Transfer of oligosaccharide from oligosaccharide pyrophosphoryl dolichol to endogenous acceptor proteins in human breast malignant and normal tissues

We have prepared dolichylpyrophosphoryl-[14C]-oligosaccharide (Dol-PP-oligosaccharide) from calf thyroid. Microsomal fractions from human breast tissues catalyzed the transfer of labeled oligosaccharide to endogenous acceptor proteins. Malignant tumors showed higher activity of the oligosaccharide transferring enzyme than normal tissue. With kojibiose (Kj), an inhibitor of (Glc3)-glucosidase, an increase in the radioactivity associated with glycoprotein was observed.     

Differences in membrane ion transport between hepatocytes from the periportal and the pericentral areas of the liver lobule

We studied the Na⁺/K⁺ pump, Na⁺/K⁺ ATPase activity, and oxygen consumption (QO₂) in hepatocytes isolated from the periportal (PH) and pericentral (CH) regions of the liver lobule, to provide an insight into the functional properties of these cells. Na⁺/K⁺ pump activity was determined using⁸⁶Rb⁺ (a functional analog of K⁺) and ouabain, a specific inhibitor of this transport system. Our results indicate the the Na⁺/K⁺, pump and Na⁺/K⁺ ATPase activity are significantly lower in CH than in PH, although basal ouabain-sensitive (OS) QO₂ was negligible in both of these cell preparations. However, OSQO₂ was significantly lower in CH than in PH when the Na⁺/K⁺ pump was activated using the ionophore nystatin in a Na⁺-containing medium. These results indicate that the differences in membrane ion transport exist between hepatocytes from different locations of the liver lobule.     

Differences in luteinizing hormone release stimulated in rat anterior pituitary cells by leukotriene C4 and by gonadotropin-releasing hormone in vitro

Summary Continuous administration of leukotriene C₄ (LTC₄, 10^–10 M) to superfused rat anterior pituitary cells increased LH release for 40 min only, whereas in a parallel experiment gonadotropin-releasing hormone (GnRH, 10^–9 M) evoked a continuous increase in hormone secretion. In contrast to GnRH, LTC₄ did not desensitize rat anterior pituitary cells. The secretory action resulting from the administration of LTC₄ (10^–10 M) was abolished for 40 min after previous stimulation. The results documented the dual action of LTC₄ on LH exocytosis.     

VIP and histamine H2 receptor activity in human fetal gastric glands

Summary Vasoactive intestinal peptide (VIP, EC₅₀=6.4×10^–10 M) and histamine (EC₅₀=3×10^–6 M) activated the cyclic AMP generating system in gastric glands isolated from two human fetuses at 23 weeks gestation. Histamine antagonism by the H₂ receptor blockers cimetidine (Ki=0.35×10^–6 M) and ranitidine (ki=0.51×10^–7 M) clearly characterized the histaminic activation as being of the H₂ type. It is suggested that these two vasoactive hormones may operate as neurocrine/paracrine regulators of the differentiation and/or function of the human gastric mucosa in utero.     

Impaired insulin release in aging rats: Metabolic and ionic events

We investigated the effect of aging on glucose uptake, glucose-induced O₂ consumption, glucose-induced⁴⁵Ca movements, and calmodulin content to elucidate age-related impairment of glucose-induced insulin release in pancreatic islets of Wistar rats. Intact pancreatic islets from old (24-month-old) rats showed impaired glucose-induced insulin release; glucose uptake and O₂ consumption were lower in old than in young (2-month-old) or adult (12-month-old) rats. Moreover,⁴⁵Ca uptake and calmodulin content were decreased in pancreatic islets from older rats, which explained the impairment in glucose-induced insulin release in aging. No major differences between the 3 age groups in glucose-induced⁴⁵Ca efflux in pancreatic islets were observed.     

Thermodynamics of bromate and iodate of potassium in dioxane-water mixtures from conductance measurements

Summary The free energy of transfer ( G _t ⁰ ) of KBrO₃ and KIO₃ from water to 10, 20 and 30% dioxane-water mixtures have been studied from conductometric measurements. The G _t ⁰ values are in the order of BrO ₃ ^– >IO ₃ ^– .     

