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1.
Patricia Horan Hand Paul F. Robbins Michael L. Salgaller Diane J. Poole Jeffrey Schlom 《Cancer immunology, immunotherapy : CII》1993,36(2):65-75
The MC-38 C57BL/6 mouse colon adenocarcinoma cell line has been transduced with a retroviral construct containing cDNA encoding the human carcinoembryonic antigen (CEA) gene [Robbins PF, Kantor JA, Salgaller M, Horan Hand P, Fernsten PD, Schlom J (1991) Cancer Res 51: 3657]. Two clones, MC-38-ceal and MC-38-cea2, expressed high levels of CEA on their cell surface. A third CEA-expressing cell line, MCA-102-cea3, was similarly derived by transduction of the MCA-102 C57BL/6 mouse fibrosarcoma cell line and is described here. In this study, the three CEA-transduced murine tumor cell lines (MC-38-cea1, MC-38-cea2, MCA-102-cea3) were evaluated for their tumorigenic potential, as well as their ability to serve as in vivo model systems for active and passive immunotherapy studies. Parameters that were investigated include tumor growth rate, the antibody response of the host to CEA, and the CEA content of the tumors. The MC-38-cea2 model appeared to be the most appropriate for immunotherapy studies. Biodistribution studies, using an125I-labeled anti-CEA mAb, demonstrated efficient tumor targeting of MC-38-cea2 tumors in C57BL/6 and athymic mice. 相似文献
2.
Characterization of the pathways for phosphatidylethanolamine biosynthesis in Chinese hamster ovary mutant and parental cell lines 总被引:3,自引:0,他引:3
A tritium suicide procedure was devised to facilitate the isolation of Chinese hamster ovary cell mutants defective in phosphatidylethanolamine biosynthesis. One mutant with a 20-50% reduction in [3H]ethanolamine incorporation was chosen for further analysis and was shown to have reduced activity of CTP: phosphoethanolamine cytidylyltransferase. Levels of phosphatidylethanolamine and rates of its biosynthesis were compared in the mutant and parent cell lines. Despite the reduced activity of the CDP-ethanolamine pathway in the mutant, levels of phosphatidylethanolamine were the same in mutant and parent cells. Rates of phosphatidylethanolamine synthesis de novo, as measured by incorporation of 32PO4 into phosphatidylethanolamine, were also the same in mutant and parent cells, as was the rate of incorporation of [3H]serine into both phosphatidylserine and phosphatidylethanolamine. After a long term labeling with [3H]serine, the specific radioactivity of phosphatidylserine was the same as that of phosphatidylethanolamine, and there was no difference in the specific radioactivities of the two lipids between mutant and parent cells. These results implicate decarboxylation of phosphatidylserine as the sole route for synthesis of phosphatidylethanolamine under normal culture conditions. 相似文献
3.
4.
Pectobacterium carotovorum (formerly Erwinia carotovora ssp. carotovora) is a phytopathogenic bacterium that causes soft rot disease, characterized by water-soaked soft decay, resulting from the action of cell wall-degrading exoenzymes secreted by the pathogen. Virulence in soft rot bacteria is regulated by environmental factors, host and bacterial chemical signals, and a network of global and gene-specific bacterial regulators. We isolated a mini-Tn5 mutant of P. carotovorum that is reduced in the production of extracellular pectate lyase, protease, polygalacturonase and cellulase. The mutant is also decreased in virulence as it macerates less host tissues than its parent and is severely impaired in multiplication in planta. The inactivated gene responsible for the reduced virulent phenotype was identified as corA. CorA, a magnesium/nickel/cobalt membrane transporter, is the primary magnesium transporter for many bacteria. Compared with the parent, the CorA(-) mutant is cobalt resistant. The mutant phenotype was confirmed in parental strain P. carotovorum by marker exchange inactivation of corA. A functional corA(+) DNA from P. carotovorum restored exoenzyme production and pathogenicity to the mutants. The P. carotovorum corA(+) clone also restored motility and cobalt sensitivity to a CorA(-) mutant of Salmonella enterica. These data indicate that CorA is required for exoenzyme production and virulence in P. carotovorum. 相似文献
5.
