Mitochondrial and plasma membrane potentials cause unusual accumulation and retention of rhodamine 123 by human breast adenocarcinoma-derived MCF-7 cells

Quantitative studies of MCF-7 cells (derived from human breast adenocarcinoma) and CV-1 cells (from normal African green monkey kidney epithelium), using the permeant cationic compound tetraphenylphosphonium (TPP), in conjunction with fluorescence microscopy using rhodamine 123 (Rh123), indicate that the mitochondrial and plasma membrane potentials affect both uptake and retention of these compounds. Under conditions that depolarize the plasma membrane, uptake and retention of TPP and Rh123, driven only by the mitochondrial membrane potential, is greater in MCF-7 than in CV-1. An ionophore that dissipates the mitochondrial membrane potential of MCF-7 cells causes them to resemble CV-1 cells by decreasing uptake and retention. Hyperpolarizing the mitochondrial membrane of CV-1 increases accumulation and prolongs retention; hyperpolarization of the plasma membrane further heightens this effect, causing the uptake of CV-1 cells to resemble that of MCF-7 cells even more closely. The greater uptake and retention by MCF-7 appears to be a consequence of elevated mitochondrial and plasma membrane potentials. The plasma membrane potential affects mitochondrial retention of TPP and Rh123 and its role in enhancing the effect of a difference in mitochondrial membrane potential is explained.     

Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles

Several novel cationic amphiphiles, based on a hydrophobic cholesteryl or dioleoylglyceryl moiety, have been prepared whose hydrophobic and cationic portions are linked by ester bonds to facilitate efficient degradation in animal cells. Dispersions combining such cationic species with phosphatidylethanolamine (PE), certain structural analogues of PE or diacylglycerol can mediate efficient transfer of both nonexchangeable lipid probes and the DNA plasmid pSV2cat into cultured mammalian (CV-1 and 3T3) cells. The abilities of different types of cationic lipid dispersions to mediate transfection of mammalian cells with pSV2cat could not be directly correlated with their abilities to coalesce with other membranes, as assessed by their ability to intermix lipids efficiently with large unilamellar phosphatidylcholine/phosphatidylserine vesicles in the presence or absence of DNA. The cytotoxicities toward CV-1 cells of dispersions combining PE with most of the degradable cationic amphiphiles studied here compare favorably with those reported previously for similar dispersions containing other types of cationic amphiphiles. Fluorescent analogues of two of the diacylglycerol-based cationic amphiphiles examined in this study are shown to be readily degraded after incorporation into CV-1 cells from PE/cationic lipid dispersions.     

A monoclonal antibody defining a lymphoma-associated antigen in man

A monoclonal antibody (Ab 89) was developed against a lymphoma-associated antigen on the tumor cells of a patient (N.B.) with a B cell, poorly differentiated lymphocytic lymphoma (D-PDL). By indirect immunofluorescence, Ab 89 was shown to react only with N.B. lymphoma cells and was unreactive with normal N.B. lymphoid cells, normal fractionated peripheral blood cells, other normal lymphoid tissues, fetal tissues, and non-lymphoid malignant cells. In addition, Ab 89 was unreactive with conventional immunoglobulins, private and public HLA antigens, or Ia-like antigens. More importantly, Ab 89 was reactive with some B cell lymphoid malignancies. Of the 57 B cell lymphomas tested, it was found that Ab 89 reacted with approximately 10% of B cell D-PDL and B cell chronic lymphocytic leukemia (CLL). Of interest was the finding that N.B. serum contained a circulating antigen which could specifically block the reactivity of Ab 89. The data obtained suggests that Ab 89 defines a tumor-associated antigen shared by a unique subgroup of B cell lymphomas. The group of patients reactive with Ab 89 did not fall into any histopathologic classification system. These data support the notion that there is greater heterogeneity of B cell lymphomas than may have been previously recognized.     

