~(125)Ⅰ标记单抗LC-I与肺腺癌细胞体内外结合特性的研究

本文用~(125)Ⅰ标记LC-1进行了一些体内外实验。实验结果表明:LC-1单抗的结合常数为4.8×10~8M~(-1),LC-1针对的SPC-A_1细胞表面抗原的位点数为7.2×10~4/细胞;LC-1与LAC-122两单抗针对的抗原决定簇没有交叉;用蛋白酶和过碘酸钠处理SPC-A_1细胞,前者对LC-1的结合抑制39%,后者抑制66%;LC- 1不但有较强的体外结合靶细胞的能力,从LC-1在荷瘤裸鼠中的组织器官分布来看,LC-1与肿瘤有较高的体内亲和性,并且是特异性的结合。     

Actin organization and fibrin-clot retractile activity of cultured mouse fibroblasts

Summary It is known that human and animal fibroblasts are able to induce the retraction of a fibrin clot. In the present study the correlation between (i) fibrinclot retractile (FCR) activity, (ii) the number of actin stress-lines in mouse fibroblasts during growth in culture, and (iii) the sensitivity of actin stress-lines to a powerful actin-depolymerizing factor (ADF), present in plasma and serum of humans and laboratory animals was investigated. Fibroblasts at early passages (2–4) were tested for these parameters at various intervals after seeding (24, 96, and 168 h). The number of actin stress-lines was progressively higher, while the sensitivity to ADF action was progressively lower in cells cultured from 24 to 168 h; the FCR capacity was significantly decreased at 168 h. These data suggest that cells containing weakly polymerized and/or stabilized actin are more active than those containing highly polymerized and/or stabilized actin in triggering fibroblast contraction.     

Pneumocystis carinii: Freeze-fracture study of stages of the organism

The morphology and life cycle of Pneumocystis carinii were studied in cortico-steroid-treated rats by ultrathin section and freeze-fracture electron microscopy. The following stages of P. carinii were noted: trophic, precyst, and cyst. The crescent-shaped cysts appeared to be intermediate forms between precyst and cyst. The cell wall of the trophic stage showed membrane structures suggestive of protozoan endocytosis, whereas the surface of the precyst stage was smooth. The cell wall of the cyst lacked the specialized structural differentiation of yeasts and resembled that of Plasmodium spp. We conclude that P. carinii belongs to the Protozoa, and is presumably Rhizopoda.     

Fibroblast heterogeneity in glucocorticoid regulation of collagen metabolism: Genetic or epigenetic?

Summary Cultured fibroblasts derived from normal human dermis show a consistent 62% inhibition of collagen synthesis by hydrocortisone, whereas cultures derived from keloids average only 30% inhibition and show a much larger strain to strain variation ranging from 75% inhibition to 49% stimulation. Examination of fibroblast clones indicates that this high variation among keloid strains is not due to differences in the proportion of normal and keloid cells in the mass culture populations. Small but significant differences in the effect of hydrocortisone on collagen deposition are also seen among these clonal populations, but are not related to the type of tissue from which cultures were derived. Two to three-fold differences among clones derived from a single individual were observed, possibly suggesting functional heterogeneity of dermal fibroblasts with regard to collagen metabolism under control conditions and in response to hydrocortisone. However, this variation among clones may simply reflect differences in clonal growth, inasmuch as both collagen synthesis and deposition, and the effect of hydrocortisone on these processes, are strongly affected by population density. This work was supported in part by PHS grants, CA-17229 from the National Cancer Institute and AG-02046 from the National Institute on Aging, DHHS; and by Grant RIM 78-17313 from the National Science Foundation.     

Relationship between excision repair and the cytotoxic and mutagenic effect of the ‘anti’ 7,8-diol-9,10-epoxide of benzo[a]pyrene in human cells

The cytotoxic and mutagenic effect of (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE) in normally excision diploid human cells treated just prior to onset of S was compared with that of cells allowed ～ 16 h for excision repair before onset of S and with that observed in excision-deficient serodema pigmentosum (SP12BE) cells. The cells were synchronized by release from density inhibition of cell replication. DNA synthesis began ～ 22 h after the cells were plated at lower density (i.e., 1.4 × 10⁴ cells/cm²). The frequency of thioguanine-resistant mutants induced in normal cells treated just prior to onset of S was ～ 12- to 16-fold higher than that observed in cells treated in early G₁ or treated in G₀ (confluence) and then plated at lower density. The frequency approximated that expected for XP12BE cells from extrapolation of data obtained at lower doses. The frequency of mutants measured in normal cells treated in exponential growth was also much higher than that in the cells treated in early G₁ or in G₀, No such difference could be seen in XP12BE cells treated in exponential growth or in G₀. In contrast to the mutagenicity data in the normal cells, there was no significant difference in the slope of the survival curve of normal cells treated at various times prior to S phase at low densities. However, normal cells treated even at the onset of S exhibited survival equal to XP12BE cells give a 4- to 5-fold lower dose. The data support the hypothesis that DNA synthesis is the cellular event which converts unexcised DNA lesions into mutations. However, they indicate that S is not the event primarily responsible for translating DNA damage into cell death. Accompanying studies on the rate of excision of anti BPDE adducts from the normal cells during the period priot to S support the conclusions.     

