Characteristics and timing of recurrence during postoperative surveillance after curative resection for lung adenocarcinoma.

PURPOSE: To establish the most effective methods of postoperative surveillance to detect early recurrence of lung adenocarcinoma. METHODS: The subjects of this retrospective study were 485 patients with p-stage I-III lung adenocarcinoma, who underwent postoperative surveillance. We examined the sites and detection modes of recurrence and calculated the recurrence-free probabilities. Patients with stage I disease were divided into low- and high-risk recurrence groups using a risk score calculated by assigning points proportional to risk factor regression coefficients. RESULTS: Of the 112 patients with recurrence, 86 had intrathoracic recurrence. Routine computed tomography (CT) revealed recurrence in 60 patients. The recurrence-free probability curves showed that 95% of recurrences were identified within the first 4 years after resection in patients with stage II/III disease. In patients with stage I disease, the predictors of recurrence included male sex, positive pleural lavage cytology, moderate-to-poor differentiation, and visceral pleural invasion. Postoperative recurrences were detected throughout the follow-up period in the high-risk group. CONCLUSIONS: Routine chest CT plays an important role in the postoperative surveillance of lung adenocarcinoma. We recommend intensive follow-up during the early post-resection period for patients with advanced stage disease and long-term follow-up for high-risk patients with stage I disease.

Hydrodynamics-based delivery of plasmid DNA encoding CTLA4-Ig prolonged cardiac allograft survival in rats.

BACKGROUND: Although gene therapy using plasmid vectors is thought to be safer compared with viral vectors, poor efficacy of gene transfer is the obstacle preventing wide application of plasmid vectors. However, high levels of foreign gene expression have been achieved by rapid tail vein injection of a large volume of a plasmid DNA solution into rats. Using this technique, we examined the effect of rat CTLA4-Ig gene transfer on prevention of cardiac allograft rejection in this animal model. METHODS: Recipient Lewis rats were injected with either plasmid pCAGGS-CTLA4-Ig-Glu-tag as a treatment vector or plasmid pCAGGS-signal peptide (SP)-Ig as a control vector by hydrodynamics-based delivery technique on the day before heart transplantation. Hearts from Brown Norway donors were transplanted into the neck of Lewis recipients and graft survival was assessed. RESULTS: The plasma level of CTLA4-Ig reached a peak of nearly 5 microg/mL 1 day after injection, and then slowly decreased but still remained above 0.9 microg/mL until 100 days after injection. The recipient rats treated with the control vector and untreated rats rejected cardiac allografts within 7 days. On the other hand, the median survival time of the grafts treated with pCAGGS-CTLA4Ig-Glu-tag was more than 100 days. Histological examination revealed that long-term survival allografts contained fewer infiltrating lymphocytes. The serum from recipients with long-term survival allograft suppressed allogenic mixed lymphocyte reaction. CONCLUSIONS: CTLA4-Ig gene transfer by means of tail vein injection of plasmid DNA into a recipient rat resulted in remarkable prolongation of cardiac allograft survival with persistent plasma level of CTLA4-Ig protein.

A case of biatrial multiple myxomas with glandular structure.

A 45-year-old male, who had been indicated by brain magnetic resonance imaging to have cerebral infarctions, was found by echocardiography to have a tumor in the left atrium. He had experienced several of the constitutional disturbances associated with myxoma. At the ages of 19 and 35 he had had two episodes associated with embolisms, and at the later one he was diagnosed as having multiple cerebral aneurysms. He received an urgent operation in which three left atrial tumors and one right atrial tumor were resected. Histologically, the tumors were myxomas, and the left atrial main tumor had glandular structure. In view of his clinical history, this patient seems to have had cardiac myxomas for a long period. The multiple growths that occurred in this case may be a good argument for allowing this condition to last for so long. To our knowledge, the present case was the first report of cardiac myxoma with glandular structure in Japan.

Ultrafiltration attenuates cardiopulmonary bypass-induced acute lung injury in a canine model of single-lung transplantation.

