Three decades of occupational individual monitoring at the University of São Paulo.

As the ionizing radiation to which workers are exposed is related to possible harmful biological effect, its dose evaluation gains relevance. Although the effects of low doses are still controversial, the radiation protection authorities assume that any dose of ionizing radiation is potentially harmful to the human health and adopt the linear non-threshold model for the dose-effect relation. The Dosimetry Laboratory of the Institute of Physics of the University of São Paulo performs the external individual monitoring of workers exposed to X- and gamma-rays since 1981, with the technique of thermoluminescence. Currently, ~500 badges are provided to the university professionals mostly working in research laboratories and hospitals. Data of individual annual dose equivalent collected from 1995 to 2015 and the performance of the monitoring service are presented in this paper.

X-ray transmission through nanostructured and microstructured CuO materials.

This study presents a comparison of the X-ray transmission through microsized and nanosized materials. For this purpose CuO nanoparticles, with 13.4 nm average grain size, and CuO microparticles, with a mean particle size of 56 µm, were incorporated separately to beeswax in a concentration of 5%. Results show that the transmission through the above material plates with microsized and nanosized CuO was almost the same for X-ray beams generated at 60 and 102 kV tube voltages. However, for the radiation beams generated at 26 and 30 kV tube voltages the X-rays are more attenuated by the nanostructured CuO plates by a factor of at least 14%. Results suggest that the difference in the low energy range may be due to the higher number of particles/gram in the plates designed with CuO nanoparticles and due to the grain size effect on the X-ray transmission.

Structural comparison of ten serotypes of staphylocoagulases in Staphylococcus aureus.

Staphylocoagulase detection is the hallmark of a Staphylococcus aureus infection. Ten different serotypes of staphylocoagulases have been reported to date. We determined the nucleotide sequences of seven staphylocoagulase genes (coa) and their surrounding regions to compare structures of all 10 staphylocoagulase serotypes, and we inferred their derivations. We found that all staphylocoagulases are comprised of six regions: signal sequence, D1 region, D2 region, central region, repeat region, and C-terminal sequence. Amino acids at both ends, 33 amino acids in the N terminal (the signal sequences and the seven N-terminal amino acids in the D1 region) and 5 amino acids in the C terminal, were exactly identical among the 10 serotypes. The central regions were conserved with identities between 80.6 and 94.1% and similarities between 82.8 and 94.6%. Repeat regions comprising tandem repeats of 27 amino acids with a 92% identity on average were polymorphic in the number of repeats. On the other hand, D1 regions other than the seven N-terminal amino acids and D2 regions were less homologous, with diverged identities from 41.5 to 84.5% and 47.0 to 88.9%, respectively, and similarities from 53.5 to 88.7% and 56.8 to 91.9%, respectively, although the predicted prothrombin-binding sites were conserved among them. In contrast, flanking regions of coa were highly homologous, with nucleotide identities of more than 97.1%. Phylogenetic relations among coa did not correlate with those among the flanking regions or housekeeping genes used for multilocus sequence typing. These data indicate that coa could be transmitted to S. aureus, while the less homologous regions in coa presumed to be responsible for different antigenicities might have evolved independently.

Decreased expression of kynurenine aminotransferase-I (KAT-I) in the substantia nigra of mice after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment.

Nerve cells in the substantia nigra pars compacta (SNPC) are known to express tyrosine hydroxylase (TH). By means of light and electron microscopical immunohistochemical techniques, we have shown that the dopaminergic neurons of SNPC express also kynurenine aminotransferase (KAT-I), the enzyme taking part in the formation of kynurenic acid, a neuroprotectant which is one of the endogeneous antagonists of N-methyl-d-aspartate receptors. It was also found that microglial cells and astrocytes express KAT-I. It has been shown that the highly selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), widely used as a model of Parkinson's disease (PD), affects not only TH of dopaminergic neurons in the SNPC but also their KAT-I immunoreactivity as well: MPTP treatment decreased the number and optical density of KAT-I immunoreactive SNPC neurons. Decrease of KAT-I after MPTP treatment has been proved also by Western blot analysis. MPTP also reduced KAT-I immunoreactivity of microglial cells, except for those involved in reactive gliosis, which were arranged in groups surrounding affected neurons of the SNPC; also the number of KAT-I immunoreactive (IR) astroglial cells was increased in SNPC. We conclude that MPTP treatment may have a dual effect: in addition to being deleterious for neurons expressing TH and KAT-I, it also affects glial cells which could exacerbate the neurodegenerative process characterizing PD.

