The herpes simplex virus type 1 vhs-UL41 gene secures viral replication by temporarily evading apoptotic cellular response to infection: Vhs-UL41 activity might require interactions with elements of cellular mRNA degradation machinery

We have previously shown that herpes simplex virus type 1 (HSV-1) infection is associated with early destabilization/degradation of infected cell mRNAs and consequent shutoff of host protein synthesis by the activity of the virion-associated host shutoff (vhs) UL41 protein. Wild-type (wt) virus destabilized/degraded the housekeeping beta-actin and alpha-tubulin mRNAs as well host stress functions, like the heat shock 70 protein induced postinfection. vhs mutants did not degrade the mRNAs. Elaborate studies by others have been concerned with the mode of mRNA degradation and the mRNAs affected. We now describe vhs activity in primary cultures of mouse cerebellar granule neurons (CGNs). Specifically, (i) upon infection in the presence of actinomycin D to test activity of input viral particles, there was a generalized inhibition of protein synthesis, which depended on the input multiplicity of infection (MOI). (ii) Low-MOI infection with vhs-1 mutant virus was associated with increased synthesis of all apparent proteins. Higher MOIs caused some shutoff, albeit significantly lower than that of wt virus. This pattern could reflect an interaction(s) of vhs-1 protein with host machinery involved in cellular mRNA destabilization/degradation, sequestering this activity. (iii) wt virus infection was associated with cell survival, at least for a while, whereas mutant virus induced apoptotic cell death at earlier times. (iv) wt virus replicated well in the CGNs, whereas there was no apparent replication of the vhs-1 mutant virus. (v) The vhs-1 mutant could serve as helper virus for composite amplicon vectors carrying marker genes and the human p53 gene. Ongoing studies test the use of vhs-1-based composite oncolytic vectors towards cancer gene therapy.     

Bisquaternary Pyridinium Oximes as Presynaptic Agonists and Postsynaptic Antagonists of Muscarinic Receptors

A study of the effects of bisquaternary pyridinium oximes on calcium-dependent potassium-evoked [3H]acetylcholine release from rat brain slices revealed that at presynaptic autoreceptors these drugs function like muscarinic agonists, as they mimic the effects of acetylcholine in their inhibition of the evoked [3H]-acetylcholine release in an atropine-sensitive and dose-dependent manner. Since the bisquaternary pyridinium oximes are mild muscarinic antagonists at postsynaptic muscarinic receptors, they constitute a category of muscarinic ligands that are characterized by inverse dual activity at pre- and postsynaptic muscarinic receptors. These drugs may have dual function on cholinergic transmission by acting as presynaptic agonists and as postsynaptic antagonists. The most potent inhibitor of the evoked [3H]acetylcholine release was 1,1'-(4-hydroxyiminopyridinium)trimethylene (TMB-4) (I50 = 8 microM) and the weakest were 1-(2-hydroxyiminoethylpyridinium) 1-(3-cyclohexylcarboxypyridinium) dimethylether (HGG-42) and 1-(2-hydroxyiminoethylpyridinium) 1-(3-phenylcarboxypyridinium) dimethylether (HGG-12) (I50 = 150 microM). As postsynaptic antagonists, the latter drugs are more potent (K1 = 1.3-3.3 microM) than TMB-4 (K1 = 50 microM). Combined therapy with two drugs such as TMB-4 and HGG-12 might be effective in blocking severe hyperactivity of the cholinergic system.     

Effect of allogeneic tumor cells,interleukin-2 and interleukin-6, on the growth of subcutaneous syngeneic tumors

In the present study we demonstrate the ability of allogeneic M3 tumor cells to induce an antitumor response against the syngeneic tumor, when injected locally together with syngeneic B16 melanoma cells. The replacement of the allogeneic tumor cells with either syngeneic or allogeneic splenocytes had no effect on the growth of the syngeneic tumor. Systemic administration of both interleukin-2 (IL-2) and IL-6 did not affect the antitumor response induced by allogeneic tumor cells. When mice, previously injected with B16 and M3 cells, were rechallenged subcutaneously with B16 tumor cells at a different anatomical site, an inhibitory effect in some, but not all, experiments was observed. Systemic injections of either IL-2 or IL-6 did not alter the antitumor effects of the allogeneic and syngeneic tumor-cell mixtures. The significance of our results in developing immunotherapy modalities based on active immunization with allogeneic tumor cells and selected cytokines is discussed.This study was supported by a grant from the Israeli Cancer Association     

Growth Factor-Like Effects Mediated by Muscarinic Receptors in PC12M1 Cells

Rat pheochromocytoma (PC12) cells stably expressing cloned m1 muscarinic acetylcholine receptors (PC12M1) undergo morphological changes when stimulated by muscarinic agonists. These changes, which include the outgrowth of neurite-like processes, are blocked by the muscarinic antagonist atropine and are not observed in PC12 cells. The observed morphological changes, which are independent of RNA and protein synthesis, are blocked by the methylation inhibitor 5'-deoxy-5'-methylthioadenosine, suggesting that methylation plays a role in this process. Analysis of cyclic AMP accumulation and phosphoinositide turnover reveals that both processes are enhanced on activation by muscarinic agonist. Our data suggest, however, that the muscarinic-dependent neurite-like outgrowth processes are not mediated by cyclic AMP, Ca2+, or protein kinase C pathways. The muscarinic-dependent neurite outgrowth effect is enhanced by nerve growth factor, with a resulting increase in both the number of neurite-extending cells and the length of the neurite. In addition, activation of muscarinic receptors in PC12M1 cells stimulates the induction of marker genes for neuronal differentiation. Muscarinic receptors may therefore mediate growth factor-like effects in these cells.     

The Fission Yeast Mitotic Regulator win1+ Encodes an MAP Kinase Kinase Kinase That Phosphorylates and Activates Wis1 MAP Kinase Kinase in Response to High Osmolarity

The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle. Although the defect is suppressed by wis1⁺ and wis4⁺, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known. We show here that win1⁺ encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1. Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions. Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1 mutations. The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain. We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50 mutant and its suppression by wis genes.     

Interaction Between Allopurinol and Copper: Possible Role in Myocardial Protection

Allopurinol, a potent inhibitor of xanthine oxidase, is known to effectively protect the heart against damage in patients undergoing cardiac bypass surgery. There is still an ambiguity concerning the presence of xanthine oxidase in the human heart. Thus, the mechanism underlying the protective effect of allopurinol is unclear. Transition metal ions, such as iron and copper, can participate in single-electron reactions and mediate the formation of oxygen-derived free radicals. In this study the interaction between allopurinol and Cu(II) was investigated. Spectrophotometric investigation shows that allopurinol (0-0.8 mM) form a 1:1 complex with Cu(II) ions (0-0.8 mM) with a specific absorbance peak at 364 nm. Also, the rate constant (k) for the copper-catalyzed aerobic oxidation of ascorbate was markedly decreased in the presence of allopurinol (from 0.068 min^-1 to 0.014min^-1). Allopurinol substantially reduced the copper-mediated and ascorbate-driven DNA breakage. Spectrophotometric measurements did not indicate a specific interaction between iron ions and allopurinol. It is suggested that the beneficial effects of allopurinol during reperfusion of the heart could stem from its chelation of copper, yielding a complex with low redox activity.