Effects of the 2,4-D herbicide on gills epithelia and liver of the fish Poecilia vivipara

The 2,4-dichlorophenoxyacetic acid, usually named 2,4-D is one of the most widely used herbicides in the world. Acute toxicity of 2,4-D herbicide was investigated through its effects on guppies (Poecilia vivipara Bloch et Schneider 1801). Fish were exposed to the herbicide at concentrations of 10, 20 and 40µl per liter of water for 24 hours to determine its effects on gills and liver epithelia. The estimated LC50 was 34.64µl of 2,4-D per liter of water. Histochemical analyses and Feulgen's reaction were conducted to detect glycoconjugates and DNA, respectively, in gills and liver epithelia. Histochemistry revealed qualitative variations of glycoconjugates present on mucous cells and granules. The four types of mucous cells contained neutral granules, acids, or both. Increasing amounts of syalomucins were observed from the control group to the group exposed to the highest concentration of 2,4-D, suggesting increased mucous viscosity and the formation of plaques that could inhibit gas exchange and osmoregulation. Lamellar fusion observed in the group exposed to 40µl of 2,4-D suggests a defense mechanism. Hepatocytes showed vacuolization in the 10 and 20µl/L groups. The 40 µl/L group showed normal hepatocytes as well as changed ones, many Ito cells, micronuclei, and nuclear swelling. These effects may be associated with toxicity or adaptative processes to cellular stress. The data from this study indicates the importance of assessing similar risks to aquatic species and suggests that Poecilia vivipara is an adequate biological model for analysis of environmental contamination.

Arc fusion of self-fluxed nickel alloys

Self-fluxed nickel alloys are usually flame fused after thermal spraying. However, due to the practical aspects of high temperatures reached during flame fusing, large structures such as the hydraulic turbines for power generation, can not be efficiently coated. An alternative is to fuse the sprayed coating with a gas tungsten electric arc. In this case, heating is much more intensive and substrate temperature during and after the fusing operation is much lower, thus reducing the possibility that any problem will occur. In this work, coatings of self-fluxed nickel alloy fused by flame and gas tungsten arc were evaluated as protection of hydraulic turbines against cavitational damage. Several tests were performed, including the ASTM ultrasonically vibration-induced cavitation, optical and scanning electronic microscopic metallography, and hardness tests. The results showed that the arc-fused coating presented better cavitation damage resistance, probably due to its finer microstructure. A field application of this new technique is also described. A self-fluxed Ni alloy was flame sprayed in critical regions of Francis-type hydraulic turbine blades and fused by a gas tungsten arc after spraying. The blades will be inspected during the next two years.

Thermalhydraulic behavior of electrically heated rod during a critical heat flux transient

In nuclear reactors, the occurrence of critical heat flux leads to fuel rod overheating with clad fusion and radioactive products leakage. To predict the effects of such phenomenon, experiments are performed using electrically heated rods to simulate operational and accidental conditions of nuclear fuel rods. In the present work, it is performed a theoretical analysis of the drying and rewetting front propagation during a critical heat flux experiment, starting with the application of an electrical power step from steady state condition. After the occurrence of critical heat flux, the drying front propagation is predicted. After a few seconds, a power cut is considered and the rewetting front behavior is analytically observed. Studies performed with various values of coolant mass flow rate show that this variable has more influence on the drying front velocity than on the rewetting one.

Concanavalin A-reactive nuclear matrix glycoprotein

The binding capacity of concanavalin A (Con A) to condensed euchromatin and heterochromatin was investigated in chicken erythrocyte nuclei (CEN), mouse liver cells, Zea mays mays meristematic cells and Drosophila melanogaster polytene chromosomes after 4 N HCl hydrolysis to determine whether binding was preferentially occurring in bands and heterochromatin. Dry mass (DM) variation was investigated in CEN by interference microscopy. Feulgen and Con A reactions were employed for all materials to correlate the loci of the two reactions. Quantifications and topological verifications were carried out by video image analysis (high performance cytometry). It was observed that 4 N HCl hydrolysis caused an important DM loss in CEN leaving a level corresponding to the average DNA DM content. In this material, Con A binding was restricted to the nuclear envelope, which reinforces the idea of the absence of a nuclear matrix in these cells. The other cell types exhibited a correspondence of Feulgen-positive and Con A-reactive areas. The Con A reaction was highly positive in the condensed chromatin areas and heterochromatin. This fact led us to speculate that Con A-positive proteins may play a role in the chromatin condensation mechanism, endowing this structure with physico-chemical stability towards acid hydrolysis and contributing to its rheological properties.

