Frequent activating HRAS mutations in trichilemmoma.

BACKGROUND: Trichilemmoma is a benign follicular epithelial tumour exhibiting outer root sheath differentiation. It is associated with Cowden syndrome and naevus sebaceus (NS), but the pathogenesis of sporadic tumours is poorly understood. Recently, NS was found to be caused by postzygotic HRAS or KRAS mutations. OBJECTIVES: We sought to determine whether NS-related and NS-unrelated trichilemmomas harbour RAS mutations. METHODS: Formalin-fixed and paraffin-embedded blocks of 12 NS-related and 15 NS-unrelated trichilemmomas from 26 individuals were retrieved and analysed to determine the presence of mutations in exons 1 and 2 of the HRAS, KRAS and NRAS genes by polymerase chain reaction and direct sequencing. Mutational hotspots of the FGFR3 and PIK3CA genes were also analysed for NS-unrelated cases. RESULTS: Among the 27 cases, mutually exclusive HRAS c.37G>C and c.182A>G mutations were observed in 17 and three tumours, respectively. Of the 12 NS-related tumours, 11 (92%) harboured the HRAS c.37G>C substitution. Of the 15 sporadic tumours, nine (60%) harboured HRAS mutations, including six c.37G>C and three c.182A>G. An HRAS c.182A>G mutation was observed only in sporadic tumours. No mutations were observed in the other genes that were tested. CONCLUSIONS: The high frequency of HRAS activating mutations, including the c.182A>G substitution, which was rather rare in NS, suggests that most trichilemmomas are authentic neoplasms.

Effects of a Chinese medical herbs complex on cellular immunity and toxicity-related conditions of breast cancer patients.

Rose geranium (Pelargonium graveolens, Geraniaceae) has anti-cancer and anti-inflammatory properties, and promotes wound healing. Similarly, Ganoderma tsugae (Ganodermataceae), Codonopsis pilosula (Campanulaceae) and Angelica sinensis (Apiaceae) are traditional Chinese herbs associated with immunomodulatory functions. In the present study, a randomised, double-blind, placebo-controlled study was conducted to examine whether the Chinese medicinal herb complex, RG-CMH, which represents a mixture of rose geranium and extracts of G. tsugae, C. pilosula and A. sinensis, can improve the immune cell count of cancer patients receiving chemotherapy and/or radiotherapy to prevent leucopenia and immune impairment that usually occurs during cancer therapy. A total of fifty-eight breast cancer patients who received chemotherapy or radiotherapy were enrolled. Immune cell levels in patient serum were determined before, and following, 6 weeks of cancer treatment for patients receiving either an RG-CMH or a placebo. Administration of RG-CMH was associated with a significant reduction in levels of leucocytes from 31·5 % for the placebo group to 13·4 % for the RG-CMH group. Similarly, levels of neutrophils significantly decreased from 35·6 % for the placebo group to 11·0 % for the RG-CMH group. RG-CMH intervention was also associated with a decrease in levels of T cells, helper T cells, cytotoxic T cells and natural killer cells compared with the placebo group. However, these differences between the two groups were not statistically significant. In conclusion, administration of RG-CMH to patients receiving chemotherapy/radiotherapy may have the capacity to delay, or ease, the reduction in levels of leucocytes and neutrophils that are experienced by patients during cancer treatment.

Space charge effects in field emission nanodevices.

Electron field emission from a single nanoemitter is a barrier tunneling, quantum mechanical process that can, therefore, be described by the well-known Fowler-Nordheim (FN) equation. At high emission current densities, however, the space charge caused by the cathode may affect the current density-voltage (J-V) characteristics predicted by the FN theory. In this study, we theoretically investigated the effect of space charge on FE nanodevices, including diode and triode structures. The J-V characteristics of FE nanodevices were obtained by analytically (diode structures) or numerically (triode structures) solving the coupled FN equation and Poisson's equation. We discuss the behavior of FE nanodiodes and nanotriodes displaying different geometries, dimensions and work functions of their emitter materials. In the high current density region, space charge plays an important role in FE nanodevices; the threshold current density of space-charge limitation is related to the electric field distributions. Besides, our theoretical results are in good agreement with the experimental results reported previously.

