Anatomical and functional pathways of rhythmogenic inspiratory premotor information flow originating in the pre-Bötzinger complex in the rat medulla.

The pre-Bötzinger complex (preBötC) of the ventrolateral medulla is the kernel for inspiratory rhythm generation. However, it is not fully understood how inspiratory neural activity is generated in the preBötC and propagates to other medullary regions. We analyzed the detailed anatomical connectivity to and from the preBötC and functional aspects of the inspiratory information propagation from the preBötC on the transverse plane of the medulla oblongata. Tract-tracing with immunohistochemistry in young adult rats demonstrated that neurokinin-1 receptor- and somatostatin-immunoreactive neurons in the preBötC, which could be involved in respiratory rhythmogenesis, are embedded in the plexus of axons originating in the contralateral preBötC. By voltage-imaging in rhythmically active slices of neonatal rats, we analyzed origination and propagation of inspiratory neural activity as depolarizing wave dynamics on the entire transverse plane as well as within the preBötC. Novel combination of pharmacological blockade of glutamatergic transmission and mathematical subtraction of the video images under blockade from the control images enabled to extract glutamatergic signal propagations. By ultra-high-speed voltage-imaging we first demonstrated the inter-preBötC conduction process of inspiratory action potentials. Intra-preBötC imaging with high spatiotemporal resolution during a single spontaneous inspiratory cycle unveiled deterministic nonlinearities, i.e., chaos, in the population recruitment. Collectively, we comprehensively elucidated the anatomical pathways to and from the preBötC and dynamics of inspiratory neural information propagation: (1) From the preBötC in one side to the contralateral preBötC, which would synchronize the bilateral rhythmogenic kernels, (2) from the preBötC directly to the bilateral hypoglossal premotor and motor areas as well as to the nuclei tractus solitarius, and (3) from the hypoglossal premotor areas toward the hypoglossal motor nuclei. The coincidence of identified anatomical and functional connectivity between the preBötC and other regions in adult and neonatal rats, respectively, indicates that this fundamental connectivity is already well developed at the time of birth.

Genetic mapping of quantitative trait loci affecting growth and carcass traits in F2 intercross chickens.

We constructed a chicken F(2) resource population to facilitate the genetic improvement of economically important traits, particularly growth and carcass traits. An F(2) population comprising 240 chickens obtained by crossing a Shamo (lean, lightweight Japanese native breed) male and White Plymouth Rock breed (fat, heavyweight broiler) females was measured for BW, carcass weight (CW), abdominal fat weight (AFW), breast muscle weight (BMW), and thigh muscle weight (TMW) and was used for genome-wide linkage and QTL analysis, using a total of 240 microsatellite markers. A total of 14 QTL were detected at a 5% chromosome-wide level, and 7 QTL were significant at a 5% experiment-wide level for the traits evaluated in the F(2) population. For growth traits, significant and suggestive QTL affecting BW (measured at 6 and 9 wk) and average daily gain were identified on similar regions of chromosomes 1 and 3. For carcass traits, the QTL effects on CW were detected on chromosomes 1 and 3, with the greatest F-ratio of 15.0 being obtained for CW on chromosome 3. Quantitative trait loci positions affecting BMW and TMW were not detected at the same loci as those detected for BMW percentage of CW and TMW percentage of CW. For AFW, QTL positions were detected at the same loci as those detected for AFW percentage of CW. The present study identified significant QTL affecting BW, CW, and AFW.

Genetic mapping of quantitative trait loci affecting body weight, egg character and egg production in F2 intercross chickens.