Extracellular Mg2+ regulates intracellular Mg2+ and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe

Effects of extracellular magnesium ions ([Mg²⁺]_o ) on intracellular free Mg²⁺ ([Mg²⁺]_i ) and its subcellular distribution in single fission yeast cells, Schizosaccharomyces pombe, were studied with digital-imaging microscopy and an Mg²⁺ fluorescent probe (mag-fura-2). Using 0.44 mM [Mg²⁺]_o , [Mg²⁺]_i in yeast cells was 0.91±0.08 mM. Elevation of [Mg²⁺]_o to 1.97 mM induced rapid (within 5 min) increments in [Mg²⁺]_i (2.18±0.11 mM). Lowering [Mg²⁺]_o to 0.06 mM, however, exerted no significant effects on [Mg²⁺]_i (0.93±0.14 mM), at least for periods of up to 30 min. Irrespective of the [Mg²⁺]_o used, the subcellular distribution of [Mg²⁺]_i remained hetero geneous, i.e. where the sub-plasma membrane region >cytoplasm >nucleus. [Mg²⁺] in all three subcellular compartments increased significantly, two- to threefold, concomitant with [Mg²⁺]_i when placed in 1.97 mM [Mg²⁺]_o . We conclude that [Mg²⁺]_i in fission yeast is maintained at a physiologic level when [Mg²⁺]_o is low, but intracellular free Mg²⁺ rapidly rises when [Mg²⁺]_o is elevated. Like most eukaryotic cells, yeast may have a Mg²⁺ transport system(s) which functions to maintain gradients of Mg²⁺ from the outside to inside the cell and among its subcellular compartments. Received 18 April 1996; received after revision 4 July 1996; accepted 26 July 1996     

Bactericidal activity againstPseudomonas aeruginosa is acquired by cultured human monocyte-derived macrophages after uptake of myeloperoxidase

Myeloperoxidase (MPO) is an enzyme located within polymorphonuclear neutrophils capable of producting cytotoxic oxidant species that are particularly active against bacteria with polysaccharide capsules.Pseudomonas aeruginosa (10⁶ bacteria per 1 ml) are killed within 1 h in vitro by a MPO/H₂O₂/Cl^– system (48 mU=132 ng of MPO). The question arose as to whether human macrophages would acquire cytotoxic activity when loaded with this enzyme. Monocytes were therefore isolated from human blood and cultured for up to ten days to induce maturation to macrophages. These cells lost endogenous MPO within five days while H₂O₂ production in response to stimulation by phorbol myristate acetate (10^–6M) decreased to 23% within ten days. On the other hand, their capacity to take up exogenous MPO increased fourfold from day three to day ten. Human macrophages cultured from eight days (when both H₂O₂ production and MPO uptake were sufficient) were therefore used to study the effects of MPO uptake on cytocidal activity againstPseudomonas aeruginosa. After a 1 h MPO loading period, macrophages (5×10⁵ cells per ml) were incubated in the presence of bacteria (0.5 to 2×10⁶ bacteria per ml) for 2 h at 37°C. At a bacteria/macrophage ratio of 1, only 34.8±7.0% of bacteria survived (compared to killing by non-loaded macrophages), while 74.4±9.3% survived at a ratio of 4. From these results, we conclude that loading macrophages with exogenous MPO could enhance their microbicidal activity, suggesting a potentially useful therapeutic application.     

Die katalytische Mutation der Ionen Al3+ und MoO

Summary The catalytic mutation of the ions Al³⁺ and MoO ₄ ^2– on the mixed carrier Cd(OH)₂/Co(OH)₂(Tr) is recognizable by the fact that the combination Tr+Al³⁺+MoO ₄ ^2– is more active in the decomposition of H₂O₂ than the combination Tr+MoO ₄ ^2– +Al³⁺.     

Prevention of enteral radiocesium absorption by hexacyanoferrates(II) in piglets

Summary The efficacy of different hexacyanoferrates(II) in preventing the enteral absorption of¹³⁴Cs was studied in piglets. As compared to the controls, oral application of¹³⁴Cs together with KFe[Fe(CN)₆], NH₄Fe[Fe(CN)₆], or Fe₄[Fe(CN)₆]₃ resulted in a strong reduction of the¹³⁴Cs-uptake by more than 97%. The decrease in enteral absorption depends on the dose of administered hexacyanoferrate(II), whereas differences between the compounds under study were small. The biological half-life of¹³⁴Cs in non-hexacyanoferrate(II) treated piglets was 21.6±3.3 days (mean±SD).     

