Effects of vasopressors on contractile and phosphatidylinositol responses of rat trachea

Purpose. Vasopressors, such as dopamine (DA), norepinephrine (NE), and phenylephrine (Phe), are commonly used during anesthesia to increase blood pressure through α₁-adrenoceptors. The present study was designed to examine the effects of DA, NE, and Phe on the contractile and phosphatidylinositol (PI) responses of the rat trachea induced by a muscarinic agonist, carbachol (CCh). Methods. A rat tracheal ring was suspended between two stainless-steel hooks in Krebs-Henseleit (K-H) solution. Contraction was induced with 0.55 μM CCh, and 30 min later DA, NE, or Phe was added. The tracheal slices were incubated in K-H solution containing LiCl, ³[H]myo-inositol, and CCh in the presence or absence of DA, NE, or Phe. The ³[H]inositol monophosphate (IP₁) formed was measured. Results. CCh caused tracheal ring contraction. NE attenuated CCh-induced contraction at a dose of 1 μM or greater and had a maximal effect at 3 μM. DA and Phe did not affect CCh-induced contraction. CCh-induced IP₁ accumulation was potentiated significantly by NE and Phe, but not by DA. Conclusion. Although NE and Phe potentiated CCh-induced IP₁ accumulation, they could not potentiate CCh-induced contraction, suggesting that in clinical settings, vasopressors such as NE, DA, and Phe might be safely used in patients with asthma. Received: February 25, 2002 / Accepted: July 2, 2002 Acknowledgments. This study was supported in part by a Grant-in-Aid for Scientific Research C (no. 10671421), from the Ministry of Education, Science, and Culture, Japan. Address correspondence to: O. Shibata     

Mechanism of superoxide anion production by hepatic sinusoidal endothelial cells and Kupffer cells during short-term ethanol perfusion in the rat

BACKGROUND/AIMS: The aim of this study was to clarify the candidate cells for and the mechanism of superoxide anion (O2*-) release into the hepatic sinusoids during short-term exposure to ethanol. METHODS: The rat liver was perfused continuously with ethanol (a substrate for alcohol dehydrogenase) or tert-buthanol (not a substrate for alcohol dehydrogenase) for 20 min at a final concentration of 40 mM. In order to detect O2*- production, MCLA (2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one), a Cypridina luciferin analogue, was simultaneously infused and MCLA-enhanced chemiluminescence was measured. The effects of gadolinium chloride (GdCL3) (a suppressor of Kupffer cells (KCs)), staurosporine (ST) (an inhibitor of serine-threonine kinases, including protein kinase C), diphenyleneiodonium chloride (DPI) (an inhibitor of NADPH oxidase), ibuprofen (IB) (an inhibitor of cyclooxygenase) and 4-methylpyrazole (4MP) (an inhibitor of ethanol metabolism) on the ethanol-induced chemiluminescence were also evaluated. Sites where O2*- could be released were determined by histochemical detection of nitro blue tetrazolium reduction. RESULTS: Both ethanol and tert-buthanol rapidly caused O2*- release. GdCL3 suppressed the ethanol-induced O2*- release by 61%. Staurosporine and DPI, but neither IB nor 4-MP, also significantly inhibited the ethanol-induced O2*- release. In the histochemical examination, ethanol-stimulated liver showed blue formazan precipitate on both sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), whereas the GdCl3-pretreated liver had the precipitate only on SECs. CONCLUSIONS: This study shows that ethanol itself stimulates both SECs and KCs to release O2*- via activation of NADPH oxidase probably involving protein kinase C (PKC).     

Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions

We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein-protein and protein-DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.     