Phenotypic measurements of chicken egg character and production traits are restricted to mature females only. Marker assisted selection of immature chickens using quantitative trait loci (QTL) has the potential to accelerate the genetic improvement of these traits in the chicken population. The QTL for 12 traits (i.e. body weight (BW), six for egg character, three for egg shell colour and two for egg production) of chickens were identified. An F2 population comprising 265 female chickens obtained by crossing White Leghorn and Rhode Island Red breeds and genotyped for 123 microsatellite markers was used for detecting QTL. Ninety-six markers were mapped on 25 autosomal linkage groups, and 13 markers were mapped on one Z chromosomal linkage group. Eight previous unmapped markers were assigned to their respective chromosomes in this study. Significant QTL were detected for BW on chromosomes 4 and 27, egg weight on chromosome 4, the short length of egg on chromosome 4, and redness of egg shell colour (using the L*a*b* colour system) on chromosome 11. A significant QTL on the Z chromosome was linked with age at first egg. Significant QTL could account for 6-19% of the phenotypic variance in the F2 population.

Quantitative trait loci analysis for growth and carcass traits in a Meishan x Duroc F2 resource population.

We constructed a pig F2 resource population by crossing a Meishan sow and a Duroc boar to locate economically important trait loci. The F2 generation was composed of 865 animals (450 males and 415 females) from four F1 males and 24 F1 females and was genotyped for 180 informative microsatellite markers spanning 2,263.6 cM of the whole pig genome. Results of the genome scan showed evidence for significant quantitative trait loci (<1% genomewise error rate) affecting weight at 30 d and average daily gain on Sus scrofa chromosome (SSC) 6, carcass yield on SSC 7, backfat thickness on SSC 7 and SSC X, vertebra number on SSC 1 and SSC 7, loin muscle area on SSC 1 and SSC 7, moisture on SSC 13, intramuscular fat content on SSC 7, and testicular weight on SSC 3 and SSC X. Moreover, 5% genomewise significant QTL were found for birth weight on SSC 7, average daily gain on SSC 4, carcass length on SSC 6, SSC 7, and SSC X and lightness (L value) on SSC 3. We identified 38 QTL for 28 traits at the 5% genomewise level. Of the 38 QTL, 24 QTL for 17 traits were significant at the 1% genomewise level. Analysis of marker genotypes supported the breed of origin results and provided further evidence that a suggestive QTL for circumference of cannon bone also was segregating within the Meishan parent. We identified genomic regions related with growth and meat quality traits. Fine mapping will be required for their application in introgression programs and gene cloning.

Pneumonitis induced by ou-gon (scullcap).

A 53-year-old Japanese man with recurrent interstitial pneumonia was referred to us. The patient had taken a traditional herb medicine, otsu-ji-to, before the onset of pneumonia. A provocation test for each herbal ingredient contained in otsu-ji-to revealed that the pneumonitis had been induced by ou-gon (scullcap). Lymphocytosis with the CD8+ T-cell subset predominance was found in the bronchoalveolar lavage fluid and lymphocytic alveolitis was noted in the transbronchial lung biopsy specimen after the provocation test. Ou-gon, or scullcap, should be included in the list of drugs with definite causal association with pneumonitis.

[A case of relapsed small cell lung cancer recognized by simple metastasis to the duodenum].

We describe a case of relapsed small cell lung cancer (SCLC) recognized by a duodenal tumor, its probable metastasis. A 69-year-old man was admitted to Keio University Hospital complaining of persistent sputum production in September, 1996. A diagnosis of SCLC of the left lung invading the mediastinum was based on a transesophageal biopsy. Chemotherapy consisting of CDDP and VP-16 followed by thoracic irradiation at a total dose of 50 Gy was performed from October 1996 to August 1997, resulting in CR (Complete Response) of the tumor. In April of 1999, a mass surrounding the duodenum was found through an abdominal CT survey for tumor relapse, but no other tumors were detected in a series of CT scans or inbone scintigraphy. Subsequent fiberscopy of the upper gastrointestinal tract revealed an ulcerative tumor extensively invading the mucosa of the duodenal bulb. Biopsy specimens obtained from the duodenal tumor showed small-cell carcinoma with features similar to those of SCLC found in 1996, suggesting that SCLC of the left lung metastasized to the duodenal wall. Chemoradiotherapy with 4 cycles of CDDP and VP-16 followed by abdominal irradiation at a dose of 30 Gy was given again from May to September 1999, producing good PR (Partial Response). Although metastasis of SCLC to the duodenum seldom occurs, this report indicates that its early detection and effective treatment may prevent serious symptoms caused by obstruction of the duodenum or the papilla Vater.

