Vaginal estriol to overcome side-effects of aromatase inhibitors in breast cancer patients.

OBJECTIVE: Aromatase inhibitors are essential as endocrine treatment for hormone receptor-positive postmenopausal breast cancer patients. Menopausal symptoms are often aggravated during endocrine treatment. We investigated whether vaginal estriol is a safe therapeutic option to overcome the urogenital side-effects of aromatase inhibitors. Serum hormone levels were used as the surrogate parameter for safety. METHODS: Fasting serum hormone levels of ten postmenopausal breast cancer patients receiving aromatase inhibitors were prospectively measured by electro-chemiluminescence immunoassays and gas chromatography/mass spectrometry before and 2 weeks after daily application of 0.5 mg vaginal estriol (Ovestin® ovula), respectively. RESULTS: Two weeks of daily vaginal estriol treatment did not change serum estradiol or estriol levels. However, significant decreases in levels of serum follicle stimulating hormone (p = 0.01) and luteinizing hormone (p = 0.02) were observed. Five out of six breast cancer patients noticed an improvement in vaginal dryness and/or dyspareunia. CONCLUSIONS: The significant decline in gonadotropin levels, indicating systemic effects, has to be kept in mind when offering vaginal estriol to breast cancer patients receiving an aromatase inhibitor.

The kinetics of binding of serum lipoproteins by immobilized heparin.

A model arterial system of heparin immobilized on an agarose gel was used to study the amount and kinetics of binding of porcine serum lipoproteins to heparin. Binding occurred to lipoproteins in the density range 1.006 less than d less than 1.062, but there was no binding with high density lipoprotein. A theoretical model of the kinetic experiments was formulated and used to demonstrate that the rate of the binding reaction could be considered instantaneous relative to the rate of transport of lipoproteins. Extrapolation of these results to arterial levels of glycosaminoglycans and lipoprotein indicate that complexes of lipoprotein and the glycosaminoglycans could account for much of the cholesterol entrapment in atherosclerotic lesions.

Influence of glycosaminoglycan content of mass transfer behavior of porcine artery wall. Part 1. Diffusive transport of 45Ca2+ and 3HHO.

The diffusion coefficients of Ca2+ and H2O in in vitro porcine arterial tissue were determined to provide a measure of vascular permeability. The effect of anatomical location and diet on the magnitude of that permeability was evaluated. The diffusion coefficient of calcium was found to vary focally and, in addition, pulmonary artery was much more permeable than thoracic aorta. Four months feeding of a high lipid diet did not affect diffusive transport.

Influence of glycosaminoglycan content on mass transfer behavior of porcine artery wall. Part 2. Differences in mass transfer rates related to variations in glycosaminoglycan content.

It has been suggested that the glycosaminoglycans (GAG) influence atherogenesis by regulating the permeability of the arterial wall. For this reason, a study has been made of the diffusive transport of Ca2+ and water across the in vitro porcine artery wall, where particular attention was focused on the influence of GAG content and distribution within the wall on the transport properties. The radioisotop-s 45Ca and 3HHO were used to measure the tracer-diffusion flux in a stirred, two chamber diffusion cell. GAG were isolated, fractionated using a cetyl pyridinium chloride-cellulose column procedure, and assayed using a colorimetric carbazole reaction for uronic acid. The biochemical analyses showed that the pulmonary artery contains significantly more hyaluronic acid and dermatan sulfate than found in two locations in the thoracic aorta. In addition, a significant regression was found for the diffusion coefficient of 45Ca2+ (99% level) and 3HHO (95% level) versus specific GAG fractions. The regression indicated an increase in permeability with increase in the ratio of sulfated: nonsulfated GAG.

A new ELISA-based assay for quantitation of human T-lymphocyte subpopulations.

In order to circumvent the problems associated with the available methods, we have developed a simple, reliable ELISA for quantitation of T-cells and their subpopulations, T-helper/inducer and T-suppressor/cytotoxic cells. Standard curves with three concentrations of sheep anti-mouse IgG-coupled beads for each T-cell population were used for the determination of the T-cell content in blood. Enzyme-labelled monoclonal antibodies against T-Pan, T-S and T-H cell surface markers were readily able to bind to such beads and the test system was calibrated with T-lymphocytes by comparing cytofluorographic and enzyme immunometric results. Purified preparations of monocytes and granulocytes were negative in the test. Lymphocytes from 50 healthy blood donors gave results which correlated closely with cytofluorograph determinations.