A new type of cell in the pulmonary epithelium of the newt Triturus alpestris Laur

Summary Single cells of a new type appear scattered among pneumocytes in the pulmonary epithelium. The surfaces of these cells communicate with the air space and display numerous finger-like microvilli. In comparison to pneumocytes, these cells have a more lucid cytoplasm and their apical parts contain large amounts of electron-lucent vesicles and electron-dense granules, which are probably released into the lumen of the lung. These secretory cells exhibit a yellow formaldehyde-induced fluorescence, which suggests that they belong to the class of APUD cells.     

Alterations in the responsiveness of diabetic fibroblasts to insulin

Fibroblastic cultures from the skin of nondiabetic and diabetic (db/db) mice have been used to investigate alterations in the biological responses of diabetic cells to insulin. Confluent cultures from the skin of both nondiabetic and diabetic animals possess specific receptors for insulin. Diabetic fibroblasts exhibit only 36% as much specific binding of insulin as nondiabetic fibroblasts, because of a decrease in the total number of binding sites, without a change in binding affinity. Insulin caused a time- and dose-dependent increase in the rate of 2-deoxy D-glucose (dGlc) uptake and in ornithine decarboxylase (ODC) activity of both nondiabetic and diabetic fibroblasts. In nondiabetic cells, half-maximal increase in dGlc uptake was obtained with 0.3 nM insulin, and a maximum increase of 120% was obtained with 4.1 nM insulin. In contrast, diabetic cultures required 0.8 nM insulin for a half-maximal increase in dGlc uptake, and maximum stimulation with 4.1 nM insulin was only 50% above control levels. With 4-fold higher insulin concentrations, ODC activity of diabetic cells was only 40% that of nondiabetic cells. In nondiabetic cells, down regulation of insulin receptors by insulin abolished the ability of insulin to stimulate dGlc uptake. These results demonstrate that cells cultured from diabetic animals, which possess a decreased number of insulin receptors, also exhibit decreased stimulation of deoxy D-glucose uptake and ornithin decarboxylase activity by insulin.     

Cystinotic and normal fibroblasts: Differential susceptibility to cysteine toxicity in vitro

Summary Extracellular cysteine concentrations between 0.5 and 2.5 mM resulted in death of normal but not cystinotic cells grown in Eagle's minimal essential medium containing supplemental fetal bovine serum and antibiotics. Differential cell survival was determined by viable cell counting using Trypan Blue dye exclusion. In cocultivation experiments of [³H]thymidine-labelled cystinotic fibroblasts with nonradioactive normal fibroblasts, autoradiography confirmed the selective survival of cystinotic cells in medium containing 1 mM cysteine. At this concentration of 1 mM cysteine, intracellular cystine content increased slightly in surviving normal cells but not in cystinotic cells, which normally contain a high level of intracellular cystine. This comparative resistance of cystinotic fibroblasts to elevated extracellular cysteine concentrations forms the basis for an in vitro selective system for these mutant human cells. Further exploration of this resistance phenomenon may well expand the understanding of the molecular defect in cystinotic cells.     

Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.Purine nucleoside phosphorylase has been purified at least a hundredfold from ³⁵S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of ³⁵S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. L. J. G. is supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Inborn errors of cobalamin metabolism: Effect of cobalamin supplementation in culture on methylmalonyl CoA mutase activity in normal and mutant human fibroblasts

We have examined the effect of addition of hydroxocobalamin to growth medium on the activity of the adenosylcobalamin-requiring enzyme methylmalonyl CoA mutase in normal human fibroblasts and in mutant human fibroblasts derived from patients with inherited methylmalonicacidemia. The mutant cell lines were assigned to four distinct genetic complementation groups (cbl A, cbl B, cbl C, and cbl D), each deficient in some step in the synthesis of adenosylcobalamin from hydroxocobalamin. After control cells were grown in cobalamin-supplemented medium, mutase holoenzyme activity increased markedly in a time- and concentration-dependent fashion. Growth in cobalamin-supplemented medium had no effect on mutase activity in some mutant lines belonging to the cbl B group, while activity increased severalfold in other cbl B mutants and in all cbl A, cbl C, and cbl D mutants examined, although mutase activity was still <10% of control. Comparison of mutase holoenzyme activity and total propionate pathway activity suggests that enhancement of mutase activity in mutant cells after cobalamin supplementation to values 5–10% of control may be sufficient to overcome the inherited metabolic block and to restore total pathway activity to normal.This work was supported in part by a research grant from the National Institutes of Health (AM 12579). H. F. W. is a recipient of a traineeship from the National Institutes of Health (T01-GM02299).