Thymoma with pemphigus foliaceus.

A 75-year-old Japanese woman was referred to us because of an anterior mediastinal mass. Crusts and shallow erosions developed 10 months earlier on her upper chest, back, and scalp. Pemphigus foliaceus was diagnosed based on histological examination of skin biopsy specimens and positivity for serum anti-desmoglein 1 antibody by enzyme-linked immunosorbent assay. Neurological examination and electromyography ruled out myasthenia gravis. Total thymectomy was performed, and the postoperative pathology studies showed mixed lymphoepithelial thymoma. One year after the resection, the eruption and alopecia improved and the serum anti-desmoglein 1 antibody titer decreased, suggesting a beneficial effect of thymectomy on thymoma-related pemphigus.

[A case of squamous cell lung cancer in a nonsmoking female, successfully treated with docetaxel and cisplatin].

A 62-year-old nonsmoking female was admitted to our hospital in May, 1998 complaining of marked cough accompanied by repeated hemosputa. Chest X-ray and CT examinations revealed a large tumor, located adjacent to a cystic lesion in the left lower lung field, in association with a clearly recognizable swelling of the ipsilateral hilar as well as the mediastinal lymph nodes. Sputum cytology after bronchofiberscopy led to the diagnosis that the patient suffered from squamous cell lung cancer of Stage IIIA with bulky N2 (T2N2M0). Chemotherapy was selected as the most reasonable treatment for this patient. The new chemotherapeutic agent docetaxel (60 mg/m2) in combination with cisplatin (CDDP: 80 mg/m2) was tried, resulting in a remarkable reduction in tumor size by 60% after the initial course of chemotherapy was completed, which fulfilled the definition of a partial response (PR). Furthermore, after 4 courses of the chemotherapy, the hilar and mediastinal lymphadenopathy had conspicuously abated and only scar tissue was visible at the site where the lung cancer was thought to have originally developed. We herein report a case in which squamous cell lung cancer sprouted in a nonsmoking female, who was successfully treated by the combined chemotherapy of docetaxel and CDDP. The present case may suggest the efficacy of newly developed docetaxel in treating non-small cell lung cancer.

Age-dependent chemosensitive pontine inhibition of medullary respiratory rhythm generation in the isolated brainstem of the neonatal rat.

Age-dependence and chemosensitivity of the pontine inhibitory effect on medullary respiratory rhythm generation were examined in the isolated brainstem-spinal cord of the neonatal rat. In early preparations (days 1-2), the increase in RR (Delta RR) induced by the pons resection was larger in 8% CO(2) (pH 7.2) than in 2% CO(2) (pH 7.8). That difference was not found in late preparations (days 3-4). Under a given pH, the Delta RR was larger in early preparations than in late preparations.

Locus coeruleus neurones in vitro: pH-sensitive oscillations of membrane potential in an electrically coupled network.

The response to hypercapnic acidosis (2-8% CO2, bath pH 7.8-7.2) was examined in whole cell recordings from neonatal (P1 to P5) rat Locus coeruleus (LC) neurones in the in vitro brainstem-spinal cord preparation exposed to low Ca2+ (0.2 mM)-high Mg2+ (5 mM). This medium suppressed chemical synaptic transmission and resulted in a pattern of subthreshold oscillations of membrane potential and rhythmic burst discharge which was synchronized throughout the network. The oscillation was suppressed, and the discharge of individual neurones desynchronized, by the gap junction uncoupler, carbenoxolone, indicating that in low Ca2+-high Mg2+ LC neurones form an electrically coupled network. Switching from 2 to 8% CO2 decreased the oscillation amplitude and increased its frequency. The oscillation was suppressed by external Cd2+ and by TTX. but persisted during injection into the cell soma of QX-314. We conclude that in LC neurones acidosis increases the frequency of a Ca2+- and Na+-dependent dendritic oscillator which is synchronized by gap junction coupling throughout the network. This coupling is retained during acidosis.

