Regulation of buccal mucosal calcium channel activity by salivary mucins

• 1.1. The effect salivary mucins on the activity of calcium channel isolated from buccal mucosal cell membranes was investigated. The uptake of ⁴⁵Ca²⁺while only moderately (15%) affected by the intact low and high molecular weight mucin forms, was significantly inhibited, by the acidic low and high molecular weight salivary mucins which evoked 64 and 60% inhibition, respectively.
• 2.2. The inhibitory effect of salivary mucins was associated with the sialic acid and sulfate ester groups of the carbohydrate chains, as the removal of either group caused partial loss in the glycoproteins inhibition, and the complete loss in the inhibitory effect occurred following desialylation and desulfation.
• 3.3. The channel in the presence of epidermal growth factor (EGF) and ATP responded by an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the phosphorylated channels showed a 46% increase in ⁴⁵Ca²⁺ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein.
• 4.4. The binding of EGF to calcium channel receptor protein was inhibited by the low and high molecular weight acidic mucins, causing 41.2 and 36.1% reduction, respectively. This reduction in binding was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoproteins' inhibitory capacity following removal of these groups.
• 5.5. The results for the first time demonstrate that salivary mucins actively participate in the modulation of the EGF-controlled buccal mucosal calcium channel activity expression, a process of importance to the preservation of oral tissue integrity.

     

Activation of gastric mucosal calcium channels by epidermal growth factor

• 1.1. Gastric mucosal calcium channel complex was isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin.
• 2.2. The complex following labeling with [³H]PN200-110 was reconstituted into phosphatidylcholine vesicles which exhibited active ⁴⁵Ca²⁺ uptake into intravesicular space as evidenced by La³⁺ displacement and osmolarity measurements. The ⁴⁵Ca²⁺ uptake was independent of sodium and potassium gradients indicating the electroneutral nature of the process.
• 3.3. The gastric mucosal channels on epidermal growth factor binding in the presence of ATP responded by an increase in protein tyrosine phosphorylation of 55 and 170 kDa subunits of calcium channel.
• 4.4. The phosphorylated channels following reconstitution into vesicles displayed at 48% greater ⁴⁵Ca²⁺ uptake, thus indicating the tyrosine kinase involvement in EGF dependent activation of calcium channel.
• 5.5. The results point towards the importance of epidermal growth factor in the maintenance of gastric mucosal calcium homeostasis.

     

Structural features of carbohydrate chains in human salivary mucins

• 1.1. The structure of carbohydrate chains in the low and high molecular weight mucus glycoprotein forms from submandibular-sublingual saliva of individuals with blood group B was investigated.
• 2.2. Alkaline borohydride reductive cleavage of the glycoproteins yielded in each case a population of neutral (55%) and acidic (45%) oligosaccharide alditols ranging in size from 3 to 16 sugar units.
• 3.3. The predominant neutral oligosaccharides in both glycoprotein forms consisted of 16 and 15 sugar units arranged in triantennary fashion, and carried blood group B and I antigenic determinants.
• 4.4. Three of the oligosaccharides in each glycoprotein contained sialic acid and ranged in size from 3 to 12 sugar units. In two oligosaccharides sialic acid was linked to C3 of galactose and in one to C6 of N-acetylgalactosamine. The sulfated oligosaccharide in both glycoproteins was identified as a pentasaccharide with the sulfate ester group at C6 of N-acetylglucosamine.
• 5.5. The results demonstrate that contrary to the earlier view the low and high molecular weight mucus glycoprotein forms of human saliva contain identical carbohydrate chains.

     

Cotranslational fatty acylation of mucus glycoprotein. Addition of palmitic acid to peptidyl-tRNA occurs prior to peptide chain completion and its release

• 1.1. The fatty acylation of mucus glycoprotein nascent peptides was investigated using [³H]palmitic acid and [³⁵S]methionine-labeled peptidyl-tRNA of rat gastric mucous cells.
• 2.2. The mucus glycoprotein peptidyl-tRNA fraction was found to contain covalently bound palmitic acid in its complexes.
• 3.3. RNase digestion of the mucus glycoprotein peptidyl-tRNA released [³H]palmitic acid labeled peptides which, on SDS-polyacrylamide gel, separated into a multitude of bands ranging in size from 2000 to 60,000 Da.
• 4.4. The analyses of low molecular weight peptides revealed that palmitic acid was present in methionine-labeled peptides containing 30–43 amino acids and those of 18–25 amino acids or larger devoid of methionine, but was not identified in methionine-labeled peptides containing 10–15 amino acids.
• 5.5. The results indicate that the N-terminal fatty acylation of mucus glycoprotein nascent peptides is a cotranslational process which is occuring in an immediate vicinity of the signal peptide fragment.

