Preservation of Haemal Nodes Fixed by Means of Two Different Aldehyde Fixatives: a Scanning Electron Microscopic Study / Preservación de Nodos Linfáticos Hemales Fijados por Dos Fijadores de Aldehído Diferentes: un Estudio de Microscopía Electrónica de Barrido

Glutaraldehyde is the fixative most commonly used in electron microscope studies of biological tissues, however it is often necessary to use samples which were not fixed in this fixative, even with the usual uncertainty of the results that may be obtained. The fixation is the more delicate step of the sample processing. Therefore in this work, the quality of preservation of haemal nodes fixed with two classic aldehyde fixatives: formaldehyde and glutaraldehyde we have compared under the scanning electron microscope. Our results showed that both fixatives were successful in preserving the morphology of haemal nodes components; however glutaraldehyde conferred satisfactory results mainly in the preservation of parenchymal cells, whereas formaldehyde was better for preservation of stromal fibres.

El glutaraldehido es el fijador que se utiliza con más frecuencia en estudios en los tejidos biológicos a través microscopía electrónica. Sin embargo, a menudo es necesario utilizar muestras que no han sido fijadas con este fijador, aún con la incertidumbre de los resultados que se puedan obtener. La fijación es el paso más importante en el procesamiento de los tejidos. Por lo anterior, hemos efectuado este estudio comparando la calidad de conservación de nodos linfáticos hemales fijados con formaldehido y glutaraldehido. Los resultados muestran que ambos fijadores conservaron adecuadamente la morfología de los componentes de los nodos linfáticos hemales, sin embargo, el glutaraldehido conservó en mejores condiciones, principalmente, las células del parénquima, pero el formaldehido conservó mejor las fibras del estroma en nodos linfáticos.

An immunohistochemical study of the gut neuroendocrine system in juvenile pejerrey Odontesthes bonariensis (Valenciennes).

In this study, several neuropeptides were identified by immunohistochemistry in neuroendocrine cells (NEC) located in the gut epithelium and nerve cell bodies of the enteric nervous system of pejerrey Odontesthes bonariensis, a species that is a promising candidate for intensive aquaculture. The neuropeptides involved in orexigenic or anorexigenic action, i.e. gastrin, cholecystokinin-8, neuropeptide Y and calcitonin gene-related peptide (CGRP), displayed a significantly higher number of immunoreactive NECs in the anterior intestine, suggesting that this region of the gut plays an important role in the peripheral control of food intake. On the other hand, leu-enkephalin and vasoactive intestinal peptide (VIP), both associated with the modulation of the enteric immune system, showed no significant variations in the mean value of immunopositive NECs between the anterior and posterior intestine. This may indicate that their activity is required at a similar level along the entire gut. In addition, CGRP and VIP-immunoreactive neurons and nerve fibres were observed in the myenteric plexus, which might exert synergistic effects with the neuropeptides immunolocalized in NECs.

Erythropoiesis and erythrophagocytosis in bovine haemal nodes / Eritropoyesis y eritrofagocitosis en nodos linfáticos hemales de bovino

Whether bovine haemal nodes are involved in turnover of red blood cells has been a subject of some controversy In this study, fluorescent and conventional optical microscopy of conventionally or immunohistochemically stained node sections, together with transmission electron microscopy, showed the presence of erythrocyte precursors and megakaryocytes, and evidence of active involvement in the destruction and replacement of old or degenerate red cells and the platelets.

Si nodos linfáticos hemales bovinos están involucrados en la cantidad de volumen de glóbulos rojos ha sido objeto de cierta controversia. En este estudio, secciones de nodos linfáticos teñidas convencionalmente o inmunohistoquimicamente fueron analizadas con microscopía óptica fluorescente y convencional, junto con microscopio electrónico de transmisión, los que revelaron la presencia de precursores eritrocíticos y megacariocitos, y la evidencia de participación activa en la destrucción y sustitución de glóbulos rojos viejos o degenerados y plaquetas.

