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PubMed1987

A comparison of two procedures used for complexing Fe(III) with human apotransferrin. II. Uptake of Fe(III) by K-562 cells from 55Fe . transferrins and Fe . [3H]transferrins.

Samples of human apotransferrin (apo . HTr) were saturated with Fe(III) by two different techniques, a method employing excess trisodium citrate to chelate Fe(III) and a nonchelating approach which involves the ferroxidase activity of ceruloplasmin to convert Fe(II)----Fe(III). The samples were radiolabelled with either 55Fe or 3H. Using an initial molar Fe/apo . HTr ratio of 2.0-2.1, preparations of human transferrin with bound Fe (Fe . HTr) using the citrate method invariably contained 2.2-2.4 atoms Fe/molecule, whereas Fe . HTr (ceruloplasmin method) contained 2.0 atoms/molecule as shown by spectrophotometric and radioactivity measurements. Uptake of Fe from these Fe . HTr preparations by K-562 cells grown in a serum-free medium was marginally, but consistently, more rapid from 55Fe . HTr (citrate) than from 55Fe . HTr (ceruloplasmin). Taking account of the different Fe contents of the Fe . HTr preparations, the rate measured over a 2-h period amounted to approximately 12,700 and 16,100 Fe atoms/(cell . min) for Fe . HTr (ceruloplasmin) and Fe . HTr (citrate), respectively. However, cell binding by the two Fe . [3H]HTr preparations did not differ significantly over the 8-h incubation period. Furthermore, from the 3H distribution, the quantities of Fe . HTr bound reversibly at the cell surface and contained within the cell were similar for the two Fe . HTr preparations. The results indicate that apo . HTr may bind Fe in different ways depending on the method of Fe presentation and that the Fe . HTr product can donate Fe to K-562 cells at a rate which may reflect the method used for Fe-complex formation.

Apoproteins↗

PubMed2003

Magnetic drug targeting--biodistribution of the magnetic carrier and the chemotherapeutic agent mitoxantrone after locoregional cancer treatment.

Magnetic Drug Targeting means the specific delivery of chemotherapeutic agents to their desired targets, e.g. tumors, by using magnetic nanoparticles (ferrofluids) bound to these agents and an external magnetic field which is focused on the tumor. This type of target directed drug injection attempts to concentrate a pharmacologic agent by enhancing its efficacy while simultaneously minimizing deleterious side effects. In previous studies, we have been able to demonstrate the efficacy of this type of localized intraarterial chemotherapy in VX2 squamous cell carcinoma among rabbits [Alexiou, C., Arnold, W., Klein, R.J., Parak, F.G., Hulin, P., Bergemann, C., Erhardt, W., Wagenpfeil, S. and Lübbe, A.S. "Locoregional cancer treatment with Magnetic Drug Targeting", Cancer Res. 60 (2000) 6641-6648]. In the present investigation, we have studied the biodistribution of ferrofluids and chemotherapeutic agent by measuring the amount in the tumor, peritumoral area, various organs and body fluids (e.g. blood and urine), with and without Magnetic Drug Targeting. We compared results to that of administering a chemotherapeutic agent soley. An external magnetic field was directed toward the tumor for 60 min. Biodistribution of ferrofluids in the tumor was investigated using histological cross sections and measured semi-quantitatively using 123I-labeled nanoparticles and quantitatively by the use of radioactive 59Fe-ferrofluids. Mitoxantrone was quantitatively measured using HPLC-analysis. The strength of the external magnetic field was 0.6 Tesla (permanent magnet) in the 123iodine study and 1.7 Tesla (electromagnet) in the 59Fe-study and HPLC-analysis. The concentration of the ferrofluids (FFs) in the tumor region i.e. the tumor tissue and the surrounding area, which was under the influence of an external magnetic field, was found to be much higher than in the absence of one. In contrast to systemic chemotherapy, a much higher concentration of mitoxantrone in the tumor and the peritumoral area (region surrounding the tumor < or = 1 cm), by using only 50% and 20% of the normal dose was seen. Thus, the higher concentration of mitoxantrone could explain the therapeutic efficacy of Magnetic Drug Targeting in treatment of VX2 squamous cell carcinoma in rabbits in our previous studies with the advantage of no adverse clinical side effects.

Animals↗

PubMed1999

The rete mirabile of the eel: a useful model for the study of transcapillary passage of MR contrast agents.