     

Epidermal growth factor alters the electrolyte profile of lactating ewes Ovis aries

• 1.1. Lactating ewes were treated with mouse epidermal growth factor (EGF) at a dose rate of 0.5 mg/day for 4 days and its effects on the electrolyte profile were observed.
• 2.2. There was no effect of EGF on plasma concentrations of sodium or potassium, although urinary and total (in urine and milk) losses of both were reduced.
• 3.3. EGF-induced hypocalcaemia was associated with reduced milk calcium secretion and increased urinary calcium excretion whereas EGF-induced hypermagnesaemia was associated with reduced urinary and total magnesium losses.
• 4.4. Glomerular filtration rate was reduced during EGF infusion.
• 5.5. Chronic intravenous EGF infusion affects the electrolyte profile by altering electrolyte secretion by the mammary gland and renal electrolyte excretion.

     

Effects of aluminium and pH on calcium fluxes,and effects of cadmium and manganese on calcium and sodium fluxes in brown trout (Salmo trutta L.)

• 1.1. Simultaneous measurement of calcium fluxes in brown trout, at low external [Ca] (20 μ mol 1⁻¹), provided evidence of active uptake of Ca from the medium.
• 2.2. At pH 4.5, calcium influx was inhibited and efflux was stimulated.
• 3.3. Cd and Mn, but not Al, at concentrations within the ranges found in acid waters experiencing fish population decline, inhibited calcium influx. Efflux was unaffected.
• 4.4. Cd and Mn stimulated sodium influx and efflux.

     

Control of mucin molecular forms expression by salivary protease: Differences with caries

• 1.1. A protease activity capable of degradation of the high mol. wt salivary mucus glycoprotein to a low mol. wt glycoprotein form was identified in human submandibular gland secretion.
• 2.2. The protease exhibited optimum activity at pH 7.0–7.4, and gave on SDS-PAGE under reducing conditions two major protein bands of 48 and 53 kDa. The enzyme showed susceptibility to PMSF, α₁antitrypsin, and egg white and soybean inhibitors, a characteristic typical to serine proteases.
• 3.3. The activity of the protease towards the high mol. wt mucus glycoprotein was found to be 3.8-fold higher in submandibular gland secretion of caries-resistant individuals than that of caries-susceptible. Furthermore, the enzyme from both groups displayed greater activity against the mucus glycoprotein of caries-resistant subjects.
• 4.4. Since the low mol. wt salivary mucus glycoprotein form is more efficient in bacterial clearance than the high mol. wt mucin, the enhanced expression of this indigenous salivary protease activity towards mucin may be the determining factor in the resistance to caries.

     

Taurine regulation of Ca2+ uptake and (Ca2+ + Mg2+)-ATPase in developing chick B-cells

• 1.1. The objective of the present study was to determine the effect of age and taurine on chick B cell calcium uptake and membrane (Ca²⁺ + Mg²⁺)-ATPase activity in 1–4-week-old chicks.
• 2.2. The calcium uptake rate decreased with age (P < 0.05) and was further decreased by taurine (P < 0.05).
• 3.3. (Ca²⁺ + Mg²⁺)-ATPase activity increased with age (P < 0.05) and was stimulated by taurine (P < 0.05).
• 4.4. The data demonstrate that the flux of calcium across the B-cell membrane changes during early post-hatch development, and that taurine regulates both the influx and efflux of calcium in chick B-cells.

