The in vitro synthesized and processed human insulin receptor precursor binds insulin.

The cell-free examination of the human insulin receptor during biogenesis may provide a greater understanding of the elements that contribute to the acquisition of receptor function. The insulin receptor precursor components were produced in a cell-free system and the insulin binding ability of the [35S]methionine-labeled translation products was determined. The processed proreceptor represented by a 190 kDa band was retained on insulin-linked biotin-streptavidin agarose or an insulin column. The insulin binding 190 kDa band migrated slower than the non-binding 190 kDa band on SDS-PAGE which suggests that covalent modifications account for these differences. The trypsin-digested product of the 190 kDa proreceptor was also retained on insulin-linked biotin-streptavidin agarose, however the alpha-subunit precursor was retained on insulin agarose to a much lesser degree. We conclude that a significant fraction of the processed, in vitro translated insulin proreceptor acquires insulin binding ability.     

An efflux pump is involved in secretion of newly synthesized siderophore by Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes the fluorescent siderophore, pyoverdine (PVD), to enable iron acquisition. Epifluorescence microscopy and cellular fractionation were used to investigate the role of an efflux pump, PvdRT-OpmQ, in PVD secretion. Bacteria lacking this efflux pump accumulated PVD, or a fluorescent precursor, in the periplasm, due to their inability to efficiently secrete into the media newly synthesized PVD. PvdRT-OpmQ is only the second system identified for secretion of newly synthesized siderophores by Gram negative bacteria.     

In vitro effects of ethanolamine on insulin secretion

The effects of ethanolamine on insulin secretion by the perfused rat pancreas were examined. During the second phase of glucose-induced insulin secretion 5-minute perfusions of ethanolamine at final concentrations of 0.1, 1 and 10 mM inhibited insulin release in a dose-related manner. When given throughout the experiment the highest dose of ethanolamine markedly suppressed both phases of glucose-induced insulin release. The inhibitory effect of ethanolamine was blunted in the presence of phentolamine. It is concluded that ethanolamine inhibits glucose-induced insulin secretion by the perfused rat pancreas and that alpha-adrenergic receptors play a role in its actions on insulin output.     

The intracellular retention of newly synthesized platelet-activating factor

The ether phospholipid platelet-activating factor (PAF) has been generally assumed to be released into the extracellular environment by the cells of origin, whereupon it effects its well-known mediator functions. However, during the generation of PAF by human neutrophils, it was noted that the majority of the measurable PAF remained associated with the cells. Accordingly, the intracellular and extracellular distribution of PAF was examined in neutrophils and several other cell types. No PAF was detected in association with unstimulated neutrophils. However, in stimulated neutrophils, PAF was produced and the majority of this material remained in association with the cells independent of the type of stimulus, dose of stimulus, or method of cell isolation used. This pattern of cell association of PAF was seen in all but one of the cell types tested. The retention of PAF by stimulated neutrophils was not due to spurious underestimates of the extracellular levels due to extracellular metabolism and inactivation of released PAF, nor to release followed by readsorption or binding of PAF to the cells. The retention of PAF also occurred in the presence of plasma and appears to be a common phenomenon. Thus, the majority of newly synthesized PAF appears to be retained within the cell and not released.     

Polarized secretion of newly synthesized lipoproteins by the Caco-2 human intestinal cell line

Lipoprotein secretion by Caco-2 cells, a human intestinal cell line, was studied in cells grown on inserts containing a Millipore filter (0.45 micron), separating secretory products from the apical and basolateral membranes into separate chambers. Under these conditions, as observed by electron microscopy, the cells formed a monolayer of columnar epithelial cells with microvilli on the apical surface and tight junctions between cells. The electrical resistances of the cell monolayers were 250-500 ohms/cm2. Both 14C-labeled lipids and 35S-labeled proteins were used to assess lipoprotein secretion. After a 24-hr incubation with [14C]oleic acid, 60-80% of the secreted triglyceride (TG) was in the basolateral chamber; 40% of the TG was present in the d less than 1.006 g/ml (chylomicron + VLDL) fraction and 50% in the 1.006 less than d less than 1.063 g/ml (LDL) fraction. After a 4-hr incubation with [35S]methionine, apolipoproteins were found to be major secretory products with 75-100% secreted to the basolateral chamber. Apolipoproteins B-100, B-48, E, A-I, A-IV, and C-III were identified by immunoprecipitation. The d less than 1.006 g/ml fraction was found to contain all of the major apolipoproteins, while the LDL fraction contained primarily apoB-100 and apoE; the HDL (1.063 less than d less than 1.21 g/ml) fraction principally contained apoA-I and apoA-IV. Mn-heparin precipitated all of the [35S]methionine-labeled apoB-100 and B-48 and a majority of the other apolipoproteins, and 80% of the [14C]oleic acid-labeled triglyceride, but only 15% of the phospholipid, demonstrating that Caco-2 cells secrete triglyceride-rich lipoproteins containing apoB. Secretion of lipoproteins was dependent on the lipid content of the medium; prior incubation with lipoprotein-depleted serum specifically reduced the secretion of lipoproteins, while addition of both LDL and oleic acid to the medium maintained the level of apoB-100, B-48, and A-IV secretion to that observed in the control cultures.     