Goiânia: 12 years after the 137Cs radiological accident.

In 1987, in the city of Goiânia, Brazil, occurred one of the worst radiological accidents ever reported. The remains of 137Cs contamination in a terrain where part of a radiotherapy unit had been manipulated in 1987 were measured in 1999-2000, and some of the results are presented here. Using the technique of gamma ray spectrometry in situ and in the laboratory, the ambient dose equivalent rate at 1 m above the ground and 137Cs concentration in soil were determined. Values higher than the ones established by the Brazilian National Nuclear Energy Commission (CNEN) as action levels in 1987, namely 0.8 microGy x h and 22.5 kBq x kg(-1), were obtained in that terrain. The 137Cs distribution profile in the soil shows high values of the specific activity in a layer located at a depth of 10-40 cm from the surface, where the soil is mixed with rubble, reaching values as high as 175 kBq x kg(-1).

Characterisation of the thermally stimulated conductivity and thermoluminescence of natural topaz.

Thermally stimulated conductivity (TSC) and thermoluminescence (TL) measurements were conducted to investigate the mechanisms of charge transfer and luminescence emission in natural samples of Brazilian topaz irradiated with beta particles from a 90Sr/90Y source or with a 1.75 MeV Van de Graaff electron beam. The luminescence and conductivity were simultaneously monitored during the heating of the samples, allowing direct comparison of the TL and TSC peaks. The results show that the three main TL peaks are accompanied by corresponding TSC peaks, usually shifted to higher temperatures. Comparison of the relative TL/TSC intensities of peaks 2 and 3 indicates that the process of thermal quenching of the luminescence is probably active, which is also supported by TL/TSC measurements at different heating rates. Results on the dose response of TL/TSC peaks also reveal an interesting feature: the TL intensity shows a monotonic increase with dose in the range of study (50 Gy-3 kGy) comprising a linear-supralinear-saturation characteristic, while the TSC peaks exhibit an increase from 50 Gy to 1 kGy, followed by a small decrease for doses greater than 1 kGy. This result is interpreted in terms of a model involving multiple traps and one recombination centre.

Measurements performed in Goiânia after a new intervention action in 2001.

Brazil's worst radiological accident took place in 1987, in the city of Goiânia. In 1999 and 2000, detailed measurements of 137Cs contamination were performed in junkyard II, one of the places involved in the accident. High values of 137Cs activity per unit mass were found in soil layers at depths between 10 and 40 cm from the surface, reaching values as high as 175 kBq x kg(-1). High values of 137Cs concentration in fruits and plants were also observed. Moreover, values of ambient dose equivalent rate at 1 m above the ground were found to be higher than the limit of 1.0 microSv x h(-1) set by the Brazilian National Nuclear Energy Commission (CNEN) in 1987. In February 2000, the CNEN was informed about the results of our measurements. As consequence, in August 2001, the CNEN performed a new intervention action in the area, covering all its extension with a concrete layer and removing some plants and trees. The new remedial action reduced the dose rate to approximately 13% of the value prior to covering the site in concrete, reaching values below the CNEN limit, as demonstrated by the measurements presented here.

Presence of kynurenic acid and kynurenine aminotransferases in the inner retina.

Kynurenine aminotransferases (KATs I and II) are pivotal to the synthesis of kynurenic acid (KYNA), the only known endogenous glutamate receptor antagonist and neuroprotectant. This study is the first to identify KYNA in the rat retina and to examine immunohistochemically the distribution of KAT isoforms. As determined by HPLC, KYNA concentration in the retina was 99.9 +/- 24.6 pmol/g wet wt. Immunohisto- chemical experiments showed that both KATs were present in the retina. KAT I was preferentially localised on Müller cell endfeet while KAT II was expressed in cells within the ganglion cell layer. In conclusion, KYNA is present and synthesised in the inner retina. This may suggest a modulatory role in glutamate-mediated retinal neurotransmission.

An archaeal protein homologous to mammalian SRP54 and bacterial Ffh recognizes a highly conserved region of SRP RNA.

The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf-SRP54) and the N-terminal G-domain and C-terminal M-domain (pf-SRP54M) of pf-SRP54 with an amino-terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non-conserved 213-nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107-nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine-tagged pf-SRP54M bound specifically to the conserved 107-nucleotide RNA in the absence of pf-SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.

