Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and "Death factor" family) are involved in regulat...Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and "Death factor" family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of "Death factor" family, the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl-2. The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis. Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.展开更多
In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice, the sustenance of their pluripotency and normal karyotype depend on the feeder layer ...In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice, the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF). Compared with the feeder layer of MEF cells, medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyo-typic normality of ES cells only in short term cell propagation. Besides, ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras. Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more convenient and efficient than the conventional microdrop method.展开更多
Some recent studies indicated that GABAergic system is involved in mammalian sperm acrosome reaction (AR), but direct evidence pertaining to the expression of gat1 in mammalian sperm is not yet demonstrated. In this s...Some recent studies indicated that GABAergic system is involved in mammalian sperm acrosome reaction (AR), but direct evidence pertaining to the expression of gat1 in mammalian sperm is not yet demonstrated. In this study, we evaluated the presence of 67kDa GAT1 protein and mRNA in rat testis by Western blotting and reverse transcription-polymerase chain reaction. Meanwhile, immunohistochemical and immunofluorescent analyses also identified GAT1 protein on the elongated spermatid and sperm. These results indicated that rat testis is a novel site of gat1 expression. Further studies should be taken to explore the role of GAT1 protein on sperm acrosome reaction.展开更多
Trichosanthin(TCS) is a potent allergen in mice. It can reproducibly induce specific IgE responses in C57BL/6J mice without the help of adjuvant alum. TCS can bring out the IgE responses to ovabumin(OVA), while OVA it...Trichosanthin(TCS) is a potent allergen in mice. It can reproducibly induce specific IgE responses in C57BL/6J mice without the help of adjuvant alum. TCS can bring out the IgE responses to ovabumin(OVA), while OVA itself could not induce IgE responses to it. How- ever, TCS only works when QVA immunization is given one day after TCS immunization. Either time lag in OVA immunization, or immunization of both antigens at the same time, or OVA immunization given first, all has no effect on the induction of IgE responses to OVA. Through analysis of the antibody specificity of hybridoma clones, it indicated that specific antibodies to TCS or OVA were secreted by independent B cell clones. The IgE antibodies showed no polyreactivity to different antigens.展开更多
Thichosanthin (TCS) is a potent allergen to mice. According to our previous experiments, it could bring out the IgE response to ovabumin (OVA) if TCS was given one day before OVA immunization, while OVA alone could no...Thichosanthin (TCS) is a potent allergen to mice. According to our previous experiments, it could bring out the IgE response to ovabumin (OVA) if TCS was given one day before OVA immunization, while OVA alone could not induce IgE to it. In this work, the kinetics of interleukin 4(IL-4) and interferon γ(IFN-γ) gene expression in the mesenteric lymph node (MLN) of TCS-immunized mice was investigated using a semi-quantitative RT-PCR method. It indicated that TCS induced significant IL-4gene expression and the peaks of IL4 gene expression were on day one after TCS immunization in both primary and secondary response. In contrast, the IFN-γ gene expression was suppressed. Furthermore, the IL-4 gene expression in the secondary response was lower than that in the primary response. Thus the presence of IgE rpemory B cells were studied. Results showed that the amount of mature IgE mRNA arose significantly and rapidly one day after TCS restimulation, while in the MLN of the mice primed 30 days before and without boost, it was almost as the same amount of the unimmunized control. These findings suggest the existence of the IgE memory B cells in the mice after the primary TCS immunization.展开更多
Epidermal growth factor (EGF) is produced primarily by Leydig cells of human testis. Expression of the EGF gene was assessed in mouse testis during the course of sexual maturation by the application of the RT-PCR meth...Epidermal growth factor (EGF) is produced primarily by Leydig cells of human testis. Expression of the EGF gene was assessed in mouse testis during the course of sexual maturation by the application of the RT-PCR method and the use of specific oligonucleotide primers. Testis EGF mRNA content increased with the developmental age of the mice, i.e., day 15 < day 30 < day 45 postnatal. The expression of the EGF gene appears to correlate with maturation of the testis and proliferation of Leydig cells.展开更多