OBJECTIVE: The purpose of this study was to investigate the effects of cardiopulmonary bypass and ultrafiltration on graft function in a canine single-lung transplantation model. METHODS: Fifteen left single-lung transplantations were done in weight-mismatched canine pairs. The animals were divided into 3 groups: group 1, in which transplantation was done without cardiopulmonary bypass; group 2, in which transplantation was done with cardiopulmonary bypass and in which the cardiopulmonary bypass flow was decreased slowly with controlled pulmonary artery pressure; and group 3, in which transplantation was done with cardiopulmonary bypass and ultrafiltration. Hemodynamic parameters and lung function were monitored for 6 hours after reperfusion. The grafts were harvested for histologic studies, myeloperoxidase assay, and real-time quantitative reverse transcription-polymerase chain reaction of mRNA encoding interleukin 6. RESULTS: The hemodynamic parameters were similar among the 3 groups. In group 1 PaO2 and alveolar to arterial gradient for O2 levels were excellent throughout the 6-hour observation period, but in group 2 they progressively deteriorated. However, ultrafiltration significantly (P = .02) improved the PaO2 level in group 3. On histology, interstitial edema and polynuclear cell infiltration were most marked in group 2 and significantly worse than in groups 1 and 3. Myeloperoxidase assay and real-time quantitative reverse transcription-polymerase chain reaction showed increased myeloperoxidase activity and interleukin 6 gene expression in group 2 grafts compared with group 1 grafts. Myeloperoxidase activity and interleukin 6 gene expression were suppressed with ultrafiltration. CONCLUSIONS: Cardiopulmonary bypass had negative effects on the graft, but ultrafiltration attenuated acute lung dysfunction by reducing the inflammatory response.

Activation of protein kinase A alters subnuclear distribution pattern of human steroidogenic factor 1 in living cells.

BACKGROUND: The aim of this study was to identify the subnuclear distribution pattern of human orphan nuclear receptor steroidogenic factor 1 (SF-1) in living cells with and without the activation of protein kinase A (PKA) signal pathway, and thus try to explain the unknown mechanism by which PKA potentiates SF-1 transactivation. METHODS: Full-length cDNAs of wild type and a naturally occurring mutant (G35E) human SF-1 were cloned and fused with green fluorescent protein (GFP). Subcellular distribution pattern of human SF-1 in living cells, whose PKA signaling was either activated or not, was studied by laser confocal microscopy after the validity of the gene sequence was confirmed. RESULTS: The transactivation ability of the GFP-SF-1 chimeric protein was highly conserved. Wild type human SF-1 diffused homogeneously within the nuclei of cells when PKA was not active, and converged to clear foci when PKA was activated. Mutant SF-1 diffused within the nuclei even in the presence of PKA activation, surprisingly aggregating as fluorescent dots inside the nucleoli, a phenomenon not altered by PKA. CONCLUSIONS: Activation of PKA causes wild type, but not mutant SF-1 to alter its subnuclear distribution pattern to a transactivationally active form (foci formation). This finding may throw new light on the mechanism by which PKA activates the orphan nuclear receptor.

Protein kinase A potentiates adrenal 4 binding protein/steroidogenic factor 1 transactivation by reintegrating the subcellular dynamic interactions of the nuclear receptor with its cofactors, general control nonderepressed-5/transformation/transcription domain-associated protein, and suppressor, dosage-sensitive sex reversal-1: a laser confocal imaging study in living KGN cells.

