Sister-chromatid exchange, chromosomal aberrations and delays in cell-cycle kinetics in human lymphocytes induced by dental composite resin eluates

We have investigated eluates derived from commercially available composite resin-based materials used for direct (Tetric Ceram/Ivoclar-Vivadent, Simile/Pentron, Filtek Z-250/3M ESPE) and indirect (Adoro/Ivoclar-Vivadent and Conquest Sculpture/Pentron) dental restorations, with respect to their genotoxic effects on human peripheral lymphocytes. Primary lymphocyte cultures obtained from blood samples of three healthy donors were exposed to eluates of freshly cured specimens of all the materials tested. Metaphases were induced with phytohaemagglutinin, collected after a 72-h treatment using colchicine and stained with the Fluorescence Plus Giemsa (FPG) procedure. Preparations were scored for sister-chromatid exchange (SCE) and chromosomal aberrations (CAs). The proliferation rate index (PRI) and the mitotic index (MI) were also calculated. Our results show that eluates derived from the three direct composites (Filtek Z-250, Simile and Tetric Ceram) increased the frequencies of SCE and CAs and markedly reduced PRI and MI. Tetric Ceram's eluate, being the most genotoxic of all eluates tested, increased the frequencies of SCE up to 24.40 per cell (control, 9.87 per cell) and of CAs up to 424 per 100 metaphases scored (control, 5). Moreover, it caused a pronounced decrease of the PRI down to 1.31 (control, 2.44) and of the MI down to 9.8 per thousand (control, 19.2 per thousand). In contrast, eluates derived from the laboratory-processed composites (Adoro and Conquest Sculpture) induced much less cytogenetic damage. Overall, the degree of genotoxicity and cytotoxicity decreased as follows: Tetric Ceram>Filtek Z-250>Simile>Adoro=Conquest Sculpture. These results indicate that composite resins used for direct and indirect dental restorations differ extensively in their cytotoxic and genotoxic potential and in their ability to affect chromosomal integrity, cell-cycle progression, DNA replication and repair. This underlines the impact of improved polymerization with respect to their biological behavior.     

Relative concentrations of polyaromatic primary amines and azaarenes in mutagenically active nitrogen fractions from a coal liquid

Thin-layer chromatography (TLC) was used to separate components in the basic and tar fractions of solvent refined coal (SRC-I) process solvent (PS) to obtain materials suitable for biological and chemical analysis. Those fractions eluted from TLC plates which were mutagenically active in the Ames/Salmonella assay were analyzed by gas chromatographic mass spectrometry (GCMS) for polycyclic azaarenes, polyaromatic primary amines (PAA) and carbazoles. In all materials tested, a strong correlation was observed between the concentration of PAAs in a given TLC region and the mutagenicity of that region in the Ames assay system. Conversely, azaarenes having 2--4 fused rings and carbazoles were present in both mutagenic and non-mutagenic TLC eluates. No PAAs were detected in mutagenically inactive TLC eluates. In comparison to the mutagenic tar fractions, the PS basic fraction contained relatively larger concentrations of 2- and 3-ringed components such as aminonaphthalenes and aminoanthracenes or aminophenanthrenes. The tar fractions, which were essentially devoid of aminonaphthalenes, had a higher average molecular weight and contained relatively higher concentrations of aminopyrenes.     

Cloning of a Heavy-Metal-Binding Protein Derived from Activated-Sludge Microorganisms

A gene of the heavy-metal-binding protein (HMBP) was newly isolated from a genetic DNA library of activated-sludge microorganisms. HMBP was produced by transformed Escherichia coli, and the copper-binding ability of HMBP was confirmed. HMBP derived from activated sludge could be available as heavy metal adsorbents in water and wastewater treatments.     

Cloning of a heavy-metal-binding protein derived from activated-sludge microorganisms

A gene of the heavy-metal-binding protein (HMBP) was newly isolated from a genetic DNA library of activated-sludge microorganisms. HMBP was produced by transformed Escherichia coli, and the copper-binding ability of HMBP was confirmed. HMBP derived from activated sludge could be available as heavy metal adsorbents in water and wastewater treatments.     

