Cytological and biochemical studies in a case of "di Guglielmo's syndrome"]

Cytochemical, biochemical and disc-elektrophoretical studies are done on a patient with Di Guglielmo-syndrom. Cytochemical tests showed the wellknown results as: activity of acid phosphatase and alpha-naphthylacetatesterase at their typical sites, deposition of PAS- and Turnbull-positive material. Biochemical findings are done on LDH, cholinesterase, alkaline and acid phosphatase, G-6-PDH and pyruvatkinase. Abnormal high level of fetal hemoglobin was found only once during the whole observation. There was also an alteration in the isoenzym pattern of the acid phosphatase. The de- or increase at the biochemical examinations, the fact of fetal hemoglobin and the variation at the isoenzymes may be due to the changing in distribution of erythropoetic and granulopoetic cells.     

Requirement for peptidoglycan synthesis during sporulation of Bacillus subtilis.

Cultures of Bacillus subtilis were treated during sporulation with antibiotics (bacitracin and vancomycin) that affect peptidoglycan synthesis. The cells were resistant to the effects of the antibiotics only when the drugs were added about 2 h after the beginning of sporulation. This was about 1 h later than the escape time of a temperature-sensitive sporulation mutant that is unable to complete prespore septation. Similar experiments were done with a mutant temperature sensitive for peptidoglycan synthesis. This showed an escape curve similar to that shown by the antibiotics. When sporulating cells were treated with antibiotics, they produced alkaline phosphatase earlier than normal. Enzyme production was unaffected by inhibition of deoxyribonucleic acid synthesis but was inhibited by chloramphenicol. Sporulation mutants that are unable to make alkaline phosphatase under normal conditions were able to make it in the presence of bacitracin. The alkaline phosphatase made under these conditions was under "sporulation-type" control since its synthesis was repressible by casein hydrolysate and unaffected by inorganic phosphate. When cells were treated with bacitracin in the growth medium as well as in the sporulation medium, alkaline phosphatase synthesis was at the same level as in an untreated control. A number of other antibiotics and surfactants were tested for the ability to cause premature production of the phosphatase of those tested, only taurodeoxycholate whowed this behavior. Moreover, incubation of cells with taurodeoxycholate in the growth medium as well as in the sporulation medium prevented premature enzyme production.     

Pathogenicity ofPseudomonas aeruginosa and its relationship to the choline metabolism through the action of cholinesterase,acid phosphatase,and phospholipase C

The increase of cholinesterase (ChE), acid phosphatase (Ac.Pase), and phospholipase C (PLC) activities byPseudomonas aeruginosa was associated with the choline consumption in growth media of varied composition (high or low P_i concentrations, presence or absence of ammonium ion, amino acids, polyamines, peptone, or tricarboxylic acid cycle intermediates). The highest production of the three enzymes occurred in the late stationary growth phase. The simultaneous presence of alkaline phosphatase (Alk.Pase) and the above enzymes was noted when the bacteria were grown in low P_i medium plus choline, in the absence of a preferred carbon source. The importance of choline in the production of ChE, Ac.Pase, and PLC was observed in either clinical isolates or collection strains ofP. aeruginosa. These enzymes catalyze the hydrolysis of acetylcholine, phosphorylcholine, and phosphatidylcholine. Through their action the bacteria may break down various compounds (e.g., acetylcholine, from the corneal epithelium; lung surfactant dipalmitoylphosphatidylcholine; phosphorylcholine, a product of the PLC action) or cell membranes through the coordinated action of PLC and Ac.Pase or Alk.Pase. The final consequence of the action of these enzymes is an increase of the free choline concentration. Extrapolated to an in vivo situation, if the stationary growth phase resembles the conditions thatP. aeruginosa encounters in its natural environments, then it is possible to include choline among the factors promoting the pathogenicity of this bacterium.     

Cholinesterases and postnatal development of the negative chronotropic effects of acetylcholine in albino rats

The negative chronotropic effects of acetylcholine were studied in the isolated atria of the hearts of albino rats aged 2, 15, 29 and 47 days and adult. In untreated preparations, i.e. with full cholinesterase activity, the strongest effects were observed in newborn animals; with advancing age the reaction grew weaker. If cholinesterase activity was inhibited with physostigmine, the differences between the various age groups were obliterated. It is thus evident that the actual acetylcholine sensitivity of the sinoatrial node tissue does alter during postnatal life, but that growing cholinesterase activity reduces the amount of acetylcholine diffusing from the medium into the acetylcholine receptor zone. The change which takes place in cholinesterase activity in the myocardial tissue during postnatal life is so great that is must be taken into account when considering the development of cholinergic control of cardiac function.     

