Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

First restriction and genetic mapping of the genomic DNA of urease-positive thermophilic campylobacters (UPTC), and small restriction fragment sequencing.

A restriction and genetic map of urease-positive thermophilic campylobacter (UPTC) CF89-12 genome DNA is constructed using a pulsed-field gel electrophoresis procedure after digestion with SalI and SmaI and Southern blot hybridisation. Each of the six gene fragments (flaA, glyA, lysS, recA, sodB and ureAB) selected are mapped in only a fragment on the restriction map. Three DNA fragments for rrn operon probes are mapped in multiple regions on the map. When two SmaI-digested neighbouring small fragments hybridised with rrn probes are cloned and sequenced, a total sequence length of 7487 bp is determined. In the sequence, part of the pnp gene (734 bp) bearing a p-independent transcriptional termination region, a cluster of five tRNA genes including the putative promoter region, a hypothetical Cj0171-like 507-bp sequence containing an internal termination codon, and a part of the rrn operon including the putative promoter region (4700 bp) are identified. The 507 bp sequence carried both putative transcriptional promoter sequences, including a ribosome binding site upstream of the ATG start codon and a characteristic G9 structure, and a possible p-independent transcriptional termination region. A hypothetical Cj0170-like 204-bp sequence containing an internal termination codon also occurred, overlapping partly with the Cj0171-like sequence. Based on nucleotide sequence alignment analysis between the UPTC rrn operon examined here and the previously reported one, two different 16S-23S ribosomal DNA (rDNA) internal spacer regions are shown to exist.

Steroid treatment based on the findings of transbronchial biopsy in idiopathic interstitial pneumonia.

This study was performed to find the rationale for administering steroids to patients with idiopathic interstitial pneumonia (IIP), which was unlikely to be usual interstitial pneumonia (UIP) but was not surgically biopsied. Among IIP patients in the file of the departments, nine patients who met the following criteria were selected for this study ("non-UIP" group): 1) transbronchial lung biopsy showed dense mononuclear cell infiltration in thickened alveolar septa; 2) chest radiograph and computed tomography showed irregular linear, reticular or ground-glass opacities with alveolar consolidation without honeycombing in the lung base; and 3) spirometry was performed before and after steroid therapy. Ten patients with pathologically confirmed nonspecific interstitial pneumonia ("NSIP" group) were also selected for the comparison. Baseline values and percentage increase of vital capacity (VC) after steroid therapy were plotted. Steroids improved VC in both groups of patients. After 1 yr of steroid therapy, percentage increase of VC in "non-UIP" was 28.8+/-7.7%, which was not significantly different from that in NSIP (30.0+/-11.7%). One "non-UIP" patient and one NSIP patient died after 6.4 and 4.3 yrs of follow-up, respectively. Patients with idiopathic interstitial pneumonia presenting cellular interstitial pneumonia in transbronchial lung biopsy, in addition to radiographic findings not typical for usual interstitial pneumonia, could expect a beneficial effect of steroids without undergoing surgical biopsy.

Overexpression of the OLE1 gene enhances ethanol fermentation by Saccharomyces cerevisiae.

The fermentation characteristics of Saccharomyces cerevisiae strains which overexpress a constitutive OLE1 gene were studied to clarify the relationship between the fatty acid composition of this yeast and its ethanol productivity. The growth yield and ethanol productivity of these strains in the medium containing 15% dextrose at 10 degrees C were greater than those of the control strains under both aerobic and anaerobic conditions but this difference was not observed under other culture conditions. During repeated-batch fermentation, moreover, the growth yield and ethanol productivity of the wild-type S. cerevisiae increased gradually and then were similar to those of the OLE1-overexpressing transformant in the last batch fermentation. However, the unsaturated fatty acid content (77.6%) of the wild-type cells was lower than that (86.2%) of the OLE1-recombinant cells. These results suggested that other phenomena caused by the overexpression of the OLE1 gene, rather than high unsaturated fatty acid content, are essential to ethanol fermentation by this yeast.

Long-term efficacy of immunization against hepatitis B virus in infants at high-risk analyzed by polymerase chain reaction.

