Adipose cells induce phospho-Thr-172 AMPK production by epinephrine or CL316243 in mouse 3T3-L1 adipocytes or MAPK activation and G protein-associated PI3K responses induced by CL316243 or aluminum fluoride in rat white adipocytes.

Responses of adipose cells to adrenoceptor regulation, including that of ß-adrenoceptor (AR), and the signalling machinery involved in these responses are not sufficiently understood; information that is helpful for elucidating the adrenoceptor (adrenergic and ß-AR)-responsive machinery is insufficient. We examined phospho-Thr-172 AMPK production in mouse-derived 3T3-L1 adipocytes treated with epinephrine or CL316243 (a ß3-AR agonist) for 15 min. We also examined MAPK activation or G protein-associated PI3K activation or -associated PI3K p85 complex formation in rat epididymal (white) adipocytes treated with CL316243 for 15 min or aluminum fluoride (a G-protein signalling activator) for 20 min. Furthermore, we examined the effect of PTX (a trimeric G-protein inactivator) on p85 complex formation induced by aluminum fluoride treatment. Western blot analysis revealed that epinephrine or CL316243 treatment increased the phospho- Thr-172 AMPK (an active form of AMPK) level in 3T3-L1 adipocytes. Activated kinase analysis with a specific substrate showed that CL316243 or aluminum fluoride treatment activated MAPK in rat adipocytes. Immunoprecipitation experiments with a G-protein ß subunit (Gß) antibody showed that treatment of rat adipocytes with CL316243 activated PI3K and increased the PI3K p85 level in the Gß antibody immunoprecipitates. Such an increase in the p85 level was similarly elicited by aluminum fluoride treatment in a PTX-sensitive manner. Our results provide possible clues for clarifying the signalling machinery involved in adrenoceptor responses, including those of ß3-AR, in mouse-derived adipocytes and rat white adipocytes. Our findings advance the understanding of responses to adrenoceptor regulation in adipose cells and of the cellular signalling machinery present in the cells.

Polymorphisms in the 5'-UTR of PTEN and other gene polymorphisms in normal Japanese individuals.

Polymorphisms are distributed differently in populations, including those of regions, ethnic groups, and diseased patients. In order to investigate variation in nucleotide sequences in normal individuals, we isolated genomic DNA from the blood of healthy Japanese individuals and sequenced the 5'-untranslated region (5'-UTR) of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene and the gene promoter, intron, and exon nucleotides of p53, p14(ARF), murine double minute 2 (MDM2), and the beta2- and beta3-adrenoceptor (-AR). We found polymorphisms in these regions, including a deletion at positions -465 to -463 and a substitution at position -404 in PTEN and a substitution at position -4924 in p14(ARF), in normal individuals. Individuals with or without the PTEN polymorphism harbored a different distribution of polymorphisms, including simultaneous alterations in nucleotides of p53, MDM2, and beta3-AR, and also harbored some polymorphic nucleotides located in the same set of associatively altered nucleotides. Our results show that multiple nucleotides, including the PTEN nucleotides, are altered in normal Japanese individuals and provide useful information for genotyping studies in individuals and populations.

Chloroform deposition in renal cyst fluid of hemodialysis patients with renal cystic disorders.

Chronic exposure to chloroform (CHCl3) induces renal neoplasms in rodents and may be carcinogenic in humans, but studies on chronic CHCl3 deposition in the human body have not been performed. In this study, we examined 27 hemodialysis patients with renal cystic diseases including acquired cystic disease of the kidney (ACDK) accompanied by renal tumors at high frequency. Intracystic and serum CHCl3 concentrations were determined using a headspace gas chromatography/mass spectrometry analysis. CHCl3 was not detected in the serum in any cases, but levels ranging from <0.1 to 0.659 mg/L were found in the cyst fluid in most cases, including patients with ACDK and autosomal dominant polycystic kidney disease. Because intracystic CHCl3 deposition was not confined to ACDK cases, we were unable to evaluate the relationship between CHCl3 accumulation and carcinogenesis in ACDK. However, our results suggest that compounds such as CHCl3 accumulate in renal cyst fluid in hemodialysis patients with renal cystic diseases.

Rat white adipocytes activate p85/p110 PI3K and induce PM GLUT4 in response to adrenoceptor agonists or aluminum fluoride.

Adipocyte responses to adrenergic and ß-adrenoceptor(-AR) (adrenoceptor) regulation are not sufficiently understood, and information helpful for elucidating the adrenoceptor-responsive machinery is insufficient. Here we show by using immunoprecipitated kinase analysis with a phosphatidylinositol 3-kinase (PI3K) p85 antibody that PI3K activation was induced by treatment with 10 or 100 µM norepinephrine (NE) for 15 min or with 10 mM aluminum fluoride (AF, a guanosine triphosphate (GTP)-binding (G) protein activator) for 20 min in white adipocytes (rat epididymal adipocytes) and that treatment with pertussis toxin (PTX, a G-protein inactivator) inhibited PI3K activation induced by the 20-min treatment with AF in the cells. In addition, western blot analysis revealed that glucose transporter 4 (GLUT4) level in the adipocyte plasma membrane (PM) fraction was increased by treatment with 10 µM NE, 100 µM dobutamine (DOB, a ß1-AR agonist), or 0.1 µM CL316243 (CL, a ß3-AR agonist) for 30 min or with 10 mM AF for 20 min. NE or AF treatment triggered 2-deoxyglucose (2-DG) uptake into adipocytes under the above conditions. Our results advance the understanding of responses to adrenoceptor regulation in white adipocytes and provide possible clues for clarifying the machinery involved in adrenergic and ß-AR responses in the cells.

CL316243 induces phosphatidylinositol 3,4,5-triphosphate production in rat adipocytes in an adenosine deaminase-, pertussis toxin-, or wortmannin-sensitive manner.

The effect of beta(3)-adrenoceptor (beta(3)-AR) agonists on adipocytes treated or not treated with signaling modulators has not been sufficiently elucidated. Using rat epididymal adipocytes (adipocytes) labeled with [(32)P]orthophosphate, we found that treatment with the selective beta(3)-AR agonist CL316243 (CL; 1 microM) induces phosphatidylinositol (PI) 3,4,5-triphosphate (PI[3,4,5]P(3)) production and that this response is inhibited by adenosine deaminase (ADA, an adenosine-degrading enzyme; 2 U/ml), pertussis toxin (PTX, an inactivator of inhibitory guanine-nucleotide-binding protein; 1 microg/ml), or wortmannin (WT, a PI-kinase inhibitor; 3 microM). The results showed that CL induced PI(3,4,5)P(3) production in intact adipocytes and that this production was affected by signaling modulators. Taken together, our findings indicate that CL produces PI(3,4,5)P(3) in an ADA-sensitive, PTX-sensitive, or WT-sensitive manner and will advance understanding of the effect of beta(3)-AR agonists on adipocytes.

Isolation and characterization of three distinct 34 kDa EDTA-extractable proteins from bovine lens.

EDTA-extractable protein (EEP) is known to be a major lens membrane protein with a molecular mass in the range 32 kDa to 38 kDa, and is also known to bind to the lens membrane and phospholipid-containing liposomes in a calcium-dependent manner. Recent results (Russell, P., Zelenka, P., Martensen, J., and Reid, T.W. (1977) Curr. Eye Res. 6, 533-538) on antibody cross-reactivity have demonstrated that a 34-35 kDa component of EEP is identical to calpactin I (lipocortin II). In this study, we have identified and purified three distinct 34 kDa components of EEP (designated as EEP-34A1, EEP-34A2 and EEP-34B) from bovine lens that inhibit phospholipase A2 activity. These proteins bind to phospholipid-containing liposome and F-actin in a calcium-dependent fashion. Two-dimensional electrophoresis demonstrates that the three proteins were distinct from one another. However, immunochemical studies and one-dimensional peptide mapping indicate that EEP-34A1 and EEP-34B are very similar. Our results also indicate that EEP-34A1 is very similar to calpactin II and that EEP-34A2 corresponds to calpactin I. The bovine lens 34-35 kDa component of EEP is a mixture of proteins rather than a single protein.

Suppression of insulin-stimulated phosphatidylinositol 3-kinase activity by the beta3-adrenoceptor agonist CL316243 in rat adipocytes.

Insulin increased 2-deoxyglucose (2-DG) uptake via the translocation of glucose transporter (GLUT) 4 to the plasma membrane fraction in rat adipocytes. The stimulatory actions of insulin were accompanied by both an increase in the immunoreactive p85 subunit of phosphatidylinositol (PI) 3-kinase in the plasma membrane fractions and PI 3-kinase activation by tyrosine phosphorylation of the p85 subunit. The beta3-adrenoceptor agonist CL316243 (CL) suppressed all the insulin actions in adenosine deaminase (ADA)-treated cells, but was without effect in non-ADA-treated cells. The inhibitory effects of CL on GLUT 4 translocation and PI 3-kinase activation were abolished by the addition of N6-phenylisopropyl adenosine. Cholera toxin treatment, which markedly increased intracellular cAMP levels, suppressed increases in the levels of GLUT 4 and PI 3-kinase in the plasma membrane fractions in response to insulin. In addition, dibutyryl (Bt2) cAMP also impaired the activation of PI 3-kinase by insulin. These results indicated that CL suppressed insulin-stimulated glucose transport under conditions where cAMP levels were markedly increased (approximately 12-fold). The inhibitory actions of PI 3-kinase activation by insulin were exerted even when cAMP, 8-bromo-cAMP, or Bt2 cAMP was added to immunoprecipitates of the p85 subunit of PI 3-kinase, after treating the cells with insulin. These results suggest that CL suppressed insulin-stimulated PI 3-kinase activity via a cAMP-dependent mechanism, at least in part, direct cAMP action in ADA-treated adipocytes, by which PI 3-kinase activation was inhibited, resulting in the decrease in GLUT 4 translocation and subsequent 2-DG uptake in response to insulin.

The effects of perioperative portal venous inoculation with donor lymphocytes on renal allograft survival in the rat. I. Specific prolongation of donor grafts and suppressor factor in the serum.

In order to investigate the in vivo functional role of the liver in the immune responses in organ transplantation, effects of perioperative portal venous p.v. administration of donor lymphocytes on renal allograft survival were tested in the rat kidney transplant model. Donor lymphocytes were prepared from BN (BN, RT-1n) or third-party DA (RT1a) rat spleens and lymph nodes and injected p.v. or intravenously to Lewis (LEW, RT-1l) hosts on the day of transplantation (day 0). Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.6 days (n = 10). Intravenous administration of 1 x 10(8) BN cells to LEW hosts on day 0 caused a slight, but not significant, prolongation of renal allograft survival (MST = 9.5 +/- 3.0 days, n = 13, NS), whereas portal venous inoculation of 1 x 10(8) BN cells on day 0 remarkably prolonged renal graft survival to 22.2 +/- 5.3 (n = 10, P less than 0.01). The prolongation of graft survival was antigen-specific; the administration of 1 x 10(8) DA cells p.v. to LEW hosts did not prolong the survival of BN renal grafts (MST = 7.4 +/- 0.8, n = 5). Spleen cells from p.v. treated LEW hosts 10 days after transplantation had no suppressor effect on the one-way MLC reaction of normal LEW responder cells toward donor BN or third-party DA stimulators. On the other hand, when serum from p.v.-treated LEW hosts was added to MLC at a concentration of 3 per cent of total volume, it suppressed the MLC reaction toward donor BN cells by 71.6 per cent, but not toward third-party DA stimulators (-8.5 per cent suppression, NS). Histological examination of p.v.-treated LEW hosts at 10 days after transplantation revealed that the liver had normal lobular architecture without expansion of portal tracts and infiltration of inflammatory cells. On the other hand, the transplanted kidney demonstrated a moderate mononuclear cell infiltration around the artery without an interstitial hemorrhage. Moreover, adoptive transfer of the serum from p.v.-treated LEW rats into the virgin secondary LEW hosts significantly prolonged the graft survival of BN kidneys from 7.8 days to 18.9 +/- 5.5 days (P less than 0.01), but not third-party DA graft survivals (MST = 7.5 +/- 0.6 days), indicating that an antigen-specific tolerogenic factor was released into the circulation through the process of allogeneic cells in the liver.

The effects of perioperative portal venous inoculation with donor lymphocytes on renal allograft survival in the rat. II. Phenotypic and functional analyses of graft-infiltrating cells.