Characterization of interactions between Escherichia coli molecular chaperones and immobilized caseins

The molecular chaperones were affinity purified with immobilized alpha-casein (45mg protein/g beads) and beta-casein columns (30 mg protein/g beads) from two heat-induced E. coli strains, NM522 and BL21. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea. The eluates from affinity columns were analyzed by SDS-PAGE and Western analysis. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES in eluates. Among samples, ATP eluates showed the highest chaperone purity of 80-87% followed by cold water eluates with 62-68% purity. The beta-casein column showed a higher chaperone binding capacity than the alpha-casein column. A higher concentration of chaperones was purified from strain BL21 than strain NM522 which may have been due to the lack of lon protease in the BL21 strain.     

Biomonitoring of DNA damage in peripheral blood lymphocytes of subjects with dental restorative fillings

Dental fillings provide a major iatrogenic exposure to xenobiotic compounds due to the high prevalence of surface restorations in developed countries. Experimental data suggest that both amalgams, which contain mercury, and resin-based dental materials cause an impairment of the cellular pro- and anti-oxidant redox balance. The aim of this study was to assess the potential genotoxicity of dental restorative compounds in peripheral blood lymphocytes of young exposed subjects compared with controls. The study examined, by use of the comet assay, 68 carefully selected subjects taking into account the major known confounding factors. In the 44 exposed subjects, the mean numbers of restored surfaces was 3.0 and 3.8 in males and females, respectively. Tail length, percentage of DNA in the tail, tail moment or Olive tail moment were twofold higher in the exposed group than in unexposed controls, with significant differences. No significant difference was observed between amalgam and composite fillings. Furthermore, as shown by multivariate analysis, the association between dental fillings and DNA damage was enhanced by the number of fillings and by the exposure time. Among the lifestyle variables, a moderate physical activity showed a protective effect, being inversely correlated to the DNA damage parameters evaluated. On the whole, the use of DNA-migration allowed us to detect for the first time the potential adverse impact on human health of both kinds of dental filling constituents, the amalgams and the methacrylates. The main mechanism underlying the genotoxicity of dental restorative materials of various nature may be ascribed to the ability of both amalgams and methacrylates to trigger the generation of cellular reactive oxygen species, able to cause oxidative DNA lesions.     

Biomonitoring of DNA damage in peripheral blood lymphocytes of subjects with dental restorative fillings

Dental fillings provide a major iatrogenic exposure to xenobiotic compounds due to the high prevalence of surface restorations in developed countries. Experimental data suggest that both amalgams, which contain mercury, and resin-based dental materials cause an impairment of the cellular pro- and anti-oxidant redox balance. The aim of this study was to assess the potential genotoxicity of dental restorative compounds in peripheral blood lymphocytes of young exposed subjects compared with controls. The study examined, by use of the comet assay, 68 carefully selected subjects taking into account the major known confounding factors. In the 44 exposed subjects, the mean numbers of restored surfaces was 3.0 and 3.8 in males and females, respectively. Tail length, percentage of DNA in the tail, tail moment or Olive tail moment were twofold higher in the exposed group than in unexposed controls, with significant differences. No significant difference was observed between amalgam and composite fillings. Furthermore, as shown by multivariate analysis, the association between dental fillings and DNA damage was enhanced by the number of fillings and by the exposure time. Among the lifestyle variables, a moderate physical activity showed a protective effect, being inversely correlated to the DNA damage parameters evaluated. On the whole, the use of DNA-migration allowed us to detect for the first time the potential adverse impact on human health of both kinds of dental filling constituents, the amalgams and the methacrylates. The main mechanism underlying the genotoxicity of dental restorative materials of various nature may be ascribed to the ability of both amalgams and methacrylates to trigger the generation of cellular reactive oxygen species, able to cause oxidative DNA lesions.     

Pentylenetetrazole-Induced Chemoshock Affects Protein Kinase C and Substrate Proteins in Mouse Brain

Abstract: Protein kinase C (PKC) activity, western blot analysis of PKCα, β, γ, ε, and ζ by isozyme-specific antibodies, and in vitro phosphorylation of endogenous substrate proteins were studied in the mice brain after pentyl-enetetrazole-induced chemoshock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified by DE-52 columns eluted with buffer A containing 100 or 200 m M KCI. This method consistently separates cytosolic and membrane proteins and various PKC isoforms. The 100 m M KCI eluates from DE-52 columns contain more PKC α and β in both cytosol and membrane than the 200 m M KCI eluates, whereas PKCγ, ε, and ζappear in equal amounts in these two eluates. The kinase activity assayed by phosphorylation of exogenous histone was increased in the chemoshocked mice in both the cytosol and membrane of 200 m M KCI eluates. In further analysis by immunoblotting, this increased activity was found to be due to the increase in content of PKC7 isozyme. As for novel-type ε and ζ isozymes, they were not altered in the chemoshocked mice. From autoradiography, the endogenous substrate 17-kDa neurogranin, which was shown below 21 kDa, was mostly eluted by 100 m M KCI from the DE-52 column, whereas 43-kDa neuromodulin, which was also demonstrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, only appeared in the 200 m M KCI eluates. The in vitro phosphorylation of neuromodulin was found to be increased in the chemoshocked mice. Therefore, the increased phosphorylation of neuromodulin and increased content of the PKCγ isoform were involved in the pentylenetetrazole-induced chemoshock.     

Isolation of fucosyl glycoproteins from human erythrocyte membranes by affinity chromatography using Aleuria aurantia lectin

Fucosyl glycoproteins were fractionated from a sialoglycoprotein preparation of human erythrocyte membrane by using Aleuria aurantia lectin (AAL) coupled to Sepharose 4B. The affinity eluates were characterized as having high fucose content and significant H activity as measured in terms of N-acetylgalactosamine (GalNAc) incorporation with A1-enzyme and hemagglutination inhibition assay with anti-H sera, and the unadsorbed fractions contained low levels of fucose and were devoid of apparent H activity. Neuraminidase treatment of the material improved the recovery of the affinity eluate. Thus, 66% of the applied asialoglycoprotein was recovered in the eluate, though only 10% of the untreated material was bound and eluted. Moreover, a fucose-rich and H-active fraction was obtained through the affinity chromatography of the previously unbound fraction after neuraminidase treatment. In sodium dodecyl sulfate-gel electrophoresis, the main component of both the unadsorbed and eluted fraction was revealed to be PAS-1 glycoprotein. These results indicate that AAL-Sepharose was effective for isolating fucose-containing compounds from the membrane glycoprotein especially after neuraminidase treatment. The reasons for the appearance of H activity in the affinity eluates are discussed.     

Solubilization and characterization of hormone- responsive phosphodiesterase activity of rat fat cells.

Fat cells particulate phosphodiesterase activity can be solubilized in high yield (80--100%) in a buffer system (30 mM Tris - HCl, pH 8.0) containing non-ionic detergents (0.1% Brij 30, 1.0% Triton X-100), salt (3.0 mM MgSO4, 5.0 mM NaBr) and dithiothreitol (5.0 mM). Polyacrylamide gel electrophoresis of the solubilized enzyme activity indicated the presence of two bands of activities of different electrophoretic mobilities, both of which hydrolyzed cyclic AMP and cyclic GMP. The solubilized activity eluted from DEAE Bio-Gel columns as a somewhat broad profile with at least two peaks of activity. Activity against both cyclic AMP and cyclic GMP eluted in similar but not identical patterns. The solubilized enzyme and DEAE column eluates wxhibited low (less than 1 micronM) Michaelis constants for cyclic AMP and cyclic GMP. In addition, the increases in phosphodiesterase activity induced by incubation of intact fat cells with insulin or adrenocorticotropic hormone are maintained in the solubilized state.     

Endocrine disrupters with (anti)estrogenic and (anti)androgenic modes of action affecting reproductive biology of Xenopus laevis: I. Effects on sex steroid levels and biomarker expression

Adult Xenopus laevis were exposed in vivo to ethinylestradiol, tamoxifen, methyldihydrotestosterone and flutamide as (anti)estrogenic and (anti)androgenic compounds, respectively, for four weeks at a concentration of 10(-8) M and to Lambro river water, a polluted river from Italy. Effects of the treatments were analysed by mRNA expression of retinol-binding protein (RBP), transferrin (TF), transthyretin (TTR) and vitellogenin (VTG) in the liver of male and female X. laevis, to analyse the potential of these genes to detect endocrine disrupting compounds (EDC) with different modes of action. In addition, plasma VTG and sex steroid levels, estradiol-17beta (E(2)) and testosterone (T), were analysed. Sex steroids were depressed by ethinylestradiol in both sexes whereas tamoxifen increased E(2) in females. The induction of VTG protein plasma levels was more pronounced at the protein level compared to hepatic VTG mRNA expression in response to estrogenic treatment but VTG mRNA expression detected both, estrogenic and antiestrogenic EDC. The mRNA expression of TF was decreased by estrogenic and increased by antiestrogenic treatment while TTR mRNA expression was down-regulated and RBP mRNA up-regulated by estrogenic exposure. The other treatments did not affect the mRNA expression of the examined genes.     

Dual-digitonin-pulse perfusion. Concurrent sampling of periportal and perivenous cytosol of rat liver for determination of metabolites and enzyme activities.

A previously described digitonin-perfusion technique [Quistorff, Grunnet & Cornell (1985) Biochem. J. 226, 289-297], by which intracellular material of rat liver could be liberated, has been refined, now allowing release of cytosol of high purity from both periportal and perivenous parts of the same liver. The cytosolic fractions are obtained by perfusing the liver for short intervals (10-20 s) with digitonin (4-5 mg/ml), first in the normal perfusion direction and then, after an interval of 1-2 min, in the retrograde direction, the eluate being collected during and after both intervals. The technique is termed 'dual-digitonin-pulse perfusion'. The eluate fractions showed a peak specific activity of the cytosolic enzymes alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and pyruvate kinase (PK) of 3-5-fold higher than obtained in a biopsy from the same liver. For glutamine synthetase (GS) a 10-fold higher specific activity was obtained. Zonation, defined as the ratio of the specific activities in periportal and perivenous eluates, of ALAT, LDH and PK was 10, 1.7 and 0.70 respectively. Zonation of GS was less than 0.01. These factors may be modified by a slight zonation of cytosolic protein of 1.2-1.3. Peak concentrations in the eluate of ATP, ADP, Pi, NAD+ and glycerol 3-phosphate were 32.5 +/- 11.4, 19.9 +/- 4.3, 71.9 +/- 25.4, 2.41 +/- 0.83 and 6.84 +/- 2.74 nmol/mg of protein for periportal eluates. There was no difference between periportal and perivenous eluates except for glycerol 3-phosphate, which was significantly higher in perivenous eluates, 12.8 +/- 4.5 nmol/mg of protein.     

Enantiomers of a nonylphenol isomer: absolute configurations and estrogenic potencies

Enantiomers of 4-(1,1,2-trimethylhexyl)phenol, a chiral isomer of the endocrine disrupting chemical nonylphenol, have been resolved and isolated by preparative chiral HPLC. The absolute configurations of the enantiomers were then determined by an X-ray crystallographic study of the (-)-camphanoyl derivative of the first eluted enantiomer NP(35)E1. The first enantiomer (NP(35)E1) and the second enantiomer (NP(35)E2) eluted were found to have the S and R absolute configurations, respectively. The estrogenic potencies of the S and R enantiomers were tested by the E-screen assay. A slight difference was observed in the relative proliferative effect between the S enantiomer and R enantiomer in the E-screen assay.     

