Adenovirus early gene products may control viral mRNA accumulation and translation in vivo

The mechanisms controlling early adenovirus gene expression in vivo have been studied using inhibitors of protein synthesis. When inhibitors were added shortly before or at the onset of infection, viral mRNA from all early regions was transcribed, spliced and accumulated over a 7 hr period. After longer pretreatment, accumulation of several early mRNAs were suppressed. Addition of inhibitors 1 hr after infection enhanced the accumulation of viral mRNA in the cytoplasm. Translation of early mRNA selected on adenovirus DNA in a cell-free system reflected the amount of viral mRNA present. A viral coded product may therefore control accumulation of viral mRNA.A different pattern emerged when inhibitors of protein synthesis were removed at 5 hr postinfection and cells were pulse-labeled in vivo. If inhibitors were introduced at or before infection, early viral proteins were synthesized only after a lag of 1–3 hr. However, if treatment was introduced 1 hr post-infection, reversion of the protein synthesis block was instantaneous. It appears that protein synthesis inhibitors reveal an in vivo translational block for viral mRNA. This block could be overcome by preinfection with a related virus. Furthermore, no block was observed in a virus-transformed human embryonic kidney cell line (293) which expresses early region 1 of the viral genome. Viral gene product(s) encoded in early region 1 may control translation of early adenovirus messenger RNA in vivo.     

Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)_n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.     

The Redox Biochemistry of Protein Sulfenylation and Sulfinylation

Controlled generation of reactive oxygen species orchestrates numerous physiological signaling events (Finkel, T. (2011) Signal transduction by reactive oxygen species. J. Cell Biol. 194, 7–15). A major cellular target of reactive oxygen species is the thiol side chain (RSH) of Cys, which may assume a wide range of oxidation states (i.e. −2 to +4). Within this context, Cys sulfenic (Cys-SOH) and sulfinic (Cys-SO₂H) acids have emerged as important mechanisms for regulation of protein function. Although this area has been under investigation for over a decade, the scope and biological role of sulfenic/sulfinic acid modifications have been recently expanded with the introduction of new tools for monitoring cysteine oxidation in vitro and directly in cells. This minireview discusses selected recent examples of protein sulfenylation and sulfinylation from the literature, highlighting the role of these post-translational modifications in cell signaling.     

S-100 protein in human lung neuroendocrine neoplasms. Immunohistochemical study of 14 cases and review of the literature

A group of lung neuroendocrine (NE) neoplasms are investigated in view of the possible presence of S-100 protein immunoreactivity in their cells. The selected tumours were classified according to Gould et al. (1983a) and Mosca et al. (1985). They comprise 5 carcinoids, 3 neuroendocrine carcinomas of the well-differentiated type, or peripheral carcinoids, 5 neuroendocrine carcinomas of the intermediate cell type, or intermediate-cell, poorly differentiated carcinomas, 3 neuroendocrine carcinomas of the microcytoma type, or small cell carcinomas-SCC and a nodal metastasis of microcytoma. All but 2 tumours were immunoreactive for neuron specific enolase (NSE). Few S-100 immunoreactive cells were detected in 4 out of 5 carcinoids, in 1 out of 3 peripheral carcinoids, in 4 out of 5 poorly differentiated carcinomas and in the 3 microcytomas examined. No S-100 positive cells were found in the SCC's nodal metastasis. The S-100 immunolabelled cells can be interpreted as dendritic reticulum cells migrating through the tumours. However, in one case of typical carcinoid, abundant S-100 positive cells were detected: their stellate morphology and their intimate relation with neoplastic cells suggest that they are part of the neoplasia as a sort of satellite cell.     

Differential permeabilization of membranes by saponin treatment of isolated rat hepatocytes. Release of secretory proteins.

Monolayer cultures of rat hepatocytes were treated with increasing concentrations of saponin (prepared from Gypsophila plants) for 30 min at 6 degrees C. Differential permeabilization of the intracellular membranes could be demonstrated: at 0.040 mg of saponin/ml the plasma membrane was permeabilized, as assessed by the release of 50% of the total cellular amount of lactate dehydrogenase, and at 0.20 mg/ml the endoplasmic reticulum was permeabilized, as measured by the release of 50% of pulse-35S-labelled albumin. The Golgi complex was permeabilized at an intermediate saponin concentration, as indicated by the release of homogeneously 35S-labelled albumin; about half the intracellular albumin is located in this organelle. At 1.0 up to 5.0 mg of saponin/ml 90-95% of the radioactively labelled albumin was released. Even at 5.0 mg/ml less than 10% of the membrane of the endoplasmic reticulum was solubilized, as judged by the degree of release of a membrane-bound enzyme specific for this organelle. These results demonstrate the usefulness of saponin as a tool for investigating the interior of different intracellular compartments.     

Specific and nonspecific effects of nucleotides on hormone-induced phosphoinositide turnover in permeabilized human pituitary tumour cells (Flow 9000)

Previous studies have shown that agonist-induced inositol phosphate formation in the human embryonic pituitary cell line Flow 9000 is regulated by guanine nucleotides, and it is likely that a guanine nucleotide-binding protein is involved in coupling receptors to phosphoinositidase C (PIC) [(1986) Biochem.Soc.Trans. 14, 1135-1136]. We have now tested the specificity of various nucleotides in regulating PIC activity in the absence or presence of the hormone cholecystokinin (CCK-8) in saponin-permeabilized [3H]inositol-labelled Flow 9000 cells. We found that all nucleotides tested (i.e. CTP, UTP, ITP, TTP, GTP, GppNHp, GTP[S], ATP, AppNHp and ATP[S]) stimulated total [3H]inositol phosphate ([3H]IP) formation in a dose-dependent manner with similar potency and efficacy. However, only guanine nucleotides significantly enhanced CCK-8 stimulation of [3H]IP production. These results indicate a physiological role for guanine nucleotides in regulating hormone-induced phosphoinositide turnover. In addition, the effects of nucleotides on calcium-dependent PIC activity are discussed.     

Energy-linked nicotinamide-nucleotide transhydrogenase. Characterization of reconstituted ATP-driven transhydrogenase from beef heart mitochondria

The interaction between pure transhydrogenase and ATPase (Complex V) from beef heart mitochondria was investigated with transhydrogenase-ATPase vesicles in which the two proteins were co-reconstituted by dialysis or dilution procedures. In addition to phosphatidylcholine and phosphatidylethanolamine, reconstitution required phosphatidylserine and lysophosphatidylcholine. Transhydrogenase-ATPase vesicles catalyzed a 20-30-fold stimulation of the reduction of NADP+ or thio-NADP+ by NADH and a 70-fold shift of the apparent equilibrium expressed as the nicotinamide nucleotide ratio [NADPH][NAD+]/[NADP+][NADH]. In both of these respects, the transhydrogenase-ATPase vesicles were severalfold more efficient than beef heart submitochondrial particles. By measuring the ATP-driven transhydrogenase and the oligomycin-sensitive ATPase activities simultaneously and under the same conditions at low ATP concentrations, i.e. below 15 microM, the ATP-driven transhydrogenase/oligomycin-sensitive ATPase activity ratio was found to be about 3. This value is consistent with the stoichiometries of three protons translocated per ATP hydrolyzed and one proton translocated per NADPH formed and with a mechanism where the two enzymes interact through a delocalized proton-motive force.