Our purpose was to study the capillary leakage of MR contrast media using a pure capillary model, the rete mirabile of the eel. The rete is a countercurrent-exchange organ composed of an arterial and a venous capillary system that can be catheterized and perfused. Substances are introduced at the arterial input by a constant infusion, and their steady-state concentrations are measured at the arterial and venous outputs. The capillary leakage of four MR contrast agents--Gd-DOTA(MW = 561 D), carboxymethyldextran-Gd-DTPA (MW = 38,900 D), albumin-Gd-DTPA (MW = 92,000 D), AMI-227 (400,000 D<MW<900,000 D)--was characterized by reference to radioactive tracers (3HHO, 22Na, 14C-sucrose, 125I-albumin) by two parameters. These parameters were the concentration ratio of the venous output over the arterial input [C(VOUT)(%)] and the permeability coefficient (P). The transcapillary pathway mechanisms for carboxymethyldextran-Gd-DTPA and albumin-Gd-DTPA were studied by electron microscopy. P values for Gd-DOTA (9.4+/-3.6 x 10(-7) cm/s) and albumin-Gd-DTPA (11.8+/-5.5 x 10(-7) cm/s) were close to P values for 14C-sucrose, while P values for carboxymethyldextran-Gd-DTPA (6.4+/-4.9 x 10(-7) cm/sec) were similar to P values for 125I-albumin. The lowest permeability was observed with AMI-227 (2.7+/-2 x 10(-7) cm/sec). Vesicular transport was demonstrated for carboxymethyldextran-Gd-DTPA and albumin-Gd-DTPA. The transcapillary passage of several MR contrast agents can be characterized with the rete mirabile model. Molecular weight is the major factor influencing transport.

Air Sacs↗

PubMed2004

X-ray absorption spectroscopy studies of reactions of technetium, uranium and neptunium with mackinawite.

Technetium, uranium and neptunium may all occur in the environment in more than one oxidation state (IV or VII, IV or VI and IV or V respectively). The surface of mackinawite, the first-formed iron sulfide phase in anoxic conditions, can promote redox changes so a series of laboratory experiments were carried out to explore the interactions of Tc, U and Np with this mineral. The products of reaction were characterised using X-ray absorption spectroscopy. Technetium, added as TcO4(-), is reduced to oxidation state IV and forms a TcS(2)-like species. On oxidation of the mackinawite in air to form goethite, Tc remains in oxidation state IV but in an oxide, rather than a sulfide environment. At low concentrations, uranium forms uranyl surface complexes on oxidised regions of the mackinawite surface but at higher concentrations, the uranium promotes surface oxidation and forms a mixed oxidation state oxide phase. Neptunium is reduced to oxidation IV and forms a surface complex with surface sulfide ions. The remainder of the Np coordination sphere is filled with water molecules or hydroxide ions.

Environmental Monitoring↗

PubMed2002

Influence of composition variations on the initial alteration rate of vitrified domestic waste incineration fly-ash.

Vitrification as a waste stabilization technology has often been considered applicable only to high-level radioactive waste for which, with the use of suitable additives, it yields a vitreous material with excellent chemical durability. It has become apparent in recent years that some waste forms-notably domestic waste incineration fly-ash purification residues--contain most of the ingredients of a vitrified material, although their composition variations are difficult to control. It is thus important to ensure not only that the materials are suitable for vitrification, but also that the resulting product exhibits acceptable long-term behavior under all circumstances. An initial study showed that, allowing for the compensation changes inherent in the melting process builtby EDF**, the residue collected by a single fly-ash dust separation defines a composition range within which the suitability of the vitrified material can be verified. "Vitrified material" refers to a melted material that contains no unmelted inclusions after cooling, but that may contain a variable fraction of crystallized phases. Five composition parameters were identified for the long-term behavior assessment: the concentrations of the three major elements (silicon, aluminum and calcium), the total alkali metal (sodium and potassium) concentration, and the sum of the concentrations of two toxic elements (zinc and lead). The other elements were assumed constant at molar ratios representative of industrial wastes. The experimentation plan methodology applied to the composition range identified fourteen materials suilable for developing and validating first-order models of the material components. The fly-ash composition had a very significant effect on the degree and kinetics and crystallization in the vitrified material within the experimental composition range; the cooling rate was the determining factor for some of the fourteen materials studied. Two crystailine phases predominated: spinels rich in chromium, zinc, aluminum, magnesium and iron formed quickly on cooling, and accounted for about 2 vol% of the final material. Gehelenite (Ca2Al2SiO7) crystallized massively in some vitrified materials, accounting for more than half the final product and giving it a rock-like appearance. The effect of composition alone must therefore be distinguished from the effect of crystallization on the leaching behavior. Soxhlet tests were conducted for 14 days according to a protocol based on that of the French AFNOR draft standard NF-M 60313 to determine the maximum alteration rate in pure water at 100 degrees C. The measured rate ranged from 4 to 40 gm(-2) day(-1), illustrating the crucial role of the silicon concentration: within the test composition range, a low silicon content (< 30 wt%) tended to result in a significantly higher initial rate. However, the initial rate alone is not sufficient to assess the chemical durability of the material. Further tests will be carried out at 25 degrees C under conditions approximating those of a proposed disposal site to highlight the role of the alteration layer and the effect of rising concentrations in solution on the decreasing alteration rate.