Polyelectrolyte precipitation of beta-galactosidase fusions containing poly-aspartic acid tails.

Protein recovery from industrial microbial processes can be very expensive, often exceeding the cost of protein production. We have genetically engineered 3 beta-galactosidase (beta-gal) fusion proteins containing poly-aspartic acid tails to test the effect of the tails on recovery by the relatively inexpensive method of polyelectrolyte precipitation. The fusion proteins, designated T1, T2, and T3, were constructed with C-terminal tails of 5, 11, and 16 aspartic acid residues, respectively. The fusion proteins were expressed in Escherichia coli, and purified by affinity chromatography. T1 and T2 had specific activities similar to that of wildtype beta-gal, whereas the specific activity of T3 was about half that of T1 and T2. The increased net charge of the fusion proteins compared to wildtype beta-gal was indicated both by ion-exchange chromatography and their migration pattern in non-denaturing polyacrylamide gel electrophoresis. All three tails enhanced polyethyleneimine (PEI) precipitation of the fusion proteins compared to wildtype beta-gal. At a low PEI/protein ratio (0.01, g g-1), recovery by precipitation of T2 and T3 was more than 2 X that of the beta-gal control, whereas that of T1 was only slightly greater than that of the control. At a higher PEI/protein ratio (0.03, g g-1) the amount of precipitation of all three fusion proteins was nearly the same, about 1.5 X that of the control.

Extraction of charged fusion proteins in reversed micelles: comparison between different surfactant systems.

Behavior of a series of fusion proteins of varying charge in reversed micellar extraction was studied. The proteins consisted of the enzyme glucoamylase from Aspergillus awamori joined to short peptides containing from 0-10 additional aspartate residues. The fusions were partitioned into two different cationic surfactant systems, one based on the surfactant trioctylmethylammonium chloride (TOMAC) and the other on cetyltrimethylammonium bromide (CTAB). These two systems differed chiefly in micelle size as measured by the water to surfactant ratio, wo. Water numbers were determined for the TOMAC system, with values of approximately 10, and as a function of pH and ionic strength for CTAB for each of the mutant enzymes. For the CTAB system, water numbers were as low as 50 with NaCl concentrations of 500 mM and as high as 68 at 300 mM NaCl (95% confidence level of 2.4). The enzyme partitioned most strongly using CTAB, with maximal recoveries approaching 95%. However, in the CTAB system, there were no significant differences in behavior between the mutants because of the relatively large micellar size, even under high salt concentrations. Extraction of the control enzyme from clarified cell broth indicated that broth components did not significantly interfere with partitioning.

Reversed micellar extraction of charged fusion proteins.

We have investigated the use of charged fusion tails with the enzyme glucoamylase in reversed micellar extraction. The addition of the charged tails increased the fraction of enzymatically active protein recovered at a given pH, with the tails containing the largest number of charges being recovered at the highest level. The series of mutations also allows for investigation of the charge-dependent behavior of reversed micellar extraction. However, in this case, the change in protein charge via fusions had a lesser impact than did the change in charge via a pH change. The difference may be due to the difficulty of partitioning the hydrodynamically larger fusion protein.

Precipitation of nucleic acids with poly(ethyleneimine).

Removal of nucleic acids from cell extracts is a common early step in downstream processing for protein recovery. We report on the precipitation of nucleic acids from a homogenate of Saccharomyces cerevisiae by addition of the cationic polyelectrolyte poly(ethyleneimine) (PEI), focusing on the effect of PEI dosage on particle size, protein loss, and extent of nucleic acid removal in both batch and continuous mode. Better than 95% removal of nucleic acids from yeast homogenates was achieved by means of precipitation with PEI with protein losses of approximately 15% with or without previous removal of cell debris. The coprecipitated protein is predominately large molecular weight material and exhibits both low and high isoelectric points. Such treatment does not aggregate the cell debris; size distribution of the precipitated particles from a continuous precipitator is very similar to that for protein precipitation.

Genetic engineering strategies for purification of recombinant proteins from canola by anion exchange chromatography: an example of beta-glucuronidase.

