Ericaceous plant-fungus network in a harsh alpine-subalpine environment.

In terrestrial ecosystems, plant species and diverse root-associated fungi form complex networks of host-symbiont associations. Recent studies have revealed that structures of those below-ground plant-fungus networks differ between arbuscular mycorrhizal and ectomycorrhizal symbioses. Nonetheless, we still remain ignorant of how ericaceous plant species, which dominate arctic and alpine tundra, constitute networks with their root-associated fungi. Based on a high-throughput DNA sequencing data set, we characterized the statistical properties of a network involving 16 ericaceous plant species and more than 500 fungal taxa in the alpine-subalpine region of Mt. Tateyama, central Japan. While all the 16 ericaceous species were associated mainly with fungi in the order Helotiales, they varied remarkably in association with fungi in other orders such as Sebacinales, Atheliales, Agaricales, Russulales and Thelephorales. The ericaceous plant-fungus network was characterized by high symbiont/host preferences. Moreover, the network had a characteristic structure called 'anti-nestedness', which has been previously reported in ectomycorrhizal plant-fungus networks. The results lead to the hypothesis that ericaceous plants in harsh environments can host unexpectedly diverse root-associated fungal taxa, constituting networks whose structures are similar to those of previously reported ectomycorrhizal networks but not to those of arbuscular mycorrhizal ones.

Effect of human adipose-derived mesenchymal stem cell conditioned medium on musculoskeletal pain.

OBJECTIVE: Several studies in animal models have shown the safety and effectiveness of mesenchymal stem cell conditioned medium (MSC-CM) in inflammatory lesions involving muscles and joints. PATIENTS AND METHODS: In this report, we retrospectively evaluated 16 patients who received local administration of the human adipose-derived mesenchymal stem cells conditioned medium (hAMSC-CM) for musculoskeletal chronic pain. Overall, 27 body locations expressing pain have been treated. The local administrated dose was 5 ml in the joint cavity and/or 2 ml in the other locations. The patients were asked to conduct self-evaluation of the degree of pain using a numeric rating scale (NRS) questionnaire and record the severity of pain before administration and at 15 min, 1 day, 1 week, and 4 weeks after administration. A second administration has been performed in 7 locations. The analysis was done considering two conditions: the "current pain status" and the "worst pain status in a week." RESULTS: The results showed statistically significant differences between before and after administration at each time point for "current pain status" and at 1-week and 4-week time points for "worst pain status in a week" after first administration (Tukey-Kramer test). After second administration, significant differences were found at 1-week and 4-week time points for "current pain status". No serious adverse effect was found. CONCLUSIONS: It was concluded that local administration of hAMSC-CM appears to be safe and could be expected to have effective therapeutic value against musculoskeletal chronic pain. Further studies are needed to clarify analgesic effects of hAMSC-CM and its underlying mechanism(s).

Impact of platinum-based chemotherapy on the progression of atherosclerosis.

OBJECTIVES: Although patients with gynecological malignancies now survive longer due to advances in early diagnosis and therapy, major issues still remain regarding the quality of life for the survivors. Surgical menopause increases the risk of atherosclerosis; however, few studies have investigated the influence of platinum-based adjuvant chemotherapy. This study was conducted to evaluate the effects of platinum-based chemotherapy on atherosclerosis. METHODS: This study enrolled 47 women (26 with ovarian cancers and 21 with endometrial cancers) who underwent surgical treatment, with or without platinum-based adjuvant chemotherapy, according to established protocols between 2007 and 2009. Arterial stiffness was measured by brachial-ankle pulse wave velocity (baPWV) performed before surgery, and subsequently at 12 months after treatment. The flow-mediated dilatation of the brachial artery was measured before and immediately following chemotherapy to evaluate the vascular endothelial damage. Human umbilical vein endothelial cells (HUVECs) were used to evaluate cisplatin-induced vascular endothelial dysfunction in vitro. RESULTS: Although there were no significant differences in the baPWV associated with surgical treatment, platinum-based chemotherapy was associated with an increased baPWV. Significant decreases of flow-mediated dilatation were observed immediately following chemotherapy. An in vitro examination demonstrated that cisplatin attenuated nitric oxide production via inhibition of Akt-eNOS cascades in HUVECs. CONCLUSIONS: This research suggests that platinum-based chemotherapy directly induces vascular endothelial dysfunction and may be a risk factor for the development of atherosclerosis. Therefore, gynecologic cancer survivors should be educated about these potential risks, and informed regarding lifestyle modifications that may benefit their general health.

Discovery of a patient with strongly suspected bullous pemphigoid in a ward by oral health care providers.

OBJECTIVES: Oral health care providers may discover systemic diseases incidentally from signs observed in the oral cavity. Here, we report a case in which oral health care providers in a hospital discovered a patient with strongly suspected bullous pemphigoid (BP), which is a relatively rare but important disease, in a ward. METHODS: The patient was a 78-year-old Japanese woman admitted to our hospital because of severe Alzheimer's disease. We discovered recurrent ulcers in the oral mucosa and skin when performing oral care in her ward. Biopsy could not be performed safely because of involuntary biting. We performed blood tests for anti-BP180-NC16a antibody, which is autoantibody specific for BP. RESULTS: The patient had a very high anti-BP180-NC16a antibody titre. We consulted a dermatologist regarding her clinical course and the clinical features of the oral mucosa and skin along with blood test results. BP was very strongly suspected. DISCUSSION: In cases in which oral health care providers suspect their patients may have BP, appropriate examination and provision of information to the doctor are important. Oral health care providers should have knowledge about systemic diseases, the signs of which appear in oral cavity to avoid missing important systemic diseases.

Cytotoxicity of amorphous silica particles against macrophage-like THP-1 cells depends on particle-size and surface properties.

Recent studies have indicated that amorphous silica particles (SPs) show cytotoxicity against various types of cells, including macrophages. However, the mechanism of cell death has not been determined, and systematic investigations of the relationship between particle characteristics and cytotoxicity are still quite limited. Here, we compared the cytotoxicity of SPs of various sizes (30-1000 nm) and surface properties against differentiated THP-1 human macrophage-like cells. We found that 300 and 1000 nm SPs showed cytotoxicity against THP-1 cells, whereas 30, 50, and 70 nm SPs did not induce cell death. We demonstrated that 1000 nm SP showed strong cytotoxicity that depended on reactive oxygen species but was independent of caspases. Furthermore, we showed that surface modification of 1000 nm SPs dramatically suppressed their cytotoxicity. Our results suggest that systematic evaluation of the association between particle characteristics and biological effects is necessary for the creation of safe SPs.

Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria.

Cyanobacteria are the simplest organisms known to have a circadian clock. A circadian clock gene cluster kaiABC was cloned from the cyanobacterium Synechococcus. Nineteen clock mutations were mapped to the three kai genes. Promoter activities upstream of the kaiA and kaiB genes showed circadian rhythms of expression, and both kaiA and kaiBC messenger RNAs displayed circadian cycling. Inactivation of any single kai gene abolished these rhythms and reduced kaiBC-promoter activity. Continuous kaiC overexpression repressed the kaiBC promoter, whereas kaiA overexpression enhanced it. Temporal kaiC overexpression reset the phase of the rhythms. Thus, a negative feedback control of kaiC expression by KaiC generates a circadian oscillation in cyanobacteria, and KaiA sustains the oscillation by enhancing kaiC expression.

Eukaryotic diversity in late Pleistocene marine sediments around a shallow methane hydrate deposit in the Japan Sea.

Marine sediments contain eukaryotic DNA deposited from overlying water columns. However, a large proportion of deposited eukaryotic DNA is aerobically biodegraded in shallow marine sediments. Cold seep sediments are often anaerobic near the sediment-water interface, so eukaryotic DNA in such sediments is expected to be preserved. We investigated deeply buried marine sediments in the Japan Sea, where a methane hydrate deposit is associated with cold seeps. Quantitative PCR analysis revealed the reproducible recovery of eukaryotic DNA in marine sediments at depths up to 31.0 m in the vicinity of the methane hydrate deposit. In contrast, the reproducible recovery of eukaryotic DNA was limited to a shallow depth (8.3 m) in marine sediments not adjacent to the methane hydrate deposit in the same area. Pyrosequencing of an 18S rRNA gene variable region generated 1,276-3,307 reads per sample, which was sufficient to cover the biodiversity based on rarefaction curves. Phylogenetic analysis revealed that most of the eukaryotic DNA originated from radiolarian genera of the class Chaunacanthida, which have SrSO4 skeletons, the sea grass genus Zostera, and the seaweed genus Sargassum. Eukaryotic DNA originating from other planktonic fauna and land plants was also detected. Diatom sequences closely related to Thalassiosira spp., indicative of cold climates, were obtained from sediments deposited during the last glacial period (MIS-2). Plant sequences of the genera Alnus, Micromonas, and Ulmus were found in sediments deposited during the warm interstadial period (MIS-3). These results suggest the long-term persistence of eukaryotic DNA from terrestrial and aquatic sources in marine sediments associated with cold seeps, and that the genetic information from eukaryotic DNA from deeply buried marine sediments associated with cold seeps can be used to reconstruct environments and ecosystems from the past.

