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PubMed2001

Strain typing of polish Leptosphaeria maculans isolates supports at the genomic level the multi-species concept of aggressive and non-aggressive strains.

47 Polish isolates of the blackleg fungus Leptosphaeria maculans (Phoma lingam) were compared with eight well-defined reference strains from Germany, France, Denmark, Australia and one Polish isolate of Phoma nigrificans. The isolates were tested (i) for growth characteristics, (ii) for their ability to form sirodesmins, (iii) for cellulolytic enzymes, and (iv) for pathotype-differentiating molecular markers generated by RAPD-PCR, PCR analysis with pathotype-specific primer pairs and PFGE. With two exceptions all Polish isolates do not form sirodesmins. grow rapidly without penetrating into the substrate and form in most cases yellow or brown pigments in Czapek-Dox liquid cultures. With respect to cellulase secretion and molecular fingerprinting Polish A strains (aggressive) fit into the general picture of the aggressive pathotype group, whereas the NA isolates (non-aggressive) display a higher degree of heterogeneity. This matches with inoculation tests on rape seedlings, which revealed a considerable number of isolates ranging in aggressivity between the conventional A and NA pathotype group. Molecular fingerprinting techniques unequivocally sorted intermediately aggressive isolates into the NA pathotype group. Isolate Ph Bial, which produces sirodesmin but groups within NA isolates according to molecular and physiological markers, may represent a novel third group besides A and NA strains with intermediate aggressivity (IA). We hybridized Southern blots of electrophoretically separated chromosomes with radioactively labelled PCR fragments used for differentiation between A and NA isolates. The specificity of diagnostic PCR amplicons is reflected at the genomic level. The A probe reveals a single hybridizing chromosome exclusively in A strains. The NA probe reveals several chromosomes and is specific for the NA pathotype group. Chromosomes from intermediately aggressive strains are equally well recognized by the NA probe as are Polish isolates with low aggressivity and give no signal with the A probe. Both diagnostic DNA sequences are highly specific for the pathotype group they were derived from. The lack of correspondence of both genetic elements between A and NA strains strongly supports the idea of ascribing the pathotype groups to different species. Whereas the A pathotype group is genetically homogeneous and congruent with the species Leptosphaeria maculans, the NA group needs to be revised taxonomically. NA isolates will presumably have to be split into several independent species.

Ascomycota↗

PubMed2003

Enhanced expression of hepatic lipogenic enzymes in an animal model of sedentariness.

The hindlimb-suspended rat was used as animal model to investigate the effects induced by immobilization of the skeletal muscle in the expression of the genes encoding hepatic lipogenic enzymes. Following a 14-day period of immobilization, rats were injected intraperitoneally with radioactive acetate, and the labeling of hepatic lipids and cholesterol was evaluated 15 min after the isotope injection. The incorporation of labeled acetate in lipids and cholesterol was almost three times higher in the liver of immobilized rats than in control animals as a consequence of the enhanced transcription of the genes encoding acetyl-CoA synthase, acetyl-CoA carboxylase, fatty acid synthase, and 3-hydroxy-3-methylglutaryl-CoA reductase. The high expression of the key enzymes for fatty acid and cholesterol synthesis induced by immobilization was not paralleled by an increase of the hepatic sterol-regulatory element binding protein (SREBP)-1 and SREBP-2 mRNA content. However, the expression of the mature form of SREBP-1 and SREBP-2 was higher in the nuclear fraction of immobilized rat liver than in controls due to a significant increase of the cleavage of the native proteins. Immobilization also affected the expression of proteins involved in lipid degradation. In fact, the hepatic content of peroxisome proliferator-activated receptor-alpha (PPARalpha) mRNA and of PPARalpha target genes encoding carnitine palmitoyl transferase-1 and acyl-CoA oxidase were significantly increased upon immobilization.

Acetates↗

PubMed1999

A yeast screen system for aromatase inhibitors and ligands for androgen receptor: yeast cells transformed with aromatase and androgen receptor.