Image analysis and definition of nuclear phenotypes

Some basic topics concerned with the extraction of textural and geometric information from cell nucleus images as well as description and characterization of chromatin supraorganization and consequent classification of nuclear phenotypes are presented.

Analysis of viral and cellular parameters which affect the fusion process of influenza viruses

In the present investigation we studied the fusogenic process developed by influenza A, B and C viruses on cell surfaces and different factors associated with virus and cell membrane structures. The biological activity of purified virus strains was evaluated in hemagglutination, sialidase and fusion assays. Hemolysis by influenza A, B and C viruses ranging from 77.4 to 97.2%, from 20.0 to 65.0%, from 0.2 to 93.7% and from 9.0 to 76.1% was observed when human, chicken, rabbit and monkey erythrocytes, respectively, were tested at pH 5.5. At this pH, low hemolysis indexes for influenza A, B and C viruses were observed if horse erythrocytes were used as target cells for the fusion process, which could be explained by an inefficient receptor binding activity of influenza on N-glycolyl sialic acids. Differences in hemagglutinin receptor binding activity due to its specificity to N-acetyl or N-glycolyl cell surface oligosaccharides, density of these cellular receptors and level of negative charges on the cell surface may possibly explain these results, showing influence on the sialidase activity and the fusogenic process. Comparative analysis showed a lack of dependence between the sialidase and fusion activities developed by influenza B viruses. Influenza A viruses at low sialidase titers (<2) also exhibited clearly low hemolysis at pH 5.5 (15.8%), while influenza B viruses with similarly low sialidase titers showed highly variable hemolysis indexes (0.2 to 78.0%). These results support the idea that different virus and cell-associated factors such as those presented above have a significant effect on the multifactorial fusion process

Patch-clamp detection of macromolecular translocation along nuclear pores

The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal 'gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleus

Association of hepatic nuclear factor-4 in the apolipoprotein B promoter: a preliminary report

Previous studies have examined the arrangement of regulatory elements along the apolipoprotein B (apoB) promoter region (-3067 to +940) and a promoter fragment extending from nucleotides -150 to +124 has been demonstrated to be essential for transcriptional activation of the apoB gene in hepatic and intestinal cells. It has also been shown that transcriptional activation of apoB requires a synergistic interaction between hepatic nuclear factor-4 (HNF-4) and CCAAT/enhancer-binding protein a (C/EBPa) transcription factors. Here, we have examined the hypothesis that HNF-4 factor binding to DNA may induce a DNA helix bend, thus facilitating the communication with a C/EBPa factor located one helix turn from this HNF-4 factor in the apoB promoter. A gel electrophoretic mobility shift assay using wild type double-stranded oligonucleotides or modified wild type duplex oligonucleotides with 10 nucleotides inserted between HNF-4 and C/EBPa factor motifs showed similar retarded complexes, indicating that HNF-4 and C/EBPa factors interact independently of the distance between binding sites. However, when only one base, a thymidine, was inserted at the -71 position of the apoB promoter, the complex shift was completely abolished. In conclusion, these results regarding the study of the mechanisms involving the interaction between HNF-4 and C/EBPa factors in the apoB promoter suggest that the perfect 5'-CCCTTTGGA-3' motif is needed in order to facilitate the interaction between the two factors.

Nuclear exclusion of transcription factors associated with apoptosis in developing nervous tissue

Programmed cell death in the form of apoptosis involves a network of metabolic events and may be triggered by a variety of stimuli in distinct cells. The nervous system contains several neuron and glial cell types, and developmental events are strongly dependent on selective cell interactions. Retinal explants have been used as a model to investigate apoptosis in nervous tissue. This preparation maintains the structural complexity and cell interactions similar to the retina in situ, and contains cells in all stages of development. We review the finding of nuclear exclusion of several transcription factors during apoptosis in retinal cells. The data reviewed in this paper suggest a link between apoptosis and a failure in the nucleo-cytoplasmic partition of transcription factors. It is argued that the nuclear exclusion of transcription factors may be an integral component of apoptosis both in the nervous system and in other types of cells and tissues.

