EC 1.1.1.49) were immobilized on the amino-substituted nylon tube through glutaraldeyde and bisimidates. 3. The effect of varying the conditions of O-alkylation and the amount of enzyme immobilized on the activity of nylon tube-hexokinase derivatives was determined. 4. The effect of varying the amount of enzyme immobilized on the activity of nylon-tube-glucose 6-phosphate dehydrogenase derivatives was determined. 5. The thermal stability of nylon-tube-hexokinase and nylon-tube-glucose 6-phosphate dehydrogenase derivatives was studied. 6. Different ratios of hexokinase and glucose 6-phosphate dehydrogenase were co-immobilized on nylon tube, and the rate of conversion of glucose into 6-phosphogluconolactone was compared with the individual activities of the immobilized enzymes. 7. Hexokinase and glucose 6-phosphate dehydrogenase co-immobilized on nylon tube were used in the automated analysis of glucose.

Alkylation

Detoxification of organophosphate pesticides using a nylon based immobilized phosphotriesterase from Pseudomonas diminuta.

A partially purified phophotriesterase was successfully immobilized onto nylon 6 and 66 membranes, nylon 11 powder, and nylon tubing. Up to 9000 U of enzyme activity was immobilized onto 2000 cm2 of a nylon 6 membrane where 1 U is the amount of enzyme necessary to catalyze the hydrolysis of 1.0 mumol of paraoxon/min at 25 degrees C. The nylon 66 membrane-bound phosphotriesterase was characterized kinetically where the apparent Km value for the immobilized enzyme was 0.35 mM. This is 5-6 times higher than that observed for the soluble enzyme. However, nylon immobilization limited the maximum rate of paraoxon hydrolysis to less than 10% of the value measured for the soluble enzyme. The addition of the cosolvent, methanol, resulted in an increase in the apparent Km value for paraoxon hydrolysis but concentrations up to 40% had no negative effect on the catalytic effectiveness with the soluble or immobilized phosphotriesterase. Based on the kinetic analysis, methanol appears to be a competitive inhibitor for both forms of enzyme. The nylon powder immobilized enzyme was shown to be stable for at least 20 mo. The immobilization of the phosphotriesterase onto nylon provides a practical method for the detoxification of organophosphate pesticides.

Aryldialkylphosphatase

Changes in the structure of intra-ocular nylon.

When using a nylon thread for the attachment of a pseudophakos to the iris, it may happen that the suture is slung tightly around the implant-lens. In such a case this constitutes a clinical experiment where is one and the same eye one part of the nylon thread is in its normal state (the cut-off ends) and the rest is in a stretched state, both under the same environmental conditions and for the same length of time. The differences in structure of the parts of the nylon thread are thus fully comparable. Clinical observatons show that when the nylon thread is under tension it will be eroded by chemical influences. It does not make any difference whether the nylon is used for corneal, scleral or intra-ocular suturing. The duration of the observations is still too shor to predict the behaviour of nylon which is not stretched.

Cataract Extraction

The preparation of several new nylon tube-glucose oxidase derivatives and their incorporation into the "reagentless" automated analysis of glucose.

Nylon tube was activated by alkylation with dimethyl sulfate and used for the immobilization of glucose oxidase. Lysine, hexamethylene diamine and polyethylene imine were also attached to activated nylon tube, and these nylon tube-spacer derivatives were reactivated with either glutaraldehyde or ethyl adipimidate for the subsequent coupling of glucose oxidase. The activities of all of the different nylon tube-glucose oxidase derivatives were compared by their incorporation into standard Technicon automated analysis systems. Activities were measured either spectrophotometrically, by following the production of hydrogen peroxide using an acid/KI assay, or polarographically by following the decrease in the oxygen concentration using a flow-through oxygen electrode assembly. The activity and stability of all of the nylon tube-glucose oxidase derivatives was such that their use in the routine estimation of glucose levels was an attractive proposition.

