N,N-Dimethylaniline (DMA, CAS No. 121-69-7) and N,N-dimethyl-p-toluidine (DMPT, CAS No. 99-97-8) belong to the N-dialkylaminoaromatics, a chemical class structurally alerting to DNA reactivity. Their ...applications may be industrial (dye and pesticide intermediates, polymerizing agents) and surgical (polymerization accelerators for the manufacture of bone cements and prosthetic devices), thus implying heterogeneous types of human exposure. Findings of carcinogenicity in rodents and some nonexhaustive genotoxicity data are available for DMA, but to our knowledge no information is available on DMPT concerning either carcinogenicity or any kind of genetic toxicity. To investigate their mechanism of action and mutagenic/carcinogenic potential, DMA and DMPT were analyzed for complementary genotoxicity endpoints, namely, gene mutation in Salmonella (Ames test), structural and numerical chromosome aberrations in hamster V79 cells (micronucleus test, matched with an immunofluorescent staining for kinetochore proteins), and in vivo DNA damage in mouse and rat liver (alkaline DNA elution test). The results essentially indicate that both chemicals are chromosome damaging agents. Indeed, at the maximum nontoxic doses, they proved nonmutagenic in Salmonella (although their toxicity did not allow concentrations > 70 micrograms/plate to be tested) and weakly positive in inducing DNA damage (increases in DNA elution rates at most approximately 2.4 times control value). Conversely, they proved clearly positive in inducing numerical chromosome alterations, with dose-dependent increases up to more than five times the control value for DMPT. At the highest dose tested, both chemicals also showed a significant clastogenic effect.
Butylated hydroxyanisole (BHA) is a food preservative with markedly contradictory effects. On one side many studies showed its antimutagenic and anticarcinogenic effects but on the other side dietary ...levels of BHA were reported to cause gastrointestinal hyperplasia in rodents. We studied the influence of BHA on cytotoxicity, mutagenicity, and DNA-damaging activity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Chinese hamster V79 cells cultured in vitro. Our results showed that BHA significantly reduced the frequency of 6-thioguanine resistant (6-TGr) mutations and micronuclei induced in V79 cells by MNNG. These antimutagenic effects of BHA were, however, accompanied by a very marked increase of MNNG toxicity and also slightly increased level of MNNG-induced DNA damage. For evaluation of toxicity we used three methods: (i) trypane blue exclusion; (ii) plating efficiency; and (iii) intensity of cellular macromolecule synthesis. The level of DNA damage was measured by the comet assay. On the basis of obtained results we suggest that BHA, which induces phase II detoxifying enzymes, probably doesn't reduce the level of DNA damage induced in time of MNNG-treatment but it reduces the level of DNA damage created during a long-term period needed for expression of 6-TGr mutations and micronuclei.
The biosynthesis of human CD8 glycoprotein in transfected rat epithelial cells produces an unglycosylated precursor, an intermediate
species only initially O-glycosylated, and a doublet mature form ...carrying neutral and sialylated O-linked oligosaccharides
with the core-2 structure (Pascale, M. C., Malagolini, N., Serafini-Cessi, F., Migliaccio, G., Leone, A., and Bonatti, S.
(1992) J. Biol. Chem. 267, 9940-9947). In this study the most relevant post-translational events: dimerization, addition of
the first O-linked GalNAc, fulfillment of O-linked chains, as well as expression of involved glycosyltransferases, have been
examined and correlated with the localization and transit rate of CD8 through the exocytic pathway. The glycosyltransferase
activities measured in rat epithelial cells transfected with human CD8 DNA are entirely consistent with the primary structure
assigned to CD8 oligosaccharides. The half-time of appearance of the initially O-glycosylated precursor and mature form was
estimated to be 4 and 14 min, respectively, and the half-time for delivery of mature CD8 to the cell surface was found to
be about 30 min, indicating a very fast routing. Pulse experiments with 35Scysteine at 37 degrees C followed by chase-periods
at low temperatures showed that folding/dimerization occurs before routing to the Golgi apparatus, whereas the addiction of
O-linked GalNAc appears to take place later, very likely in cis-Golgi cisternae. Treatment of cells with monensin accumulated
the intermediate CD8 form carrying non-elongated O-linked GalNAc, whereas brefeldin A treatment produced a sialylated glycoprotein
species with a mobility faster than the mature CD8. These results indicate that the two drugs affect assembly of O-linked
chains at different time of their processing.
Newly synthesized membrane proteins are exported from the endoplasmic reticulum to the Golgi complex through an intermediate compartment. Incubation at low temperature (15 degrees C) arrests the ...proteins in the intermediate compartment and prevents the entry into the Golgi complex. We have studied, in living cells, the effect of dithiothreitol (DTT) and ATP depletion on the transport to the Golgi complex of proteins accumulated either in the endoplasmic reticulum or in the intermediate compartment after a temperature block. The morphological results obtained with vesicular stomatitis virus ts-O45 G glycoprotein and the biochemical analysis performed with human CD8 protein, an O-glycosylated protein, showed that: 1) ATP depletion blocks the export to the Golgi complex of proteins located either in the endoplasmic reticulum or in the intermediate compartment and ii) DTT interferes with the folding and export of proteins located in the endoplasmic reticulum, but it does not prevent the transfer from the intermediate compartment to the Golgi complex.
