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Biomedical subjects

Y Ke

Publications and source records attributed to Y Ke.

At least 109 records · Page 6Linked to original sources
PubMed1995

Antigen recognition by CTL is dependent upon ectoATPase activity.

Alloantigen-specific and OVA-specific CD8+ CTL were shown here to express an ectoATPase. These CTL also express an ectoADPase, but do not express detectable levels of an ectoAMPase. CD8+ CTL transported adenosine into their cytoplasm at a rate of 2.3 x 10(-11) mmol/min/10(5) cells. In contrast, adenosine uptake was 34-fold lower when ATP was used as the source of the nucleoside. This was consistent with the lack of ectoAMPase and suggests that the role of ectoATPase is not in the salvage of extracellular nucleotides. 5'-p-(fluorosulfonyl)benzoyl adenosine (5'-FSBA) is an ATP analogue affinity label that irreversibly inhibits CTL ectoATPase. Cells made ectoATPase activity deficient by modification with 5'-FSBA were not susceptible to potential lytic effects of extracellular ATP with less than 20% specific lysis at 20 mM of exogenous ATP. However, cells modified by 5'-FSBA were unable to kill their respective target cells. Complete inhibition of cell-mediated killing was observed with 1 mM 5'-FSBA. CTL modified by 5'-FSBA also failed to secrete TNF-alpha and IFN-gamma after activation by the appropriate Ag. Killing was also inhibited by 5'-adenylylimidodiphosphate (a nonhydrolyzable ATP analogue), but not by ATP, ADP, alpha, beta-methylene ADP (a nonhydrolyzable ADP analogue), AMP, or adenosine. Blockage of CTL activity by 5'-FSBA was not reversed by addition of ADP, suggesting that hydrolysis of ATP is an essential ectoATPase-mediated signal for CTL activation. These results suggest that ectoATPase is essential for Ag recognition and/or effector activities of CTL.

Adenosine↗
PubMed1995

SV40-induced immortalization and ras-transformation of human bronchial epithelial cells.

Non-tumorigenic SV40-immortalized human cells may be transformed to tumorigenicity by activated oncogenes, but the molecular genetics of this process are still poorly understood. We describe here 4SV40-transformed bronchial epithelial (BE) cell lines that became immortalized after a period of crisis, and then transfection of 6 BE lines or sub-lines with an activated c-Ha-ras (EJ-ras) oncogene. pSV2neo-transfected cells did not form any tumors in athymic nude mice. Even though each of the EJ-ras-transfected lines was shown to be expressing the mutant ras gene, only one cell line, BEAS-2B, and 2 of its sub-lines were tumorigenic after transfection. We conclude that immortalization is not sufficient for BE cells to be transformed by the EJ-ras oncogene. Thus there are at least 2 unknown genetic events in this in vitro model of carcinogenesis: escape from crisis (immortalization), and development of ability to cooperate with activated ras in tumorigenic transformation. We found no evidence that either immortalization or ability to complement ras is related to abnormalities of the SV40 T antigens, of p110RB or of p53.

Animals↗
PubMed1995

Ovalbumin injected with complete Freund's adjuvant stimulates cytolytic responses.

CD8+ cytotoxic T lymphocytes (CTL) recognize antigens (Ag) associated with class I major histocompatibility complex (MHC) molecules. Endogenously synthesized protein Ag are processed into peptides in the cytoplasm and transported to the endoplasmic reticulum where they are bound by class I proteins. Exogenous Ag do not enter the class I processing pathway of most cells and thus do not activate CD8+ CTL. Nevertheless, several investigators have reported that immunization with exogenous Ag can activate CD8+ T cells that have immunoregulatory activity. To determine how exogenous Ag entered the class I pathway in vivo and whether immunosuppressive CD8+ T cells were cytolytic, we have shown in this report that injection with OVA emulsified in the complete Freund's adjuvant (CFA) primed CD8+, class I MHC-restricted, OVA-specific CTL in mice. These CTL recognize the OVA257-264 epitope, produce tumor necrosis factor-alpha and interferon-gamma upon activation. Both oil and mycobacteria components in CFA were required for inducing CTL responses. Priming was not attributed to direct sensitization of class I-bearing cells by contaminating peptides. Rather, phagocytic cells, but not CD4+ helper T cells, were required for priming CD8+ CTL by OVA-CFA. Thus, OVA in CFA is taken up by antigen-presenting cells and processed into the class I pathway by phagocytic cells in vivo. In addition, CTL induced by OVA-CFA suppressed the antibody response to OVA in adoptive recipients. These results suggest that CD8+ CTL specific for exogenous proteins might be routinely stimulated by injecting proteins in conventional adjuvants and that such cells have the potential to regulate immune responses in vivo.