Remains of 137Cs contamination in the city of Goiânia, Brazil.

The results of measurements, performed in 1999, of the remaining 137Cs contamination in some of the sites where fragments of a radioactive source of a teletherapy unit had been manipulated in 1987 are presented. This episode occurred in the city of Goiânia, during Brazil's worst radiological accident ever reported. Using the technique of gamma ray spectrometry, analyses of both surface and profile soil and vegetable samples were made. High values of 137Cs activity per unit mass were found in soil layers at depths between 10 and 40cm from the surface. Some values exceeded by up to eight times the action level of 22.5 kBq x kg(-1) proposed by the Brazilian National Nuclear Energy Commission (CNEN) during the decontamination process at the time of the accident, for the first year after the accident. Absorbed dose rates at 1 m above the ground were calculated from the data of 137Cs concentration in the soil and compared with those obtained from in situ gamma ray spectrometry and from thermoluminescence dosimetry.

Ambient dose equivalent rate in Goiânia 12 years after the 137Cs radiological accident.

This paper describes the situation of ambient dose equivalent rates in four of the main foci of 137Cs contamination in the city of Goiânia, Brazil, in 1999, 12 y after one of the worst radiological accidents in the world. During the decontamination, all the buildings of the three highly contaminated sites were demolished and the top soil removed. Afterwards, the soil of two of these lots was covered with concrete, and they remain vacant today. The soil of the third of these lots, identified here as E, previously known as junkyard II, was covered only with clean soil. Three to four years after the accident, new houses were constructed on this lot, and some very poor people live and work there collecting recyclable material. Gamma ray spectrometry, with a portable survey meter, was performed in the quoted places along with outdoor measurements in many other locations of Goiânia. The average ambient dose equivalent rate due to natural background radiation from radionuclides in the soil and cosmic radiation in non-contaminated areas of the city of Goiânia is 62 nSv h(-1). In most of the highly contaminated sites during the accident, the average ambient dose equivalent rate ranged from around 100 to 1,000 nSv h(-1). The only exception was site E, where values of ambient dose equivalent rate as high as 2.6 microSv h(-1) were found.

Effects of in vivo sodium azide administration on the immunohistochemical localization of kynurenine aminotransferase in the rat brain.

Endogenous excitotoxins that act on receptors of cerebral excitatory amino acids play important roles in the pathogenesis of excitotoxic brain diseases. Activation of excitatory amino acid receptors results in neuronal death characteristic of these disorders. Kynurenic acid, a powerful endogenous excitatory amino acid receptor antagonist, which is therefore widely regarded as a potent neuroprotective agent, is produced from its biological precursor, L-kynurenine, by the action of the enzyme kynurenine aminotransferase-I. The chemical hypoxia induced by mitochondrial toxins produces a secondary excitotoxicity, leading to the activation of N-methyl-D-aspartate receptors. Accordingly, sodium azide, an inhibitor of cytochrome oxidase, induces the release of excitotoxins via an energy impairment and this, in turn, results in neurodegeneration. Since energy-dependent secondary excitotoxic mechanisms also account for the pathogenesis of neurodegenerative diseases, a study was made of the effects of sodium azide on the immunohistochemical localization of kynurenine aminotransferase-I. After in vivo administration of sodium azide for five days, a markedly decreased glial kynurenine aminotransferase-I immunoreactivity was found by immunohistochemical techniques in the glial cells of the striatum, hippocampus, dentate gyrus and temporal cortex; at the same time, kynurenine aminotransferase-I started to be expressed by nerve cells which had not been immunoreactive previously. The accumulation of kynurenine aminotransferase-I reaction product around the ribosomes of neuronal endoplasmic reticulum suggests de novo synthesis of kynurenine aminotransferase-I in the reactive nerve cells.

Sequestration of human muscarinic acetylcholine receptor hm1-hm5 subtypes: effect of G protein-coupled receptor kinases GRK2, GRK4, GRK5 and GRK6.

Sequestration of porcine muscarinic acetylcholine receptor m2 subtypes (m2 receptors) expressed in COS-7 cells is facilitated by coexpression of G protein-coupled receptor kinases 2 (GRK2). We examined the effect of coexpression of GRK2, GRK4 delta, GRK5 and GRK6 on sequestration of human m1-m5 receptors expressed in COS-7 cells, which was assessed as loss of [3H]N-methylscopolamine binding activity from the cell surface. Sequestration of m4 receptors as well as m2 receptors was facilitated by coexpression of GRK2 and attenuated by coexpression of the dominant negative form of GRK2 (DN-GRK2). Sequestration of m3 and m5 receptors also was facilitated by coexpression of GRK2 but not affected by coexpression of DN-GRK2. On the other hand, proportions of sequestered m1 receptors were not significantly different with coexpression of GRK2 and DN-GRK2. GRK4 delta, GRK5 and GRK6 did not facilitate sequestration of m1-m5 receptors in COS-7 cells, except that the sequestration of m2 receptors tended to be facilitated by coexpression of GRK4 delta, GRK5 and GRK6. However, coexpression of GRK4 delta, GRK5, but not GRK6, in BHK-21 cells facilitated sequestration of m2, but not m3, receptors. These results indicate that the effect of GRK2 to facilitate receptor sequestration is not restricted to m2 receptors but is generalized to other muscarinic receptors except m1 receptors and that other kinases, including GRK4 delta, GRK5 and endogenous kinase(s) in COS-7 cells, also contribute to sequestration of m2 and m4 receptors.

Characterization of rat brain kynurenine aminotransferases I and II.

The endogenous neuroprotectant kynurenic acid (KYNA) is produced by irreversible transamination of L-kynurenine (KYN). In the brain, two distinct kynurenine aminotransferases (KAT I and KAT II) are responsible for the formation of KYNA. The present experiments were designed to examine the respective roles of the two KATs in the normal rat brain. To this end, the two enzymes were partially purified, and their characteristics were examined. KAT I (identical with glutamine transaminase K) had an optimal pH of 9.5, preferred pyruvate as a cosubstrate and was potently inhibited by glutamine. KAT II (identical with L-alpha-aminoadipate transaminase) had a neutral optimal pH, showed no preference for pyruvate, and was essentially insensitive to inhibition by glutamine. KAT II was selectively inhibited by quisqualic acid (IC50: 520 microM). The endogenous substrate 3-hydroxykynurenine had an approximately 10-fold preference for KAT II. The distinct properties of the two enzymes made it possible to measure brain KAT I and KAT II in parallel by using dialyzed tissue homogenate (to remove interfering endogenous amino acids). Under these conditions, both enzymes presented essentially the same apparent Km values as the partially purified enzymes. In lesioned, neurondepleted brain tissue and in brain regions other than the cerebellum, KYNA derived primarily from KAT II at physiologic pH. In summary, the present study describes a simple methodology for the simultaneous determination of the two KYNA-producing enzymes in small rat brain tissue samples and provides baseline values for future work in experimentally challenged animals.

How safe is ERCP to the endoscopist?

BACKGROUND: Interventional techniques in endoscopy such as endoscopic retrograde cholangiopancreatography (ERCP) have greatly increased since laparoscopic cholecystectomy has become widespread; mainly these techniques deal with common bile duct stones. Fluoroscopy is usually employed, and chronic exposure to X-ray, in spite of the relative low dose, can lead to potentially unhealthy conditions such as malignancies like bone marrow and other solid cancers. A median of 18 years of life is lost per fatal cancer, including the time of latency since exposure. Nor should one forget benign condition such as cataracts that can lead to partial or complete blindness and which surely impair life's quality. METHODS: Simulated examinations were carried at the University Hospital (São Paulo, Brazil) using an anthropomorphic phantom in place of the physician. Four sets of dosimeters were placed in the forehead, neck, torso, and lower abdomen (with and without a lead apron) and standard ERCP fluoroscopic techniques were employed. RESULTS: The dose equivalents were calculated and compared to the recommended exposure doses of national and international boards of radiation protection. CONCLUSIONS: Based on the results found and compared to standards, working safely means: (1) A lead (0.5 mm thickness) apron is fundamental. Without it less than one ERCP\month should be performed. (2) With an apron, 23 examinations/month are allowed. (3) No thyroid protection grants only 19 exams/month. (4) Performing ERCP without lead glasses is hazardous to the eye, allowing only seven ERCPs monthly.

Cloning and recombinant expression of rat and human kynureninase.

Kynureninase [E.C.3.7.1.3.] is one of the enzymes involved in the biosynthesis of NAD cofactors from tryptophan through the kynurenine pathway. By tryptic and CNBr digestion of purified rat liver kynureninase, we obtained about 28% of the amino acid sequence of the enzyme. The rat kynureninase cDNA, isolated by means of reverse-transcribed polymerase chain reaction and hybridization screening, codes for a polypeptide of 464 amino acids. Northern blot analysis revealed the synthesis of a 2.0 kb rat kynureninase mRNA. A cDNA encoding human liver kynureninase was also isolated. The deduced amino acid sequence is 85% identical to that of the rat protein. COS-1 cells were transfected with both cDNAs. The Km values of the rat enzyme, for L-kynurenine and DL-3-hydroxykynurenine, were 440 +/- 20 microM and 32 +/- 5 microM and of the human enzyme 440 /- 20 microM and 49 +/- 6 microM, respectively. Interestingly, COS-1 cells transfected with the cDNA coding for rat kynureninase also display cysteine-conjugate beta-lyase activity.

Immuno-localization of kynurenine aminotransferase (KAT) in the rat medulla and spinal cord.

In the mammalian brain, kynurenine aminotransferase (KAT) is pivotal to the synthesis of kynurenic acid, a preferential antagonist at the strychnine-insensitive NMDA-glycine site. As NMDA receptors are involved in autonomic function, we have examined the immunohistochemical localization of KAT in the medulla and spinal cord of the rat. KAT immunoreactivity (KAT-li) was found throughout these areas, in both glia and neurons. Unlike the mainly astrocytic localization in forebrain structures, KAT-li was predominantly neuronal, notably in areas important for blood pressure and heart rate regulation: ventral medulla, nucleus ambiguus, nucleus of the solitary tract and intramediolateral cell column of the spinal cord. The presence of KAT in these nuclei supports a neuromodulatory role for kynurenic acid in NMDA-mediated autonomic function.

Kynurenine aminotransferases in the rat. Localization and characterization.

Kynurenine-2-oxoglutarate aminotransferase (kynurenine specific, designated here KAT-II) and kynurenine pyruvate aminotransferase (designated here KAT-I) activities were detected in the kidney using 2 microM kynurenine. The major activities were performed by KAT-II and the contributi-n of KAT-I is about 1/15. KAT-I activity was detected in all organs tested. The liver showed the highest KAT-I activity, however, the highest activity of glutamine transaminase-K (GTK) was detected in the kidney. KAT-I activity was well corresponded with GTK activity in all organs except liver. KAT-I or GTK activity of crude extract didn't inhibited by addition of glutamine either kynurenine. KAT-I or GTK activity of purified preparation, however, inhibited strongly addition of glutamine either kynurenine. KAT-I and GTK showed different pH optimum profile, but purified and crude preparation of those were similar. Phenylpyruvate or 2-oxo-4-methiolbutyrate reduced the inhibition of purified KAT-I activity by glutamine using 2 microM kynurenine. Phenylpyruvate changed the Km value for kynurenine and Vmax, suggesting conformational change of the enzyme.

Molecular cloning and sequencing of a cDNA encoding alanine-glyoxylate aminotransferase 2 from rat kidney.

Alanine-glyoxylate aminotransferase (AGT) 2 is a pyridoxal 5'-phosphate dependent, mitochondrial enzyme which, in the rat, is expressed at a high level in the kidney. The amino acid sequences of nine tryptic and seven CNBr peptides of the rat kidney AGT2 were determined. Three overlapping cDNAs encoding the AGT2 were cloned on the basis of its partial amino acid sequences by means of a polymerase chain reaction-based approach involving rat kidney poly(A)+ RNA. The complete cDNA sequence comprised 1,919 bases, and contained a 1,536-base open reading frame which encodes a polypeptide of 512 amino acid residues with a putative presequence consisting of 39 amino acid residues at the amino terminus, giving a precursor protein with a molecular mass of 57,150 Da. The sequence of AGT2 exhibits significant homology with neither peroxisomal AGT1 from human liver nor mitochondrial AGT1 from rat liver. However, the sequence of AGT2 exhibited 30.8, 29.2, and 27.1% identity with those of Escherichia coli 4-aminobutyrate aminotransferase, rat ornithine aminotransferase, and Pseudomonas cepacia 2,2-dialkylglycine decarboxylase, respectively. The active site sequences were also well conserved among these aminotransferases. AGT2, thus, is more similar to the other aminotransferases than to AGT1. The results suggest that the rat kidney AGT2 may play a biological role in amino acid metabolism distinct from that of AGT1.