Energy transduction at the catalytic site of enzymes: hydrolysis of phosphoester bonds and synthesis of pyrophosphate by alkaline phosphatase

Alkaline phosphatase from mouse intestinal epithelial cells catalyzes the synthesis of pyrophosphate from Pi during hydrolysis of either glucose 6-phosphate, ATP, ADP, inorganic pyrophosphate or p-nitrophenylphosphate. The rate of pyrophosphate synthesis is increased by MgCl2 and by decreasing the pH of the medium from 8.5 to 6.0. The data presented indicate that at the catalytic site of alkaline phosphatase the energies of hydrolysis of the phosphoserine residue and of pyrophosphate are different from those measured in aqueous solutions.     

Coordinate regulation of collagen and alkaline phosphatase levels in chick embryo chondrocytes

Chick embryo tibial chondrocytes release into their extracellular matrix several species of proteochondroitin sulfate and collagen as well as matrix vesicles that are rich in Ca2+ and alkaline phosphatase and that appear to play a role in the calcification of cartilage. To determine whether there was any parallel regulation of the production of these products, the rates of collagen synthesis by cultured chick embryo tibial chondrocytes were altered, and the resulting changes in proteochondroitin sulfate synthesis and alkaline phosphatase levels in the cells were measured. As the rate of collagen synthesis was increased by adding increasing amounts of ascorbic acid to the culture medium, there was a parallel increase in the level of alkaline phosphatase. Similarly, when the rate of collagen synthesis was inhibited by adding 3,4-dehydroproline to the culture medium, the levels of alkaline phosphatase fell. The alkaline phosphatase in the culture medium was associated with vesicles which appeared to be matrix vesicles. It was recovered quantitatively by filtration through membranes with a pore size of 0.1 mu and measured by solubilizing the alkaline phosphatase from the membrane with detergent and assaying with 4-methylumbelliferyl phosphate as the substrate. When the matrix vesicles from the culture medium were analyzed for collagen types, it was found that only Type X collagen was recovered in this fraction. The implications of the association of Type X collagen and the matrix vesicles, both of which are found primarily in growth plate cartilage in the zone of hypertrophied chondrocytes which is in the process of mineralization, are discussed.     

Effect of cobalt on synthesis and activation of Bacillus licheniformis alkaline phosphatase.

The effect of CO2+ on the synthesis and activation of Bacillus licheniformis MC14 alkaline phosphatase has been shown by the development of a defined minimal salts medium in which this organism produces 35 times more (assayable) alkaline phosphatase than when grown in a low-phosphate complex medium or in the defined medium without cobalt. Stimulation of enzyme activity with cobalt is dependent on a low phosphate concentration in the medium (below 0.075 mM) and continued protein synthesis. Cobalt stimulation resulted in alkaline phosphate production being a major portion of total protein synthesized during late-logarithmic and early-stationary-phase culture growth. Cells cultured in the defined medium minus cobalt, or purified enzyme partially inactivated with a chelating agent, showed a 2.5-fold increase in activity when assayed in the presence of cobalt. Atomic spectral analysis indicated the presence of 3.65 +/- 0.45 g-atoms of cobalt associated with each mole of purified active alkaline phosphatase. A biochemical localization as a function of culture age in this medium showed that alkaline phosphatase was associated with the cytoplasmic membrane and was also found as a soluble enzyme in the periplasmic region and secreted into the growth medium.     

Astrocyte-mediated induction of alkaline phosphatase activity in human umbilical cord vein endothelium: an in vitro model

The blood-brain barrier, localized in the endothelium of the cerebral capillaries, is characterized by the existence of tight junctions, a low mitochondrial density, a low number of vesicles and a high activity of certain enzymes like alkaline phosphatase and gamma-glutamyl transpeptidase. Astroglial cells secrete a product that induces brain microvessel endothelial cells to differentiate into endothelial cells with blood-brain barrier properties. If rat astrocytes were grown together with human umbilical cord vein endothelial cells in a co-culture system in which there is no cellular contact between both cell types, alkaline phosphatase activity was induced in the endothelial cells after three days of co-culturing. If the endothelial cells were cultured in astrocyte conditioned medium, alkaline phosphatase activity was also induced, and preliminary results showed that formation of tight junctions occurred after five days. These observations support the hypothesis that astrocytes induce the differentiation of non-blood-brain barrier endothelial cells into endothelial cells with blood-brain barrier properties, in this study based on alkaline phosphatase-activity induction and induction of tight junction formation. These inductive processes are produced by a soluble factor released by the astrocytes.     

Alkaline phosphatase activity of normal and transformed cultured human cells

A study was made of the relationship between the activity of alkaline phosphatase and the proliferation of cultured human cells with different replicative potentials. It is shown that alkaline phosphatase plays a role as one of endogenic stimulators of cellular proliferation. The ageing of diploid cells is accompanied by a decrease in the enzyme activity. Maximum activity was observed during a period of logarithmic cell growth. Addition of placental alkaline phosphatase to the synchronized diploid cells stimulated DNA synthesis in the S-phase of the cell cycle. Heteroploid cells with a high growth rate possessed a 30-100 times higher alkaline phosphatase activity than in the diploid cells. Under certain conditions alkaline phosphatase may presumably function as a proteinkinase.     

Requirement of serum pretreatment for induction of alkaline phosphatase activity with prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCl in human liver cells continuously cultured in serum-free medium

A cell line (HuL-1) derived from normal fetal human liver was adapted to grow continuously in a modified Eagle's minimum essential medium without serum or hormones. The population doubling time of this adapted cell line (HuL-1-317) was about 72 h and the modal number of chromosomes was 54. The morphology of HuL-1-317 cells was round in the absence of serum, but at 37 degrees C with the addition of serum (1-10%), the cells flattened. HuL-1-317 cells had a low level of alkaline phosphatase activity. However the enzyme activity was slightly enhanced by the combination of prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and a hypertonic concentration of NaCl after 3 days of incubation at 37 degrees C. The increase in alkaline phosphatase activity with the four agents was further amplified dose-dependently by the pretreatment of the cells with serum. The stimulatory effect of the serum was evident at concentrations as low as 1%, and was maximal at 20%. The half life of the effect of serum on alkaline phosphatase induction was 48 h at 37 degrees C. Serum alone could not enhance the enzyme activity without the four agents. The present results indicate that serum contributes to the regulation of alkaline phosphatase induction by the combination of prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCl in fetal human liver cells (HuL-1-317).     

Influence of phosphate ions and alkaline phosphatase activity of cells on survival ofEscherichia coli in seawater

When grown in a minimal medium and suspended for 2 hours in distilled water, seawater, phosphate buffer or a polyphosphate solution,E. coli MC4100 cells with high alkaline phosphatase activity survived in seawater for longer periods than cells with low or no activity. However, mutant cells totally deprived of alkaline phosphatase activity held in phosphate-containing media before transfer to seawater showed survival almost as high as the wild type strain, indicating that alkaline phosphatase activity is not the only factor influencing survival. Alkaline phosphatase activity also increased the protection of cells provided by glycine betaine. Survival was enhanced when cells were preincubated in the presence of phosphate or polyphosphate. Thus, the transfer of cells in wastewater could influence their subsequent survival in seawater.     

Culture of osteogenic cells from human alveolar bone: a useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of approximately 120 kDa that could be solubilized by phospholipase C or Polidocanol.     

Effects of extracellular calcium on the proliferation and differentiation of porcine osteoblasts in vitro

We examined the effects of various extracellular calcium concentrations on DNA content, procollagen type I carboxy-terminal propeptide (PICP) release (reflects type I collagen synthesis), and alkaline phosphatase activity of porcine osteoblasts. Osteoblasts seeded in control medium (2.2 mM calcium) were transferred to low (0.5 or 1 mM) calcium medium or to high (3, 5, 7, or 10 mM) calcium medium at different stages of the culture period and for different incubation times. When osteoblasts were transferred to low or high (3 or 5 mM) calcium medium 1 or 2 days after plating and kept in that medium until the end of the culture period, PICP release was inhibited, but DNA content and alkaline phosphatase activity were unchanged, except in 5 mM calcium, which inhibited alkaline phosphatase activity. Short-term culture of subconfluent and near-confluent osteoblasts in 7 or 10 mM calcium for 48 h inhibited DNA content. DNA content returned to normal levels when cells were transferred back to control medium, whereas alkaline phosphatase inhibition induced by 5, 7, or 10 mM calcium was not reversible. Short-term culture in high calcium media did not affect PICP release. Thus, in porcine osteoblasts, low and high extracellular calcium concentrations affect DNA content, PICP release, and the expression of osteoblastic phenotype markers (alkaline phosphatase activity). These effects are dependent on the duration of calcium treatment and the state of differentiation of the osteoblasts.     

Cortisol modification of HeLa 65 alkaline phosphatase. Decreased phosphate content of the induced enzyme.

Alkaline phosphatase activity of HeLa cells is increased 5-20-fold during growth in medium with cortisol. The increase in enzyme activity is due to an enhanced catalytic efficiency rather than an increase in alkaline phosphatase protein in induced cells. In the present study the chemical composition of control and induced forms of alkaline phosphatase were investigated to determine the enzyme modification that may be responsible for the increased catalytic activity. HeLa alkaline phosphatase is a phosphoprotein and the induced form of the enzyme has approximately one-half of the phosphate residues associated with control enzyme. The decrease in phosphate residues of the enzyme apparently alters its catalytic activity. Other chemical components of purified alkaline phosphatase from control and induced cells are similar; these include sialic acid, hexosamine and sulfhydryl residues.     

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2′-deoxyuridine

Summary Growth of choriocarcinoma cells in the presence of 5-bromo-2′-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effect of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing: BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.     

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2'-deoxyuridine.

Growth of choriocarcinoma cells in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effects of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.     

CELL CYCLE EVENTS IN THE HYDROCORTISONE REGULATION OF ALKALINE PHOSPHATASE IN HELA S3 CELLS

The increase in alkaline phosphatase in asynchronous cultures of HeLa S₃ cells grown in medium supplemented with hydrocortisone is characterized by a lag period of 10–12 hr. Present studies utilizing synchronous cell populations indicate: (a) a minimum of 8–10 hr of incubation with hydrocortisone is necessary for maximum induction of alkaline phosphatase; (b) the increase in enzyme activity produced by hydrocortisone is initiated exclusively in the synthetic phase of the cell cycle; (c) alkaline phosphatase activity does not vary appreciably over a normal control cell cycle. Radioactive hydrocortisone is rapidly distributed into HeLa cells irrespective of their position in the cell cycle, indicating that inductive effects are not governed by selective permeability during the cell cycle. Hydrocortisone-1,2-[³H] diffuses back from the cell into the medium when the cells are incubated in fresh medium containing no hydrocortisone, and the alkaline phosphatase induction, under these conditions, is completely reversible.     

Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate-resistant and -sensitive acid phosphatases, and non-specific esterases were assayed kinetically and histochemically. In asynchronous subconfluent cultures, less than 15% of the cells stained positive and assayed activity was 0.04 IU/10(6) cells/cm2. After 1 week, the percent of alkaline phosphatase positive-staining cells increased 5-fold, while activity increased 10-fold. Non-specific esterases and tartrate-sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate-resistant acid phosphatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25-30 h. In synchronous cultures induced by a thymidine-aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate-sensitive acid phosphatase and non-specific esterase levels were relatively stable throughout the cell cycle, while tartrate-resistant acid phosphatase activity was not assayable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.     

Relation between the content of acetyl-coenzyme A and acetylcholine in brain slices.

Slices of rat caudate nuclei were incubated in vitro in media containing, among other constituents, three different concentrations of glucose (0.5, 2 and 10 mM), 0.2 mM-choline, paraoxon as an inhibitor of cholinesterase, and 5 mM- or 30 mM-K+. After 30 and 60 min of incubation, the concentrations of acetyl-CoA, acetylcholine and choline in the tissue and of acetylcholine in the incubation medium were measured. The content of acetyl-CoA in the sliced varied in direct relation to the concentration of glucose in the incubation medium. The content of acetylcholine in the slices and, in experiments with high K+, also the amount of acetylcholine released into the incubation medium varied in direct relation to the concentration of glucose in the incubation medium and to the concentration of acetyl-CoA in the slices; the relation between the concentrations of acetyl-CoA and of acetylcholine in the slices was linear. It was concluded that the availability of acetyl-CoA had a decisive influence on both the rate of synthesis of acetylcholine and its steady-state concentration. The observations accord with the view that, at the ultimate level, the synthesis of acetylcholine is controlled by the Law of Mass Action.