Phenotype, donor-specific cytolytic activity, and helper activity to release cytokines of cells infiltrating within renal allografts of hosts rendered unresponsive by perioperative administration of donor lymphocytes via the portal vein (p.v.) were investigated in order to analyze the mechanism of prolongation of allograft survival. Graft-infiltrating cells (GIC) were obtained from Lewis (LEW, RT-1l) hosts inoculated perioperatively with 1 x 10(8) donor Brown-Norway (BN, RT-1n) lymphocytes p.v., a group that displays prolonged renal allograft survival (MST: 22.2 +/- 5.3 days, n = 10) compared with an uninoculated control group (MST: 7.8 +/- 0.6 days, n = 10, P less than 0.01). The percentages of cytotoxic/suppressor T cells (OX-8+) and Ia-positive cells (OX-6+) in GIC (23.1 +/- 4.4% and 9.0 +/- 2.0%, respectively) and in spleen cells (7.5 +/- 2.6% and 8.5 +/- 1.1%, respectively) from p.v.-inoculated LEW hosts on day 6 postgrafting were significantly lower than those of uninoculated control recipients (GIC: OX-8; 39.4 +/- 8.2%, OX-6; 23.0 +/- 1.9%. SP cell: OX-8; 21.6 +/- 9.9%, OX-6; 12.7 +/- 0.4%, P less than 0.05). Cytolytic activity of GIC from tolerant hosts on day 6 postgrafting toward donor blastoid lymphocytes was significantly decreased (19.0 +/- 1.2% at E/T = 50), compared with that from control allografts during ongoing rejection (51.5 +/- 5.3%, P less than 0.01). The amounts of in vitro cytokine production of GIC from tolerant hosts after mitogen stimulation were remarkably decreased (IL-2: 8.7 +/- 1.4 U/ml, IL-3: 15.4 +/- 0.6 U/ml, and BSF-2: 24.6 +/- 3.5 U/ml) than those of uninoculated control hosts during ongoing rejection (IL-2: 19.6 +/- 2.9 U/ml, IL-3: 22.2 +/- 2.7 U/ml, and BSF-2: 67.5 +/- 13.2 U/ml, P less than 0.05). These results demonstrated that activation of both Tc cells and Th cells was inhibited in the spleen and in situ in renal allografts following administration of donor lymphocytes through the portal vein.

Prolongation of renal allograft survival in the rat treated with amniotic fluid.

To assess the role of amniotic fluid (AMF) in the maintenance of pregnancy, immunosuppressive effects of AMF were studied in vivo, and the mechanisms of suppressor activity were analyzed immunologically in vitro in the rat. Female Lewis (LEW, RT-1l) rats mated with Brown-Norway (BN, RT-1n) rats for 14 days were sacrificed and cell-free AMF was obtained. AMF was diafiltered with PBS (PH 7.2) and reconstituted to 2 OD units measured at 280 nm. Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.2 days (n = 10). Five days of intravenous inoculation of AMF into LEW hosts remarkably enhanced BN graft survivals (MST = 20.3 +/- 4.4 days, n = 12) compared with controls (P less than 0.01), and slightly prolonged third-party DA (RT-1a) graft survivals (MST = 9.4 +/- 0.8 days, n = 7) compared with control LEW hosts engrafted with a DA kidney (MST = 7.6 +/- 0.2 days, n = 6). Five days of intravenous inoculation of pregnant sera into LEW hosts had no effect on BN graft survival. The AMF suppressed the proliferative response of LEW lymphocytes against not only irradiated BN stimulator cells but also irradiated third-party DA stimulators. The AMF also suppressed allokiller T cell generation of normal LEW lymphocytes against BN cells by 70.1% and 51.3%, and against DA cells by 64.9% and 38.9% at concentrations of 25% and 12.5%, respectively (P less than 0.01). To dissect the immunosuppressive activity of AMF, the effect of AMF on cytokine production and interleukin 2 (IL-2) receptor expression of concanavalin A-stimulated lymphocytes were investigated. AMF suppressed interferon and IL-2 production. Interestingly, however, AMF did not suppress interleukin 3 (IL-3) and interleukin 6 (IL-6) production, as well as IL-2 receptor expression. These results demonstrated that rat AMF displayed a strong immunosuppression in vivo as well as in vitro, and that AMF might play an important role in the maintenance of pregnancy.

