Purification and properties of the beta-fructofuranosidase from Kluyveromyces fragilis.

The beta-fructofuranosidase from Kluyveromyces fragilis was purified to one band on electrophoresis by 3 different methods. Two of the preparations were found to be impure by isoelectric focusing. This demonstrates the need for more than one criteria of homogeneity when purifying this enzyme. The enzyme was found to be a glycoprotein, stable at 50 degrees C, with a pH optimum of 4.5. The cations Hg2+, Ag+, Cu2+ and Cd2+ exhibited a marked inhibition of the enzyme. Competitive inhibition was observed with the fructose analog 2,5-anhydro-D-mannitol suggesting that the enzyme is inhibited by the furanose form of fructose.

A simple inulin assay for renal clearance determination using an immobilized beta-fructofuranosidase.

Current hospital practice for testing renal function is to use the creatinine clearance test. Inulin clearance, an inherently more accurate procedure, currently is only carried out by specialized laboratories because there is not a simple biochemical assay for inulin, that is, an assay that could be carried out by any laboratory without special facilities. We have developed a simple enzymatic assay system for measuring inulin in plasma and urine. The procedure uses a beta-fructofuranosidase immobilized on Concanavalin A to convert inulin to fructose. Fructose is then measured by measuring the NADH----NAD conversion produced when fructose is converted to sorbitol by the enzyme sorbitol dehydrogenase. Kinetic parameters, binding capacities, and operating conditions for the immobilized beta-fructofuranosidase were determined as well as general operating parameters for the complete assay system. This system offers the potential for replacing the creatinine clearance test as the assay of choice for renal function.

Microbial sterility testing of oil-formulated bovine somatotropin using Tween 80 dispersion.

The Food and Drug Administration requires that sterile pharmaceutical products be free of viable microorganisms. Sterility testing of pharmaceutical products provides added assurance that the product is sterile. Sterility testing is typically done by inoculating the drug product into microbial growth media followed by visual inspection for growth during incubation for a specified time period. A lack of visual growth indicates that the drug product samples tested were sterile. Formulated Posilac bovine somatotropin consists of protein particles suspended in an oil-based excipient. The product formulation is immiscible in aqueous media due to the excipient's water insolubility and the insolubility of the protein particles at near neutral pH values. Because the formulation is packaged and sold as a sterile product, it is critical that a sensitive microbial sterility test method be used for this key quality test. A sterility test method was developed for Posilac that utilized Tween 80 (i.e. polysorbate 80) as a dispersant. Dispersion of the product using Tween 80 produced a homogeneous suspension of bovine somatotropin particles and oil droplets in the micron size range. The suspension did not appreciably settle out with time, attesting to the homogeneous nature of the mixture. This method was found to be compatible with survival, recovery, and growth from low numbers of the test organisms required by the U.S. Pharmacopeia XXIII as well as from two additional test cultures.

Tritium concentrations inside the homes of occupationally exposed workers: dosimetric implications.

The average tritiated water concentration in the indoor air of the occupationally exposed worker's residence (55 Bq m(-3), range 53-59 Bq m(-3)) was higher than the indoor air of control residences (0.7 Bq m(-3), range 0.4-0.8 Bq m(-3)). The worker had an average concentration of tritium-in-urine of 30 kBq L(-1) from chronic intakes of occupational levels of tritiated water. Higher residential concentrations of tritiated water vapor were due to tritium transferred by the worker. Urine samples from an adult co-occupant were collected and had tritiated water concentrations between 89 and 345 Bq L(-1). These concentrations were higher than for individuals (range, 6-32 Bq L(-1)) living in other residences having similar outdoor and indoor concentrations of tritiated water in air. The range of measured tritiated water in urine was in agreement with the prediction of biokinetic models for tritium intakes as recommended by the International Commission on Radiological Protection Publication 56. The tritiated water vapor in the indoor air of the exposed worker's residence contributed about 96% of the daily tritium intakes. The annual average tritium dose to the family member (7 microSv) was well below the International Commission on Radiological Protection Publication 60 recommended annual dose limit (1 mSv) for members of the public. We conclude that, for a few members of the public living near a heavy-water research reactor facility, daily intakes of tritium will relate to tritiated water dispersed by the exposed worker, as well as to tritium transported by the atmosphere from the reactor site.

