Genotoxic activity of the insecticide Nuvacron (Monocrotophos) detected by the micronucleus test in bone marrow erythrocytes of mice and in CHO cells

The organophosphorus insecticide Nuvacron (Monocrotophos) is a very toxic agent widely utilized in Brazilian agriculture. To evaluate the clastogenic potential of this insecticide, in vivo and in vitro micronucleus (MN) assay experiments were carried out on Swiss mice and on Chinese hamster ovary (CHO) cells, respectively. Nuvacron administered at doses of 2.5 and 5.0 mg/kg induced a statistically significant increase in the frequencies of MN detected in polychromatic bone marrow erythrocytes from animals (six/group) treated ip 24 h before. Exponentially growing CHAO cells were treated continuously (16h) with Nuvacron diluted in water to final concentrations of 1, 10, 100, 200, and 400 mug/ml. Three experiments were carried out using the cytokinesis-block method and a total of 6000 binucleated cells were scored to determine MN frequencies. A statistically significant increase in the frequencies of MN was observed for the cells treated with 1 and 10 mug/ ml Nuvacron. A marked decrease in cell proliferation rates was observed for CHO cultures treated with higher concentrations. These data demonstrate that Nuvacron has a genotoxic effect on both in vivo and in vitro mammalian test systems.

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.

Heparan sulfate and control of cell division: adhesion and proliferation of mutant CHO-745 cells lacking xylosyl transferase

We have examined the role of cell surface glycosaminoglycans in cell division: adhesion and proliferation of Chinese hamster ovary (CHO) cells. We used both wild-type (CHO-K1) cells and a mutant (CHO-745) which is deficient in the synthesis of proteoglycans due to lack of activity of xylosyl transferase. Using different amounts of wild-type and mutant cells, little adhesion was observed in the presence of laminin and type I collagen. However, when fibronectin or vitronectin was used as substrate, there was an enhancement in the adhesion of wild-type and mutant cells. Only CHO-K1 cells showed a time-dependent adhesion on type IV collagen. These results suggest that the two cell lines present different adhesive profiles. Several lines of experimental evidence suggest that heparan sulfate proteoglycans play a role in cell adhesion as positive modulators of cell proliferation and as key participants in the process of cell division. Proliferation and cell cycle assays clearly demonstrate that a decrease in the amount of glycosaminoglycans does not inhibit the proliferation of mutant CHO-745 cells when compared to the wild type CHO-K1, in agreement with the findings that both CHO-K1 and CHO-745 cells take 8 h to enter the S phase.

Antibody recognition of Plasmodium falciparum infected red blood cells by symptomatic and asymptomatic individuals in the Brazilian Amazon

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.

Effect of taxol on chromosome aberrations induced by gamma radiation or by doxorubicin in Chinese hamster ovary cells

Combined therapy with radiation and chemotherapy has being increasingly used in cancer treatment. The effect of combinations of taxol (0.08 mug/ml) with doxorubicin (DXR, 0.5 or 1.0 mug/ml) or gamma radiation (20 or 40 cGy) was examined in two different treatment schedules (pretreatment or simultaneous treatment) using Chinese hamster ovary (CHO) cells treated at the G2 phase of the cell cycle. The results showed that taxol did not have a radiosensitizing effect on the chromosomal aberrations induced by gamma radiation nor did it have a potentiating effect on the chromosomal aberrations induced by DXR in CHO cells treated in the G2 phase of the cell cycle

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

Citocalasinas produzidas por Xylaria sp., um fungo endofítico de Piper aduncum (piperaceae)

A chemical study on the EtOAc extract produced by Xylaria sp., an endophytic fungus from Piper aduncum, resulted in the isolation of a new cytochalasin 1, along with five known 19,20-epoxycytochalasin D (2), C (3), N (4), Q (5), and R (6). The 1-6 were evaluated against the fungi C. cladosporioides and C. sphaerospermum and only 5 showed weak activity. The cytotoxicity in vitro against HeLA and CHO cells lines were investigated and the cytochalasins 2-4, and 6 showed a strong activity against HeLA. The DNAdamaging activity of 1-6 were also investigated against mutant strains of S. cerevisiae.

