RESUMO
OBJECTIVES: To estimate the frequency of the cystathionine beta-synthase deficiency caused by c.1105C>T mutation in Central Europe compared to Norway, and to examine the pathogenicity of the corresponding p.R369C mutant enzyme. STUDY DESIGN: Mutation c.1105C>T was analyzed in 600 anonymous Czech newborn blood spots. Catalytic activity and quaternary structure of the p.R369C mutant was evaluated after expression in 2 cellular systems. RESULTS: Population frequency of the c.1105C>T mutation was 0.005, predicting the birth prevalence of homocystinuria of 1:40000, which increased to 1:15500 in a model including 10 additional mutations. In Escherichia coli the p.R369C mutant misfolded, and its activity was severely reduced, and expression in Chinese hamster ovary cells enabled proper folding with activity decreased to 63% of the wild-type enzyme. This decreased activity was not due to impaired K(m) for both substrates but resulted from V(max) lowered to 55% of the normal cystathionine beta-synthase enzyme. CONCLUSIONS: The c.1105C>T (p.R369C) allele is common also in the Czech population. Although the p.R369C mutation impairs folding and decreases velocity of the enzymatic reaction, our data are congruent with rather mild clinical phenotype in homozygotes or compound heterozygotes carrying this mutation.
Assuntos
Cistationina beta-Sintase/genética , Frequência do Gene , Homocistinúria/epidemiologia , Homocistinúria/genética , Mutação/genética , Animais , Células CHO/enzimologia , Cricetinae , Cricetulus , República Tcheca/epidemiologia , Escherichia coli/enzimologia , Expressão Gênica , Genótipo , Homocistinúria/enzimologia , Humanos , Recém-Nascido , Prevalência , Dobramento de ProteínaRESUMO
GEM (glycosphingolipid-enriched microdomains) are specialized detergent-resistant domains of the plasma membrane in which some gangliosides concentrate. Although genesis of GEM is considered to occur in the Golgi complex, where the synthesis of gangliosides also occurs, the issue concerning the incorporation of ganglioside species into GEM is still poorly understood. In this work, using Chinese hamster ovary K1 cell clones with different glycolipid compositions, we compared the behaviour with cold Triton X-100 solubilization of plasma membrane ganglioside species with the same species newly synthesized in Golgi membranes. We also investigated whether three ganglioside glycosyltransferases (a sialyl-, a N-acetylgalactosaminyl- and a galactosyl-transferase) are included or excluded from GEM in Golgi membranes. Our data show that an important fraction of plasma membrane G(M3), and most G(D3) and G(T3), reside in GEM. Immunocytochemical examination of G(D3)-expressing cells showed G(D3) to be distributed as cold-detergent-resistant patches in the plasma membrane. These patches did not co-localize with a glycosylphosphatidylinositol-anchored protein used as GEM marker, indicating a heterogeneous composition of plasma membrane GEM. In Golgi membranes we were unable to find evidence for GEM localization of either ganglioside glycosyltransferases or newly synthesized gangliosides. Since the same ganglioside species appear in plasma membrane GEM, it was concluded that in vivo nascent G(D3), G(T3) and G(M3) segregate from their synthesizing transferases and then enter GEM. This latter event could have taken place shortly after synthesis in the Golgi cisternae, along the secretory pathway and/or at the cell surface.
Assuntos
Detergentes/química , Gangliosídeos/biossíntese , Gangliosídeos/metabolismo , Glicosiltransferases/metabolismo , Complexo de Golgi/química , Membranas Intracelulares/química , Animais , Células CHO/química , Células CHO/enzimologia , Células CHO/metabolismo , Extratos Celulares/química , Linhagem Celular , Membrana Celular/química , Cricetinae , Complexo de Golgi/enzimologia , Humanos , Membranas Intracelulares/enzimologia , Microdomínios da Membrana/química , Octoxinol/metabolismo , Sialiltransferases/biossínteseRESUMO
This paper deals with the relationship between the polyamine metabolism and apoptosis in the different phases of the cell cycle in a Chinese hamster ovary (CHO) cell line. Synchronously growing cells were obtained by the addition of 1.2 mM hydroxyurea and the progression through the cell cycle was monitored by determining the incorporation of 3H-thymidine in the DNA. Ornithine decarboxylase (ODC) activity showed a peak in S phase, while intracellular putrescine and spermine contents increased constantly, reaching to a maximum level at G2 phase; spermidine content doubled during G2 and increased four times during M, compared to G1. The increment in the endogenous polyamine content was associated to a diminished uptake from the medium. The apoptotic index was higher in G2 phase, coinciding with the maximum level observed in putrescine content. The results support the idea that intracellular putrescine level is closely related to apoptosis
Assuntos
Animais , Cricetinae , Apoptose , Células CHO/citologia , Células CHO/enzimologia , Divisão Celular/fisiologia , Ornitina Descarboxilase , PoliaminasRESUMO
This paper deals with the relationship between the polyamine metabolism and apoptosis in the different phases of the cell cycle in a Chinese hamster ovary (CHO) cell line. Synchronously growing cells were obtained by the addition of 1.2 mM hydroxyurea and the progression through the cell cycle was monitored by determining the incorporation of 3H-thymidine in the DNA. Ornithine decarboxylase (ODC) activity showed a peak in S phase, while intracellular putrescine and spermine contents increased constantly, reaching to a maximum level at G2 phase; spermidine content doubled during G2 and increased four times during M, compared to G1. The increment in the endogenous polyamine content was associated to a diminished uptake from the medium. The apoptotic index was higher in G2 phase, coinciding with the maximum level observed in putrescine content. The results support the idea that intracellular putrescine level is closely related to apoptosis
Assuntos
Animais , Cricetinae , Apoptose/fisiologia , Células CHO/citologia , Células CHO/enzimologia , Divisão Celular/fisiologia , Ornitina Descarboxilase/metabolismo , Poliaminas/metabolismoRESUMO
This paper deals with the relationship between the polyamine metabolism and apoptosis in the different phases of the cell cycle in a Chinese hamster ovary (CHO) cell line. Synchronously growing cells were obtained by the addition of 1.2 mM hydroxyurea and the progression through the cell cycle was monitored by determining the incorporation of 3H-thymidine in the DNA. Ornithine decarboxylase (ODC) activity showed a peak in S phase, while intracellular putrescine and spermine contents increased constantly, reaching to a maximum level at G2 phase; spermidine content doubled during G2 and increased four times during M, compared to G1. The increment in the endogenous polyamine content was associated to a diminished uptake from the medium. The apoptotic index was higher in G2 phase, coinciding with the maximum level observed in putrescine content. The results support the idea that intracellular putrescine level is closely related to apoptosis.