Distinct role of cAMP and cGMP in the cell cycle control of vascular smooth muscle cells: cGMP delays cell cycle transition through suppression of cyclin D1 and cyclin-dependent kinase 4 activation

cAMP and cGMP are known to suppress vascular smooth muscle cell (SMC) proliferation. In this study, our aim was to delineate the molecular mechanism underlying cAMP and cGMP suppression of cell cycle transition in human SMCs. cAMP inhibits both platelet-derived growth factor-stimulated cyclin-dependent kinase (cdk) 2 and cdk4 activation through upregulation of the cdk2 inhibitor p27(Kip1) and downregulation of cyclin D1 expression, which leads to a complete arrest of the cells in phase G(1). In contrast, cGMP inhibits cyclin D1 expression, inhibits cdk4 activation, and delays platelet-derived growth factor-mediated cdk2 activation, resulting in a delay in G(1)/S transition. A transient increase in p27(Kip1) in cdk2 immunoprecipitates, without changes in total cellular p27(Kip1) levels, correlates with the delay in cdk2 activation caused by cGMP. Thus, cAMP and cGMP differentially affect cell cycle through distinct regulation of cell cycle molecules in human SMCs.     

Polyamines in 1 alpha, 25-dihydroxycholecalciferol-induced differentiation of human promyelocytic leukemia cells, HL-60

The human promyelocytic leukemia cell line, HL-60, differentiated into macrophage/monocytes in the presence of 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3], as assessed by the percentage of morphologically mature cells and their ability to reduce nitroblue tetrazolium. In this study of the mechanism involved, the activities of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase (SAT), the rate-limiting enzymes of polyamine metabolism, as well as the cellular levels of polyamine were measured. ODC activity reached a peak 24 h after the addition of 1 alpha,25(OH)2D3 and then decreased, while SAT activity gradually increased as differentiation commenced. An increase in putrescine and decreases in spermidine and spermine were also observed. Addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODC, with or without methylglyoxalbis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, caused no effect on 1 alpha,25(OH)2D3-induced cell differentiation, although the cellular levels of putrescine and spermidine decreased markedly. Addition of alpha-difluoromethylornithine markedly suppressed cell proliferation; this effect was reversed by the addition of exogenous putrescine. Addition of exogenous spermidine or spermine to overcome activation of SAT also had no effect on 1 alpha,25(OH)2D3-induced cell differentiation. These results suggest both that polyamine metabolism is not important in 1 alpha,25(OH)2D3-induced differentiation of HL-60 cells, but that it is intimately involved in the proliferation of these cells.     

Is it necessary to adjust the replacement dose of thyroid hormone to the season in patients with hypothyroidism?

Hypothalamo-pituitary-thyroid activity varies with the temperature of the environment; we therefore measured variables involved with thyroid function in summer and winter in normal controls and in patients with primary hypothyroidism. All seven patients had impalpable thyroid glands and had received a set replacement dose of thyroxine for over a year. In the patients, serum T3 and FT4 levels were slightly but significantly lower in winter, and TSH levels and delta TSH at 30 minutes in the TRH tests were significantly higher. In the controls, there were no significant differences between summer and winter in these values. These findings suggest that the dose required for replacement of thyroid hormone in patients with hypothyroidism may be higher in winter than in summer.     

p53 point mutations in primary human gastric carcinomas

Summary p53 point mutations in primary gastric carcinomas were analyzed by performing cDNA deoxynucleotide sequencing of the gene. Out of 16,9 (56.3%) primary gastric carcinoma cases, including early cancer, showed one or more p53 point mutations in their open-reading frame, and 4 out of 9 cases had a p53 point mutation within highly conserved domains. The characteristics of the p53 mutation spectrum observed in primary tumors were (a) frequent mutation at an A:T pair (50%, 7 out of 14 mutations), (b) high transversion incidence (29%, 4 out of 14 mutations), (c) no transition at CpG, and (d) no G:C to T:A transversion. Our results suggest that p53 mutation is a common event in gastric carcinoma occurring from the early stage of progression with its specific mutation spectrum.Abbreviation PCR-SSCP polymerase chain reaction single-strand conformation polymorphism     

Rare somatic inactivation of the multiple endocrine neoplasia type 1 gene in secondary hyperparathyroidism of uremia

The molecular pathway of autonomous growth of the parathyroid glands in uremic patients is poorly understood. Loss of heterozygosity at the recently identified multiple endocrine neoplasia type 1 (MEN1) gene locus on chromosome 11q13 has been found in a subset of parathyroid glands from patients with refractory hyperparathyroidism. To clarify the role of the MEN1 gene in parathyroid tumorigenesis, we analyzed 81 parathyroid glands from 22 Japanese uremic patients for allelic loss on chromosomal arm 11q13 DNA using 3 flanking markers (PYGM, D11S4946, and D11S449) and for mutations of the MEN1-coding exons by PCR-based single strand conformation polymorphism analysis and sequencing. Allelic loss on 11q13 was observed in 6 glands (7%), and 1 of 6 demonstrated a previously unrecognized somatic frameshift deletion (331delG) of the MEN1 gene. This mutation would probably result in a nonfunctional menin protein, consistent with a tumor suppressor mechanism. Clinical and pathological characteristics of hyperparathyroidism were unrelated to the presence or absence of loss of heterozygosity on 11q13 and MEN1 gene mutations. These observations indicate that somatic inactivation of the MEN1 gene contributes to the pathogenesis of uremia-associated parathyroid tumors, but its role in this disease appears to be very limited.     

Ticlopidine alone versus ticlopidine plus aspirin for preventing recurrent stroke

OBJECTIVE: To compare the efficacy and safety of two antiplatelet regimens, ticlopidine alone (200 mg daily) and ticlopidine (100 mg daily) plus aspirin (81 mg daily), in patients with ischemic stroke from the Tokai district of Japan. METHODS: A randomized comparative study was performed from April 1992 until December 1995, with follow-up for an average of 1.59 years (maximum: 3 years). Statistical analysis was done on 270 eligible patients (138 treated with ticlopidine alone and 132 treated with ticlopidine plus aspirin). PATIENTS: A total of 276 patients who had cerebral infarction within the previous 1 to 6 months, or one or more transient ischemic attacks within the previous 3 months. RESULTS: The incidence of ischemic and hemorrhagic stroke, myocardial infarction, and other vascular events was 10.1% (n = 14) in the ticlopidine group and 9.8% (n = 13) in the ticlopidine plus aspirin group, showing no significant difference (p = 0.933). There was also no significant difference in the event-free rate between the two groups (p = 0.5003, Kaplan-Meier analysis and log-rank test). Regarding serious adverse reactions, neutropenia occurred in one patient from the ticlopidine group, while gastric ulcer and thrombocytopenia occurred in one patient each from the ticlopidine plus aspirin group. CONCLUSION: We conclude that both antiplatelet regimens are comparable in efficacy and safety for preventing the recurrence of ischemic stroke.     

Quantitative Monitoring of Circulating Epstein-Barr Virus DNA for Predicting the Development of Posttransplantation Lymphoproliferative Disease

Epstein-Barr virus (EBV)-DNA was quantitatively measured to assess posttransplantation virus reactivation by real-time polymerase chain reaction (PCR). In the first retrospective analysis of a 7-year-old boy with lymphoproliferative disease (LPD) after an unrelated cord blood transplantation, serum EBV-DNA progressively increased to 4 x 10(5) copies/mL. EBV load was then prospectively monitored in peripheral blood from posttransplantation patients. The second case was an 8 year-old boy with aplastic anemia who received a CD34+ cell transplantation. This patient died of LPD with the progression of pulmonary nodules. EBV-DNA increased to 4 x 10(4) copies/mL after the control of cytomegalovirus reactivation. On the other hand, EBV-DNA was undetectable (<200 copies/mL) in the series of all 58 samples from 10 patients who did not develop LPD after hematopoietic stem cell transplantation. Sequential monitoring of circulating EBV-DNA by quantitative PCR may be a useful indicator for predicting the development of posttransplantation LPD.     

Does coronary artery morphology predict favorable results of intracoronary thrombolysis in patients with unstable angina pectoris?

The efficacy of intracoronary thrombolysis (ICT) for unstable angina pectoris (UAP) has been limited, despite the similar pathogenesis between UAP and acute myocardial infarction. To ascertain the subset of UAP suitable for ICT, the clinical responses to ICT were assessed in patients with UAP. Eighty-2 patients with medically refractory angina were divided into 2 groups according to the coronary artery morphology of the culprit lesion before ICT: (1) lesions with acute cut off and/or filling defects (AC) and (2) lesions with a tapered shape (TA). The TIMI flow grade was determined from coronary angiograms before and immediately after ICT. The diameter stenosis (%DS) and minimal lumen diameter (MLD) of the culprit lesion were determined using quantitative coronary angiographic analysis before and immediately after ICT. In addition, inhospital cardiac event rates including urgent/emergency coronary angioplasty or bypass surgery, nonfatal myocardial infarction or cardiac death were compared between the 2 groups. Multivariate logistic regression analysis was performed using 13 clinical factors contributing to successful ICT. The results showed that all 3 coronary angiographic parameters (TIMI flow, %DS, and MLD) significantly improved in the AC group (p<0.01, p<0.01 and p<0.05, respectively), whereas none of these parameters improved in the TA group. The inhospital cardiac event rate after ICT was significantly higher in the TA group (76%) than in the AC group (48%; p=0.016). Odds ratio predicting successful ICT was 7.09 (p<0.01) for the AC lesion, and 2.54 (p<0.01) for new angina. In conclusion the AC lesions are more commonly associated with coronary thrombosis that responds to ICT than are the TA lesions. Thus, the coronary angiographic morphology may be an important predictor for a successful ICT in patients with medically refractory UAP.     

Human anti-murine antibodies interfere with CPR assays performed with commercial kits

Quantitation of C-peptide is important for the assessment of insulin secretion, in particular in patients receiving insulin therapy. Since the CPR levels become much higher than the concentration of C-peptide for several reasons, such as the high concentration of proinsulin, CPR values sometimes need to be assessed carefully. We have had two diabetic patients whose CPR values were abnormally high when determined with a Daiichi C-peptide kit III (method 1). CPR values determined by other methods were from two to ten times lower, indicating considerable interference when method 1 was used. Since method 1 uses mouse monoclonal antibodies (mmab) for detection antibodies, we suspected that human anti-murine antibodies (HAMA) were responsible for the interference. HAMA were detected in serum from both patients (45 and 460 ng/ml in case 1 and case 2 (at peak), respectively). Removal of HAMA from serum eliminated the interference. Modification of method 1 to exclude mmab from the assay system removed all interference. HAMA were, therefore, considered to be the cause of the interference. In case 2, the peak concentration of HAMA was recorded 16 months earlier than the maximum of interference. Further analysis revealed that HAMA with high affinities were responsible for the interference.