Identification, localisation and functional implication of 26RFa orthologue peptide in the brain of zebra finch (Taeniopygia guttata).

Several neuropeptides with the C-terminal Arg-Phe-NH(2) (RFa) sequence have been identified in the hypothalamus of a variety of vertebrates. The present study was conducted to isolate novel RFa peptides from the zebra finch brain. Peptides were isolated by immunoaffinity purification using an antibody that recognises avian RFa peptides. The isolated peptide consisted of 25 amino acids with RFa at its C-terminus. The sequence was SGTLGNLAEEINGYNRRKGGFTFRFa. Alignment of the peptide with vertebrate 26RFa has revealed that the identified peptide is the zebra finch 26RFa. We also cloned the precursor cDNA encoding this peptide. Synteny analysis of the gene showed a high conservation of this gene among vertebrates. In addition, we cloned the cDNA encoding a putative 26RFa receptor, G protein-coupled receptor 103 (GPR103) in the zebra finch brain. GPR103 cDNA encoded a 432 amino acid protein that has seven transmembrane domains. In situ hybridisation analysis in the brain showed that the expression of 26RFa mRNA is confined to the anterior-medial hypothalamic area, ventromedial nucleus of the hypothalamus and the lateral hypothalamic area, the brain regions that are involved in the regulation of feeding behaviour, whereas GPR103 mRNA is distributed throughout the brain in addition to the hypothalamic nuclei. When administered centrally in free-feeding male zebra finches, 26RFa increased food intake 24 h after injection without body mass change. Diencephalic GPR103 mRNA expression was up-regulated by fasting for 10 h. Our data suggest that the hypothalamic 26RFa-its receptor system plays an important role in the central control of food intake and energy homeostasis in the zebra finch.

Cytoskeletal abnormalities in relation with meiotic competence and ageing in porcine and bovine oocytes during in vitro maturation.

We investigated the frequencies of cytoskeletal anomalies in metaphase-II (M-II) and incompetent [arrested at an immature metaphase (IM) stage] porcine and bovine oocytes during in vitro maturation (IVM) in relation with ageing by immunostaining and confocal microscopy. In porcine oocytes, meiotic arrest at the IM stage was associated with abnormalities of cortical actin but not with abnormal spindles. Prolongation of IVM culture to 52 h did not affect microfilament and spindle abnormalities, but reduced the microfilament-rich area overlaying the spindle. Meiotic arrest of bovine oocytes at the IM stage was associated with degenerations of microfilaments, and the frequencies of abnormal spindles were also higher than those of M-II oocytes. Ageing of bovine oocytes (IVM for 30 h) did not affect cortical microfilaments but increased the frequency of spindle alterations in both M-II and IM bovine oocytes. These results suggest that, in both species, altered ability of oocytes to polymerize F-actin might be a possible reason for the failure of polar body extrusion during IVM. Also, there seem to be differences between the two species in the sensitivity of oocytes to suffer ageing-related spindle damages.

Development of single blastomeres derived from two-cell embryos produced in vitro in pigs.

The objective was to investigate development of single blastomeres derived from IVP two-cell porcine embryos. There was no difference (P > 0.05) in blastocyst rates among intact two-cell embryos (IN), zona-free two-cell embryos (ZF), and single blastomere (SB) groups (50.0 ± 9.7, 57.4 ± 5.7, and 45.1 ± 7.2%, respectively; mean ± SEM). However, blastocyst yield for the SB group (90.2 ± 14.4%, based on the original number of two-cell embryos before blastomere separation) was higher (P < 0.05) than those of IN and ZF groups. Although the number of inner cell mass (ICM) and trophectoderm (TE) cells in SB blastocysts (6.2 ± 0.8 and 15.5 ± 1.1, respectively) was lower (P < 0.05) than those in IN (12.4 ± 1.3 and 26.0 ± 3.8) and ZF blastocysts (10.7 ± 1.6 and 26.4 ± 3.4), ICM:TE ratios did not differ significantly among groups. Expressions of transcripts associated with cellular organization (TUBA1 and TUBB) were reduced (P < 0.05) in SB versus IN blastocysts. However, there was no significant difference among groups for expression of transcripts associated with responses to stress (HSPE1, HSPD1, and HSPCA) or glucose catabolism (ENO1, COX6C, COX7B, NDUFA4, NDUFA13, UCRC, and UQCRFS1) in blastocysts. The percentage of the sister blastomere pairs in which both cells developed to blastocysts (36.6 ± 5.3%) or both degenerated (46.3 ± 10.3%) were higher (P < 0.05) than that of the pairs in which one developed to blastocyst while the other degenerated (17.1 ± 7.8%). When both pairs developed to blastocysts, one blastocyst had more (P < 0.05) ICM and TE cells (8.2 ± 1.2 and 20.2 ± 2.1, respectively) than the other (5.2 ± 0.9 and 13.5 ± 1.1), although ICM:TE cell ratios were not significantly different. In conclusion, blastomere separation at the two-cell stage significantly increased blastocyst yield from IVP porcine embryos. This might be a useful approach for conservation of rare pig breeds, in which low numbers of embryos limited the success of embryo transfer.