Amines

The reversible immobilization of proteins on nylon activated through the formation of a substituted imidoester, and its unusual properties.

Alkylation of nylon produces nylon imidate, which is used for the covalent coupling of enzymes and other proteins and ligands. Nylon imidate is unusually stable when stored wet, with a half-life of about 60 days. Reaction with enzymes has an optimum about pH 7-8 and is extremely rapid, with about 60% of the reaction being complete in the first 10 min. The amidine formed in reaction with an amino group can be displaced by another nucleophile. In view of this finding that nylon amidine is capable of exchanging proteins, it is now concluded that nylon-tube reactors containing immobilized enzymes made by this method may not be used as extracorporeal shunts or as on-line monitors on patients, since the enzyme in the reactor may be released into the circulation by nucleophiles in the blood. This can lead to complications. The chemistry of this displacement reaction is discussed.

Amidines

Running nylon suture dissolution after penetrating keratoplasty.

We attempted to evaluate whether the low degrees of astigmatism achieved early in the postkeratoplasty period with the combined interrupted/running suturing technique were maintained for long periods of time. For 13 to 70 months (mean, 30.2 months), we monitored a group of patients (25 eyes) who had previously undergone the combined interrupted/running suturing technique (12 interrupted 10-0 nylon sutures and one running 11-0 nylon suture). Nine running sutures broke spontaneously, causing a significant increase of the keratometric astigmatism of the entire population from 1.7 +/- 1.6 to 3.4 +/- 2.6 diopters (mean +/- standard deviation). The mean vector-corrected change in astigmatism after suture breakage was 4.9 +/- 2.6 diopters. Surgical procedures to reduce astigmatism were required in many of the eyes in which the 11-0 running nylon suture broke. Our results suggested that 11-0 nylon is not an ideal material for the running suture because its high rate of spontaneous disruption leads to undesired, statistically significant increases in postkeratoplasty astigmatism. Further, our results indicated that the selective suture technique can maintain low degrees of astigmatism only if the sutures remain intact. Studies of the effect of keratoplasty suturing techniques on astigmatism should probably include a follow-up that is sufficiently long to indicate its long-term value to the patient.

Adult

Degradation of circulating DNA by extracorporeal circulation over nuclease immobilized on nylon microcapsules.

Studies were undertaken to determine whether deoxyribonuclease I, (DNase I) once immobilized on activated nylon microspheres, would be capable of degrading circulating DNA in vitro and in vivo in an extracorporeal circulation system in dogs. Nylon microspheres were prepared and after gentle hydrolysis and glutaraldehyde treatment, demonstrated a retention of up to 4.73 mg of Dnase I. In vitro studies showed that DNase I immobilized on microspheres degreded a significant percentage of 125I-native DNA (nDNA) within 15 min. Mongrel dogs were injected with 125I-nDNA and a variation in initial t 1/2 in individual animals was observed. Therefore, for experimental studies, 125I-nDNA was injected and decay was recorded during a control period in which untreated microcapsules were utilized in the extracorporeal system. DNase I microspheres were then introduced into the extracorporeal circuit which resulted in an acceleration of degradation of acid precipitable 125I-nDNA. When 200 mug of unlabeled DNA with 125I-nDNA was injected, a similar augmentation of DNA degradation was noted after extracorporeal circulation over DNase I microcapsules. This effect could not be attributed to release of DNase I from the microspheres since no 131I-DNase was detected in the serum or organs of the dogs at the conclusion of the experiments. 125I-nDNA:anti-DNA complexes were passively injected into dogs and after a similar control period of circulation over untreated microcapsules. DNase I microspheres were introduced. Results showed a rapid acceleration in the degradation rate of 125I-nDNA:anti-DNA complexes precipitable with (NH4)2SO4. Extracorporeal circulation over nylon microspheres resulted in no significant alteration of the host's hematocrit or platelet count, and little residual cellular debris on the microcapsules. These data suggest that DNAase immobilized on nylon microspheres may have a potential role in the specific therapy of systemic lupus erythematosus, when it is desirable to hydrolyze DNA circulating free or in combination with antibody.