Respiration-modulated membrane potential and chemosensitivity of locus coeruleus neurones in the in vitro brainstem-spinal cord of the neonatal rat.

1. The activity of locus coeruleus (LC) neurones (n = 126) was examined in whole-cell (conventional and amphotericin B-perforated patch) recordings, and the relationship of this activity to the respiratory discharge recorded on the C4 or C5 phrenic nerve roots was determined at different CO2 concentrations (2 and 8 %; bath pH 7. 8 and 7.2) in the in vitro brainstem-spinal cord preparation of the neonatal rat (1-5 days old). 2. In most neurones (n = 105) ongoing activity was modulated at respiratory frequency. Typically, this consisted of a phase of depolarization and increased discharge frequency synchronous with the phrenic burst, followed by a phase of hyperpolarization and inhibition of discharge (n = 94 of 105). The incidence of respiratory modulation decreased from 91 % on P1 to 57 % on P5. 3. Bath application of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5 microM) or the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV; 100 microM) abolished both phases of respiratory modulation. The hyperpolarizing phase alone was abolished by the adrenoceptor antagonists idazoxan (5 microM) or phentolamine (0.8 microM). These results indicate that excitatory amino acid pathways are involved in the transmission of both the excitatory and inhibitory components and that the latter involves in addition an alpha2-adrenoceptor-mediated pathway. 4. Increasing the CO2 concentration from 2 to 8 % resulted in a shortening of expiratory duration and weakening or loss of respiratory-phased inhibition; this was accompanied by depolarization, increased discharge frequency and, in those neurones where they were initially present (60 %), an increase in the frequency of subthreshold membrane potential oscillations. The depolarizing response was retained in the presence of tetrodotoxin (TTX, 0.2-1.0 microM). 5. These results indicate that in this neonatal preparation LC neurones form part of the synaptically connected brainstem respiratory network, and that the LC constitutes a site of CO2- or pH-dependent chemoreception.

Effects of active vasoconstriction and total flow on perfusion distribution in the rabbit lung.

We analyzed the effects of hypoxic vasoconstriction and total flow on the distribution of pulmonary perfusion in 38 isolated left rabbit lungs perfused under zone 3 conditions. Lungs were suspended in an upright position, oriented to the apicobasal line. Distributions of regional perfusion rates (RPR) along the vertical and horizontal axes were determined using nonradioactive microspheres labeled with heavy metal elements, which were detectable with X-ray fluorescence spectrometry. Changing the O2 concentration of a respirator and an extracorporeal membrane oxygenator independently, respective influences of active vasoconstriction induced by alveolar hypoxia and pulmonary artery hypoxia (PA hypoxia) on the RPR distribution were examined at a flow rate of 0.4 ml x min(-1) x g wet lung tissue(-1). To analyze the effects of changes in total flow, we investigated the RPR distribution at a perfusion rate of 1.2 ml x min(-1) x g wet lung tissue(-1). The RPR distribution in the absence of hypoxia was inhomogeneous and was augmented in the lower lung fields, whereas alveolar hypoxia shifted the RPR upward and significantly diminished the RPR in the lung base. RPR distributions along the horizontal axes under alveolar hypoxia conditions demonstrated that remarkable hypoxic pulmonary vasoconstriction (HPV) takes place in medial regions at the lung base. PA hypoxia altered the RPR distribution in qualitatively the same manner as alveolar hypoxia. Increased flow rate augmented the RPR in the lung, except in the dorsobasal region. These results suggest that the occurrence of HPV and the vascular conductance are not uniform throughout the lung.

Changes in the expression of gap junction proteins (connexins) in hamster tongue epithelium during wound healing and carcinogenesis.