A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins

Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5))()and CHO-745 (2 x 10(5) and 5 x 10(5)) cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications.

Caspase dependence of the death of neonatal retinal ganglion cells induced by axon damage and induction of autophagy as a survival mechanism

We examined the degeneration of post-mitotic ganglion cells in ex-vivo neonatal retinal explants following axon damage. Ultrastructural features of both apoptosis and autophagy were detected. Degenerating cells reacted with antibodies specific for activated caspase-3 or -9, consistent with the presence of caspase activity. Furthermore, peptidic inhibitors of caspase-9, -6 or -3 prevented cell death (100 µM Ac-LEDH-CHO, 50 µM Ac-VEID-CHO and 10 µM Z-DEVD-fmk, respectively). Interestingly, inhibition of autophagy by 7-10 mM 3-methyl-adenine increased the rate of cell death. Immunohistochemistry data, caspase activation and caspase inhibition data suggest that axotomy of neonatal retinal ganglion cells triggers the intrinsic apoptotic pathway, which, in turn, is counteracted by a pro-survival autophagic response, demonstrated by electron microscopy profiles and pharmacological autophagy inhibitor.

Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE). As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80) than the latter (1:80 to 1:160). Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

Effect of scorpion toxin on the enterochromaffin-like cells in normal and Trypanosoma cruzi-infected rats: a morphological study

Intravenous injection of scorpion toxin (Tityus serrulatus) in normal and Trypanosoma cruzi infected rats did not cause ultrastructural morphologic changes on enterochromaffin-like (ECL) cells of the stomach, although it induced a significant increase of the gastric secretion. Our data seem to indicate that gastric ECL cells structure is not affected by stimulation with scorpion toxin or by acute infection with T. cruzi in the rat.

OBTENTION OF RABIES ANTIGEN THROUGH BHK21 CELLS ADHERED TO MICROCARRIERS

Four rabies antigen batches were produced from virus suspensions resulting from BHK21 cells adhered to microcarriers (Cytodex 1), inoculated and cultured in a bioreactor. In parallel the methodology of production of rabies virus through cultures of BHK21 cells in monolayers in bottles was used. The results obtained showed that infecting titles were 106.69 DL50/mL and 107.28 DL50/mL for suspensions cultured in bottles and in the bioreactor, respectively. The viral suspension volumes collected were on average 11,900 per batch from the bioreactor and 800mL per bottle. Ten horses were immunized with the antigen produced in the bioreactor. The means of antirabies antibody titers found were 240 and 212 IU/mL after the initial and the first booster doses, respectively. Rabies antigen with satisfactory infecting titers can be obtained on a large scale by culturing in a bioreactor inoculated BHK21 cells adhered to microcarriers.

IL-2 AND IFN-g, BUT NOT IL-4 SECRETION BY PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) ARE RELATED TO CD4+ T CELLS AND CLINICAL STATUS IN BRAZILIAN HIV-1-INFECTED SUBJECTS

It has been reported that production of IL-2 and IFN-g, known as T-helper type 1 cytokines, by peripheral mononuclear cells (PBMC) decreases with progression of HIV infection. In contrast, IL-4 and IL-10 production, Th2 cytokine profile, increases with HIV disease progression. PBMC were evaluated from 55 HIV-infected subjects from Divisão de Imunologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, to "in vitro" cytokines production after 24 hours of stimulation with PHA. Low levels of IL-4 production in both HIV- infected patients and normal subjects, were detected. The patients with CD4+ T cell counts <200 showed a significant decrease of IL-2 and IFN-g production compared to controls. Patients with higher counts of CD4+ T cells (either between 200-500 or >500 cells/mm3) also showed decreased production of IL-2 that was not statistically significant. There was a correlation between IL-2 and IFN-g release with CD4+ T cells counts. HIV-1-infected individuals with CD4+ T cells >500 cells/mm3 showed increased levels of IL-2 and IFN-g, than individuals with CD4+ T cells <500 cells/mm3. In conclusion, we observed a decline of IL-2 and IFN-g production at advanced HIV disease. IL-4 production was not affected during HIV infection. Taken together, these findings suggest that the cytokine profile might be influenced by the HIV infection rather than the cause of disease progression.