Mad protein has been shown as an antagonist of cMyc protein in some cell lines. The effect of Mad protein to the malignant phenotype of human hepatoma BEL7404 cell line was investigated experimentally. An eukarryotic ...Mad protein has been shown as an antagonist of cMyc protein in some cell lines. The effect of Mad protein to the malignant phenotype of human hepatoma BEL7404 cell line was investigated experimentally. An eukarryotic vector pCDNA Ⅲ containing full ORF fragmentof mad cDNA was transfected into targeted cells. Under G418 selection, stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells. DNA synthesis, cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cel1s were partially inhibited in comparison to control cells.Flow Cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase, resulting in the retardation of cell proliferation. RT-PCR detected a marked inhibition of the expression of cdc25A, an important regulator gene of G0/G1to S phase in cell cycle. It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-7404-M1 cells in the absence of serume.Thus, Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL7404 cells.madoverexpression and regulation of cell growth and apoptosis展开更多
Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye, Lucifer Yellow. Close correlation existed between the incidences of dye coupling and quan...Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye, Lucifer Yellow. Close correlation existed between the incidences of dye coupling and quantitative evaluation of gap junctions. High incidences of dye coupling and of gap junctions occurred at a stage when notochord cells were active in the change of cell shape and cell arrangement. With the subsidence of cell movements, both dye coupling and gap junctions were reduced to lower levels. It was, therefore, suggested that intercellular communication via gap junctions played an important role in the coordination of notochord cell movements.Gap junctions of altered configuration occurred in notochord cells in late tailbud stage. The comparison of incidences of dye coupling at this stage with those at other stages strongly suggested that the gap junctions of altered configuration functioned just as those of generalized type.展开更多
In order to elucidate the molecular mechanisms of globin gene expression during embryonic development, the nuclear extracts from mouse hematopoietic tissue at different stages of development have been prepared. By usi...In order to elucidate the molecular mechanisms of globin gene expression during embryonic development, the nuclear extracts from mouse hematopoietic tissue at different stages of development have been prepared. By using DNase I footprinting and gel mobility shift assays, the binding of protein factors in these extracts to the human βglobin promoter was analyzed. The differences in the binding patterns of protein factors during development were observed. An erythroid-specific and stage-specific nuclear protein in the nuclear extract from d 18 mouse fetal liver was identified, which can bind to the sequence (from -66bp to -90bp) of human β-globin promoter. We therefore speculate that the function of this cis-acting element may be similar to stage selector element (SSE) in chieken βA- promoter.展开更多
The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes ...The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.展开更多
The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5'-flanking regulatory elements of human β-globin gene, two negative control regions(NCR1,-610 to -490...The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5'-flanking regulatory elements of human β-globin gene, two negative control regions(NCR1,-610 to -490 bp;NCR2, -338 to-233bp), was identified.Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2.These data provided evidence that the cis acting elements of the 5'-flanking region might be involved in the developmental control of β-globin gene and NCR2 might be responsible in part for the silence of β-globin gene in the embryonic and fetal stages.展开更多
The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled, inpart, by the silencer (-392bp～-177bp) upstream of thisgene. In order to elucidate its role, the nuclear ex...The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled, inpart, by the silencer (-392bp～-177bp) upstream of thisgene. In order to elucidate its role, the nuclear extractfrom the human fetal liver has been prepared and the interactions between trans-acting factors and this silencerelement have been examined. By using DNasel footprinting assay, a major protected region from -278bp to -235bpwithin this silencer element was identified. Furthermore,we found in gel mobility shift assay and Southwestern blotting assay that there were at least four trans-acting factors (MW ≈ 32, 28, 26 and 22kD) in the nuclear extractisolated from the human fetal liver, which could specifically bind to this region. Our results suggested that thesetrans-acting factors might play an important role in silencing the human embryonic ε-globin gene expression at thefetal stage through the interactions with this silencer.展开更多