Members of the Hyposoter didymator Ichnovirus repeat element gene family are differentially expressed in Spodoptera frugiperda.

BACKGROUND: The abundance and the conservation of the repeated element (rep) genes in Ichnoviruses genomes suggest that this gene family plays an important role in viral cycles. In the Ichnovirus associated with the wasp Hyposoter didymator, named HdIV, 10 rep genes were identified to date. In this work, we report a relative quantitative transcription study of these HdIV rep genes in several tissues of the lepidopteran host Spodoptera frugiperda as well as in the H. didymator wasps. RESULTS: The data obtained in this work indicate that, in the early phases of infection (24 hours), HdIV rep genes each display different levels of transcripts in parasitized 2nd instar or HdIV-injected last instar S. frugiperda larvae. Only one, rep1, is significantly transcribed in female wasps. Transcript levels of the HdIV rep genes were found as not correlated to their copy number in HdIV genome. Our results also show that HdIV rep genes display different tissue specificity, and that they are primarily transcribed in S. frugiperda fat body and cuticular epithelium. CONCLUSION: This work is the first quantitative analysis of transcription of the ichnovirus rep gene family, and the first investigation on a correlation between transcript levels and gene copy numbers in Ichnoviruses. Our data indicate that, despite similar gene copy numbers, not all the members of this gene family are significantly transcribed 24 hours after infection in lepidopteran larvae. Additionally, our data show that, as opposed to other described HdIV genes, rep genes are little transcribed in hemocytes, thus suggesting that they are not directly associated with the disruption of the immune response but rather involved in other physiological alterations of the infected lepidopteran larva.

Morphological changes in the mink area postrema during growth and under different stages of sexual activity.

The histological features of the area postrema (AP) of mink brains of both sexes were investigated at different ages and physiological conditions with light and electron microscopy. The mink AP was a twin-winged structure located at the dorsal surface of the medulla oblongata and consisted of neurons, glial cells, and both continuous and fenestrated capillaries enmeshed in a rich neuropil. The ventricular surface of the mink AP was covered by a single layer of tanycytes except at its most caudal part that was covered by a basal membrane derived from the pia mater. Supraependymal cells and intraventricular axons were also a common finding over the apical poles of tanycytes. However, our study demonstrates that the mink AP acquires the above general features at an advanced postnatal time and that, once fully developed, it undergoes morphological changes that can be directly linked to the aging process and sexual activity of the animals.

Identification of positive cells to interleukin-4 in bovine haemal nodes.

The bovine haemal nodes are lymphatic organs located in the haemal circulation. Their parenchyma is represented by plasma cells, macrophages and B and T lymphocytes. The T helper type 2 (Th2) CD4 lymphocyte can be found within the T lymphocytes. The activated Th2 CD4 lymphocyte produces interleukin-4 (IL-4), a peptidic hormone involved in the acute-phase immune response. This interleukin can promote either B-lymphocyte differentiation and T-lymphocyte proliferation or it can promote the type of immunoglobulin that can be liberated. Our results have shown, by immunostaining with anti-IL-4, not only the presence and localization of these lymphocytes in bovine haemal nodes but also the participation of polymorphonuclear cells (neutrophils) in the storage of IL-4. These results give value to the humoral and cellular immunological importance of haemal nodes in bovines and they can serve as a contribution to determine the cross-reactivity of bovine IL-4 with the human anti-serum used in this work.

Related RNAs in lepidopteran cells after in vitro infection with Hyposoter didymator virus define a new polydnavirus gene family.