Identification of the Simian Virus 40 Which Replicates When Simian Virus 40-Transformed Human Cells Are Fused with Simian Virus 40-Transformed Mouse Cells or Superinfected with Simian Virus 40 Deoxyribonucleic Acid

Simian virus 40 (SV40) was rescued from heterokaryons of transformed mouse and transformed human cells. To determine whether the rescued SV40 was progeny of the SV40 genome resident in the transformed mouse cells, the transformed human cells, or both, rescue experiments were performed with mouse lines transformed by plaque morphology mutants of SV40. The transformed mouse lines that were used yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used did not release SV40 spontaneously or after fusion with CV-1 cells. From each mouse-human fusion mixture, only the SV40 resident in the transformed mouse cells was recovered. Fusion mixtures of CV-1 and transformed mouse cells yielded much more SV40 than those from transformed human and transformed mouse cells. The rate of SV40 formation was also greater from monkey-mouse than from human-mouse heterokaryons. Deoxyribonucleic acid (DNA) from SV40 strains which form fuzzy, largeclear, or small-clear plaques on CV-1 cells was also used to infect monkey (CV-1 and Vero), normal human, and transformed human cell lines. The rate of virion formation and the final SV40 yields were much higher from monkey than from normal or transformed human cells. Only virus with the plaque type of the infecting DNA was found in extracts from the infected cells. Two uncloned sublines of transformed human cells [W18 Va2(P363) and WI38 Va13A] released SV40 spontaneously. Virus yields were not appreciably enhanced by fusion with CV-1 cells. However, clonal lines of W18 Va2(P363) did not release SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of WI38 Va13A cells did continue to shed SV40 spontaneously.     

Phospholipases and cationic peptides inhibit Cryptosporidium parvum sporozoite infectivity by parasiticidal and non-parasiticidal mechanisms

The apicomplexan parasite Cryptosporidium parvum is an important cause of diarrhea in humans and cattle, and it can persistently infect immunocompromised hosts. No consistently effective parasite-specific pharmaceuticals or immunotherapies for control of cryptosporidiosis are presently available. The innate immune system represents the first line of host defense against a range of infectious agents, including parasitic protozoa. Several types of antimicrobial peptides and proteins, collectively referred to herein as biocides, constitute a major effector component of this system. In the present study, we evaluated lactoferrin, lactoferrin hydrolysate, 5 cationic peptides (lactoferricin B, cathelicidin LL37, indolicidin, β-defensin 1, β-defensin 2), lysozyme, and 2 phospholipases (phospholipase A2, and phosphatidylinositol-specific phospholipase C) for anti-cryptosporidial activity. The biocides were evaluated either alone or in combination with 3E2, a monoclonal antibody (MAb) against C. parvum that inhibits sporozoite attachment and invasion. Sporozoite viability and infectivity were used as indices of anti-cryptosporidial activity in vitro. All biocides except lactoferrin had a significant effect on sporozoite viability and infectivity. Lactoferrin hydrolysate and each of the 5 cationic peptides were highly parasiticidal and strongly reduced sporozoite infectivity. While each phospholipase also had parasiticidal activity, it was significantly less than that of lactoferrin hydrolysate and each of the cationic peptides. However, each phospholipase reduced sporozoite infectivity comparably to that observed with lactoferrin hydrolysate and the cationic peptides. Moreover, when 3 of the cationic peptides (cathelicidin LL37, β-defensin 1, and β-defensin 2) were individually combined with MAb 3E2, a significantly greater reduction of sporozoite infectivity was observed over that by 3E2 alone. In contrast, reduction of sporozoite infectivity by a combination of either phospholipase with MAb 3E2 was no greater than that by 3E2 alone. These collective observations suggest that cationic peptides and phospholipases neutralize C. parvum by mechanisms that are predominantly either parasiticidal or non-parasiticidal, respectively.     

Benzo[a]pyrene diol epoxide induces viral reactivation at concentrations that block DNA elongation in mammalian cells

The survival of UV-irradiated Simian virus 40 (SV40) in CV-1P African green monkey kidney cells treated with (+/-)7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP-diol epoxide I) was studied. Enhanced survival of UV damaged SV40 was detected when CV-1P cells were treated with dose levels of BP-diol epoxide I corresponding to the exponential portion (0.33-1.11 microM) of a CV-1P cell survival curve. Dose levels of BP-diol epoxide I corresponding to the shoulder region (less than or equal to 0.16 microM) of a CV-1P survival curve did not induce viral reactivation. The shoulder region concentrations of BP-diol epoxide I selectively inhibited DNA initiation while the concentrations on the exponential portion of the curve preferentially inhibited DNA elongation. It was shown in a time course of enhanced viral survival at 0.66 microM BP-diol epoxide I that the reactivation response was fully induced by 24 h. In conclusion, the viral reactivation response was associated with concentrations of BP-diol epoxide I which induced lethal damage and preferentially inhibited DNA elongation.     