Glycoprotein misfolding in the endoplasmic reticulum: identification of released oligosaccharides reveals a second ER-associated degradation pathway for Golgi-retrieved proteins

Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process whereby misfolded proteins are removed from the endoplasmic reticulum (ER) for subsequent degradation by the ubiquitin/proteasome system. In the present work, analysis of the released, free oligosaccharides (FOS) derived from all glycoproteins undergoing ERAD, has allowed a global estimation of the mechanisms of this pathway rather than following model proteins through degradative routes. Examining the FOS produced in endomannosidase-compromised cells following α-glucosidase inhibition has revealed a mechanism for clearing Golgi-retrieved glycoproteins that have failed to enter the ER quality control cycle. The Glc₃Man₇GlcNAc₂ FOS species has been shown to be produced in the ER lumen by a mechanism involving a peptide: N-glycanase-like activity, and its production was sensitive to disruption of Golgi-ER trafficking. The detection of this oligosaccharide was unaffected by the overexpression of EDEM1 or cytosolic mannosidase, both of which increased the production of previously characterised cytosolically localised FOS. The lumenal FOS identified are therefore distinct in their production and regulation compared to FOS produced by the conventional route of misfolded glycoproteins directly removed from the ER. The production of such lumenal FOS is indicative of a novel degradative route for cellular glycoproteins that may exist under certain conditions.     

Cholinesterases are down-expressed in human colorectal carcinoma

The aberrations of cholinesterase (ChE) genes and the variation of ChE activity in cancerous tissues prompted us to investigate the expression of ChEs in colorectal carcinoma. The study of 55 paired specimens of healthy (HG) and cancerous gut (CG) showed that acetylcholinesterase (AChE) activity fell by 32% and butyrylcholinesterase (BuChE) activity by 58% in CG. Abundant AChE-H, fewer AChE-T, and even fewer AChE-R and BuChE mRNAs were observed in HG, and their content was greatly diminished in CG. The high level of the AChE-H mRNA explains the abundance of AChE-H subunits in HG, which as glycosylphosphatidylinositol (GPI)-anchored amphiphilic AChE dimers (G₂^A) and monomers (G₁^A) account for 69% of AChE activity. The identification of AChE-T and BuChE mRNAs justifies the occurrence in gut of A₁₂, G₄^H and PRiMA-containing G₄^A AChE forms, besides G₄^H, G₄^A and G₁^H BuChE. The down-regulation of ChEs might contribute to gut carcinogenesis by increasing acetylcholine availability and overstimulating muscarinic receptors. Received 19 May 2006; received after revision 5 June 2006; accepted 5 July 2006     

Calcium and the contractile response to prostaglandin in the smooth muscle of guinea-pig stomach

Summary In the longitudinal smooth muscle of guinea-pig stomach, verapamil (10^–5 M) which showed marked suppression of high K-induced contractures, did not suppress the contractile response to PGE₁ (1.5×10^–9 to 10^–6 M) markedly. These results suggest that the contractile mechanism of PGE₁ in guinea-pig stomach may mainly depend on a release of bound Ca in the cell and partly depend on a Ca influx from the extracellular origin.     

17β-estradiol in vitro affects Na-dependent and depolarization-induced Ca2+ transport in rat brain synaptosomes

Effects of 17-estradiol (E₂) in vitro on Na-dependent Ca²⁺ efflux from, and depolarization-induced Ca²⁺ uptake into, the nerve cell were studied with the use of synaptosomes isolated from the brain stem, mesencephalic reticular formation (MRF), caudate nucleus and the hippocampus of long-term ovariectomized adult female rats. It was found that E₂ (1) at a concentration of 10 nM or lower, stimulates Na-dependent Ca²⁺ efflux in the caudate nucleus and hippocampus, and does not affect the efflux in MRF and brain stem; (2) at concentrations above 10 nM has no effect on the Ca²⁺ efflux in any of the four structures investigated; and (3) produces a biphasic effect on the depolarization-induced Ca²⁺ uptake, increasing it in all structures except MRF at 10 nM concentration, and decreasing it at concentrations higher than 10 nM, irrespective of the structure investigated. These results suggest that E₂, acting at extranuclear sites, modulates synaptic transmission via alterations of Ca²⁺ transport mechanisms in nerve endings.     

Effect of potassium on isolated bovine facial and human saphenous veins

Summary A moderate elevation of external (K ₀ ⁺ ) (5–10 mM) induces relaxation in bovine facial and human saphenous veins. A further increase of (K ₀ ⁺ ) leads to biphasic reactions (relaxation followed by contraction). Concentrations of (K ₀ ⁺ ) higher than about 15 mM cause contractions only. The potassium-induced relaxation may be explained by the stimulation of an electrogenic sodium pump.     