A cDNA encoding the mouse GABA transporter has been isolated and sequenced. The results show that the mouse GABA transporter cDNA differs from that of the rat by 60 base pairs at the open reading frame region but the ...A cDNA encoding the mouse GABA transporter has been isolated and sequenced. The results show that the mouse GABA transporter cDNA differs from that of the rat by 60 base pairs at the open reading frame region but the deduced amino acid sequences of the two cDNAs are identical and both composed of 599 amino acids. However,the amino acid sequence is different from the sequence deduced from a recently published mouse GABA transporter cDNA.展开更多
The characteristics of the particulate mouse centromere enriched fraction from isolated nuclei obtained in our laboratory were investigated by indirect immunoflu-orescence, test of the activity of microtubule organizi...The characteristics of the particulate mouse centromere enriched fraction from isolated nuclei obtained in our laboratory were investigated by indirect immunoflu-orescence, test of the activity of microtubule organizing center(MTOC), SDS-PAGE, and fluorescence in situ hybridization. Most of the particles of the fraction are complexes of DNA and kinetochore proteins and show MTOC activity. The DNA isolated from the fraction can hybridize with DNA in the regions of the primary constrictions of all chromosomes of ascites cells. The kinetochore proteins isolated from the fraction are mainly those with molecular weight of 55 KD and 59 KD. Results suggested that the fraction obtained is a centromere enriched nuclear fraction as indicated in our previous report.展开更多
The effect of peripheral nerve (PN) on neurite outgrowth from retinal explants of adult hamsters was examined. Cultures of retinal explants, and co-cultures of retinal explants and PN were performed using chick retina...The effect of peripheral nerve (PN) on neurite outgrowth from retinal explants of adult hamsters was examined. Cultures of retinal explants, and co-cultures of retinal explants and PN were performed using chick retinal basement membrane (BM) as substrate. The presence of PN increases the number and length of neurite outgrowth. In addition, a high proportion of neurites situated close to PN tend to grow towards it. Since there was no contact between retinal ex-plants and PN, we suggest that PN might secrete diffusible substances to attract the neurites to grow towards it.展开更多
In order to analyze the mechanism of immunomodulation by LPS on murine peritoneal suppressor macrophages, we have, using RNase protection assay,checked the changes of mRNA expression pattern of several cytokine genes ...In order to analyze the mechanism of immunomodulation by LPS on murine peritoneal suppressor macrophages, we have, using RNase protection assay,checked the changes of mRNA expression pattern of several cytokine genes during the immuno-modulation.It has been found that, after treating peritoneal suppressor macrophages with LPS, mRNAs of IL-12 p35, IL-12 p40,IL-6 and IFN-γ are newly appeared, while those of IL-1α, IL-1β and IL-1Ra are increased and those of other cytokines, like TGF-β1 and MIF are not changed at all.It seems certain that those cytokines, whose expression is increased by LPS stimulation, may be responsible for the functional changes of suppressor macrophages during immuno-modulation. Among these changes, the appearance of IL-12 mRNA may play a critical role, and, in this regard, the synergetic effect between IFN-γ and LPS on the increase of IL-12 p35 and IL-12 p40 mRNA expression is an interesting finding.展开更多
The restriction fragment length polymorphisms distribution and frequency of dystrophin gene in Chinese were studied by using 14 subclones of the entire 14kb cDNA for the dystrophin as hybridization probes. Allelic fra...The restriction fragment length polymorphisms distribution and frequency of dystrophin gene in Chinese were studied by using 14 subclones of the entire 14kb cDNA for the dystrophin as hybridization probes. Allelic fragments were detected in hybridization patterns of PvuII/1a, Taq I/2b-3, Taq I/5b-7, and Xba I/10. Among them, the allelic fragments (26kb and 3.8kb) in PvuII/2b-3 pattern and the allelic fragments (10.0kb and 8.4kb) in Taq I/5b-7 patterns had never been reported previously. Compared with the data from Caucasians and Japanese, it indicated that there was a significant difference (P<0.01) of the allelic fragment frequency in Taq I/2b-3 and Xba I/10 patterns between Chinese and Caucasians. The frequencies of allelic fragments A2 (5.6kb) in Taq I/8 and A2 (10.7kb) in EcoR V/9 were high in Caucasians, yet had not been detected in Chinese, the differences were also highly significant. But in Chinese and Caucasians, the B1B2 allelic frequencies in Taq I/5b-7 are the same. As to the frequency of the allelic fragments A1A2 and B1B2 in Pvu II/1a, there was no significant difference between Chinese and Japanese.展开更多