Endoplasmic reticulum stress causes insulin resistance by inhibiting delivery of newly synthesized insulin receptors to the cell surface

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates a signaling network known as the unfolded protein response (UPR). Here we characterize how ER stress and the UPR inhibit insulin signaling. We find that ER stress inhibits insulin signaling by depleting the cell surface population of the insulin receptor. ER stress inhibits proteolytic maturation of insulin proreceptors by interfering with transport of newly synthesized insulin proreceptors from the ER to the plasma membrane. Activation of AKT, a major target of the insulin signaling pathway, by a cytosolic, membrane-bound chimera between the AP20187-inducible F_V2E dimerization domain and the cytosolic protein tyrosine kinase domain of the insulin receptor was not affected by ER stress. Hence, signaling events in the UPR, such as activation of the JNK mitogen-activated protein (MAP) kinases or the pseudokinase TRB3 by the ER stress sensors IRE1α and PERK, do not contribute to inhibition of signal transduction in the insulin signaling pathway. Indeed, pharmacologic inhibition and genetic ablation of JNKs, as well as silencing of expression of TRB3, did not restore insulin sensitivity or rescue processing of newly synthesized insulin receptors in ER-stressed cells.     

The protective effect of newly synthesized compounds against the action of UV-C irradiation on human lymphocytes in vitro

In this work we present our data about the protective effect of the newly synthesized compound 1-(4-fluorphenyltioureido)-4-methyl-piperazyne (FTMP) against high doses UV-C irradiation using human lymphocytes in vitro as a model system. The endpoint used was chromosome aberrations. The genotoxic effect of different UV-C doses in the range from 10 J/m2 to 200 J/m2 was evaluated. Studying the protective effect of FTMP, it was obtained that a low, adaptive concentration (10(-6) mol/l) applied before harmful doses of UV-C irradiation (100 J/M2 and 150 J/M2) induces the yield of chromosome aberrations lower than theoretical, estimated as a sum of single effects of both agents. A tendency for reducing the damage effect of UV-C irradiation was established. The effect is the most clear when a 4-hour interval between the treatments was used. The mitotic index was not affected. These results pointed out the ability of FTMP to decrease the damaging effect of UV-C irradiation in this type of cells and possess the potential to induce the adaptive response against the damaging effect of UV-C irradiation.     

The sites of deposition of newly synthesized histone.

The chromosomal fragments produced by nuclease digestion of freshly replicated chromatin migrate more rapidly relative to bulk chromatin when analyzed in nucleoprotein gels. The cause of the anomalous migration has been studied and the evidence indicates that rather than reflecting a shorter nucleosomal repeat in vivo that it may be a consequence of nucleosome sliding during the digestion itself. The distinct electrophoretic characteristics of nucleosomal material containing newly replicated DNA have enabled us to examine their histone composition by two dimensional electrophoresis. We find that nucleosomes containing new DNA also contain newly synthesized histones H3 and H4. In contrast more than 50% of newly synthesized H2A and H2B, and essentially all of new H1, are deposited at sites on the bulk chromatin distinct from that material containing newly replicated DNA. In addition we show that newly synthesized histones H3 and H4 are bound unusually weakly when they first become associated with the chromatin.     

In vitro enhancement of insulin secretion by growth hormone

Using the isolated perfused hamster pancreas bovine growth hormone at a concentration of 7.5 ug per ml was recycled for 1 hour prior to stimulation by glucose. Under these conditions the total insulin secreted was roughly doubled compared to the controls during 1 hour of glucose stimulation.