Surface membrane redistribution and stabilization of concanavalin A-specific receptors following Yaba tumor poxvirus infection

Monkey kidney cells productively infected with Yaba tumor poxvirus clearly exhibit plasma membrane alterations when treated with both fluorescein-labeled and unlabeled concanavalin A. The convanavalin A-mediated cytoagglutination reaction for Yaba-infected Jinet and CV-1 cells increased linearly from 12 to 16 h post-infection, reaching a maximum by 24-28 h. Treatment of either Yaba-infected CVC-1 or Jinet cells with methyl-D-glucopyranoside before or after addition of concanavalin A completely blocked or reversed the cytoaglutination response. Trypsin treatment of uninfected CV-1 or Jinet cells enhanced concanavalin A-mediated cytoagglutination properties. Conversely, trypsin treatment of Yaba-infected Jinet cells resulted in a reduced cytoagglutination response. Increasing temperature and lectin concentration enhance concanavalin A-mediated cytoagglutination for uninfected, trypsin-treated and Yaba-infected CV-1 cells. Cytosine arabinoside has little or no effect on the Yaba-induced cell cytoagglutination reaction while cycloheximide blocks the cytoagglutinatin response if added prior to 12 h post-infection. Fluorescein-labeled concanavalin A binding studies have revealed that at 4 degrees C, Yaba-infected CV-1 cells display a predominantly 'patchy' pattern of topological fluorescence, while trypsin-treated and uninfected CV-1 cells at 4 degrees C display a uniform pattern of fluorescence binding. Patchy fluorescence, indicative of concanavalin A-suspeptible, receptor-site clustering on the surface membrane, was reduced 50% if Yaba-infected CV-1 cells were treated with glutaraldehyde (2.5%) before addition of fluorescein-labeled concanavalin A at 4 degrees C. Similar pre-fixatin of trypsin-treated CV-1 cells resulted in uniform, fluorescent labelling patterns at all assay temperatures.     

In vivo effects of cis- and trans-diamminedichloroplatinum(II) on SV40 chromosomes: differential repair, DNA-protein cross-linking, and inhibition of replication

The mechanism of action of the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, was investigated by using the approximately 5200 base pair (bp) chromosome of simian virus 40 (SV40) as an in vivo chromatin model. Comparative studies were also carried out with the clinically ineffective isomer trans-DDP. Although 14 times more trans- than cis-DDP in the culture medium is required to inhibit SV40 DNA replication in SV40-infected green monkey CV-1 cells, the two isomers are equally effective at inhibiting replication when equimolar amounts are bound to SV40 DNA in vivo. Since both isomers are transported into CV-1 cells at similar rates, differential uptake cannot account for the greater ability of cis-DDP to inhibit SV40 DNA replication. Rather, this result is explained by the finding that cis-DDP-DNA adducts accumulate continuously over the incubation period, whereas trans-DDP binding to DNA reaches a maximum at 6 h and thereafter decreases dramatically. We suggest that the different accumulation behavior of cis-DDP and trans-DDP on DNA is due to their differential repair in CV-1 cells. A variety of non-histone proteins, including SV40 capsid proteins but virtually no histones, are cross-linked to SV40 DNA in vivo by either cis- or trans-DDP. More DNA-protein cross-links are formed by trans-DDP than by cis-DDP at equivalent amounts of DNA-bound platinum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and properties of new rubomycin derivatives

Condensation of rubomycin (daunorubicin) with respective hydrazides yielded novel substituted hydrazones: 13-cyanoacetyl hydrazone rubomycin, 13-L-phenylalanyl hydrazone rubomycin, 13-BOC-3-(uracilyl-1)-DL-alanyl hydrazone rubomycin and 13-BOC-3-(adenylyl-9)-DL-alanyl hydrazone rubomycin. With successive treatment of rubomycin with hydrazine hydrate and respective ketones novel asymmetric azines were prepared: 13-cyclopentylidene hydrazone rubomycin, 13-alpha,alpha'-dimethyl-cyclopentylidene hydrazone rubomycin and 13-(1-phenylethylidene-1) hydrazone rubomycin. 14-Adenylyl-N9-rubomycin was synthesized by interaction of 14-bromorubomycin with adenine and hydrogenation of its analog, 14-N-imidazolyl rubomycin by sodium borhydrite yielded 13-dihydro-14-N-imidazolyl rubomycin. There was observed correlation between the antimicrobial activity of the derivatives against B. mycoides and their cytostatic effect on the cells of murine leukemia NK/LI. The high in vitro activity of 13-cyclopentylidene hydrazone rubomycin showed satisfactory correlation with the results of the study on the antitumor effect in animals.     