The elution behavior of native canola proteins from different anion-exchange resins was determined. The elution profiles showed the potential for simplified recovery of acidic recombinant proteins from canola. When Q-sepharose fast flow was used, there were three optimal salt elution points at which a recombinant protein would have minimal contamination with native proteins. The feasibility of exploiting this advantage was examined for recovery of the acidic protein beta-glucuronidase (GUS/GUSD0 from the Escherichia coli gene) along with three polyaspartate fusions to the wild-type GUS. The fusions contained 5 (GUSD5), 10 (GUSD10), or 15 (GUSD15) aspartic acids fused to the C-terminus and were chosen to extend the elution time. The three fusions and the wild-type enzyme were produced in E. coli, purified, and added to canola extracts before chromatography. The equivalence of this spiking experiment to that of extracting a recombinant protein from transgenic canola was determined in a control experiment using transgenic canola expressing the wild-type enzyme. Behavior in the transgenic and spiked experiments was equivalent. GUSD0 eluted at the earliest optimal elution point; the addition of polyaspartate tails resulted in longer retention times and better selective recovery. If one assumes binding through a single fusion (the protein is a tetramer), there is a nearly linear shift in elution within the salt gradient of 17 mM per added charge up to 10, with a reduced increment from 10 to 15. The fusions and their enzymatic activity proved very stable in the canola extracts through 7 days in cold storage, providing flexibility in process scheduling.

Characterization and polyelectrolyte precipitation of beta-galactosidase containing genetic fusions of charged polypeptides.

Genetically engineered versions of beta-galactosidase were constructed through the addition of charged polypeptide fusion tails for the purpose of enhancing polyelectrolyte precipitation. Negatively charged aspartic acid tails and positively charged poly(arginine) tails were added to beta-galactosidase from Escherichia coli. These fusion proteins were all shown to possess specific activity equal to that of the native enzyme. Gel permeation and ion-exchange chromatography provided evidence concerning the integrity of the tails as well as their altered charge characteristics. All enzymes containing charged tails displayed enhanced polyelectrolyte precipitation over the native enzyme. An optimal number of charged residues, beyond which no further enhancement of precipitation was observed, was found to be approximately 10 residues for each type of tail. No interference from nucleic acids was observed in the precipitation of positively tailed beta-galactosidase.

Enhanced recovery and purification of Aspergillus glucoamylase from Saccharomyces cerevisiae by the addition of poly(aspartic acid) tails.

Poly(aspartic acid) tails of different lengths were fused to the glucoamylase (GA) of Aspergillus awamori by genetic engineering techniques. Tails consisting of 5, 7, and 10 aspartate residues were fused to the N-terminus of the full-length mature GA (aa 1-616) downstream from the intact leader peptide to produce fusion proteins designated GAND5, GAND7, and GAND10, respectively. Three fusion proteins with C-terminal tails were also constructed, designated GACD0, GACD5, and GACD10 (0, 5, and 10 aspartate residues, respectively). For the C-terminal fusion proteins, the tails were fused to a catalytically active but truncated form of GA (aa 1-484). All of the charged tails had the general sequence Met-Ala-Aspn-Tyr, where n = 0, 5, 7, or 10. The modified genes were expressed in the yeast Saccharomyces cerevisiae and the proteins secreted into the culture medium. The enzymes were subsequently purified by affinity chromatography. The specific activity of each purified enzyme was found to be comparable to the wild-type enzyme. The C-terminal tails did not interfere with expression, whereas decreased extracellular glucoamylase activities corresponding to increased tail length were found for the N-terminal fusion proteins. Amino-terminal amino acid sequence analysis of the purified GAND proteins confirmed the authenticity of the amino termini of the modified proteins and showed that both the leader peptidase and KEX2 protease cleavages had occurred faithfully. The increased net negative charge of the GAND and GACD proteins was indicated by both nondenaturing PAGE and isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of chlortetracycline potentiation with citric acid in the chicken.

Serum concentrations of chlortetracycline (CTC) in healthy chickens were determined for the 24-hour period after they were given CTC (with and without citric acid) as an oral (25 mg/kg) or IV (0.9 mg/kg) dose. The oral time-course drug data were fitted adequately by a 2-compartment pharmacokinetic model with absorption. The resulting absorption rate constant (Ka) for the birds orally given CTC with citric acid was nearly equal to that for the birds given CTC alone. Although the uptake of orally administered CTC was rapid, only a small fraction of the dose was absorbed. The administration of citric acid-CTC significantly increased the mean serum concentration of CTC and the fraction of the dose absorbed. The citric acid-CTC mixture also produced significantly higher elimination (Kel) and distribution (K12) rate constants for CTC.

Oral absorption of chlortetracycline in turkeys: influence of citric acid and Pasteurella multocida infection.