Über die Wirkung blutzuckersenkender Sulfonylharnstoffe auf das isolierte Rattenzwerchfell

Summary The action of blood sugar depressing sulfonylureas on glucose and oxygen uptake, as well as on glycogen content and formation of C¹⁴O₂ from uniformly labelled C¹⁴-glucose was investigated in rat hemidiaphragms incubated in phosphate buffer. The following results were obtained: (1) Tolbutamide and Carbutamide increased the glucose uptake. (2) Tolbutamide decreased the glycogen-content. (3) Oxygen uptake as well as formation of C¹⁴O₂ were increased by Tolbutamide. (4) The action of Tolbutamide and insuline was equal with respect to glucose uptake but different as regarding the glycogen content, oxygen uptake and CO₂-formation.It is concluded that sulfonylureas increase glucose oxidation in the rat hemidiaphragm probably without increasing insulin sensitivity. To our knowledge this mechanism of action has hitherto not been described.     

Heavy metal hydrolysis of polyisoprenoid-phosphate mono- and oligosaccarides

Summary Mono- and oligosaccharide derivatives of dolichol phosphate can be hydrolyzed by heavy metal, preferably Zn⁺⁺ at 100°C or 65°C. The reactions follow first order kinetics. The reaction proceeds through hydrolysis of the sugar phosphate bond.This work was supported by GB No. 38335.     

Biosynthesis of 1-aminocyclopropanecarboxylic acid: Steric course of the reaction at the C-4 position

Summary Under the action of the appropriate synthase from ripe tomatoes a 11 mixture of (3S, 4R)-[3,4-²H₂] and (3R, 4S)-[3,4-²H₂]-(2S)-adenosylmethionine is transformed into a 11 mixture of the two meso forms of [²H₂]-1-aminocyclopropanecarboxylic acid, a result which proves the operation of an inversion mechanism and which is consistent with direct nucleophilic displacement of the leaving group in the substrate.     

Complex modulation of Cav3.1 T-type calcium channel by nickel

Nickel is considered to be a selective blocker of low-voltage-activated T-type calcium channel. Recently, the Ni²⁺-binding site with critical histidine-191 (H191) within the extracellular IS3–IS4 domain of the most Ni²⁺-sensitive Ca_v3.2 T-channel isoform has been identified. All calcium channels are postulated to also have intrapore-binding site limiting maximal current carried by permeating divalent cations (PDC) and determining the blockade by non-permeating ones. However, the contribution of the two sites to the overall Ni²⁺ effect and its dependence on PDC remain uncertain. Here we compared Ni²⁺ action on the wild-type “Ni²⁺-insensitive” Ca_v3.1_w/t channel and Ca_v3.1_Q172H mutant having glutamine (Q) equivalent to H191 of Ca_v3.2 replaced by histidine. Each channel was expressed in Xenopus oocytes, and Ni²⁺ blockade of Ca²⁺, Sr²⁺, or Ba²⁺ currents was assessed by electrophysiology. Inhibition of Ca_v3.1_w/t by Ni²⁺ conformed to two sites binding. Ni²⁺ binding with high-affinity site (IC₅₀ = 0.03–3 μM depending on PDC) produced maximal inhibition of 20–30 % and was voltage-dependent, consistent with its location within the channel’s pore. Most of the inhibition (70–80 %) was produced by Ni²⁺ binding with low-affinity site (IC₅₀ = 240–700 μM). Q172H-mutation mainly affected low-affinity binding (IC₅₀ = 120–160 μM). The IC₅₀ of Ni²⁺ binding with both sites in the Ca_v3.1_w/t and Ca_v3.1_Q172H was differentially modulated by PDC, suggesting a varying degree of competition of Ca²⁺, Sr²⁺, or Ba²⁺ with Ni²⁺. We conclude that differential Ni²⁺-sensitivity of T-channel subtypes is determined only by H-containing external binding sites, which, in the absence of Ni²⁺, may be occupied by PDC, influencing in turn the channel’s permeation.     

The action of the peptidoleukotriene LTD4 on intracellular calcium in rat mesangial cells

The dose-dependent effect of CGP 45715A on the LTD₄-induced Ca²⁺ response of glomerular mesangial cells has been studied. Our results demonstrate that the LTD₄-dependent increase in the cytosolic Ca²⁺ concentration primarily involves an InsP₃-mediated release of Ca²⁺ from intracellular storage sites and to a minor extent an enhanced influx of Ca²⁺ through receptor-operated Ca²⁺ channels located in the plasma membrane. The action of CGP 45715A on the Ca²⁺ response is an inhibitory one and is convincingly explained by a displacement of LTD₄ from its receptor site(s). The contractile effect of LTD₄ on pulmonary smooth muscle is proposed to be mainly caused by a receptor-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate.