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21.
Alfonso Pompella Caterina Cambiaggi Silvia Dominici Aldo Paolicchi Roberto Tongiani Mario Comporti 《Histochemistry and cell biology》1996,105(3):173-178
Confocal laser scanning fluorescence microscopy coupled to image analysis was employed in order to develop and evaluate procedures for the appraisal at the single-cell level of: (1) protein-bound 4-hydroxynonenal, the specific product of membrane peroxidation (by means of immunocytochemistry with biotin-avidin revelation); (2) protein oxidation (by reaction of protein carbonyls with 2,4-dinitrophenyl-hydrazine followed by immunocytochemistry of dinitrophenyl moieties); and (3) cellular protein thiols (by direct alkylation of sulfhydryl groups with thiol-specific fluorescent reagents possessing different cell permeabilities). The procedures proved able to reveal the subcellular distribution of cytochemical parameters useful as indices of oxidative stress conditions, and may allow redox phenotyping of isolated cells, which would provide an efficient tool in selected experimental models. 相似文献
22.
Alfonso Pompella Aldo Paolicchi Silvia Dominici Mario Comporti Roberto Tongiani 《Histochemistry and cell biology》1996,106(3):275-282
A number of studies indicate that cell proliferation can be modulated by changes in the redox balance of (soluble and protein)
cellular thiols. Free radical processes, including lipid peroxidation (LPO), can affect such a balance, and a role for LPO
in multistage carcinogenesis has been envisaged. The present study was aimed to assess the relationships between the protein
thiol redox status and the LPO process in chemically induced preneoplastic tissue. The Solt-Farber's initiation-promotion
model of chemical carcinogenesis in the rat liver was used. In fresh cryostat sections, preneoplastic lesions were identified
by the reexpression of γ-glutamyltranspeptidase (GGT) activity. In serial sections, different classes of protein thiols were
stained; in additional sections, LPO was elicited by various prooxidant mixtures and determined thereafter by the hydroxynaphthoic
hydrazide-Fast Blue B procedure. The incubation of sections in the presence of chelated iron plus substrates for GGT activity
leads to the development of LPO in selected section areas closely corresponding to GGT-positive lesions, indicating the ability
of GGT activity to initiate LPO. Protein-reactive thiols, as well as total protein sulfur, were decreased by 20–25% in cells
belonging to GGT-positive preneoplastic nodules, suggesting the occurrence of oxidative conditions in vivo. The incubation
of additional adjacent sections with the prooxidant mixture H2O2 plus iron(II), in order to induce the complete oxidation of lipid present in the section, showed a decreased basal concentration
of oxidizable lipid substrate in GGT-rich areas. The decreased levels of both protein thiols and lipid-oxidizable substrate
in GGT-positive nodules suggest that the observed GGT-dependent path-way of LPO initiation can be chronically operative in
vivo during early stages of chemical carcinogenesis, in cells expressing GGT as part of their transformed phenotype. 相似文献
23.
24.
Caenorhabditis Elegans Mutants Resistant to Inhibitors of Acetylcholinesterase 总被引:4,自引:2,他引:2 下载免费PDF全文
We characterized 18 genes from Caenorhabditis elegans that, when mutated, confer recessive resistance to inhibitors of acetylcholinesterase. These include previously described genes as well as newly identified genes; they encode essential as well as nonessential functions. In the absence of acetylcholinesterase inhibitors, the different mutants display a wide range of behavioral deficits, from mild uncoordination to almost complete paralysis. Measurements of acetylcholine levels in these mutants suggest that some of the genes are involved in presynaptic functions. 相似文献
25.
Alfonso Calvo Luis M. Pastor Ramon Horn Jacinto Pallares 《The Histochemical journal》1995,27(9):670-680
Summary The glycoconjugates of hamster epididymis were investigated with conventional and lectin histochemistry. A zone of the caput
epididymis, with particular histochemical characteristics, has been differentiated. β-Elimination in combination with lectins
was used to establish the presence and distribution of N- and O-linked glycoconjugates. The epithelium, spermatozoa and the
intertubular matrix were rich in glycoconjugates. The Golgi apparatus and stereocilia of the principal cells were intensely
positive with HPA, PNA and SBA lectins. β-limination indicated that these cells contained abundant O-linked glycoconjugates.