Specificity of monoclonal antibodies to an EBV transformed B-cell line

Monoclonal antibodies were produced against an Epstein Barr virus (EBV) transformed human B-cell line with the following HLA specificities: HLA A2, B27, Cw2, Dr3,2. Antibodies from three clones, Mab B1, Mab B2 and B3 reacted with human Ia-like molecules on peripheral blood B cells, some monocytes, CLL cells, lymphoblastoid B-cell lines and some mixed leukocyte culture (MLC)-activated T cells, but were unreactive with leukoagglutinin (LA) and Concanavalin A (Con A)-activated T blasts and T-cell lines. Antibodies obtained from three other clones (Mab 4, 5 and 6) reacted with a mw 80,000 protein present on peripheral lymphocytes and on most of the lymphoblastoid T-and B-cell lines tested.     

六种阳离子助剂对阿维菌素的增效作用

采用浸虫法和叶片药膜法比较了6种阳离子助剂与阿维菌素混用对小菜蛾Plutella xylostella 3龄幼虫的增效作用,并通过测定助剂对阿维菌素药液物理性状的影响以及助剂对小菜蛾体内超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalases,CAT)和过氧化物酶(peroxidases,POD) 3种保护酶活性的影响等方面对其增效机制进行了初步分析,为阳离子助剂在杀虫剂领域的开发提供一定的依据。结果表明： 在400 mg/L以下,各阳离子助剂单独使用对小菜蛾3龄幼虫均无生物活性,但可明显提高阿维菌素对小菜蛾的药效。两种方法中,浸虫法的增效作用大于叶片药膜法,其中松香酸铜、1427和412103对阿维菌素的增效倍数分别为3.71、2.82和2.72。6种助剂均可明显降低阿维菌素药液的表面张力和接触角及增加药液在甘蓝叶片上的沉积量,但不同助剂间及相同助剂不同浓度间差异虽大多显著; 与增效比结果综合分析表明,助剂的润湿性能与其增效作用之间关系不大。阿维菌素药液分别加入松香酸铜、1427和412103后使试虫体内POD和SOD的活性明显提高,从侧面说明这3种助剂对虫体更具渗透性。     

Purification of an ultraviolet-inducible, damage-specific DNA-binding protein from primate cells.

A UV-inducible, damage-specific DNA-binding (DDB) protein with high affinity for double-stranded UV-irradiated DNA has been identified recently in monkey kidney (CV-1) cells (Hirschfeld, S., Levine, A. S., Ozato, K., and Proti?, M. (1990) Mol. Cell. Biol. 10, 2041-2048). We have now purified the DDB protein from extracts of CV-1 cells using hydroxylapatite, phosphocellulose, Mono S, and DNA-affinity column chromatography. The DDB activity, either from mock-treated or UV-induced cells, is heterodisperse in column chromatography, and separation of three forms of the protein was obtained on a phosphocellulose column. Analysis of purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that greater than 90% of all three forms is a protein of approximately 126 kDa. The size of the native DDB protein was deduced from gel filtration and native polyacrylamide gel electrophoresis to be approximately 210 kDa, which suggests that the native DDB protein in solution is a homodimer. Preparations of partially purified DDB protein from UV-treated cells have enhanced levels of DDB activity and the protein when compared with similar preparations from mock-treated cells. This damage-recognition protein, alone or in conjunction with other subunits, may be of general importance for the initial recognition of DNA damage in mammals.     

