Anti-helix-loop-helix domain antibodies: discovery of autoantibodies that inhibit DNA binding activity of human centromere protein B (CENP-B).

Centromere protein B (CENP-B) is one of the centromere DNA binding proteins constituting centromeric heterochromatin of human chromosomes. This protein was originally identified as the target antigen in autoimmune disease patients (often with scleroderma). In this study, we cloned a human CENP-B cDNA which was longer than the previously isolated one and expressed functional recombinant CENP-B in Escherichia coli. The DNA binding domain was finely located within the N-terminal 134-amino-acid residues covering a predicted helix-loop-helix (HLH) structure, by using a set of recombinant products with stepwise deletions from the C-terminus. From the analysis of their reactivity to anti-centromere sera from autoimmune disease patients, four epitopes were mapped on CENP-B antigen. In addition to two epitopes at the C-terminus, two were found on the HLH region at the N-terminus. In the analysis of the interaction between the antigen and autoantibodies, we found that the DNA binding activity of CENP-B was distorted by the attack of the anti-HLH domain antibodies in in vitro binding reactions. Our results suggest that the direct inhibition of the DNA binding activity by the autoantibodies might be involved in patients' autoimmune reactions in vivo.     

Nucleic acid of flacherie viruses of the silkworm, Bombyx mori

It was reported previously that two spherical flacherie viruses of silkworm, FVS I and FVS II, had been isolated from flacherie silkworm larvae and the nucleic acid of FVS II was RNA as suggested by the experiments of incorporation of [³H]-uracil. In this paper, it has been confirmed by biochemical methods that the nucleic acid of FVS I and FVS II is RNA. FVS I and FVS II were labeled with ³²P in flacherie silkworms, and the viruses were analyzed by sucrose density gradient centrifugation. When the ³²P-labeled compound in the viruses was treated with 0.5 n KOH, the acid-insoluble ³²P-labeled compound changed to acid-soluble compounds. It was determined by paper chromatography and ion-exchange column chromatography that the alkali-decomposed compounds included four ribonucleotides. Therefore, the viral nucleic acid of FVS I and FVS II was determined to be RNA. The correlations between FVS I and FVS II particles were discussed, and it was suggested that FVS I and FVS II might be closely related or were the same viral species.     

Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process

Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3′-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3′-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3′-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences.     

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.     

A neural-specific F-box protein Fbs1 functions as a chaperone suppressing glycoprotein aggregation

Fbs1 is an F-box protein present abundantly in the nervous system. Similar to the ubiquitously expressed Fbs2, Fbs1 recognizes N-glycans at the innermost position as a signal for unfolded glycoproteins, probably in the endoplasmic reticulum-associated degradation pathway. Here, we show that the in vivo majority of Fbs1 is present as Fbs1-Skp1 heterodimers or Fbs1 monomers but not SCF(Fbs1) complex. The inefficient SCF complex formation of Fbs1 and the restricted presence of SCF(Fbs1) bound on the endoplasmic reticulum membrane were due to the short linker sequence between the F-box domain and the sugar-binding domain. In vitro, Fbs1 prevented the aggregation of the glycoprotein through the N-terminal unique sequence of Fbs1. Our results suggest that Fbs1 assists clearance of aberrant glycoproteins in neuronal cells by suppressing aggregates formation, independent of ubiquitin ligase activity, and thus functions as a unique chaperone for those proteins.     

Induction of steady-state glomeruloid sphere by self-assembly from human embryonic kidney cells

The glomerulus is a network of capillaries known as a tuft, located at the beginning of a nephron in the kidney. Here we describe a novel method for the induction of a macroscopically visible three-dimensional glomerulus-like sphere (GLS). This procedure did not require any additional cytokines and completed the formation of spheres within 24?h. After the formation was complete, GLS maintained a steady state for at least five days without proliferation and without a decrease in viability. Therefore, this procedure assists various assays for a prolong period of time. Overall, our protocol allows for a very simple mixing of cells from different sources to obtain fine-grained and highly dispersed GLSs. The kidney filtration barrier is a unique structure characterized by a complex three-dimensional framework of podocytes and endothelial cells. GLS exhibited the induction of many podocyte-specific gene profiles similar to those in adult human kidneys, suggesting that the sphere formation process is important for the maturation of podocytes. Focal segmental glomerulosclerosis (FSGS) is one of the major causes of steroid-resistant nephrotic syndrome, and some circulating permeability factors in the patient's serum FSGS have been implicated in the pathogenesis of the disease. Serum from patients with FSGS induced the collapse of GLS, which imitates the appearance of glomerulosclerosis in patients. In conclusion, the investigation and use of GLS may provide a novel method to elucidate the molecular mechanisms underlying complicated and unexplained events in glomeruli in a similar condition in adult kidneys.     

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.