Menstrual history in altitude chamber trainees.

Previous studies have determined a higher rate of altitude-induced decompression sickness (DCS) in women than in men. Women are reportedly at higher risk for developing DCS during menses. A study of menstrual history in women completing altitude chamber training without developing DCS has never been accomplished. The purpose of this study was to collect and analyze menstrual history in these women. Thirteen U.S. Air Force Aerospace Physiology Units participated in a USAF-approved survey for 1 year. After completing altitude chamber flights, data on age, day of menstrual cycle (DMC), birth control pill use (BCP), and mean durations of menstrual cycle and menses were collected. There were 508 responses analyzed. There was no differences between mean duration of menstrual cycle and menses in the Yes (Y) and No (N) BCP groups. Y and N BCP groups were equally distributed across the menstrual cycle. Women completing altitude chamber training without developing DCS appear to be evenly distributed across their menstrual cycle, with use of BCPs not affecting their susceptibility to DCS.

Type II altitude decompression sickness (DCS): U.S. Air Force experience with 133 cases.

Type II altitude-related decompression sickness (DCS), due to its wide spectrum of symptoms, is often difficult to diagnose. This difficulty sometimes leads unnecessarily to the permanent grounding of an experienced aviator. So that this condition could be better understood, a total of 133 cases of Type II altitude DCS (on file at the United States Air Force Hyperbaric Medicine Division, School of Aerospace Medicine, Brooks AFB, TX) were reviewed. Most cases (94.7%) followed altitude chamber training. The most common manifestation was joint pain (43.6%), associated with headache (42.1%), visual disturbances (30.1%), and limb paresthesia (27.8%). The next most common symptoms were, in order of decreasing frequency: mental confusion (24.8%), limb numbness (16.5%), and extreme fatigue (10.5%). Spinal cord involvement, chokes, and unconsciousness were rare (6.9%, 6%, and 1.5%, respectively). Hyperbaric oxygen treatment produced fully successful results in 97.7% of the cases. Only 2.3% of the cases resulted in residual deficit; no deaths occurred. A thorough knowledge of the differential diagnosis and predisposing factors is essential to narrow the margins of error in the diagnosis and prevention of decompression sickness in the operational or training environment. A recommendation for favorable consideration of waiver action for those aviators who suffered Type II DCS is presented. These recommendations are based on a unique classification of the severity of symptoms.

Hyperbaric oxygen therapy and combat casualty care: a viable potential.

Extreme interest exists among the United States and Soviet military forces regarding the efficacy of hyperbaric oxygenation (HBO) for the treatment of wartime casualties. Specific areas of potential usefulness include the treatment of chemical agent exposure, crush injuries, and burns with HBO. Future direction dictates immediate scientific and medical support for the use of this powerful treatment modality.

Field measurements of key parameters associated with nocturnal OBT formation in vegetables grown under Canadian conditions.

The objective of this study was to provide the parameter values required to model OBT formation in the edible parts of plants following a hypothetical accidental tritium release to the atmosphere at night. The parameters considered were leaf area index, stomatal resistance, photosynthesis rate, the photosynthetic production rate of starch, the nocturnal hydrolysis rate of starch, the fraction of starch produced daily by photosynthesis that appears in the fruits, and the mass of the fruit. Values of these parameters were obtained in the summer of 2002 for lettuce, radishes and tomatoes grown under typical Canadian environmental conditions. Based on the maximum observed photosynthetic rate and growth rate, the fraction of starch translocated to the fruit was calculated to be 17% for tomato fruit and 14% for radish root.

Tritium profiles in snowpacks.

