RESUMO
BACKGROUND: We evaluated whether baseline maternal heart rate variability (HRV), including the Analgesia Nociception Index (ANI), is associated with maternal hypotension and fetal heart rate (FHR) abnormalities following combined spinal-epidural (CSE) labor analgesia. METHODS: Laboring women were enrolled in this prospective observational study. The primary endpoint was maternal hypotension. The secondary endpoint was FHR abnormalities within 30â¯min following CSE analgesia initiated with intrathecal plain bupivacaine 1.0â¯mg and fentanyl 20⯵g. The maternal ANI, electrocardiogram, blood pressure, heart rate, oxygen saturation, and FHR tracings were recorded 15â¯min before and 30â¯min after CSE. Parturients were grouped based on presence of hypotension and FHR abnormalities. Patient demographics and HRV metrics were compared. Receiver operating characteristics (ROC) curves were constructed for the prediction of hypotension and FHR abnormalities. RESULTS: No significant intergroup differences were detected in patient characteristics. Several baseline HRV metrics and ANI differed significantly between the normotensive (nâ¯=â¯50) and hypotensive (nâ¯=â¯31) groups and between parturients showing FHR abnormalities (nâ¯=â¯19) and those showing reassuring FHR traces (nâ¯=â¯62). The area under the ROC curve (AUC) for predicting hypotension of the baseline low-frequency (LF)/high-frequency (HF) ratio was 0.677 (95% CI 0.55 to 0.80), and that of the ANI was 0.858 (95% CI 0.78 to 0.94). For predicting non-reassuring FHR patterns, the AUC of the LF/HF ratio was 0.77 (95% CI 0.65 to 0.89), and that of the ANI was 0.833 (95% CI 0.72 to 0.94). CONCLUSIONS: The ANI can predict the propensity for maternal hypotension and non-reassuring FHR patterns following CSE.
Assuntos
Analgesia Epidural , Analgesia Obstétrica , Hipotensão , Trabalho de Parto , Gravidez , Feminino , Humanos , Frequência Cardíaca Fetal , Trabalho de Parto/fisiologia , BupivacaínaRESUMO
We have reported previously that highly metastatic LuM1 cells derived from colon carcinoma colon 26 secrete larger amounts of gelatinase B than NM11 cells with poor metastatic potential, and that an increase in this gelatinase B secretion can be induced by autocrine factors (Hyup et A, Cancer Res., 54: 3611-3616, 1994). In the present study, a partial characterization was achieved by comparison of the autocrine factor preparation (fraction G) from serum-free medium conditioned with metastatic LuM1 cells with soluble factors known to stimulate gelatinase B secretion. Secretion of gelatinase B by LuM1 cells was augmented by tumor necrosis factor alpha, transforming growth factor beta1 (TGF-beta1), interleukin 1beta, or epidermal growth factor, and specific neutralizing antibodies abolished the induced increases. Platelet-derived growth factor and insulin-like growth factor 1 had no effect on gelatinase B secretion by LuM1 cells. The enhancement of gelatinase B secretion by fraction G was partially inhibited by the antibody to TGF-beta1. TGF-beta1 was detected in both active and latent forms in serum-free medium conditioned with LuM1 or NM11 cells, with the amount of TGF-beta1 higher in the former case. Gelatinase B secretion by LuM1 cells was enhanced by the addition of TGF-beta1 to the culture medium, but that by NM11 cells was not seriously affected, although the latter bound more of the factor. These results indicate the involvement of this growth factor in the autocrine stimulation of gelatinase B secretion by LuM1 cells. However, the autocrine factor effect was not fully explained by TGF-beta1 in the medium, and the involvement of some other unknown factor(s) was thus indicated.
Assuntos
Carcinoma/patologia , Colagenases/metabolismo , Neoplasias do Colo/patologia , Metástase Neoplásica , Fator de Crescimento Transformador beta/fisiologia , Animais , Carcinoma/enzimologia , Colagenases/genética , Neoplasias do Colo/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Substâncias de Crescimento/farmacologia , Interleucina-1/fisiologia , Metaloproteinase 3 da Matriz , Metaloproteinase 9 da Matriz , Metaloendopeptidases/genética , Camundongos , RNA Mensageiro/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Células Tumorais CultivadasRESUMO
We previously reported that murine tumor cells with a high spontaneous metastatic potential to the lung secrete higher amounts of M(r) 95,000 gelatinase (matrix metalloproteinase 9, MMP9) than do poorly metastatic cells. The present study, conducted to clarify the mechanisms underlying the increase in MMP9, revealed an autocrine factor that enhances the secretion of M(r) 95,000 gelatinase (MMP9). The secretion of MMP9 by highly metastatic colon carcinoma LuM1 cells, detected by zymography, was augmented 10-fold when cultured in medium supplemented with serum-free medium conditioned with LuM1 cells. Because the secretion of M(r) 60,000 gelatinase (MMP2), as well as total protein, by the same cells was not affected under these conditions, the augmentation appears specific for MMP9. The steady-state level of MMP9 mRNA was elevated in LuM1 cells cultured in the presence of the supernatant. The amount of the factor in the culture medium increased with time in culture, indicating that it was produced by the LuM1 cells. It was found to be heat stable but sensitive to trypsin digestion. Conditioned medium from poorly metastatic NM11 cells did not stimulate the secretion of gelatinases by NM11 cells, suggesting that autocrine stimulation of MMP9 secretion is a characteristic of metastatic cells. This factor could account for the augmented secretion of MMP9 by murine tumor cells with spontaneous metastatic potential to the lung.
Assuntos
Colagenases/metabolismo , Neoplasias do Colo/metabolismo , Gelatinases/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Metaloendopeptidases/metabolismo , Proteínas de Neoplasias/análise , Animais , Meios de Cultura Livres de Soro , Feminino , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Camundongos , Camundongos Endogâmicos BALB C , Células Tumorais CultivadasRESUMO
The molecular mechanism of organ-specific metastasis to the liver remains largely unknown. However, it is conceivable that paracrine growth factors produced by a target organ induce migration and proliferation of malignant cells to that organ, and this is the cause of organ-specific metastasis. In this study, we investigated the effect of hepatocyte growth factor/scatter factor (HGF/SF) and activin A, which are known to be produced by the liver, on the motility and growth of liver-metastatic cell line FBJ-LL. HGF/SF and activin A induced motility synergistically, but they did not affect the proliferation of FBJ-LL cells. Expression of the HGF/SF receptor, the c-met gene, and the activin-receptor type IA, type IB, and type IIA genes in FBJ-LL cells was detected by reverse transcription polymerase chain reaction. These findings suggest that both HGF/SF and activin A promote organ-specific metastasis to the liver by induction of migration through their specific receptors on liver-metastatic cells.
Assuntos
Fator de Crescimento de Hepatócito/fisiologia , Inibinas/fisiologia , Neoplasias Hepáticas/secundário , Receptores de Ativinas Tipo I , Ativinas , Animais , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-met/análise , Receptores de Fatores de Crescimento/análise , Células Tumorais CultivadasRESUMO
Selective glycopeptide mapping of recombinant human erythropoietin (rhEPO) used as a model glycoprotein was successfully carried out by on-line high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) using a Vydac C18 column eluted in acetonitrile-1 mM ammonium acetate, pH 6.8. rhEPO expressed in a Chinese hamster ovary clone was exhaustively digested into four glycopeptides and nine peptides with endoproteinase Glu-C. Both glycopeptides and peptides were eluted with trifluoroacetic acid as the eluent, whereas only glycopeptides were eluted selectively with ammonium acetate in the following order: N38, N24, 0126, and N83. Furthermore, many glycoforms included in each glycopeptide were found to be separated by differences in the numbers of sialic acid and N-acetyllactosaminyl repeats. Twenty, 16 and 22 different N-linked oligosaccharides were determined at Asn24, 38, and 83, respectively, and two different O-linked oligosaccharides were observed at Ser126. Our method is simple, rapid, and useful for determining the carbohydrate structures at each glycosylation site and for elucidating the site-specific carbohydrate heterogeneity.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Eritropoetina/química , Glicopeptídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Mapeamento de PeptídeosRESUMO
A simple, rapid, and accurate assay using surface plasmon resonance (SPR) apparatus with anti-follistatin antibody (SPR immunoassay) has been developed for the quantitation of recombinant follistatin. This assay can be performed with a direct injection of conditioned medium; results were obtained within 10 min. The quantitation component of this assay was precise and accurate with a limit of quantitation of 62.5 ng/ml in Ham's F12 medium containing 2% fetal bovine serum. These results demonstrate that SPR immunoassay is a powerful technique for several researches, especially for screening of gene transfectant and monitoring of protein production.