Perinatal transmission of hepatitis B virus (HBV) is a common cause of chronic infection. In the present study, we evaluated the long-term efficacy of immunization against HBV in infants at high-risk, by using polymerase chain reaction (PCR). Two hundred and fifty-one infants received hepatitis B immunoglobulin at birth and a course of hepatitis B vaccine within 6 months of age between 1981 and 1993. Of 251 infants, 203 (81%) and 97 (39%) were followed until 1 and 4-6 years of age, respectively. HBV-DNA was detected by PCR in 74 (36%) of 203 children at 1 year of age, while the prevalence rate of children positive for HBV-DNA decreased to 14 (14%) of 97 children at 4-6 years of age, including 2 children who had the breakthrough variants of HBV. Our results indicate that most of HBV infections occur early, during the first year, and that immunization against HBV effectively protects infants at high-risk against viral transmission, at least up to 4-6 years of age.

Most of the patients with cirrhosis in Japan die from hepatocellular carcinoma.

A total of 424 patients with cirrhosis were entered into a registry in Japan. One hundred and seven patients were hepatitis B virus (HBV) associated cirrhosis while 252 were hepatitis C virus (HCV) associated cirrhosis. Patients were followed for a period of 3.3+/-3. 3 years. Fifty-six (80%) of 70 deaths in HBV patients and 151 (90%) of 161 deaths in HCV patients were due to complications associated with hepatocellular carcinoma (HCC). The complication rate of HCC in HCV group was significantly higher than in HBV group. In conclusion, most of the patients with cirrhosis died from HCC.

Association of mutations in the core promoter and precore region of hepatitis virus with fulminant and severe acute hepatitis in Japan.

It was recently reported that mutations in the precore and core promoter region of hepatitis B virus (HBV) are associated with fulminant hepatitis. The aim of this study was to investigate the association of mutations in the precore and core promoter region of HBV with fulminant and severe acute hepatitis. We studied Japanese patients with acute HBV infection, including seven patients with fulminant hepatitis, 12 with severe acute hepatitis and 41 with acute self-limited hepatitis. The presence of HBV mutants was examined by using a point mutation assay to detect a G to A transition at position 1896 in the precore region and an A to T transition at position 1762 and a G to A transition at position 1764 in the core promoter region. Significant differences in the proportion of mutations in the precore or core promoter region were present between patients with fulminant hepatitis and self-limited acute hepatitis (7/7 (100%) vs 4/41 (9.8%), P<0.01) and between severe acute hepatitis and self-limited acute hepatitis (6/12 (50.0%) vs 4/41 (9.8%), P<0.01). The frequency of mutation increased proportionately with the severity of disease in patients with acute HBV infection. Fulminant hepatitis B in Japan is closely associated with mutations in the core promoter and precore gene of HBV. Point mutation assays for HBV precore and core promoter analysis may be useful to predict the outcome of liver disease in patients with acute HBV infection.

[Effect of hyaluronidase on porcine pancreatic elastase-induced lung injury].

The present study was conducted to investigate the effects of hyaluronidase on elastase-induced lung injury in rats. Male Wistar rats were divided into 4 groups: 1) Control group, single tracheal instillation of 0.3 ml saline; 2) PPE group, single tracheal instillation of 120 micrograms porcine pancreatic elastase (PPE); 3) HD group, intravenous injection of 240 mg/kg hyaluronidase (HD); and 4) HE + PPE group, intravenous injection of HD before single tracheal instillation of PPE. Cellular component and protein concentration in bronchoalveolar lavage fluid (BALF), myeloperoxidase (MPO) activity and hyaluronan content in lungs were examined 24 hours after treatment. After 8 weeks of treatment, hyaluronan content in the lungs and physiological parameters such as total lung capacity (TLC), functional residual capacity (FRC) and static compliance (Cst) were examined. The number of BALF nucleated cells in the HD + PPE group 6 h after treatment, and lung MPO activity in the HD group after 1 h and 3 h. were both higher than the corresponding measurements in the PPE group. After 6 h of treatment, the number of BALF nucleated cells in the HD + PPE group was significantly higher than in the HD group. The BALF albumin concentrations in the HD + PPE and HD groups were also significantly higher than in the control group. After 8 weeks of treatment, the measurements of TLC and Cst indicated greater emphysematous changes in the HD + PPE group. These results suggest that decreased interstitial HA accelerates acute inflammation and emphysematous changes in elastase-induced lung injury.