In the present study, we describe the isolation and the characterization of three different Hyposoter didymator virus (HdV) lepidopteran host-expressed genes, the products of which might interfere with the host physiology during parasitism. In this report, we study the expression of HdV genes in Sf9 cells infected with HdV since results indicate that the Sf9 model mimics to some extent the in vivo model and may be utilized to study expression of HdV genes in lepidopteran host cells. This system allowed us to isolate three HdV-specific cDNAs, termed M24, M27, and M40. cDNA nucleotide sequence analysis demonstrated significant regions of homology. The three cDNAs displayed repeated sequences arranged in tandem array that might have evolved through domain duplication. Similar to other previously described polydnavirus host-expressed genes, two intron positions have been found in the M24 leader region. The cDNAs corresponded to RNAs of 1.5, 1.6, and 2.3 kb that are also detected in parasitized Spodoptera littoralis larvae. They are encoded by different genes likely located on different HdV DNA molecules. Corresponding RNAs are detected early postinfection and remain detectable for at least 10 days postinfection. They encode secreted glycine- and proline-rich proteins. An antiserum raised against a baculovirus recombinant M24-encoded protein detected similar proteins in the culture medium of infected lepidopteran cells and in parasitized host hemolymph. We propose that the three cloned genes belong to an HdV gene family specifically expressed in parasitized lepidopteran hosts.

A scanning and immunohistochemical study in bovine haemal node.

The bovine haemal nodes are lymphatic organs with haemal circulation. The blood circulates inside of them through virtual cavities named sinuses. In these sinuses there is passage of cells and particulate materials from parenchyma to sinuses and vice versa. For this passage temporal overtures exist in the sinus walls. The sinus wall is composed of endothelial cells, basal membrane and reticular cells. Our results have demonstrated that type IV collagen was one of the basal membrane constituents in bovine haemal nodes and by this constitution a dynamic equilibrium of the sinus walls was permitted.

High glucose induces antioxidant enzymes in human endothelial cells in culture. Evidence linking hyperglycemia and oxidative stress.

It has been suggested that oxidative stress may play an important role in the pathogenesis of diabetic complications. Hyperglycemia may cause increased production of free radicals, and evidence supports a prominent role for these reactive molecules as mediators of endothelial cell dysfunction in diabetes. It has been demonstrated that active oxygen species induce antioxidant enzyme expression in some tissues, and this phenomenon is considered proof of an existing oxygen-dependent toxicity. In this study, human endothelial cells from umbilical vein, immortalized human endothelial cells, and immortalized human endothelial cells transfected to express high glutathione peroxidase levels were grown in normal and high-glucose conditions. High glucose delayed replication after 7 and 14 days of culture of human endothelial cells, both from umbilical vein and immortalized, while transfected cells were not affected. The activity and the mRNA expression of the antioxidant enzymes CuZn-superoxide-dismutase, Mn-superoxide-dismutase, catalase, and glutathione peroxidase were evaluated after 2, 7, and 14 days of culture. High glucose at days 7 and 14 induced an overexpression of CuZn-superoxide-dismutase, catalase, and glutathione peroxidase in both human endothelial cells from umbilical vein and immortalized human endothelial cells, while in transfected cells it did not. This study demonstrates that high glucose induces an increase in antioxidant enzyme levels in human endothelial cells, suggesting that elevated glucose levels may produce an oxidative stress in the cells.

Ultraviolet-B-light-induced mutagenesis of C-H-ras codons 11 and 12 in human skin fibroblasts.

Mutations in the ras oncogene are detected with a high frequency in non-melanoma skin cancer. Approximately half of the squamous-cell carcinomas (SCC) and one third of the basal-cell carcinomas (BCC) carry mutations at the second position of Ha-ras codon 12 (GGC to GTC), whereas mutations in Ki-ras codon 12 occur less frequently. Since the mutations in the Ha-ras and Ki-ras oncogenes are located opposite potential pyrimidine dimer sites (C-C), it is likely that the mutations are induced by ultraviolet radiation present in sunlight. We studied the capacity of ultraviolet B (UVB) light to induce base-pair changes in Ha-ras codons 11 and 12 in human skin fibroblasts. UVB induced mostly C to T and G to A transitions and C to A and G to T transversions. The base-pair change with the highest relative abundance was C to T in the middle position of codon 11 followed by (in diminishing relative abundance) C to A in the middle position of codon 11, G to A and G to T in the middle position of codon 12. The C to T and G to A transitions are compatible with pyrimidine photodimers as pre-mutagenic lesions, whereas the C to A and G to T transversions could be generated due to the formation of 8-hydroxyguanine, which is the major oxidation product of guanine. The relative abundance of mutations induced by UVB in Ha-ras codons 11 and 12 does not correlate with mutations observed in the DNA from non-melanoma skin cancer, where the G to T transversion in the middle position of codon 12 is selected.

