Regulation of inositol monophosphatase in Saccharomyces cerevisiae

Inositol monophosphatase is a key enzyme in the de novo biosynthesis of inositol and in the phosphoinositide second-messenger signalling pathway. Inhibition of this enzyme is a proposed mechanism for lithium's pharmacological action in bipolar illness (manic depression). Very little is known about how expression of this enzyme is regulated. Because the yeast Saccharomyces cerevisiae has been shown to be an excellent model system in which to understand the regulation of inositol metabolism, we characterized inositol monophosphatase in this yeast. Lithium inhibited monophosphatase activity in vitro . Growth in the presence of inositol resulted in increased expression of the enzyme in vivo , although inositol had no effect on enzyme activity in vitro . The inositol effect was apparent when cells were grown in glucose but not in glycerol/ethanol. Monophosphatase activity was derepressed as cells entered stationary phase. This effect was apparent only during growth in glucose plus inositol. The results demonstrate that S. cerevisiae monophosphatase is inhibited by lithium and regulated by factors affecting phospholipid biosynthesis.     

Simultaneous determination of NO and NO in fisheries wastewaters high in chloride concentration by capillary ion electrophoresis with direct UV detection

The simultaneous determination by capillary ion electrophoresis of nitrate and nitrite in wastewaters with chloride concentrations of 15 to 23g/l is described. Chloride concentrations over 200mg/l hampered the determination; thus, lead, mercuric and silver acetate were used to precipitate chloride. Silver acetate added in 1.5 times the stoichiometric amount for AgCl formation gave the best results in terms of nitrate and nitrite peak resolution.     

Proliferation of pulmonary endothelial cells: Time-lapse cinematography of growth to confluence and restitution of monolayer after wounding

A fundamental characteristic of vascular endothelium is that it exists as a monolayer, a condition that must be met in both vascular growth and repair. Maintenance of the monolayer is important both for the exchange of nutrients and for interactions between blood solutes and endothelial enzymes and transport systems. We have used time-lapse cinematography to compare proliferative behavior of bovine pulmonary endothelial cells in (1) establisment of a monolayer from a low-density seed (7.5 × 10⁴ cells in a 60 mm dish) and (2) restitution of a confluent monolayer (approx. 2.9 × 10⁶ cells in a 60 mm dish) following a mechanical wound (removal of cells from an area 5 × 15 mm by scraping). Culture 2 was not refed after wounding. In culture 2, approx. 30% of the cells accounted for repopulation (confluence in 40 hr). In culture I, all cells entered into division. Participating cells of culture 2 began division immediately (69 divisions/filmed area in 10 hr, vs. four divisions in culture I). Interdivision times (IDT) were longer and relatively constant in culture I until near confluence; none were < 10 h, whereas in 2, 24% of the IDT's were ≤ 10 hr. Remarkably, IDTs of culture 2 decreased steadily until confluence was re-established. Cell migration in culture 1 was multidirectional while direction of migration in culture 2 was always into the wound area. Mean migration rate (MIG) in culture 2 was related to the site of origin of the cells, those dividing farthest from the unwounded area had fastest MIGs. Neither culture formed more than a single layer of cells. Although the cell kinetics of cultures 1 and 2 differed, the same goal, confluence, was achieved in either case.     

Repressor and int synthesis of bacteriophage lambda in the E. coli host mutant ER437.

Summary Analysis of phage infection of the host mutant ER437 by SDS polyacrylamide gel electrophoresis and autoradiography has revealed altered expression of repressor and integration function (Int). We show that in this host Int as well as repressor synthesis is not dependent upon the cIII gene product in the usual manner, nor is their synthesis turned off in the normal way.     

Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

Vascular smooth muscle cell detachment from elastin and migration through elastic laminae is promoted by chondroitin sulfate-induced “shedding” of the 67-kDa cell surface elastin binding protein

