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Rationale We present the first case of a patient with severe aortic stenosis who developed anaphylactic shock and was successfully treated with adrenaline and landiolol, a highly selective β1-receptor blocker, to prevent disruption of the myocardial oxygen supply-demand balance caused by tachycardia. Patient concerns An 86-year-old woman was scheduled for simultaneous anterior-posterior fixation for a burst fracture of the 12th thoracic vertebra; 200 mg sugammadex, a neuromuscular blocking agent antagonist, was administered postoperatively, and she was extubated without complications. However, 6 min after extubation, her blood pressure decreased abruptly to 55/29 mm Hg, and her heart rate increased to 78 bpm. Then, we intervened with fluid loading, an increased dose of noradrenaline, and phenylephrine administration. However, her blood pressure did not increase. Diagnoses A general observation revealed urticaria on the lower leg; thus, we suspected anaphylactic shock due to sugammadex administration. Interventions We carefully administered 2 doses of 0.05 mg adrenaline and simultaneously administered landiolol at 60 μg/kg/min to suppress adrenaline-induced tachycardia. Adrenaline administration resulted in a rapid increase in blood pressure to 103/66 mm Hg and a maximum heart rate of 100 bpm, suppressing excessive tachycardia. Outcomes The patient's general condition was stable after the intervention, and circulatory agonists could be discontinued the following day. She was discharged from the intensive care unit on the fourth postoperative day. Lessons Landiolol may help control the heart rate of patients with aortic stenosis and anaphylactic shock. The combined use of landiolol and adrenaline may improve patient outcomes; however, their efficacy and risks must be evaluated by studying additional cases.

Objective: To characterize the pre- and postoperative coagulation profiles of patients undergoing adult-to-adult living donor liver transplantation (LDLT), using various coagulation tests and rotational thromboelastometry (ROTEM). Methods: This single-center observational study evaluated the various coagulation profiles of 22 patients (13 men and 9 women). Blood samples were obtained immediately after the induction of anesthesia (PRE) and on postoperative days (PODs) 1, 3, 5, and 7 after LDLT surgery. Results: Most procoagulant factors (fibrinogen, platelet, and coagulation factors II, VII, VIII, and IX) improved to levels equal to or greater than the PRE levels on POD 7. The levels of von Willebrand factor significantly increased after surgery, whereas those of disintegrin-like and metalloproteinase with thrombospondin type 1 motif 13 decreased. Although the thrombin-antithrombin III complex increased immediately after surgery, the plasmin-α 2 plasmin inhibitor complex increased only on POD 7. The level of plasminogen activator inhibitor-1 increased on POD 1, returning to PRE levels on POD 3. Almost all ROTEM parameters were decreased or prolonged, compared to the PRE levels, on POD 7. Conclusions: The values of most coagulation tests showed the improvement or acceleration of coagulability on POD 7 than at PRE, with almost all the ROTEM parameters decreased or prolonged. Therefore, it cannot be concluded whether ROTEM reflects the net effect of hemostatic balance after liver transplantation., Transplantation Reports, 5(1), art.no.100037; 2020

Small molecules complement genetic mutants and can be used to probe pigment cell biology by inhibiting specific proteins or pathways. Here, we present the results of a screen of active compounds for those that affect the processes of melanocyte and iridophore development in zebrafish and investigate the effects of a few of these compounds in further detail. We identified and confirmed 57 compounds that altered pigment cell patterning, number, survival, or differentiation. Additional tissue targets and toxicity of small molecules are also discussed. Given that the majority of cell types, including pigment cells, are conserved between zebrafish and other vertebrates, we present these chemicals as molecular tools to study developmental processes of pigment cells in living animals and emphasize the value of zebrafish as an in vivo system for testing the on- and off-target activities of clinically active drugs.

Coordination of cell proliferation and differentiation is crucial for tissue formation, repair and regeneration. Some tissues, such as skin and blood, depend on differentiation of a pluripotent stem cell population, whereas others depend on the division of differentiated cells. In development and in the hair follicle, pigmented melanocytes are derived from undifferentiated precursor cells or stem cells. However, differentiated melanocytes may also have proliferative capacity in animals, and the potential for differentiated melanocyte cell division in development and regeneration remains largely unexplored. Here, we use time-lapse imaging of the developing zebrafish to show that while most melanocytes arise from undifferentiated precursor cells, an unexpected subpopulation of differentiated melanocytes arises by cell division. Depletion of the overall melanocyte population triggers a regeneration phase in which differentiated melanocyte division is significantly enhanced, particularly in young differentiated melanocytes. Additionally, we find reduced levels of Mitf activity using an mitfa temperature-sensitive line results in a dramatic increase in differentiated melanocyte cell division. This supports models that in addition to promoting differentiation, Mitf also promotes withdrawal from the cell cycle. We suggest differentiated cell division is relevant to melanoma progression because the human melanoma mutation MITF4TΔ2B promotes increased and serial differentiated melanocyte division in zebrafish. These results reveal a novel pathway of differentiated melanocyte division in vivo, and that Mitf activity is essential for maintaining cell cycle arrest in differentiated melanocytes.

