Bombyx mori cell line as a model of immune-system organs.

We tested 11 Bombyx mori cell lines for induction of cecropin B gene (CecB) expression. After the immune challenge, CecB expression was induced in seven cell lines. A mixture of the cell-free supernatant from the immune-responsive cell lines and lipopolysaccharide activated a promoter of CecB in the non-immune-responsive cell line, indicating that secreted factor(s) is involved in CecB activation. The expressed sequence tags of one of the immune-responsive cell lines, NISES-BoMo-Cam1, contained genes encoding proteins similar to Relish, Cactus, clip-domain serine protease, serpin, lectin, peptidoglycan recognition protein, 6tox and gloverin, in addition to seven known B. mori immune-inducible genes. These results show that NISES-BoMo-Cam1 cells can be used as an in vitro model of the immune system organs of B. mori.

A novel lipopolysaccharide response element in the Bombyx mori cecropin B promoter.

Cecropin B is one of the major antibacterial peptides in the silkworm, Bombyx mori. Transcription of the cecropin B gene (CecB) occurs rapidly after bacterial invasion. Using 235 base pairs (bp) of the CecB promoter region, a kappaB-related protein and two additional DNA-binding complexes (designated F2BPI and F4BP) were identified in nuclear extracts from immunized larval fat body by the electrophoretic mobility shift assay (EMSA) (1). Further EMSA analyses indicated that the F2BPI-binding site was CATTA, and that F2BPI translocated from the cytoplasm to the nucleus after infection. In a recently established B. mori cell line, NISES-BoMo-DZ, 235 bp of CecB promoter linked to a reporter luciferase was activated 6-fold by stimulation with lipopolysaccharide (LPS), which is a major trigger of CecB expression in larvae. Truncation of the F2BPI-binding site from the promoter reduced the activation 2-fold. Deletion of either of two kappaB motifs also reduced promoter activation 2-fold. Elimination of both the F2BPI-binding site and the kappaB motifs resulted in the complete loss of LPS inducibility. These results indicate that the F2BPI-binding site is an LPS-responsive cis-element that is necessary for full activation of CecB.

A case of rectal Dieulafoy's ulcer and successful endoscopic sclerotherapy.

A 54-year-old woman presented a massive hematochezia 7 days after sigmoidectomy. Repeated colonoscopy and angiography failed to locate the site of bleeding and Hartman's operation was performed. Rebleeding from the rectum on the day of operation occurred and pulsate arterial bleeding with minimal surrounding ulcer 1 cm above the pectinate line was observed. Screlotherapy with ethanol and electro coagulation was successfully performed to achieve permanent hemostasis. The importance of detailed rectal examination and an awareness of this clinical entity in life-threatening lower intestinal bleeding is discussed.

cDNA cloning and gene expression of cecropin D, an antibacterial protein in the silkworm, Bombyx mori.

We have isolated a cDNA clone encoding a cecropin D precursor from the fat body of Bombyx mori larvae immunized with bacteria by means of differential display. The cDNA contains 298 bp with a coding region of 183 bp for 61 amino acids plus a termination codon (TAG), a 5'-untranslated region of 36 bp, and a 3'-untranslated region of 79 bp including the poly(A) tail. There is a polyadenylation signal sequence of AATAAA at position 266, 43 nucleotides downstream from the termination codon TAG. The homology of the deduced amino acids is greater to the cecropin D precursor from Hyalophora cecropia (67% identity) than to the precursors of cecropins A and B from B. mori (49% to both). Northern blotting analyses reveal that the gene expression of cecropin D is detectable by 4 h after the bacterial injection and reaches the maximal level at 24 h. That high level is maintained up to 48 h post-immunization. Additionally, the gene is expressed mainly in the fat body and slightly in hemocytes, but it is undetectable in other tissues such as the midgut, the Malpighian tubule and silk gland.

Induction of gene expression of antibacterial proteins by chitin oligomers in the silkworm, Bombyx mori.

Activation of antibacterial protein genes by various chitin oligomers (from dimer to hexamer) was investigated by Northern blot analysis using cDNAs encoding cecropin B, attacin and lebocin from Bombyx mori as probes. All chitin oligomers tested were found to strongly trigger expression of cecropin B, attacin and lebocin genes. Furthermore, gene expression triggered by chitin oligomers was confirmed to occur specifically in the fat body and haemocytes. These results suggest that chitin oligomers have the same characteristics as those of lipopolysaccharide (LPS) and peptidoglycan in triggering gene expression of insect antibacterial proteins.

Isolation, cDNA cloning and gene expression of an antibacterial protein from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros.