There is evidence that a carboxyl-terminal valine residue is an anterograde transport signal for type I transmembrane proteins. Removal of the signal would either delay glycosylation in the Golgi ...complex of proteins destined to recycle to the endoplasmic reticulum or determine accumulation in the endoplasmic reticulum of newly synthesized proteins destined for the plasma membrane. We used the human CD8 alpha glycoprotein to investigate the role of the carboxyl-terminal valine in the exocytic pathway. Using immunofluorescence light microscopy, metabolic labeling, and cell fractionation, we demonstrate that removal of the carboxyl-terminal valine residue delays transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment. Removal of the residue did not affect the other steps of the exocytic pathway or the folding/dimerization and glycosylation processes. Therefore, it is likely that this signal plays a role in the transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment either before or during the formation of the transport vesicles that drive the exit the protein from the endoplasmic reticulum.
ERGIC-53 is a lectin-like transport receptor protein, which recirculates between the ER and the Golgi complex and is required
for the intracellular transport of a restricted number of glycoproteins. ...We show in this article that ERGIC-53 accumulates
during the heat shock response. However, at variance with the unfolded protein response, which results in enhanced transcription
of ERGIC-53 mRNA, heat shock leads only to enhanced translation of ERGIC-53 mRNA. In addition, the half-life of the protein
does not change during heat shock. Therefore, distinct signal pathways of the cell stress response modulate the ERGIC-53 protein
level. Heat shock also affects the recycling pathway of ERGIC-53. The protein rapidly redistributes in a more peripheral area
of the cell, in a vesicular compartment that has a lighter sedimentation density on sucrose gradient in comparison to the
compartment that contains the majority of ERGIC-53 at 37 °C. This effect is specific, as no apparent reorganization of the
endoplasmic reticulum, intermediate compartment and Golgi complex is morphologically detectable in the cells exposed to heat
shock. Moreover, the anterograde transport of two unrelated reporter proteins is not affected. Interestingly, MCFD2, which
interacts with ERGIC-53 to form a complex required for the ER-to-Golgi transport of specific proteins, is regulated similarly
to ERGIC-53 in response to cell stress. These results support the view that ERGIC-53 alone, or in association with MCFD2,
plays important functions during cellular response to stress conditions.
The leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effectors, some of which are essential for cell ...transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double‐stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA‐binding protein specifically induced by BCR/ABL. The peptide sequence of the affinity‐purified protein was identical to that of the RNA‐binding protein FUS (also called TLS). Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKCβII‐dependent FUS phosphorylation. Moreover, suppression of PKCβII activity in BCR/ABL‐expressing cells by treatment with the PKCβII inhibitor CGP53353, or by expression of a dominant‐negative PKCβII, markedly impaired the ability of FUS to bind DNA. Suppression of FUS expression in myeloid precursor 32Dcl3 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte‐colony stimulating factor receptor (G‐CSFR) and downregulation of interleukin‐3 receptor (IL‐3R) β‐chain expression, and accelerated G‐CSF‐stimulated differentiation. Downregulation of FUS expression in BCR/ABL‐expressing 32Dcl3 cells was associated with suppression of growth factor‐independent colony formation, restoration of G‐CSF‐induced granulocytic differentiation and reduced tumorigenic potential in vivo. Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression.
The biosynthesis, post-translational modifications, and oligosaccharide structure of human CD8 glycoprotein have been studied
in transfected rat epithelial cells. These cells synthesized and ...expressed on the plasma membrane high amounts of CD8 in a
homodimeric form stabilized by a disulfide bridge. Three different CD8 forms were detected by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis analysis after metabolic labeling and immunoprecipitation: a newly synthesized, unglycosylated 27-kDa
(CD8u), a palmitylated and initially O-glycosylated 29-kDa (CD8i), and the mature, terminally glycosylated 32-34-kDa doublet
(CD8m). CD8i is a transient intermediate form between CD8u and CD8m: characterization of carbohydrate moiety of 3Hglucosamine-labeled
CD8i showed that it comprises for the vast majority non-elongated O-linked GalNAc closely spaced on the peptide backbone.
Structural analysis of oligosaccharides released by mild alkaline borohydride treatment from the 3Hglucosamine-labeled CD8
34-kDa form showed that the neutral tetrasaccharide Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAcOH, and an homologous monosialylated
pentasaccharide, predominate; the disialylated NeuAc2,3Gal beta 1,3(NeuAc alpha 2,6) GalNAcOH tetrasaccharide appeared to
be poorly present. In the CD8 32-kDa form the neutral tetrasaccharide was by far the prominent O-linked chain, and no disialyloligosaccharides
were identified. These results indicate that the maturation of CD8 glycoprotein in transfected rat epithelial cells results
in the formation of branched O-linked oligosaccharides and that a higher degree of sialylation is responsible for the production
of the heavier 34-kDa form.
The use of symbolic computation is proposed here as an effective tool for studying electric circuits. Using well established methods to write electric circuit equations, a student can generate a set ...of equations involving only state variables. With the help of symbolic computation, these equations can be rewritten in a normal form. They then can be solved for any input-output pair. Furthermore, the influence of a certain parameter can be analyzed in detail via analytic expressions.
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