Plasma and tissue concentrations, following the oral administration of the antibiotic chlortetracycline (CTC) alone or with citric acid, were determined in healthy and infected (Pasteurella multocida) turkeys. The principal results were: 1) The dose (of CTC) versus plasma level relationship was nearly linear. 2) Addition of citric acid to an oral preparation produced significantly higher plasma levels when divalent cations Ca2+ (.3 g/liter) and Mg2+ (.1 g/liter) were present in the drinking water and dosage solution than when citric acid was omitted. 3) The concentration of CTC was considerably higher in the liver and kidney than in the muscle and brain. 4) Birds infected with P. multocida had significantly higher plasma levels than healthy birds. 5) Oral administration of CTC increased the survival rate of the birds infected with P. multocida.

Production and characterization of microbial biosurfactants for potential use in oil-spill remediation.

Two biosurfactants, surfactin and fatty acyl-glutamate, were produced from genetically-modified strains of Bacillus subtilis on 2% glucose and mineral salts media in shake-flasks and bioreactors. Biosurfactant synthesis ceased when the main carbohydrate source was completely depleted. Surfactin titers were ∼30-fold higher than fatty acyl-glutamate in the same medium. When bacteria were grown in large aerated bioreactors, biosurfactants mostly partitioned to the foam fraction, which was recovered. Dispersion effectiveness of surfactin and fatty acyl-glutamate was evaluated by measuring the critical micelle concentration (CMC) and dispersant-to-oil ratio (DOR). The CMC values for surfactin and fatty acyl-glutamate in double deionized distilled water were 0.015 and 0.10 g/L, respectively. However, CMC values were higher, 0.02 and 0.4 g/L for surfactin and fatty acyl-glutamate, respectively, in 12 parts per thousand Instant Ocean®[corrected].sea salt, which has been partly attributed to saline-induced conformational changes in the solvated ionic species of the biosurfactants. The DORs for surfactin and fatty acyl-glutamate were 1:96 and 1:12, respectively, in water. In Instant Ocean® solutions containing 12 ppt sea salt, these decreased to 1:30 and 1:4, respectively, suggesting reduction in oil dispersing efficiency of both surfactants in saline. Surfactant toxicities were assessed using the Gulf killifish, Fundulus grandis, which is common in estuarine habitats of the Gulf of Mexico. Surfactin was 10-fold more toxic than fatty acyl-glutamate. A commercial surfactant, sodium laurel sulfate, had intermediate toxicity. Raising the salinity from 5 to 25 ppt increased the toxicity of all three surfactants; however, the increase was the lowest for fatty acyl-glutamate.

Mechanisms of aqueous extraction of soybean oil.

Aqueous extraction processing (AEP) of soy is a promising green alternative to hexane extraction processing. To improve AEP oil yields, experiments were conducted to probe the mechanisms of oil release. Microscopy of extruded soy before and after extraction with and without protease indicated that unextracted oil is sequestered in an insoluble matrix of denatured protein and is released by proteolytic digestion of this matrix. In flour from flake, unextracted oil is contained as intact oil bodies in undisrupted cells, or as coalesced oil droplets too large to pass out of the disrupted cellular matrix. Our results suggest that emulsification is an important extraction mechanism that reduces the size of these droplets and increases yield. Protease and SDS were both successful in increasing extraction yields. We propose that this is because they disrupt a viscoelastic protein film at the droplet interface, facilitating droplet disruption. An extraction model based on oil droplet coalescence and the formation of a viscoelastic film was able to fit kinetic extraction data well.

Task-dependent differences in subjective fatigue scores.

The aim of the present study was to evaluate time-on-task effects on subjective fatigue in two different tasks of varying monotony during night-time testing (20:00 to 4:00 hours) in a sleep deprivation intervention. The experiment included eight test runs separated by breaks of approximately 20 min. Twenty healthy volunteers performed a driving simulator and the Mackworth clock vigilance task in four of the test runs each. Sequence of tasks was varied across subjects. Before and after each task, subjective sleepiness was assessed by means of the Karolinska sleepiness scale and subjective fatigue was rated on the Samn-Perelli checklist. Fatigue and sleepiness significantly increased over the course of the night. Both tasks led to an increase in fatigue and sleepiness across test runs. However, this time-on-task effect was larger in the vigilance than in the driving simulator task. It is important to note that fatigue and sleepiness in one test run were not influenced by the task performed in the preceding test run, that is there were no cross-over effects. The results suggest that time-on-task effects superimpose circadian and sleep-related factors affecting fatigue. They depend on the monotony of the task and can be quantified by means of a design including separate test runs divided by breaks.