An antibacterial protein, designated rhinocerosin, was purified to homogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros immunized with Escherichia coli. Based on the amino acid sequence of the N-terminal region, a degenerate primer was synthesized and reverse-transcriptase PCR was performed to clone rhinocerosin cDNA. As a result, a 279-bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended rhinocerosin cDNA clone by 5' rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids without cystein residues and was shown to be rich in glycine (11.1%) and proline (11.1%) residues. Comparison of the deduced amino acid sequence of rhinocerosin with those of other antibacterial proteins indicated that it has 77.8% and 44.6% identity with holotricin 2 and coleoptrecin, respectively. Rhinocerosin had strong antibacterial activity against E. coli, Streptococcus pyogenes, Staphylococcus aureus but not against Pseudomonas aeruginosa. Results of reverse-transcriptase PCR analysis of gene expression in different tissues indicated that the rhinocerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gene expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the midgut.

A novel member of lebocin gene family from the silkworm, Bombyx mori.

We screened genomic clones encoding lebocin, an antibacterial peptide from the silkworm, Bombyx mori. Two positive clones were obtained and their nucleotide sequences indicated that they contain no introns. The deduced amino acid sequences revealed that one clone (Leb 3) encoded lebocin 3 and another (Leb 4) is a new member of the lebocin gene family. Gene expression of both Leb 3 and Leb 4 was shown to be induced by lipopolysaccharide and to occur tissue-specifically in the fat body and hemocytes. Our results suggest that lebocin as well as cecropin forms a multiple gene family in B. mori.

Bone metabolism and daily physical activity in women undergoing hemodialysis.

BACKGROUND: We investigated the relationships between bone mineral density (BMD) and bone metabolic markers in 41 Japanese women (age range, 23 to 85 years; mean, 61; SD, 16) undergoing hemodialysis, and the relationship between BMD and daily physical activity with nutritional state in those patients. METHOD: The patients were divided into a group with hyperparathyroidism (intact parathyroid hormone [PTH] > or = 300 pg/mL, n = 9) and a group without hyperparathyroidism (intact PTH < 300 pg/mL, n = 32). Total BMD and spinal BMD (regional evaluation) were measured using dual-energy X-ray absorptiometry. We determined serum concentrations of intact PTH, osteocalcin (bone Gla protein) and alkaline phosphatase as markers of bone formation, and serum tartrate-resistant acid phosphatase (TRAP) as a marker of bone resorption. Mean energy expenditure per day was measured by calorie counters worn by the patients, and serum levels of total protein, albumin, cholesterol, ferritin, and blood urea nitrogen were measured as indices of nutritional status. RESULTS: In the group without hyperparathyroidism, 9 patients had a spinal BMD < 0.85 g/cm2 and were considered to be at risk for bone fracture. In this group, a significant correlation was observed between total BMD and TRAP (r = -0.52, p = 0.004). Age, duration of hemodialysis, dry body weight, energy expenditure per day, and serum total protein levels were correlated with total BMD and spinal BMD, using multiple regression analysis (p < 0.0001). CONCLUSIONS: In patients with low BMD, the bone turnover tended toward bone resorption rather than formation, without strong influence by parathyroid function. Energy expenditure showed a strong relationship to the total BMD (r = 0.32, F = 18.5), which appeared to improve bone turnover.

In vitro phagocytosis of Escherichia coli and release of lipopolysaccharide by adhering hemocytes of the silkworm, Bombyx mori.

A primary culture containing adhering hemocytes mainly granular cells from the silkworm, Bombyx mori, was used to investigate in vitro phagocytosis of Escherichia coli. Phagocytosis was confirmed to occur in this system by microscopic observation. Lipopolysaccharide (LPS) concentration in the culture medium was measured by a Limulus test and a higher LPS concentration was detected in phagocytosis-occurred samples than in control samples, which omitted either E. coli cells or adhering hemocytes. Moreover, it was found that LPS containing sample but not control samples strongly induces gene expression of cecropin B, an antibacterial protein. These results suggest that bacterial cell wall components like LPS released by phagocytosis play an important role in the induction of insect antibacterial proteins.

Elicitors triggering the simultaneous gene expression of antibacterial proteins of the silkworm, Bombyx mori.

Various elicitors were examined by Northern blot analysis to investigate the simultaneous induction of gene expression of antibacterial proteins such as cecropin B, attacin and lebocin from the silkworm, Bombyx mori. Lipopolysaccharide (LPS), lipid A, 2-keto-3-deoxyoctonate (KDO) and peptidoglycan (PG) triggered efficiently and simultaneously the gene expression of antibacterial proteins. Effects of inhibitors for signal transduction on the gene expression of Bombyx mori (Bm) cecropin B triggered by lipid A were observed using isolated adherent hemocytes consisting of granular cells and plasma cells. H-7, H-89 but not W-7 inhibited gene expression, suggesting that protein kinase C and A but not myosine light chain kinase may participate in signal transduction.