Octopus gonadotrophin-releasing hormone: a multifunctional peptide in the endocrine and nervous systems of the cephalopod.

The optic gland, which is analogous to the anterior pituitary in the context of gonadal maturation, is found on the upper posterior edge of the optic tract of the octopus Octopus vulgaris. In mature octopus, the optic glands enlarge and secrete a gonadotrophic hormone. A peptide with structural features similar to that of vertebrate gonadotrophin-releasing hormone (GnRH) was isolated from the brain of octopus and was named oct-GnRH. Oct-GnRH showed luteinising hormone-releasing activity in the anterior pituitary cells of the Japanese quail Coturnix coturnix. Oct-GnRH immunoreactive signals were observed in the glandular cells of the mature optic gland. Oct-GnRH stimulated the synthesis and release of sex steroids from the ovary and testis, and elicited contractions of the oviduct. Oct-GnRH receptor was expressed in the gonads and accessory organs, such as the oviduct and oviducal gland. These results suggest that oct-GnRH induces the gonadal maturation and oviposition by regulating sex steroidogenesis and a series of egg-laying behaviours via the oct-GnRH receptor. The distribution and expression of oct-GnRH in the central and peripheral nervous systems suggest that oct-GnRH acts as a multifunctional modulatory factor in feeding, memory processing, sensory, movement and autonomic functions.

[The evaluation of the complications and its management in 800 patients who underwent the thoracoscopic surgery].

Thoracoscopy has been applied to almost all thoracic surgery as shown (table 1). Besides an opportunity to follow and encounter a trouble during surgery using thoracoscopy, so that it is necessary to become skilled in the prediction and management of complication. Nowadays, there are two options on surgery using thoracoscopy, thoracoscopic surgery (TS) is performed only on in a port and on a mini-thoracotomy with a few ports (video-assisted thoracoscopic surgery: VATS). When changing into emergency or converting into open thoracotomy, a muscle sparing thoracotomy could be available. The symptoms which occurred in the thorax are fundamentally similar during both TS and VATS procedure. However, peculiar dangerous factor existed according to the difference in internal organs, procedures, and diseases. Critical complications during surgery were as follows, i.e., injury of pulmonary vessels and bronchus, tension pneumothorax of the opposite lung and tumor implantation of the chest wall. Basically, standard practice is the most important to avoid an accident and determine whether converting to open thoracotomy should be employed. In the present commentary, we would like to present our experiences and propose regarding management.

Comparison of changes in hemodynamics between unilateral and bilateral lung volume reduction for pulmonary emphysema.

This study was aimed to compare changes in hemodynamics between unilateral (UL) or simultaneous bilateral (BL) lung volume reduction surgery (LVRS) for chronic obstructive lung disease. Sixteen patients underwent LVRS by stapler resection with neodymium: yttrium-alminum-garnet (Nd: YAG) laser ablation; five underwent BL-LVRS (four by median sternotomy and one by thoracoscopy) and 11 underwent UL-LVRS by thoracoscopy. Four patients had multiple bullae within pulmonary emphysema. At preoperation and 6, 12, 24, and 48 hours postoperatively, hemodynamics and right ventricular performance were evaluated. UL- and BL-LVRS reduced afterload of the right and left ventricle postoperatively. Although the pulmonary arterial resistance increased after surgery, the total pulmonary resistance decreased (p=0.001) in association with the reduced systemic vascular resistance (p=0.001). These reductions improved cardiopulmonary circulation, resulting in increased stroke volume and cardiac output (p=0.003). The right ventricular ejection fraction showed minimal change 48 hours postoperation. Two patients died of pneumonia caused by persistent air leakage. In conclusion, both the UL- and BL-LVRS showed similar effectiveness in terms of improvement in the systemic and cardiopulmonary circulation after LVRS, if there were no postoperative complications. We concluded that we had to reduce and repair the persistent air leakage after LVRS.