Assuntos
Ativinas/análise , Imunoensaio/métodos , Ressonância de Plasmônio de Superfície , Ativinas/biossíntese , Animais , Biotecnologia , Células CHO/metabolismo , Cricetinae , Meios de Cultura/química , Folistatina , Proteínas Recombinantes/análise , Tecnologia FarmacêuticaRESUMO
We reported previously that peptide mapping using high-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS) are useful for determination of the glycosylation sites, carbohydrate structure, and site-specific carbohydrate heterogeneity of glycoproteins. Here, with intention to enhance the sensitivity and shorten the time-span of analysis to characterize glycoproteins, especially biotechnological products with carbohydrate moieties, we studied the introduction of HPLC with a microbore column to LC/MS with recombinant erythropoietin (rh-EPO). In addition, we evaluated the ability of LC/MS/MS precursor-ion scanning to make identification of glycopeptides and facilitate the analysis of carbohydrate moieties. We found that the peptide mapping with microbore HPLC is highly sensitive and more rapid than the previous method, and the precursor-ion scanning is helpful for identifying glycopeptides. Our results indicate that these methods are very useful for characterization and quality control of the carbohydrate moieties of biotechnological products.
Assuntos
Eritropoetina/análise , Glicopeptídeos/análise , Controle de Qualidade , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Eritropoetina/química , Glicopeptídeos/química , Espectrometria de Massas , Mapeamento de Peptídeos/métodos , Proteínas Recombinantes , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Using recombinant human erythropoietin (rh-EPO) from three different sources, the usefulness of HPAEC-PAD (high-pH anion exchange chromatography with pulsed amperometric detection) for evaluation of carbohydrate moieties of rh-EPO products was evaluated. It is well known that in vivo bioactivity and metabolic fate of EPO are dependent on the number of sialic acids and the degree of branching in the carbohydrate moieties. Here we show that HPAEC analysis reveals differences in the number of sialic acids as well as in the structure of desialylated N-glycans among the rh-EPO products. Therefore, HPAEC is useful for evaluation of the quality of rh-EPO products.
Assuntos
Cromatografia por Troca Iônica/métodos , Eritropoetina/normas , Sequência de Carboidratos , Eritropoetina/química , Humanos , Ácido N-Acetilneuramínico/química , Controle de Qualidade , Proteínas Recombinantes/químicaRESUMO
For clarification of the functions of gangliosides on tumor metastasis, examination was made of the ganglioside patterns of poorly metastatic FBJ-S1 and highly metastatic FBJ-LL cells. FBJ-S1 cells expressed GM3 and GD1a, whereas FBJ-LL cells expressed GM3 and slightly expressed GD1a. The capacity for FBJ-LL cells to migrate was ten times that of FBJ-S1 cells, but decreased by a half by pretreatment with GD1a. GD1b or GT1b had the same effect as GD1a, and synthetic sialyl compounds to a lesser extent, suggesting that gangliosides contain more than two sialyl residues to inhibit the migration of FBJ-LL cells.
Assuntos
Gangliosídeos/fisiologia , Animais , Cromatografia em Camada Fina , Gangliosídeos/genética , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica/genética , Células Tumorais CultivadasRESUMO
High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.
Assuntos
Carboidratos/análise , Cromatografia Líquida de Alta Pressão/métodos , Glicoproteínas/química , Espectrometria de Massas/métodos , Animais , Sequência de Carboidratos , Carboidratos/química , Bovinos , Dados de Sequência Molecular , Oligossacarídeos/análise , Oligossacarídeos/química , Ribonucleases/química , Álcoois Açúcares/análiseRESUMO
High-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS) were applied to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin (EPO) used as a model of the sialylated glycoprotein. N-linked oligosaccharides were released from recombinant human EPO expressed in Chinese hamster ovary cells enzymatically and reduced with NaBH(4). Many different sialylated oligosaccharides of EPO were separated and characterized by LC/MS equipped with a graphitized carbon column (GCC). Glycosylation sites and the preliminary glycosylation pattern at each glycosylation site were determined by LC/MS of endoproteinase Glu-C-digested EPO. The detailed site-specific carbohydrate heterogeneity caused by the differences in the molecular weight, branch, linkage, and sequence was elucidated by GCC-LC/MS of the N-linked oligosaccharides released from the isolated glycopeptides. Structural details of the isomers were analyzed by LC/MS/MS, and it was indicated that di- and trisialylated tetraantennary oligosaccharides are attached to Asn24, 38, and 83, whereas their isomers, di- and trisialylated triantennary oligosaccharides containing N-acetyllactosamines, are combined with Asn24. Our method is useful for the determination of glycosylation sites, the site-specific carbohydrate heterogeneity of glycoproteins, and the carbohydrate structure.