Apical and clear cells presented a common lectin affinity; their reactivities towards WGA and UEA-I were very positive. These
cells probably contain abundant N-glycoconjugates. The spermatozoa were stained by periodic acid-Schiff (PAS) and by all the
lectins (especially in the acrosome), except by those with an affinity for α-l-fucosyl residues; the most intense reaction was found with HPA, WGA, PNA and SBA. Changes in the sperm lectin binding along
the ductus were observed: sperm flagellum abruptly acquired WGA and PNA labelling from the posterior caput, and HPA reactivity
was negative only in the zone between the caput and the corpus. 相似文献
26.
Characterization of SKM1, a Saccharomyces cerevisiae gene encoding a novel Ste20/PAK-like protein kinase 总被引:1,自引:0,他引:1
Humberto Martín Alfonso Mendoza Jose M. Rodríguez-Pachón María Molina & César Nombela 《Molecular microbiology》1997,23(3):431-444
Ste20/PAK serine/threonine protein kinases have been suggested as playing essential roles in cell signalling and morphogenesis as potential targets of Cdc42 and Rac GTPases. We have isolated and characterized the Saccharomyces cerevisiae SKM1 gene, which codes for a novel member of this family of protein kinases. The amino acid sequence analysis of Skm1p revealed the presence of a PH domain and a putative p21-binding domain near its amino terminus, suggesting its involvement in cellular signalling or cytoskeletal functions. However, deletion of SKM1 produced no detectable phenotype under standard laboratory conditions. Moreover, disruption of each of the two other S. cerevisiae Ste20/PAK-like kinase-encoding genes, STE20 and CLA4 , in skm1 backgrounds, showed that Skm1p is not redundant with Ste20p or Cla4p. Interestingly, overexpression of SKM1 led to morphological alterations, indicating a possible role for this protein in morphogenetic control. Furthermore, overproduction of Skm1p lacking its N-terminus caused growth arrest. This effect was also seen when similarly truncated versions of Ste20p or Cla4p were overexpressed. We further observed that overproduction of this C-terminal fragment of Skm1p complements the mating defect of a ste20 mutant strain. These results suggest that the N-terminal domains of S. cerevisiae Ste20/PAK-like protein kinases share a negative regulatory function and play a role in substrate specificity. 相似文献
27.
Influence of the culture medium on the expression of surface polypeptides of Yersinia enterocolitica
Three polypeptides (200, 46, and 25 kDal) encoded by the virulence plasmid were detected by SDS-PAGE in the outer membrane of Yersinia enterocolitica 09 grown at 37°C in brain-heart infusion medium. Bacteria grown at the same temperature in the tissue culture medium RPMI 1640 expressed five additional polypeptides (170, 135, 118, 100, and 98 kDal), but the 25-kDal band was not seen. The protein profile in RPMI 1640 resembles the expression pattern displayed by yersiniae when grown in vivo. The immunoblot of total membrane proteins of bacteria grown in brain-heart infusion medium revealed eight plasmid-encoded polypeptides, four of which were also in the outer membrane preparations, including a 28-kDal polypeptide. These peptides do not coincide with known plasmid-encoded outer membrane proteins. 相似文献
28.
The cattle of Doñana (139 individuals in four social groups in 1989) have lived under free-ranging conditions for centuries. Their ranging behaviour was analysed during a three-year period. A total of 17,603 locations corresponding to 247 different animals allowed both for the estimation of global and seasonal home ranges of individuals and social groups and for the comparison of movement patterns. Cattle ranging behaviour was not affected by human interference, and was shown to be regulated by a complex interaction of environment, individual and social factors. Habitat structure and seasonal fluctuations in abundance and distribution of resources determined general patterns of ranging behaviour: the greater the concentration of resources, the smaller the home ranges of individuals and social groups. These patterns were modified at an individual level by the sex of the animal and its reproductive status if male. Social influences on ranging behaviour were important because these implied the segregation of home ranges among dominant bulls and among social groups. As a result, there was a great variability in space use and home-range behaviour. 相似文献
29.
Marta Elisa Vázquez-Memije Alfonso Cárabez-Trejo Graciela Gallardo-Trillanes Graciela Delhumeau-Ongay 《Archives of biochemistry and biophysics》1984,232(2):441-449
Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N′-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F1 to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few “knobs” or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was in liver mitochondria, whereas in the testis it was . In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown. 相似文献
30.