Effects of silymarin on the resolution of liver fibrosis induced by carbon tetrachloride in rats.

Silymarin, a standardized extract of the milk thistle (Silybum marianum), has a long tradition as a herbal remedy, and was introduced as a hepatoprotective agent a few years ago. However, the therapeutic effects of silymarin remain undefined. Carbon tetrachloride (CCl4) is a xenobiotic used extensively to induce oxidative stress and is one of the most widely used hepatic toxins for experimental induction of liver fibrosis in the laboratory. In this study, we investigated the restoration of the CCl4-induced hepatic fibrosis by high dose of silymarin in rats. After treatment with oil (as normal group; n = 6) or CCl4 [as model (n = 7) and therapeutic (n = 7) groups] by intragastric delivery for 8 weeks for the induction of liver fibrosis, the rats in the normal and model group were administered orally normal saline four times a week for 3 weeks whilst the therapeutic group received silymarin (200 mg/kg). The histopathological changes were observed with Masson staining. The results showed that the restoration of the CCl4-induced damage of liver fibrosis in the therapeutic group was significantly increased as compared to that in the model group. Moreover, silymarin significantly decreased the elevation of aspartate aminotransferase (AST), alanine aminotransferase, and alkaline phosphatase in serum, and also reversed the altered expressions of alpha-smooth muscle actin in liver tissue. Therefore, these findings indicated that silymarin may have the potential to increase the resolution of the CCl4-induced liver fibrosis in rats.

Synthesis design of artificial magnetic metamaterials using a genetic algorithm.

In this article, we present a genetic algorithm (GA) as one branch of artificial intelligence (AI) for the optimization-design of the artificial magnetic metamaterial whose structure is automatically generated by computer through the filling element methodology. A representative design example, metamaterials with permeability of negative unity, is investigated and the optimized structures found by the GA are presented. It is also demonstrated that our approach is effective for the synthesis of functional magnetic and electric metamaterials with optimal structures. This GA-based optimization-design technique shows great versatility and applicability in the design of functional metamaterials.

Biology and molecular biology of viruses in the genus Tenuivirus.

Viruses in the genus Tenuivirus (Tenuiviruses) cause a number of important diseases in economically important crop plants including rice and maize. Tenuiviruses are transmitted from plant to plant by specific planthopper vectors, and their transmission relationship is circulative-propagative. Thus, Tenuiviruses have host ranges including plants and animals (planthoppers). Four or five characteristic, circular ribonucleoprotein particles (RNPs), each containing a single Tenuivirus genomic RNA, can be isolated from Tenuivirus-infected plants. The genomic RNAs range in size from ca 9.0 kb to 1.3 kb and together give a total genome size of ca 18-19 kb. The genomic RNAs are either negative-sense or ambisense, and expression of the ambisense RNAs utilizes cap-snatching during mRNA transcription. The combination of characteristics exhibited by Tenuiviruses are quite different than those found for most plant viruses and are more similar to vertebrate-infecting viruses in the genus Phlebovirus of the Bunyaviridae.

Elevation of transforming growth factor alpha and its relationship to the epidermal growth factor and alpha-fetoprotein levels in patients with hepatocellular carcinoma.

A radioimmunoassay for transforming growth factor alpha (TGF-alpha) using synthetic rat sarcoma transforming growth factor and its rabbit polyclonal antibody has been developed. Using radioimmunoassays, the urinary TGF-alpha and epidermal growth factor (EGF) concentrations in 31 patients with hepatocellular carcinoma (HCC), 15 probable HCC, four metastatic liver cancer, and 33 age, sex-matched healthy controls were determined. For the first time, we have shown that the average TGF-alpha concentration for HCC patients was 21.5 +/- 20.3 micrograms per g creatinine, significantly higher than that of healthy subjects, 4.9 +/- 2.8 micrograms per g creatinine (P less than 0.001). There was no statistical difference in the level of EGF between HCC patients and controls (40.9 +/- 29.3 versus 46.2 +/- 16.6 micrograms per g creatinine; P greater than 0.05). The ratio of EGF/TGF-alpha between HCC patients (3.37 +/- 4.42) and controls (15.5 +/- 13.0) was significantly different (P less than 0.001). Among patients, 65% (20 of 31) of HCC cases and 87% (13 of 15) of probable HCC cases showed a marked elevation of TGF-alpha levels. We found only 16% (five of 31) of HCC cases with increased EGF level. EGF excretion was inversely age related. Serum total protein concentration and alkaline phosphatase activity were positively correlated to EGF concentration (r = 0.522, P less than 0.01 and rt = 0.393, P less than 0.05, respectively). There was no correlation between biochemical functions of liver and TGF-alpha concentration in HCC patients. Our results also suggested that TGF-alpha may be a useful complementary tumor marker for management of patients with clinical manifestation of HCC who have low alpha-fetoprotein levels.

