Nucleic Acids Synthesis of Nuclear Polyhedrosis Virus in Cultured Embryonic Cells of Silkworm

Embryos of the silkworm, Bombyx mori L., were dispersed by trypsin and the dissociated cells were cultured for infection with nuclear polyhedrosis virus (NPV) of the silkworm. The monolayer and suspension cultures were infected with NPV. RNA and DNA syntheses in the normal and NPV-infected cells were measured by incorporation of ³²P into RNA and DNA fractions. RNA and DNA syntheses in the cells after infection significantly increased over those in control cells (mock infection). The effects of actinomycin D, chloramphenicol and mitomycin C on RNA and DNA syntheses in infected cells were examined. The syntheses were inhibited by the antibiotics. It was suggested that the cellular DNA synthesis was inhibited by the viral infection, because the mitomycin C-resistant DNA synthesis was found in the normal cells but not in the infected cells treated with mitomycin C. The rate of DNA synthesis induced by NPV was immediately dropped to that of control cells by addition of chloramphenicol, while the RNA synthesis induced by NPV was not affected for 6 hr after the addition of chloramphenicol. If the antibiotic did not affect the size of precursor pools, this event suggested that the RNA polymerase concerned with viral RNA synthesis was more stable than the DNA polymerase participating in the viral DNA synthesis. The viral DNA as templates for RNA and DNA syntheses was decomposed by mitomycin C.     

Mechanism of Action of Bacillus thuringiensis Insecticidal Delta-endotoxin on Insect Cells in Vitro

Cultured insect cells, TN-368 from the cabbage looper, swelled and burst upon treatment with the enzyme-digested delta-endotoxin of Bacillus thuringiensis var. aizawai. The cytotoxic sweelling was depended upon the amount of the delta-endotoxin added and the concentration of NaCl or KC1 in the isotonic solution. The concentration of Na⁺ in the swollen cells approximately doubled in isotonic NaCl, while that of K⁺ decreased to 10% of the original cellular concentration. The cell swelling was inhibited by tetrodotoxin and also by ouabain in only KC1 isotonic solution. On the other hand, 4-aminopyridine stimulated the swelling in the isotonic KC1 solution, These results indicate that the delta-endotoxin induces the stimulation of Na+ influx and K+ efflux in the isotonic NaCl solution, and also stimulates the Na⁺, K⁺-ATPase in the isotonic KC1 solution. The cytotoxic swelling was also blocked by cAMP, AMP, ATP, GTP, and NAD, but not by adenosine and GMP. These results suggest the participation of nucleotide derivatives in the action of delta-endotoxin.     

Functional domain structure of human heterochromatin protein HP1(Hsalpha): involvement of internal DNA-binding and C-terminal self-association domains in the formation of discrete dots in interphase nuclei

Human heterochromatin protein HP1(Hsalpha) possesses two evolutionarily conserved regions in the N- and C-terminal halves, so-called chromo and chromo-shadow domains, and DNA-binding domain in the internal non-conserved region. Here, to examine its in vivo properties, we expressed HP1(Hsalpha) as a fusion product with green fluorescent protein in human cells. HP1(Hsalpha) was observed to form discrete dots in interphase nuclei and to localize in the centromeric region of metaphase chromosomes by fluorescence microscopy. Interestingly, this dot-forming activity was also found in the N-terminal half retaining the chromo and DNA-binding domains and in the C-terminal chromo-shadow domain. However, the chromo domain alone stained nuclei homogeneously. To correlate this dot-forming activity with self-associating activity in vitro, the chromo and chromo-shadow domain peptides were independently expressed in Escherichia coli, affinity purified, and chemically cross-linked with glutaraldehyde. In a SDS-polyacrylamide gel, the former mainly produced a dimer, while the latter produced a ladder of bands up to a tetramer. When passed through a gel filtration column in a native state, these peptides were exclusively separated as a dimer and a tetramer, respectively. These results suggested that the internal DNA-binding and C-terminal chromo-shadow domains are both involved in heterochromatin formation in vivo.     