CV-1 cell recipients of the mouse ouabain resistance gene express a ouabain-insensitive Na,K-ATPase after growth in cardioactive steroids

The cation-transporting activity and Na,K-ATPase activity of CV-1 cell recipients of the mouse ouabain resistance gene (ouaR6, or OR6 cells; see Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1489-1493) have been further characterized. OR6 cells grown in strophanthidin (a cardiac aglycon which may be removed rapidly from the Na,K-ATPase) possess both ouabain-sensitive and -insensitive 86Rb+ uptake activities. The ouabain-sensitive 86Rb+ uptake activity of these cells (OR6-S cells) exhibits the same Ki for ouabain as that of the CV-1 parent cells (Ki(app) = 3 x 10(-7) M ouabain), but accounts for only approximately 30% of total 86Rb+ uptake into Na+-loaded OR6-S cells, compared to 80% for CV-1 cells. Most of the ouabain-resistant 86Rb+ uptake in OR6-S cells is dependent on internal Na+ and is insensitive to furosemide, suggesting that it is due to an ouabain-resistant Na,K pump. In OR6-S cell lysates, 50% of Na+-dependent ATPase activity is insensitive to 1 mM ouabain, compared to less than 5% in CV-1 cell lysates. In addition, purified plasma membranes from OR6-S cells contain a 100-kDa protein which is transiently phosphorylated by ATP in an Na+-dependent, K+-sensitive manner, like the alpha subunit of the CV-1 Na,K-ATPase and the canine renal Na,K-ATPase, but which is unaffected by preincubation in 1 mM ouabain. All of these data suggest that OR6-S cells possess a ouabain-insensitive Na,K pump with characteristics similar to the ouabain-sensitive pump of CV-1 parent cells. Since the mouse ouabain resistance gene does not encode either subunit of the Na,K-ATPase, these results suggest that the ouabain resistance gene product may modify the ouabain sensitivity of the endogenous CV-1 Na,K pump.     

Two Arabidopsis cytochrome P450 monooxygenases, CYP714A1 and CYP714A2, function redundantly in plant development through gibberellin deactivation

The rice gene ELONGATED UPPERMOST INTERNODE1 (EUI1) encodes a P450 monooxygenase that epoxidizes gibberellins (GAs) in a deactivation reaction. The Arabidopsis genome contains a tandemly duplicated gene pair ELA1 (CYP714A1) and ELA2 (CYP714A2) that encode EUI homologs. In this work, we dissected the functions of the two proteins. ELA1 and ELA2 exhibited overlapping yet distinct gene expression patterns. We showed that while single mutants of ELA1 or ELA2 exhibited no obvious morphological phenotype, simultaneous elimination of ELA1 and ELA2 expression in ELA1-RNAi/ela2 resulted in increased biomass and enlarged organs. By contrast, transgenic plants constitutively expressing either ELA1 or ELA2 were dwarfed, similar to those overexpressing the rice EUI gene. We also discovered that overexpression of ELA1 resulted in a severe dwarf phenotype, while overexpression of ELA2 gave rise to a breeding-favored semi-dwarf phenotype in rice. Consistent with the phenotypes, we found that the ELA1-RNAi/ela2 plants increased amounts of biologically active GAs that were decreased in the internodes of transgenic rice with ELA1 and ELA2 overexpression. In contrast, the precursor GA(12) slightly accumulated in the transgenic rice, and GA(19) highly accumulated in the ELA2 overexpression rice. Taken together, our study strongly suggests that the two Arabidopsis EUI homologs subtly regulate plant growth most likely through catalyzing deactivation of bioactive GAs similar to rice EUI. The two P450s may also function in early stages of the GA biosynthetic pathway. Our results also suggest that ELA2 could be an excellent tool for molecular breeding for high yield potential in cereal crops.     

Cationic polypeptides are required for anti-HIV-1 activity of human vaginal fluid

Mucosal surfaces of the vagina are the portals for heterosexual transmission of HIV-1 and therefore play a fundamental role in the pathogenesis of primary infection. In the search for direct biological evidence for the role of human vaginal fluid in innate host defense, we characterized the anti-HIV-1 function of cationic polypeptides within minimally manipulated vaginal fluid. In the current study we revealed that vaginal fluid confers intrinsic anti-HIV-1 properties against both X4 and R5 strains of HIV-1 and could protect against HIV-1 infection and reduce proviral genome integration in organotypic cultures of human cervicovaginal tissue. The majority of this activity was contained in the cationic polypeptide fraction, and the depletion of cationic polypeptides using a selective cation exchange resin ablated most of the intrinsic activity against HIV-1. By adding the cationic polypeptide fraction to depleted vaginal fluid, we were able to restore activity against HIV-1. Using a proteomic approach, we identified 18 cationic polypeptides within vaginal fluid, nearly all of which are either known antimicrobials or have other purported roles in host defense. Interestingly, physiologic concentrations of 13 of the cationic polypeptides were not active alone against HIV-1, yet in concert they partially restored the anti-HIV-1 activity of cation-depleted vaginal fluid. These results suggest that synergism between cationic polypeptides is complex, and full anti-HIV-1 activity probably involves the aggregate of the cationic peptides and proteins in vaginal fluid.     