Increased urinary endothelin-1-like immunoreactivity excretion in NIDDM patients with albuminuria.

OBJECTIVE: To determine the urinary endothelin-1-like immunoreactivity (ET-1-LI) in non-insulin-dependent diabetes mellitus (NIDDM) patients and to investigate whether urinary ET-1-LI excretion is elevated in patients with albuminuria. RESEARCH DESIGN AND METHODS: Urinary ET-1-LI substance was determined by radioimmunoassay in 45 normoalbuminuric and 28 albuminuric NIDDM patients. RESULTS: The mean amounts of 24-h ET-1-LI excretion in NIDDM patients with normoalbuminuria and albuminuria were 154.9 +/- 13.5 and 174.4 +/- 12.9 ng/day, respectively. The latter was significantly higher than that of the 40 normal control subjects (111.8 +/- 7.9 ng/day, P < 0.01). CONCLUSIONS: The increase in urinary ET-1-LI substance excretion in patients with albuminuria suggests that ET-1 may be a pathogenetic factor in diabetic nephropathy.

Increased urinary atrial natriuretic peptide-like immunoreactivity excretion but decreased plasma atrial natriuretic peptide concentration in patients with hyperosmolar-hyperglycemic nonketotic syndrome.

OBJECTIVE: This study was undertaken to measure urinary atrial natriuretic peptide-like immunoreactivity (ANP-LI) and plasma ANP concentration in patients with hyperosmolar-hyperglycemic nonketotic syndrome (HHNS) to investigate the change of renal ANP-LI and cardiac ANP synthesis in volume-depleted diabetic patients. RESEARCH DESIGN AND METHODS: The urine ANP-LI:creatinine ratio, plasma ANP level, and plasma renin activity (PRA) were measured in 12 patients with HHNS during the acute stage and after recovery, in 28 oral hypoglycemic agent (OHA)-treated type 2 diabetic patients, and in 23 normal subjects. ANP and PRA were measured by radioimmunoassay. RESULTS: These HHNS patients had severe hyperglycemia and hyperosmolality as well as increased blood urea nitrogen, creatinine, and PRA levels, as compared with normal subjects and OHA-treated type 2 diabetic patients. In these patients, the urinary ANP-LI:creatinine ratio (11.69 +/- 2.11 pmol/mmol) was significantly increased in comparison with the normal group (1.78 +/- 0.11 pmol/mmol) and OHA-treated diabetic patients (2.43 +/- 0.45 pmol/mmol), whereas plasma ANP concentration (5.12 +/- 0.72 pmol/l) was significantly lower than the corresponding values of the normal group (7.39 +/- 0.85 pmol/l) and OHA-treated diabetic patients (8.43 +/- 1.05 pmol/l). All of these abnormalities were significantly ameliorated after insulin, fluid, and electrolyte replacement. CONCLUSIONS: Our data show that urinary ANP-LI was significantly increased, whereas plasma ANP concentration was decreased, in the face of raised PRA in HHNS patients. This study indicates that renal ANP-LI substances and cardiac ANP may exhibit different responsiveness in diabetic patients with HHNS.

Up-regulation of adrenal cortical and medullary atrial natriuretic peptide and gene expression in rats with deoxycorticosterone acetate-salt treatment.