Legionnaires' disease associated with habitual drinking of hot spring water.

A 57-year-old man presented with pneumonia, respiratory distress, and myelodysplastic syndrome. A diagnosis of Legionnaires' disease due to Legionella pneumophila (L. pneumophila) was established. The patient had long been drinking tap water via a conduit from a hot spring resource, from which L. pneumophila was also isolated. Both the patient's strain and the water strain of L. pneumophila were identified as serogroup 1, and the genetic relatedness between the two strains as seen by pulsed-field gel electrophoresis was 87%. The patient was successfully treated with erythromycin, fluoroquinolone, and rifampicin. This case raises an important issue on public health represented by legionellosis in Japan.

Transcriptional regulation of Mesp1 and Mesp2 genes: differential usage of enhancers during development.

Mesp1 and Mesp2 encode bHLH-type transcription factors, Mesp1 and Mesp2, respectively. The expression of both genes is observed in the nascent mesoderm, and subsequently in the rostral presomitic mesoderm. To determine the regulatory mechanism for gene expression, we attempted to identify enhancer elements by transient transgenic analysis. At least two enhancers, which are responsible for the expression of the two genes in the early mesoderm (early mesodermal enhancer, EME) and the presomitic mesoderm (PSM enhancer, PSME), and one suppressor, which is responsible for the rostrally restricted expression in the presomitic mesoderm, were identified. Deletion studies of these enhancer elements indicate that either gene may use the same enhancer for early mesoderm development, whereas both genes may utilize separate enhancers to regulate their expression in the presomitic mesoderm.

High survival rate of 6 cases of pulmonary large cell neuroendocrine carcinoma formerly classified as small cell carcinoma.

UNLABELLED: In the revised WHO classification of lung cancer, published in 1999, large cell neuroendocrine carcinoma (LCNEC) was employed as a new histological entity. LCNEC is generally considered a high-grade malignant lung cancer, and appropriate treatment remains to be determined. Before its new classification, LCNEC had long been classified into several entities. Advancing the review of previous cases in Nippon Medical School Hospital, we noticed that some LCNEC patients were formally diagnosed as having small cell lung cancer (SCLC), and they showed long-term survival. MATERIAL AND METHODS: All histological specimens of surgically resected SCLC in Nippon Medical School Hospital were reclassified according to the 1999 WHO classification manual. Their neuroendocrine differentiations were confirmed by the use of immunostainings with chromogranin A and synaptophysin. RESULTS: Fourteen cases satisfied the qualifications for both histological and clinical reevaluation. Among them, 6 patients were reclassified as LCNEC, and their stage distribution was as follow: IA; 1, IB; 2, IIIA; 2, and IIIB; 1. Their survival term ranged from 33.8 to 78.0 months; 5 were still alive, and 1 (IIIB) died 57.6 months after surgery. DISCUSSION: According to this study, all the LCNEC patients who were treated as SCLC patients showed more favorable prognoses than patients described in published studies, even overall lung cancer. Therefore, it is suggested that multimodality therapy for SCLC may improve the prognoses of patients with LCNEC.

A retroviral-derived immunosuppressive peptide activates mitogen-activated protein kinases.

The highly conserved region within the retroviral transmembrane envelope proteins has been implicated in a number of retrovirus-associated mechanisms of immunosuppression. CKS-17, a synthetic peptide representing the prototypic sequence of the immunosuppressive domain, has been found to suppress numerous immune functions, disregulate cytokines, and elevate intracellular cAMP. In this report we show that using a human monocytic cell line THP-1, CKS-17 activates mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase 1 and 2 (ERK1/2). Kinetic studies show that CKS-17 induces an acute increase of ERK1/2 activity followed by a rapid decrease and then a second sustained increase of ERK1/2. CKS-17 also activates MAP kinase/ERK kinase (MEK) with a similar induction pattern. Mutant THP-1 cells isolated in our laboratory, in which CKS-17 exclusively fails to activate cAMP, did not show the transient decrease of CKS-17-induced ERK1/2 phosphorylation. Pretreatment of THP-1 cells or mutant THP-1 cells with cAMP analog or forskolin followed by treatment with CKS-17 showed no activation of MEK or ERK1/2. These results indicate that CKS-17 activates the MEK/ERK cascade and that there is a cross-talk between CKS-17-mediated MEK/ERK cascade and cAMP in that the MEK/ERK cascade is negatively regulated by cAMP. These data present a novel molecular mechanism(s) by this highly conserved retroviral immunosuppressive component.

Immunologic reconstitution following bone marrow transplantation for X-linked hyper IgM syndrome.

X-linked hyper IgM syndrome (XHIM), caused by mutations of the CD40 ligand (CD40L) gene, is characterized by recurrent bacterial and opportunistic infections, an increased incidence of autoimmunity and malignancies, and immunodeficiency due to abnormal T/B cell interaction. Because of poor long-term prognosis, bone marrow transplantation (BMT) has been proposed as an alternative treatment. An 8-month-old boy with XHIM and a splice site mutation of CD40L underwent BMT using a fully matched sibling donor. Markers of engraftment and immunologic reconstitution were measured serially. After BMT, activated T cells expressed functional CD40L, and genomic DNA obtained from circulating white cells contained predominantly wild-type CD40L sequences. Serum immunoglobulin levels including IgE and antibody responses to recall antigens normalized, and immunization with the T-cell-dependent neoantigen, bacteriophage φX174, demonstrated amplification of the response and isotope switching. BMT provides a permanent cure for XHIM if a fully matched sibling donor is available and the procedure is performed before complications have occurred.

Involvement of a herbimycin A-sensitive protein tyrosine kinase in extracellular action of HIV-1 Nef.

Extracellular Nef which has been implicated in disease progression and development of AIDS induces IL-10, a potent immunosuppressive cytokine, in vitro. The present study was designed to examine whether the action of extracellular Nef is a protein tyrosine kinase (PTK)-dependent event. Anti-phosphotyrosine immunoblotting reveals that recombinant HIV-1 Nef induces rapid tyrosyl phosphorylation of several cellular proteins in human peripheral blood mononuclear cells. Pre-treatment of cells with herbimycin A, but not with genistein, significantly abolishes the Nef-induced tyrosine phosphorylation of cellular proteins. Furthermore, ELISA and RNase protection assays show that herbimycin A significantly blocks Nef-induced production of IL-10 at both the protein and the mRNA level. Genistein and aminogenistein have a much less blocking effect on the ability of Nef to induce IL-10. These results provide evidence for the involvement of a herbimycin A-sensitive PTK in the signal transduction pathway for exogenous HIV-1 Nef.

Prediction of the postoperative pulmonary function and complication rate in elderly patients.

We analyzed the risk factors predisposing elderly patients to develop postoperative respiratory complications (PRCs) and investigated the possibility of predicting the postoperative pulmonary function and PRC rate. The postoperative pulmonary functions were predicted according to a simplified system, which we developed using plain chest roentgenograms from patients with primary lung cancer. Both univariate and multivariate analyses of PRCs were performed in 39 elderly patients with lung cancer from July 1982 to March 1991 (the early period). Based on the results obtained, the permissible extent of lung resection to achieve a predicted postoperative % forced expiratory volume in 1 s (ppo%FEV1.0) and a predicted postoperative % vital capacity (ppo%VC) of more than 55% was selected as the basic criteria for undergoing such an operation after April 1991 (the recent period). A ppo%FEV1.0 and/or ppo%VC of 55% or less was the most significant risk factor for developing PRCs. The PRC rate decreased from 33.3% to 9.8% (P = 0.0251) and the operative mortality rate decreased from 10.3% to 0%. The survival rates for stage I, II. and III cases were not significantly different between the early and recent periods. Decisions made on the operability and the permissible extent of lung resection based on our system using plain chest roentgenograms therefore appeared to reduce the PRC rate and operative mortality rate in elderly patients.

Effectiveness of prostaglandin E1 on pulmonary hypertension and right cardiac function induced by single-lung ventilation and hypoventilation.