Probable effect of photoperiod on seasonal variation in the nuclear volume of the adrenal cortex of viscacha (Lagostomus maximus maximus)

The neuroendocrine system regulates several organic functions such as reproduction, metabolism and adaptation to the environment. This system shows seasonal changes linked to the environment. The experimental model used in the present study was Lagostomus maximus maximus (viscacha). The reproduction of males of this species is photoperiod dependent. Twenty-four adult male viscachas were captured in their habitat at different times during one year. The adrenal glands were processed for light microscopy. Serial cuts were stained with hematoxylin-eosin for the morphometric study, and 100 nuclei of each zone of the adrenal cortex were counted per animal. Data were analyzed statistically by ANOVA and the Tukey test. The cells of the glomerulosa zone are arranged in a tube-shaped structure. The fasciculata zone has large cells with central nuclei and clearly visible nucleoli and with a vacuolar cytoplasm. In the reticularis zone there are two of types of cells, one with a nucleus of fine chromatin and a clearly visible nucleolus and the other with nuclear pycnosis. Morphometric analysis showed maximum nuclear volumes during the February-March period with values of 133 ± 7.3 µm3 for the glomerulosa, 286.4 ± 14.72 µm3 for the fasciculata, and 126.3 ± 9.49 µm3 for the reticularis. Minimum nuclear volumes were observed in August with values of 88.24 ± 9.9 µm3 for the glomerulosa, 163.7 ± 7.78 µm3 for the fasciculata and 64.58 ± 4.53 µm3 for the reticularis. The short winter photoperiod to which viscacha is subjected could inhibit the adrenal cortex through a melatonin increase which reduces the nuclear volume as well as the cellular activity.

alpha-Smooth muscle actin and proliferating cell nuclear antigen expression in focal segmental glomerulosclerosis: functional and structural parameters of renal disease progression

The aim of the present study was to investigate the expression of alpha-smooth muscle actin (alpha-SM-actin) and proliferating cell nuclear antigen (PCNA) in renal cortex from patients with focal segmental glomerulosclerosis (FSGS) and their correlations with parameters of renal disease progression. We analyzed renal biopsies from 41 patients with idiopathic FSGS and from 14 control individuals. The alpha-SM-actin immunoreaction was evaluated using a score that reflected the changes in the extent and intensity of staining in the glomerular or cortical area. The PCNA reaction was quantified by counting the labeled cells of the glomeruli or renal cortex. The results, reported as median ± percentile (25th; 75th), showed that the alpha-SM-actin scores in the glomeruli and tubulointerstitium from the renal cortex were 2.0 (2.0; 4.0) and 3.0 (3.0; 4.0), respectively, in patients with FSGS, and 0.5 (0.0; 1.0) and 0.0 (0.0; 0.5) in the controls. The number of PCNA-positive cells per glomerulus and graded field of tubulointerstitium from the renal cortex was 0.2 (0.0; 0.4) and 1.1 (0.3; 2.2), respectively, for patients with FSGS, and 0.0 (0.0; 0.5) and 0.0 (0.0; 0.0) for controls. The present data showed an increase of alpha-SM-actin and PCNA expression in glomeruli and renal cortex from FSGS patients. The extent of immunoreaction for alpha-SM-actin in the tubulointerstitial area was correlated with the intensity of proteinuria. However, there was no correlation between the kidney expression of these proteins and the reciprocal of plasma creatinine level or renal fibrosis. These findings suggest that the immunohistochemical alterations may be reversible.

Ligand-induced endocytosis and nuclear localization of angiotensin II receptors expressed in CHO cells

A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization.

Presence of autoantibodies against HeLa small nuclear ribonucleoproteins in chagasic and non-chagasic cardiac patients

We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95% of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31% reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.

A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

c-erbB-2 expression and nuclear pleomorphism in canine mammary tumors

The objective of the present investigation was to study the expression of c-erbB-2 and MIB-1 and try to associate them with morphological features of the cell such as nuclear pleomorphism, mitotic count and histological grade in a series of 70 canine mammary gland tumors, 22 of them benign and 48 malignant. Tumors were collected at the Veterinary Hospital of UFMG (Brazil) and the Veterinary Faculty of Porto University (Portugal). c-erbB-2 expression was determined according to the guidelines provided by the manufacturer of the HercepTest system and nuclear pleomorphism, mitotic count and histological grade according the Elston and Ellis grading system. The HercepTest is the FDA-approved in vitro diagnostic test marketed by Dako. It is a semi-quantitative immunohistochemical assay used to determine overexpression of HER2 protein (human epidermal growth factor receptor) in breast cancer tissue. MIB-1 expression was also evaluated in 28 malignant tumors. Seventeen (35.4%) of the malignant tumors were positive for c-erbB-2 expression, which was positively associated with nuclear pleomorphism (P < 0.0001), histological grade (P = 0.0017) and mitotic count (P < 0.05). Nuclear pleomorphism also showed a positive association with MIB-1 index (P < 0.0001). These results suggest that some of the biological and morphological characteristics of the tumor are associated in canine mammary gland tumors, as also reported for human breast cancer. It was also possible to show that the immunoexpression of c-erbB-2 can be a factor in mammary carcinogenesis. This fact opens the possibility of using anti-c-erbB-2 antibodies in the treatment of canine mammary tumors.