The defence against free radicals protects endothelial cells from hyperglycaemia-induced plasminogen activator inhibitor 1 over-production.

It has been shown that hyperglycaemia may stimulate plasminogen activator inhibitor 1 (PAI-1) over-production in human endothelial cells in culture. At the same time, it has been shown that glucose may enolize, producing free radicals. In this study, the possibility that hyperglycaemia stimulates PAI-1 over-production in human endothelial cells in culture by generating free radicals has been evaluated. For this purpose two experimental models were used: human endothelial cells transfected to express high glutathione peroxidase levels cultured in hyperglycaemic media, and human endothelial cells cultured in hyperglycaemic media with the antioxidant GSH. Cells grown in 20 mM glucose produced higher values of PAI-1 with respect to controls. The production of PAI-1 was not influenced by hyperglycaemia in transfected cells. GSH in the medium reduced hyperglycaemia-induced PAI-1 over-production, but also reduces the basal production of PAI-1 in the cells grown in normal glucose concentration. These data show that antioxidant defences may reduce hyperglycaemia-induced PAI-1 over-production in human endothelial cells in culture. The hypothesis that oxidative stress may play an important role in the pathogenesis of diabetic complications is then supported by this study.

[Psychological, behavioral and occupational status changes after an aortocoronary bypass intervention]. / Variazioni psicologiche, comportamentali e di status professionale dopo intervento di bypass aortocoronarico.

BACKGROUND. Studies on the quality of life after coronary artery by-pass grafting (CABG) have yielded discordant results. Several studies have described psychological and social improvements while others have reported a lack of change in behavioural risk factors and return to work. There have been no reports on Italian patients, and, because of the wide range of psychological measures used in previous studies, it is difficult to draw any general conclusions. The aim of this study was to assess the psychological sequelae of CABG. METHODS. A total of 164 patients (142 men and 22 women, aged 60 years) with myocardial ischemia, completed the CBA-H Questionnaire 3-5 days before elective CABG and again after 6 months. RESULTS. State anxiety scores were lower after surgery (p < .000) as were health fears (p < .000), depression (p < .009) and life stress (p < or = .000) scores. There were also improvements in well-being (p < .003), affective relationships (p < .000) and sexual relations (p < .0007). There was a decline in behavioural risk factors, namely: smoking behaviour (p < .09), alcohol consumption (p < .002), over-eating (p < .0000) and sedentary life-style (p < .02). Clinical post-operative complications did not negatively influence patients' psychological state and return to work. Preoperative health fears (p < .04) and social anxiety (p < .02) did influence patients' return to work. CONCLUSIONS. In conclusion, psychosocial function, health state and quality of the life generally improved after elective CABG. Return to work was found to be an unreliable measure of the success of surgery. Pre- and post-operative data revealed a general denial trait which identifies patients at greater risk of cardiovascular events after CABG.

Transfection with human copper-zinc superoxide dismutase induces bidirectional alterations in other antioxidant enzymes, proteins, growth factor response, and paraquat resistance.