Animals

Comparative adherence of granulocytes to endothelial monolayers and nylon fiber.

Adherence of granulocytes to tissue culture monolayers of endothelium averaged 26.2 +/- 1.3% SEM, which was similar to their adherence on 50-mg nylon fiber columns (27.7 +/- 3.6%). In contrast, adherence to epithelial cells, fibroblasts, kidney cells, and plastic Petri dishes without monolayers was only 12.4, 9.9, 11.1, and 4.3%, respectively. Cyclic nucleotides and adherence-modifying plasma factors induced changes of adherence to endothelium similar to those in nylon fiber columns. Adherence of granulocytes in whole blood was the same as for purified granulocytes in Hank's balanced salt solution. Exposure of endothelial monolayers to 0.18% trypsin for 10 min reduced subsequent granulocyte adherence to 25.2% of control values. Incubation of trypsin-treated monolayers with nutrient medium for 4 h did not improve adherence, but values returned to normal or above by 24 h, with or without serum proteins present in the nutrient medium. The similarity of granulocyte adherence to nylon fiber and to endothelial monolayers in vitro suggests that results with the nylon fiber assay reflect in vivo granulocyte-endothelium interaction. Furthermore, the endothelial monolayer offers a new model for studying this cell-cell relationship in vitro.

Adhesiveness

Nylon-fiber-induced neutrophil fragmentation.

We have investigated the effects of mechanical elution of neutrophils from nylon-wool fiber (NWF) using the scanning electron microscope and biochemical analysis of elution fractions. We have determined that mechanical removal of neutrophils from nylon-wool fiber disrupts neutrophils adherent to nylon-wool fiber and augments release of granules, release of peripheral cytoplasmic fragments, and release of lactic dehydrogenase, a soluble cytoplasmic enzyme. Mechanical shearing of the adherent cell, and not adherence per se, causes the fragmentation. The extent of fragmentation is proportional to the NWF surface area available to neutrophils and is maximal at the temperature for optimal adherence and spreading. Agents that decrease cell spreading (n-ethylmaleimide and cold) diminish fragmentation. Cytochalasin B, an agent that destabilizes the neutrophil cortex, increases fragmentation. Fragmentation may be an important contributing cause of the abnormal morphology, function, and in vivo survival of nylon-wool-fiber procured human neutrophils. The prevention of fragmentation would appear to be necessary to insure the procurement of optimally functioning cells. Elution of NWF-adherent neutrophils in the cold might be a practical way to diminish neutrophil damage during clinical filtration leukapheresis.

Cell Adhesion

Extraction of 'nylon' teeth and associated abnormalities in Tanzanian children.

The aim of this study is to determine the prevalence of extraction of 'nylon' teeth and its long term adverse effects on the permanent dentition of Tanzanians. A total of 1890 subjects, aged between 3 and 5 years, and 1377 subjects aged between 12 and 19 years, in different parts of the country were examined. The prevalence of missing teeth in the primary dentition due to 'nylon' teeth practice ranged from 0% to 16.9%, with a weighted mean of 9.5%. Canines were the most involved teeth, accounting for 95% of all missing teeth. Mutilated permanent teeth, a result of traditional extraction of deciduous tooth germs, were observed only in Singida district in 8% of the examined subjects. 22.8% of these mutilations presented as malformations, while the remaining 77.2% presented as missing teeth. Most missing teeth and all the malformed teeth were seen in the mandible. The mandibular canines were the most affected, followed by the mandibular lateral incisors. It is suggested that 'nylon' teeth practice in Tanzania may be a recent invention rather than a traditional tribe related custom. The precariously high prevalence of 'nylon' teeth practice in Tanzania, and the people's perception of this malpractice, requires that particular attention be paid to this issue during oral health educational programmes in the affected communities.