Our previous study demonstrated that human adrenal medulla is a site of atrial natriuretic peptide (ANP) synthesis. To further evaluate the role of adrenal ANP in body fluid homeostasis, we investigated the changes in adrenal ANP in rats receiving deoxycorticosterone acetate (DOCA)-salt treatment. In situ hybridization and immunohistochemical study showed that adrenal ANP messenger RNA (mRNA) and ANP-like immunoreactivities (ANP-LI) were mainly localized in the zona glomerulosa and medulla of vehicle-treated rats. DOCA-salt treatment activated ANP mRNA and peptide expression in all adrenal zones, especially in the zona fasciculata/reticularis from 12 h to the entire 8-day study period. Using a semiquantitative RT-PCR technique, the relative quantities of ANP mRNA in the adrenals of the DOCA-salt-treated group were significantly increased from 1 to 8 days, whereas the adrenal weights of DOCA-salt-treated rats were significantly decreased from day 2 to day 8. Our results are the first to indicate that ANP is synthesized not only in the adrenal medulla but also in the adrenal cortex and their syntheses are markedly increased in DOCA-salt-treated rats. These results imply that adrenal ANP may participate in the intraadrenal regulation of adrenal function on water-electrolyte homeostasis in an autocrine or paracrine manner.

Mutations of the p53 gene in human functional adrenal neoplasms.

To clarify gene alterations in functional human adrenal tumors, we performed molecular analysis for p53 abnormalities in 23 cases with adrenal neoplasms. The immunohistochemical study with anti-p53 monoclonal antibody pAb1801 demonstrated that 10 of 23 (43.5%) cases overexpressed p53 protein in the tumor cells. Using a polymerase chain reaction-single strand conformation polymorphism study, 5 of 6 (83.3%) pheochromocytoma tissues (1 malignant and 5 benign) and 11 of 15 (73.3%) adrenocortical adenomas (2 with Cushing's syndrome and 13 with primary aldosteronism, all benign) showed an apparent electrophoretic mobility shift between the tumor and its paired adjacent normal adrenal tissue. Such differences were detected in exon 4 (12 cases), exon 5 (2 cases), and exon 7 (3 cases). The types of these mutations in exon 4 were a substitution from threonine (ACC) to isoleucine (ATC) at codon 102 in 5 cases, from glutamine (CAG) to histidine (CAC) at codon 104 in 1 case, from glycine (GGG) to alanine (CGG) at codon 117 in 1 case, from glutamate (GAG) to glutamine (CAG) at codon 68 in 1 case, and single base changes resulting in a premature stop codon at codon 100 in 2 cases. A 2-basepair deletion at codon 175 in exon 5 resulting in a frame shift was identified in 1 case. A single point mutation was identified, resulting in the substitution of glutamine (CAG) for arginine (CGG) at codon 248 of exon 7 in 1 case. A single basepair deletion at codon 249 resulted in a frame shift in 2 cases. There was 1 case with malignant pheochromocytoma that combined a single point mutation in exon 4 and a single base deletion in exon 7. Only 2 of 23 cases showed a loss of a normal allele encoding in the p53 gene. Northern blot analysis with 1.8-kilobase p53 cDNA revealed that p53 mRNA was overexpressed in 6 cases. Our results indicate that high frequencies of p53 gene mutation, especially in exon 4, exist in functional adrenal tumors. As p53 protein is a regulator of guanine nucleotide synthesis, the loss of normal inhibitory regulation by the p53 mutation would serve to increase the availability of GTP for the transduction of signals essential for increased cell growth and hormone expression in the adrenal tumors. These findings suggest that the p53 gene mutation may play a role in the tumorigenesis of benign and functional human adrenal tumors.

Increased adrenal medullary atrial natriuretic polypeptide synthesis in patients with primary aldosteronism.