Exponentially growing solutions in homogeneous Rayleigh-Bénard convection.

It is shown that homogeneous Rayleigh-Bénard flow, i.e., Rayleigh-Bénard turbulence with periodic boundary conditions in all directions and a volume forcing of the temperature field by a mean gradient, has a family of exact, exponentially growing, separable solutions of the full nonlinear system of equations. These solutions are clearly manifest in numerical simulations above a computable critical value of the Rayleigh number. In our numerical simulations they are subject to secondary numerical noise and resolution dependent instabilities that limit their growth to produce statistically steady turbulent transport.

Radioresistance of cancer stem-like cell derived from canine tumours.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye-based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage-independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct-4 and CD133 gene than those of adherent-cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent-cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent-cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.

Vascular action of circulating and local natriuretic peptide systems is potentiated in obese/hyperglycemic and hypertensive rats.

Hypertension is commonly associated with diabetes mellitus. The aim of the present study was to explore the pathophysiological significance of the natriuretic peptide (NP) system in hypertension associated with genetically obese/hyperglycemic Wistar fatty rats. The messenger RNA (mRNA) levels of the two biologically active NP receptors, NP-A receptor [more specific for atrial natriuretic peptide (ANP)] and NP-B receptor [more specific for C-type natriuretic peptide (CNP)], and CNP mRNA levels were determined in the aorta and kidney by ribonuclease protection assay. Plasma ANP levels were determined by RIA. Both NP-A and NP-B receptor mRNA levels in the aortae of Wistar fatty rats were double those in Wistar lean rats. Plasma ANP levels and CNP mRNA levels in the aorta of Wistar fatty rats were also significantly higher than those in Wistar lean rats. In contrast, there was no significant difference in renal levels of the mRNA for both NP receptors and CNP between the two strains. Administration of a NP-A and -B receptor antagonist, HS-142-1, to Wistar fatty rats resulted in a significant increase in systolic blood pressure and a larger decrease in plasma cGMP level than that in Wistar lean rats, with no difference in the extents of decrease in urine volume and urinary sodium excretion between the two strains. These results suggest that both the ANP/NP-A system and the CNP/NP-B system in vessels are up-regulated at the level of gene expression and may, thus, play an important role in counteracting the hypertension associated with diabetes mellitus.

Modulation of vascular natriuretic peptide receptor gene expression in hypertensive and obese hyperglycemic rats.

Receptors for natriuretic peptide (NP) consist of three subtypes: NP-A, NP-B, and NP-C. Recent studies in cultured aortic cells have suggested a phenotype-related switching of the vascular NP receptor from NP-A to NP-B. To ascertain the biological significance of the phenomenon in vivo, we developed a sensitive and reproducible ribonuclease protection assay and determined each receptor messenger RNA (mRNA) level in the vascular vessels of stroke-prone spontaneously hypertensive rats, deoxycorticosterone acetate-salt hypertensive rats, and genetically hyperglycemic. Wistar fatty rats and in cultured aortic smooth muscle cells. The aortic NP-A receptor mRNA level was significantly up-regulated in both types of hypertensive rats, whereas the NP-B receptor mRNA level did not show any significant change. Both NP-A and NP-B receptor mRNA levels were significantly up-regulated in Wistar fatty rats compared with the control values. There was no significant up-regulation of NP-A receptor mRNA in the inferior vena cava of the stroke-prone spontaneously hypertensive rats. Although the NP-A receptor was always the predominant subtype in rat aortic tissue, NP-B receptor was the predominant subtype in aortic smooth muscle cells in culture. These findings suggest that up-regulation of the NP-A receptor, but not the subtype switching, is the major modulation of receptor gene expression in both hypertensive and diabetic rats.

Angiotensin II-dependent down-regulation of vascular natriuretic peptide type C receptor gene expression in hypertensive rats.