SUMMARY Hypopigmentation is a feature of copper deficiency in humans, as caused by mutation of the copper (Cu2+) transporter ATP7A in Menkes disease, or an inability to absorb copper after gastric surgery. However, many causes of copper deficiency are unknown, and genetic polymorphisms might underlie sensitivity to suboptimal environmental copper conditions. Here, we combined phenotypic screens in zebrafish for compounds that affect copper metabolism with yeast chemical-genetic profiles to identify pathways that are sensitive to copper depletion. Yeast chemical-genetic interactions revealed that defects in intracellular trafficking pathways cause sensitivity to low-copper conditions; partial knockdown of the analogous Ap3s1 and Ap1s1 trafficking components in zebrafish sensitized developing melanocytes to hypopigmentation in low-copper environmental conditions. Because trafficking pathways are essential for copper loading into cuproproteins, our results suggest that hypomorphic alleles of trafficking components might underlie sensitivity to reduced-copper nutrient conditions. In addition, we used zebrafish-yeast screening to identify a novel target pathway in copper metabolism for the small-molecule MEK kinase inhibitor U0126. The zebrafish-yeast screening method combines the power of zebrafish as a disease model with facile genome-scale identification of chemical-genetic interactions in yeast to enable the discovery and dissection of complex multigenic interactions in disease-gene networks.

Non-invasive positive pressure ventilation (NPPV) reduces the incidences of ventilator-associated pneumonia, the duration of ICU stay and the mortality rate compared with conventional respiratory management of the patients with acute respiratory failure (ARF). Recently, helmet NPPV equipment became available. Because of the high tolerability, the helmet seems to be the best NPPV interface when prolonged and continuous assistance is needed. In this study, we analyzed several factors related to failure of helmet NPPV in ARF patients in intensive care unit (ICU), retrospectively.Institutional Research Committee of Nagasaki Rosai Hospital approved this study. We studied consecutive patients with ARF who needed ventilator support in ICU from February 2012 to February 2013. We excluded the patients whose trachea had been intubated before admission to ICU and comatose patients. After admission to ICU, all ARF-patients received helmet NPPV and conventional intensive care therapy including sedation with dexmedetomidine and vasoactive agents. General clinical data including blood gas analysis were recorded at admission to ICU and during ICU stay. Patient's tracheas were intubated if they met at least one of the following criteria, as judged after they had received helmet NPPV: lack of improvement in arterial blood pH or PaCO2; changes in mental status, in patients unable to tolerate noninvasive ventilation; a decrease in SaO2 to less than 85% despite the use of a high FIO2. The final decision of endotracheal intubation was made by a staff intensivist. We defined the failure of helmet NPPV as the execution of endotracheal intubation. The data were presented as median (IQR), and statistical analysis was performed using Mann-Whitney U-test and Fisher's exact probability test at the P0.05 level of significance.The subjects were 36 patients (25 males and 11 females) aged 27 to 94 years, including 6 patients with acute heart failure (AHF), 8 with pneumonia, 6 with aspiration pneumonia, 2 with hemothorax, 10 with acute respiratory distress syndrome (ARDS), 1 with asthma, and 3 with acute exacerbation of chronic obstructive pulmonary disease (COPD). NPPV was successful in 29 (19 males and 10 females), but unsuccessful in 7 patients (6 males and 1 female). There were no significant differences in demographic data and the variables before induction of NPPV between the successful and unsuccessful groups. The P/F ratio was improved from 133 (99,167) to 209 (143,274) in the successful group, and from 93 (81,157) to 188 (129,271) in the unsuccessful group after the induction of NPPV, but there was no significant difference between the two groups. In the patients with unsuccessful NPPV, expiratory positive airway pressure, inspiratory positive airway pressure, respiratory rate, body temperature and FIO2 before removing NPPV were significantly higher, and ICU stay was longer compared with the patients with successful NPPV. Furthermore, marked excretion of sputum was observed in 4 of the 7 patients with unsuccessful NPPV.Helmet NPPV improved oxygenation in ARF patients immediately after induction of NPPV. Although there were no significant predictable parameters of unsuccessful NPPV before induction of NPPV, a lot of excretion of sputum might be suggested as a risk factor.

The myc target gene Mina53 was reported to be overexpressed in esophageal cancer with a poor prognosis. The purpose of the present study was to examined Mina53 expression and its relationship to clinicopathological parameters in human renal cell carcinoma (RCC). Mina53 and Ki-67 expression was examined on immunohistochemistry for 64 surgically resected RCC and non-cancerous tissue. In addition, the relationship between Mina53 expression and clinicopathological prognostic factors of RCC such as age, stage, microvenous invasion (MVI), histological subtype, Ki-67 labeling index (LI), and prognosis, was examined. Mina53 was expressed in the nuclei of tumor cells and tubular nuclei of normal renal tissue. The expression level of Mina53 was significantly higher in patients with poor prognostic factors (stage IV, MVI-positive, and sarcomatoid RCC, and high Ki-67 LI). The prognosis of high Mina53-expressing tumors was significantly poorer than that of non-Mina53-high tumors (P < 0.0001). In conclusion, Mina53 is overexpressed in RCC tissue from patients with poor prognostic factors, suggesting that Mina53 overexpression is one of the factors for poor prognosis in RCC.