Serological studies of a host range mutant of a lactic streptococcal bacteriophage. 总被引:12,自引:9,他引:3
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A W Jarvis 《Applied microbiology》1978,36(6):785-789
A host range mutant was isolated from a bacteriophage that attacked Streptococcus cremoris 114. The mutant was able to adsorb and grow on S. cremoris 266, where the parent phage could not. The mutant phage was unable to adsorb to the original bacterial host, S. cremoris 114. The change in host range was accompanied by an alteration in the neutralization antigen as shown by a change in neutralization rate by an anti-phage serum. Serum-blocking experiments confirmed the difference in neutralization antigen between parent and mutant phages. The two phages nevertheless had similar complement fixation antigens, confirming that one was a mutant derived from the other. A distinction between complement fixation and neutralization antigens, similar to that found for the coliphages and staphylococcal phages, has therefore been demonstrated for two lactic streptococcal phages. 相似文献
6.
Williams TJ Zhang CL Scott JH Bazylinski DA 《Applied and environmental microbiology》2006,72(2):1322-1329
Strain MC-1 is a marine, microaerophilic, magnetite-producing, magnetotactic coccus phylogenetically affiliated with the alpha-Proteobacteria. Strain MC-1 grew chemolithotrophically with sulfide and thiosulfate as electron donors with HCO3-/CO2 as the sole carbon source. Experiments with cells grown microaerobically in liquid with thiosulfate and H14CO3-/14CO2 showed that all cell carbon was derived from H14CO3-/14CO2 and therefore that MC-1 is capable of chemolithoautotrophy. Cell extracts did not exhibit ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO) activity, nor were RubisCO genes found in the draft genome of MC-1. Thus, unlike other chemolithoautotrophic, magnetotactic bacteria, strain MC-1 does not appear to utilize the Calvin-Benson-Bassham cycle for autotrophy. Cell extracts did not exhibit carbon monoxide dehydrogenase activity, indicating that the acetyl-coenzyme A pathway also does not function in strain MC-1. The 13C content of whole cells of MC-1 relative to the 13C content of the inorganic carbon source (Deltadelta13C) was -11.4 per thousand. Cellular fatty acids showed enrichment of 13C relative to whole cells. Strain MC-1 cell extracts showed activities for several key enzymes of the reverse (reductive) tricarboxylic acid (rTCA) cycle including fumarate reductase, pyruvate:acceptor oxidoreductase and 2-oxoglutarate:acceptor oxidoreductase. Although ATP citrate lyase (another key enzyme of the rTCA cycle) activity was not detected in strain MC-1 using commonly used assays, cell extracts did cleave citrate, and the reaction was dependent upon the presence of ATP and coenzyme A. Thus, we infer the presence of an ATP-dependent citrate-cleaving mechanism. These results are consistent with the operation of the rTCA cycle in MC-1. Strain MC-1 appears to be the first known representative of the alpha-Proteobacteria to use the rTCA cycle for autotrophy. 相似文献
7.
Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis. 总被引:5,自引:0,他引:5
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Arginine-specific cysteine proteinase (Arg-gingipain [RGP], a major proteinase secreted from the oral anaerobic bacterium Porphyromonas gingivalis, is encoded by two separate genes (rgpA and rgpB) on the P. gingivalis chromosome and widely implicated as an important virulence factor in the pathogenesis of periodontal disease (K. Nakayama, T. Kadowaki, K. Okamoto, and K. Yamamoto, J. Biol. Chem. 270:23619-23626, 1995). In this study, we investigated the role of RGP in the formation of P. gingivalis fimbriae which are thought to mediate adhesion of the organism to the oral surface by use of the rgp mutants. Electron microscopic observation revealed that the rgpA rgpB double (RGP-null) mutant possessed very few fimbriae on the cell surface, whereas the number of fimbriae of the rgpA or rgpB mutant was similar to that of the wild-type parent strain. The rgpB+ revertants that were isolated from the double mutant and recovered 20 to 40% of RGP activity of the wild-type parent possessed as many fimbriae as the wild-type parent, indicating that RGP significantly contributes to the fimbriation of P. gingivalis as well as to the degradation of various host proteins, disturbance of host defense mechanisms, and hemagglutination. Immunoblot analysis of cell extracts of these mutants with antifimbrilin antiserum revealed that the rgpA rgpB double mutant produced small amounts of two immunoreactive proteins with molecular masses of 45 and 43 kDa, corresponding to those of the precursor and mature forms of fimbrilin, respectively. The result suggests that RGP may function as a processing proteinase for fimbrilin maturation. In addition, a precursor form of the 75-kDa protein, one of the major outer membrane proteins of P. gingivalis, was accumulated in the rgpA rgpB double mutant but not in the single mutants and the revertants, suggesting an extensive role for RGP in the maturation of some of the cell surface proteins. 相似文献
8.