Biological actions of natriuretic peptide (NP) are determined by the condition of the receptor as well as that of the hormone. Although we previously demonstrated in hypertensive rats the up-regulation of NP-A receptor that mediates various biological actions of NPs, the pathophysiologic significance of NP-C receptor, another subtype thought to be related to clearance of NPs and possibly to biological actions, remains unknown. In the present study, we determined NP-C receptor messenger RNA (mRNA) level in the aortic tissue of stroke-prone spontaneously hypertensive rats (SHR-SP/Izm) and in cultured aortic smooth muscle cells by ribonuclease protection assay. The aortic NP-C receptor mRNA level in SHR-SP/Izm was significantly lower than that in the control WKY/Izm. Oral administration of an angiotensin (Ang) II receptor (AT1) antagonist, TCV-116, but not a calcium channel blocker, manidipine, reversed the down-regulated NP-C receptor mRNA in SHR-SP/Izm to the level in WKY/Izm, whereas the latter was more potent in decreasing the blood pressure. In cultured aortic smooth muscle cells, the NP-C receptor was the predominant subtype. Ang II decreased the NP-C receptor mRNA level in a dose-dependent manner, but this effect was reversed by an AT1 antagonist, CV-11974. Neither the NP-A nor NP-B receptor mRNA level was altered by Ang II. These findings indicate that vascular NP-C receptor is down- regulated via Ang-II-mediated mechanism in SHR-SP/Izm. The phenomenon, together with the up-regulation of the NP-A receptor, may play an important role in counteracting hypertension by enhancing the action of NPs.

Potentiation of natriuretic peptide action by the beta-adrenergic blocker carvedilol in hypertensive rats: a new antihypertensive mechanism.

Treatment with a beta-adrenergic blocker (beta-blocker) in hypertension is associated with increased plasma atrial natriuretic peptide (ANP) levels despite a decrease in cardiac overload. The mechanism and pathophysiological significance of the phenomenon remain unclear. To clarify the role of the ANP system in the antihypertensive effects of the beta-blocker, we investigated the effects of carvedilol (30 mg/kg x day, orally, for 4 weeks) on the ANP system in stroke-prone spontaneously hypertensive rats (SHR-SP/Izm). Plasma ANP levels showed a significant increase despite a significant decrease in blood pressure and heart rate in the carvedilol group. Although ANP messenger RNA levels in the heart did not change, messenger RNA levels of the natriuretic peptide-C (NP-C) receptor as a clearance receptor showed a significant decrease in both the aorta and lung in the carvedilol group. NP-C receptor densities were also significantly decreased in the lung in this group. The biological half-life of exogenous ANP in circulating blood was prolonged in the carvedilol group compared with that in the control group. Administration of the ANP receptor antagonist, HS-142-1, resulted in a greater increase in systolic blood pressure in the carvedilol group than in the control group. In addition, both basal and ANP-stimulated cGMP contents in the aorta were significantly higher in the carvedilol group. These results suggest that carvedilol potentiates the hypotensive action of ANP by increasing plasma ANP levels and enhancing the vascular response to ANP. These effects were closely related to the down-regulation of the NP-C receptor. The newly found mechanism seems to account for a sizable portion of the antihypertensive effects of carvedilol and could be of potential importance in the treatment of cardiovascular disease with beta-blockers.

Does plasma immunoreactive ouabain originate from the adrenal gland?

It was reported recently that the endogenous digitalis-like factor ouabain may mainly originate from the adrenal gland. To ascertain the pathophysiological significance of endogenous ouabain and to examine if it originates in the adrenal gland, we determined plasma immunoreactive ouabain levels in patients with various cardiovascular and endocrine diseases. Plasma immunoreactive ouabain levels were also determined in the adrenal venous blood by adrenal venous sampling. Plasma immunoreactive ouabain levels were significantly increased in patients with essential hypertension, primary aldosteronism, Cushing's syndrome, pheochromocytoma, acromegaly, and chronic renal failure. Plasma immunoreactive ouabain levels were decreased in patients with primary aldosteronism after unilateral adrenalectomy, acromegaly after pituitary adenomectomy, and chronic renal failure after hemodialysis. Plasma immunoreactive ouabain levels in patients after bilateral adrenalectomy were similar to those in healthy subjects. There was no significant step-up of immunoreactive ouabain levels in the adrenal vein from the peripheral vein in three patients, whereas one patient with hypertension and right adrenal tumor but without any known adrenal hormone excess showed higher plasma immunoreactive ouabain levels in the right adrenal vein than those in the peripheral vein. These results suggest an important pathophysiological significance of endogenous ouabain in various cardiovascular and endocrine diseases. It is unlikely that the adrenal gland is a major source of plasma ouabain, although a possible excess production of ouabain by the adrenal tumor remains to be elucidated.

