Importance of the anti-interferon capacity of Sendai virus C protein for pathogenicity in mice

The Sendai virus (SeV) C protein blocks signal transduction of interferon (IFN), thereby counteracting the antiviral actions of IFN. Using HeLa cell lines expressing truncated or mutated SeV C proteins, we found that the C-terminal half has anti-IFN capacity, and that K(151)A, E(153)A, and R(154)A substitutions in the C protein eliminated this capacity. Here, we further created the mutant virus SeV Cm*, in which K(151)A, E(153)K, and R(157)L substitutions in the C protein were introduced without changing the amino acid sequence of overlapped P, V, and W proteins. SeV Cm* was found to lack anti-IFN capacity, as expected. While the growth rate and final yield of SeV Cm* were inferior to those of the wild-type SeV in IFN-responsive, STAT1-positive 2fTGH cells, SeV Cm* grew equivalently to the wild-type SeV in IFN-nonresponsive, STAT1-deficient U3A cells. SeV Cm* was thus shown to maintain multiplication capacity, except that it lacked anti-IFN capacity. Intranasally inoculated SeV Cm* could propagate in the lungs of STAT1(-/-) mice but was cleared from those of STAT1(+/+) mice without propagation. It was found that the anti-IFN capacity of the SeV C protein was indispensable for pathogenicity in mice. Conversely, the results show that the innate immunity contributed to elimination of SeV in early stages of infection in the absence of anti-IFN capacity.     

Effects of nitrogen supply restriction on gas exchange and photosystem 2 function in flag leaves of a traditional low-yield cultivar and a recently improved high-yield cultivar of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The effects of nitrogen (N) supply restriction on the CO₂ assimilation and photosystem 2 (PS2) function of flag leaves were compared between two contrastive Japanese rice cultivars, a low-yield cultivar released one century ago, cv. Shirobeniya (SRB), and a recently improved high-yield cultivar, cv. Akenohoshi (AKN). Both cultivars were solution-cultured at four N supply levels from N4 (control) to N1 (the lowest). With a reduction in N-supply, contents of N (LNC), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO), and chlorophyll (Chl) in flag leaves decreased in both cultivars. In parallel with this, the net photosynthetic rate (P _N), mesophyll conductance (g _m), and stomatal conductance (g _s) decreased. P _N was more dominantly restricted by g _m than g _s. The values of P _N, g _m, and RuBPCO content were larger in AKN than SRB at the four N supply levels. The content of Chl greatly decreased with N deficiency, but the reduction in the maximum quantum yield of PS2 was relatively small. Quantum yield of PS2 (Φ_PS2) decreased with N deficiency, and its significant cultivar difference was observed between the two cultivars at N1: a high value was found in AKN. The content ratio of Chl/RuBPCO was also significantly low in AKN. The low Chl/RuBPCO is one of the reasons why AKN maintained a comparatively high P _N and Φ_PS2 at N deficiency. The adequate ratio of N distribution between Chl and RuBPCO is the important prerequisite for the efficient and sustainable photosynthesis in a flag leaf of rice plant under low N-input.     

Testicular development in growth-retarded mice.

To assess delayed fertility in male growth-retarded (grt) mice with congenital primary hypothyroidism, their testes were chronologically examined. The testicular weight in grt mice was significantly lower than age-matched normal mice until 8 weeks but was comparable at 13 and 26 weeks. While normal mice had mature sperm cells in both testes and epididymides at 5 weeks, age-matched grt mice did not. The size of the seminiferous tubules in testes of grt mice was smaller than that of normal mice before 13 weeks but was comparable at 26 weeks. These findings suggest that male grt mice might need more than 13 weeks to develop mature testes.     

Infiltration of H-2d-specific cytotoxic macrophage with unique morphology into rejection site of allografted meth A (H-2d) tumor cells in C57BL/6 (H-2b) mice

It is assumed that CD8(+) cytotoxic T lymphocytes (CTLs) mediate direct lysis of allografts and that their growth, differentiation, and activation are dependent upon cytokine production by CD4(+) helper T lymphocytes. In the present study, the effector cells responsible for the rejection of i.p. allografted, CTL-resistant Meth A tumor cells from C57BL/6 mice were characterized. The cytotoxic activity was associated exclusively with peritoneal exudate cells and not with the cells in lymphoid organs or blood. On day 8, when the cytotoxic activity reached a peak, 3 types of cells (i.e., lymphocytes, granulocytes, and macrophages) infiltrated into the rejection site; and allograft-induced macrophages (AIM) were cytotoxic against the allograft. Bacterially-elicited macrophages also exhibited cytotoxic activity (approximately 1/2 of that of AIM) against Meth A cells, whereas the cytotoxic activity of AIM against these cells but not that of bacterially-elicited macrophages was completely inhibited by the addition of donor (H-2(d))-type lymphoblasts, suggesting H-2(d)-specific cytotoxicity of AIM against Meth A cells. In contrast, resident macrophages were inactive toward Meth A cells. Morphologically, the three-dimensional appearance of AIM showed them to be unique large elongated cells having radiating peripheral filopodia and long cord-like extensions arising from their cytoplasmic surfaces. The ultrastructural examination of AIM revealed free ribosomes in their cytoplasm, which was often deformed by numerous large digestive vacuoles. These results indicate that AIM are the H-2(d)-specific effector cells for allografted Meth A cells and are a more fully activated macrophage with unique morphological features.     

ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element

Arginine rich, mutated in early stage of tumors (ARMET) was first identified as a human gene highly mutated in a variety of cancers. However, little is known about the characteristics of the ARMET protein and its expression. We identified ARMET as a gene upregulated by endoplasmic reticulum (ER) stress. Here, we show that the mouse homologue of ARMET is an 18-kDa soluble ER protein that is mature after cleavage of a signal sequence and has four intramolecular disulfide bonds, including two in CXXC sequences. ER stress stimulated ARMET expression, and the expression patterns of ARMET mRNA and protein in mouse tissues were similar to those of Grp78, an Hsp70-family protein required for quality control of proteins in the ER. A reporter gene assay using a mouse ARMET promoter revealed that the unfolded protein response of the ARMET gene is regulated by an ERSE-II element whose sequence is identical to that of the HERP gene. ARMET is the second fully characterized ERSE-II-dependent gene and likely contributes to quality control of proteins in the ER.     

Role of the ompT mutation in stimulated decrease in colony-forming ability due to intracellular protein aggregate formation in Escherichia coli strain BL21

Recently we found that the cells of Escherichia coli strain BL21 producing a fusion protein, GST-Sup35NM, show a much more rapid decrease in colony-forming ability in the stationary phase than control cells. In this study, it was found that an extract of the cells producing GST-Sup35NM forms fibrous protein polymers containing GST-Sup35NM. In the course of the study, we realized that strain BL21 carried the ompT mutation. We suspected that the deficiency in OmpT protease was responsible for the observed phenotype. To test this, we introduced the wild-type ompT gene into strain BL21, and found that the transformed cells recovered the wild-type phenotype. We concluded that OmpT protease, though known to localize on the cell surface, is involved in protein quality control within the cell.