The mechanism through which protein kinase A (PKA) potentiates the transactivation ability of adrenal 4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) is currently unclear. In the present study, we investigated the mechanism by applying laser confocal microscopy and fluorescence recovery after photobleaching technique. In KGN cells, forskolin (a PKA stimulator) could reorganize wild-type Ad4BP/SF-1, but not mutant Ad4BP/SF-1 (G35E), from a diffuse distribution pattern to foci formation in the nucleus. The subcellular distributions of GCN5 (general control nonderepressed) and TRRAP (transformation/transcription domain-associated protein), both of which were recently proved to be working in the same complex as the third class of nuclear receptor coactivators, were unexpectedly diffuse inside and outside the nucleus, respectively, when they were separately transfected. However TRRAP was translocated into the nucleus in the presence of GCN5, and together with GCN5 colocalized with Ad4BP/SF-1 in the same foci when PKA was activated. A luciferase assay also indicated that these two cofactors enhanced Ad4BP/SF-1 transactivation.Dosage-sensitive sex reversal (DAX-1) interacts with and thus inhibits Ad4BP/SF-1 transactivation. The coexistence of the two proteins dramatically altered their respective subnuclear distributions. They colocalized extensively, suggestive of binding, and Ad4BP/SF-1 was sharply immobilized when DAX-1 was coexpressed, whereas PKA could maintain mobility, as evidenced by Fluorescence Recovery After Photobleaching showing that Ad4BP/SF-1 mobility recovered after forskolin treatment.Therefore, the PKA signal pathway may modify the interaction between Ad4BP/SF-1 and its activators and repressor (GCN5 and TRRAP are integrated, whereas DAX-1 is disassociated), and thus stimulate the Ad4BP/SF-1 transactivation.

Correlation of oxidative stress with activity of matrix metalloproteinase in patients with coronary artery disease. Possible role for left ventricular remodelling.

AIMS: Oxidative stress is implicated in the progression of heart failure, and matrix metalloproteinase (MMP) activity is increased in patients with congestive heart failure. We examined the role of oxidative stress on MMP activity in humans. METHODS AND RESULTS: We measured the MMP activity and the level of 8-iso-prostagandin F2alpha (8-iso-PGF2alpha), a specific and quantitative maker of oxidant stress, in the pericardial fluid (PF) in 47 consecutive patients with coronary artery disease who underwent coronary artery bypass surgery. Zymography of PF showed bands at 92-85kDa (MMP-9) and 72-65kDa (MMP-2). The MMP activity was expressed as the ratio to MMP-2 standard. MMP-2, MMP-9 and total gelatinolysis activities were positively correlated with left ventricular end-diastolic volume index (LVEDVI), and MMP-2 and total gelatinolysis activities were also positively correlated with LV end-systolic volume index. Moreover, MMP-2, MMP-9 and total gelatinolysis activities were all positively correlated with pericardial level of 8-iso-PGF2alpha. Also, LVDEVI was positively correlated with pericardial level of 8-iso-PGF2alpha. CONCLUSIONS: Oxidative stress may play an important role in the regulation of MMP activity. Augmented MMP activity may be involved in the development of ventricular remodelling in patients with coronary artery disease.

Plasma concentration of coupling factor 6 and cardiovascular events in patients with end-stage renal disease.

BACKGROUND: Plasma asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is an independent predictor of overall mortality and cardiovascular outcome in hemodialysis patients. However, not only ADMA but also traditional risk factors account for only part of the high cardiovascular morbidity and mortality in these patients. We investigated cross-sectionally the association between coupling factor 6 (CF6), an endogenous inhibitor of prostacyclin synthesis, and cardiovascular events in 95 hemodialysis patients. METHODS: Plasma CF6 level was measured by radioimmunoassay, whereas plasma ADMA level by high-performance liquid chromatography (HPLC). RESULTS: Plasma levels of CF6 and ADMA were threefold higher in hemodialysis patients than in control individuals, and there was a positive correlation between these two compounds (r=0.25, P < 0.05). Plasma CF6 level was positively correlated with serum creatinine level (r=0.36, P < 0.01) and was reduced after dialysis (P < 0.05). Plasma CF6 and ADMA levels were both higher in hemodialysis patients complicating ischemic heart disease (myocardial infarction and/or angina) than in those free of cardiovascular events. In a multiple regression model, plasma CF6 level (r=0.24, P=0.023) and ADMA level (r=0.26, P=0.023) were independently related to the occurrence of ischemic heart disease in hemodialysis patients. CONCLUSION: CF6 is a novel risk factor for ischemic heart disease in end-stage renal disease (ESRD). Synergism of this peptide and ADMA might contribute to its occurrence presumably by inhibition of prostacyclin and nitric oxide production. A prospective study is needed to evaluate this issue more precisely.

Effect of shear stress on asymmetric dimethylarginine release from vascular endothelial cells.

We demonstrated recently that plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, are increased by high salt intake concomitantly with a decrease in plasma levels of NO in human hypertension. We investigated the effect of shear stress on ADMA release in 2 types of cells: transformed human umbilical vein endothelial cells (HUVECs; cell line ECV-304) and HUVECs. Exposure of ECV-304 cells and HUVECs to shear stress with the use of a cone-plate viscometer enhanced gene expression of protein arginine methyltransferase (PRMT-1), ADMA synthase. In HUVECs, the ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase mRNA was increased by 2-fold by a shear stress of > or =15 dyne/cm2. A dominant-negative mutant of IkappaB kinase alpha and troglitazone at 8 micromol/L, an activator of peroxisome proliferator-activated receptor gamma, abolished the shear stress-induced increase in PRMT-1 gene expression in parallel with the blockade of nuclear factor (NF)-kappaB translocation into the nucleus. The activity of dimethylarginine dimethylaminohydrolase, the degradation enzyme of ADMA, was unchanged after shear stress < or =15 dyne/cm2 and was enhanced by 1.48+/-0.06-fold (P<0.05) by shear stress at 25 dyne/cm2. The release of ADMA was increased by 1.64+/-0.10-fold (P<0.05) by shear stress at 15 dyne/cm2 but was not affected by shear stress at 25 dyne/cm2. These results indicate that shear stress enhances gene expression of PRMT-1 and ADMA release via activation of the NF-kappaB pathway. Shear stress at higher magnitudes facilitates the degradation of ADMA, thus returning ADMA release levels to baseline.

High plasma level of asymmetric dimethylarginine in patients with acutely exacerbated congestive heart failure: role in reduction of plasma nitric oxide level.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is elevated in congestive heart failure (CHF) concomitantly with the higher levels of nitric oxide (NO) and cytokines. We investigated the association among ADMA, NO, and cytokines in human CHF. Blood was collected from 25 patients with acutely exacerbated chronic CHF (acute CHF, mean age 61 +/- 3 years), 23 patients with chronic compensated CHF (chronic CHF, mean age 62 +/- 2 years), and 26 control subjects (mean age 51 +/- 1 years). ADMA was measured by high-performance liquid chromatography. Tumor necrosis factor-alpha (TNF-alpha) was measured by enzyme-linked immunosorbent assay. Nitrate plus nitrite (NOx) was measured by the Griess method. The plasma levels of ADMA and TNF-Alpha were higher in patients with acute CHF than in those with chronic CHF and control subjects (both P < 0.05). The plasma level of NOx was higher in patients with chronic CHF than in those with acute CHF and control subjects (both P < 0.01). The plasma level of TNF-Alpha was positively correlated with that of ADMA in combination with patients with acute and chronic CHF (r = 0.31, P < 0.01). The plasma level of ADMA was, furthermore, negatively correlated with that of NOx (r = -0.29, P < 0.05). These findings indicate that ADMA is related to exacerbation of chronic CHF by suppression of the compensatory higher level of plasma NO.

Relationship between salt intake, nitric oxide and asymmetric dimethylarginine and its relevance to patients with end-stage renal disease.

Patients with essential hypertension (n = 24) were administered a low-salt diet (2 g NaCl/day), a high-salt diet (20-23 g) and then a low-salt diet for 7 days, and plasma levels of nitrate and nitrite (NOx) and asymmetric dimethylarginine (ADMA) were examined. There was a negative correlation between the percent changes in mean blood pressure and the plasma NOx concentration after salt loading and restriction. The percent change in plasma ADMA concentration was negatively correlated with that in the plasma NOx concentration after salt loading and restriction. In patients with end-stage renal disease (n = 51), the plasma ADMA concentration was positively correlated with the duration of dialysis treatment. The frequency of cardiovascular events was greater in patients with a plasma ADMA level of >/=3 microM than in those with a plasma AMDA level of <3 microM. The results indicate that ADMA is not only a modulator of salt sensitivity in hypertension but also a cardiovascular risk factor in end-stage renal disease.

Activation function-1 domain of androgen receptor contributes to the interaction between subnuclear splicing factor compartment and nuclear receptor compartment. Identification of the p102 U5 small nuclear ribonucleoprotein particle-binding protein as a coactivator for the receptor.

In the androgen receptor (AR), most of its transactivation activity is mediated via the activation function-1 (AF-1). By employing yeast two-hybrid assay, we isolated a cDNA sequence encoding a protein binding to AR-AF-1. This protein, named ANT-1 (AR N-terminal domain transactivating protein-1), enhanced the ligand-independent autonomous AF-1 transactivation function of AR or glucocorticoid receptor but did not enhance that of estrogen receptor alpha. In contrast, the ANT-1 did not enhance any ligand-dependent AF-2 activities. Furthermore, the ligand-independent interaction between AR-AF-1 and ANT-1 was confirmed in vivo and in vitro. The ANT-1 sequence was identical to that of a protein that binds to U5 small nuclear ribonucleoprotein particle, a human homologue of yeast splicing factor Prp6p, involved in spliceosome. ANT-1 was compartmentalized into 20-40 coarse splicing factor compartment speckles against the background of the diffuse reticular distribution. AR colocalized with ANT-1 only in the diffusely distributed area, whereas the ANT-1 speckles were spatially distinct from but surrounded by the AR compartments. The active gene transcription has been shown to couple simultaneously with pre-mRNA processing at the periphery of the splicing factor compartment. The molecular interaction between two spatially distinct subnuclear compartments mediated by ANT-1 may therefore recruit AR into the transcription-splicing-coupling machinery.

The presence of both the amino- and carboxyl-terminal domains in the AR is essential for the completion of a transcriptionally active form with coactivators and intranuclear compartmentalization common to the steroid hormone receptors: a three-dimensional imaging study.

To clarify the physiological significance of the intranuclear speckled distribution, or foci formation, of liganded steroid receptors, the subnuclear distribution of green (GFP), yellow (YFP), and cyan (CFP) fluorescent protein-tagged receptors and coactivators was investigated. The foci formation of 5 alpha-dihydrotestosterone (DHT)-bound AR-GFP in COS7 cells was abolished by the cotransfection of a CBP Delta (118-2393) fragment eliciting a dominant negative effect on the transactivation capacity of the AR. The N-terminal AR fragment (AR-AF-1-YFP), which has a strong constitutive transactivation function, formed foci without DHT, whereas the C-terminal AR fragment (AR-AF-2-CFP), which has a quite low transactivation function, was distributed homogeneously even in the presence of DHT. The reporter gene assay showed a synergism between the transactivation functions of AR-AF-1 and AR-AF-2. This synergism was not reflected by the above two-dimensional imaging. In contrast, a three-dimensional imaging method clearly showed a difference in the intranuclear spatial distribution. The DHT-bound wild-type AR-GFP alone or AR-AF-1-YFP plus DHT-bound AR-AF-2-CFP was distributed as approximately 300 discrete spots in one nucleus, whereas AR-AF-1-YFP alone was distributed as one volume in a reticular pattern. Furthermore, not only AR but also the glucocorticoid receptor-YFP, ER alpha -GFP, and YFP-tagged SRC-1, TIF2, and CBP were found to be accumulated in identical spots in the presence of ligand. All of the above results indicate that CBP is one of the factors essential for foci formation of the AR, and may propose the hypothesis that transcriptionally activated steroid receptors, regardless of the type of receptor, are transferred to common compartments (foci) and form a complex with coactivators, and this process is essential to full transactivation.