Transfection of a pSV2 human copper-zinc superoxide dismutase expression vector into murine fibroblasts resulted in stable transgenic clones producing increased amounts of copper-zinc superoxide dismutase. Two classes of transfectants were observed and were characterized by the presence or absence of an increase in endogenous glutathione peroxidase activity. In addition, increases and decreases in individual clones in the activities of manganese superoxide dismutase, glutathione reductase, and NADPH-reductase were detected. In general, these alterations in enzyme activity correlated to the cellular glutathione peroxidase/copper-zinc superoxide dismutase ratio. Parameters of cellular physiological functions were also altered, including cell division time, FGF and EGF response, fibronectin content, paraquat resistance, hydrogen peroxide release into media, and sensitivity to radiation. Some of these cellular parameters were also bidirectional and reflected the cellular glutathione peroxidase/copper-zinc superoxide dismutase ratio. Our results indicate that small deviations from the normal physiological copper-zinc superoxide dismutase/seleno-glutathione peroxidase ratios can have pronounced effects on other antioxidant enzymes, growth rate, growth factor response, and expression of proteins normally not associated with oxygen metabolism.

The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B.

BACKGROUND: The inducible, higher eukaryotic transcription factor NF-kappa B is activated by a variety of stimuli. Several lines of evidence have suggested that reactive oxygen intermediates (ROIs) serve as messengers for most if not all of these stimuli. To identify the relevant ROI species and to gain more direct evidence for an involvement of ROIs as messengers, we investigated whether changes in the levels of enzymes that control intracellular ROI levels affect the activation of NF-kappa B. RESULTS: Cell lines stably overexpressing the H2O2-degrading enzyme catalase were deficient in activating NF-kappa B in response to tumor necrosis factor alpha (TNF) or okadaic acid. The catalase inhibitor aminotriazol restored NF-kappa B induction. In contrast, stable overexpression of cytoplasmic Cu/Zn-dependent superoxide dismutase (SOD), which enhances the production of H2O2 from superoxide, potentiated NF-kappa B activation. The level of cytoplasmic NF-kappa B-I kappa B complex was unchanged, indicating that synthesis of NF-kappa B was not affected. CONCLUSIONS: Our data show that one ROI species, H2O2 acts as a messenger in the TNF- and okadaic acid-induced post-translational activation of NF-kappa B. Superoxide is only indirectly involved, as a source for H2O2. These data explain the inhibitory effects of many antioxidative compounds on the activation of NF-kappa B and its target genes. H2O2 is overproduced in response to various stimuli, and normal levels of catalase appear insufficient to remove it completely. H2O2 can therefore accumulate and act as an intracellular messenger molecule in the response to pathogens.

The role of the cellular antioxidant defense in oxidant carcinogenesis.

Oxidant carcinogens interact with multiple cellular targets including membranes, proteins, and nucleic acids. They cause structural damage to DNA and have the potential to mutate cancer-related genes. At the same time, oxidants activate signal transduction pathways and alter the expression of growth- and differentiation-related genes. Indeed, the carcinogenic action of oxidants results from the superposition of these genetic and epigenetic effects. All cells possess elaborate antioxidant defense systems that consist of interacting low and high molecular weight components. Among them, superoxide dismutases (SOD), glutathione peroxidases (GPx), and catalase (CAT) play a central role. Our studies with mouse epidermal cells demonstrate that the balance between several antioxidant enzymes rather than the activity of a single component determines the degree of protection. Unexpectedly, increased levels of Cu,Zn-SOD alone in stable transfectants resulted in sensitization to oxidative chromosomal aberrations and DNA strand breaks. However, a concomitant increase in CAT or GPx in double transfectants corrected or overcorrected the hypersensitivity of the SOD clones depending on the ratios of activities CAT/SOD or GPx/SOD. The cellular antioxidant capacity also affected oxidant induction of the growth-related immediate early protooncogene c-fos. Increases in CAT or SOD reduced the accumulation of c-fos message, albeit for different reasons. The cellular antioxidant defense also affects the action of UVB light (290-320 nm) that represents the most potent carcinogenic wavelength range of the solar spectrum. UVB light is known to exert its action in part through oxidative mechanisms. Increases in CAT and GPx protected mouse epidermal cells from UVB-induced DNA breakage.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential cytotoxicity in mouse epidermal JB6 cells: a potential mechanism for oxidant tumor promotion.