The present study was designed to determine whether atrial natriuretic polypeptide (ANP) is synthesized in the human adrenal gland and, if so, to investigate the ANP content of adrenal tissue and the ANP mRNA changes in patients with primary aldosteronism. A considerable amount of human alpha ANP-like immunoreactive substances was extracted from the remnant adrenal glands of three patients with primary aldosteronism (1.44, 1.0, and 0.77 pmol/g wet tissue; mean +/- SD, 1.07 +/- 0.28 pmol/g) and the adrenal glands of three kidney donors for transplantation (0.93, 0.58, and 0.27 pmol/g wet tissue; mean +/- SD, 0.59 +/- 0.27 pmol/g). High performance gel permeation chromatographic analysis coupled with a RIA of the tissue extract showed that the molecular form of ANP in the adrenal gland was the precursor form, i.e. human gamma ANP. An in situ hybridization study using an ANP cRNA probe indicated that the ANP mRNA was localized mainly in the medullary area of the gland. Northern blot analysis, using ANP cDNA as a probe, detected ANP mRNA in the adrenal gland. Furthermore, the level of ANP mRNA in the adrenal glands of patients with primary aldosteronism was obviously elevated compared to that in the kidney donors. Our results were the first to indicate that ANP is synthesized in the human adrenal medulla, and such medullary ANP synthesis increases in patients with hypermineralocorticoidism. These facts support the proposal that extraatrial (medullary) ANP synthesis might act in a paracrine or endocrine manner to regulate water and electrolyte homeostasis.

Brain natriuretic peptide is synthesized in the human adrenal medulla and its messenger ribonucleic acid expression along with that of atrial natriuretic peptide are enhanced in patients with primary aldosteronism.

The present study was designed to determine whether brain natriuretic peptide (BNP) is synthesized in the human adrenal gland and, if so, to investigate the BNP content of adrenal tissue and the changes in BNP messenger ribonucleic acid (mRNA) in patients with primary aldosteronism. A considerable amount of BNP-like immunoreactive substances was extracted from the adrenal glands of kidney donors for transplantation (0.21 +/- 0.02 pmol/g wet tissue; n = 3) and the remnant nontumorous adrenal glands of patients with primary aldosteronism (0.20 +/- 0.05 pmol/g wet tissue; n = 3; mean +/- SEM). Immunohistochemical study with a specific antihuman BNP antibody revealed that BNP-like immunoreactivity was localized in the adrenal medullary area, and an in situ hybridization study indicated that the BNP mRNA was mainly expressed in the cells of adrenal medulla. Using a reverse transcription and polymerase chain reaction technique, BNP complementary DNA was cloned from the human adrenal gland, and the sequence was identical to that of BNP identified in the atria. The level of BNP mRNA in the adrenal glands of patients with primary aldosteronism (n = 4) was obviously elevated compared to that in the kidney donors (n = 4), as determined by Northern blot analysis. Quantitative polymerase chain reaction measurements of BNP and atrial natriuretic peptide (ANP) mRNAs showed that both of the adrenomedullary natriuretic peptide gene transcriptions were enhanced in patients with primary aldosteronism, but the amount of ANP mRNA was far higher than that of BNP mRNA in the human adrenal gland. Our results are the first to indicate that BNP is synthesized in the human adrenal medulla, and that such medullary BNP synthesis increases in patients with primary aldosteronism. These facts support the proposal that adrenomedullary BNP along with ANP may play some role in water and electrolyte homeostasis or act in a paracrine manner to regulate adrenocortical functions.

Clinical relevance of transforming growth factor-beta 1 in the urine of patients with hepatocellular carcinoma.

To assess the clinical relevance of transforming growth factor-beta 1 (TGF-beta 1) in the urine of patients with hepatocellular carcinoma (HCC), TGF-beta 1 was measured, by radioimmunoassay, in 140 patients with HCC, 50 cirrhotic patients, 30 patients with chronic active hepatitis, and 50 healthy controls. The results indicate that there were significantly increased urinary TGF-beta 1 levels in patients with HCC. Raised TGF-beta 1 levels were associated, in a dose-related fashion, with increased risk for development of HCC (odds ratio, 1.05, 95% confidence interval, 1.03-1.07). HCC patients with raised TGF-beta 1 levels had shorter survival than those with normal TGF-beta 1 levels (p = 0.038). TGF-beta 1 levels decreased after successful anticancer therapy (p < 0.0001). There was an inverse correlation between TGF-beta 1 and serum alpha-fetoprotein (AFP) (r = -0.199, p < 0.04). Receiver operating characteristics (ROC) curve analysis indicated that parallel determination of TGF-beta 1 and AFP significantly increased the sensitivity and diagnostic accuracy, with a high specificity. In conclusion, raised urinary TGF-beta 1 was associated with HCC development. It is a predictor of poor prognosis, and a tumor marker for diagnosis and therapeutic follow-up of HCC.

