A tracer method to study unidirectional fluxes of lithium: Application to frog skin

We describe a new tracer method to measure unidirectional fluxes of Li⁺, despite the lack of any utilizable radioisotope of lithium. This method uses the purified stable isotopes, ⁶Li and ⁷Li, detected with an ion-probe microanalyser. The accuracy is comparable to that obtained for other ions (e.g., Na⁺) with radiotracers.The method has been applied to frog skin with both faces bathed in a 20% lithium/80% sodium medium. Sodium and lithium unidirectional fluxes have been measured simultaneously. The results are consistent with lithium being actively pumped, the outflux of lithium being, however, much larger than that of sodium.     

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Dynamic features of cells expressing macrophage properties in tissue cultures of dissociated cerebral cortex from the rat

Summary Two previously identified forms of macrophage were investigated in primary cultures of cerebral cortical cells. Dynamic features were revealed through time-lapse video recording and aspects of macrophage function were assessed. The two cell forms were shown to be different pre-mitotic stages of a single cell type. The cell cycle for these cells involved an initial large, flat, quiescent cell which retracted to yield a slightly rounded form with numerous processes. This latter form lost processes and developed profuse filopodia as it became very rounded just prior to division; both resulting daughter cells then regained the initial large flat appearance. These cells possessed several properties of macrophages, including phagocytosis, nucleoside diphosphatase enzyme, and CR3 receptors. These properties were transient, expressed just before and after mitosis, but subsequently down-regulated in the flat daughter cells. Because of this feature, it was difficult to determine the exact size of this cell population; however, the observed rate of proliferation suggests it may be substantial. It is suggested that these cells correspond to non-microglial macrophages of brain tissue and, because of their significant down-regulation, they may be difficult to detect. This may be important in studies of brain accessory immune cells in tissue culture.     

Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity

Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the prosurvival IGF-I/AKT/FOXO3 pathway by redox activation of the stress kinases p38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes, IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that, in astrocytes exposed to oxidative stress by hydrogen peroxide (H₂O₂), p38 activation did not inhibit AKT (protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and more sustained in astrocytes than in neurons either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H₂O₂-exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury.     

Phosphorylation of elF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells

Mitogenic stimulation of protein synthesis is accompanied by an increase in elF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition. However, the phosphorylation state, as well as the turnover of phosphate on elF-4E, remained unchanged. Apparently, the change in protein synthesis after RA addition is regulated by another mechanism than elF-4E phosphorylation. By using P19 cells overexpressing the EGF receptor, we show that the signal transduction pathway that leads to phosphorylation of elF-4E is present in P19 cells; the EGF-induced change in phosphorylation of elF-4E in these cells is likely to be regulated by a change in elF-4E phosphatase activity. These results suggest that the onset of retinoic acid-induced differentiation is triggered by a signal transduction pathway which involves changes in protein synthesis, but not elF-4E phosphorylation. © 1995 Wiley-Liss, Inc.     

Time course of pump inhibition by ouabain in amphibian epithelia

Inhibition by ouabain of rheogenic Na⁺ transport across the basolateral membranes of frog skin is found to be manifest within 3–4 min. This rate of pump inhibition is not different from the rate of diffusion through extracellular tissue layers between the serosal bath and the actual site of action, i.e., the epithelial cell layers. It is concluded that the well-known slow time course of decrease in transepithelial current flow is due ionic redistribution and conductance changes of the epithelial membranes secondary to pump inhibition.     

An Ecteola-cellulose chromatography assay for 3′-phosphoadenosine 5′-phosphosulfate: Phenol sulfotransferase

A new assay procedure for phenol sulfotransferase which employs [35S]-3'-phosphoadenosine-5'-phosphosulfate as a sulfate donor and a variety of phenols as sulfate acceptors was developed. The appearance of the 35S-sulfated products or the disappearance of the [35S]-3'-phosphoadenosine-5'-phosphosulfate are determined simultaneously by chromatography of the assay incubation mixtures on Ecteola-cellulose columns, eluting with an NH4HCO3 step gradient. Various acidic, neutral, and basic phenols can be employed as substrates for phenol sulfotransferase using this procedure.     

Add-on therapy with anagliptin in Japanese patients with type-2 diabetes mellitus treated with metformin and miglitol can maintain higher concentrations of biologically active GLP-1/total GIP and a lower concentration of leptin

Metformin, α-glucosidase inhibitors (α-GIs), and dipeptidyl peptidase 4 inhibitors (DPP-4Is) reduce hyperglycemia without excessive insulin secretion, and enhance postprandial plasma concentration of glucagon-like peptide-1 (GLP-1) in type-2 diabetes mellitus (T2DM) patients. We assessed add-on therapeutic effects of DPP-4I anagliptin in Japanese T2DM patients treated with metformin, an α-GI miglitol, or both drugs on postprandial responses of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), and on plasma concentration of the appetite-suppressing hormone leptin. Forty-two Japanese T2DM patients with inadequately controlled disease (HbA1c: 6.5%–8.0%) treated with metformin (n = 14), miglitol (n = 14) or a combination of the two drugs (n = 14) received additional treatment with anagliptin (100 mg, p.o., b.i.d.) for 52 weeks. We assessed glycemic control, postprandial responses of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), and on plasma concentration of leptin in those patients. Add-on therapy with anagliptin for 52 weeks improved glycemic control and increased the area under the curve of biologically active GLP-1 concentration without altering obesity indicators. Total GIP concentration at 52 weeks was reduced by add-on therapy in groups treated with miglitol compared with those treated with metformin. Add-on therapy reduced leptin concentrations. Add-on therapy with anagliptin in Japanese T2DM patients treated with metformin and miglitol for 52 weeks improved glycemic control and enhanced postprandial concentrations of active GLP-1/total GIP, and reduce the leptin concentration.