We investigated the effects of interferon-beta (IFN-beta) on the growth of human liver cancer cells. The effects of IFN-beta with or without 5-fluorouracil (5-FU) on the proliferation of 13 liver cancer cell lines were investigated in vitro. Chronologic change in IFN-alpha receptor 2 (IFNAR-2) expression was monitored in hepatocellular carcinoma (HCC) cells (HAK-1B) cultured with IFN-beta. After HAK-1B cells were transplanted into nude mice, various doses of IFN-beta were administered, and the tumor volume, weight, histology, tumor blood vessel, and angiogenesis factor expression were examined. IFN-beta inhibited the growth of 11 cell lines with apoptosis in a dose-dependent and time-dependent manner. With IFN-beta, IFNAR-2 expression in HAK-1B cells was significantly downregulated from 6 to 12 h. IFN-beta induced a dose-dependent decrease in tumor volume and weight and a significant increase of apoptosis in the tumor. Both basic fibroblast growth factor (bFGF) and blood vessel number in the tumor decreased only in mice receiving the lowest dose (1000 IU) of IFN-beta. IFN-beta with 10 muM of 5-FU frequently induced synergistic antiproliferative effects. IFN-beta with or without 5-FU induces strong antitumor effects in HCC cells, and we conclude that IFN-beta is useful for the prevention and treatment of HCC.

We investigated the effects of pegylated (PEG)-IFN-alpha2b on alpha-fetoprotein (AFP) expression as demonstrated by protein and mRNA levels in six human hepatocellular carcinoma (HCC) cell lines. The number of KIM-1 cells in culture with PEG-IFN-alpha2b decreased between 24 amd 240 h, whereas the levels of intracellular and secreted AFP per cellular protein increased (except at 192 h), with levels 1.9-fold and 2.9-fold higher at maximum, respectively, than cells without PEG-IFN-alpha2b (control). The mRNA level increased between 72 and 192 h, when the level was 3-fold higher than that of the control. In the 72-h culture with 40-5000 IU/mL PEG-IFN-alpha2b, there were dose-dependent increases in AFP protein and mRNA expression and dose-dependent decrease in cell number resulting from apoptosis and blockage of the cell cycle at the S-phase. The rate of fucosylated AFP in the cell lysate decreased in a dose-dependent and time-dependent manner. In the PEG-IFN-alpha2b culture of the other five HCC cell lines, cell proliferation was suppressed, but the expressions of AFP protein and mRNA increased in only two cell lines, and suppression of cell proliferation was not related to the increase in AFP expressions. Our findings demonstrated that PEG-IFN-alpha2b induces an increase in AFP expression at both the protein and mRNA levels.

PURPOSE We investigated the effects of pegylated IFN-alpha2b (PEG-IFN-alpha2b) on the growth of human liver cancer cells. METHODS The effect of PEG-IFN-alpha2b on the proliferation of 13 liver cancer cell lines was investigated in vitro. Chronological changes in growth and IFN-alpha receptor-2 (IFNAR-2) expression were monitored in hepatocellular carcinoma (HCC) cells (HAK-1B) cultured with PEG-IFN-alpha2b. After HAK-1B cells were transplanted into nude mice, various doses of PEG-IFN-alpha2b or IFN-alpha2b were administered, and tumor volume, weight, histology, and IFNAR-2 expression were examined. RESULTS PEG-IFN-alpha2b inhibited the growth of nine cell lines with apoptosis in a dose- and time-dependent manner. Continuous contact with PEG-IFN-alpha2b induced time-dependent growth inhibition and down-regulation of IFNAR-2 expression. PEG-IFN-alpha2b induced a dose-dependent decrease in tumor volume and weight, a significant increase of apoptotic cells, and a decrease in IFNAR-2 expression in the tumor. The clinical dose for chronic hepatitis C was also effective. The antitumor effect of PEG-IFN-alpha2b was significantly stronger than that of non-PEG-IFN-alpha2b in vivo. CONCLUSIONS Continuous contact with PEG-IFN-alpha2b induces strong antitumor effects and the down-regulation of IFNAR-2 in HCC cells. The data suggest potential clinical application of PEG-IFN-alpha2b for the prevention and treatment of HCC.

Molecular characterization of the brain secretory peptides, PTTH and bombyxin, of Bombyx mori is reviewed. PTTH is a 30-kDa homodimeric glycoprotein, the monomer of which consists of 109 amino acids. Two monomers are held together by a disulfide bond. cDNA and gene coding for PTTH were cloned and the precursor protein for PTTH monomer was deduced. A novel 5-kD brain secretory peptide named bombyxin has been discovered from Bombyx brain. Bombyxin is highly homologous to vertebrate insulin-family peptides and possesses the prothoracicotropic activity when injected into brain-removed pupae of a heterologous moth, Samia cynthia ricini, though inactive to Bombyx from which it was derived. cDNA and gene coding for bombyxin were cloned, preprobombyxin protein was deduced, and posttranslational processing to generate mature bombyxin was suggested. The Bombyx genome contains highly multiple copies of the gene coding for bombyxins. Immunohistochemically, PTTH- and bombyxin-producing brain neurosecretory cells were identified. (Communicated by Saburo TAMURA, M.J.A.)