The release of tritiated water (HTO) to the atmosphere during the winter can contribute significantly to snow contamination and to water-soil-plant contamination after the spring thaw. The dose significance of such a release depends on the persistence of tritiated water in the snowpack, which is primarily controlled by the HTO diffusion process in snow and the rate of re-emission into the atmosphere from the snowpack surface. Monitoring data collected after an acute winter release at Chalk River Laboratories and data obtained in winter over a chronically contaminated area were analyzed to estimate the diffusion coefficient of HTO in the snowpack. Under conditions of cold and dry snow, the diffusion coefficient lay in the range 1-2x10(-10)m(2)s(-1), an order of magnitude lower than diffusion in water but an order of magnitude higher than self-diffusion in ice. These results confirm the theoretical predictions (Bales, 1991). Values up to six times higher were found for warmer periods and just before spring melt, when other processes contribute to profile evolution. The low diffusion rate of tritium in cold, dry snow means that tritium remains in the snowpack throughout the winter, to be released during spring thaw to potentially contaminate surface water, soil and crops.

Major issues in quality assessment and improvement of clinical hyperbaric services: an international perspective.

The continued rapid worldwide diffusion of clinical hyperbaric facilities has substantially increased interest in clinical quality assessment and service improvement. This paper examines major issues, perspectives, and methods integral to the measurement and improvement of the quality of care provided to hyperbaric patients and their relevance and applicability across different societies. Special focus is directed toward the importance of quality assessment and improvement of clinical hyperbaric care, multiple stakeholder perspectives on improved clinical quality, measurement of clinical outcomes of hyperbaric care, importance of facility accreditation, process improvement methods, and the future importance of quality management in clinical hyperbaric facilities.

Purification and Properties of beta-Glucosidase from Aspergillus terreus.

A beta-glucosidase (EC 3.2.1.21) from the fungus Aspergillus terreus was purified to homogeneity as indicated by disc acrylamide gel electrophoresis. Optimal activity was observed at pH 4.8 and 50 degrees C. The beta-glucosidase had K(m) values of 0.78 and 0.40 mM for p-nitrophenyl-beta-d-glucopyranoside and cellobiose, respectively. Glucose was a competitive inhibitor, with a K(i) of 3.5 mM when p-nitrophenyl-beta-d-glucopyranoside was used as the substrate. The specific activity of the enzyme was found to be 210 IU and 215 U per mg of protein on p-nitrophenyl-beta-d-glucopyranoside and cellobiose substrates, respectively. Cations, proteases, and enzyme inhibitors had little or no effect on the enzyme activity. The beta-glucosidase was found to be a glycoprotein containing 65% carbohydrate by weight. It had a Stokes radius of 5.9 nm and an approximate molecular weight of 275,000. The affinity and specific activity that the isolated beta-glucosidase exhibited for cellobiose compared favorably with the values obtained for beta-glucosidases from other organisms being studied for use in industrial cellulose saccharification.

The cell wall-associated inulinase of Kluyveromyces fragilis.

The yeast Kluyveromyces fragilis (ATCC 12424) was grown on a 2% inulin-1% yeast extract medium for 36 h and subsequently fixed with 0.5% glutaraldehyde. The glutaraldehyde treatment did not affect the beta-fructofuranosidase (inulinase, EC 3.2.1.7) activity of the cells but it did make the cells resistant to chemical and physical treatments that normally release beta-fructofuranosidase from untreated cells. The enzyme in the treated cells exhibited Km values for sucrose and raffinose identical to those obtained for the free enzyme. The cell wall of the treated cells exhibited the same diffusion properties for sucrose, raffinose, and inulin as those observed for untreated cells. The beta-fructofuranosidase was not bound covalently to the cell by the glutaraldehyde treatment. The results support the permeability barrier model for the enzyme retention in the yeast cell wall.

Enzymatic hydrolysis of inulin to fructose by glutaraldehyde fixed yeast cells.