Cold-stimulated increase in a regulatory subunit of cAMP-dependent protein kinase in human hepatoblastoma cells.

Although cold-stress responses in bacteria and plants have been well studied and hypothermic conditions are used in clinical treatments, there has been little investigation of cold-stress responses in human cells, and there has been no report on the involvement of signal transduction modulators in the cold-stress response in human cells. We therefore investigated alterations in the expression of genes involved in the signal transduction system and the mechanisms of cold-stimulated increases in the expression of genes in human hepatoblastoma (HepG2) cells. Using a cDNA expression array method, we found that a transcript encoding a regulatory subunit Ibeta (RIbeta) of cyclic AMP-dependent protein kinase (PKA) was increased in cold-stressed cells. Western blot analysis revealed that the amount of PKA RIbeta protein was increased by cold treatment, while that of a PKA catalytic subunit (C) was unchanged. The protein level of PKA RIbeta was increased in cells treated with low concentrations of actinomycin D, whereas that of PKA C was not, implying that the increase was caused by the suppression of transcription at low temperatures. In addition, degradation of the PKA RIbeta protein was not stimulated by cold treatment, unlike that of the PKA C protein. The results suggest that signal transduction through PKA also participates in cold-stress responses in human cells and that multiple mechanisms are involved in the increase in the level of the PKA RIbeta protein.

Efficacy of perioperative portal venous inoculation with donor lymphocytes on skin graft survivals in the rat.

Although the administration of donor lymphocytes via portal vein (PV) on the day of transplantation significantly prolongs rat renal allograft survivals and the unresponsive state is mediated by an antigen-specific suppressor factor in the serum, significant variations exist among rodent models in terms of immunogenicity and mechanism of antigen presentation. The present studies sought to assess the effect of perioperative PV inoculation with donor lymphocytes on skin allograft survivals. Donor lymphocytes were prepared from Brown-Norway (BN, RT-1n) or third-party DA (RT-1a) rat spleens and lymph nodes and injected via PV or intravenously to Lewis (LEW, RT-1l) hosts on the day of skin grafting. Untreated LEW hosts rejected BN skin grafts at 9.0 +/- 1.4 days (n = 10). Intravenous administration of 1 x 10(8) BN cells into LEW hosts on day 0 did not prolong the skin graft survivals (MST = 8.6 +/- 1.2 days, n = 7, NS), whereas PV inoculation of 1 x 10(8) BN cells prolonged skin graft survival to 13.4 +/- 3.9 (n = 8, P < .01). PV administration of 1 x 10(8) DA cells to LEW hosts did not prolong the survival of BN skin grafts (MST = 8.6 +/- 1.5 days, n = 6). PV inoculation with BN cells inhibited the generation of anti-BN delayed-type hypersensitivity (DTH) response in the hosts, whereas untreated control hosts or hosts inoculated with third-party DA cells could not inhibit the anti-BN DTH response.(ABSTRACT TRUNCATED AT 250 WORDS)

[The analysis of surgically treated pulmonary tuberculosis].

Between January 1990 and December 1999, thirteen patients with pulmonary tuberculosis underwent surgical management in our hospital. The purpose of surgery was classified into three groups: drug-resistant or persistent disease (7 patients), hemoptysis (3), and the others. We have no operative death, but have two late deaths due to postoperative persistent positive sputum and progressive tuberculosis infection. One patient relapsed one year and six months after operation and medical treatment was done successfully. Pulmonary resection is useful for localized pulmonary tuberculosis even drug-resistant cases.

[Postoperative interstitial pneumonia: 2 cases after lobectomy].

Two patients with postoperative interstitial pneumonia are reported. Preoperative diagnosis was primary lung cancer without idiopathic interstitial pneumonia (IIP). Within one week after operation acute interstitial pneumonia (AIP) occurred on the nonoperated side and developed. Steroid therapy was performed but one was dead. AIP is a fatal complication after pulmonary resection and steroid therapy may be useful in some cases of postoperative AIP.

[Surgical management of pulmonary disease due to nontuberculous mycobacteria].

