Expression of adrenocorticotropin-releasing hormone precursor gene in placenta and other nonhypothalamic tissues in man

Adrenocorticotropin-releasing hormone (CRH) is a peptide originally isolated from the hypothalamus. Immunocytochemical and RIA studies have revealed that CRH-like peptide is also localized in human nonhypothalamic tissues and some tumors. To see if CRH is synthesized in these nonhypothalamic tissues and tumors, we examined preproCRH mRNA in these tissues by Northern blot analysis using a cloned human preproCRH gene as a probe. PreproCRH mRNA was detected in human hypothalamus, cerebral cortex, adrenal gland, placenta, pheochromocytoma, and thymic carcinoid. The content of preproCRH mRNA in placenta was apparently greater than that in the whole hypothalamus.     

Human joining peptide: a proopiomelanocortin product secreted as a homodimer

The human (h) POMC gene sequence predicts a 30 amino acid joining peptide (JP) separating the N-terminal fragment [POMC(1-76) or hNT] and ACTH within their common precursor. We used an anti-serum directed against the amidated COOH-terminal end of mouse JP to develop a RIA for the predicted hJP molecule. Immunoreactive JP was detected in tissue extracts from human normal pituitary, ACTH-secreting pituitary- and nonpituitary tumors, and in plasma from patients with ACTH hypersecretory syndromes. Its molar concentration was of the same order of magnitude as, and correlated with, that of the other POMC peptides. Gel exclusion chromatography in 1% formic acid and 6 M guanidine-HCl revealed a predominant immunoreactive material with an apparent mol wt of ca. 6000. After reduction with dithiothreitol this material was recovered in an elution volume identical to that of purified hJP and corresponding to a mol wt of ca. 3000. These data show that POMC processing generates a COOH terminally amidated hJP predominantly secreted as a homodimer, probably through disulfide bonding between the single Cys9 residue of two molecules.     

Induction of LAK cells by high density dialyzing culture device and its cytotoxicity

Lymphokine activated killer (LAK) cells are generated by culture of lymphocytes with interleukin 2 (IL-2) in short term culture (3 to 5 days) and are used for adoptive immunotherapy for advanced cancer patients. The culture condition hitherto reported are essentially based on the rotating culture system, in which the maximum cell density was at 2 X 10(6) cell/ml and the cell recovery was usually less than 100%. The inability to induce LAK cells efficiently in vitro made the culturing of cells for therapy rather difficult and costly work because the mean infusion dose of LAK cells of one patient requires more than 1 X 10(10)/ml. We have therefore attempted to culture lymphocytes in 10 times higher concentration comparing with conventional methods. By using a new dialyzing culture system under continuous regulation of the amount of infused IL-2, nutrition medium, and pO2 and pCO2, we could culture cells at 2 X 10(7)/ml for more than 21 days and the resulted LAK cells showed a 100 times increase of activity on a per cell basis. By limiting dilution procedure, these killer cells mostly express T cell markers such as CD3 and CD8 but dose not express CD16.     

Human leukemia cell lines--clinical and theoretical significances

Establishment and total characterization of permanent growth-factor independent human leukemia cell lines are reviewed. Among others, a few significant contributions in the utility of leukemia cell lines described include establishment of immunodiagnosis of human leukemias and lymphomas, discovery of EB virus and of human retroviruses, proposed model of normal hematopoietic cell differentiation scheme, lymphokine-monokine production, identification and characterization, studies on genes responsible for immunobiological and growth regulatory molecules, and pharmacotoxicological and kinetic research. It is conceivable that the future leads by the utility of human leukemia cell lines to the advancement of research are indeed unlimited.     

Effects of antipyretics and anticancer drug on human cells at high temperature condition

The effects of antifebriles and anticancer drug on human vascular endothelial cells (HVE) and several cultured human cells were investigated. The HVE were isolated from umbilical cord veins by enzyme treatment and cultured successively in aerated synthetic medium, RPMI-1640, with 20% preclostrum new born calf serum. The presence of factor VIII antigen in the HVE was determined by enzyme-labeled antibody method. Cell count and protein amount were examined at regular intervals. At 3 hour-expose, sulpyrine was more toxic to the cultured cells than aspirin at 37 degrees C. The cytotoxicity of sulpyrine was markedly enhanced at 40 degrees C than at 37 degrees C. However, there was no enhancement in the cytotoxicity of aspirin at 40 degrees C. Cultured HVE and normal human fetal lung (HAIN-55) cells at 37 degrees C were sensitive to sulpyrine, and their sensitivity of the cells to the drug were markedly enhanced when they were incubated at 41 degrees C. In contrast, sensitivity of malignant human cells (HeLa cells) to sulpyrine was not found at 37 degrees C, however sensitivity of the cells to the drug was manifested at 41 degrees C of incubation. There was no effect of 5-fluorouracil (FU) on the growth of HVE and HAIN-55 cells at 41 degrees C, while HeLa cells showed high susceptibility to FU at the same temperature. The results showed the possibility that normal human cells may be sensitive to antifebrile drugs but not to anticancer drug at ordinary and high temperature, whereas malignant human cell may be susceptible to both antifebrile drugs and anticancer drug at high temperature.     