Animals↗
PubMed1995

Cadmium-113 NMR studies of bovine and human alpha-lactalbumin and equine lysozyme.

The high-affinity calcium-binding sites of bovine and human alpha-lactalbumin as well as equine lysozyme were analyzed by 113Cd NMR spectroscopy. In the case of equine lysozyme, the addition of isotopically enriched 113Cd2+ results in a signal at delta = -75.9 ppm corresponding to the metal ion bound to the lone Ca(2+)-binding site of the protein. A peak at virtually the identical resonance position (delta = -77.1 ppm) was observed in the analogous experiment with bovine alpha-lactalbumin. In addition, a signal upfield of these (delta = -94.7 ppm) was observed for 113Cd(2+)-substituted human alpha-lactalbumin. The chemical shifts of these proteins are in the vicinity of those reported for other Ca(2+)-binding proteins. The field dependence of the 113Cd signals for all three proteins and bovine calmodulin were compared. At each field, the 113Cd signal linewidths for the alpha-lactalbumins and the lysozyme are somewhat broader than those observed for the EF-hand protein. In addition, the 113Cd linewidths for the lactalbumins and the lysozyme, especially bovine alpha-lactalbumin, increase dramatically with the square of the magnetic field strength, indicative of the presence of nuclear relaxation via chemical shift anisotropy and chemical exchange. The protein-bound 113Cd signals for the alpha-lactalbumins are also markedly affected by changes in the amount of K+ present, since Cd2+ and K+ can compete for occupation of the high-affinity Ca(2+)-site. Their linewidths also to some extent depend on the concentration of the protein itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

[Esophagogastrostomy covered by sero-muscular flap of gastric wall in the treatment of gastric cardia cancer in 100 cases].

A new operative procedure-esophagogastrostmy covered by sero-muscular flap of gastric wall to treat cancer of gastric cardia was designed by the authors in order to prevent anastomotic complications. One hundred cases with cases of gastric cardiac 97 cases with adenocarcinoma and 3 cases with squamous cell carcinoma of esophago-cardiac iunction were treated with the new anastomotic method without operative mortality and anastomotic complicatins within one year after surgery.

Adenocarcinoma↗
PubMed1994

Delivery of exogenous antigen into the major histocompatibility complex class I and class II pathways by electroporation.

Exogenous, nonreplicating protein antigens (Ags) are usually taken up by antigen-presenting cells (APCs) via endocytosis or pinocytosis and enter the major histocompatibility complex (MHC) class II processing and presentation pathway. Although exogenous Ags are not processed and presented in the class I pathway by most cells, soluble proteins can enter the class I processing and presentation pathway if they are introduced directly into the cytoplasm of APCs. The purpose of these studies was to determine whether exogenous proteins could be processed and presented to T cells if they were delivered into cells by electroporation. The conditions for electroporation were optimized so that the viability of the electroporated cells was high, and the majority of electroporated cells had protein incorporated. Electroporated B cells not only presented exogenous ovalbumin to CD8+, class I MHC-restricted T cells but also stimulated CD4+, class II MHC-restricted T cells. Electroporated cells also primed Ag-specific cytotoxic T lymphocytes (CTLs) in vivo, stimulated CTL precursors in vitro, and served as target cells for lysis by Ag-specific CTLs, indistinguishable from transfected cells. Thus, electropermeabilized cells were structurally intact, and the introduced exogenous protein was processed and presented in association with both class I and class II MHC molecules. This approach is as efficient and reproducible as other techniques of delivering exogenous proteins into the intracellular processing pathways. These studies suggest that electroporation could be employed for the study of cell-mediated immunity to various exogenous proteins.