Six new mycetophilid species from three genera belonging to the tribe Exechiini are described: Anatella prominens on a fungus of the Polyporaceae, Allodia bipexa and Allodia conifera on fungi of the Russulaceae, Allodia quadrata on fungi of the Hygrophoraceae, Tricholomataceae, Rhodophyllaceae, Russulaceae and Fistulinaceae, Allodia laccariae on a fungus of the Tricholomataceae, and Cordyla bidenticulata on a fungus of the Boletaceae. Their characteristic male genitalia are illustrated. Cordyla flaviceps Staeger, Cordyla fusca Meigen and Cordyla pusilla Edwards are newly recorded from Japan.

Synthesis and secretion of the insect molting hormone ecdysteroid in the prothoracic glands (PGs) are stimulated by the prothoracicotropic hormone (PTTH) secreted by the brain. Bombyxins, insulin-like peptides of the silkworm Bombyx mori, show prothoracicotropic activity when administered to the saturniid silkworm Samia cynthia ricini, but they are inactive to B. mori itself. Recently, the genes for the bombyxin homologs of S. cynthia ricini (referred to as Samia bombyxin-related peptides, SBRPs) were cloned. To examine the prothoracicotropic activity of SBRPs on S. cynthia ricini, we synthesized two representative molecules, SBRP-A1 and -B1. They promoted pupa-to-adult development with ED(50) of 50 and 10 ng/pupa (EC(50) of 5 and 1 nM), respectively.

The effects of an insect insulin-related peptide, bombyxin, on carbohydrate metabolism were investigated in the silkworm Bombyx mori. Bombyxin lowered the concentration of the major hemolymph sugar, trehalose, in a dose-dependent manner when injected into neck-ligated larvae. Bombyxin also caused elevated trehalase activity in the midgut and muscle, suggesting that bombyxin induces hypotrehalosemia by promoting the hydrolysis of hemolymph trehalose to glucose and thereby facilitating its transport into tissues. In addition, bombyxin reduced the glycogen content in the fat body and concurrently raised the percentage of active glycogen phosphorylase in this tissue. Because hemolymph trehalose is also a major storage form of carbohydrate in insects, our results indicate that bombyxin reduces the amount of both principal storage carbohydrates in B. mori larvae. It is therefore suggested that although bombyxin is involved in the control of carbohydrate metabolism like insulin, the physiological role of bombyxin in insects is different from that of insulin in mammals.

Thirty-eight genes that encode bombyxin, the insulin-related brain secretory peptide of the silkmoth Bombyx mori, have been cloned and characterized. These genes have been classified into four families, A, B, C and D, according to their sequence similarity. All the bombyxin genes lack introns. Five of them have structural features of pseudogenes. The 38 genes cluster in the three DNA segments of Bombyx in unique distribution patterns. Their arrangement has been classified into three categories: gene pairs, gene triplets and single genes. In the pairs, two bombyxin genes belonging to families B and A (B/A) or to families B and C (B/C) are apposed with opposite transcriptional orientation. All triplets are arranged in the order of the family-B, family-C and family-A genes, and the transcriptional directions of the family-C and family-A genes are opposite to the direction of the family-B gene. The bombyxin gene triplets may have been generated by an unequal crossing-over between two gene pairs, B/A and B/C. Crossing-over may have occurred in the bombyxin family-B genes to increase their structural diversity. Duplications may have served to multiply the bombyxin gene triplets. These genomic rearrangements are thought to have led to the generation of multiple bombyxin gene copies and their diversity in structure and genomic organization.

Prothoracicotropic hormone (PTTH) is a brain neurosecretory protein that controls insect development. PTTH of the silkmoth Bombyx mori is a homodimeric protein, the subunit of which consists of 109 amino acids. Clear-cut sequence similarity to any other proteins has not been observed. By disulfide-bond pattern analysis and modeling of the PTTH structure based on the known three-dimensional (3D) structures of growth factor family with cystine-knot motif, we propose that the PTTH protomer adopts the fold unique to the structural superfamily of the growth factors, beta-nerve growth factor (beta-NGF), transforming growth factor-beta 2 (TGF-beta 2), and platelet-derived growth factor-BB (PDGF-BB). The insect neurohormone PTTH appears to be a member of the growth factor superfamily, sharing a common ancestral gene with the three vertebrate growth factors, beta-NGF, TGF-beta 2 and PDGF-BB.

The three-dimensional solution structure of bombyxin-II, an insulin-like two-chain peptide produced by the brain of the silkworm Bombyx mori, has been determined by simulated annealing calculations based on 535 distance constraints and 24 torsion-angle constraints derived from NMR data and three distance constraints of the disulfide bonds. To our knowledge, this is the first three-dimensional structure determined for an invertebrate insulin-related peptide. The root-mean-square deviations between the best 10 structures and the mean structure are 0.58(+/- 0.15) A for the backbone heavy atoms (N, C alpha, C) and 1.03(+/- 0.18) A for all non-hydrogen atom if less well-defined N and C termini (A1, A20, B(-2) to B4 and B23 to B25) are excluded. The overall main-chain structure of bombyxin-II is similar to that of insulin. However, there are significant conformational and functional differences in their B-chain C-terminal parts. The B-chain C-terminal part of bombyxin-II adopts an extension of the B-chain central helix like that of relaxin and is not required for bombyxin activity, while the corresponding part of insulin adopts a sharp turn and a beta-strand and is essential for insulin activity. This structure demonstrates that bombyxin-II is more closely related to relaxin than to insulin, and suggests that insulin might have evolved the additional receptor-recognition site in the B-chain C-terminal beta-strand to distinguish itself from bombyxin and relaxin. The structure of bombyxin-II thus provides novel insights into the receptor recognition and divergent molecular evolution of insulin-superfamily peptides.