Identification of an immunodominant epitope on RNA polymerase III recognized by systemic sclerosis sera: application to enzyme-linked immunosorbent assay

OBJECTIVE: To characterize an immunodominant epitope on RNA polymerase III (RNAP III) recognized by systemic sclerosis (SSc) sera and to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of serum anti-RNAP I/III antibodies. METHODS: RNAP III-specific subunits RPC62 and RPC155 were generated in a bacterial expression system as a series of recombinant fragments. Reactivities to these recombinant fragments were examined by immunoblots and/or ELISA in 16 SSc sera containing anti-RNAP I/III antibodies, 89 SSc sera lacking anti-RNAP I/III antibodies, 61 systemic lupus erythematosus (SLE) sera, and 61 healthy control sera. RESULTS: Anti-RNAP I/III-positive SSc sera recognized several distinct epitopes on RPC62 and RPC155 in various combinations, but the fragment encoding amino acids at positions 732-1166 of RPC155 was recognized by all 11 anti-RNAP I/III-positive SSc sera tested. Carboxyl- and amino-terminal deletion studies showed that at least 130 amino acids at positions 891-1020 of RPC155 were necessary for the antibody binding, but strong reactivity required an additional amino-terminal extension. When a purified recombinant fragment containing the immunodominant epitope was used as the antigen source in an ELISA, elevated antibody reactivity was detected in all 16 anti-RNAP I/III-positive SSc sera, but in no anti-RNAP I/III-negative SSc, SLE, or healthy control sera, representing a sensitivity of 100% and a specificity of 100%. CONCLUSION: A major epitope commonly recognized by SSc sera containing anti-RNAP I/III antibodies was identified on RPC155. The ELISA using a recombinant fragment expressing the immunodominant epitope should be a valuable tool for routine screening for anti-RNAP I/III antibodies in clinical diagnostic laboratories.     

Induction of arginase II in livers of bile duct-ligated rats

Nitric oxide (NO) has been implicated in playing a role in liver cirrhosis, but the regulatory mechanisms are still unclear. As arginase shares a common substrate with NO synthase (NOS), the aim of this study was to investigate the expression of arginase I and II in cirrhotic liver. Liver cirrhosis was induced in rats by chronic bile duct ligation (BDL). Controls were sham-operated. Competitive polymerase chain reaction was performed to assay the expression of messenger RNA of arginase I and II. Protein expression was detected by immunohistochemistry and western-blotting. The level of arginine in plasma was lower in BDL rats, while the ornithine level in plasma was correspondingly higher (r= -0.96, P<0.0001). Arginase I messenger RNA was reduced significantly in BDL rats (3.34+/-0.32 vs. 1.32+/-0.21 x 10(4) attomole/microg of total RNA, sham vs. BDL, P<0.001), as well as arginase I protein. In contrast, arginase II mRNA was induced in the livers of BDL rats, with negligible expression in controls (0.35+/-0.11 vs. 3.64+/-0.54 attomole/microg of total RNA, sham vs. BDL, P<0.001). Arginase II protein was localized in some hepatocytes and hyperplastic bile ductular epithelial cells of cirrhotic livers but not in control livers. In conclusion, arginase II was induced in BDL livers, while the expression of arginase I was down-regulated. These data suggest that arginase I and II are regulated differently and may have different functions in the livers of BDL rats. Reduction of arginase I in BDL livers may be responsible for the lowering of arginine levels in the plasma, while induction of arginase II could be important in regulating NO synthesis as well as other important mechanisms involved in liver cirrhosis.     

Multiplex Mutation Screening of the BRCA1 Gene in 1000 Japanese Breast Cancers

To detect BRCA1 mutations in Japanese breast cancer patients, we screened 1,000 unselected primary cancers for mutations in exon 11, which accounts for 61% of the entire BRCA1 coding sequence. Using a method based on multiplex single-strand conformational polymorphism (SSCP) analysis of multiple restriction fragments generated by restriction-enzyme digestion of amplified DNA, we identified eight mutations. All eight were germline mutations; four of them were non-sense mutations or small deletions resulting in premature stop codons, and the other four were missense mutations. The Japanese carriers of these mutant BRCA1 alleles had developed breast cancers at ages ranging from 45 to 62, five of them bilaterally.     