We examined changes in the expression and localization of connexin proteins and transcripts by means of immunofluorescence and in situ hybridization in normal conditions, wound healing and carcinogenesis using hamster tongue epithelium, in which differentiation, migration and growth of keratinocytes takes place physiologically and pathologically. In normal hamster tongue epithelium, immunofluorescent staining showed that Cx26 and Cx43 proteins were localized differently during differentiation of keratinocytes, but in in situ hybridization, the localization of Cx26 and Cx43 transcripts overlapped considerably, suggesting that the different localization of Cx26 and Cx43 proteins in squamous epithelium is largely regulated at post-transcriptional levels. During wound healing, the expression and localization of connexin proteins and transcripts were changed drastically. Shortly (6 h) after injury the expression of Cx26 and Cx43 proteins decreased at wound edges, but by 1-3 days after injury the expression of both proteins increased and both proteins co-localized to the same spots in the epithelium near wound edges. During carcinogenesis, the increased expression of Cx26 and Cx43 proteins and their transcripts and co-localization of both proteins occurred in papillomas, and the expression of Cx26 was reduced as cancer cells became morphologically less differentiated. We also found, that during wound healing in papillomas, squamous cell carcinomas and keratinocytes, Cx26 and Cx43 proteins were localized aberrantly in the cytoplasm, especially around nuclei, rather than on plasma membranes. These results indicate that quantitative and qualitative changes in connexin expression are associated with differentiation, migration and proliferation of keratinocytes in squamous epithelium.

Inhomogeneities of ventilation and the diffusing capacity to perfusion in various chronic lung diseases.

Although impairment of gas exchange caused by ventilation-perfusion (VA/Q) mismatch has been extensively analyzed, there have been no systematic studies focused on determining the distributions of diffusion properties in dose connection with those of VA/Q. We attempted to clarify the simultaneous distributions of VA/Q and diffusion capacity to perfusion (D/Q) in patients with idiopathic pulmonary fibrosis (IPF) or chronic obstructive pulmonary disease (COPD). To assess pathologic determinants causing functional abnormalities, we compared VA/Q and D/Q distributions with the findings on high-resolution computed tomography. O2, CO2, and CO together with six foreign inert gases were used as indicator gases. We transformed the measured data on indicator gases in arterial blood into a continuous distribution of Q in the VA/Q-D/Q field. In IPF, active alveolitis or acinitis played a major role in producing low D/Q regions impeding gas exchange via a diffusion limitation, whereas extensive fibrosis with minimal inflammation accounted for low D/Q as well as low VA/Q regions. In COPD, no regions with low D/Q ratios were observed, but an abnormality in the VA/Q distribution with low or high VA/Q ratios was identified. Emphysematous lesions produced high VA/Q regions, whereas peripheral airway involvement yielded low VA/Q regions. These findings suggest that hypoxemia in patients with IPF is caused by inhomogeneous distributions of D/Q in combination with those of VA/Q. Hypoxemia in patients with COPD is attributable primarily to inhomogeneities in VA/Q rather than in D/Q distributions.

Effect of hyperoxia on adhesion molecule expression in human endothelial cells and neutrophils.

To investigate the pathogenesis of pulmonary oxygen toxicity, we examined the effect of hyperoxia on adhesion molecule expression in cultured human pulmonary artery endothelial cells (HPAEC) and human umbilical vein endothelial cells (HUVEC). Endothelial cell monolayers were exposed to either hyperoxic (90% O(2)-5% CO(2)) or normoxic (21% O(2)-5% CO(2)) conditions for various periods. The level of intercellular adhesion molecule (ICAM)-1 expression had increased in hyperoxia-exposed HPAEC and HUVEC at 48 h (194 +/- 38 and 233 +/- 56%, respectively; P < 0.001) and at 72 h (200 +/- 43 and 223 +/- 52%, respectively; P < 0.001) compared with normoxic conditions. These hyperoxia-induced ICAM-1 expressions were dose dependently attenuated by a protein kinase C inhibitor (H-7). In contrast, the levels of P-selectin and E-selectin expression in HPAEC and HUVEC were unchanged. The levels of ICAM-1 mRNA and the numbers of adherent neutrophils were increased in HPAEC and HUVEC at 48 and 72 h of hyperoxia. On the other hand, hyperoxia caused neutrophil H(2)O(2) production without affecting the level of CD11/CD18 expression. These results suggest that increased ICAM-1 expression in endothelial cells plays an important role in neutrophil accumulation during hyperoxia.

Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli.

The outer membrane protein OpcP1 of Burkholderia (formerly Pseudomonas) cepacia is one of the subunits forming the porin oligomer OpcPO by non-covalent association. OpcP1 was cleaved with lysyl endopeptidase and the N-terminal amino acid (aa) sequences of the polypeptide fragments were determined. Based on the sequence information, we cloned the opcP gene on a 10-kb EcoRI DNA fragment of the B. cepacia ATCC25416 chromosome. Nucleotide (nt) sequencing revealed a 1086-bp open reading frame (ORF), encoding a 361-aa polypeptide with a signal sequence of 20 residues. The predicted opcP gene encoded a mature protein of Mr 35,696, which agrees well with the value observed previously on SDS-PAGE. The opcP was sub-cloned into pTrc99A and introduced into Escherichia coli. Immunoblot analysis using murine antiserum specific to OpcP1 visualized the protein expressed in the E. coli cells after induction by isopropyl beta-D-thiogalactopyranoside (IPTG).

Mixed-cryoglobulinemia associated with cutaneous vasculitis and pulmonary symptoms.

A 45-year-old Japanese man with Sjögren's syndrome developed recurrent skin ulcers, palpable purpura, and dyspnea. Serum mixed-type cryoglobulin level was elevated. A biopsy of his skin lesion showed the characteristic leukocytoclastic vasculitis of mixed-type cryoglobulinemia. Dyspnea, skin ulcers, and purpura resolved along with a reduction in the serum cryoglobulin level after prednisolone administration. This patient demonstrated cryoglobulinemia-associated vasculitis, as well as possible cryoglobulinemia-associated pulmonary symptoms.

Effect of antioxidants on hyperoxia-induced ICAM-1 expression in human endothelial cells.

The regulating mechanism of hyperoxia-induced ICAM-1 expression has not been elucidated. We studied the effect of antioxidants, including superoxide dismutase (SOD), catalase and N-acetylcysteine (NAC), on hyperoxia-induced ICAM-1 expression in human pulmonary artery endothelial cells (HPAEC) and human umbilical vein endothelial cells (HUVEC). Cells were cultured to confluence and exposed to either hyperoxic or normoxic gas with or without various kinds of antioxidants. The levels of ICAM-1 expression in the endothelial cells and the concentrations of reduced (GSH) and oxidized glutathione (GSSG) in the media were examined by flow cytometry and by spectrophotometry, respectively. After 48-hour exposure to hyperoxia, ICAM-1 expression was increased (HPAEC; 161 +/- 21% and HUVEC; 163 +/- 16%) and total glutathione concentration in the media was decreased as compared with normoxia. SOD did not change the GSH and GSSG concentrations in the media. Catalase dose-dependently decreased the supernatant GSSG concentration in both HPAEC and HUVEC, while the GSH concentration was nearly constant. NAC dose-dependently increased the supernatant GSH concentrations in both HPAEC and HUVEC. There was no difference in the supernatant GSSG concentrations between the NAC-treated HPAEC and HUVEC. There was no difference in ICAM-1 expression in either HPAEC or HUVEC with SOD treatment. ICAM-1 expressions in 100 U/ml (236 +/- 20%) and 1,000 U/ml (315 +/- 36%) of catalase were increased in HPAEC, and that in 1,000 U/ml (440 +/- 209%) of catalase was increased in HUVEC. Five and 10 U/ml of NAC decreased ICAM-1 expression in HPAEC (141 +/- 26% and 113 +/- 11%) and HUVEC (119 +/- 23% and 106 +/- 7%), respectively. These results suggest that extracellular glutathione may play a role in regulating hyperoxia-induced ICAM-1 expression in HPAEC and HUVEC.