Animals↗
PubMed1994

Effects of sterigmatocystin and T-2 toxin on the induction of unscheduled DNA synthesis in primary cultures of human gastric epithelial cells.

Primary cultures of human gastric epithelial cells were tested for induction of unscheduled DNA synthesis (UDS) by sterigmatocystin (ST) and T-2 toxin. Autoradiographic results indicated that ST (10(-6)-10(-4)M) induced UDS in the presence of S9 activation system. The repair rates were 24-91% (net grains > or = 3) and 2-71% (net grains > or = 5). T-2 toxin did not induce UDS in this study.

Animals↗
PubMed1994

[Establishment and characterization of a SV40 transformed human fetal gastric epithelial cell line-GES-1].

9 month human fetal gastric epithelial cells were primarily cultured and infected with SV40 virus. About 3 weeks after infection the transformed-like cells showed up and were isolated when most of the normal cells senesced. One transformed clone named GES-1 were analyzed biologically. The results indicated integration of the SV40 T gene and its expression in the cell nuclei and the cells became hyperploid in chromosomes. This cells also maintained a normal cytoskeleton, positive in PAS reaction and were non-tumorigenic in nude mice. The established cell line may be served as an important model system in study of human gastric carcinogenesis.

Animals↗
PubMed1993

Stimulation of gene expression by introns: conversion of an inhibitory intron to a stimulatory intron by alteration of the splice donor sequence.

Efficient expression of many mammalian genes depends on the presence of at least one intron. We previously showed that addition of almost any of the introns from the mouse thymidylate synthase (TS) gene to an intronless TS minigene led to a large increase in expression. However, addition of intron 4 led to a reduction in minigene expression. The goal of the present study was to determine why TS intron 4 was unable to stimulate expression. Insertion of intron 4 into an intron-dependent derivative of the ribosomal protein L32 gene did not lead to a significant increase in expression, suggesting that its inability to stimulate expression was due to sequences within the intron. Deleting most of the interior of intron 4, improving the putative branch point, removing purines from the pyrimidine stretch at the 3' end of the intron, or removing possible alternative splice acceptor or donor sites within the intron each had little effect on the level of expression. However, when the splice donor sequence of intron 4 was modified so that it was perfectly complementary to U1 snRNA, the modified intron 4 stimulated expression approximately 6-fold. When the splice donor site of TS intron 1 (a stimulatory intron) was changed to that of TS intron 4, the modified intron 1 was spliced very inefficiently and lost the ability to stimulate mRNA production. Our observations support the idea that introns can stimulate gene expression by a process that depends directly on the splicing reaction.

Animals↗
PubMed1993

Ectopic production of heparin-binding growth factors and receptors for basic fibroblast growth factor by rat mammary epithelial cell lines derived from malignant metastatic tumours.

A rat mammary (Rama) epithelial cell line, Rama 704, derived from normal rat mammary gland does not possess any detectable cell-surface receptors for basic fibroblast growth factor (bFGF), produces a barely detectable level of bFGF mRNA and does not contain detectable levels of bFGF-like activity. Similar results have been obtained with the Rama 37 epithelial cells derived from benign tumours. However, 4 independently isolated epithelial cell lines derived from malignant rat mammary tumours and their metastases possess receptors for bFGF and contain between 2 ng and 9 ng heparin-binding, growth-stimulatory activity per 10(6) cells. The weakly metastatic Rama 600 cells possess high- and low-affinity receptors for bFGF, (KD 20 pM and 8 nM, respectively), while the moderately metastatic Rama 800 cells possess only high-affinity receptors (KD 40 pM). The moderately metastatic C18PLN and 267LU cells, derived from metastases arising from benign Rama 37 cells which had been transfected with DNA from the malignant Rama 800 cells, also possess only high-affinity receptors (KD 36 pM and 80 pM, respectively). Our results show that within the Rama system there is a correlation between the appearance of heparin-binding growth factors and of high-affinity but not low-affinity receptors for bFGF with the malignant phenotype.