Atrial and brain natriuretic peptides in cardiovascular diseases.

The human heart secretes both atrial natriuretic peptide and brain natriuretic peptide. This study attempts to clarify the pathophysiological significance of the peptides in cardiovascular diseases. Using immunoradiometric assay, plasma brain natriuretic peptide and atrial natriuretic peptide levels in essential hypertension, various secondary hypertension, chronic renal failure, chronic heart failure during cardiac pacing, and acute myocardial infarction were determined. Mean plasma brain natriuretic peptide and atrial natriuretic peptide levels in healthy subjects were 3.7 +/- 0.3 and 5.7 +/- 0.3 pmol/L, respectively, and increased as a function of age. Plasma brain natriuretic peptide levels showed a larger increase than atrial natriuretic peptide levels in various cardiovascular diseases. In chronic renal failure, whereas plasma atrial natriuretic peptide levels decreased significantly after hemodialysis and were correlated with the changes in body weight, changes in plasma brain natriuretic peptide levels were less prominent and did not show such a correlation. In chronic heart failure, both basal plasma brain natriuretic peptide and atrial natriuretic peptide levels were also significantly elevated. However, in response to acute ventricular or atrial pacing, brain natriuretic peptide levels did not show any increase in contrast to the marked increase of atrial natriuretic peptide levels. In acute myocardial infarction, brain natriuretic peptide levels showed more prominent changes than atrial natriuretic peptide levels and were correlated with serum levels of creatine kinase and cardiac myosin light chain I in most patients. These results suggest that both brain and atrial natriuretic peptides play an important role in the regulation of cardiovascular homeostasis.(ABSTRACT TRUNCATED AT 250 WORDS)

Two nuclear localization signals are required for nuclear translocation of nuclear factor 1-A.

Nuclear factor 1 (NF1) proteins are encoded by at least four genes (NF1-A, B, C, X). Although DNA-binding and the transcription regulation domains of these proteins are well characterized, the nuclear localization signals (NLSs) are still unknown in all NF1s. We have identified two NLSs in NF1-A, and both are required for full translocation to the nucleus, although one of them itself has a partial translocation ability. These two NLSs are conserved in all four NF1s. Interestingly, three isoforms of NF1-A (NF1-A1, A2, A4) have two NLSs and translocate completely to the nucleus. In contrast, NF1-A3 lacks the second NLS and partially stays in the cytoplasm. Since NF1s construct homodimer and heterodimer, these findings indicate the differential regulations of the NF1 translocation.

Vasculo-protective effects of insulin sensitizing agent pioglitazone in neointimal thickening and hypertensive vascular hypertrophy.

A novel insulin sensitizing agent, thiazolidine, has been demonstrated to inhibit the growth of cultured vascular smooth muscle cells (VSMC) in vitro. This study was undertaken to examine the in vivo effects of the thiazolidine compound pioglitazone (PIO) on carotid neointimal thickening, after endothelial injury in Wistar rats and vascular hypertrophy in stroke-prone spontaneously hypertensive rats (SHR-SP/Izm). PIO treatment (3 mg/kg/day for 1 week prior to endothelial injury and 2 weeks postendothelial injury) remarkably decreased neointimal cross-sectional areas in treated animals (63.8 +/- 4.9 x 10(3) microm2) versus controls (196 +/- 7.6 x 10(3) microm2, P < 0.05). Bromodeoxyuridine uptake in the neointima, a marker of DNA synthesis, was also decreased after treatment compared with controls. In SHR-SP/Izm but not in Wistar rats, PIO treatment decreased blood pressure and plasma insulin levels. PIO treatment in SHR-SP/Izm (3 mg/kg/day from 4 weeks of age for 7 weeks) significantly decreased the medial wall thickness of the mesenteric artery (10.4 +/- 1.2 x 10(3) microm2 versus control, 21.2 +/- 2.4 x 10(3) microm2, P < 0.05). In addition, PIO treatment significantly decreased the expression of EIIIA fibronectin both in the carotid neointima of Wistar rats and the media of the mesenteric artery in SHR-SP/Izm compared with their respective controls (P < 0.05). These results suggest that PIO has vasculo-protective effects in both acute and chronic vascular injury in vivo through inhibition of VSMC proliferation.