Endocrine disruptors are hormone mimics that modify hormonal action in humans and animals. It is thought that some endocrine disruptors modify estrogen and androgen action in humans and animals by suppressing aromatase activity. Aromatase cytochrome P450 is the key enzyme that converts C19 androgens to aromatic C18 estrogenic steroids. We have developed a novel aromatase inhibitor screening method that allows us to identify antiaromatase activity of various environmental chemicals. The screen was developed by coexpressing the human aromatase and the mouse androgen receptor in yeast cells, which carry the androgen-responsive ss-galactosidase reporter plasmid. Functional expression of aromatase in yeast has been demonstrated using the [3H]-water release assay with intact cells as well as with yeast microsomes. The aromatase activity could be blocked by known aromatase inhibitors such as aminoglutethimide (AG). Yeast-produced androgen receptors were able to transactivate a yeast basal promoter linked to an androgen-responsive element in response to androgens. The resultant triple yeast transformant responded to the treatment of testosterone, androstenedione, or 5 alpha-dihydrotestosterone (5 alpha-DHT). In the absence of the aromatase inhibitor AG, transcriptional activation was observed only for the nonaromatizable androgen 5 alpha-DHT. However, the two aromatizable androgens (testosterone and androstenedione) induced the reporter activity in the presence of AG. Using this yeast-based assay, we confirmed that two flavones, chrysin and alpha-naphtholflavone, are inhibitors of aromatase. Thus, this yeast system allows us to develop a high-throughput screening method, without using radioactive substrate, to identify aromatase inhibitors as well as new ligands (nonaromatizable androgen mimics) for the androgen receptors. In addition, this screening method also allows us to distinguish nonandrogenic aromatase inhibitors from inhibitors with androgenic activity. This yeast screening method will be useful to screen environmental chemicals for their antiaromatase activity and for their interaction with androgen receptor.

Animals↗

PubMed2006

Minimum detectable activity concentration in direct alpha spectrometry from outdoor air samples: continuous monitoring versus separate sampling and counting.

Rapid method for identifying the presence of alpha particle emitting radionuclides in outdoor air is of paramount importance should a nuclear or radiological incident occur. Minimum detectable activity concentrations of U, U, Pu, and Pu in outdoor air are calculated for two direct alpha spectrometry methods: continuous air monitoring is compared with separate sampling and subsequent alpha particle counting in a vacuum chamber. The radon progeny activity concentration typical for outdoor air and the effects for the alpha particle spectra caused by the properties of the filter and the aerosol particles are taken into account using measurements and Monte Carlo simulations. Continuous air monitoring is a faster method for identifying the presence of (trans)uranium elements when their activity concentration is considerably higher than the typical detection limit. Separate sampling and counting in a vacuum chamber is a more sensitive method when concentrations are close to the detection limit and when the duration of the sampling-counting cycle is greater than approximately 2 h. The method may serve as a tool for rapid field measurements.

Air Pollutants, Radioactive↗

PubMed1989

Potassium-evoked efflux of [3H]purines from the rat submaxillary gland.

1. Normal, atrophied and denervated submaxillary glands were incubated with [3H]adenine for 1 h. The accumulation of [3H]adenine, expressed as microCi/g tissue, did not differ significantly when the sympathetically denervated glands were compared with the control group. The radioactivity retained in both control and denervated tissues was also similar. 2. In atrophied glands 3H-accumulation as well as 3H-retention were 2-fold higher than these obtained in controls per unit weight, but 30% lower when expressed per gland. 3. The spontaneous efflux of radioactivity, expressed as fractional release, from normal, atrophied and denervated glands prelabelled with [3H]adenine was similar. 4. The outflow of radioactivity was enhanced by exposure of the tissues to 60 mM K+ during 2.5 min. 5. In all three groups, the purine release induced by K+ was the same. 6. Phentolamine 3.1 microM enhanced the K+-induced release of [3H]purine compounds in control and atrophied glands but not in denervated glands. 7. Propranolol 0.3 microM produced no changes among the three experimental groups. 8. Atropine 1 microM and phentolamine 3.1 microM plus atropine 1 microM did not modify the release of tritiated purine compounds in control and denervated glands. 9. Our results cannot discriminate between neuronal or non-neuronal elements as the source of purines released by depolarization but suggest that classical pharmacological tools such as phentolamine and atropine may affect purine metabolism in a complex fashion.

