Identification of hemolytic granules isolated from human myocardial cells

Human myocardial cells from fresh autopsy material contained granules which possessed hemolytic activity against guinea pig and rabbit erythrocytes. The hemolytic granules, which had a density of 1.02 and a diameter of 200-300 nm, were recovered as a microsome fraction from subcellular homogenates of human myocardial cells by differential centrifugation in 300 mM sucrose containing 0.1 mM PMSF and 10 mM EDTA. The membrane lesions caused by the granules were ring-like structures with an internal diameter of about 10-17 nm, analogous to that caused by perforin- and complement-induced lysis. However, the requirement for divalent cation differed from that for perforin-induced lysis, since the microsome-mediated lysis occurred in the presence of EDTA.     

Relationship between lectin-affinity granules in anuran embryos and formation of primordial germ cells

Summary Embryos of the anuran,Rana nigromaculata, contained granules with a specific affinity for Con A and GS-I. Larvae derived from embryos injected with these lectins had a noticeable reduction in both the number and size of primordial germ cells (PGCs). This observation suggests that the granules are somehow involved in the formation of PGCs.     

Anti-lipopolysaccharide activity of histatins,peptides from human saliva

Histatins are histidine-rich polypeptides secreted in human saliva. They were found to inhibit lipopolysaccharide (LPS)-mediated gelation of Limulus amoebocyte lysate, and to reverse the anti-complement action of LPS or lipid A. Histatins also gave precipitate bands in agarose gels with various LPS. The results indicate that histatins neutralized the activity of LPS by binding to the lipid A moiety of LPS.     

Identification of cancer stem cell-like cells from human epithelial ovarian carcinoma cell line

Cancer stem cells (CSCs) play an important role in the development, invasion, and drug resistance of carcinoma, but the exact phenotype and characteristics of ovarian CSCs are still disputable. In this study, we identified cancer stem cell-like cells (CSC-LCs) and investigated their characteristics from the ovarian adenocarcinoma cell line 3AO. Our results showed that CSC-LCs were enriched in sphere-forming test and highly expressed CD44⁺CD24⁻. The spheres and CD24⁻ cells possessed strong tumorigenic ability by transplantation into nonobese diabetic/severe combined immunodeficient mice. CD44⁺CD24⁻ cells expressed stem cell markers and differentiated to CD44⁺CD24⁺ cells by immunofluorescence assay and fluorescence-activated cell-sorting analysis. In vitro experiments verified that CD44⁺CD24⁻ cells were markedly resistant to carboplatin and paclitaxol. In conclusion, our study identifies the CD44⁺CD24⁻ phenotype, self-renewal, high tumorigenicity, differentiation potential, and drug resistance of ovarian CSC-LCs. Our findings may provide the evidence needed to explore a new strategy in the treatment of ovarian cancer.     

Analysis of OCT4 expression in an extended panel of human tumor cell lines from multiple entities and in human mesenchymal stem cells

OCT4 is considered a main regulator of embryonic stem cell pluripotency and self renewal capacity. It was shown that relevant OCT4 expression only occurs in cells of embryonic pluripotent nature. However, several recent publications claimed to have demonstrated OCT4 expression in human somatic tumor cells, human adult stem or progenitor cells and differentiated cells.We analysed 42 human tumor cell lines from 13 entities and human bone marrowderived mesenchymal stem cells (MSC). To validate OCT4 expression we used germ cell tumor (GCT) cell lines, derived xenografts and GCT samples. Analysis by RT-PCR, western blotting, immunocytochemistry and immunohistochemistry was performed. With exception of typical embryonal carcinoma cells, we did not observe reliable OCT4 expression in somatic tumor cell lines and MSC. We suggest that a high level of expression of the OCT4 protein together with its nuclear localization still remains a reliable and definitive feature of cells with embryonic pluripotent nature. Received 30 September 2008; received after revision 05 November 2008; accepted 10 November 2008     

Gap junctional intercellular communication of cultured rat liver parenchymal cells is stabilized by epithelial cells and their isolated plasma membranes

The gap junctional intercellular communication (GJIC) determined by measuring dye coupling with Lucifer yellow, decreased within 3 d from 66% to 28% in monocultures of rat liver parenchymal cells. Coculturing of the parenchymal cells with a nonparenchymal epithelial cell line from rat liver resulted in increased and stabilized intercellular communication (83% after 3 d). The presence of isolated plasma membrane vesicles of the nonparenchymal epithelial cells also stabilized the intercellular communication between the liver parenchymal cells (70% after 3 d). When liver parenchymal cells were cocultured with a rat liver fibroblast cell line the gap junctional communication between the parenchymal cells was not stabilized (43% after 3 d), and isolated plasma membrane vesicles of the fibroblast were also unable to support the GJIC in parenchymal cells (35% after 3 d). It is concluded that plasma membrane constituents of the nonparenchymal epithelial cells were responsible for the stabilization of the GJIC between parenchymal cells. A heterotypic gap junctional communication between parenchymal and nonparenchymal cells was not observed.     