Crystallization↗

PubMedDate not supplied

[The effect of environmental factors on the concentration of tumor markers in the blood serum and on the indices characterizing thyroid function].

Combined versus separate exposure of male organism to cesium-137 and chemical agents results in a more pronounced hyperferritinemia in the former case. In female chemists the ferritin level is dependent to a considerable extent on the menstrual cycle showing a tendency for the iron-containing protein level to decrease because of the action of a number of chemical agents. The rise of the level of cancer embryonic antigen is more readily seen in persons with high levels of cesium-137. Concentrations of carbohydrate antigen (CA-125) and mucin-like antigen are appreciably higher in female chemists incorporating cesium-137, and in those within the 30-km radius of the ChNPP. The level of thyroglobulin was raised in the chemists having a background incorporation of cesium, the liquidators of the aftermaths, and particularly in those happened to be in the 30-km zone. Each of the unfavourable factors taken separately (chemical agent or cesium-137) had lesser effect on the degree of elevation of TG content and hormone-forming function of the thyroid gland. An additional information has been obtained concerning the risk groups, which, however, serves as an indirect measure of carcinogenic effect various environmental factors exert on the organism.

Adult↗

PubMed1990

Indoor air radon.

This review concerns primarily the health effects that result from indoor air exposure to radon gas and its progeny. Radon enters homes mainly from the soil through cracks in the foundation and other holes to the geologic deposits beneath these structures. Once inside the home the gas decays (half-life 3.8 d) and the ionized atoms adsorb to dust particles and are inhaled. These particles lodge in the lung and can cause lung cancer. The introduction to this review gives some background properties of radon and its progeny that are important to understanding this public health problem as well as a discussion of the units used to describe its concentrations. The data describing the health effects of inhaled radon and its progeny come both from epidemiological and animal studies. The estimates of risk from these two data bases are consistent within a factor of two. The epidemiological studies are primarily for hard rock miners, although some data exist for environmental exposures. The most complete studies are those of the US, Canadian, and Czechoslovakian uranium miners. Although all studies have some deficiencies, those of major importance include uranium miners in Saskatchewan, Canada, Swedish iron miners, and Newfoundland fluorspar miners. These six studies provide varying degrees of detail in the form of dose-response curves. Other epidemiological studies that do not provide quantitative dose-response information, but are useful in describing the health effects, include coal, iron ore and tin miners in the UK, iron ore miners in the Grangesburg and Kiruna, Sweden, metal miners in the US, Navajo uranium miners in the US, Norwegian niobian and magnitite miners, South African gold and uranium miners, French uranium miners, zinc-lead miners in Sweden and a variety of small studies of environmental exposure. An analysis of the epidemiological studies reveals a variety of interpretation problem areas. The major and almost universal problem is in estimating exposure levels. In many cases there were no direct measurements of radon or radon progeny and the exposure levels are estimates based on irregular measurements and known levels in nearby mines. Perhaps the most important variable or complicating factor in the determination of the risk due to radon exposure is the confounding factor of exposure to cigarette smoke. The general scientific concensus is that, although the interaction could be somewhere between linear and supramultiplicative, it is likely a combination, and closer to multiplicative. A number of other complexities contribute to the uncertainty in the risk estimates, likely to a lesser degree than those of exposure measurements and cigarette smoke confounding.(ABSTRACT TRUNCATED AT 400 WORDS)

Air Pollutants↗

PubMed2002

U(VI) adsorption to heterogeneous subsurface media: application of a surface complexation model.