Adenine↗

PubMed1989

Effects of diabetes on myocardial perfusion in the atherosclerotic monkey.

We examined the influence of diabetes on the severity of coronary atherosclerosis, as judged by coronary morphometric and blood flow studies. Cynomolgus monkeys were fed a coconut-peanut oil mixture plus cholesterol to induce the plaque lesions of atherosclerosis in nondiabetic (group 2) and diabetic monkeys (group 3). Group 1 consisted of chow-fed controls. After 18 months of hypercholesterolemia, the animals were anesthetized to assess myocardial blood flow, by use of radioactive microspheres. Transmural and inner/outer wall flow ratios were normal in the two lipid-fed groups in the basal state. Vasodilation after adenosine (0.45 mg/kg/min IV) elicited a more than threefold rise of transmural blood flow in group 1, and a significantly reduced increment in groups 2 and 3. The blood flow ratio was not different from unity in group 1 but declined to 0.69 +/- 0.03 in group 2 and 0.69 +/- 0.06 in group 3, with similar decrements of heart rate and aortic pressure. In contrast to results in group 1, left ventricular filling pressure rose and velocity of contractile elements declined to a similar extent in groups 2 and 3 after adenosine, consistent with myocardial ischemia. Morphometric measurements as well as chemical analyses were performed on the three major coronary arteries. The degree of intimal thickening and increase of cholesterol and collagen content were comparable in groups 2 and 3. Thus, in this model the plaque lesions of atherosclerosis did not appear to be intensified by diabetes. Moreover, the responses to adenosine in terms of myocardial underperfusion and mechanical dysfunction were comparable in the two experimental groups.

Adenosine↗

PubMed1990

The double-stranded RNA associated with the '447' cytoplasmic male sterility in Vicia faba is packaged together with its replicase in cytoplasmic membranous vesicles.

The 447 male sterility trait in Vicia faba is strictly correlated with the presence of well-defined membranous vesicles or 'cytoplasmic spherical bodies' not found in fertile isogenic maintainer plants, and by the occurrence of a discrete high molecular weight double-stranded RNA. We have purified these cytoplasmic membranous vesicles and find that they contain the dsRNA together with an RNA-dependent RNA polymerase whose activity depends upon the presence of Mg2+, requires the four-nucleoside triphosphates and is unaffected by inhibitors of cellular transcriptases, e.g. alpha-amanitin and Actinomycin D. The dsRNA can be labelled in vitro by incubating the cytoplasmic vesicles with radioactive NTPs, and the RNA synthesized in vitro is also in a double-stranded form as judged by its resistance to RNase digestion at high salt and its behaviour upon CF-11 chromatography. Treatment of the vesicles with a non-ionic detergent releases the dsRNA in the form of a complex with the RNA-dependent RNA polymerase. The enzyme can still carry out the specific synthesis of dsRNA in these solubilized complexes. The cytoplasmic vesicles therefore isolate this vertically transmitted, self-replicating dsRNA from the cellular milieu: the possible mode of action and relevance of this novel genetic element to the 447 cytoplasmic male sterility trait are discussed.

Cytoplasm↗

PubMed1991

Biological half-life of gaseous elemental iodine deposited onto rice grains.

In order to obtain the biological half-life (Tb) of iodine deposited on rough rice grains, rice plants of four different growing stages--heading, milky, dough, and yellow ripe--were exposed to elemental gaseous iodine. After the exposure, the rough rice samples were collected at different intervals and analyzed for iodine to estimate the value of Tb. The average value of Tb obtained by the experiments at the dough and yellow ripe stages was about 200 d. This value is considerably larger than those for pasture grass and leafy vegetables.

Food Contamination, Radioactive↗

PubMed1988

Interaction of heat and salt shock in cultured tobacco cells.