Characterization of a Tetrahymena thermophila mutant strain unable to develop normal thermotolerance. 总被引:2,自引:1,他引:1
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For Tetrahymena thermophila cells to survive extended periods of time at 43 degrees C, they must continuously synthesize heat shock proteins. For its translational machinery to function at 43 degrees C, T. thermophila requires either prior nonlethal heat shock treatment or brief treatment with partially inhibiting doses of cycloheximide or emetine. We have identified and characterized a mutant strain of T. thermophila (MC-3) in which prior nonlethal heat shock does not prevent protein synthesis inactivation at 43 degrees C. In addition, treatment of MC-3 cells with either of the antibiotics that normally confer 43 degrees C thermoprotection on wild-type cells elicited no similar thermoprotective response in these cells. Despite these phenotypic characteristics, by other criteria MC-3 synthesized a normal, functional array of heat shock proteins at 40 degrees C, a nonlethal heat shock protein-inducing temperature. The mutation in MC-3 which prevents the thermostabilization of protein synthesis by nonlethal heat shock is, by genetic criteria, most likely the same one which prevents the induction of thermotolerance by drug treatments. We present evidence that this mutation may affect some ribosome-associated functions. 相似文献
9.
The effect of castanospermine on the oligosaccharide structures of glycoproteins from lymphoma cell lines. 总被引:2,自引:0,他引:2
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The effect of castanospermine on the processing of N-linked oligosaccharides was examined in the parent mouse lymphoma cell line and in a mutant cell line that lacks glucosidase II. When the parent cell line was grown in the presence of castanospermine at 100 micrograms/ml, glucose-containing high-mannose oligosaccharides were obtained that were not found in the absence of inhibitor. These oligosaccharides bound tightly to concanavalin A-Sepharose and were eluted in the same position as oligosaccharides from the mutant cells grown in the absence or presence of the alkaloid. The castanospermine-induced oligosaccharides were characterized by gel filtration on Bio-Gel P-4, by h.p.l.c. analysis, by enzymic digestions and by methylation analysis of [3H]mannose-labelled and [3H]galactose-labelled oligosaccharides. The major oligosaccharide released by endoglucosaminidase H in either parent or mutant cells grown in castanospermine was a Glc3Man7GlcNAc, with smaller amounts of Glc3Man8GlcNAc and Glc3Man9GlcNAc. On the other hand, in the absence of castanospermine the mutant produces mostly Glc2Man7GlcNAc. In addition to the above oligosaccharides, castanospermine stimulated the formation of an endoglucosaminidase H-resistant oligosaccharide in both cell lines. This oligosaccharide was characterized as a Glc2Man5GlcNAc2 (i.e., Glc(1,2)Glc(1,3)Man(1,2)Man(1,2)Man(1,3)[Man(1,6)]Man-GlcNAc-GlcNAc). Castanospermine was tested directly on glucosidase I and glucosidase II in lymphoma cell extracts by using [Glc-3H]Glc3Man9GlcNAc and [Glc-3H]Glc2Man9GlcNAc as substrates. Castanospermine was a potent inhibitor of both activities, but glucosidase I appeared to be more sensitive to inhibition. 相似文献
10.