Quantitative analysis of interferon alpha/beta receptor mRNA in the liver of patients with chronic hepatitis C: correlation with serum hepatitis C virus-RNA levels and response to treatment with interferon.

We analysed the expression of interferon (IFN) alpha/beta receptor mRNA in the liver of patients with chronic hepatitis C and examined the relationship between the expression of this receptor gene and the level of hepatitis C virus (HCV)-RNA as well as the response to 16 weeks of 6 x 10(6) units IFN. The mean level of IFN alpha/beta receptor mRNA in patients with chronic HCV infection (expressed as delta cycle; 10.8 +/- 1.9 (mean +/- SD); n = 39) was significantly higher than that of control subjects (9.4 +/- 0.5; n = 6; P < 0.01). There was a significant negative correlation between the level of IFN alpha/beta receptor mRNA and serum HCV-RNA in 39 patients with chronic hepatitis C (R = -0.546; P < 0.01). The mean level of IFN alpha/beta receptor mRNA in six patients who showed a complete response to IFN therapy (12.3 +/- 1.6) was higher than that of 15 patients who failed to respond to therapy (10.1 +/- 1.5; P < 0.01). Our results are consistent with the suggestion that the anti-viral activity of IFN depends on the level of the IFN alpha/beta receptor on hepatocytes in patients with chronic hepatitis C.

[Levels of alpha-elastin and of desmosine as indexes of elastase-induced lung injury in rats].

Two markers of elastin degradation, alpha-elastin and desmosine levels, were measured in sera from rats after single tracheal instillations of porcine pancreatic elastase (PPE). Biochemical and pathophysiological changes in the lung were also studied. Male Wistar rats were divided into three groups. The control group received one intratracheal dose of saline (0.3 ml). The PPE 60 group received one intratracheal dose of 60 U of PPE. The PPE 120 group received one intratracheal dose of 120 of PPE. Blood samples were collected 0, 1, 3, 5, 7, 14, 28, and 56 days after the intervention, and levels of alpha-elastin and desmosine were measured by enzyme-linked immunosorbent assay. At the end of the study (day 56), lung volumes and static pressure-volume curves were measured by body pletysmography. Then the rats were killed and the mean linear intercept was measured as defined by Thurlbeck. The serum alpha-elastin level in the PPE 120 group on day 7 was significantly higher (p < 0.05) than those in other groups. Desmosine values on day 1 in the PPE 60 group and on days 1 and 7 in the PPE 120 group were significantly higher (p < 0.05) than those in the control group. Functional and morphometric analyses revealed that the PPE 120 group had more emphysematous changes than the control group (p < 0.01) and more than the PPE 60 group (p < 0.05). These results suggest that levels of alpha-elastin and of desmosine reflect changes that occur in the early stages of lung injury, and that these changes are related to abnormalities found in rat lungs on day 56.

[The role of interstitial hyaluronan in acute lung injury].

The pulmonary interstitium may affect the movement of water, macromolecules, and inflammatory cells between capillaries and lymphatics. Hyaluronan (hyaluronic acid), a glycosaminoglycan of the interstitial matrix, helps to retain water in the lung interstitium and to exclude proteins. Marked reduction of interstitial hyaluronan by infusion of hyaluronidase decreased the resistance to fluid transport and decreased water retention in the interstitium, and thus accelerated lymphatic removal of fluid filtered across capillary walls. However, reduction of hyaluronan increased the accumulation of neutrophils in the lung and exacerbated acute lung injury caused by pancreatic elastase. Interstitial hyaluronan may protect against acute inflammatory changes in elastase-induced lung injury.

[Effect of theophylline on disturbances of ventilation and of gas exchange during exercise in patients with pulmonary emphysema].

Factors limiting exercise were studied in 23 patients with pulmonary emphysema, and the effects of theophylline on exercise tolerance and on hypercapnic ventilatory response were studied in 16 patients with pulmonary emphysema. Exercise in these patients was limited by inability to increase tidal volume and by disturbances of gas exchange, as indicated by low PaO2, high AaDO2, and high PaCO2 during exercise, and by low diffusing capacity. Theophylline increased tidal volume during exercise, but it did not improve oxygenation during exercise. Theophylline improved exercise tolerance and hypercapnic ventilatory response. The percent increases in VEmax and in VO2max after theophylline administration correlated significantly with the percent increase in the ratio of mouth occlusion pressure to PACO2 during rebreathing. These results indicate that exercise tolerance in patients with pulmonary emphysema can be improved by theophylline, probably because of an increase in ventilatory drive.