Cultured tobacco cells (Nicotiana tabacum L. var Wisconsin-38) developed tolerance to otherwise nonpermissive 54 degrees C treatment when heat-shocked at 38 degrees C (2 h) but not at 42 degrees C. Heat-shocked cells (38 degrees C) exhibited little normal growth when the 54 degrees C stress came immediately after heat shock and normal growth when 54 degrees C stress was administered 8 hours after heat shock. Heat shock extended the length of time that the cells tolerated 54 degrees C. Tobacco cells developed tolerance to otherwise lethal 2% NaCl treatment when salt-shocked (1.2% NaCl for 3 hours). The time course for salt tolerance development was similar to that of thermotolerance. Heat-shocked cells (38 degrees C) developed tolerance of nonpermissive salt stress 8 hours after heat shock. Alternatively, cells heat-shocked at 42 degrees C exhibited immediate tolerance to lethal salt stress followed by a decline over 8 hours. Radioactive methionine incorporation studies demonstrated synthesis of heat shock proteins at 38 degrees C. The apparent molecular weights range from 15 to 115 kilodaltons with a protein complex in the 15 to 20 kilodalton range. Synthesis of heat shock proteins appeared to persist at 42 degrees C but with large decreases in incorporation into selected heat shock protein. During salt shock, the synthesis of normal control proteins was reduced and a group of salt shock proteins appeared 3 to 6 h after shock. Similarities between the physiology and salt shock proteins/heat shock proteins suggest that both forms of stress may share common elements.

Journal Article↗

PubMed1988

Role of plasminogen activator in degradation of extracellular matrix protein by live human alveolar macrophages.

Recent evidence indicates that human alveolar macrophages can degrade purified elastin in vitro by a cell contact-dependent process involving acidic proteinases of the cysteine proteinase class. It is unclear to what extent these cells can degrade elastin within a natural extracellular matrix. To address this question, we cultured live human alveolar macrophages on elastin-rich, 3H-lysine-labeled, extracellular matrices deposited by rat smooth muscle cells in vitro. Under various culture conditions, we then measured release of total radioactivity from the matrices during co-culture with cells as well as net loss of desmosine/isodesmosine as a specific marker of elastin degradation. Live macrophages adhered to and progressively solubilized matrix protein at a slow rate (approximately 5 micrograms/10(6) cells/24 h) but the rate of solubilization increased more than 15-fold in the presence of plasminogen. The elastin component of the complicated matrix was not measurably degraded in the absence of plasminogen, but in medium containing plasminogen, 3.5 X 10(6) macrophages degraded 25 +/- 8 micrograms of elastin in 72 h. After pretreatment of matrices with trypsin to remove glycoprotein elements, live cells degraded 16 +/- 4 micrograms of elastin under plasminogen-free conditions. The addition of serum to the medium (1 to 5%) inhibited degradation of elastin within whole matrices (approximately 50% compared to serum-free medium containing plasminogen) but had no effect on degradation of elastin in trypsin-pretreated matrices. An active site inhibitor of cysteine proteinases, Z-phenylalanine-phenylalanine-diazomethylketone, blocked approximately 50% of the elastin degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1983

[Experimental model for hemodynamic and metabolic studies of extracorporeal circulation. Apropos of 14 cases].

The authors present an homogeneous and stable experimental model, the basic characteristics of which are: 14 extracorporeal bypass with: total bypass; normothermia; total haemodilution; beating heart; continuous flow in dogs anesthetised with chloralose. Three methods were employed: continuous recording of the venous lactate concentration; invasive electromagnetic measurement of the arterial blood flow; measurement of the tissular capillary blood flow with radioactive labelled microspheres. The statistic treatment of the informations gathered confirms that those experimentations took place in normothermia and constant: pH, venous and arterial pressure, cardiac pacing and arterial carotid blood flow. We noticed no modification of the body electrolytic repartition; blood gas variations do not modify haemodynamic conditions. Increase of the lactate concentration, as noticed in the literature, is in relation with a catecholamine hypersecretion and not with an anaerobic tissular function. Tissular capillary blood flow is constant if reported to cardiac index. This model will allow us to compare those results with new series where only one of the basic elements will be modified.

Animals↗

PubMed1996

Formation of reversible disulfide bonds with the protein matrix of the endoplasmic reticulum correlates with the retention of unassembled Ig light chains.