大肠杆菌BL21(DE3)磷酸转乙酰基酶缺陷变株的发酵研究 总被引:3,自引:1,他引:2
研究了E.coliBL21(DE3)及其磷酸转乙酰基酶(PTA)缺陷变株FR55发酵过程中菌体生长和有机酸产生情况,并以肿瘤坏死因子(TNF)为外源蛋白表达的模型考察了pta基因缺陷对外源蛋白表达的影响。在摇瓶培养条件下,pta变株TNF的表达水平比亲株提高了23%。在5L发酵罐中进行了补料分批培养试验,在不限制比生长速率的条件下pta变株能够以较长时间和较高比生长速率保持对数生长,最终达到32.5g(DCW)/L的菌密度,TNF的总表达量达2.8g/L;而在相同条件下,以BL21(DE3)为受体菌的对照组最高菌密度为19.5g(DCW)/L,TNF总表达量只有0.84g/L。表明pta变株对于提高工程菌外源蛋白的表达和实现高密度培养具有一定应用价值。分析了补料分批培养过程中发酵液有机酸组成和含量的动态变化情况,发现pta变株乙酸累积水平明显降低(为亲株乙酸累积水平的42%)的同时,其他几种有机酸(丙酮酸、乳酸、琥珀酸)的累积有显著增加的趋势,使发酵液中总有机酸浓度增加了123%,其中乳酸的累积是影响菌体进一步生长的主要因素。 相似文献
11.
E Cavalieri R Roth J Althoff C Grandjean K Patil S Marsh D Mclaughlin 《Chemico-biological interactions》1978,22(1):69-81
Trapping of 3-methylcholanthrene (MC) radical cation by nucleophilic compounds occurs specifically at the 1-carbon atom. With the purpose of providing more evidence for the hypothesis that the critical mechanism of activation of MC is one-electron oxidation, the carcinogenicity of MC was compared to that of 1-hydroxy-3-methylcholanthrene (MC-1-OH), 3-methylcholanthrene-1-one (MC-1-one), 2-hydroxy-3-methylcholanthrene (MC-2-OH), 3-methylcholanthrene-2-one (MC-2-one) and 3-methylcholanthrylene (MCL) by repeated application on mouse skin. Seven-week-old female Swiss mice in 6 groups of 30 were treated on the back with 0.2 mumol of compound in acetone twice weekly for 20 weeks. In addition, the metabolism of MC and its derivatives was studied using mouse skin homogenates. The compounds tested were classified according to carcinogenicity in 4 groups: MC and MC-2-OH, the strongest carcinogens; MC-2-one and MCL, weaker than MC and MC-2-OH; MC-1-OH, the weakest carcinogen; and MC-1-one, non-carcinogenic. These results support the hypothesis that one-electron oxidation for MC, MC-2-OH and MC-1-one might be the critical mechanism of carcinogenic activation, with C-1 the binding site to cellular nucleophiles. The carcinogenic effect of MC-1-OH is speculated to be the formation of an ester bearing a good leaving group, which might be the ultimate alkylating compound in the in vivo reaction. The lack of carcinogenic activity for MC-1-one may be attributed to absence of nucleophilic trapping at C-1 via the radical cation pathway as well as the inability of mouse skin to reduce MC-1-one to the carcinogenic MC-1-OH. 相似文献
12.
Insertional inactivation of a chromosomal locus that modulates expression of potential virulence determinants in Staphylococcus aureus. 总被引:2,自引:1,他引:1
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A single insertion of transposon Tn551 into a unique chromosomal locus of Staphylococcus aureus ISP479C has resulted in a pleiotropic effect on the expression of both extracellular and cell wall proteins. In particular, the expression of cell wall protein A and clumping activity with fibrinogen were rendered undetectable in the mutant 1E3 compared with the parent. The secretion of alpha-hemolysin in mutant 1E3 was modestly increased. Southern blot and phenotypic analyses indicated that this locus is distinct from agr, xpr, and sar, three previously described global regulatory loci. Transduction experiments demonstrated that the genotype associated with mutant 1E3 could be transferred back into the parental strain ISP479C. The transductant 1E3-2 displayed a phenotypic profile similar to that of the original mutant. Northern (RNA) blot studies showed that this locus may be involved in modulating target genes at the mRNA level. In the rabbit endocarditis model, there was a significant decrease in both the infectivity rate and intravegetation bacterial density with mutant 1E3 compared with the parent at an inoculum of 10(3) CFU. Since protein A and the fibrinogen-binding protein(s) are major surface proteins that may mediate bacterial adhesion to host tissues, this locus may be an important genetic element involved in the expression of virulence determinants in S. aureus. 相似文献
13.