[Pathogenic bacteria isolated from the sputum of the patients with pulmonary emphysema].

Isolated pathogenic bacteria from sputum of the patients with pulmonary emphysema who were admitted in our hospital from 1984 to 1994 were examined to elucidate the relationship between isolated bacteria from sputum and pulmonary functions including vital capacity (VC), forced expiratory volume (FEV1.0), PaO2 and PaCO2. VC of the patients from whom MSSA (methicillin-sensitive Staphylococcus aureus) or Enterococcus faecalis (E. faecalis) were isolated was significantly lower than that of the patients from whom Streptococcus pneumoniae (S. pneumoniae), Branhamella catarrhalis (B. catarrhalis) or Haemophilus influenza (H. influenza) were isolated. FEV1.0 had a similar tendency as VC in terms of isolated organisms from the patients with emphysema. Similarly, PO2 of the patients from whom MSSA or E. cloacae were isolated was significantly lower than that of the patients from whom S. pneumoniae, B. catarrhalis or H. influenzae were isolated, and PCO2 of the patients from whom S. pneumoniae, B. catarrhalis or H. influenza were isolated. There was also impaired respiratory function in the patients from whom MSSA, Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Xanthomonas maltophilia (X. maltophilia) or Enterobacter cloacae (E. cloacae) were isolated, compared with those in the patients from whom S. pneumonia, B. catarrhalis or H. influenzae were isolated. These results suggest that isolated pathogenic bacteria are shifted from S. pneumoniae, B. catarrhalis or H. influenza to MSSA, E. coli, P. aeruginosa, X. maltophilia or E. cloacae in the course of impairment of respiratory function in pulmonary emphysema. The treatment and prophylaxis for acute exacerbation in pulmonary emphysema should be based on these results.

[Effect of E. coli endotoxin and D-galactosamine on pathophysiology in rat lungs].

The effects of repeated intravenous injection of E. coli endotoxin (ETX) and intraperitoneal injection of D-galactosamine (GAL), which decreases the circulating level of alpha 1-antitrypsin, on the pathophysiology of chronic lung injury was studied in rats. Four groups were prepared as follows for 8 weeks. Group 1 (control): Intravenous injection of saline. Group 2: Intravenous injection of ETX (2 mg/kg) once a week. Group 3: Intraperitoneal injection of GAL (200 mg/kg), 2 times daily on 3 consecutive days each week. Group 4: Injection of both ETX and GAL, at the same dosages as used in groups 2 and 3. Total lung capacity and static lung compliance divided by weight were high in the ETX group and the ETX + GAL group, comparative when compared with those in the control and GAL groups, even though weight gain rates in the ETX + GAL group was less than in other groups. Mean linear intercept of rats in the ETX + GAL group was significantly greater than in other groups. These results suggest that ETX + GAL-treated rats have more emphysematous changes in pulmonary function and structure.

[Effects of liposome-entrapped E. coli endotoxin on lung pathophysiology in rats].

The effects of repeated intravenous injections of E. coli endotoxin (ETX)-containing liposomes (LP) on the development of emphysematous change in rats were examined. Male Wistar rats, weighting 150-200 g, were divided into 3 groups. Group 1; rats treated with phosphate buffered saline (PBS), Group 2; rats treated with liposome-entrapped PBS, Group 3; rats treated with liposome-entrapped ETX (10 mg/ml). In each group, 0.5 ml was injected intravenously once a week for 8 consecutive weeks. After 8 weeks, functional and morphometrical analyses of lungs were performed. Functional residual capacity (FRC), total lung capacity (TLC), and static compliance (Cst.) were measured using a pressure plethysmograph for small animals. Mean linear intercept (MLI) and internal surface area (ISA) were also determined. TLC did not differ among the 3 groups. In rats treated with liposome-entrapped ETX, Cst. corrected by body weight (Cst./body weight) was significantly increased compared with that in liposome-entrapped PBS-treated rats. Further more, MLI was larger than that of the other 2 groups and ISA was less than that of the liposome-entrapped PBS-treated rats. These results suggest that liposome-entrapped ETX induces emphysematous changes in rats.