The pH-dependent adsorption of U(VI) onto three heterogeneous, subsurface media from the Department of Energy (DOE) Oak Ridge Reservation, Savannah River Site, and Hanford Reservation was investigated. The three materials contained significant quantities of iron and manganese oxides with nearly identical extractable iron oxide contents (25.3-25.8 g/kg). A model independently developed for the adsorption of U(VI) to synthetic ferrihydrite (Waite, T. D.; Davis, J. A.; Payne, T. E.; Waychunas, G. A.; Xu, N. Geochim. Cosmochim. Acta 1994, 58, 5465-5478) was able to predict the major features of the pH-dependent U(VI) adsorption to the materials under the assumption that all the dithionite-citrate-bicarbonate extractable iron oxide was present as ferrihydrite. Further experiments with the Oak Ridge soil as a function of carbonate and U(VI) concentration indicated that the model could predict pH-dependent U(VI) adsorption to within a root mean square error of 0.163-0.408, even under conditions outside of those for which the model was developed. These results indicate that this model could be used as a first approximation in predicting U(VI) adsorption and transport in the subsurface. U(VI) adsorption also decreased at pH >10, even in the absence of carbonate, which is of potential importance to U(VI) mobility in extreme environments present in the subsurface at some DOE facilities. The pH-dependent adsorption of U(VI) was fundamentally different in systems with a constant CO2 partial pressure as compared to a constant total carbonate concentration. Experiments at constant CO2 partial pressure may not be representative of the conditions present in the subsurface, and a constant carbonate concentration does not always result in decreased U(VI) adsorption at higher pH values.

Adsorption↗

PubMed1981

[Problems in using radioactively labeled bacteria in animal experiments. 4. Distribution of activities in the mouse following intratracheal administration of labeled Pasteurella multocida].

Intratracheal administration of 3H-labelled, 14C-labelled, 59Fe-labelled or 125J-Labelled Pasteurella multicida germs to mice resulted in more or less differentiated, nuclide-dependent, distributions of radioactivity in blood, spleen, liver, lung, kidney, and gastro-intestinal tract. All distributions were comparable to those following subcutaneous application. Elimination of antigen from lungs and other organs could be characterised by an e-function, once a certain level of distribution had been reached. Some of the antigen was persistent in the lung not less than 14 days. Extremely high activity concentration and persistence was recordable, following the use of 59Fe complete antigen. Phagocytosis of Pasteurella multicida germs through alveolar macrophages of the lung was secured by autoradiography. Most of the antigen seemed to be discharged from the lungs through the digestive tract. Antigen distributions recorded from immunised and non-immunised mice seemed to suggest that the fate of antigen applied was affected by the kind of immunisation. No difference in antigen distribution between non-immunised and subcutaneously immunised animals were recordable, following intratracheal antigen application, but is was clearly recordable, following intratracheal immunisation. Elimination of antigen from the lungs of intratracheally immunised animals was found to occur faster than it did from non-immunised animals.

Animals↗

PubMed2001

Determination of radionuclides induced by fast neutrons from the JCO criticality accident in Tokai-mura, Japan for estimating neutron doses.

A criticality accident occurred at a uranium conversion facility in Tokai-mura, Japan on September 30, 1999, and fission neutrons were continuously emitted for about 20 hours. Materials of stainless steel or iron, and chemical reagents were collected at places between 2 m and 270 m from the criticality accident site on October 25 and 26, 1999, November 27, 1999 and February 11, 2000. Neutron-induced radionuclides. such as 54Mn and 58Co, in the materials exposed to fast neutrons from the accident were measured to estimate the neutron fluences and energy distributions. Highly sensitive y-ray spectrometry with a well-type Ge detector was performed after radiochemical separation of Mn and Co from the materials. An instrumental neutron activation analysis was mainly applied for determinations of the target elements and chemical yields. The concentrations of 54Mn and 58Co in a mesh screen of stainless steel collected at a location 2.0 m from the accident site were determined. The total number of fission events was evaluated to be 2.5 x 10(18) by Monte-Carlo calculations of neutron transfer by considering the observed values of 54Mn and 58Co. The results presented here are fundamental to estimate the neutron doses at various distances.