     

The effects of heavy metals and metabolic inhibitors on calcium uptake by gills and scales of Fundulus heteroclitus in vitro

• 1.1. Cadmium (Cd) and zinc (Zn) were inhibitory to calcium uptake by isolated gills of Fundulus heteroclitus in vitro. The metals appeared to act by displacing Ca²⁺ ions from protein carriers involved in facilitated diffusion.
• 2.2. In saltwater fish, transport of calcium across the serosal membrane of gill chloride cells is partly energy dependent and is likely mediated by Ca²⁺-ATPase. However, much of the calcium transport through the gill epithelium appears to occur by passive processes.
• 3.3. Cd (10⁻⁵M—10⁻³M) and Zn (10⁻⁷M—10⁻³ M) inhibited calcium uptake by isolated scale patches incubated in a physiological saline.
• 4.4. Cyanide, oubain, and quercetin treatment of scale patches produced results similar to those of the Cd and Zn treatments suggesting that metal-induced inhibition of ATPases may be responsible for reduced calcium transport by scale osteoblasts.

     

Biomines-stimulated phosphorylation and (Na+, K+-ATPase in the gills of the chinese crab,Eriocheir sinensis

• 1.1. Subcellular distribution of (NA⁺, K⁺-ATPase and ouabain-insensitive ATPase (Mg²⁺-ATPase) are compared in branchial tissues of the euryhaline crab, Eriocheir sinensis, acclimated to fresh water.
• 2.2. Both the anterior and posterior gills contain cAMP-dependent protein kinase and endogenous protein substrate for phosphorylation.
• 3.3. Phosphorylation occurs in both “particulate” and “soluble” subcellular fractions but its stimulation by cAMP is restricted to the “soluble” fraction.
• 4.4. serotonin (5-HT) and dopamine receptors are present only in the “light particulate” fraction isolated from the posterior gills.

• 1.(a) Serotonin and dopamine have no effect on the phosphorylation observed in a subcellular fraction alone.
• 2.(b) Activation of the phosphorylation by serotonin and dopamine is found when the soluble fraction (source of cAMP-dependent protein kinase) is added to the fraction P₃ from the posterior gills.
• 3.(c) No activation occurs with the fractions P₃ as well as P₁ or P₂ (not shown) from anterior gills of fresh water crab.
• 4.(d) Cyproheptadine, a serotonin receptor antagonist, inhibits the 5-HT dependent increase in phosphorylation.
• 5.(e) The dopamine receptor antagonist, chlorpromazine, inhibits dopamine-stimulated phosphorylation.
• 6.5. Ouabain mimics the effect of cyproheptadine on the serotonin-stimulated phosphorylation found in the posterior gills.

     

Initial characterization of human thymocyte sialidase activity: Evidence that this enzymatic system is not altered during the course of T-cell maturation

• 1.1. The sialidase activity of human thymocyte was examined by a fluorogenic assay.
• 2.2. These studies revealed that human thymocyte sialidase activity is essentially acid-active and membrane-bound since 59.6% and 33% of the total activity was recovered in the lysosome-enriched and microsomal fractions, respectively.
• 3.3. A weak activity was also detected in the cytosolic fraction.
• 4.4. However, the acidic optimum pH of this soluble sialidase was at variance with the general concept of mammalian soluble sialidases which are known to be optimally active at more neutral pH.
• 5.5. This acidic soluble sialidase seems to be a general characteristic of the human T-cell lineage since examination of mature circulating T-cells revealed that they contain a soluble sialidase activity similar to that observed in thymocytes.
• 6.6. Analysis of mature and immature thymocyte subpopulation obtained by differential PNA agglutination indicated that this enzymatic system was not altered during the course of thymic maturation.
• 7.7. These results suggest that unlike in T-cell activation where changes in the level of sialidase activity were shown to influence the extent of cell surface sialylation and thereby the cell physiology, this enzymatic system seems not to be involved in the fluctuation of cell surface sialic acid content observed during thymic maturation.

     

Serotonin,histamine and somatostatin modulation of PMA-stimulated phosphorylation of 130kDa Ca2+ pump-like protein from rat cerebellum synaptosomal membranes

• 1.1. Influence of some neurotransmitters and neuromodulators on the PMA-stimulated phosphorylation in vitro of calcium pump-like protein from rat cerebellum synaptosomal membranes was examined.
• 2.2. The prolonged time (up to 6 min) of synaptosomal membranes preincubation with 1 and 10 μM serotonin results in the increase of phosphorylation. The decrease of phosphorylation up to 80% of control value was observed for 100 μM serotonin.
• 3.3. The most stimulating effect on 130kDa protein phosphorylation was observed with 1μM of histamine (160% of control value).
• 4.4. 1 and 0.1 μM somatostatin triggered a short-time transient increase of 130 kDa phosphorylation (up to 135% of control value).