Isolation and characterization of a new member of the insect defensin family from a beetle, Allomyrina dichotoma.

A new family member of insect defensin, an antibacterial peptide, has been isolated from larvae of a beetle, Allomyrina dichotoma. The peptide consisted of 43 amino acids and 6 cystein residues were conserved in the same position as that of other insect defensins. The new defensin was found to be inducible by bacterial injection. Analysis of the antibacterial spectrum of A. dichotoma defensin indicated that this peptide showed antibacterial activity against Gram-positive bacteria like Staphylococcus aureus and Bacillus subtilis but not against Gram-negative bacteria like Escherichia coli and Pseudomonas aeruginosa, indicating a typical spectrum of the insect defensin family. In addition, A. dichotoma defensin also exhibited antibacterial activity against methicillin-resistant S. aureus (MRSA) isolated from a patient.

Nucleotide sequence of 5'-upstream region and expression of a silkworm gene encoding a new member of the attacin family.

A genomic clone encoding a new member of attacin, an insect antibacterial protein, was isolated from a genomic library of the silkworm, Bombyx mori, and the nucleotide sequence of the 5'-upstream region was determined. The region contained Bm 1, a highly repetitive element of B. mori and a lipopolysaccharide (LPS) response element (RE)(NF-kappaB binding site), CAAT box and TATA box. Northern blot analysis showed that the attacin gene expression was rapidly induced by bacterial cell wall components such as LPS from Escherichia coli and peptidoglycan (PG) from Micrococcus luteus, suggesting that attacin plays an important role in an early phase of the self-defense system upon bacterial infection.

Structure of two cecropin B-encoding genes and bacteria-inducible DNA-binding proteins which bind to the 5'-upstream regulatory region in the silkworm, Bombyx mori.

Two genomic DNAs encoding cecropin B (CecB), an antibacterial protein from Bombyx mori, were cloned and sequenced. The number of CecB genes was estimated to be more than four copies per haploid by genomic Southern blotting. Two genes, CecB1 and CecB2, were located tandemly within 12 kb in the same orientation. These two genes encoded identical amino acids, though 15 nucleotides (nt) were different in the coding region and the intron size varied. About 90% of the nt spanning 800 bp in the 5'-untranslated region (UTR) were identical between the two genes. This 5'-flanking region contained characteristic sequences such as a repetitive element of B. mori (Bm1), an interleukin-6 response element (IL-6 RE), and two putative lipopolysaccharide (LPS) response elements (LPS RE). An electrophoretic mobility shift assay (EMSA) showed that the fat body contains at least three different nuclear proteins inducible by bacteria which bind to the 5'-UTR, suggesting that these proteins may be involved in CecB expression triggered by bacteria.

cDNA cloning and gene expression of lebocin, a novel member of antibacterial peptides from the silkworm, Bombyx mori.

A cDNA encoding lebocin, a novel member of insect antibacterial peptides, was isolated from the fat body cDNA library of Bombyx mori larvae immunized with Escherichia coli. The cDNA was 844 bp long and had an open reading frame (ORF) containing a probable signal peptide (16 amino acids), a putative prosegment (104 amino acids) and a mature peptide (32 amino acids) followed by 27 additional amino acids at its carboxyl-terminus. Northern blot analysis showed that lebocin gene expression was inducible by bacterial injection, occurred tissue-specifically in the fat bodies and continued at least for 48 h post-infection. These results suggest that lebocin has a unique precursor structure and shows typical gene expression pattern as insect antibacterial peptide.

Baculovirus-mediated production of the human growth hormone in larvae of the silkworm, Bombyx mori.

To express the cDNA encoding human growth hormone (hGH) in larvae of Bombyx mori, B. mori nuclear polyhedrosis virus (BmNPV) was employed as an expression vector. For the construction of the recombinant virus, the hGH cDNA was inserted into the downstream of the strong polyhedrin promoter to achieve a high level expression. Immunoblot analysis revealed that the virus-mediated hGH was synthesized in the larvae and secreted into the hemolymph. The yield of the recombinant hGH synthesized in the larvae reached to a level of 160 micrograms/ml of hemolymph after purification. The purified recombinant hGH was confirmed to have both the same molecular weight and amino acid sequence at its N-terminal region as those of the natural counterpart. In addition, the biological activity of the recombinant hGH was comparable to that of the natural hGH in the growth-stimulating effect on rat Nb 2 Node lymphoma cells.