Increased IgM-containing circulating immune complexes in patients co-infected with hepatitis C and hepatitis B.

To assess the role of circulating immune complexes (CIC) in chronic hepatitis C virus (HCV) infection, the relative frequency of CIC was determined in 60 patients with chronic hepatitis C alone, 19 patients co-infected with hepatitis B and C, 15 asymptomatic HCV carriers, and 54 healthy controls. Levels of CIC were determined with immunoglobulin-specific C1q-binding and conglutinin (K)-binding immune complex assays. Although there was no statistical difference in the levels of each type of CIC between asymptomatic HCV carriers and healthy controls, elevated levels of CIC containing IgM and IgG were common in patients with chronic HCV infection. Compared to patients with hepatitis C alone, patients co-infected with hepatitis B and C have a higher frequency of abnormal IgM-containing CIC and significantly higher levels of IgM-containing CIC. CIC levels in patients with chronic active hepatitis were significantly higher than those in patients with chronic lobular hepatitis or chronic persistent hepatitis. In conclusion, although CIC containing IgM and IgG were common in patients with chronic hepatitis C, abnormal IgM-containing CIC are the major types of CIC in patients co-infected with hepatitis B and C. An immune-mediated mechanism may play a role in the pathogenesis of chronic hepatitis C.

Alteration in the expression of protein kinase C isoforms in human hepatocellular carcinoma.

This study was designed to investigate the alterations of individual protein kinase C (PKC) isoforms in human liver cancer. Surgical specimens of hepatocellular carcinoma and adjacent normal tissues were extracted into cytosolic and membranous fractions. The level of membrane-bound PKCalpha in the cancer tissue was significantly lower than that in the adjacent normal tissue and consistent with the change in PKC activity. In addition, there was a significant negative correlation between PKCalpha and tumor size. In both cytosolic and membrane fractions, levels of PKCdelta and PKCzeta was significantly higher in the cancer tissue than those in the adjacent normal liver tissue. The alterations in the PKC isoforms signify their roles in the hyperproliferation in liver cancer.

Increased C-type natriuretic peptide mRNA expression in the kidney of diabetic rats.

To investigate the responsiveness of renal-synthesized C-type natriuretic peptide (CNP) to changes in water and electrolyte balance, we measured renal CNP mRNA levels, plasma CNP concentrations and urinary CNP excretion rates in streptozotocin-induced diabetic rats eating a normal (0.26% NaCl) or low (0.04% NaCl) salt diet. Using reverse transcription-PCR followed by Southern blot analysis, we found that renal cortical and medullary CNP mRNA levels were markedly enhanced in diabetic rats from the 14th day and remained enhanced with an accompanying elevation of urinary CNP excretion rates for the entire 60-day study period. All increases of renal CNP mRNA and urinary CNP excretion rates in diabetic rats were attenuated in low salt diet-treated diabetic rats as well as insulin-treated diabetic rats. These results demonstrate that renal CNP synthesis is enhanced in diabetic rats and the increase of renal CNP mRNA is ameliorated by salt restriction and insulin treatment. These results imply that renal-synthesized CNP is responsive to the alteration of water and electrolyte homeostasis in diabetic rats.

Fatal outcome after ingestion of star fruit (Averrhoa carambola) in uremic patients.

Clinical outcome of dialysis patients after eating star fruit (Averrhoa carambola) varies, but it may be fatal. In the past 10 years, 20 such patients were treated in our hospital when they developed clinical symptoms after eating the fruit or drinking star fruit juice. Their initial presentations included sudden-onset limb numbness, muscle weakness, intractable hiccups, consciousness disturbance of various degrees, and seizure. No other major events that might be responsible for these symptoms could be identified. Eight patients died, including one patient with a serum creatinine level of 6.4 mg/dL who had not yet begun dialysis. The clinical manifestations of the survivors were similar to those who died except for consciousness disturbance and seizure. Death occurred within 5 days despite emergent hemodialysis and intensive medical care. The survivors' symptoms usually became less severe after supportive treatment, and these patients subsequently recovered without obvious sequelae. The purpose of this article is to report that patients with renal failure who ingest star fruit may develop neurological symptoms and also run the risk for death in severe cases. Mortality may also occur in patients with chronic renal failure not yet undergoing dialysis.