Fast Neutrons↗

PubMedDate not supplied

Embryo toxicity and teratogenicity of formaldehyde.

C-14 formaldehyde crosses the placenta and enters fetal tissues. The incorporated radioactivity is higher in fetal organs (i.e., brain and liver) than in maternal tissues. The incorporation mechanism has not been studied fully, but formaldehyde enters the single-carbon cycle and is incorporated as a methyl group into nucleic acids and proteins. Also, formaldehyde reacts chemically with organic compounds (e.g., deoxyribonucleic acid, nucleosides, nucleotides, proteins, amino acids) by addition and condensation reactions, thus forming adducts and deoxyribonucleic acid-protein crosslinks. The following questions must be addressed: What adducts (e.g., N-methyl amino acids) are formed in the blood following formaldehyde inhalation? What role do N-methyl-amino adducts play in alkylation of nuclear and mitochondrial deoxyribonucleic acid, as well as mitochondrial peroxidation? The fact that the free formaldehyde pool in blood is not affected following exposure to the chemical does not mean that formaldehyde is not involved in altering cell and deoxyribonucleic acid characteristics beyond the nasal cavity. The teratogenic effect of formaldehyde in the English literature has been sought, beginning on the 6th day of pregnancy (i.e., rodents) (Saillenfait AM, et al. Food Chem Toxicol 1989, pp 545-48; Martin WJ. Reprod Toxicol 1990, pp 237-39; Ulsamer AG, et al. Hazard Assessment of Chemicals; Academic Press, 1984, pp 337-400; and U.S. Department of Health and Human Services. Toxicological Profile of Formaldehyde; ATSDR, 1999 [references 1-4, respectively, herein]). The exposure regimen is critical and may account for the differences in outcomes. Pregnant rats were exposed (a) prior to mating, (b) during mating, (c) or during the entire gestation period. These regimens (a) increased embryo mortality; (b) increased fetal anomalies (i.e., cryptochordism and aberrant ossification centers); (c) decreased concentrations of ascorbic acid; and (d) caused abnormalities in enzymes of mitochondria, lysosomes, and the endoplasmic reticulum. The alterations in enzymatic activity persisted 4 mo following birth. In addition, formaldehyde caused metabolic acidosis, which was augmented by iron deficiency. Furthermore, newborns exposed to formaldehyde in utero had abnormal performances in open-field tests. Disparities in teratogenic effects of toxic chemicals are not unusual. For example, chlorpyrifos has not produced teratogenic effects in rats when mothers are exposed on days 6-15 (Katakura Y, et al. Br J Ind Med 1993, pp 176-82 [reference 5 herein]) of gestation (Breslin WJ, et al. Fund Appl Toxicol 1996, pp 119-30; and Hanley TR, et al. Toxicol Sci 2000, pp 100-08 [references 6 and 7, respectively, herein]). However, either changing the endpoints for measurement or exposing neonates during periods of neurogenesis (days 1-14 following birth) and during subsequent developmental periods produced adverse effects. These effects included neuroapoptosis, decreased deoxyribonucleic acid and ribonucleic acid synthesis, abnormalities in adenylyl cyclase cascade, and neurobehavioral effects (Johnson DE, et al. Brain Res Bull 1998, pp 143-47; Lassiter TL, et al. Toxicol Sci 1999, pp 92-100; Chakraborti TK, et al. Pharmacol Biochem Behav 1993, pp 219-24; Whitney KD, et al. Toxicol Appl Pharm 1995, pp 53-62; Chanda SM, et al. Pharmacol Biochem Behav 1996, pp 771-76; Dam K, et al. Devel Brain Res 1998, pp 39-45; Campbell CG, et al. Brain Res Bull 1997, pp 179-89; and Xong X, et al. Toxicol Appl Pharm 1997, pp 158-74 [references 8-15, respectively, herein]). Furthermore, the terata caused by thalidomide is a graphic human example in which the animal model and timing of exposure were key factors (Parman T, et al. Natl Med 1999, pp 582-85; and Brenner CA, et al. Mol Human Repro 1998, pp 887-92 [references 16 and 17, respectively, herein]). Thus, it appears that more sensitive endpoints (e.g., enzyme activity, generation of reactive oxygen species, timing of exposure) for the measurement of toxic effects of environmental agents on embryos, fetuses, and neonates are more coherent than are gross terata observations. The perinatal period from the end of organogenesis to the end of the neonatal period in humans approximates the 28th day of gestation to 4 wk postpartum. Therefore, researchers must investigate similar stages of development (e.g., neurogenesis occurs in the 3rd trimester in humans and neonatal days occur during days 1-14 in rats and mice, whereas guinea pigs behave more like humans). Finally, screening for teratogenic events should also include exposure of females before mating or shortly following mating. Such a regimen is fruitful inasmuch as environmental agents cause adverse effec