For making it clear whether GABAA-like receptor is in cells or rat testis and how itsgene expresses in tissue cells, the mRNA of rat testis was microinjected into Xenopusoocyte. The membrane electrobiological results ...For making it clear whether GABAA-like receptor is in cells or rat testis and how itsgene expresses in tissue cells, the mRNA of rat testis was microinjected into Xenopusoocyte. The membrane electrobiological results showed that an inward 30 nA micro-currentwas elicited, under two-electrode voltageclamped configuration, by extracellular GABA(500 μmol/L), and both Bicuculline and Picrotoxin could inhibit this effect. It is an indication that the mRNA of rat testis can express GABA receptor in the membrane of Xenonpus oocyte. Using Muscimol as a excitant of GABA receptor type A, the micro--currentwas also elicited to generate; however, using Baclofen as a excitant of GABA receptortype B, the micro-current could not be induced. These results indicate that GABA receptorexpressed in Xenopus oocyte membrane is not type B but for type A. On the basis of the research result above, 24-mer oligonucleotides to be complementary to the high conservativeregion of the cDNAs of neur-GABA receptors were synthesized as probes to hybridize respectively with the RNAs Of brain and testis of adult rat, and of rat testes of the variousages. The dot hybridizations showed that the hybrid signal of rat testis was stronger thanthat of rat brain, meaning that, compared with rat brain, the RNA of rat testis has morehomologous regions with probes, and also implying that the GABA receptor from theRNA Of rat testis may be similar to neural-GABA receptor. This receptor therefore iscalled as GABA_A-like receptor. The dot blot analysis above also showed the testis GABA_Alike receptor. The dot blot analysis above also showed testis GABA_A like receptor mRNAcontent changed with the develOPmental age of rats. There is a very low level of gene expression in the rat testis on day 5 postnatal; there is the highest expression of GABA_A-likereceptor gene in rat testes on day 30to 100 after birth; the expression begins to drop on day200 after birth.展开更多
We found T-type calcium channel blocker Ni2+ can efficiently induce the formation of cement gland in Xenopus laevis animal cap explants. Another T-type specific calcium channel blocker Amiloride can also induce the fo...We found T-type calcium channel blocker Ni2+ can efficiently induce the formation of cement gland in Xenopus laevis animal cap explants. Another T-type specific calcium channel blocker Amiloride can also induce the formation of cement gland, while L-type specific calcium channel blocker Nifedipine has no inductive effect. These results may offer us an new approach to study the differentiation of cement gland through the change of intracelluax calcium concentration.展开更多
A 550 bp cDNA fragment of HSD I coding for an extracellular domain of human sperm membrane protein (hSMP 1) was ligated with an Adapter containing the universal stop codon, and the ligated fragment cDNA was then ...A 550 bp cDNA fragment of HSD I coding for an extracellular domain of human sperm membrane protein (hSMP 1) was ligated with an Adapter containing the universal stop codon, and the ligated fragment cDNA was then cloned into the MAS of pUC19. The desired plasmid with correct open reading frame was obtained, and was cut with EcoR I.The insert was purified and then cloned into the two asd + Salmonella expression vectors (the low copy number plasmid pYA292 and the high copy number plasmid pYA3137). The recombinant plasmid containing the insert with the correct orientation was selected by restriction enzyme digestion analysis. The recombinant plasmids were transferred into the non pathogenic Salmonella typhimurium χ4550, which was deletion of the Δcya, Δcrp and Δasd genes. Western blot analysis of the whole cell lysate of the two recombinants of S. typhimurium showed a predominant protein band at 21 KD, which reacted with the anti hSMP 1 antiserum. The result indicated that two recombinants of S. typhimurium containing the 550 bp cDNA of HSD I were constructed and the characteristics of their growth in vitro were determined. They may be used as new potential mucosal immunization antifertility.展开更多
基金Major State Basic Reaearch (973) Program of China.
文摘Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and "Death factor" family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of "Death factor" family, the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl-2. The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis. Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.
文摘In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice, the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF). Compared with the feeder layer of MEF cells, medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyo-typic normality of ES cells only in short term cell propagation. Besides, ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras. Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more convenient and efficient than the conventional microdrop method.