Characterization of a Bombyx mori cDNA encoding a novel member of the attacin family of insect antibacterial proteins.

A Bombyx mori cDNA was cloned that hybridized with Hyalophora cecropia attacin probe and its nucleotide sequence was determined. This cDNA consisted of 846 nucleotides and the deduced amino acid sequence showed that the cDNA encodes an attacin precursor protein. The putative mature protein of B. mori attacin had 70.4, 68.3 and 18.8% identity in amino acid sequences with that of H. cecropia acidic and basic attacins and Sarcophaga peregrina sarcotoxin IIA, respectively. B. mori and H. cecropia attacins and S. peregrina sarcotoxin IIA had two subdomains in each G domain, suggesting that common amino acid residues in the subdomains are conserved during evolution and plays an important role in the activity of the antibacterial proteins. Expression of B. mori attacin gene was rapidly induced by the injection of Escherichia coli cells into B. mori larvae and continued at least for 48 h mainly in fat bodies and hemocytes.

Cloning and expression of the gene of hemocytin, an insect humoral lectin which is homologous with the mammalian von Willebrand factor.

Invertebrate lectins play an important role in a non-specific self-defense mechanism, as invertebrates do not synthesize specific antibodies. We report the cloning of several overlapping cDNAs encoding the entire silkworm (Bombyx mori) lectin, which we propose to call hemocytin. The sequence (10477 bp) encoded 3133 amino acids. The characteristics features of the carbohydrate-recognition domain of C-type animal lectin were revealed at C-terminal sequence of hemocytin. When cDNA encoding this region was introduced into baculovirus vector, hemagglutinating activities were detected in the culture fluid of a recombinant virus-infected cells. These activities were inhibited by D-mannose, N-acetyl-D-galactosamine, and D-maltose which are haptenic saccharides of authentic hemocytin. Analysis of dot and Northern blot hybridization revealed that hemocytin gene was transcribed in hemocytes of the silkworm at larval-pupal metamorphosis and/or after the injection of Escherichia coli and lipopolysaccharide. After silkworm larvae were injected with C-terminal portion of hemocytin, aggregation of hemocytes was observed in the hemolymph. Hemocytin has significant homology with mammalian von Willebrand factor which involves in platelet adhesion to subendothelium. Also, hemocytin has a homologous region with coagulation factor V and VIII. These results suggest that hemocytin molecule is an adhesive protein and relates to hemostasis or encapsulation of foreign substances for self-defense.

Occurrence of novel types of nitric oxide synthase in the silkworm, Bombyx mori.

Nitric oxide (NO) synthase activity was detected in fat body and the Malpighian tubles of the silkworm, Bombyx mori. Main NO synthase activity in the fat body was Ca(2+)/calmodulin-dependent, inducible by bacterial lipopolysaccharide (LPS) and required NADPH, FAD, FMN, dithiothreitol (DTT) and tetrahydrobiopterin (BH4) as cofactors for the full expression of the activity. The Malpighian tubles contained two types of NO synthase. One was Ca(2+)-independent, calmodulin-dependent and constitutive and the other was Ca(2+)-dependent and constitutive. The former NO synthase required the same cofactors as fat body NO synthase. The activity of Malpighian tuble NO synthases increased dramatically at the end of the last instar period, just prior to spinning. These results indicate that B. mori contains new types of NO synthase, suggesting the wide distribution and different characteristics of this enzyme among vertebrates and invertebrates.

Lipopolysaccharide-lipophorin complex formation in insect hemolymph: a common pathway of lipopolysaccharide detoxification both in insects and in mammals.

The formation of the lipophorin-lipopolysaccharide (LPS) complex in Bombyx mori hemolymph and its role in LPS detoxification were explored. LPS, an antibacterial protein inducer in insects, was injected into B. mori larvae. Analytical density gradient ultracentrifugation revealed that after injection the LPS peak shifts to a zone of lower density with time. The shifted peak was identified as the lipophorin-LPS complex. This complex formation was also achieved in an in vitro mixture of cell-free hemolymph and LPS at 25 degrees C but not at 1 degree C. The lipophorin-LPS complex had a significantly lower capacity to elicit the mRNA of cecropin B, an antibacterial protein. The biological activity of reextracted LPS from the complex was slightly reduced in the Limulus test and no structural modification was observed in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggested that the formation of lipophorin-LPS strikingly reduces the cecropin inducibility of LPS without any structural change in LPS. Similar serum lipoprotein-LPS complex formation and reduction of biological activities of LPS were also observed in mammals. We, therefore, suggest that the formation of the serum lipoprotein-LPS complex is a common pathway to inactivate LPS both in insects and in mammals.