It has been suggested that superior antioxidant defense systems protect promotion-sensitive (p+t) mouse epidermal JB6 clone 41 cells from excessive deleterious effects of oxidants, allowing their clonal expansion in contrast to that of promotion-resistant (p-) clone 30 cells. In support of this concept, we report that oxidants produced by xanthine/xanthine oxidase cause more cytotoxicity, cellular damage, and cell death in p-cells. Cell surface blebbing, an early morphological consequence of oxidative injury, was detected in cultures grown on glass coverslips. While a rise in cytosolic ionized calcium ([Ca2+]i) preceding bleb formation was observed in both p+ and p- cells by digital imaging fluorescence microscopy, elevated levels of [Ca2+]i were sustained longer in p- cells. This increase was dependent on the levels of extracellular ionized calcium ([Ca2+]e) in p+ but not p- cells. We conclude that the superior antioxidant defense or improved Ca2+ buffering of promotable clone 41 cells protects them from more severe deregulation of [Ca2+]i and, as a consequence, from excessive cytotoxicity after exposure to oxidant promoters.

Oxy-radical induced mutagenesis of hotspot codons 248 and 249 of the human p53 gene.

Oxidants are suspected to represent important human carcinogens. They are mutagenic and may participate in the activation of proto-oncogenes and the inactivation of tumor suppressor genes. We have studied the capacity of hydrogen peroxide plus ferric chloride (FeCl3) to induce base pair changes in the hotspot codons 248 and 249 of the p53 tumor suppressor gene in human fibroblasts. In codon 248 (CGG) H2O2/FeCl3 only induced the transversion of G to C in the second position and the transition of G to A in the third position. No evidence was obtained for spontaneous or oxidant-induced deamination of 5-methylcytosine in the CpG dinucleotide of codon 248 since neither C to T transitions in the first position nor G to A transitions in the middle position were observed. H2O2/FeCl3 efficiently induced G to T transversions at both G-residues of codon 249 (AGG) and C to A transversions at the first position of codon 250 (CCC). It is evident that H2O2/FeCl3 possesses essentially the same mutagenic specificity for codons 249 and 250 of p53 as bulky carcinogens such as aflatoxin B1, benzo(a)pyrene or heterocyclic amines. In particular, it is not possible to eliminate oxidants from the list of candidate carcinogens which may be responsible for the high incidence of p53 codon 249 AGT mutations in hepatocellular carcinoma from certain areas of the world.

Ultraviolet B light-induced mutagenesis of p53 hotspot codons 248 and 249 in human skin fibroblasts.

Mutations in the p53 tumor suppressor gene are detected in approximately half of non-melanoma skin cancers. The type of base-pair changes observed strongly suggests solar radiation as the causative mutagen. Mutations are distributed nonrandomly and form moderate hotspots. We studied the capacity of ultraviolet B light (UVB, 280-320 nm) to induce base-pair changes into the p53 exon 7 sequence extending from nt 14067 to 14075 in human skin fibroblasts. This sequence contains hotspot codon 248. UVB induced mostly C-->A and G-->T transversions. The base-pair change with the highest relative abundance was C-->A in the first position of codon 250 (CCC-->ACC), followed by (in diminishing relative abundance) G-->T in the third position of codon 249 (AGG-->AGT), C-->A in the first position of codon 248 (CGG-->AGG), and C-->A in the third position of codon 247 (AAC-->AAA). The C-->T transition in the third position of codon 247 (AAC-->AAT) occurred with moderate efficiency. These base-pair changes are compatible with pyrimidine photodimers as premutagenic lesions, but they could also form opposite 8-hydroxyguanine, which is the major oxidation product of guanine. No evidence was obtained for the presence of tandem double CC-->TT transitions in the untranscribed strand at codons 247/248 and 250. The relative abundance of mutations induced by UVB in the p53 sequence extending from codon 247 to 250 in human fibroblasts does not correlate with mutations observed in the DNA from non-melanoma skin cancer. This lack of correlation suggests that the mutability of this p53 sequence at the DNA level plays only a minor role in the pathogenesis of non-melanoma skin cancer in humans.