The ability of Helicobacter pylori to colonize the stomach requires that it combat oxidative stress responses imposed by the host. The role of methionine sulfoxide reductase (Msr), a methionine repair enzyme, in H. pylori stress resistance was evaluated by a mutant analysis approach. An msr mutant strain lacked immunologically detectable sulphoxide reductase protein and also showed no enzyme activity when provided with oxidized methionines as substrates. The mutant strain showed diminished growth compared to the parent strain in the presence of chemical oxidants, and showed rapid viability loss when exposed to oxidizing conditions. The stress resistance and enzyme activity could be recovered by complementing the mutant with a functional copy of the msr gene. Upon fractionation of parent strain and the complemented mutant cells into membranes and cytoplasmic proteins, most of the immunologically detectable Msr was localized to the membrane, and this fraction contained all of the Msr activity. Qualitative detection of the whole cell protein pattern using 2,4-dinitro phenyl hydrazine (DNPH) showed a far greater number of oxidized protein species in the mutant than in the parent strain when the cells were subjected to oxygen, peroxide or s-nitrosoglutathione (GSNO) induced stress. Importantly, no oxidized proteins were discerned in either strain upon incubation in anaerobic conditions. A mutant strain that synthesized a truncated Msr (corresponding to the MsrA domain) was slightly more resistant to oxidative stress than the msr strain. Mouse colonization studies showed Msr is an important colonization factor, especially for effective longer-term (14 and 21 days) colonization. Complementation of the mutant msr strain by chromosomal insertion of a functional gene restored mouse colonization ability. 相似文献
14.
In this study, the proliferative effects of retinoids were examined in the MC-26 and LoVo colon adenocarcinoma cell lines. The proliferation of the LoVo cell line was not altered in the presence of the retinoidsall trans-retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA). Both retinoids, however, stimulated the growth, as measured by cell proliferation, of MC-26 cells.atRA and 9-cis-RA were equipotent in increasing MC-26 cell proliferation, suggesting that the growth stimulation is mediated by one or more retinoic acid receptors (RARs). To determine the RAR which might be responsible for this growth stimulatory effect, we characterized the RAR subtypes which were present in both cell lines. mRNA for the RARα, RARβ, and RARγ were detected in the MC-26 cell. Of the RARs present in MC-26 cells, the RARα does not mediate the growth stimulatory effects of retinoids, for a selective RARα antagonist was unable to prevent the retinoid-induced increase in MC-26 cell growth. RARα, RARβ, and RARγ mRNA are also expressed in the LoVo cell line; the lack of growth-stimulation by retinoids in LoVo cells, therefore, does not seem to be due to the absence of RARs. The results obtained in these experiments demonstrate that the growth response elicited by retinoids can vary between colon cancer cells and that the differences in response may not be solely determined by the RAR subtypes which are expressed in a colon cancer cell line. 相似文献
15.
16.
Phospholipase activity in bacteriophage-infected Escherichia coli. III. Phopholipase A involvement in lysis of T4-infected cells. 总被引:4,自引:1,他引:3
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Bacteriophage studies with Escherichia coli K-12 (gamma)DR-DS-, a mutant lacking the major known fatty acyl hydrolases (phospholipases), and its wild-type parent showed equivalent phage infection with regard to phage production and time of phage release. Further examination of the DR-DS- mutant, however, revealed that the progeny bacteriophage were released without complete dissolution of the host cell. Prolonged cell integrity of the infected mutant was noted by spectrophotometry and supported by direct microscope examination. The phage release occurred at normal "lysis" time with phage yields comparable to that of the wild-type bacteria. Inner membrane degradation was indicated by the release of beta-galactosidase, a cytoplasmic enzyme, and of trichloracetic acid-precipitable RNA. Thus, outer membrane degradation is required for dissolution of phage-infected cells, and this degradation is at least partly dependent on activation of host phospholipases. 相似文献
17.
