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41.
Sequences near the 3'' secretion-specific polyadenylation site influence levels of secretion-specific and membrane-specific IgG2b mRNA in myeloma cells. 总被引:11,自引:2,他引:9
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The expressed immunoglobulin gamma 2b (IgG2b) heavy-chain gene of 4T001 was cloned into the shuttle vector pSV2-gpt and transfected into myeloma J558L and lymphoma A20.2J. Northern blots indicated that the transfected gamma 2b gene was processed in a manner similar to the endogenous heavy chain in both lymphoma and myeloma cells. To identify sequences important for immunoglobulin mRNA processing, we constructed deletions around the secretion-specific polyadenylation site and introduced the deleted genes into J558L cells. The BAL deletion lacked 670 base pairs of intervening sequence between secreted and membrane regions; the Kpn deletion lacked 830 base pairs in this region. J558L cells transfected with either the entire gamma 2b gene or the delta BAL vector produced predominantly secretion-specific gamma 2b mRNA and protein. J558L cells transfected with the delta Kpn vector produced approximately equimolar amounts of secretion-specific and membrane-specific gamma 2b mRNA. Both 55,000-dalton secreted and 62,000-dalton putative surface IgG2b proteins were detected in the delta Kpn transfectants. We conclude that sequences absent in the Kpn deletion but present in the BAL deletion exert an important role in the production of secretion-specific mRNA. The Kpn deletion removes the normal site of cleavage and poly(A) addition, and it is possible that it is the absence of this site which changes the processing pattern. Alternatively, it is possible that sequences absent in the Kpn deletion but present in the BAL deletion function in regulating the production of predominantly secretion-specific mRNA in myeloma cells. The possible role of a highly conserved sequence found in this region is discussed. 相似文献
42.
Transforming growth factor-alpha in the adult bovine ovary: identification in growing ovarian follicles 总被引:1,自引:0,他引:1
The pituitary gonadotropins and gonadal steroids are required for normal follicular growth and development but neither has been shown to act directly as a granulosa cell mitogen in vitro. A number of polypeptide growth factors, however, are known to have pronounced mitogenic effects on the cells of the follicle. We have localized transforming growth factor-alpha (TGF-alpha), a potent mitogen, in bovine thecal cells via immunoperoxidase staining using a monoclonal antibody for TGF-alpha that does not cross-react with epidermal growth factor. TGF-alpha staining is most intense in the theca of follicles at the discrete physiological stages known to show rapid granulosa cell growth (small follicles of 0.7-2.0 mm diameter). Staining intensity for TGF-alpha declines in large preovulatory follicles, coincident with the known decline in granulosa cell mitosis. These studies provide further evidence for paracrine interactions in the ovary and show that TGF-alpha may play an important role in the regulation of follicular development in the adult bovine ovary. 相似文献
43.
Chromosomal localization of seven members of the murine TGF-beta superfamily suggests close linkage to several morphogenetic mutant loci 总被引:7,自引:0,他引:7
M E Dickinson M S Kobrin C M Silan D M Kingsley M J Justice D A Miller J D Ceci L F Lock A Lee A M Buchberg 《Genomics》1990,6(3):505-520
Chromosomal locations have been assigned to seven members of the TGF-beta superfamily using an interspecific mouse backcross. Probes for the Tgfb-1, -2, and -3, Bmp-2a and -3, and Vgr-1 genes recognized only single loci, whereas the Bmp-2b probe recognized two independently segregating loci (designated Bmp-2b1 and Bmp-2b2). The results show that the seven members of the TGF-beta superfamily map to eight different chromosomes, indicating that the TGF-beta family has become widely dispersed during evolution. Five of the eight loci (Tgfb-1, Bmp-2a, Bmp-2b1, Bmp-2b2, Vgr-1) mapped near mutant loci associated with connective tissue and skeletal disorders, raising the possibility that at least some of these mutations result from defects in TGF-beta-related genes. 相似文献
44.
Akihiro Sakai Jiahuai Han Andrew CB Cato Shizuo Akira Jian-Dong Li 《BMC molecular biology》2004,5(1):2-16
Background
Despite the importance of glucocorticoids in suppressing immune and inflammatory responses, their role in enhancing host immune and defense response against invading bacteria is poorly understood. Toll-like receptor 2 (TLR2) has recently gained importance as one of the major host defense receptors. The increased expression of TLR2 in response to bacteria-induced cytokines has been thought to be crucial for the accelerated immune response and resensitization of epithelial cells to invading pathogens. 相似文献45.
BACKGROUND: GM-CSF-secreting, allogeneic cell-based cancer vaccines have shown promise for the treatment of a variety of solid tumors. We have now applied this approach to breast cancer. The aim of these studies was to optimize expansion parameters, qualify the manufacturing process, and establish expected outcomes for cGMP-compliant manufacturing of two GM-CSF-secreting breast tumor cell lines. METHODS: The variables affecting the efficiency of expanding and formulating two allogeneic GM-CSF-secreting cell lines, 2T47D-V and 3SKBR3-7, were systematically evaluated. Production criteria investigated included alternative cell culture vessels (flasks vs. cell factories), centrifugation time and speed variables for large volume cell concentration, cell seeding density, the minimal concentration of FBS required for maximal cell expansion, and the dose and timing of irradiation in relation to cryopreservation. RESULTS: These studies demonstrate that, in comparison with standard 150-cm2 tissue culture flasks, Nunc 10-Stack Cell Factories are a more efficient and practical cell culture vessel for vaccine cell line manufacture. Centrifugation optimization studies using the COBE 2991 Cell Processor established that a speed of 2000 r.p.m. (450 g) for 2 min reliably concentrated the cells while maintaining acceptable viability and bioactivity. Radiation studies established that lethal irradiation prior to cryopreservation does not compromise the quality of the product, as measured by post-thaw cell viability and GM-CSF cell line-specific secretion levels. Finally, studies aimed at optimizing the production of one vaccine cell line, 3SKBR3-7, demonstrated that seeding the cells at a higher density and maintaining them in half the initial concentration of FBS maximized the yield of bioactive cells, resulting in significant cost savings. DISCUSSION: A manufacturing process that simultaneously maximizes cell yield, minimizes cell manipulation and maintains vaccine cell potency is critical for producing cell-based cancer vaccines in an academic setting. These studies define a feasible, reproducible and cost-effective methodology for production of a GM-CSF-secreting breast cancer vaccine that is cGMP compliant. 相似文献
46.
The aim of this research was to discriminate and categorize isolateral stress-strain characteristics contained within the musculocutaneous compartment (MCC) using the model of the first dorsal interosseous muscle (FDI) in vivo. In our approach, the musculocutaneous compartment of the dorsal interosseous muscle was progressively isokinetically compressed by a solid vertical bar with a 0.25 mm incremental step. During each step, the force and deflection were measured and recorded electronically. The subject maintained a constantly relaxed position. Twenty-two strongly right-handed young males returned three data acquisition sequences from each hand. From the sequences, the elastic modulus and the specific energy of deformation were determined for both the total musculocutaneous compartment structure and separately for the discrete cutaneous and muscular compartments. No unilateral pattern of dominance was interpreted from the analysis of the range of indices. There was a highly variable individual pattern of bilateral dominance with no specific indication or predictability represented by the data: only one subject illustrated a data profile confirming the classical approach to right-handed dominance. As the muscle was relaxed and the integument layers were under minimal cortical influence, we suggest that the classical theory of primary cortical influence in motor lateralization does not adequately explain our recorded patterns of mechanical response of the musculocutaneous compartment analyzed in this study. 相似文献
47.
A molecular and structural analysis of the VH and VK regions of monoclonal antibodies bearing the A48 regulatory idiotype 总被引:3,自引:0,他引:3
C Victor-Kobrin Z T Barak F A Bonilla B Kobrin I Sanz D French J Rothe C Bona 《Journal of immunology (Baltimore, Md. : 1950)》1990,144(2):614-624
The results presented in this paper explore the molecular basis for expression of the A48 regulatory Id (RI). A48 RI+ mAb derived from idiotypically manipulated mice molecularly resembled the A48 and UPC 10 prototypes of this system by utilizing a VHX24-Vk10 combination. Id expression by these antibodies was not restricted by a particular D region sequence, JH, or JK segment, but quantitative differences in Id expression were associated with utilization of different members of the VK10 germ-line gene families. The VL sequences of these A48 RI+ mAb has identified amino acid residues lying in four different idiotope-determining regions which may contribute to the structural correlate of this Id. A comparative sequence analysis of the VH regions of these VHX24 utilizing A48 RI+ mAb with several A48 RI+ mAb utilizing VHJ558 or VH7183 VH genes as well as a hybrid transfectoma antibody derived from two A48 RI-, VHJ558 utilizing hybridomas, all suggested that four nonconsecutive positions which lie outside the idiotope-determining regions may contribute structural elements toward expression of this Id. The VH and VL regions of the A48RI+, VHX24-Vk 10+ mAb showed low to moderate levels of somatic mutation which showed different patterns of distribution between the complementary determining region (CDR) and framework regions in the H and L chains. Although the VK sequences contained 50% of the replacement mutations in the CDR, with a replacement/silent mutation ratio of 10, the CDR of the VH sequences contained only 31% of the replacement mutations with a replacement/silent mutation ratio of 0.69. 相似文献
48.