Fate of aluminium and nickel in soil. Evaluation through lysimeters under laboratory conditions

ABSTRACT

Nanomaterials (Nms) applications and environmental deposition are continuously increasing. Aluminum (Al) and nickel (Ni) fate in soil, both from gamma alumina-based Nms and as chloride salts were evaluted through lysimeters. After 85 days of treatment, which included irrigation and collection of eluates, the soil of each lysimeter was divided into four sections. The metal concentration was analyzed in eluates, soil samples, and extracts. Al and iron (Fe) present in soil eluted from Control lysimeter. Al from Nms suspension treatment was quantified in the eluates since 30 days on. Ni eluted upon solid salt deposition on top of one device. These results indicate that Al and Ni applied under certain conditions on soil, could leach and reach groundwater. The total concentration and bioavailability (extractable metals) of Al and Fe in soils showed similar patterns. Ni was retained only in the soil of devices treated with chloride salts. Bioavailability % results were of concern for Ni under certain conditions of treatment: 15.57% and 11.08% in two chloride salt-treated lysimeters versus 0.55% and 0.47% of those in control and treated with Nms lysimeters. Conducting studies with different kinds of soil and longer treatment periods should be useful to understand Nms-metals fate in the environment. The results presented here constitute important evidences both for significant metal release from Nms and elution and for considerable Ni bioavailability, after deposition on soil in the form of Nms or as a chloride salt, respectively. Then, possible toxic effects could occur through exposure of aquatic and terrestrial organisms.     

An evaluation of elution techniques in the study of immune complex glomerulonephritis.

Antigen and antibody from glomerular immune complex deposits in rabbits with experimental bovine serum albumin-(BSA) induced chronic serum sickness (CSS) were quantitated in elutes from kidneys in which a portion of the antigen and antibody had been radiolabeled. The largest quantities of 125I BSA eluted with 1 M roprionic acid at pH 2.7 (86%) and 0.1 M borate buffer at pH 11.25 (80%). However, these buffers yielded less functional anti-BSA antibody than 0.02 M citrate buffer at pH 3.2 (344 mug/g kidney). Citrate buffer-eluted anti-BSA antibody was reactive in immunodiffusion, immunofluorescence, and radiolabeled BSA binding test systems, but complement fixation was impaired relative to chaotropic ion-eluted antibody. It was found that up to 75% of the eluted antibody was lost to further study by recombination with eluted BSA. This could be prevented by fractionation of the dissociated eluate before neutralization. IgG fractionated eluates were successfully fluorescein conjugated or radiolabeled for use as reagents. Elution of cryostat sections of CSS kidney was also studied; BSA, IgG, and complement (C3) eluted in parallel, and sub-microgram quantities of anti-BSA antibody were recovered.     

Microbial transformations 59: first kilogram scale asymmetric microbial Baeyer-Villiger oxidation with optimized productivity using a resin-based in situ SFPR strategy

This study is demonstrating the scale up of asymmetric microbial Baeyer-Villiger oxidation of racemic bicyclo[3.2.0]hept-2-en-6-one (1) to the kilogram scale using a 50 L bioreactor. The process has been optimized with respect to bottlenecks identified in downscaled experiments. A high productivity was obtained combining a resin-based in situ substrate feeding and product removal methodology (in situ SFPR), a glycerol feed control, and an improved oxygenation device (using a sintered-metal sparger). As expected both regioisomeric lactones [(-)-(1S,5R)-2 and (-)-(1R,5S)-3] were obtained in nearly enantiopure form (ee > 98%) and good yield. This represents the first example of such an asymmetric Baeyer-Villiger biooxidation reaction ever operated at that scale. This novel resin-based in situ SFPR technology therefore clearly opens the way to further (industrial) upscaling of this highly valuable (asymmetric) reaction.     

Bisphenol a inhibits follicle growth and induces atresia in cultured mouse antral follicles independently of the genomic estrogenic pathway

Bisphenol A (BPA) is an estrogenic chemical used to manufacture many commonly used plastic and epoxy resin-based products. BPA ubiquitously binds to estrogen receptors throughout the body, including estrogen receptor alpha (ESR1) in the ovary. Few studies have investigated the effects of BPA on ovarian antral follicles. Thus, we tested the hypothesis that BPA alters cell cycle regulators and induces atresia in antral follicles via the genomic estrogenic pathway, inhibiting follicle growth. To test this hypothesis, we isolated antral follicles from 32- to 35-day-old control and Esr1-overexpressing mice and cultured them with vehicle control (dimethylsulfoxide [DMSO]) or BPA (1-100 μg/ml). Additionally, antral follicles were isolated from 32- to 35-day-old FVB mice and cultured with DMSO, BPA (1-100 μg/ml), estradiol (10 nM), ICI 182,780 (ICI; 1 μM), BPA plus ICI, or BPA plus estradiol. Follicles were measured for growth every 24 h for 96-120 h and processed either for analysis of estrogen receptor, cell cycle, and/or atresia factor mRNA expression, or for histological evaluation of atresia. Results indicate that estradiol and ICI do not protect follicles from BPA-induced growth inhibition and that estradiol does not protect follicles from BPA-induced atresia. Furthermore, overexpressing Esr1 does not increase susceptibility of follicles to BPA-induced growth inhibition. Additionally, BPA up-regulates Cdk4, Ccne1, and Trp53 expression, whereas it down-regulates Ccnd2 expression. BPA also up-regulates Bax and Bcl2 expression while inducing atresia in antral follicles. These data indicate that BPA abnormally regulates cell cycle and atresia factors, and this may lead to atresia and inhibited follicle growth independently of the genomic estrogenic pathway.     

Stability of viruses in waste water sludge eluates

The survival of indigenous enteric viruses in samples of unconcentrated and concentrated waste water sludge eluates, which had been prepared using a combination beef extract elution - organic flocculation concentration procedure, was studied at 2, 23, and -70 degrees C. Changes of virus titer occurring in the samples were followed during an 84-day observation period, with rates of change then calculated by least-squares regression. Virus survival in both types of eluates was statistically dependent (p less than or equal to 0.05) upon storage temperature. Based upon the observed rates of inactivation the average times which would be required for a 90% decrease (one log10 unit) in virus titer for unconcentrated eluates are 27 days at 23 degrees C, 198 days at 2 degrees C, and 375 days at -70 degrees C. The calculated average times required for a 90% decrease in virus titer for concentrated eluates are 22 days at 23 degrees C, 132 days at 2 degrees C, and 246 days at -70 degrees C. In both types of eluates the rates of virus inactivation at 2 degrees C were statistically different from those observed at 23 degrees C, but not different from those observed at -70 degrees C. The three study temperatures were selected to approximate holding of samples in an air-conditioned room, fluid on wet ice (H2O), and frozen on dry ice (CO2).     

The separation of bovine brain beta-N-acetyl-D-hexosaminidases. Abnormal gel-filtration behaviour of beta-N-acetyl-D-glucosaminidase C.

Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the beta-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.     

Anti-RNA polymerase I antibodies: potential role in the induction and progression of murine lupus nephritis

Antibodies against RNA polymerase I were detected in plasma and kidney eluates of NZB/W mice. Plasma concentrations of the antibodies were the highest in mice with incipient nephritis and the lowest in mice with progressive nephritis. Mice with attenuated nephritis due to immunosuppressive therapy had intermediate plasma concentrations of the antibodies. The specific concentrations (ng/microgram IgG) of anti-RNA polymerase I antibodies in kidney eluates were significantly (10- to 70-fold) greater than the corresponding plasma concentrations. These results indicated that the decreased plasma concentration of the antibodies in mice with more advanced disease was at least partially due to selective concentration of anti-RNA polymerase I antibodies in the kidneys. The degree of this selective concentration was directly proportional (R2 = 0.9962) to the severity of renal disease, as reflected by the concentration (microgram/g tissue) of IgG eluted from the kidneys. The concentration (microgram/g tissue) of anti-RNA polymerase I eluted from the kidneys also was increased in mice with more severe renal disease. Further, the extent of this increase was greater than that of total IgG, again suggesting that anti-RNA polymerase I antibodies had been selectively concentrated in the kidneys. These findings are strongly suggestive of an important role for the RNA polymerase I/anti-RNA polymerase I antibody system in the pathogenesis of murine lupus nephritis.