Assuntos
Carboidratos/química , Cromatografia Líquida/métodos , Eritropoetina/química , Espectrometria de Massas/métodos , Animais , Células CHO , Configuração de Carboidratos , Cricetinae , Glicosilação , Humanos , Proteínas RecombinantesRESUMO
We previously demonstrated that high-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) is useful for the structural analysis of carbohydrates in glycoproteins. Using LC/MS with GCC, sulfated N-linked oligosaccharides were found in erythropoietin (EPO) expressed in baby hamster kidney cells. Sulfation occurs in a part of the N-linked oligosaccharides in the EPO. Sulfated monosaccharide residue in the sulfated N-linked oligosaccharide was determined by exoglycosidase digestion followed by sugar mapping by LC/MS. The linkage position and branch-location of the sulfate group in the tetraantennary oligosaccharide were analyzed by (1)H-nuclear magnetic resonance. It was suggested that sulfation occurs on the C-6 position of GlcNAc located in the GlcNAcbeta1-4Manalpha1-3 branch.
Assuntos
Eritropoetina/química , Oligossacarídeos/química , Enxofre/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Linhagem Celular , Cromatografia Líquida , Cricetinae , Glicosídeo Hidrolases/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Oligossacarídeos/isolamento & purificação , Proteínas Recombinantes , Espectrometria de Massas por Ionização por Electrospray , Álcoois Açúcares/químicaRESUMO
Ganglioside GD1a, which is highly expressed in poorly metastatic FBJ-S1 cells, has been shown to inhibit the serum-induced migration capability of highly metastatic FBJ-LL cells. In the present study, the capacity of FBJ-S1 cells to adhere to vitronectin was found to be about half that of FBJ-LL cells. Pre-treatment of FBJ-LL cells with GD1a decreased this capacity by 30% that of the control, whereas GM1-pre-treatment caused only a 10% decrease, indicating that GD1a specifically inhibits FBJ-LL cell adhesion to vitronectin. Since FBJ-LL cells contain almost no GD1a, transfectants capable of expressing GD1a to varying degrees were produced in this study by transfection of FBJ-LL cells with GM2/GD2-synthase cDNA. Decrease in the serum-induced migration capacity of these transfectants was accompanied by an increment in GD1a expression. Adhesion of the transfectants to vitronectin decreased by 30% as compared with mock-transfected cells. Within 4 to 5 weeks after GD1a-expressing transfectant and mock-transfected cells were transplanted into mice, metastatic nodules were observed in liver, lung, kidney and adrenal glands of mock-transplanted mice, but not in those with GD1a-expressing transfectants, indicating that GD1a suppresses the metastasis of FBJ-osteosarcoma cells, possibly by inhibiting cell migration and cell adhesion. The involvement of the ganglioside in the suppression of metastasis is clearly demonstrated in the present study.
Assuntos
Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Gangliosídeos/farmacologia , Metástase Neoplásica , Animais , DNA Complementar , Fibronectinas , Gangliosídeo G(M1)/farmacologia , Gangliosídeo G(M3)/metabolismo , Gangliosídeos/genética , Laminina , Camundongos , Camundongos Endogâmicos BALB C , N-Acetilgalactosaminiltransferases/genética , Transfecção , Células Tumorais Cultivadas , Vitronectina , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
We previously reported that ganglioside GD1a, which is highly expressed in poorly metastatic FBJ-S1 cells, inhibits the serum-induced motility of FBJ-LL cells and that the metastatic potential of FBJ-LL cells is completely suppressed by enforced GD1a expression (Hyuga et al., Int J Cancer 1999;83:685-91). We recently discovered that hepatocyte growth factor (HGF) induces FBJ-LL cell motility. In the present study, the HGF-induced motility of FBJ-S1 cells was found to be one-thirtieth that of FBJ-LL cells. This motility of GD1a-expressing transfectants, which were produced by transfection of FBJ-LL cells with GM2/GD2 synthase cDNA, decreased with increases in their GD1a expression and HGF induced almost no motility in GD1a-pretreated FBJ-LL cells, indicating that GD1a inhibits the HGF-induced motility of FBJ-LL cells. The expression of the HGF receptor c-Met on FBJ-S1 cells, FBJ-LL cells, transfectants and a mock-transfectant was almost the same. The level of tyrosine phosphorylation of c-Met after HGF stimulation in FBJ-S1 cells, GD1a-pretreated FBJ-LL cells and a GD1a-expressing transfectant was significantly lower than in FBJ-LL cells and a mock-transfectant. These findings suggested that GD1a inhibits the HGF-induced motility of FBJ-LL cells through suppression of tyrosine phosphorylation of c-Met. HepG2 cells, a human hepatoma cell line, were used to investigate whether GD1a interferes with other cancer cells expressing c-Met. HepG2 cells did not express GD1a. HGF induced cell scattering of HepG2 cells and the scattering was inhibited by pretreating the cells with GD1a. The c-Met in the cells was autophosphorylated by stimulation with HGF, but after treating the cells with GD1a, the HGF-induced autophosphorylation of c-Met was suppressed. These results suggest that GD1a acts as a negative regulator of c-Met in cancer cells.