Animals↗
PubMed1993

Development of tumorigenicity in simian virus 40-immortalized human bronchial epithelial cell lines.

Of five SV40-transformed clonal human bronchial epithelial cell lines previously shown to be nontumorigenic at early passages (R. R. Reddel et al., Cancer Res., 48: 1904-1909, 1988), two lines (BES-1A1 and BEAS-2B) from different donors have become weakly tumorigenic with further passaging. BES-1A1 passage 26 cells formed tumors in 3 of 9 athymic nude mice given s.c. injections, whereas BEAS-2B cells of > or = 32 passages formed highly cystic tumors at 8 of 58 injection sites after long latency periods [17 +/- 7 (SD) weeks]. These tumors took a total of 36 +/- 8 weeks to reach a diameter of 1.0 cm. Tumor cell lines were established from four BEAS-2B tumors, and these are resistant to the growth-inhibitory effects of serum, an inducer of squamous differentiation in BEAS-2B and normal bronchial epithelial cells. This finding supports the hypothesis that development of resistance to inducers of terminal squamous differentiation may be a step in the process of bronchial carcinogenesis. One of these tumor cell lines, B39-TL, is significantly more tumorigenic than the others and has a deletion from the short arm of chromosome 3 as has been described previously for some naturally occurring human bronchial carcinomas. Thus, from the clonally derived BEAS-2B cell line, cell populations with various degrees of tumorigenicity have developed. Analysis of the changes in these cells may yield insights into the multiple events involved in acquisition of the tumorigenic phenotype.

Animals↗
PubMed1993

Localized detection of glioma glycolysis using edited 1H MRS.

In vivo 1H MRS can be used to detect and quantify the lactate resonance at 1.3 ppm provided that overlapping lipid resonances are eliminated. A homonuclear spectral editing method was developed to acquire uncontaminated 1H spectra of lactate with adiabatic pulses. An advantage of the adiabatic pulse sequence is the ability to induce uniform flip angles and to maximize sensitivity in applications employing surface coil transmitters which produce highly inhomogeneous B1. Glycolytic activity in an intracerebral C6 glioma in rats was monitored by using adiabatic editing sequences to observe [3-13C]lactate produced from infused [1-13C]glucose. Acute hyperglycemia (serum glucose > 22 mM, n = 10) had no significant effect (P = 0.08) on the total ([12C] + [13C]) tumor lactate signal intensity.

Animals↗
PubMed1993

A serum-free medium for hybridoma cell culture.

The effects of several different substances, including insulin, transferrin, ethanolamine, selenite and butyrate on the growth of murine hybridoma 2F7 cells, which secrete monoclonal antibody against small cell lung cancer, were investigated, and a serum-free medium SFM I was formulated. The effects of taurine, spermidine, progesterone and adenine on the cell growth were tested further on the basis of the medium SFM I, and a modified serum-free medium SFM II was established. On the basis of medium SFM II, the substitution tests of ferric citrate for transferrin were carried out, and it was found that transferrin could be replaced. The experiments suggested that the formulated serum-free medium was suitable for 2F7 cell growth and monoclonal antibody secretion, and thus facilitated subsequent purification of monoclonal antibody.

Animals↗
PubMed1993

Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.