Biophysical properties of porin pores from mitochondrial outer membrane of eukaryotic cells

Summary The matrix space of mitochondria is surrounded by two membranes. The mitochondrial inner membrane contains the respiration chain and a large number of highly specific carriers for the mostly anionic substrates of mitochondrial metabolism. In contrast to this the permeability properties of the mitochondrial outer membrane are by far less specific. It acts as a molecular sieve for hydrophilic molecules with a defined exclusion limit around 3000 Da. Responsible for the extremely high permeability of the mitochondrial outer membrane is the presence of a pore-forming protein termed mitochondrial porin. Mitochondrial porins have been isolated from a variety of eukaryotic cells. They are basic proteins with molecular masses between 30 and 35 kDa. Reconstitution experiments define their function as pore-forming components with a single-channel conductance of about 0.40 nS (nano Siemens) in 0.1 M KCl at low voltages. In the open state mitochondrial porin behaves as a general diffusion pore with an effective diameter of 1.7 nm. Eukaryotic porins are slightly anion-selective in the open state but become cation-selective after voltage-dependent closure.     

The potency of heparin-like activity of glycosaminoglycans released by human endothelial cells in culture

Summary Glycosaminoglycans isolated from culture medium conditioned by human endothelial cells showed heparin-like antithrombin III cofactor activity measured by Xa inhibition. Their activity was relatively weak, 0.1% of the potency of heparin, but was approximately 5-fold more potent than that of glycosaminoglycans derived from vascular smooth muscle cells.     

Effect of human serum on alkaline phosphatase induction in cultured human tumor cells

Summary The continuous cell lines T 24 and HT-29, derived from human bladder and colon carcinomas, produce term-placental and intestinal alkaline phosphatase, respectively. Growth in hyperosmolar medium or exposure to prednisolone or sodium butyrate induces increased enzyme levels, and combinations of inducers elicit synergistic activity increases. The effect of the inducing agents is strikingly diminished when cells are grown in the presence, of high concentrations of human serum, and the synergistic increases are essentially abolished. Major human serum protein fractions do not affect alkaline phosphatase induction.     

Induction of lymphokine-activated killer cells from nude mouse spleen cells by interleukin-4

The induction of lymphokine-activated killer (LAK) cells from natural killer (NK) lineage cells by interleukin-4 (IL-4) was studied in vitro. Activation of nude mouse spleen cells by IL-4 generated cytotoxic cells, capable of killing NK-sensitive as well as NK-resistant tumor cells. The induction of peak lytic activity was demonstrated after 3 days of culture with IL-4. Surface marker analysis indicated that the majority of precursor cells were aGM1⁺, Thy1^–, and the majority of effector cells were aGM1⁺, Thy1⁺, suggesting that IL-4 induced LAK cells from nude mouse spleen cells were similar to those from normal mouse spleen cells. The induction of nude mouse LAK cells by IL-4 was partially inhibited by anti-IL-4 or anti-interferon (IFN)-, antibody, and it was further inhibited by the combination of two antibodies, suggesting that IFN-, production was associated with LAK induction of NK lineage cells by IL-4.     

Immunocytochemical demonstration of contractile cells in the human ovarian follicle

Summary Actin-and myosin-like immunoreactivity is found in cells located in the theca externa of the follicle wall of the human ovary, and corresponding to previously observed myoid cells. The immunocytochemical observation provides direct structural evidence that non-vascular contractile cells are also present in the follicle wall in humans. As expected, perifollicular blood vessels showed a positive immunoreaction for actin and myosin in their smooth muscle walls.This work was supported by the Swedish Medical Research Council, grant No. 14X-732/5680.     

Retroviruses released from a human tumor xenograft in nude mice induce colony-stimulating factor (CSF) activity in human fibroblastic cells

Summary A human colony-stimulating factor (CSF)-producing tumor transplanted into athymic nude mice released retroviruses in vitro. The viruses induced CSF activity in human fibroblastic cell lines.     

Changes in brush-border enzyme activities of intestinal epithelial cells isolated from the villus-crypt axis during the early phase of alloxan diabetes in rats

Summary The sucrase activity in enterocytes isolated from the villus crypt axis was found to increase in all regions of the villus from day 2 after induction of diabetes, and the increase continued until day 4. In contrast, alkaline phosphatase activity increased mainly in the apical one-third of the villus-crypt column, and the increase occurred abruptly on day 4 with increase in food intake.     

