RESUMO
Epimerase-deficiency galactosemia results from impairment of the human enzyme UDP galactose-4'-epimerase (GALE). We report a rapid, internally controlled PCR-based method for detection of nine naturally occurring point mutations in human GALE associated with epimerase deficiency. These mutations were derived from patients whose clinical presentations ranged from mild to severe; all but one of these mutations have been reported previously. The tests described here work well on both cDNA and genomic samples and require no specialized equipment beyond a thermal cycler and an agarose gel electrophoresis system. Finally, although these tests in no way replace the need for biochemical diagnosis in epimerase-deficiency galactosemia, they do provide the possibility of additional molecular information to support a biochemical diagnosis and facilitate the possibility of more accurate carrier testing, should that option be desired.
Assuntos
Galactosemias/genética , Mutação/genética , Reação em Cadeia da Polimerase/métodos , UDPglucose 4-Epimerase/genética , Sequência de Bases , Primers do DNA/genética , Humanos , Dados de Sequência MolecularRESUMO
Fibroblast growth factor 7 (FGF7/KGF) is synthesized exclusively by fibroblasts in normal tissues; it acts as a potent mitogen on epithelial cells, through interaction with the FGF7-specific receptor FGFR2/IIIb. To examine the importance of this growth factor both to prostate physiology and to prostate-cancer progression, we have tested the exogenous effect of FGF7. Thus, by mimicking the paracrine pathway (on proliferation, growth in soft agar and invasion) on the human prostatic epithelial cell line PNT1A positively checked for FGFR2/IIIb expression, FGF7 significantly enhanced cell proliferation at an optimal concentration of 7.5 x 10(-11) M, but no significant invasion or growth in soft agar were observed. To confirm FGF7 properties on human prostatic epithelial cells, we constitutively expressed FGF7 by transfecting PNT1A cells with FGF7-cDNA. The FGF7-transfected clones, PNT1A/ FGF7-T5 and PNT1A/FGF7-T6, were stable and expressed FGF7. Analysis of the FGF7-autocrine loop on the non-tumorigenic epithelial cells PNT1A showed acquired invasive potential in in vitro extracellular-matrix migration assays, specifically inhibited by an FGF7-neutralizing antibody, and over-expressed factors implicated in the migration process: the metalloproteinase MMP-1 and the plasminogen activator uPA. Taken together, these results demonstrate a role for FGF7 in triggering invasion of human prostatic epithelial cells. Furthermore, these FGF7-transfected clones exhibited functional and physiological differences from the original PNT1A cell line: anchorage-independent growth, growth in serum-free media and increased proliferation. These data confirm the oncogenic function of FGF7 in prostate progression potentially acting through paracrine and/or autocrine regulatory pathways.
Assuntos
Transformação Celular Neoplásica/induzido quimicamente , Fatores de Crescimento de Fibroblastos , Substâncias de Crescimento/toxicidade , Próstata/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/genética , Progressão da Doença , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Humanos , Masculino , Metaloendopeptidases/análise , Mitógenos/toxicidade , Invasividade Neoplásica , Reação em Cadeia da Polimerase , Próstata/enzimologia , Próstata/patologia , Transfecção , Ativador de Plasminogênio Tipo Uroquinase/análiseRESUMO
Heterocysts, cells specialized in nitrogen fixation in Anabaena sp. PCC 7120, lose the potential for cell division once fully differentiated. This suggests that cell division activity is differentially regulated in heterocysts and vegetative cells. FtsZ has been shown to play a crucial role in bacterial cell division. Two degenerate oligonucleotide primers were designed to detect, by polymerase chain reaction (PCR), an ftsZ homologue from the heterocystous cyanobacterium Anabaena sp. PCC 7120. A PCR-amplified DNA fragment was cloned and used as a probe to isolate the entire ftsZ gene of Anabaena sp. PCC 7120. The deduced amino acid sequence shares strong similarities with other FtsZ proteins, suggesting remarkable conservation of the FtsZ protein during evolution. An ORF downstream of ftsZ, which would be transcribed in the opposite direction compared to ftsZ, could encode a polypeptide with significant sequence similarity to the glutathione synthetase from Escherichia coli. Inactivation experiments in vivo for both ftsZ and the glutathione synthetase gene did not yield any double recombinants either in the presence or in the absence of combined nitrogen, suggesting that both genes are essential for cell growth under these conditions.