Inulin, a polyfruction, is found as the reserve carbohydrate in the roots and tubers of various plants (i.e. Jerusalem artichoke, chicory, and dahlia tubers). The beta-fructofuranosidase (inulase) from the yeast Kluyveromyces fragilis is of interest because of its industrial potential in fructose syrup and alcohol production from inulin containing plants. We have found that the inulase of K. fragilis can be immobilized in the yeast cells by glutaraldehyde treatment. These cells are resistant to physical and enzymatic destruction. Although the exact nature of the immobilization is not fully understood, the kinetic parameters of the immobilized enzyme are similar to those of the soluble enzyme. No reduction of enzyme activity was observed after glutaraldehyde treatment and glutaraldehyde concentration did not affect enzyme activity. A 96% hydrolysis of dahlia inulin was achieved in 10.5 h with a 9.5% (w/v) fixed enzyme suspension. A Jerusalem artichoke extract containing 16.8%polyfructan was completely hydrolyzed in 3.5 h with a 0.24% (w/v)fixed enzyme suspension. This is a time frame feasible for industrial consideration.

Immunological interrelationships of Hong Kong, Asian and equi-2 influenza viruses in man.

Sera from an elderly population drawn prior to the 1968 Hong Kong influenza epidemic were found to have a high prevalence of Hong Kong antibody, indicating the circulation of a Hong Kong-like virus before 1900. This report compares an elderly (birth dates before 1892) and a young adult population with regard to serum antibody response to Hong Kong variant vaccine, avidity of Hong Kong antibody, and antibody absorption with Asian, A/Equi-2, and Hong Kong viruses.The demonstration of doubly-absorbable Hong Kong and A/Equi-2 antibodies in sera where both are present and of doubly-absorbable Hong Kong and Asian antibodies in young adult sera after Hong Kong variant vaccine lends new support to the view that there is an immunological relationship between these viruses.The difference in the response of the two age-groups to Hong Kong variant vaccine-specifically the anamnestic boosting of Hong Kong antibody together with the lack of Asian antibody rise in the elderly population in contrast to the striking Asian antibody response in the young adult population-leads to the conclusion that a Hong Kong-like virus may have been the original antigenic sin in the elderly population with initial Asian exposure in 1957. The excellent avidity of Hong Kong antibody in pre- and post-vaccine sera from the elderly suggests a close similarity if not identity of the haemagglutinins of the Hong Kong-like virus that circulated in human populations prior to 1900 and of the 1968 Hong Kong virus.

Biosynthesis and incorporation into protein of norleucine by Escherichia coli.

The methionine analog norleucine was produced during the synthesis of bovine somatotropin by Escherichia coli strain W3110G containing the recombinant plasmid pBGH1. Norleucine was generated by the leucine biosynthetic pathway from pyruvate or alpha-ketobutyrate in place of alpha-ketoisovalerate as the initial substrate. The intracellular level of norleucine was high enough to permit the analog to compete successfully with methionine for incorporation into protein. Two ways were found to prevent either the formation of norleucine or its incorporation into protein. The endogenous synthesis of norleucine was eliminated by deleting the leucine operon. The addition of sufficient methionine or 2-hydroxy-4-methylthiobutanoic acid, a precursor of methionine, to the culture medium prevented any norleucine from being incorporated into protein.

Cloning and Expression of an Insecticidal k-73 Type Crystal Protein Gene from Bacillus thuringiensis var. kurstaki into Escherichia coli.

A 75-kilobase plasmid from Bacillus thuringiensis var. kurstaki (HD-244) was associated with the k-73 type insecticidal crystal protein production by mating into B. cereus and subsequent curing of excess plasmids. This plasmid was partially digested with endonuclease R . Sau3A and the fragments were cloned into Escherichia coli (HB101) on vector pBR322. Candidate clones were screened for plasmid vectors which contained the expected insert size (at least 3 kilobases) and then with an enzyme-linked immunosorbent assay, using antisera prepared against electrophoretically purified, solubilized insecticidal crystal protein of 130,000 daltons. Several positive clones were isolated and were analyzed for expression, toxicity, and genetic content by restriction enzyme analysis. Electrophoretic transfer blots of proteins from a candidate E. coli clone, analyzed by enzyme-linked immunosorbent assay, demonstrated a predominant cross-reacting protein of about 140,000 daltons. Ouchterlony analysis also showed a single precipitin band. Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity (50% lethal dose per microgram of cross-reacting protein) equivalent to that of the parental B. thuringiensis strain or a B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.