Frequent Development of Murine T-Cell Lymphomas with TcRα/β+, CD4-/8 Phenotype after Implantation of Human Inflammatory Breast Cancer Cells in BALB/c Nude Mice

Tumors developed quite frequently in some of the visceral organs, including spleen and liver, in BALB/c nude mice upon subcutaneously xenografting surgical specimens from five different inflammatory breast cancer patients. All of these tumors developed within two and a half months to one year after the subcutaneous inoculation of surgical specimens. From these tumors, five independent transplantable tumors, including tMK-2, tHK-1, tYK-1, tYK-2 and tTY-1 have been established. Chromosome analysis, morphologic studies by light and electron microscopy and phenotype analysis indicated that these tumors are of mouse origin. The tMK-2 tumor was highly metastatic to the spleen and liver when it was subcutaneously transplanted into the right scapular region. In addition, the region where the tMK-2 tumor cells were subcutaneously inoculated showed an apparently inflammatory process represented by erythema. After subcutaneous inoculation into the right scapular region, tHK-1, tYK-1,2, and tTY-1 tumors also metastasized to some of the visceral organs, including spleen and liver. From these tumors, in vitro cell lines were established. The cells grew in a stromal-cell dependent manner under in vitro culture conditions. The cells were again tumorigenic at the inoculated region and metastasized to various organs, including liver and spleen, of BALB/c nude mice. Histological examination revealed that the tumors showed features of malignant lymphoma. Phenotypically, these five tumors expressed early T lymphocyte markers as revealed by anti-mouse anti-TcR4aL/β, anti-CD3, CD4 and CDS monoclonal antibodies. To our knowledge, these cell lines are the first T-cell lines showing the phenotype of extrathymically differentiated T-cells in the liver.     

Renal Osteodystrophy in Patients on Continuous Ambulatory Peritoneal Dialysis

Twelve patients on continuous ambulatory peritoneal dialysis (CAPD) were studied in order to evaluate the progression of renal osteodystrophy (ROD). All patients received doses of 0.01 - 0.02 μg/kg of 1 alpha vitamin D₃(1α - D₃) and 0.1 - 0.15 g/kg of calcium carbonate for 12 - 18 months. Serum total protein, total calcium (Ca), creatinine, inorganic phosphate alkaline phosphatase (ALP), and n-terminal parathyroid hormone were measured regularly. The radiological bone appearance for ROD or rickets and the height standard deviation score were compared between the outset and the end of this study. An increase of Ca values and a decrease of ALP values correlated with a suppression of hyperparathyroidism, and the hyperphosphatemia was controlled in the majority of our patients throughout this study. Two patients had ROD and rickets at the outset of this study, and all patients but one had neither ROD nor rickets at the end of this study. Growth retardation improved or remained stable in 10 patients. Our results indicate that adequate doses of 1α - D₃ and calcium carbonate are effective in the prevention of ROD and rickets in patients on CAPD.     

Association of the -381T/C promoter variation of the brain natriuretic peptide gene with low bone-mineral density and rapid postmenopausal bone loss

Osteoporosis is believed to result from interplay among multiple environmental and genetic determinants, including factors that regulate bone-mineral density (BMD). Recent quantitative trait locus analysis in human suggested a possible involvement of chromosomal region 1p36.2-p36.3 for determination of BMD. The brain natriuretic peptide (BNP, also named NPPB) gene lies within this candidate region for BMD determination. Overexpression of the BNP resulted in skeletal overgrowth in transgenic mice. Association analysis between nucleotide variations of the BNP gene and radial BMD in 378 Japanese postmenopausal women revealed a significant association of the -381T/C variation of the BNP gene with radial BMD (r = 0.17, P = 0.01). Homozygous T-allele carriers had the lowest BMD values (0.395 +/- 0.056 g/cm(2)), homozygous C-allele carriers had the highest (0.429 +/- 0.051 g/cm(2)), and heterozygous individuals had intermediate radial BMD values (0.405 +/- 0.048 g/cm(2)), indicating a dosage effect. Accelerated bone loss also correlated with the -381 T allele in a 5-year follow-up study (r = 0.21, P = 0.017). These results suggest that variation of BNP may be an important determinant of postmenopausal osteoporosis, in part through the mechanism of accelerated postmenopausal bone loss.