     

Purification and partial characterization of 200 kDa oncofetal antigen from radiation induced murine lymphocytic lymphoma

• 1.1. A 200 kDa glycoprotein (gp200) oncofetal antigen was purified from solubilized membranes of a radiation-induced murine lymphomcytic lymphoma cell line (XR11-5T), grown in syngeneic RFM mice, by successive gel chromatography of the active fraction on lentil lectin agarose, Q- and S-Sepharose and Superose-12 using an FPLC system.
• 2.2. A murine monoclonal antibody 115, produced by the syngeneic immunization of adult male C57BL/6N mice with 12-day mouse fetal cells, was used in a slot blot antibody assay to follow up the active fractions.
• 3.3. The purified glycoprotein has a pi of 5.4.
• 4.4. Treatment of radiolabeled gp200 with neuraminidase caused a slight reduction in size due to the removal of sialic acid groups and a shift in pI to 6.3.
• 5.5. Treatment of gp200 with different glycosidases shows that gp200 is susceptible to N- and O-glycanase but not to endoglycosaminidase H.
• 6.6. On extraction of gp200 with Triton X-114 it partitions exclusively into the detergent-rich fraction consistent with being an integral membrane protein.

     

Responses of channel catfish (Ictalurus punctatus) swim-up fry to dietary calcium in soft and hard water

• 1.1. Responses of channel catfish (Ictalurus punctatus) swim-up fry to dietary calcium in soft (< 1 mg/1 as CaCO₃) and hard (> 100 mg/1 as CaCO₃) water were determined by feeding purified egg-white diets containing 0, 0.5, 1.0, or 2.0% calcium from CaCO₃ for 8 weeks.
• 2.2. Catfish fry fed the basal diet (0.03% Ca) in hard and soft water had lower whole-body ash and whole-body calcium concentrations but higher weight gain and survival than those fed calcium-supplemented diets.
• 3.3. Fry in soft water generally had lower whole-body ash, whole-body calcium, and survival, as well as a higher incidence of spinal deformities than fry in hard water.
• 4.4. Feeding higher levels of calcium to fry reared in soft water did not increase whole-body calcium levels or decrease spinal deformities to the levels observed for fry reared in hard water and fed supplemental calcium.
• 5.5. These data indicate that calcium derived solely from dietary or environmental sources was not sufficient for optimum health of channel catfish fry.

     

Modulation of the ATP induced [Ca2+]c increase in as-30D hepatoma cells

• 1.1. The regulation of the increase in the cytosolic calcium concentration ([Ca²⁺]c) induced by extracellular ATP in AS-30D hepatoma cells was studied.
• 2.2. Homologous desensitization involving the refilling of intracellular calcium pools and the participation of protein kinase C was found.
• 3.3. Isoproterenol, forskolin and dibutyril-cyclic AMP also induced an increase in [Ca²⁺]c.
• 4.4. Interestingly, synergism was found for isoproterenol or forskolin and ATP.
• 5.5. The results suggest that there are two pathways for mobilizing [Ca²⁺] in AS-30D hepatoma cells; one is activated by ATP receptors and the other by cyclic AMP.

     

Glycoprotein effect on the osmotic permeability of liposomes

• 1.1. Proteoliposomes with incorporated α₁-acid glycoprotein were found to be osmotically sensitive to alkali metal salts.
• 2.2. The apparent permeabilities of monovalent metal cations were determined. They were compared with those of pure liposomes in the presence of amphoterecin B.
• 3.3. It was found that proteoliposomes showed a selective permeability to K⁺ in spite of the fact that pure liposomes in the presence of amphotericin B are more permeable to Rb⁺.
• 4.4. It was assumed that this difference arises apparently from the modification of lecithin bilayer in the presence of glycoprotein molecule.