Circadian rhythm of urinary endothelin-1 excretion in mild hypertensive patients.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide with diverse physiologic actions and has been considered to be involved in the pathogenesis of hypertension. We sought to investigate the role of renal synthesis of ET-1 in the regulation of daily sodium homeostasis and the possible contribution of renal synthesized ET-1 in the pathogenesis of essential hypertension (EHT). Urinary ET-1-like immunoreactivity (ET-1-L1) was measured by a radioimmunoassay after extraction in 23 EHT patients without detectable target organ damage, and in 11 normotensive controls. All study subjects received a controlled diet during an 8-day study period. Urinary and blood samples were collected by four sampling periods/day from the 4th to 6th days, and on the 7th day, study subjects were given an intravenous infusion of 1250 mL normal saline over 2 h. In the basal state, the urinary sodium and ET-1-L1 excretions showed diurnal patterns in both the normal and hypertensive groups, and urinary ET-1-L1 excretion rate correlated well with urinary sodium excretion rate. There were no differences found in plasma ET-1 levels, urinary ET-1-L1, and sodium excretion rates between the control and hypertensive groups. After saline infusion, ten hypertensive patients showed nonexaggerated natriuresis, and the 24-h urinary ET-1-L1 excretion (47.0+/-4.0 pmol/day), collected during the day of saline infusion, was significantly lower than that of the control group (86.3+/-10.0 pmol/day) or the exaggeratedly natriuretic hypertensive patients (91.7+/-8.4 pmol/ day). Our results suggest that renal ET-1 may be responsible for the renal handling of sodium homeostasis, and alteration of renal ET-1 synthesis may be a contributory factor in the pathogenesis of essential hypertension and salt sensitivity.

A significant decrease of the transcriptional activity of p53 mutants deriving from human functional adrenal tumors.

Recently, our laboratory has found a high incidence (77%) of p53 gene mutations in human functional adrenal tumors. Furthermore, the majority of mutant sites were assembled at codons 100, 102, and 249. These mutation sites are not common, and there have been no studies addressing whether or not these mutants points or mutant styles cause the p53 protein to lose function. It has been well known that p53 is a transcription factor. To examine the transcriptional activities of these mutant p53 genes from patients with functional adrenal tumors, we constructed p53 expression plasmids from tumors and paired adjacent normal adrenal gland tissues, using a transient co-transfection assay with a reporter gene in H358 cells. Wild-type p53 from normal adrenal gland tissues specifically trans-activates the expression of a chloramphenicol acetyltransferase (CAT) reporter gene in H358 cells. Three mutant p53 proteins (at codons 100, 102, and 249, respectively) from tumors showed a >90% loss of transcriptional activity. One mutant at codon 68, other than at hot spots, remained at approximately 65% transcriptional activity. An immunoprecipitation assay showed that the mutant proteins of codon 68 and codon 102 could respond to the three monoclonal antibodies (PAbDO-1, PAb1620, and PAb421), indicating that there were no obvious changes in the antigenicity of the proteins. However, the mutant protein of codon 249 could not respond to the carboxy-terminus-specific antibody PAb421 and conformation-specific antibody PAb1620, indicating that there were some obvious changes in the conformation of the mutant proteins. The mutant protein of codon 100 could not be detected by immunoprecipitation assay but could be analyzed by Western blot. In a further study using a DNA-binding assay, it was shown that the loss of transcriptional activity was caused by the loss of DNA-binding ability. These results show that the p53 mutants, derived from functional adrenal tumors, actually lost DNA-binding ability and decreased the transcriptional activity. However, the role of the mutant protein in the tumorigenesis of functional adrenal tumors requires further investigation.