Animals↗

PubMed2005

Uranium contents and (235)U/(238)U atom ratios in soil and earthworms in western Kosovo after the 1999 war.

The uranium content and (235)U/(238)U atom ratio were determined in soils and earthworms of an area of Kosovo (Djakovica garrison), heavily shelled with depleted uranium (DU) ammunition during the 1999 war. The aim of the study was to reconstruct the small-scale distribution of uranium and assess the influence of the DU added to the surface environment. The total uranium concentration and the (235)U/(238)U ratio of topsoils showed great variability and were inversely correlated. The highest uranium levels (up to 31.47 mg kg(-1)) and lowest (235)U/(238)U ratios (minimum 0.002147) were measured in topsoils collected inside, or very close to, the clusters of DU penetrator holes. Regarding the fractionation of uranium in the surface soils, the uranium concentrations in the soluble and exchangeable fractions increased as the total uranium concentration of the topsoils increased. High and rather uniform percentage contents of uranium (24-36%) were associated with the poorly crystalline iron oxide phases of soils. In the U-enriched soils the elevated levels of the element were probably due to the presence of very small, unevenly distributed oxidized DU particles. The total uranium concentration in earthworms was in the range 0.142-0.656 mg kg(-1), with the highest concentrations in Lumbricus terrestris. The juveniles of all three studied species seemed to accumulate uranium more than adults, probably due to age-related differences in metabolism. The (235)U/(238)U ratio in the earthworms was variable (0.005241-0.007266) and independent of both the total uranium contents in soils and the absolute uranium levels in the animals. Bioconcentration was greater at lower U concentrations in soil, probably due to an increasing rate of elimination of uranium by the earthworms as the soil contents of the element increase. The results of this study clearly indicate that DU was added to the soil of the study area. Nevertheless, the phenomenon was very limited spatially and the total uranium concentrations fell within the natural range of the element in soils. Moreover, the absolute uranium concentrations indicate that there was no contamination of the earthworm species studied.

Animals↗

PubMed1999

[Utilization of a transferred arc-plasma rotating furnace to melt and found oxide mixtures at around 2000 degrees C (presentation of the film VULCANO)].

Unless security measures are taken, a hypothetical accident resulting from the loss of the cooling circuit in a pressurized water nuclear reactor could cause the heart of the reactor to melt forming a bath, called the corium, mainly composed of uranium, zirconium and iron oxides as well as the structural steel. This type of situation would be similar to the Three Mile Island accident in 1979. In order to limit the consequences of such an accident, the Atomic Energy Commission has implemented a large study program [1] to improve our understanding of corium behavior and determine solutions to stabilize it and avoid its propagation outside the unit. The VULCANO installation was designed in order to perform the trials using real materials which are indispensable to study all the phenomena involved. A film on the VULCANO trials was presented at the Henri Moissan commemorative session organized by the French National Academy of Pharmacy. The rotating furnace used to melt and found the mixture simulating the corium is a direct descendant of the pioneer work by Henri Moissan. An electrical arc is directed at the center of the load to melt which is maintained against the walls by centrifugal force. After six high-temperature trials performed with compositions without uranium oxide, the first trial with real corium showed that the magma spread rather well, a result which is quite favorable for cooling.

France↗

PubMed1997

Heterologous expression of biphenyl dioxygenase-encoding genes from a gram-positive broad-spectrum polychlorinated biphenyl degrader and characterization of chlorobiphenyl oxidation by the gene products.