基金grants of National "Pan Deng" program LMCBand National Natural Science FOundation!No: 39770776
文摘Some recent studies indicated that GABAergic system is involved in mammalian sperm acrosome reaction (AR), but direct evidence pertaining to the expression of gat1 in mammalian sperm is not yet demonstrated. In this study, we evaluated the presence of 67kDa GAT1 protein and mRNA in rat testis by Western blotting and reverse transcription-polymerase chain reaction. Meanwhile, immunohistochemical and immunofluorescent analyses also identified GAT1 protein on the elongated spermatid and sperm. These results indicated that rat testis is a novel site of gat1 expression. Further studies should be taken to explore the role of GAT1 protein on sperm acrosome reaction.
文摘Trichosanthin(TCS) is a potent allergen in mice. It can reproducibly induce specific IgE responses in C57BL/6J mice without the help of adjuvant alum. TCS can bring out the IgE responses to ovabumin(OVA), while OVA itself could not induce IgE responses to it. How- ever, TCS only works when QVA immunization is given one day after TCS immunization. Either time lag in OVA immunization, or immunization of both antigens at the same time, or OVA immunization given first, all has no effect on the induction of IgE responses to OVA. Through analysis of the antibody specificity of hybridoma clones, it indicated that specific antibodies to TCS or OVA were secreted by independent B cell clones. The IgE antibodies showed no polyreactivity to different antigens.
文摘Thichosanthin (TCS) is a potent allergen to mice. According to our previous experiments, it could bring out the IgE response to ovabumin (OVA) if TCS was given one day before OVA immunization, while OVA alone could not induce IgE to it. In this work, the kinetics of interleukin 4(IL-4) and interferon γ(IFN-γ) gene expression in the mesenteric lymph node (MLN) of TCS-immunized mice was investigated using a semi-quantitative RT-PCR method. It indicated that TCS induced significant IL-4gene expression and the peaks of IL4 gene expression were on day one after TCS immunization in both primary and secondary response. In contrast, the IFN-γ gene expression was suppressed. Furthermore, the IL-4 gene expression in the secondary response was lower than that in the primary response. Thus the presence of IgE rpemory B cells were studied. Results showed that the amount of mature IgE mRNA arose significantly and rapidly one day after TCS restimulation, while in the MLN of the mice primed 30 days before and without boost, it was almost as the same amount of the unimmunized control. These findings suggest the existence of the IgE memory B cells in the mice after the primary TCS immunization.
文摘Epidermal growth factor (EGF) is produced primarily by Leydig cells of human testis. Expression of the EGF gene was assessed in mouse testis during the course of sexual maturation by the application of the RT-PCR method and the use of specific oligonucleotide primers. Testis EGF mRNA content increased with the developmental age of the mice, i.e., day 15 < day 30 < day 45 postnatal. The expression of the EGF gene appears to correlate with maturation of the testis and proliferation of Leydig cells.
文摘Mad protein has been shown as an antagonist of cMyc protein in some cell lines. The effect of Mad protein to the malignant phenotype of human hepatoma BEL7404 cell line was investigated experimentally. An eukarryotic vector pCDNA Ⅲ containing full ORF fragmentof mad cDNA was transfected into targeted cells. Under G418 selection, stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells. DNA synthesis, cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cel1s were partially inhibited in comparison to control cells.Flow Cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase, resulting in the retardation of cell proliferation. RT-PCR detected a marked inhibition of the expression of cdc25A, an important regulator gene of G0/G1to S phase in cell cycle. It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-7404-M1 cells in the absence of serume.Thus, Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL7404 cells.madoverexpression and regulation of cell growth and apoptosis
文摘Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye, Lucifer Yellow. Close correlation existed between the incidences of dye coupling and quantitative evaluation of gap junctions. High incidences of dye coupling and of gap junctions occurred at a stage when notochord cells were active in the change of cell shape and cell arrangement. With the subsidence of cell movements, both dye coupling and gap junctions were reduced to lower levels. It was, therefore, suggested that intercellular communication via gap junctions played an important role in the coordination of notochord cell movements.Gap junctions of altered configuration occurred in notochord cells in late tailbud stage. The comparison of incidences of dye coupling at this stage with those at other stages strongly suggested that the gap junctions of altered configuration functioned just as those of generalized type.