Effects of two calcium channel blockers on messenger RNA expression of endothelin-1 and nitric oxide synthase in cardiovascular tissue of hypertensive rats.

OBJECTIVE: The aim of this study was to investigate the effects of calcium channel blockers on messenger RNA expression of endothelin-1 and endothelial-type nitric oxide synthase in the cardiovascular tissue of stroke-prone spontaneously hypertensive rats (SHRSP). MATERIALS AND METHODS: The calcium channel blocker nilvadipine (1.0 or 3.2 mg/kg per day) was subcutaneously administered to two groups of SHRSP, from 4 or 8 weeks of age, for 8 weeks and 4 weeks, respectively. For comparison, nifedipine (3.2 mg/kg per day) was similarly administered to SHRSP from 4 weeks of age for 8 weeks. Kidney, heart, aorta and brain tissue samples were obtained when the rats were 12 weeks old. Messenger RNA expression of endothelin-1 and endothelial-type nitric oxide synthase was determined by reverse transcription-polymerase chain reaction followed by Southern blotting and a ribonuclease protection assay, respectively. Results were compared with those in untreated SHRSP and Wistar-Kyoto rats at 12 weeks of age. RESULTS: Both nilvadipine and nifedipine significantly decreased blood pressure in SHRSP. Although there were no changes in the weights of the kidney and brain, there was a significant decrease in the weight of the left ventricle of the groups treated with nilvadipine (1.0 mg/kg per day: mean +/- SEM 0.282 +/- 0.003 g; 3.2 mg/kg per day: 0.269 +/- 0.005 g) and nifedipine (1 mg/kg/day: 0.281 +/- 0.012 g) for 8 weeks compared with untreated SHRSP (0.301 +/- 0.004 g). Endothelin-1 messenger RNA expression, which was significantly increased by about twofold in the kidney, heart and brain of SHRSP compared with Wistar-Kyoto rats, was normalized by both calcium blockers. Endothelin-1 messenger RNA expression, which was decreased in the aorta of SHRSP, was further decreased by both calcium blockers. While there was no significant difference in endothelial-type nitric oxide synthase messenger RNA expression in the kidney, heart and aorta between the untreated SHRSP and Wistar-Kyoto rats, expression in the aorta was significantly increased in the group treated with these calcium blockers for 8 weeks from 4 weeks of age. CONCLUSIONS: These results suggest that, in addition to their potent antihypertensive effects, calcium channel blockers may exhibit cardiovasculoprotective and renoprotective effects by modifying mRNA expression of endothelin-1 and endothelial-type nitric oxide synthase in tissue.

The relatively high frequency of p53 gene mutations in multiple and malignant phaeochromocytomas.

To explore the clinical significance of p53 in the pathogenesis of adrenal neoplasms, we investigated the incidence of p53 gene mutations in functioning human adrenal tumours using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to screen p53 exons 4 to 9. We examined 29 adrenocortical adenomas (primary aldosteronism, n=17; Cushing's syndrome, n=12, all benign), and 33 phaeochromocytomas (benign solitary, n=18; benign multiple, n=5; malignant, n=10) in Japanese and Chinese patients. PCR-SSCP did not show any abnormal band-shifts in any of the adrenocortical adenoma and benign solitary phaeochromocytoma tissues. In contrast, six phaeochromocytoma tissues (two cases benign multiple, four cases malignant) showed PCR-SSCP band-shifts. Subsequent DNA sequencing analysis of the shifted bands revealed six cases with nine mutations or intronic sequence alterations: three cases contained sequence alterations within intronic regions, three cases with silent mutation (sequence alteration in codon without amino acid alteration), and three cases contained missense mutations (one case each in exons 5, 6 and 9). Immunohistochemical staining demonstrated that two of three cases with missense mutations and one case with an intronic sequence alteration over-expressed p53 protein in tumour cell nuclei. We observed no association between p53 gene mutation and p21/WAF1/Cip-1 expression. The relatively high incidence of p53 gene mutations or intronic sequence alteration in multiple and malignant phaeochromocytomas, but not in benign solitary cases, suggests that p53 mutation could play some role in the pathogenesis of multiple and/or malignant phaeochromocytomas.