The bphA1A2A3A4 gene cluster, encoding a biphenyl dioxygenase from Rhodococcus globerulus P6, a gram-positive microorganism able to degrade a wide spectrum of polychlorobiphenyls (PCBs), was expressed in Pseudomonas putida, thereby allowing characterization of chlorobiphenyl oxidation by this enzyme. While P6 biphenyl dioxygenase activity was observed in P. putida containing bphA1A2A3A4, no activity was detected in Escherichia coli cells containing the same gene cluster. In E. coli, transcription of genes bphB and bphCl, located downstream of bphA1A2A3A4, was shown to be driven solely by a vector promoter, which indicated that the lack of biphenyl dioxygenase activity was not due to a lack of mRNA synthesis. Radioactive labelling of bph gene products in E. coli implied inefficient translation of the bphA gene cluster or rapid degradation of the gene products. The biosynthesis of functional P6 biphenyl dioxygenase in P. putida cells containing the same plasmid construct that yielded no activity in E. coli emphasizes the importance of the host strain for heterologous expression and shows that synthesis, correct folding, and assembly of a Rhodococcus biphenyl dioxygenase can be achieved in a gram-negative organism. Dioxygenation of six mono- and dichlorinated PCB congeners by P. putida containing the P6 bphA gene cluster indicates the following ring substitution preference for this reaction (from most to least preferred): un-, meta-, para-, and ortho-substitution. No indications were found for dioxygenation of meta/para carbon pairs, or for hydroxylation of chlorinated carbons at any position of a monochlorinated ring, suggesting a strict specificity of this biphenyl dioxygenase for attack at nonhalogenated ortho/meta vicinal carbons. This contrasts the properties of an analogous enzyme from Pseudomonas sp. strain LB400, which can both dioxygenate at meta and para positions and dehalogenate substituted ortho carbons during ortho and meta dioxygenation.

Biodegradation, Environmental↗

PubMed1982

Technique for determination of human zinc absorption from measurement of radioactivity in a fecal sample or the body.

The intestinal absorption of an oral dose of zinc chloride was determined from the ratio of 65Zn and a nonabsorbed radioactive marker, 51Cr, present in a single stool specimen or the body 24-72 h later. Chromic chloride had no effect on [65Zn]zinc chloride absorption and 51Cr and 65Zn had similar intestinal transit times. In 17 healthy control subjects given 92 mumol ZnCl2 labeled with 0.5 microCi 65Zn, 52 +/- 14% (SD) of the dose was taken up from the lumen. Intestinal absorption of 65Zn at 24 h correlated closely with 65Zn body retention of zinc measured by whole-body counting 7 days later, r = 0.995. Neither zinc absorption nor zinc retention correlated with blood leukocyte zinc levels. An average of 55% of 65Zn was retained in the body from doses of 18-90 mumol ZnCl2 but a progressively smaller proportion of zinc was absorbed from doses of 180-900 mumol. The average absorption and body retention of 65Zn were significantly reduced in 7 patients with mucosal disease of the proximal intestine but they were not affected by resection of the lower jejunum, ileum, and colon. Thus the absorption of ZnCl2 from a 92-mumol dose predominantly takes place by a rate-limited mechanism in the duodenum and upper jejunum.

Adolescent↗

PubMed1981

Formation of a cytochrome c-like species from horse apoprotein and hemin catalyzed by yeast mitochondrial cytochrome c synthetase.

Cytochrome c synthetase in yeast mitochondria catalyzes the formation of a yeast cytochrome c-like species from the apoprotein and hemin (Basile, G., DiBello, C., and Taniuchi, H. (1980) J. Biol. Chem. 255, 7181-7191). To test the specificity of this enzyme, 125I-labeled horse apocytochrome c was incubated with the yeast mitochondrial fraction in the presence of hemin, NADPH, and an ethanol extract of the postmitochondrial fraction. A radioactive 125I-labeled cytochrome c-like species was formed in yields of up to 26%. This 125I-labeled species is indistinguishable from horse cytochrome c by ion exchange chromatography (under the conditions which allow separation of horse and yeast cytochrome c), resistance in its reduced form to digestion by trypsin, resistance against autoxidation, reduction by cytochrome b2, and generation of the apoprotein after treatment with silver sulfate and dithiothreitol. With unlabeled horse apoprotein and [59Fe]hemin, the yield of a [59Fe-labeled horse cytochrome c-like species was up to 7% with respect to the apoprotein incubated. The yield of the 59Fe-labeled species was not altered by the addition of unlabeled FeCl3. Conversely, synthesis of the 59Fe-labeled species was not detectable after incubation of yeast mitochondria with unlabeled horse apoprotein, unlabeled hemin, and 59FeCl3. The formation of both 125I- and 59Fe-labeled cytochrome c-like species was sensitive to heat. Thus, we conclude that cytochrome c synthetase catalyzes direct bonding of heme (or hemin) to the apoprotein. Since the amino acid sequences of horse and yeast cytochromes c differ considerably, cytochrome c synthetase may recognize only a limited region(s) of the apoprotein.

