High levels of deoxyribonuclease I (DNase I) were detected in the cytoplasm of hormone-secreting cells in anterior and intermediate lobes of human and rat pituitary glands. Tissue homogenate fractions of anterior and intermediate pituitary lobes showed strong DNase I-speciflc enzymatic activity. Immunofluorescence, with several specific antibodies against human and rat DNase I, indicated strong immunoreactivity in the cytoplasm of hormone-secreting cells within the gland. In situ hybridization demonstrated high levels of DNase I mRNA in the cells. By immunoelectron microscopy, immunogold particles were found in the rough endoplasmic reticulum, Golgi apparatus and secretory granules. Exocytotic secretory granules were also immunopositive. These results indicate that endocrine cells in the pituitary gland synthesize and secrete DNase I together with their respective pituitary hormones.

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During myogenic differentiation of quail myoblasts transformed with a temperature sensitive mutant of Rous sarcoma virus (QM-RSV cells), it was observed that myogenin was continuously expressed in myotubes. In contrast, in myotubes derived from quail primary cells (parent cells of QM-RSV cells), myogenin expression was seen only in the myotubes not having striated structures comprised of myoflbrils, but not in myotubes having the structures. The fact that there are not striated structures of myofibrils formed in myotubes derived from QM-RSV cells suggests that these myotubes stop at an immature state prior to the final differentiation. These results also suggest that myogenin is not only required for early steps during differentiation but also maturation steps of myotubes. To clarify the roles of myogenin after myotube formation and maturation, myotubes derived from QM-RSV cells were treated with N, N'-hexamethylene bisacetamide (HMBA) or incubated at 35.5°C, a permissive temperature of RSV for suppression of myogenin expression. On treatment with HMBA, myogenin expression disappeared and myotubes began to incorporate 5-bromo-2'-deoxyuridine (BrdU) into the nuclei, whereas the expression remained in many myotubes on culture at 35.5°C. These results suggest that immature myotubes can return to an up step of differentiation, prior to the commitment step with HMBA treatment, but not with culture at 35.5°C.

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In a previous paper, we demonstrated that cultured human fibroblasts synthesize transferrin (Tf). Two types of Tf are present; one is produced by the cells and the other is internalized from the culture medium. To study the metabolism of intracellular Tf, we investigated the subcellular localization of the two types of Tf in human fibroblasts by immunocytochemical and fluorescence-labeling techniques. The internalized Tf was found to be localized in the perinuclear area, and the synthesized Tf was associated with microtubules, forming a fibrous structure in the cytoplasm. When the cells were treated with colchicine which depolymerizes microtubules irreversibly, the synthesized Tf lost its fibrous structure and spread out in cytoplasm, but the internalized Tf remained around the nucleus. These results suggest that the two types of Tf are regulated differently in the cells.

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The distribution of basal actin bundles and cytokeratin intermediate filaments in the rabbit kidney was examined along isolated renal tubules by confocal laser microscopy. Perfusion of collagenase and subsequent manipulation of kidney tissues yielded well preserved renal tubules free of connective tissue components often extending from glomeruli to collecting tubules. Such isolated renal tubules were fluorescently stained with phalloidin and with anti-cytokeratin antibody and then observed as whole-mount preparations by confocal laser microscopy. Epithelial cells of Bowman's capsules showed conspicuous actin bundles which were arranged parallel to each other in individual cells. In proximal ducts, thick actin bundles were seen running in the basal regions of epithelial cells perpendicular to the tubule axis. Basal actin bundles in proximal tubules appeared to be somewhat thinner and run more irregularly in more distal parts of the tubule. Thin limbs of Henle's loop showed bright but very short rod or dot-like stainings of actin, while in thick limbs, there were thick and short actin bundles. In distal tubules basal actin bundles wre somewhat longer than those in thick limbs of Henle's loop. In the collecting ducts in the renal cortex, a few short actin bundles as well as dotty or patchy stainings were shown in the basal region. In both whole-mount preparations and cryostat sections, cytokeratin immunoreactivities were almost exclusively detected in epithelial cells of Henle's loops and collecting ducts but poorly in proximal and distal tubules. Epithelial cells of Henle's loops showed dense networks of cytokeratin immunoreactivities throughout the cytoplasm, whereas cell boundaries were immunostained in collecting ducts. The actin and cytokerating distribution in such isolated renal tubules was confirmed by immunofluorescence of cryostat sections and by thin-section electron microscopy. The observations that actin and intermediate filaments varied in distribution in different segments of renal tubules are interesting in view of the segmental differentiation of renal tubule functions.