Between 1983 and 1998, 9 patients with nontuberculous mycobacteriosis (NTM) underwent pulmonary resection. 8 patients were men and 1 was woman. Mean length of preoperative period was 20.3 months (range 6 months to 51 months). Most operative indication was localized NTM resistant to multidrug therapy. Lobectomy was performed in 7 patients, segmentectomy in one, partial resection in one. We have no operative mortality but air leaks occurred in one and he needed thoracoplasty. Mean postoperative follow up period was 67.7 months. Only one patients relapsed 34 months after the first operation. For localized NTM, early surgical management results good outcome.

[Prediction of the need for mechanical ventilation after transsternal thymectomy in patients with myasthenia gravis].

Between June 1992 and May 2000, transsternal extended thymectomy was performed for 70 patients with myasthenia gravis in our hospital. We were able to evaluate 64 of them in terms of prediction of the need for postoperative mechanical ventilation using the score systems reported by Leventhal et al., Kimura et al. and the criteria of Adachi et al.. For these systems, the rates of agreement between predictions and results were 85.9%, 82.8%, and 64.1%, respectively. The two former systems had some false negative cases (i.e., they predicted that ventilation would not be needed when in fact it was), but the last one gave no false negatives. We recommend Adachi's criteria for clinical safety. In our cases the patients whose value of %VC multiplied by FEV1.0% was less than 7,000 (Adachi's criterion is less in 8,300), especially, needed careful management with regard to respiratory crisis.

[The effect of perioperative portal venous inoculation with donor lymphocytes in combination with immunosuppressive agents on rat cardiac allograft survivals].

The synergistic effects of inoculation with donor lymphocytes via portal vein (PV) in combination with FK506 or monoclonal anti-T lymphocyte antibody (OX19) on cardiac allograft survival were investigated in rat heart transplant model (Lewis engrafted BN heart). The recipients were divided into the six groups. A) Control B) FK506 group C) PV group D) FK506+PV group E) OX19 group F) OX19+PV group. The recipients of PV group and FK506 group displayed a prolongation of cardiac allograft survival [MST: 11.2 +/- 1.0 and 12.4 +/- 1.0 days] compared with control group [MST: 7.3 +/- 0.3 days]. However, MST of FK506+PV group was 10.6 +/- 0.8 days, showing no synergistic effect of FK506 with PV administration. Significant prolongation of the graft survivals was not observed between OX19 group and OX19+PV group [MST: 17.2 +/- 2.1 and 15.0 +/- 1.2 days]. Serum from PV-treated hosts on day 7 after transplantation had suppressor effect (92.7%) on the MLR of Lewis responder cells toward donor BN cells. On the other hand, suppressor effect of serum from FK506+PV group was significantly reduced (5.8%) compared with PV alone. These results indicate that FK506 might inhibit the generation of suppressor factor which was induced in the serum after administration of donor lymphocytes, and that generation of suppressor factor might be dependent on T cell.

[The effect of combination therapy of cyclosporine with steroid on the production of gamma-interferon and interleukin-1 in kidney transplant recipients].

The present study examined the effects of CsA administered with steroid in vivo on the capacity of kidney transplant recipient mononuclear cells to generate cytokines and their gene expression at the level of messenger RNA (mRNA). Peripheral blood mononuclear cells (PBMC) from CsA-Prednisolone (Pred) treated recipients displayed 66.9% inhibition (54.3 + 12.4 IU/ml, n = 42, p less than 0.01) of gamma-IFN production compared with normal individuals (134.6 + 18.6 IU/ml, n = 23). Azathioprine (Az)-Pred treated recipients displayed significantly less inhibition of gamma-IFN generation (96.0 + 16.1 IU/ml, n = 22, p less than 0.05) than CsA treated patients. Macrophages (m phi) from CsA-Pred treated recipients displayed 60.0% inhibition (5.1 + 0.7 U/ml, n = 20, p less than 21). These result were confirmed by the experiments using cDNA probe for gamma-IFN or IL-1 (alpha, beta). High levels of gamma-IFN mRNA in PHA-stimulated PBL or IL-1 (beta) mRNA in LPS-stimulated m phi were present in normal individuals, but not in CsA treated recipients as judged by hybridization to a cloned human gamma-IFN or IL-1 (beta) cDNA probe. These studies demonstrated that combination therapy of CsA with steroid inhibits both gamma-IFN and IL-1 gene expression at the level of mRNA at physiological concentration.