Animals↗

PubMed1970

Partial purification and kinetics of oestriol 16 alpha-glucuronyltransferase from the cytosol fraction of human liver.

An enzyme that conjugates the 16alpha-hydroxyl group of oestriol with glucuronic acid was found in the cytosol fraction of human liver. The enzymic activity could not be sedimented when the cytosol fraction was centrifuged at 158000g(av.) for 120min. The oestriol 16alpha-glucuronyltransferase was purified 100-fold by 0-30% saturation of the cytosol fraction with ammonium sulphate followed by filtration of the precipitate through Sephadex G-200. The activity was eluted at the void volume. The product of the reaction, oestriol 16alpha-monoglucuronide, was identified by paper chromatography and by crystallization of radioactive product to constant specific radioactivity. The optimum temperature was 37 degrees C, and the activation energy was calculated to be 11.1kcal/mol. The apparent Michaelis-Menten constants for oestriol and UDP-glucuronic acid were 13.3 and 100mum respectively. Cu(2+), Zn(2+) and Hg(2+) inhibited, whereas Mg(2+), Mn(2+) and Fe(2+) stimulated the enzyme. Substrate-specificity studies indicated that the amount of oestradiol-17beta, oestradiol-17alpha and oestrone conjugated was not more than about 5% of that found for oestriol. Oestriol 16alpha-monoglucuronide, a product of the reaction, did not inhibit the 16alpha-oestriol glucuronyltransferase; in contrast, UDP, another product of the reaction, inhibited the enzyme competitively with respect to UDP-glucuronic acid as the substrate, and non-competitively with respect to oestriol as the substrate. ATP and UDP-N-acetylglucosamine did not affect the oestriol 16alpha-glucuronyltransferase. 17-Epioestriol acted as a competitive inhibitor and 16-epioestriol as a non-competitive inhibitor of the glucuronidation of oestriol. 5alpha-Pregnane-3alpha,20alpha-diol also inhibited the enzyme non-competitively. It is most likely that the oestriol 16alpha-glucuronyltransferase described here is bound to the membranes of the endoplasmic reticulum.

Adenosine Triphosphate↗

PubMed1988

Fever and regional blood flows in wethers and parturient ewes.

To determine whether the reported absence of fever in full-term-pregnant ewes might be associated with shifts of regional blood flows from thermogenic tissues to placenta during this critical period, fevers were induced twice by injections of Escherichia coli lipopolysaccharide (LPS, 0.25 microgram/kg iv) into each of six Merino ewes from 8 to 1 days prepartum, and their regional blood flow distribution was measured with radioactive, 15-microns-diam microspheres before and during the rise in fever (when their rectal temperature had risen approximately 0.4 degree C). Unexpectedly, fever always developed, rising to heights not significantly different at any time before parturition [4-8 days prepartum = 0.81 +/- 0.23 degree C (SE); 1-3 days prepartum = 0.75 +/- 0.17 degree C) and similar to those in three wethers treated similarly (0.90 +/- 0.10 degree C). Generally, during rising fever, blood flow in the ewes shifted away from heat loss tissues (e.g., skin, nose) to heat production tissues (e.g., shivering muscle, fat) and cardiac output increased; blood flow through redistribution organs (e.g., splanchnic bed) decreased. The reverse occurred during defervescence. Utero-placental blood flow remained high in the febrile ewes. These regional blood flow distributions during febrigenesis and lysis are essentially the same as those during exposures to ambient cold and heat, respectively. Some differences in the responses of cardiac output and its redistribution, however, were apparent between wethers and pregnant ewes. We conclude that 1) the previously reported "absence of fever in the full-term-pregnant sheep" should not be regarded as a general phenomenon and 2) full-term-pregnant sheep support fever production without sacrificing placental blood flow.

Animals↗