Disposition of 125I-labeled recombinant human tumor necrosis factor in the tumor-bearing mouse

Disposition of [125I]rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation. After i.v. administration, [125I]rHu-TNF measured by radioactivity and immunoreactivity biphasically decreased in plasma. Tumor level of [125I]rHu-TNF was the maximum at 1 h, then decreased and finally remained essentially constant. After i.t. administration, plasma level reached the maximum at 1 h. Tumor level decreased quickly and then became essentially constant. [125I]rHu-TNF was suggested to be degraded to small fragments in the tumor. Significant distribution of [125I]rHu-TNF was found in the kidney, lung, liver and tumor. Most tissue levels decreased with time in parallel with plasma levels. [125I]rHu-TNF radioactivity was found in proximal convoluted tubules of kidney and in those areas of tumor consisting of degenerating cells with pyknotic nuclei. Urine contained most of administered radioactivity, which being neither immunoreactive nor protein-bound.     

Accumulation of collagen III at the cleft points of developing mouse submandibular epithelium

The distribution of collagens I, III, IV and V was studied by immunoperoxidase staining of early developing mouse submandibular glands. Collagen I was always present in the extracellular matrices of the mesenchyme and at the epithelial-mesenchymal interfaces of the 12-day gland with no clefts and of the 13-day gland with a few definite clefts. Collagen III was found in a similar fashion to that of collagen I in the mesenchyme, but the distribution at the epithelial-mesenchymal interfaces was very different. In the mid 12-day gland with a round lobule, collagen III was distributed at every slightly indented site of basal epithelial surfaces. At the late 12-day stage, a few initial signs of cleft appeared on the surface, at which accumulation of collagen III became evident. Intense immunoreaction of collagen III in the early 13-day gland was seen at the bottom of every narrow cleft. No specific accumulation of collagens IV and V was observed in clefts of the late 12-day and early 13-day glands. Staining of collagen III in the 12-day gland cultured for 10 h in the presence of bovine dental pulp collagenase inhibitor, which has been shown to stimulate cleft initiation, was very prominent at the bottom of every narrow cleft. These observations suggest that collagen III works as a key substance for either in vitro or in vivo cleft initiation of the mouse embryonic submandibular epithelium.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. II. Effect of the steel locus on spermatogenesis

Mutant mice of Sl/Sld genotype are deficient in melanocytes, erythrocytes, mast cells and germ cells. Deficiency of melanocytes, erythrocytes and mast cells is not attributable to an intrinsic defect in their precursor cells but to a defect in the tissue environment that is necessary for migration, proliferation and/or differentiation. We investigated the mechanism of germ cell deficiency in male Sl/Sld mice by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent white stripes were obtained. Two of four such chimaeras were fertile and the phenotypes of resulting progenies showed that some Sl/Sld germ cells had differentiated into functioning sperms in the testis of the chimaeras. In cross sections of the testes of chimaeras, both differentiated and nondifferentiated tubules were observed. However, the proportions of type A spermatogonia to Sertoli cells in both types of tubules were comparable to the values observed in differentiated tubules of normal +/+ mice. We reconstructed the whole length of four tubules from serial sections. Differentiated and nondifferentiated segments alternated in a single tubule. The shortest differentiated segment contained about 180 Sertoli cells and the shortest nondifferentiated segment about 150 Sertoli cells. These results suggest that Sertoli cells of either Sl/Sld or +/+ genotype make discrete patches and that differentiation of type A spermatogonia does not occur in patches of Sl/Sld Sertoli cells.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. I. Different distribution patterns between melanocytes and mast cells in the skin

In spite of their different origin, both melanocytes and mast cells are deficient in the skin of mutant mice of the Sl/Sld genotype. Since the neural crest and the liver of Sl/Sld embryos contain normal precursors of melanocytes and mast cells, respectively, the deficiency is attributed to a defect in tissue environment necessary for migration and/or differentiation of precursor cells. We investigated whether the tissue environment used for differentiation of melanocytes and mast cells was identical by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent pigmented and nonpigmented stripes were obtained. In the nonpigmented stripes of these Sl/Sld in equilibrium with +/+ chimaeras, melanocytes were not detectable in hair follicles but were detectable in the dermis. In contrast, melanocytes were detectable neither in hair follicles nor in the dermis of nonchimaeric Sl/Sld mice. Concentrations of mast cells were comparable in the pigmented and nonpigmented stripes of Sl/Sld in equilibrium with +/+ chimaeras, but the average concentration of mast cells significantly varied in the chimaeras (from 8% to 74% of the value observed in control +/+ mice). The present result suggests that mesodermal cells that support the migration and differentiation of both melanocyte precursors and mast-cell precursors mix homogeneously in the dermis and that ectodermal cells that influence the invasion of differentiating melanocytes into hair follicles make discrete patches.