Adolescent

Specific removal of bovine serum albumin (BSA) antibodies in vivo by extracorporeal circulation over BSA immobilized on nylon microcapsules.

Studies were undertaken to determine whether BSA, once immobilized on activated nylon microspheres, would be capable of specifically removing circulating BSA antibody in vitro and in vivo in an extracorporeal circulation system in dogs. Nylon microspheres were prepared and, after gentle hydrolysis and glutaraldehyde treatment, demonstrated a retention of up to 34.5 mg of BSA. In vitro studies showed that BSA immobilized on microspheres removed a significant percentage of BSA-binding activity. For in vivo studies, an extracorporeal circulation system was established and mongrel dogs were then injected with anti-BSA and anti-HSA antibodies. After an equilibration period, BSA microspheres were introduced into the extracorporeal circulation system. After the insertion of BSA microcapsules, serum exhibited a sharp reduction in BSA binding over the next 15 min, with a more gradual diminution over the ensuing 60 to 90 min. There was no significant reduction in anti-HSA binding over the same time frame. This effect could not be attributed to release of BSA from the microspheres since no 125I-BSA was detected in the serum or organs of the dogs at the conclusion of the experiments. After extracorporeal circulation over nylon microspheres, there were only minimal changes in the host's hematocrit or leukocyte counts and no significant thrombotic material or cellular debris was recoverable on the capsules. These data suggest that antigen immobilized on nylon microspheres may specifically withdraw circulating antibodies in vivo with minimal release of its antigenic material and little alteration in the host's hematologic status.

Animals

Intradermal sutures in facial lacerations. Comparative study of clear monofilament nylon and polyglycolic acid.

A blind study comparing the clinical inflammatory response of intradermal clear monofilament nylon sutures with polyglycolic acid sutures was carried out on 106 patients with facial lacerations. There were no clinically apparent infections in either group of sutures. Intradermal monofilament nylon was associated with significantly less inflammatory response than similarly placed polyglycolic acid sutures. The lesser inflammatory response, the tolerance to infection, and the greatly prolonged tensile strength of monofilament nylon are factors that lend support to its being the superior intradermal suture in facial lacerations.

Adolescent

The influence of penicillin on experimental wound contamination with staphylococci: studies with chromic catgut and monofilament nylon closure.

This study examines the ability of penicillin to eradicate or modify the pattern of infection in experimental wounds contaminated with Staphylococcus pyrogenes in the presence of chromic catgut or monofilament nylon suture. Penicillin therapy initiated within 4 days of the onset of infection was successful in eradicating infection when compared with untreated controls (P less than 0-01). Infection was modified but not eradicated in penicillin was administered from the seventh day. The response of infection to penicillin was identical in wounds containing monofilament nylon or chromic catgut over a 21-day period. Antibiotics given before wound drainage permitted healing of all chronically infected wounds even in the presence of monofilament nylon. When antibiotics were begun at the time of drainage, wound healing occurred in 50 per cent (P less than 0-01).

Animals

Adjustable nylon ties for abdominal wall closure.

A study in 64 patients with a high risk of wound dehiscence or eviscerated wounds or in whom repeat laparotomies for peritoneal lavages due to sepsis were planned was accomplished using adjustable nylon ties for the closure of the abdominal wall, either as retention sutures or as one-layer sutures. The adjustable nylon ties are described, as well as the technique for placing and adjusting them. It is concluded that the adjustable nylon ties can substitute advantageously for the conventional retention sutures since the former are wider, easy to place, and can be readjusted to the point that they can be opened completely if it is necessary and then closed again. This is especially useful when repeat laparotomies for peritoneal lavages are required in patients with intra-abdominal sepsis.

Abdominal Muscles

Altered surface topology and membrane functions of rat thymocytes eluted from nylon wool columns.