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Metabolism in cultured cells strongly depends on cell density, thus cell density may be an important factor in biotechnological maintenance of cell cultures. Therefore the energy metabolism of three related cell types with different proliferation activity has been characterised: density controlled primary or secondary flbroblasts [pmf], immortalized, but still density controlled 3T3 cells, and SV40-transformed 3T3 lacking growth control. The investigations revealed the decrease of oxygen consumption, net lactate production, ATP-content, NAD-content and NAD-redox potential, and F-actin content with increasing culture density in pmfs, less distinct in 3T3 cells and in SV40-3T3 cells. The main decrease of these factors is related to cell-cell contacts rather than to proliferation which ceases at least two division cycles after most cells contacted a neighbouring cell. SV40-3T3 cells also at preconfluent densities exhibit relatively low metabolic activity as revealed by the above mentioned factors. Supply with metabolites for catalytic processes seems to be the rate limiting factor as deduced from a decreasing NADH/total NAD ratio.
SV40-3T3 cells lack a contact mediated reduction of energy metabolism which is in accord with the missing contact inhibition of motility and proliferation in SV40-3T3 cells. Because of a possible association of glycolytic enzymes with actin, F-actin content has been determined. No correlation, however, was revealed by the F-actin/lactate ratio. This may be due to the fact that about 50% of the lactate released into the culture medium originated to from glutaminolysis rather than glycolysis. Only in SV40-3T3 cells were respiration and lactate production insensitive to glutamine deprivation and in these cells both these parameters did not change significantly, therefore they did not allow testing as to whether f-actin content and glycolytic activity are correlated.
Fractionated release of adeninnucleotides revealed that energy charge in the cytosolic fraction is the only factor which strictly correlates with the cessation of proliferation. Thus energy charge of the cytosol seems to be the only factor of these studied here, which can be used as an indicator of a culture's commitment for further proliferation. To compare energy metabolic parameters among different cell types one always has to take into account the density dependence of these factors.

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Metal ions such as Ca²⁺, Mg²⁺, or Zn²⁺, are important for many cell functions, for example, signal transduction and the modulation of enzyme activity. The relationship between apoptosis and metal cations, especially Ca²⁺, has been described in many reports. We have investigated the role of metal cations in the regulation of apoptosis in the mouse neuroblastoma cell line, Neuro-2A. When Neuro-2A cells were treated with ethylene diaminetetraacetic acid (EDTA), apoptosis was detected as growth inhibition, DNA fragmentation with a ladder pattern in agarose gel electrophoresis, and nuclear decomposition. However, in case of the treatment with ethylene glycol bis- (β-aminoethyl ether) N, N, N', N'-tetraacetic acid (EGTA), which has a higher chelating specificity for Ca²⁺ than EDTA, DNA fragmentation was not detected. Moreover, the apoptosis induced by EDTA was inhibited by exogenous Zn²⁺. The membrane permeable Zn²⁺ chelator N, N, N', N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) also induced apoptosis of the Neuro-2A cells, and addition of equimolar exogenous Zn²⁺ or Cu²⁺, but not Mn²⁺ or Fe²⁺, prevented TPEN-induced apoptosis. The results suggest that Zn²⁺ may be a key regulator of apoptosis in Neuro-2A cells.

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Brains from 7 p53-deficient mice on fetal day 13 were divided into 4 regions: cerebral cortex, cerebellum, upper spinal cord, and the rest of brain. They were trypsinized and cultured in medium containing 10% fetal calf serum. By dilution culture, 138 clonal lines were established from every regions. The lines were characterized morphologically and immunocytochemically as neuronal, glial, myogenic, or unidentified. They were cultured for more than a year, indicating that the lines are immortalized. p53-deficiency alone is sufficient for establishing clonal cell lines of the central nervous system.

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