The thymidylate synthase (TS) gene is expressed at much higher levels in proliferating cells than in quiescent cells. We have been studying the sequences that are important for regulating the mouse TS gene. We previously showed that DNA sequences upstream of the essential promoter elements as well as downstream of the ATG codon are both necessary (but neither is sufficient) for normal regulation in growth-stimulated cells. In the present study, we examined the possible roles of the coding region, polyadenylation signal, and introns as downstream regulatory elements. Minigenes consisting of 1 kb of the TS 5'-flanking region, the coding region (with or without various introns at their normal locations), and polyadenylation signals from the TS gene, the human beta-globin gene, and the bovine growth hormone gene were stably transfected into wild-type mouse 3T6 cells. Minigenes that contained introns 5 and 6, 1 and 2, or 1 alone were regulated regardless of which polyadenylation signal was included. A minigene that contained an internally deleted version of intron 1 was also regulated in response to growth stimulation. However, when all introns were omitted, there was little if any change in the level of minigene expression as cells progressed from G1 through S phase. These observations indicate that TS introns contain sequences that are necessary for normal growth-regulated expression of the mouse TS gene. These sequences appear to be associated with sequences that are important for splicing and to function in cooperation with upstream regulatory elements to bring about normal S-phase-specific expression.

Animals↗
PubMed1993

The expression of basic fibroblast growth factor and its receptor in cell lines derived from normal human mammary gland and a benign mammary lesion.

mRNA for basic Fibroblast Growth Factor (bFGF) was expressed in a series of SV40-transformed human mammary cell lines as molecules of 7.1, 3.6, 2.0 and 1.2 kb. This expression was much weaker in those lines of epithelial morphology than in myoepithelial-like cell lines derived from them. It was confirmed, using northern hybridization to single-stranded RNA probes, that the multiple mRNAs were transcribed from the coding strand for bFGF. bFGF activity was detected in extracts of the cells and the relative amounts of activity corresponded in general to the amounts of mRNA found. Similar results were obtained from spontaneously transformed cell lines derived from a human benign breast lesion. The presence of bFGF protein in the extracts was confirmed by western blotting, which showed a band of 18-19 kDa, migrating in the same position as authentic bFGF; in addition, the myoepithelial-like cells showed prominent bands of bFGF at 24 and 26 kDa. No FGF receptor was detectable by the binding of 125I-bFGF to the SV40-transformed cell lines or to the epithelial cell lines from the benign breast lesion, but both high- and low-affinity receptors were found on myoepithelial-like cells derived from the latter. The results indicate that differentiation to the human myoepithelial-like phenotype in culture is associated with the enhanced expression of bFGF, and it is suggested that bFGF, immunocytochemically detected in the basement membrane of the human breast, may arise, at least in part, from the myoepithelial cells of the mammary parenchyma.

Breast↗
PubMed1992

Calcium-43 NMR studies of calcium-binding lysozymes and alpha-lactalbumins.

The calcium-binding properties of equine and pigeon lysozyme as well as those of bovine and human alpha-lactalbumin were investigated by 43Ca NMR spectroscopy. All proteins were found to contain one high-affinity calcium-binding site. The chemical shifts, line widths, relaxation times (T1 and T2), and quadrupole coupling constants for the respective 43Ca NMR signals were quite similar; this is indicative of a high degree of homology between the strong calcium-binding sites of these four proteins. The measured chemical shifts (delta approximately -3 to -7 ppm) and quadrupole coupling constants (chi approximately 0.7-0.8 MHz) are quite distinct from those observed for typical EF-hand calcium-binding proteins, suggesting a different geometry for the calcium-binding loops. The correlation times for bound calcium ions in these proteins were on the order of 4-8 ns, indicating that the flexibilities of these binding sites are limited. The apparent pKa values for the high-affinity sites ranged from 3.4 to 4.7, confirming the participation of carboxylate-containing residues in the coordination of the calcium ion. Competition experiments with EDTA showed that the affinities of these proteins for calcium follow the series bovine alpha-lactalbumin approximately human alpha-lactalbumin greater than pigeon lysozyme greater than equine lysozyme (KD approximately 5 x 10(-8) to 10(-6) M). Evidence for the existence of a second weak calcium-binding site (KD = 3 x 10(-3) M) was obtained for bovine alpha-lactalbumin, but not for the other proteins studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