Various human Burkitt lymphoma and LCL lines established in vitro and their derived somatic cell hybrids were tested for their comparative EBV receptor levels in a virus binding assay. Their graded C3b and C3d complement receptor expression was estimated simultaneously by means of isotope labeled rosette marker cells. The receptor concentration of each cell line was related to Raji as the standard of comparison, K 562, P3HR-1, and YACUT were used as negative controls. In general, the charging curves for EBV and C3d receptors parallelled each other (r = 0.97) while C3b receptor charging showed no correlation (r < 0.60). In the Raji hybrids between the C3b receptor positive Raji parent and various patents that were negative for this receptor, C3b receptor expression was low or negative. In contrast, the C3d negative P3HR-1 line gave rise to hybrids, after fusion with receptor-positive cells, that were intermediate with regard to their C3d receptor expression. The host range restriction of the Epstein-Barr virus is determined at the receptor level. The close relationship between the EBV receptor and the C3d receptor, a B-lymphocyte-specific moiety, suggests that the moderate interaction with EBV with the B lymphocytes may have had a selective advantage, favoring the presence of EBV. Since EBV causes lytic infections after artificial introduction into nonnatural host cells, it may represent a B-lymphocyte-specific host range mutant, derived from an originally lytic herpesvirus with a much broader target cell range. 相似文献
18.
Growth-promoting effects of gastrin on mouse colon cancer cells in vitro: Absence of autocrine effects 总被引:2,自引:0,他引:2
Yan-Shi Guo Madhulika Baijal Gui-Fang Jin James C. Thompson Courtney M. Townsend Jr. Pomila Singh 《In vitro cellular & developmental biology. Plant》1990,26(9):871-877
Summary We have previously demonstrated trophic effects of gastrin on mouse colon cancer (MC-26) cells, in vivo, and demonstrated
the presence of gastrin receptors (GR) on these cells. The cellular and intracellular mechanism by which gastrin expresses
trophic effects on colon cancer cells is, however, as yet unknown. For us to start investigating the possible mechanisms involved,
it was important that we first develop an in vitro model, in which gastrin expresses its trophic effects directly on the MC-26
cells. The growth-promoting effects of gastrin on the MC-26 cells were examined in various in vitro culture models, in terms
of [3H]thymidine incorporation and cell number. A significant trophic effect of gastrin could be demonstrated on quiescent cells
in culture, in the absence of serum. The optimal cell-culture conditions for observing trophic effects of gastrin were defined
and included a 24-h period of rapid growth of MC-26 cells in serum-supplemented normal growth medium, followed by a 24-h period
of culture in serum-free medium containing an optimal dose (1.0 mM) of thymidine, to achieve growth-arrest of the cells. Addition of gastrin (0.5 to 25 nM) to the quiescent, growth-arrested cells resulted in significant dose-dependent increases in both the incorporation of [3H]thymidine uptake by the cells, and a significant increase in cell number. The concentration of GR on the growth-arrested
quiescent MC-26 cells in culture was significantly increased compared to the GR concentration on the control, asynchronized
cells. The increased presence of GR on the growth-arrested, synchronized MC-26 cells may have allowed us to observe a significant
trophic effect of gastrin on the MC-26 cells, in vitro itself. To determine if gastrin was functioning as an autocrine growth
factor for MC-26 cells, we examined the effect of gastrin antibodies on the growth of MC-26 cells; no significant effect of
the antigastrin IgG on the growth of MC-26 cells was observed.
Supported by grants from the National Institutes of Health, Bethesda, MD (PO1 DK 35608 and CA 38651, and a grant from the
American Cancer Society, Washington, DC (PDT-220). 相似文献
19.
A mutant of the uniformly lethal L5178Y lymphoma, called the L5178Y/Manitoba (L5178Y/M), was rejected after subcutaneous challenge in syngeneic DBA/2 mice. Karyotypic analysis revealed that the parent L5178Y lymphoma had four chromosome markers, with the mutant L5178Y/M sharing one of them as well as possessing two distinguishing markers. One diploid and two hypotetraploid clones were isolated from the L5178Y/M; they contained all the marker chromosomes and were also rejected by the syngeneic host. In addition to the shared chromosome markers, the L5178Y/M possessed antigens in common with the parent L5278Y. DBA/2 mice made immune to the mutant by subcutaneous immunization were able to slow the growth of the parent tumor but not the unrelated P-815-X2 mastocytoma. 相似文献
20.