Exposed thiols act as intracellular retention elements for unassembled secretory molecules. Yet, some free Ig lambda light chains are secreted despite the presence of an unpaired cysteine (Cys214). This is due largely to the presence of a flanking acidic residue: substitution of Asp213 for Gly or Lys increases pre-Golgi retention and degradation of free lambda. Secretion is restored by exogenous reducing agents or by assembly with heavy chains. In the endoplasmic reticulum (ER), lambda chains form covalent complexes with many proteins through Cys214. These complexes are absent from the Golgi. They are more abundant in transfectants expressing the lambdaGly2I3 and lambdaLys213 mutants that are poorly secreted. Radioactive N-ethylmaleimide labels some monomeric lambda chains isolated from the ER, but not from the Golgi or from the medium, indicating that the Cys214 thiol is masked during ER-Golgi transport. Mass spectrometry reveals the presence of a free cysteine residue disulfide-linked to Cys214. We suggest that thiol-mediated retention involves the formation of reversible disulfide bonds with the protein matrix of the ER. The presence of an acidic residue next to the critical cysteine may allow the masking of the thiol and transport to the Golgi.

Base Sequence↗

PubMed2006

A simple in vitro RNA editing assay for chloroplast transcripts using fluorescent dideoxynucleotides: distinct types of sequence elements required for editing of ndh transcripts.

RNA editing is found in various transcripts from land plant chloroplasts. In tobacco chloroplasts, C-to-U conversion occurs at 36 specific sites including two sites identified in this work. Our RNA editing assay system using chloroplast extracts facilitated biochemical analyses of editing reactions but required mRNAs labeled with (32)P at specific sites. Here, we have improved the in vitro system using fluorescence-labeled chain terminators, ddGTP and ddATP, and have measured the editing activity at 19 sites in ndh transcripts. Editing activities varied from site to site. It has been reported that one editing site in ndhA mRNAs is present in spinach but absent in tobacco, but a corresponding editing capacity had been found in vivo in tobacco using biolistic transformation. We confirmed biochemically the existence of this activity in tobacco extracts. Using the non-radioactive assay, we examined sequences essential for editing within a 50-nt mRNA region encompassing an editing site. Editing of the ndhB-2 site requires a short sequence in front of the editing site, while that of the ndhF mRNA requires two separate regions, a sequence surrounding the editing site and a 5' distal sequence. These results suggest that distinct editing mechanisms are present in chloroplasts.

Chloroplasts↗

PubMed1997

A comparison of independently conducted dose assessments to determine compliance and resettlement options for the people of Rongelap Atoll.

Rongelap Island was the home of Marshallese people numbering less than 120 in 1954; 67 were on the island and severely exposed to radioactive fallout from an atomic weapons test in March of that year. Those resident on Rongelap were evacuated 50 h after the test, returned 3 y later, then voluntarily left their home island in 1985 due to their ongoing fear of radiation exposure from residual radioactive contamination. Following international negotiations in 1991, a Memorandum of Understanding (MOU) was signed in early 1992 between the Republic of the Marshall Islands Government, the Rongelap Atoll Local Government, the U.S. Department of Energy, and the U.S. Department of the Interior. In this MOU it was agreed that the Republic of the Marshall Islands, with the aid of the U.S. Department of Energy, would carry out independent dose assessments for the purpose of assisting and advising the Rongelap community on radiological issues related to a safe resettlement of Rongelap. The MOU enacted two action levels which were agreed to be used to establish whether mitigation should be considered as a condition for resettlement of Rongelap Island: (1) no individual should receive an annual dose in the future of 1 mSv or more, above that from natural background radiation, assuming that his/her diet consists of only locally produced foods, and (2) the total surface soil concentration of plutonium and other transuranic elements must be less than 629 Bq kg(-1) (averaged over the top 5 cm). Environmental radiological data and dietary information were collected over two years (1992-1993) for the purpose of predicting future potential doses to Rongelapese who might resettle. In 1994, four independent assessments were reported, including one from each of the following entities: Marshall Islands Nationwide Radiological Study; Lawrence Livermore National Laboratory; an independent advisor from the United Kingdom (MCT); and a committee of the National Research Council. All four assessments concluded that possibly more than 25% of the adult population could exceed the 1 mSv y(-1) dose level based on strict utilization of a local food diet. The purpose of this report is to summarize the methodology, assumptions, and findings from each of four assessments; to summarize the recommendations related to mitigation and resettlement options; to discuss unique programmatic aspects of the study; and to consider the implications of the findings to the future of the Rongelap people.