Cathepsin L is crucial for a Th1-type immune response during Leishmania major infection

Prior to the activation of CD4+ T cells, exogenous proteins are digested by endo/lysosomal enzymes in antigen-presenting cells (APCs) to produce antigenic peptides that are presented on MHC class II molecules. In the studies described here, the functional significance of cathepsin L for antigen processing and Th1/Th2 differentiation in experimental leishmaniasis was investigated. We first demonstrated that cathepsin L is one of the candidates for endo/lysosomal enzymes in the processing of soluble Leishmania antigen (SLA) by using CLIK148, a specific inhibitor of cathepsin L. Treatment of BALB/c or DBA/2 mice with CLIK148 exacerbated the disease by enhancing an SLA-specific Th2-type response such as IL-4 production. CLIK148 did not exert any direct influence on Leishmania major promastigotes themselves or on the course of L. major infection in SCID mice. Taken together, these findings suggest that treatment of host mice with CLIK148 affects the processing of SLA in APCs, resulting in the potentiation of Th2-type immune responses and thus leading to exacerbation of the disease. Furthermore, endo/lysosomal cathepsin L was found to be functionally distinct from previously described cathepsins B and D.     

Localized compliance of small airways in excised rat lungs using microfocal X-ray computed tomography.

Airway compliance is a key factor in understanding lung mechanics and is used as a clinical diagnostic index. Understanding such mechanics in small airways physiologically and clinically is critical. We have determined the "morphometric change" and "localized compliance" of small airways under "near"-physiological conditions; namely, the airways were embedded in parenchyma without dehydration and fixation. Previously, we developed a two-step method to visualize small airways in detail by staining the lung tissue with a radiopaque solution and then visualizing the tissue with a cone-beam microfocal X-ray computed tomography system (Sera et al. J Biomech 36: 1587-1594, 2003). In this study, we used this technique to analyze changes in diameter and length of the same small airways ( approximately 150 microm ID) and then evaluated the localized compliance as a function of airway generation (Z). For smaller (<300-microm-diameter) airways, diameter was 36% larger at end-tidal inspiration and 89% larger at total lung capacity; length was 18% larger at end-tidal inspiration and 43% larger at total lung capacity than at functional residual capacity. Diameter, especially at smaller airways, did not behave linearly with V(1/3) (where V is volume). With increasing lung pressure, diameter changed dramatically at a particular pressure and length changed approximately linearly during inflation and deflation. Percentage of airway volume for smaller airways did not behave linearly with that of lung volume. Smaller airways were generally more compliant than larger airways with increasing Z and exhibited hysteresis in their diameter behavior. Airways at higher Z deformed at a lower pressure than those at lower Z. These results indicated that smaller airways did not behave homogeneously.     

Antibodies specific for heat shock proteins in human and murine malaria

Heat shock proteins (HSPs) are immunodominant antigens recognized by the host immune system in various infectious diseases. We analyzed HSP-specific antibodies, including immunoglobulin G (IgG), IgM and IgA, in sera from malaria patients in Thailand by using an enzyme-linked immunosorbent assay. All of the antibodies to HSP90 were remarkably increased in the patients compared with those in controls, while only IgM to HSP70 or IgA to HSP65 was significantly elevated. Further experiments showed that anti-HSP IgG was significantly increased in C57BL/6 mice infected with a non-lethal strain of Plasmodium yoelii, with anti-HSP90 IgG being the most elevated. These results suggest that the antigenic potential of HSP90 is higher than those of HSP70 and HSP65 in malaria infection.     

Endothelin-1 binding to endothelin receptors in the rat anterior pituitary gland: possible formation of an ETA-ETB receptor heterodimer

