Stathmokinetic Analysis of Human Epidermal Cells in vitro

Proliferation kinetics of cultured human epidermal cells is characterized in quantitative terms. Three distinct subpopulations of keratinocytes, two of which are cycling have been discriminated by two parameter DNA/RNA flow cytometry. Based on mathematical modelling, the cell cycle parameters of the cycling subpopulations have been assessed from stathmokinetic data collected at different time points after initiation of cultures (7–15 days). the first subpopulation is composed of low-RNA cells which resemble basal keratinocytes of epidermis and which show some characteristics of stem cells; these cells have a mean generation time of approximately 100 hr. the second subpopulation consists of high-RNA cells, resembling stratum spinosum cells of epidermis, which have an average generation time of approximately 40 hr. the third subpopulation consists of non-cycling cells with G_o/G₁ DNA content, with cytochemical features similar to those of cells in granular layer of epidermis. The results based on modelling can reproduce with acceptable accuracy the actual growth curve of the cultured cell population. Analysis of kinetics and differentiation of human keratinocytes is of interest in view of the recent application of cultured epidermal cell sheets for transplantation onto burn wounds. the results of this study also reveal the existence of regulatory mechanisms associated with proliferation and differentiation in the cultured epidermal cell population.     

Crystallographic studies of the chicken gizzard G-actin X DNase I complex at 5A resolution

The structure of the chicken gizzard G-actin X DNase I complex has been determined at 5 A resolution by an X-ray diffraction method. Protein phases were computed by the multiple isomorphous replacement method using four heavy atom derivatives. The mean figure of merit was 0.65. Dimensions of the three molecular species, the complex, G-actin and DNase I, were determined based on the "cypress wood" models derived from the electron density map. The natures of the heavy atom binding sites are discussed in relation to the distinction between the two component molecules. The pattern of successive contacts between actin molecules observed in the present crystal seems unrelated to that found in F-actin.     

Measurement of receptor-independent metabolism of low-density lipoprotein. An application of glycosylated low-density lipoprotein

Incubation of human low-density lipoprotein (LDL) with glucose results in a nonenzymatic formation of a Schiff base between the monosaccharide and lysyl residues of apolipoprotein B. Increasing the percentage of lysyl residues of apolipoprotein B modified by glycosylation decreases the fractional catabolic rate of the glycosylated LDL, and decreases the metabolism of the glycosylated LDL by human skin fibroblasts. The glycosylated LDL, containing 20-40% of total lysyl residues of apoprotein B modified, was metabolized at a slow rate by both human skin fibroblasts and mouse peritoneal macrophages. These results led to the suggestion that glycosylated LDL is primarily catabolized via a receptor-independent process. Assuming LDL catabolism occurs via receptor-dependent and receptor-independent processes, the ratio of (fractional catabolic rate of glycosylated LDL)/(fractional catabolic rate of native LDL) should be an estimate of the percentage of LDL catabolism via the receptor-independent process. From the fractional catabolic rates of glucose-LDL (20-40% of lysyl residues modified) and galactose-LDL (30-60% of lysyl residues modified) 41% and 30% respectively, of LDL catabolism occurred by a receptor-independent process.     

Phytohemagglutinin induces rapid degradation of phosphatidylinositol 4,5-bisphosphate and transient accumulation of phosphatidic acid and diacylglycerol in a human T lymphoblastoid cell line, CCRF-CEM

The human T lymphoblastoid cell line designated CCRF-CEM responds to phytohemagglutinin with a 3.7-fold enhancement of the 32PO4 incorporation into phosphatidylinositol. In myo-[2-3H]inositol-prelabeled CCRF-CEM cells, phytohemagglutinin induced a 3.3-fold accumulation of myo-[2-3H]inositol phosphate during 15 min incubation at 37 degrees C in the presence of 5 mM LiCl. Since Li+ is a potent inhibitor of myo-inositol-1-phosphatase, the results indicate that phytohemagglutinin induces the hydrolysis of inositol lipids in CCRF-CEM cells. In 32PO4-prelabeled CCRF-CEM cells, phytohemagglutinin induced a breakdown of 28% of [32P]phosphatidylinositol 4,5-bisphosphate 40-60 s after the stimulation. The decrease of [32P]phosphatidylinositol 4,5-bisphosphate was found as early as 10 s after the stimulation. This decrease was followed by an increased 32P-labeling of phosphatidic acid. In [2-3H]glycerol-prelabeled CCRF-CEM cells, phytohemagglutinin induced a transient accumulation of [3H]phosphatidic acid and [3H]diacylglycerol. The amount of [3H]phosphatidic acid in the stimulated cells was 3.7-times the control value at 2 min after the stimulation, whereas the amount of [3H]diacylglycerol in the stimulated cells was 1.5-times the control value at 5 min after the stimulation. In [3H8]arachidonate-prelabeled CCRF-CEM cells, phytohemagglutinin induced a transient accumulation of [3H]phosphatidic acid; the amount was 2.5-times the control value at 2 min after the stimulation. Quinacrine (1 mM) caused 41% reduction in the amount of [3H]phosphatidic acid accumulated by the stimulation in [2-3H]glycerol-prelabeled cells. Stimulation in a Ca2+-free saline containing 1 mM EGTA caused 53% reduction in the amount of [3H]phosphatidic acid accumulated by the stimulation. The results presented in this paper indicate that a human T lymphoblastoid cell line, CCRF-CEM, responds to phytohemagglutinin with a rapid turnover of inositol lipids.     