     

Regulatory properties of rabbit liver phosphorylase kinase

• 1.1. Purified native rabbit liver phosphorylase kinase becomes activated during the assay of its activity while low molecular weight forms of the same enzyme do not.
• 2.2. The activation requires ATP and maganesium ions, suggesting the phosphorylation of the enzyme by a protein kinase as the mechanism involved.
• 3.3. The activation of the enzyme can be reverted by the action of a type 1 protein phosphatase isolated from the same tissue.
• 4.4. The activation can also be catalyzed by the catalytic subunit of cAMP-dependent protein kinase in a process that requires a much lower ATP concentration to proceed.
• 5.5. The activation is believed to be due to an autocatalytic phosphorylation of phosphorylase kinase itself. In support of this hypothesis are the regulation of the process through calcium ions, the low levels of endogenous protein kinase detected in the purified preparation, the high ATP concentrations required in the absence of cAMP dependent protein kinase and the fact that the process cannot be blocked by an excess of the heat stable inhibitor specific for the later enzyme.
• 6.6. The low molecular weight forms of the enzyme on their side are not affected by the action of neither protein phosphatase 1 nor cyclic AMP dependent protein kinase.
• 7.7. Both activated and nonactivated phosphorylase kinase are partially dependent on calcium ions, the affinity of the former being higher than that of the latter. The low molecular forms do not require calcium ions to express their activity.

     

Crayfish retinular cells: Influence of extracellular sodium and calcium upon receptor potential

• 1.1. In crayfish, light stimulation of the retinular cells induces a depolarizing receptor potential.
• 2.2. Experiments were designed to determine the role of Na⁺ and Ca²⁺ on receptor potential during dark And light states.
• 3.3. Depolarization depends on Na⁺ and Ca²⁺ availability to the retinular cell.
• 4.4. Repolarization velocity and response duration depend on extracellular Ca²⁺ availability.
• 5.5. Light adaptation increases receptor potential dependence on calcium and sodium ions.
• 6.6. We analyse these results with respect to other invertebrate photoreceptors.

     

A Curated Resource for Phosphosite-specific Signature Analysis

1. Download : Download high-res image (123KB)
2. Download : Download full-size image

Highlights

• •Database of PTM site-specific phosphorylation signatures of kinases, perturbations and signaling pathways (PTMsigDB).
• •PTM signature enrichment analysis (PTM-SEA) outperformed gene-centric analysis in detection of EGF induced phospho signaling events.
• •PI3K perturbation signatures were readily detected in PI3Ka inhibited human breast cancer cells.
• •PTMsigDB and PTM-SEA can be freely accessed at https://github.com/broadinstitute/ssGSEA2.0.

     

Comparison of metabolic effects of EGF,TGF-α, and TGF-β1 in primary culture of fetal bovine myoblasts and rat L6 myoblasts

• 1.1. Comparative studies of EGF, TGF-α, and TGF-βl action on the synthesis of DNA and cellular proteins in rat L6 myogenic cells and fetal bovine myoblasts demonstrated considerable differences between particular growth factors, dependent on dose and target cells.
• 2.2. Among examined growth factors only EGF exerted mitostimulatory action, more pronounced at lower concentrations. EGF, progressively with dose, stimulated protein synthesis much more effectively in fetal bovine myoblasts than in L6 cells.
• 3.3. The dynamics of stimulation of protein synthesis by TGF-α was greater than by EGF in both examined types of cell cultures.
• 4.4. The maximal response of fetal bovine myoblasts to TGF-α in a concentration of 100 ng/ml reached 370%, whereas EGF in a 10 times higher concentration stimulated protein synthesis only to 123% of control.
• 5.5. In contrast to EGF, TGF-α significantly inhibits DNA synthesis. Inhibition of the mitogenic response with simultaneous stimulation of protein synthesis by TGF-α may indicate changes toward cell differentiation.
• 6.6. TGF-β 1 in smallest concentration inhibits both DNA and protein synthesis. The suppressive action of TGF-β 1 was more distinct in fetal bovine myoblasts than in the L6 cell line.
• 7.7. Increasing concentrations of TGF-β l diminished its inhibitory effect, even leading to stimulation of protein synthesis at higher doses in L6 myoblasts.