1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ET_A and ET_B receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds.2. In saturation binding studies with increasing concentrations (0.77–200 pM) of ¹²⁵I-ET-1 (nonselective bivalent radioligand), ¹²⁵I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a K _D of 71 pM and a B _max of 120 fmol mg^–1. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of K _D and 8.0 fmol mg^–1 of B _max, whereas 10 nM sarafotoxin S6c (ET_B agonist) exerted little change in these binding parameters (K _D, 72 pM; B _max, 110 fmol mg^–1).3. Competition binding studies with a fixed amount (3.8 pM) of ¹²⁵I-ET-1 revealed that when 1.0 M BQ-123 was present in the incubation buffer, ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ET_B agonist), and BQ-788 (ET_B antagonist) competitively inhibited ¹²⁵I-ET-1 binding with K _is of 140, 18, 350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for ¹²⁵I-ET-1 binding in the case of absence of BQ-123.4. In cold-ligand saturation studies with a fixed amount (390 pM) of ¹²⁵I-IRL 1620 (ET_B radioligand), IRL1620 bound to a single population of the ET_B receptor, and no change was observed in binding characteristics in the presence of 1.0 M BQ-123. ¹²⁵I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with K _is of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 M BQ-123.5. Two nonbivalent ET_A antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for ¹²⁵I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c.6. Taken together with the present finding that mRNAs encoding the rat ET_A and the ET_B receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ET_A and ET_B receptors with a functional binding capability for ET receptor-ligands, the ET_B receptor does not independently recognize ET-1 without the aid of the ET_A receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ET_A–ET_B receptor heterodimer.     

SOCS-3 regulates onset and maintenance of T(H)2-mediated allergic responses

Members of the suppressor of cytokine signaling (SOCS) family are involved in the pathogenesis of many inflammatory diseases. SOCS-3 is predominantly expressed in T-helper type 2 (T(H)2) cells, but its role in T(H)2-related allergic diseases remains to be investigated. In this study we provide a strong correlation between SOCS-3 expression and the pathology of asthma and atopic dermatitis, as well as serum IgE levels in allergic human patients. SOCS-3 transgenic mice showed increased T(H)2 responses and multiple pathological features characteristic of asthma in an airway hypersensitivity model system. In contrast, dominant-negative mutant SOCS-3 transgenic mice, as well as mice with a heterozygous deletion of Socs3, had decreased T(H)2 development. These data indicate that SOCS-3 has an important role in regulating the onset and maintenance of T(H)2-mediated allergic immune disease, and suggest that SOCS-3 may be a new therapeutic target for the development of antiallergic drugs.     

Lysosomal cysteine protease, cathepsin B, is targeted to lysosomes by the mannose 6-phosphate-independent pathway in rat hepatocytes: site-specific phosphorylation in oligosaccharides of the proregion

Cathepsin B, a lysosomal cysteine protease, is synthesized as a glycoprotein with two N-linked oligosaccharide chains, one of which is in the propeptide region while the other is in the mature region. When cultured rat hepatocytes were labeled with [(32)P]phosphate, (32)P-labeled cathepsin B was immunoprecipitated only in the proform from cell lysates and medium. Either Endo H or alkaline phosphatase treatment of (32)P-labeled procathepsin B demonstrated the acquisition of a mannose 6-phosphate (Man 6-P) residue on high mannose type oligosaccharides. To identify the site of phosphorylation, immunoisolated (35)S- or (32)P-labeled procathepsin B was incubated with purified lysosomal cathepsin D, since cathepsin D cleaves 48 amino acid residues from the N-terminus of procathepsin B, in which one N-linked oligosaccharide chain was also included [Kawabata, T. et al. (1993) J. Biochem. 113, 389-394]. Treatment of intracellular (35)S-labeled procathepsin B with a molecular mass of 39-kDa with cathepsin D resulted in the production of the 31-kDa intermediate form, but the (32)P-label incorporated into procathepsin B disappeared after treatment with cathepsin D. These results indicate that the phosphorylation of procathepsin B is restricted to an oligosaccharide chain present in the propeptide region. Interestingly, cathepsin B sorting to lysosomes was not inhibited by NH(4)Cl treatment and about 90% of the intracellular procathepsin B initially phosphorylated was secreted into the medium without being dephosphorylated intracellularly, and did not bind significantly to cation-independent-Man 6-P receptor, suggesting the failure of Man 6-P-dependent transport of procathepsin B to lysosomes. Additionally, about 50% of the newly synthesized (35)S-labeled cathepsin B was retained in the cells in mature forms consisting of a 29-kDa single chain form and a 24-kDa two chain form, while part of the procathepsin B was associated with membranes in a Man 6-P-independent manner. Taken together, these results show that in rat hepatocytes, cathepsin B is targeted to lysosomes by an alternative mechanism(s) other than the Man 6-P-dependent pathway.