Bombyxin-II, a brain-secretory peptide of the silkmothBombyx mori, shares 40% sequence identity and the characteristics core structure with human insulin. In spite of the structural similarity, no cross-activity is observed between them. To localize the active region of bombyxin-II, we have synthesized chimeric molecules of bombyxin-II and human insulin, and examined their bombyxin activity. Two chimeric molecules, which were sequentially identical except for the B-chain central part, showed significantly different potencies in bombyxin activity. Solution structure determination of these chimeric molecules revealed that their B-chain central parts took similar main-chain conformation, but formed dissimilar patches on their molecular surfaces. Therefore, the surface patch formed by the central part of the bombyxin-II B-chain is of critical importance for recognition of the bombyxin receptor. The above results, together with other data on the structure-activity relationships of bombyxin, indicate that the receptor-recognition surface of bombyxin-II includes the A-chain N and C, termini in addition to the B-chain central part. Though bombyxin-II, human insulin and human relaxin 2 use the common surface as their receptor-recognition sites, each of the surface patches is characterized by the variety of involved side-chains. Insulin and relaxin involve additional parts for receptor recognition, particularly the B-chain C-terminal part and the extended A-chain N-terminal helix, respectively. In conclusion, these ligands have evolved their own specific mechanisms for receptor recognition while retaining the major recognition surface.

Summary Understanding how drugs work in vivo is critical for drug design and for maximizing the potential of currently available drugs. 5-nitrofurans are a class of prodrugs widely used to treat bacterial and trypanosome infections, but despite relative specificity, 5-nitrofurans often cause serious toxic side effects in people. Here, we use yeast and zebrafish, as well as human in vitro systems, to assess the biological activity of 5-nitrofurans, and we identify a conserved interaction between aldehyde dehydrogenase (ALDH) 2 and 5-nitrofurans across these species. In addition, we show that the activity of nifurtimox, a 5-nitrofuran anti-trypanosome prodrug, is dependent on zebrafish Aldh2 and is a substrate for human ALDH2. This study reveals a conserved and biologically relevant ALDH2-5-nitrofuran interaction that may have important implications for managing the toxicity of 5-nitrofuran treatment., Graphical Abstract Highlights ► Zebrafish provide a viable assay for the biological toxicity of 5-nitrofurans ► ALDH2 inhibitors prevent 5-nitrofuran toxicity in zebrafish and yeast ► Genetic dependence on ALDH2 for 5-nitrofuran toxicity in zebrafish and yeast systems ► 5-Nitrofurans bind to and are substrates of human ALDH2, 5-nitrofurans are antibiotics activated by pathogen specific enzymes, however, less is known about what happens in the host. Zhou et al. identify aldehyde dehydrogenase 2 as a 5-nitrofuran activating enzyme that has implications for managing some of the toxicity associated with 5-nitrofuran treatment.

The disulfide bond location of a homodimeric peptide, prothoracicotropic hormone (PTTH) of the silkworm, Bombyx mori, was determined by a combination of partial reduction and sequence analysis of peptide fragments generated through a partial reduction of PTTH followed by alkylation and enzyme digestion. The partial reduction and S-alkylation broke the interchain disulfide bond but did not affect the intrachain disulfide bonds, generating monomeric PTTH whose intrachain disulfide bonds were kept intact. This monomeric PTTH has about one-half the biological activity of intact PTTH. Sequence analysis of the fragments generated by lysyl endopeptidase digestion of this monomeric PTTH after complete reduction and S-alkylation by another S-alkylating reagent showed that only the Cys15 residue was reduced and S-alkylated by the foregoing partial reduction, indicating that this residue formed the interchain disulfide bond. The other disulfide bonds which formed intrachain bridgings were determined by sequence and mass analyses of the fragments generated by two successive enzyme digestions of the monomeric PTTH. In conclusion, the disulfide bond location of PTTH was assigned to Cys15-Cys15' as an interchain disulfide linkage and Cys17-Cys54, Cys40-Cys96, and Cys48-Cys98 as intrachain disulfide linkages.

We cloned and characterized two allelic variants of the gene for the Bombyx mori prothoracicotropic hormone (PTTH), a homodimeric 30-kDa brain secretory protein. These PTTH genes contain five exons that encode a precursor protein consisting of 224 amino acid residues whose C-terminal 109 residues represent the PTTH subunit. The Bombyx haploid genome contains a single copy of the PTTH gene. The major site of PTTH expression is the brain but expression at a very low level occurs in the gut. One Bombyx brain at day 0 of the fifth larval instar contained 2.4-2.8 pg PTTH mRNA, and this amount did not change markedly during larval-pupal development.