(1) Following incubation of thymocytes with nylon wool at 37 degrees C, the eluted cells showed an increase in the number of microvilli per cell and a concominant elongation of the microvilli (0.22 mum versus 1.15 mum. (2) Cyclic adenosine monophosphate (cylic AMP) levels were lowered by 30-50% in nylon wool-treated thymocytes. (3) Nylon wool-treated cells showed an impaired Na+-dependent amino acid transport system (2-aminoisobutyrate) whereas the Na+-independent amino acid transport system (1-aminocyclopentane-1-carboxylate) was unaffected.

Amino Acids

Electrophoretic mobility of human lymphocytes purified by nylon wool columns and spontaneous sheep red blood cells rosetting techniques correlation with immunofluorescence.

Cell electrophoresis allows separation of normal human lymphocytes into two principal groups which are a function of their relative rates of migration. In 42 healthy adults, 19.9% of the lymphocytes have a slower migration rate, and 80.1%, a faster migration rate than the reference speed (1 mum - sec-1 - cm). Two methods are used for the selection of the lymphocytic populations: spontaneous rosetting with sheeps red blood cells, a property of T-lymphocytes, and adherence to nylon wool columns, which preferentially selects B-lymphocytes. The cells which do not form spontaneous rosettes, but adhere to nylon wool columns show mainly slow migration. Cells which do not adhere to nylon columns show a faster migration rate. These findings affirm the T-nature of the rapidly migrating lymphocytes, and the B-nature of the slow-migrating lymphocytes. Results by the immunofluorescence technique confirm this.

Adolescent

A new nylon oligomer degradation gene (nylC) on plasmid pOAD2 from a Flavobacterium sp.

Flavobacterium sp. strain KI725 harbors plasmid pOAD21, a derivative of nylon oligomer-degradative plasmid pOAD2, in which all of nylA (the gene for 6-aminohexanoate cyclic dimer hydrolase [EI]) was deleted but nylB (the gene for 6-aminohexanoate dimer hydrolase [EII]) was retained. KI725 showed no growth on unfractionated nylon oligomers (Nom1) obtained from a nylon factory as a sole carbon and nitrogen source (Nom1 minimum plate). Extracts of KI725 cells possessed hydrolytic activity for Nom1 (approximately 5% of the activity of KI72), but pOAD2-cured strains (KI722 and KI723) showed no activity. KI725R strains which grew on the Nom1 minimum plate were spontaneously isolated from KI725 at a frequency of 10(-7) per cell. Activity toward Nom1 was enhanced in KI725R strains (10 to 30% of the activity of KI72). This new Nom1 degrading enzyme (EIII, the nylC gene product) hydrolyzed not only Nom1 but also the N-carbobenzoxy-6-aminohexanoate trimer, a substrate which was not hydrolyzed by either EI or EII. Cloning and sequence analysis showed that the nylC gene is located close to nylB on pOAD21 and is a 1,065-bp open reading frame corresponding to 355 amino acid residues. The nucleotide sequence of the nylC gene and the deduced amino acid sequence of EIII had no detectable homology with the sequences of nylA (EI) and nylB (EII).

Amidohydrolases

Antibacterial multifilament nylon sutures.

Multifilament nylon fibers were made antibacterial by dopping with iodine. Nylon fibers were immersed in acetone solution of iodine for 48 hours at room temperature for dopping of iodine. It was observed that iodine uptake by the nylon fibers increased with the increase in concentration of iodine in the solution. Antibacterial activity of these iodine dopped samples was evaluated by measuring the zone of inhibition. The bacterial species used for this study were Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae. Iodine dopped fibers exhibited good antibacterial activity against these bacterial species. Release of iodine in distilled water is sustained for about 30 days. Antibacterial activity of the fibers decreases with the release of iodine in water. Ultra-violet and visible spectroscopic studies showed that tri-iodide ions were released from the dopped samples in the aqueous medium. These I3- ions might be responsible for the observed antibacterial activity. Fiber shrinks on iodine dopping leading to increase in the denier of the fiber. However effect of iodine dopping on the breaking load of fibers is not significant.

Anti-Infective Agents, Local