Impaired elastin fiber assembly is observed in the fetal ductus arteriosus (DA), associated with a reduced concentration of elastin binding protein (EBP), a 67-kDa galactolectin. It is also seen in cultured aortic (Ao) smooth muscle cells (SMC) following the release of the EBP by glycosaminoglycans rich in AN-acetylgalactosamine, such as chondroitin sulfate (CS). In the DA, impaired elastin fiber assembly is observed in conjunction with intimal thickening associated with increased migration of SMC into the subendothelium, a feature we previously related to increased production of fibronectin. In this report, we determined whether SMC use the EBP to attach to an elastin substrate, whether shedding of the EBP promotes SMC migration through a threedimensional network of pure elastic laminae prepared from sheep aorta, and whether the latter is associated with increased production of fibronectin. We observed reduced attachment to elastin-coated surfaces of DA SMC deficient in EBP compared to Ao SMC. Addition of CS but not heparan sulfate (a glycosaminoglycan which does not induce EBP shedding) decreased Ao SMC attachment to elastin, as did preincubation with VGVAPG elastin-derived peptides which saturate the EBP. The immunolocalization of cell surface EBP suggested that cells can quickly replace EBP released from their surfaces by CS treatment. The magnitude of CS-induced impaired attachment of SMC to elastin was dose dependent and could be further increased by the administration of cyclohexamide and sodium azide. Also, the reversibility of CS-induced detachment was prevented by monensin. This suggests that a process of new synthesis and intracellular transport of the EBP was necessary to replace the EBP molecules released from the cell surface by CS treatment. In the migration assay, both DA and Ao SMC attached to the top of an elastin membrane, but only DA SMC deficient in EBP migrated through the laminae. Addition of CS, which induced shedding of EBP, resulted in Ao SMC migration associated with increased synthesis of fibronectin. We postulate that CS-induced release of EBP from SMC surfaces causes cell detachment from elastin and an increase in fibronectin synthesis, processes which may be critical in promoting SMC migration associated with intimal thickening developmentally in the DA and perhaps also in vascular disease.     

Scanning electron microscopy ofMycoplasma pneumoniae on the membrane of individual ciliated tracheal cells

Summary Cell monolayer cultures were prepared from hamster tracheal explants by a collagenase exposure and subsequent incubation in Waymouth’s MAB 87/3 medium. The epithelial outgrowth occurred on glass cover slips. Cilia on the monolayers continued to beat normally after the “parent” explant was removed. Monolayer cultures infected withMycoplasma pneumoniae had significant amounts of attachment. A morphological analysis of the attachment was conducted with scanning electron microscopy. Clusters, cocci, and filaments ofM. pneumoniae all attached to the epithelial cells, but the filaments were especially common. Mycoplasmas were seen in association with both ciliated and nonciliated cell membranes. On ciliated cells, mycoplasmas were on the ciliary strands and on the cell membrane. When located immediately adjacent to or in between cilia, mycoplasmas were oriented vertically with the constricted attachment tip oriented down toward the host cell membrane. When located more than a micron away from the ciliary fibers, mycoplasmas lay horizontally along the epithelial cell membrane. The photographic data suggest that clusters or “sperules” of mycoplasmas may liberate individual mycoplasmas that attach to the cell membrane. It appears that the receptor sites forM. pneumoniae are rather uniformly distributed along the ciliated cell membrane, and are not restricted to the interciliary areas. Electron microscopy was done with the cooperation of Dr. R. Macleod and the staff of the Center for Electron Microscopy at the University of Illinois. Critical editorial review was provided by C. Dayton. This investigation was supported in part by grants to M. G. G. from the National Institute of Allergy and Infectious Diseases (AI 12559) and the National Heart, Lung, and Blood Institute (HL 23806), Bethesda, Maryland.     

Random recirculation of small T lymphocytes from thoracic duct lymph in the mouse

The migration of ⁵¹Cr-labeled nylon-wool separated mouse thoracic duct T cells has been followed in order to determine whether there is a circulation of small (nondividing) T cells through the small intestine. Approximately 6% of the injected dose of T-TDL localized in the small intestine (minus Peyer's patches). Experiments revealed that this gut-localizing cell population consisted almost entirely, if not exclusively, of lymphoblasts present in mouse T-TDL. When lymphoblasts and small lymphocytes from mouse T-TDL were separated by velocity sedimentation, and the migration of separated fractions was studied, we found large cells (66% blasts) migrated well to the gut but poorly to the lymph nodes, whereas small cells (2% blasts) showed minimal migration to the gut but localized randomly in lymph nodes and spleen. The in vivo distribution of small cells from T-TDL was similar to that of T-PLN. Furthermore, the recirculatory patterns of both ⁵¹Cr-labeled T-TDL and T-PLN were found to be identical as accessed by their rate of recovery in the thoracic duct lymph of recipient mice. These results support the notion that the vast majority of T-TDL and T-PLN are part of a common pool of recirculating T cells which recirculate randomly through lymph nodes and spleen and not the small intestine.     

Cytochemistry and freeze-fracture of membranes isolated from the electrocyte of Electrophorus electricus (L.)

Summary Membranes were isolated from the main electric organ of Electrophorus electricus and studied by means of cytochemistry and freezefracture. The membrane fractions consisted of vesicles inside-in as determined by localization of anionic sites using colloidal iron and cationized ferritin particles. The anionic sites were not homogeneously distributed on the surface of the vesicle. Freeze-fracture showed the presence of intramembranous particles associated with either protoplasmic (P) or extracellular (E) faces of the membrane. Regions of the membrane without particles were observed. The results are discussed in relation to the existence of association between intramembranous particles and membrane receptors.For all correspondence