~~ Monoclonal antibodies were raised against pure, native bombyxin-ll (bombyxin-ll antibody) and against a synthetic nonapeptide corresponding to the amino-terminus of the C-peptide of the bombyxin precursor protein (C-peptide antibody). The bombyxin-ll antibody recognized both bombyxin and probombyxin. A radioimmunoassay for bombyxin using the bombyxin-ll antibody was developed, and developmental change in the titer of bombyxin immunoreactivity in the Bombyx hemolymph was investigated. The titer was low and almost constant during the fourth and early fifth instars. In the male, the titer rose abruptly 3 days after the beginning of wandering. One day after pupation it rose again steeply to reach the maximal level which lasted until the middle of the developing adult stage. The titer decreased thereafter and increased again at adult emergence. In the female, the pattern of titer fluctuation was similar to that in the male, but the female titers during pupal-adult development were 2-3 times higher than the male titers.

From the genomic DNA of the moth Samia cynthia ricini, we cloned and characterized six clustered genes that encode precursor molecules for peptides structurally related to bombyxin, a Bombyx mori brain secretory peptide that is structurally like insulin and functionally like the prothoracicotropic hormone. The precursor molecules deduced from these genes have the domain organization of signal peptide/B-chain/C-peptide/A-chain, as in preprobombyxins and preproinsulins. The Samia bombyxin-related peptide (SBRP) genes are classified into families A and B according to their sequence homology. Two genes belonging to different families are arranged close to each other to form a pair with opposite transcriptional orientations (A-1/B-1, A-2/B-2, and A-3/B-3). None of these genes have introns, and gene B-3 has an in-frame stop codon representing a pseudogene. Four genes, A-1, A-3, B-1, and B-2, are expressed in Samia brain. Genomic Southern hybridization suggests that the Samia genome contains many other SBRP genes.

The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6-A11, A7-B10, and A20-B22. An intermolecular bond of A20-B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6-A11, A7-B10, A20-B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6-A7, A11-B10, A20-B22-cystine]- and [A6-B10, A7-A11, A20-B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.

We investigated the effects of pegylated IFN-alpha2b (PEG-IFN-alpha2b) alone and PEG-IFN-alpha2b plus 5-fluorouracil (5-FU) in vitro on the proliferation of renal cell carcinoma (RCC) cell lines. After the transplantation of RCC cells into nude mice, we administered IFN (PEG-IFN-alpha2b or IFN-alpha2b) alone, 5-FU alone, or IFN (PEG-IFN-alpha2b or IFN-alpha2b) plus 5-FU; and investigated tumor volume, tumor weight, the numbers of apoptotic cells and artery-like blood vessels, relative mRNA expression levels of enzymes which relate to 5-FU metabolism, angiogenesis factor, and type I interferon receptor. RCC cells in vitro were generally and relatively resistant to the anti-proliferative effects of PEG-IFN-alpha2b, but the addition of 5-FU augmented IFN-induced anti-proliferative effects with the induction of apoptosis. PEG-IFN-alpha2b in vivo presented stronger anti-tumor effects than IFN-alpha2b, and its combination with 5-FU augmented the effects. The significant anti-tumor effect of the combination treatment was the increase in apoptotic cell number, but there were no significant differences in the suppression of angiogenesis, expression of IFN receptor, and the actions of metabolic enzymes of 5-FU. In conclusion, PEG-IFN-alpha2b presents stronger anti-tumor effects than non-pegylated IFN, and the effects are augmented in the combination with 5-FU. Our findings suggest the clinical usefulness of PEG-IFN-alpha2b in the treatment of RCC.

A monoclonal antibody that recognized the Bombyx prothoracicotropic hormone (PTTH) was produced by immunizing mice with a synthetic pentadecapeptide corresponding to the amino-terminal portion of Bombyx PTTH. The antibody recognized both intact and reduced forms of PTTH. Immunohistochemistry with this antibody has demonstrated that PTTH is produced by two pairs of dorso-lateral neurosecretory cells of the brain and transported to the corpora allata by axons running through the contralateral hemisphere of the brain. Immunoreactive axon terminals in the corpora allata were localized between the glandular cells, suggesting that PTTH is released at the inner part of this organ.

Four medial neurosecretory cells (MNC) and 4 lateral neurosecretory cells (LNC) in each brain hemisphere, and one pair of cells in each thoracic ganglion (TG) of Galleria larva react with antibodies against bombyxin and insulin. Material secreted from the MNC and LNC is released mainly in the corpora allata, and that from the TG through the ventral median nerves. Intrinsic secretory cells of the corpora cardiaca (CC) also contain bombyxin-like, but not insulin-like material. The immunoreactivities all disappear during molts and reappear with resumption of feeding. In the MNC and TG they reappear for less than a day, but in cells of the CC immunoreactivity reappears for the whole feeding period. Before pupation, the LNC become temporarily immunopositive towards the end of feeding period, and the MNC and TG during the wandering period, i.e. at the time of prothoracic gland stimulation. Immunoreactivity disappears during the pupal molt. In pupae it is present in the 4 pairs of MNC and 1–2 pairs of LNC 12–48 hr after ecdysis, and in cells of the CC from 12 hr after ecdysis until the end of the pupal instar. In adult, immunoreactivity is restricted to 2 pairs of the LNC and to CC cells.