Adult↗

PubMed1991

Chemical speciation and bioavailability of elements in the environment and their relevance to radioecology.

The commonly used terms 'chemical speciation' and 'bioavailability' are discussed and an attempt made to produce both qualitative and quantitative descriptions of their influence on the transfer of elements in the environment. The importance of considering the presence of the many different species of radionuclides which may occur in the environment is emphasized. The limitations of using concentration ratios, which take no account of the differing bioavailability of various chemical species of radionuclides, are discussed.

Accidents↗

PubMed1990

Gamma-detecting probe and autoradiographic studies of radiolabeled antibody B72.3 in CX-1 colon xenografts.

Nude mice bearing CX-1 colon tumors were injected with 50 microCi 125I-labeled monoclonal antibody (MAb) B72.3. Radioactivity in tumors was studied with the gamma detecting probe (GDP) on days 1, 3, 7, and 10 after MAb injection. On each day, two mice were sacrificed and sections were examined with autoradiography (ARG), immunoperoxidase methods (IMP), and routine stains. Mean probe counts showed increasing tumor to background ratios and ARG demonstrated a progressive increase in radionuclide in the tumors. The distribution of 125I was primarily around the vascular spaces on day 1, but by day 3 and progressively it appeared in tumor gland lumina and necrotic areas. A regional correlation was shown between radionuclide in vascular spaces and its sequestration in tumor elements.

Animals↗

PubMed1983

Sequence organisation in nuclear DNA from Physarum polycephalum. Arrangement of highly-repeated sequences.

Recombinant plasmids containing highly repetitive Physarum DNA segments were identified by colony hybridisation using a radioactively-labelled total Physarum DNA probe. A large number of these clones also hybridised to a foldback DNA probe purified from Physarum nuclear DNA. The foldback DNA probe was characterised by reassociation kinetic analysis. About one-half of this component was shown to consist of highly repeated sequences with a kinetic complexity of 1100 bp and an average repetition frequency of 5200. Direct screening of 67 recombinant plasmids for foldback sequences using the electron microscope revealed that about one-half were located in segments of DNA containing highly repetitive sequences; the remainder were present in clones containing low-copy number repeated elements. Analysis of two DNA clones showed that they contained repetitive elements located in over half of all DNA segments containing highly repetitive DNA and that the foci containing these highly repetitive sequences had different sequence arrangements. The results are consistent with the hypothesis that the most highly repeated DNA sequence families in the Physarum genome are few in number and are clustered together in different arrangements in about one-sixth of the genome. Over one-half of the foldback DNA complement in the Physarum genome is derived from these segments of DNA.

DNA, Fungal↗

PubMed1987

Post-translational regulation of Lcr plasmid-mediated peptides in pesticinogenic Yersinia pestis.

The low calcium response of wild type Yersinia pestis, the causative agent of bubonic plague, and of enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica is known to be mediated by a shared Lcr plasmid of about 70 kb. At 37 degrees C in Ca2+-deficient medium, this element promotes restriction of growth with concomitant production of virulence functions including the common V antigen and a set of yersiniae outer membrane peptides termed YOPs (Lcr+). The latter are expressed by the enteropathogenic species but not by wild type Y. pestis which possesses a unique 10 kb Pst plasmid associated with pesticinogeny (Pst+). We show in this report that, after pulse with 35S-methionine, peptides with molecular weights corresponding to YOPs of 78, 47, 45, 44, 36, and 26 kDa are synthesized during the low calcium response by both Lcr+, Pst+ and Lcr+, Pst- cells of Y. pestis. Although stable in the latter, radioactivity in YOPs of wild type was rapidly chased into lower molecular weight degradation products. At least four soluble peptides, including V, were also labeled during starvation for Ca2+; these structures were stable in both Lcr+, Pst+ and Lcr+, Pst- yersiniae. These findings suggest that a product encoded by the Pst plasmid of Y. pestis is required for post-translational regulation of outer membrane but not soluble peptides mediated by a second unrelated Lcr plasmid.

Autoradiography↗