Synergistic effects of low doses of histatin 5 and its analogues on amphotericin B anti-mycotic activity

The increase in the use of antifungal agents for prophylaxis and therapy has led to the development of antifungal drug resistance. Drug combinations may prevent or delay resistance development. The aim of the present study was to investigate whether naturally and designed cationic antifungal peptides act synergistically with commonly used antimycotics. No enhanced activity was found upon addition of dhvar4, a designed analogue of the human salivary peptide histatin 5, or PGLa to fluconazole or 5–flucytosine, respectively. In contrast, strong synergism of amphotericin B with the peptides was found against several Aspergillus, Candida, and Cryptococcus strains, and against an amphotericin B-resistant C. albicans laboratory mutant in the standardised broth microdilution assays according to the NCCLS standard method M27–T. Amphotericin B showed synergism with dhvar5, another designed analogue of histatin 5, and with magainin 2 against all seven tested strains. Combinations of amphotericin B with histatin 5, dhvar4, and PGLa showed synergism against four of the seven strains. The growth inhibitory activity of amphotericin B was enhanced by sub-MIC concentrations of peptide, but its haemolytic activity remained unaffected, suggesting that its cytotoxicity to host cells was not increased and that peptides may be suitable candidates for combination therapy.     

阳离子助剂1227和C8-10及有机硅助剂Breakthru S240对三种杀虫剂的增效作用

为提升杀虫剂防效, 减少杀虫剂用量, 提高农产品安全, 本研究采用虫体浸渍法研究了阳离子助剂1227和C_8-10及有机硅助剂Breakthru S240对高效氯氰菊酯、 毒死蜱和溴虫腈在甜菜夜蛾Spodoptera exigua幼虫和棉铃虫Helicoverpa armigera幼虫上的增效作用, 并测定了3种助剂对清水物理性状的影响。结果表明： （1）供试助剂在两试虫上对3种杀虫剂均有显著增效作用, 且增效比与助剂浓度存在显著相关关系。相同浓度下, 除Breakthru S240对毒死蜱在棉铃虫上增效作用高于1227外, 3种助剂在两试虫上对3种杀虫剂增效作用均为C_8-10＞1227＞Breakthru S240。3种助剂在甜菜夜蛾上对3种杀虫剂的最佳增效浓度均为900 mg/L。3种助剂在棉铃虫上对3种杀虫剂的最佳增效浓度分别为900, 300 和900 mg/L。（2）3种助剂对清水物理性状均有显著影响。相同浓度下, 供试助剂对清水表面张力的降低能力、 对扩展直径和最大稳定持留量的增强能力均为 Breakthru S240＞1227＞C_8-10。综上所述, 阳离子助剂1227和C_8-10对3种杀虫剂在两试虫上的增效作用显著高于有机硅助剂Breakthru S240, 但后者对于提高杀虫剂在叶表沉积量的能力高于前者, 研究结果为3种助剂在杀虫剂领域的应用提供了一定的依据。     

Production of monoclonal antibodies against principal cells of the renal collecting duct by in vitro immunization with unsolubilized antigens

We report the production of monoclonal antibodies (MAb) by an in vitro technique which react with principal cells of the renal collecting duct. Spleen cells were directly simulated in vitro with unsolubilized antigens, i.e., by direct contact with the apical site of cultivated principal cells or by contact with cell fragments. Out of several others two antibodies, IV1 and IV2, were selected, which specifically reacted with the principal cells of the collecting duct. MAbIV1 also reacted with Type A intercalated cells, indicating the existence of a common antigen in the apical membrane of both cell types. Type B intercalated cells were consistently unreactive. All other parts of the uriniferous tubule were also unreactive. In Western blot analysis MAb IV1 showed immunoreactivity with a 40 KD and a 43 KD antigen. Our experiments demonstrate the possibility of producing antibodies against unsolubilized antigens by a simple in vitro technique. The activity of particular lymphocyte in this in vitro system is shown by the specificity of the antibodies.