A novel gene C-1 encoding bombyxin, an insulin-family peptide which is produced by the brain neurosecretory cells of the silkmoth Bombyx mori, has been cloned and characterized. The nucleotide sequence of this gene and the deduced amino acid sequence are definitely different from those previously characterized for the A- and B-family bombyxin genes. Southern hybridization indicates that the Bombyx genome contains multiple gene copies closely related to this gene comprising a novel family C. Tertiary structural and functional features of bombyxins are speculated from their primary structure. Inspection of the sequences of the bombyxin genes so far characterized suggests that they may be the functional processed genes.

Accumulation of bombyxin in the brain of Bombyx mori was examined throughout post-embryonic development. Immunohistochemically, four pairs of large dorsomedial neurosecretory cells of brain were shown to contain bombyxin throughout the stages from larval hatching to adult eclosion. The bombyxin content in the brain that was estimated by immunohistochemistry, immunoblotting and bioassay remarkably changed during larval-larval and larval-pupal development. The bombyxin level in penultimate-instar larvae was high in the first half while low in the second half of the stage. It became high again at the time of ecdysis to the final instar, but gradually decreased afterward until the larvae began wandering. After wandering the bombyxin content increased gradually until pupation when a level as high as that in the newly ecdysed, final-instar larvae was regained. Immunoblotting of brain extracts demonstrated that bombyxin consisted of many molecular forms, all of which were present through larval-larval development. Based on the developmental changes in the bombyxin content in the brain, the physiological role of this neuropeptide is discussed.

Epidermal cysts of the upper urinary tract are extremely rare. Only three cases have been reported in the published work written in English, Italian or German. We encountered a case of an epidermoid cyst in the ureter of a 72-year-old male. Findings on urine analysis and radiological examination were useful for establishing a correct diagnosis of epidermoid cyst of the urinary tract.

We investigated the effects of pegylated (PEG)-IFN-alpha2b on alpha-fetoprotein (AFP) expression as demonstrated by protein and mRNA levels in six human hepatocellular carcinoma (HCC) cell lines. The number of KIM-1 cells in culture with PEG-IFN-alpha2b decreased between 24 amd 240 h, whereas the levels of intracellular and secreted AFP per cellular protein increased (except at 192 h), with levels 1.9-fold and 2.9-fold higher at maximum, respectively, than cells without PEG-IFN-alpha2b (control). The mRNA level increased between 72 and 192 h, when the level was 3-fold higher than that of the control. In the 72-h culture with 40-5000 IU/mL PEG-IFN-alpha2b, there were dose-dependent increases in AFP protein and mRNA expression and dose-dependent decrease in cell number resulting from apoptosis and blockage of the cell cycle at the S-phase. The rate of fucosylated AFP in the cell lysate decreased in a dose-dependent and time-dependent manner. In the PEG-IFN-alpha2b culture of the other five HCC cell lines, cell proliferation was suppressed, but the expressions of AFP protein and mRNA increased in only two cell lines, and suppression of cell proliferation was not related to the increase in AFP expressions. Our findings demonstrated that PEG-IFN-alpha2b induces an increase in AFP expression at both the protein and mRNA levels.

K. NAGATA1,2 , H. HATANAKA1 , D. KOHDA1 , H. ISHIZAKI3 , K. MOMOMURA4 , K. TAMORI4 , T. KADOWAKI4 , M. TANAKA2 , H. KATAOKA2 , H. NAGASAWA2 , A. ISOGAI2 , A. SUZUKI2 and F. INAGAKI1 1 Department of Molecular Physiology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan 2 Department of Agricultural Chemistry, The University of Tokyo, Tokyo, Japan 3 Department of Biology, Nagoya University, Nagoya, Japan 4 The Third Department of Internal Medicine, The University of Tokyo, Tokyo, Japan

Interferon (IFN)-alpha directly inhibits proliferation of liver cancer cells by inducing apoptosis, but the molecular mechanisms by which IFN-alpha induces apoptosis in these cells are not fully understood. We examined the effect of broad spectrum caspase inhibitor, Z-VAD-fmk, and the caspase activation in IFN-alpha-mediated apoptosis by using 4 liver cancer cell lines that were sensitive or resistant to IFN-alpha-mediated apoptosis. Involvement of apoptosis-related mitochondrial proteins and Bcl-2 family proteins in IFN-alpha-mediated apoptosis was further examined in 1 sensitive cell line (KIM-1). The Z-VAD-fmk completely or moderately inhibited IFN-alpha-mediated apoptosis in the sensitive cells. IFN-alpha induced time-dependent activation of caspase-3 in the sensitive cells, while the resistant cells showed mild or no activation. Activation of caspase-9, caspase-8, and caspase-7, and the cleavage of poly(ADP-ribose)polymerase were identified in either or both of the sensitive cell lines, but not in the resistant cells. In KIM-1 cells, the release of cytochrome c and Smac/DIABLO from mitochondria to cytosole was confirmed. Meanwhile, Bcl-xL was upregulated, and Bid activation or translocation, or conformational changes of Bax were not identified. In conclusion, our results suggest IFN-alpha-mediated apoptosis in liver cancer cells involves the mitochondrial apoptotic pathway and is induced by activating various caspases.