文摘In order to elucidate the molecular mechanisms of globin gene expression during embryonic development, the nuclear extracts from mouse hematopoietic tissue at different stages of development have been prepared. By using DNase I footprinting and gel mobility shift assays, the binding of protein factors in these extracts to the human βglobin promoter was analyzed. The differences in the binding patterns of protein factors during development were observed. An erythroid-specific and stage-specific nuclear protein in the nuclear extract from d 18 mouse fetal liver was identified, which can bind to the sequence (from -66bp to -90bp) of human β-globin promoter. We therefore speculate that the function of this cis-acting element may be similar to stage selector element (SSE) in chieken βA- promoter.
文摘The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.
基金gramts from shanghai Joint Laboratory of Life sciences,Academia Sinica,and the National National sciences Foundation,
文摘The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5'-flanking regulatory elements of human β-globin gene, two negative control regions(NCR1,-610 to -490 bp;NCR2, -338 to-233bp), was identified.Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2.These data provided evidence that the cis acting elements of the 5'-flanking region might be involved in the developmental control of β-globin gene and NCR2 might be responsible in part for the silence of β-globin gene in the embryonic and fetal stages.
文摘The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled, inpart, by the silencer (-392bp～-177bp) upstream of thisgene. In order to elucidate its role, the nuclear extractfrom the human fetal liver has been prepared and the interactions between trans-acting factors and this silencerelement have been examined. By using DNasel footprinting assay, a major protected region from -278bp to -235bpwithin this silencer element was identified. Furthermore,we found in gel mobility shift assay and Southwestern blotting assay that there were at least four trans-acting factors (MW ≈ 32, 28, 26 and 22kD) in the nuclear extractisolated from the human fetal liver, which could specifically bind to this region. Our results suggested that thesetrans-acting factors might play an important role in silencing the human embryonic ε-globin gene expression at thefetal stage through the interactions with this silencer.
文摘A cDNA encoding the mouse GABA transporter has been isolated and sequenced. The results show that the mouse GABA transporter cDNA differs from that of the rat by 60 base pairs at the open reading frame region but the deduced amino acid sequences of the two cDNAs are identical and both composed of 599 amino acids. However,the amino acid sequence is different from the sequence deduced from a recently published mouse GABA transporter cDNA.
基金China National"863 Project"for Biotechmoloigt Development
文摘The characteristics of the particulate mouse centromere enriched fraction from isolated nuclei obtained in our laboratory were investigated by indirect immunoflu-orescence, test of the activity of microtubule organizing center(MTOC), SDS-PAGE, and fluorescence in situ hybridization. Most of the particles of the fraction are complexes of DNA and kinetochore proteins and show MTOC activity. The DNA isolated from the fraction can hybridize with DNA in the regions of the primary constrictions of all chromosomes of ascites cells. The kinetochore proteins isolated from the fraction are mainly those with molecular weight of 55 KD and 59 KD. Results suggested that the fraction obtained is a centromere enriched nuclear fraction as indicated in our previous report.
文摘The effect of peripheral nerve (PN) on neurite outgrowth from retinal explants of adult hamsters was examined. Cultures of retinal explants, and co-cultures of retinal explants and PN were performed using chick retinal basement membrane (BM) as substrate. The presence of PN increases the number and length of neurite outgrowth. In addition, a high proportion of neurites situated close to PN tend to grow towards it. Since there was no contact between retinal ex-plants and PN, we suggest that PN might secrete diffusible substances to attract the neurites to grow towards it.
文摘In order to analyze the mechanism of immunomodulation by LPS on murine peritoneal suppressor macrophages, we have, using RNase protection assay,checked the changes of mRNA expression pattern of several cytokine genes during the immuno-modulation.It has been found that, after treating peritoneal suppressor macrophages with LPS, mRNAs of IL-12 p35, IL-12 p40,IL-6 and IFN-γ are newly appeared, while those of IL-1α, IL-1β and IL-1Ra are increased and those of other cytokines, like TGF-β1 and MIF are not changed at all.It seems certain that those cytokines, whose expression is increased by LPS stimulation, may be responsible for the functional changes of suppressor macrophages during immuno-modulation. Among these changes, the appearance of IL-12 mRNA may play a critical role, and, in this regard, the synergetic effect between IFN-γ and LPS on the increase of IL-12 p35 and IL-12 p40 mRNA expression is an interesting finding.