Interferon-alpha in serum and carcinomatous pleural effusion after repeated intrapleural injections of antitumor agents

Pleural effusions and sera of two patients with lung cancer were tested after intrapleural injection of OK-432 as an anticancer drug for IFN-alpha activity by biological assay and for IFN-alpha as an antigen by radioimmunoassay. The titers by radioimmunoassay were fairly consistent with those by biological assay, but were usually higher. In Case 1, IFN-alpha was observed fairly early after administration of OK-432 and only in pleural effusions. In Case 2, induction of IFN-alpha at low level was observed late after the first administration of OK-432 both in the pleural effusion and serum and was detected only by radioimmunoassay.     

Reversal of X-inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro

A series of near-diploid embryonal carcinoma-like hybrid cells were obtained from polyethylene glycol mediated cell fusion between murine embryonal carcinoma cells (PSA-6TG1 or OTF9-63) having one X chromosome and thymocytes or bone marrow cells from female mice carrying Cattanach's or Searle's translocation. Prior to fusion with EC cells the somatic cells are presumed to contain only one active X chromosome. Following hybrid formation, the chronology of X chromosome replication and the expression of X-linked gene Pgk-1 indicated that all X chromosomes contributed by both parents were active in these hybrids. Experiments were performed to rule out the possibility that the hybrids were formed by fusion of EC cells with rare somatic cells in which both X chromosomes were active. Taken together the data indicate that within four days of fusion there is reactivation of the entire inactive X chromosome.     

CAM-idling in Hoya carnosa (Asclepiadaceae)

In the leaf succulent Asclepiad Hoya carnosa (L.) R. Br., CAM photosynthesis occurred under well-watered conditions, as characterized by diurnal gas exchange and changes in titratable acidity. Following 10–12 days of severe water stress, the plants shifted from CAM to a modified CAM-idling mode of metabolism. CAM-idling was characterized by complete or almost complete stomatal closure accompanied by CAM-like diurnal changes in titratable acidity. H. carnosa plants maintained this CAM-idling mode of photosynthesis for at least 8 weeks. Upon reirrigation, the plants returned to the original CAM mode within 1 week. These results suggested that CAM-idling is a reversible, intermediate form of sustained metabolism which enables plant survival under conditions of extended drought.This work was supported in part by NSF Grant PCM 8200366 and in part by the Science and Education Administration of the United States Department of Agriculture under Competitive Grant 5901-0420-8-0018-0.     

Cortical vesicle exocytosis in isolated cortices of sea urchin eggs: Description of a turbidometric assay and its utilization in studying effects of different media on discharge

Cortices of unfertilized sea urchin eggs can be isolated in suspension and will discharge the attached cortical vesicles (CVs) in response to calcium. We describe a simple turbidometric assay for monitoring the Ca²⁺-induced discharge of these vesicles and also compare the discharge of vesicles isolated in a high salt medium (primarily KCl) with a medium more closely simulating the internal milieu of the cell (primarily potassium gluconate and glycine). Discharge in response to calcium is similar in both media, requiring approximately 6 μM calcium for one-half maximal discharge. There are, however, significant differences in morphology and protein composition of the two types of preparations (more proteins present in the glycine cortices) and also in the rate of discharge of the vesicles in response to calcium (KCl cortices with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ 6 sec as opposed to 30 sec in the glycine cortices). The glycine cortices gradually lose their ability to respond to calcium but retention of calcium sensitivity is considerably aided by inclusion of ATP in the media; ATP has no apparent effect on discharge of the KCl cortices. The glycine cortices, as opposed to the KCl cortices, exhibited variation in calcium sensitivity during the breeding season and in the number of vesicles which would not break down in response to added calcium (referred to as refractory vesicles). The question of which type of cortex preparation most closely simulates the in vivo situation is discussed, and the view is presented that the glycine cortices most closely resemble the in vivo situation.     

Topographical difference in taste organ density and its sensitivity of frog tongue

Distribution density of the taste disks of the fungiform papillae in the frog tongue was larger at the proximal portion than at the apical and middle portions. The number of myelinated afferent nerve fibres and taste cells per cm2 area of the tongue increased in the order of proximal greater than middle greater than apical portion. The amplitudes of gustatory neural responses for 0.5 M NaCl, 0.5 M KCl, 0.5 M NH4Cl, 0.05 M CaCl2, 1 mM acetic acid and 1 mM quinine-HCl (Q-HCl) were significantly larger with lingual stimulation of the proximal region than with the stimulation of the apical region. With these stimuli the mean ratio of the apical response to the proximal response was 1.00:1.54. On the other hand, this ration with deionized water was 1.00:5.00. The mean magnitudes of receptor potentials in taste cells for 1 mM acetic acid and 10 mM Q-HCl were the same among the apical, middle and proximal portions of the tongue. The mean magnitudes of receptor potentials for 0.5 M NaCl were significantly larger at the apical portion than at the other portions, whereas those for deionized water tended to be the largest at the proximal portion. It is concluded that the larger magnitude of the gustatory neural responses at the proximal portion of the tongue is due to morphological and physiological properties of the taste organ.     

Role and induction of 2,3-bisphosphoglycerate synthase

Molecular and Cellular Biochemistry - 2,3-Bisphosphoglycerate accumulates in mammalian erythrocytes, where it facilitates the supply of oxygen to the tissues by binding to hemoglobin. Regulatory...