It is known that prostaglandin E1 (PGE1) is a potent vasodilator and improves red cell deformability. Single lung-ventilation sometimes occurs under lung transplantation, lung cancer surgery and traumatic pneumonectomy, and may result in increased pulmonary resistance, right heart failure and severe hypoxemia. The present experimental study was undertaken to examine the effects of PGE1 on these states induced by single-lung ventilation and hypoventilation. Fourteen pigs weighing 32-33 kg were anesthetized, intubated and ventilated using a respirator and then randomly assigned to two groups, the control group and the PGE1 treated group, 7 pigs each. After median sternotomy to induce severe hypoxemia hypoventilation was induced and then the right hilus pulmonis was cross clamped. Mean blood pressure, mean pulmonary arterial pressure (PAP), pulmonary vessel resistance (PVR), right ventricular stroke work (RVSW) and arterial blood gases (PaO2 and SatO2) were measured at baseline, in the hypoventilation state, and 15 min, 1 hour, and 2 hours after the right hilus pulmonis clamping with hypoventilation. PGE1 (250 microg/20 ml saline) was administered via the central vein starting 15 min after right hilus cross clamping for 1 hour and 45 min in the PGE1 group. PGE1 significantly reduced PAP and PVR, normalized RVSW, and improved PaO2. PGE1 may be useful for the condition of increased pulmonary hypertension during single-lung ventilation and hypoventilation.

Monocytes are target cells for IL-10 induction by HIV-1 Nef protein.

IL-10 plays a pivotal role in the pathogenesis of several diseases and is elevated in sera of HIV-infected patients. Recently, we demonstrated that HIV Nef induces IL-10 mRNA expression as well as IL-10 production using PBMCs, H9 or U937 cells. This induction of IL-10 is inhibited by a calmodulin antagonist, W-7. In the present study, T or B lymphocytes or monocytes were isolated from PBMCs of healthy HIV-negative donors. Production of IL-10 and mRNA gene expression were analyzed on each isolated cell population after treatment with Nef or SEA for 3-24 h. The results show that Nef induces IL-10 production as well as mRNA expression significantly using monocytes but not with T or B lymphocytes. By contrast, SEA induced IL-10 production as well as mRNA expression using T lymphocytes but not with monocytes or B lymphocytes.

Pulmonary carcinosarcoma: immunohistochemical and ultrastructural studies.

A case of pulmonary carcinosarcoma in a 68-year-old male patient is reported. The tumor in the resected left upper lobe extended mainly endobronchially, invading the normal bronchial lumina and mucosa. The carcinomatous component consisted of poorly differentiated squamous cell carcinoma and was mainly located in the periphery of the tumor nests. The sarcomatous component consisted of chondrosarcoma and was mainly located in the center of the tumor nests. Tumor cells in the sarcomatous component reacted with anti-S-100 protein antibody and were surrounded with abundant homogeneous extracellular matrix staining positively with Alcian blue. The transition from the carcinomatous component to the sarcomatous component appeared to be very smooth. The tumor cells in both the carcinomatous and sarcomatous components reacted with anti-epithelial membrane antigen antibody. Ultrastructurally, the tumor cells with tonofibrils in the carcinomatous component were apposed and connected to each other by desmosomes. By contrast, in the sarcomatous component, the tumor cells had well-developed and dilated rough endoplasmic reticulum and were arranged loosely in a myxomatous matrix. Some tumor cells in the sarcomatous component had occasional tonofibrils, and were apposed and connected to each other by desmosome-like structures. It is shown for the first time, ultrastructurally and immunohistochemically, that the tumor cells in the sarcomatous component of pulmonary carcinosarcomas have features of both epithelial and mesenchymal cells. It is suggested that the sarcomatous component in the present case is derived from the carcinomatous component.

Expression of alpha-smooth muscle actin in small bronchioloalveolar adenocarcinoma of the lung: assessment and comparison with noguchi criteria.

The Noguchi criteria are useful in assessing the prognosis of patients with small lung adenocarcinoma. Although there is a significant difference in prognosis between type A or B and type C, it is difficult in some cases to distinguish these types accurately by microscopy. In this study, we used immunohistochemistry to examine alpha-smooth muscle actin (alpha-SMA) produced by active fibroblasts in 25 pulmonary adenocarcinomas less than 2 cm in diameter. Eleven of type C (61%) showed positive staining for alpha-SMA, whereas no positive cases were seen in type A or B. The incidence of cancerous blood vessel and lymphatic invasion were significantly higher in alpha-SMA positive cases than in negative cases, and the positive cases showed poorer prognosis. These findings indicate that immunohistochemical detection of alpha-SMA is useful and essential for histological typing by the Noguchi criteria.