文摘The restriction fragment length polymorphisms distribution and frequency of dystrophin gene in Chinese were studied by using 14 subclones of the entire 14kb cDNA for the dystrophin as hybridization probes. Allelic fragments were detected in hybridization patterns of PvuII/1a, Taq I/2b-3, Taq I/5b-7, and Xba I/10. Among them, the allelic fragments (26kb and 3.8kb) in PvuII/2b-3 pattern and the allelic fragments (10.0kb and 8.4kb) in Taq I/5b-7 patterns had never been reported previously. Compared with the data from Caucasians and Japanese, it indicated that there was a significant difference (P<0.01) of the allelic fragment frequency in Taq I/2b-3 and Xba I/10 patterns between Chinese and Caucasians. The frequencies of allelic fragments A2 (5.6kb) in Taq I/8 and A2 (10.7kb) in EcoR V/9 were high in Caucasians, yet had not been detected in Chinese, the differences were also highly significant. But in Chinese and Caucasians, the B1B2 allelic frequencies in Taq I/5b-7 are the same. As to the frequency of the allelic fragments A1A2 and B1B2 in Pvu II/1a, there was no significant difference between Chinese and Japanese.
文摘For making it clear whether GABAA-like receptor is in cells or rat testis and how itsgene expresses in tissue cells, the mRNA of rat testis was microinjected into Xenopusoocyte. The membrane electrobiological results showed that an inward 30 nA micro-currentwas elicited, under two-electrode voltageclamped configuration, by extracellular GABA(500 μmol/L), and both Bicuculline and Picrotoxin could inhibit this effect. It is an indication that the mRNA of rat testis can express GABA receptor in the membrane of Xenonpus oocyte. Using Muscimol as a excitant of GABA receptor type A, the micro--currentwas also elicited to generate; however, using Baclofen as a excitant of GABA receptortype B, the micro-current could not be induced. These results indicate that GABA receptorexpressed in Xenopus oocyte membrane is not type B but for type A. On the basis of the research result above, 24-mer oligonucleotides to be complementary to the high conservativeregion of the cDNAs of neur-GABA receptors were synthesized as probes to hybridize respectively with the RNAs Of brain and testis of adult rat, and of rat testes of the variousages. The dot hybridizations showed that the hybrid signal of rat testis was stronger thanthat of rat brain, meaning that, compared with rat brain, the RNA of rat testis has morehomologous regions with probes, and also implying that the GABA receptor from theRNA Of rat testis may be similar to neural-GABA receptor. This receptor therefore iscalled as GABA_A-like receptor. The dot blot analysis above also showed the testis GABA_Alike receptor. The dot blot analysis above also showed testis GABA_A like receptor mRNAcontent changed with the develOPmental age of rats. There is a very low level of gene expression in the rat testis on day 5 postnatal; there is the highest expression of GABA_A-likereceptor gene in rat testes on day 30to 100 after birth; the expression begins to drop on day200 after birth.
文摘We found T-type calcium channel blocker Ni2+ can efficiently induce the formation of cement gland in Xenopus laevis animal cap explants. Another T-type specific calcium channel blocker Amiloride can also induce the formation of cement gland, while L-type specific calcium channel blocker Nifedipine has no inductive effect. These results may offer us an new approach to study the differentiation of cement gland through the change of intracelluax calcium concentration.
文摘A 550 bp cDNA fragment of HSD I coding for an extracellular domain of human sperm membrane protein (hSMP 1) was ligated with an Adapter containing the universal stop codon, and the ligated fragment cDNA was then cloned into the MAS of pUC19. The desired plasmid with correct open reading frame was obtained, and was cut with EcoR I.The insert was purified and then cloned into the two asd + Salmonella expression vectors (the low copy number plasmid pYA292 and the high copy number plasmid pYA3137). The recombinant plasmid containing the insert with the correct orientation was selected by restriction enzyme digestion analysis. The recombinant plasmids were transferred into the non pathogenic Salmonella typhimurium χ4550, which was deletion of the Δcya, Δcrp and Δasd genes. Western blot analysis of the whole cell lysate of the two recombinants of S. typhimurium showed a predominant protein band at 21 KD, which reacted with the anti hSMP 1 antiserum. The result indicated that two recombinants of S. typhimurium containing the 550 bp cDNA of HSD I were constructed and the characteristics of their growth in vitro were determined. They may be used as new potential mucosal immunization antifertility.