V I Kobrin E E Ignatova V A Vostrikov 《Biulleten' eksperimental'no? biologii i meditsiny》1991,112(10):339-340
The duration of periods of the spontaneous reversible fibrillation of heart ventricles of different masses of guinea pigs, rabbits, pigeons and chickens was investigated. It was demonstrated, that a heart mass doesn't play an important role in the process of spontaneous heart ventricles defibrillation. Only the number of strong contacts and the level of the parasympathetic control are determined. 相似文献
49.
A comparison of apparent mRNA half-life using kinetic labeling techniques vs decay following administration of transcriptional inhibitors. 总被引:9,自引:0,他引:9
Several different techniques were used to determine the apparent half-lives of immunoglobulin gamma 2b heavy chain and kappa light chain mRNA's in mouse myeloma 4T001 and a mutant derived from 4T001, i.e., mutant I17. The mutant I17 Ig heavy chain mRNA lacks CH1 and has fused CH2 and CH3 domains resulting in a truncated protein. By all four techniques the Ig heavy chain mRNA from mutant I17 displays a half-life that is approximately 70% the half-life of Ig mRNA in 4T001 cells. However, the absolute values of apparent half-life varied by greater than twofold for both lines among several of the techniques employed. The half-life of Ig gamma 2b mRNA in 4T001 cells was found to be 6.4 h by measuring decay following administration of the adenosine analog DRB to block new mRNA synthesis and 5.7 hr by measuring accumulation in an approach to steady-state labeling protocol. In contrast, the observed Ig mRNA half-lives determined by measuring decay following administration of actinomycin D to block new mRNA synthesis, or in a pulse-chase analysis were 2.9 and 3.8 h, respectively. The apparent half-life for Ig kappa light chain mRNA was the same in the 4T001 and I17 lines using any one technique but the value varied depending on the technique from a high value of 5.9 h following DRB to a low value of 2.4 h with actinomycin decay. Approach to steady-state is theoretically the most accurate method to measure mRNA half-life when that value is less than the doubling time of the cells. Pulse-chase analyses are accurate for measuring mRNA half-life when that value is longer than the effective chase period. Measuring preformed message decay following administration of drugs to block new mRNA synthesis is adaptable over a range of half-lives, but the cells must be shown to retain correct RNA metabolism over the time frame of the experiment. Determining a correct half-life for a particular mRNA may not be feasible using only one method and may, in fact, require several different approaches until a consensus value emerges. 相似文献
50.
The aim of this research was to discriminate and categorize isolateral stress-strain characteristics of the musculocutaneous compartment (MCC) using the model of the first dorsal interosseous muscle (FDI) in vivo. In our approach the MCC was progressively isokinetically compressed with a solid vertical bar in 0.25 mm steps, the force and deflection being measured and recorded electronically. The subject maintained a constantly relaxed position. Twenty-two strongly right-handed young males each yielded three data acquisition sequences from each hand. The elastic modulus and the specific energy of deformation were determined both for the total MCC and separately for the skin and muscle moieties. No unilateral pattern of dominance could be interpreted from the analysis of the range of indices. There was a highly variable individual pattern of bilateral dominance with no specific indication or predictability; only one subject yielded a data profile confirming the classical approach to right-hand dominance. As the muscle was relaxed and the integument layers were under minimal cortical influence, we think that the classical theory of primary cortical influence in motor lateralization does not adequately explain our recorded patterns of mechanical response of the MCC under study. 相似文献