[Effects of theophylline on ventilatory regulation in patients with chronic obstructive pulmonary disease].

The effects and the mechanism of single and long-term administration of theophylline on ventilatory regulation were investigated in patients with chronic obstructive pulmonary disease (COPD). The ventilatory and mouth occlusion pressure (P0.1) responses during CO2 rebreathing were measured before and 4 hours after oral, single administration of theophylline in 15 patients with pulmonary emphysema, 15 patients with asthma and 10 healthy subjects. Ventilatory response to CO2 (VE/PCO2) was increased in patients with asthma and P0.1 response to CO2 (P0.1/PaCO2) was increased in patients with pulmonary emphysema after single theophylline administration. In patients with pulmonary emphysema, the increase in P0.1 was more marked than that in maximum inspiratory pressure static (MIPS). The effect of long-term administration of theophylline was studied in 8 patients with COPD and 8 patients with restrictive lung disease. In patients with COPD, P0.1/PaCO2 was significantly increased and the increase was maintained until three months later. These findings suggest that theophylline is effective in improving ventilatory and occlusion pressure responses to CO2 in patients with asthma probably by its bronchodilating effect, and in patients with pulmonary emphysema by its direct effect on the ventilatory control system.

[Pathological examination of 11 autopsy cases of acute onset interstitial pneumonia of unknown etiology].

In 11 autopsy cases of acute onset interstitial pneumonia of unknown etiology, all cases had thickening of alveolar septa with various degrees of lymphocytic infiltration and fibroblast proliferation. Only a few cases had edematous alveolar septa, which were weakly positive in alcian-blue staining. Intra-alveolar, intra-alveolar duct and intra-bronchiolar organization was a characteristic finding. These findings may represent the repair phase rather than the exudative phase of diffuse alveolar damage. Localized bacterial pneumonia was superimposed in 3 cases, and neither Pneumocystis carinii nor viral inclusion bodies were identified in any case. The severity of fibrosis was not obviously correlated with the period of artificial ventilation. From these findings, terminal infection and oxygen therapy did not seem to significantly modify the course of acute-onset interstitial pneumonia. Further examination of autopsy cases as well as open-lung biopsy is important to elucidate the time course of acute-onset interstitial pneumonia.

Effect of aminophylline on ventilatory response to hypercapnea in patients with asthma.

The nature of ventilatory response to CO2 in patients with airway obstruction is controversial. Although the bronchodilating effect of aminophylline has been well documented, its effect on direct stimulation of breathing has not been systematically evaluated. Therefore, the effect of intravenous aminophylline on ventilatory response to CO2 of patients with asthma was studied by comparing with that of the healthy subjects. Ventilation (VE) at rest was not significantly different between in asthmatics and healthy subjects, and mouth occlusion pressures (P0.1) at rest was significantly higher in asthmatics than in healthy group (p less than 0.05). P0.1 after aminophylline increased significantly (p less than 0.05) compared to control value in asthmatics. Changes in VE and P0.1 relatively correlated well in both groups before and after aminophylline (r = 0.58). Ventilatory response to CO2 (VE/PA CO2) and P0.1 response to CO2 (P0.1/PACO2) in asthmatics were slightly increased, but not significantly different from those in the healthy group before and after aminophylline. These findings suggest that aminophylline might be effective in enhancing ventilatory response to CO2 in asthmatics than in healthy subjects, because of its bronchodilating effect and stimulation to the respiratory neurons.

[Clinical studies on flomoxef in respiratory infection].

Clinical efficacy, bacteriological effect and safety of a new antibiotic flomoxef (FMOX, 6315-S) in respiratory infections were studied. Efficacy of FMOX in 6 patients with infectious diseases including 2 cases with pneumonia, 3 cases with acute exacerbation by respiratory infection, 1 case with obstructive pneumonia were clinically evaluated. Two strains of Haemophilus influenzae, 1 strain of Streptococcus pneumoniae and 1 strain of Staphylococcus aureus which were detected as causative organisms in 2 cases disappeared or decreased after treatment with FMOX. Assessing both clinical and bacteriological findings, effects of FMOX were good in 5 cases and fair in 1 case. No adverse effects were observed in clinical or laboratory findings. Consequently, FMOX is considered to be a very useful antibiotic in the treatment for respiratory infectious diseases.