Evidence of a direct action of triiodothyronine (T3) on the cell membrane of GH3 cells: an electrophysiological approach

Summary Electrophysiological experiments demonstrate that triiodothyronine (T₃) exerts a direct effect on the membrane of a strain of cultured rat pituitary tumor cells, GH₃/B₆. These cells respond to pressure application of T₃ (2–5 nl, concentration 1·10^–10 M) with an increase in the membrane resistance (R_m) and a hyperpolarization. Spontaneously firing cells become silent.     

Different biological behavior of AKR lymphoma cells from primary and metastatic tumors

Summary AKR lymphoma cells derived from primary s.c. tumors (PT) and cells from their metastases (MT) were inoculated into recipient mice in order to compare their malignant behavior. A higher malignant potential of MT compared to PT cells was found. The results support the hypothesis that metastasis is a process of selection of cells possessing a potential to metastasize, which preexist in the primary tumor. In the model used, both the selection of variants of malignancy and the assay of malignancy were as close as possible to natural tumor progression.The authors are thankful to Mrs Ora Gal-Mor and Mrs Elinora Miron for skillful technical assistance.     

Shortening velocity of single muscle cells isolated from a molluscan smooth muscle

Summary The maximal unloaded shortening velocity (V_max) of smooth muscle cells isolated from the pedal retractor muscle ofMytilus was more than twice as large as that of the whole muscle, suggesting the presence of extracellular components which resist the contraction of the whole muscle. The V_max of the isolated cells was almost constant at cell lengths ranging between 0.5 and 0.831₀ (1₀, optimal length for tension generation) indicating that the intracellular resistance to contraction is negligible within this range of lengths.     

Effect of temperature on in vitro proliferative activity of human umbilical vein endothelial cells

Human umbilical vein endothelial cells, skin fibroblasts, and retinal pigment epithelial cells are cultivated in medium supplemented with 15 to 20% serum in our laboratory. The effects of various incubation temperatures on the proliferation of these cells was examined. Our study shows that the mitogenic response of the endothelial cells to a change of temperature differed markedly from that of the fibroblasts and epithelial cells. Cultivation of human umbilical vein endothelial at 37°C required seeding densities as high as 1–2×10⁴ cells/cm², and yet resulted in a low growth rate and premature senescence. However, under the same culture conditions, but at 33°C, the proliferative capacity of these endothelial cells was potentiated. The results were striking; at 33°C the cells grew actively and the life span was extended. The number of cumulative population doublings increased fourfold compared with that for the same cells cultivated at 37°C. The inoculum size could be reduced, since at 33°C the endothelial cells were able to replicate at seeding densities as low as 20 cells/cm². The cells serially subcultured at 33°C retained morphological features and specific immunological markers of endothelial cells.     

Morphogenesis of human colon cancer cells with fetal rat mesenchymes in organ culture

Summary The morphogenesis and cytodifferentiation of human colon cancer cells (LS174T and HT29) were examined by combining cancer cells with fetal rat digestive-tract mesenchyme in organ culture. LS174T cells migrated into the mesenchyme to form glandular structures composed of single columnar cells with their nuclei oriented basally, while HT29 cells formed cell masses with little lumen formation. Immunohistochemical studies with antibodies against carcinoembryonic antigen and secretory components showed that the composition of cell surface glycoproteins was not necessarily reversed to the normal type, even when neoplastic cells exhibited normal glandular structures.This work was supported by grants-in-aid for Cancer Research from the Ministry of Education, Science, and Culture, Japan, and by the Veterans Administration Medical Research Service, USA. Y.S. Kim is the recipient of a Medical Investigator Award of the Veterans Administration.     

Smallest zinc quantities affect the histamine release from peritoneal mast cells of the rat

Summary Seven individual 0.025-mg doses of zinc administered as lactose tablets on consecutive days, significantly increase histamine release from peritoneal mast cells of the rat. Seven individual doses of 0.25 g cause a somewhat smaller, though still very pronounced increase in the release in comparison with zero control.Acknowledgments. We thank Ms T. L. Pham and Ms K. Löppen for their technical assistance.     

Spontaneous rhythmic contractions of human saphenous veins isolated from old subjects are sensitive to cyclooxygenase inhibitors

Summary Spontaneous rhythmic contractions were observed in some preparations of human isolated saphenous veins from old (>60 years) subjects. These contractions were insensitive to adrenergic and histaminergic blockers, but were abolished by the cyclooxygenase inhbitors, aspirin and indomethacin, indicating the participation of endogenous eicosanoids.