Interferon (IFN)-alpha directly inhibits proliferation of liver cancer cells by inducing apoptosis, but the molecular mechanisms by which IFN-alpha induces apoptosis in these cells are not fully understood. We examined the effect of broad spectrum caspase inhibitor, Z-VAD-fmk, and the caspase activation in IFN-alpha-mediated apoptosis by using 4 liver cancer cell lines that were sensitive or resistant to IFN-alpha-mediated apoptosis. Involvement of apoptosis-related mitochondrial proteins and Bcl-2 family proteins in IFN-alpha-mediated apoptosis was further examined in 1 sensitive cell line (KIM-1). The Z-VAD-fmk completely or moderately inhibited IFN-alpha-mediated apoptosis in the sensitive cells. IFN-alpha induced time-dependent activation of caspase-3 in the sensitive cells, while the resistant cells showed mild or no activation. Activation of caspase-9, caspase-8, and caspase-7, and the cleavage of poly(ADP-ribose)polymerase were identified in either or both of the sensitive cell lines, but not in the resistant cells. In KIM-1 cells, the release of cytochrome c and Smac/DIABLO from mitochondria to cytosole was confirmed. Meanwhile, Bcl-xL was upregulated, and Bid activation or translocation, or conformational changes of Bax were not identified. In conclusion, our results suggest IFN-alpha-mediated apoptosis in liver cancer cells involves the mitochondrial apoptotic pathway and is induced by activating various caspases.

This paper presents an updated estimation of the total factor productivity (TFP) of the U.S. economy following the two preceding empirical studies, Basu et al. (2001) and Burnside et al. (1995). Based on these two estimation approaches, both of which carefully handle the potential estimation bias stemming from cyclical utilization, we verified that the TFP growth in the late 90s was, to some extent, higher than that in the 80s. Further, our estimation results support the following views; (i) the effect of the higher growth of TFP on the level of output was permanent, however, (ii) the acceleration of growth rate of output was transitory in the sense that the TFP growth rates after 1999 turn out to be nearly the same as those in the 80s and early 90s.

After a decade of expansion, the US economy entered recession in March 2001. What characterizes the latest recession more than anything else is the fact that corporate sector demand, as represented by capital investment, is undergoing a deeper correction than seen in past recessions, while household sector spending (consumption expenditures and housing investment) is unusually strong. Because of this, the recession itself has been extremely shallow.

Prothoracicotropic hormone (PTTH), a brain secretory polypeptide of insects, stimulates the prothoracic glands to produce and release ecdysone, the steroid essential to insect development. The complementary DNAs encoding PTTH of the silkmoth Bombyx mori were cloned and characterized, and the complete amino acid sequence was deduced. The data indicated that PTTH is first synthesized as a 224-amino acid polypeptide precursor containing three proteolytic cleavage signals. The carboxyl-terminal component (109 amino acids) that follows the last cleavage signal represents one PTTH subunit. Two PTTH subunits are linked together by disulfide bonds, before or after cleavage from prepro-PTTH, to form a homodimeric PTTH. When introduced into Escherichia coli cells, the complementary DNA directed the expression of an active substance that was functionally indistinguishable from natural PTTH. In situ hybridization showed the localization of the prepro-PTTH mRNA to two dorsolateral neurosecretory cells of the Bombyx brain.

Bombyxin-IV, a molecular species of bombyxin, which is a member of insulin-like heterodimeric peptides of the silkworm Bombyx mori with prothoracicotropic hormone activity, was synthesized. The A- and B-chains of bombyxin-IV containing four and two Cys residues, respectively, were first synthesized separately by solid phase chemistry using Boc protocol. Then they were coupled by stepwise removal of two different protecting groups at the cysteinyl thiols for semiselective formation of disulfide bridges to give bombyxin-IV in 8% yield. The synthetic bombyxin-IV was shown to have chromatographic and biological properties identical with those of natural bombyxin-IV.

Epidermal cysts of the upper urinary tract are extremely rare. Only three cases have been reported in the published work written in English, Italian or German. We encountered a case of an epidermoid cyst in the ureter of a 72-year-old male. Findings on urine analysis and radiological examination were useful for establishing a correct diagnosis of epidermoid cyst of the urinary tract.

The insect brain neuropeptide, prothoracicotropic hormone (PTTH), elicits the molting process by stimulating ecdysteroidogenesis in the prothoracic glands. Changes in the subcellular distribution of PTTH during the fifth larval instar and larval-pupal metamorphosis of Bombyx were investigated using immunogold electron microscopy and computerized image analysis. A time-resolved fluoroimmunoassay was employed to quantify the hemolymph PTTH titer and the hemolymph ecdysteroid titer was determined by radioimmunoassay. Immunoreactivity was apparent only in the cytoplasm of two pairs of dorsal-lateral neurosecretory cells (La) in the brain, the prothoracicotropes, and their axons that traverse the peripheral area of the brain neuropil and then enter the neurohemal lobes of the corpora cardiaca and end in the corpora allata. Immunospecificity was evident as noted by the restriction of the 5-nm gold particles to the neurosecretory granules. Synaptic contacts were noted frequently between the dendritic collaterals of the prothoracicotropes and neighboring collaterals from other neurons. The morphological data suggest that PTTH is synthesized and released throughout the fifth larval instar. The concentration of gold particles in the neurosecretory granules and the number of neurosecretory granules increased five times during the fifth instar, and these increases were correlated with surges in the hemolymph PTTH and ecdysteroid titers. The data suggest that PTTH biosynthesis, secretory granule formation, and secretion are three continuous and almost synchronized events.