Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome). We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53. We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele. Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.     

Can we use erythrocytes for the study of the activity of the ubiquitous Na+/H+ exchanger (NHE-1) in essential hypertension?

Both Na+/Li+ countertransport and electrochemical proton gradient (delta mu(H+))-induced Na+ and H+ fluxes are increased in erythrocytes from patients with essential hypertension. It was assumed that these abnormalities are related to ubiquitous (housekeeping) forms of the Na+/H+ exchanger (NHE-1). To examine this hypothesis, we compared kinetic and regulatory properties of erythrocyte Na+/Li+ countertransport and delta mu(H+)-induced Na+ and H+ fluxes with data obtained for cloned isoforms of the Na+/H+ exchanger. In human erythrocytes, Na+/Li+ countertransport exhibited a hyperbolic dependence on [Na+]0 with a K0.5 of approximately 30 to 40 mmol/L. The activity of this carrier was increased by two-fold in the fraction of erythrocytes enriched with the old cells, was inhibited by 0.1 mmol/L phloretin, and was insensitive to both 1 mmol/L amiloride and ATP depletion. In contrast, delta mu(H+)-induced 22Na influx was exponentially increased at [Na+]0 > 60 mmol/L, was insensitive to phloretin, was partly decreased by both 1 mmol/L amiloride and ATP depletion, and was the same in total erythrocytes and in the old cells. The values of Na+/Li+ countertransport and delta mu(H+)-induced Na+ influx in erythrocytes from different species were not correlating and their ratio in human, rat, and rabbit erythrocytes was 10:1:170 and 1:5:1 for Na+/ Li+ countertransport and delta mu(H+)-induced Na+ influx, respectively. In contrast to the majority of nonepithelial cells and cells transfected with an ubiquitous isoform of Na+/H+ exchanger, both delta mu(H+)-induced Na+ influx and Na+/Li+ countertransport in human erythrocytes were completely insensitive to ethylisopropyl amiloride (20 micromol/L) and cell shrinkage. Thus, our data strongly suggest that human erythrocyte Na+/Li+ countertransport and delta mu(H+)-induced Na+/H+ exchange are mediated by the distinct transporters. Moreover, because the properties of these erythrocyte transporters and NHE-1 are different, it complicates the use of erythrocytes for the identification of the mechanism for activating the ubiquitous form of Na+/H+ exchanger in primary hypertension.     

The phospholipase C inhibitor U73122 increases cytosolic calcium in MDCK cells by activating calcium influx and releasing stored calcium

The effects of the phospholipase C (PLC) inhibitor U73122 on intracellular calcium levels ([Ca2+]i) were studied in MDCK cells. U73122 elevated [Ca2+]i dose-dependently. Ca2+ influx contributed to 75% of 20 microM U73122-induced Ca2+ signals. U73122 pretreatment abolished the [Ca2+]i transients evoked by ATP and bradykinin, suggesting that U73122 inhibited PLC. The Ca2+ signals among individual cells varied considerably. The internal Ca2+ source for the U73122 response was the endoplasmic reticulum (ER) since the response was abolished by thapsigargin. The depletion of the ER Ca2+ store triggered a La3+-sensitive capacitative Ca2+ entry. Independently of the internal release and capacitative Ca2 entry, U73122 directly evoked Ca2+ influx through a La3+-insensitive pathway. The U73122 response was augmented by pretreatment of carbonylcyanide m-chlorophynylhydrozone (CCCP), but not by Na+ removal, implicating that mitochondria contributed significantly in buffering the Ca2+ signal, and that efflux via Na+/Ca2+ exchange was insignificant.     

Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations

Denaturing high performance liquid chromatography (DHPLC) has been described recently as a method for screening DNA samples for single nucleotide polymorphisms and inherited mutations. Thirty-eight DNAs, 22 of which were heterozygous for previously characterized rearranged transforming gene (RET) or cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations or polymorphisms, were examined using DHPLC analysis to assess the accuracy of this scanning method. Ninety-one per cent (20/22) of the PCR amplicons from specimens with heterozygous RET or CFTR sequence showed elution profiles distinct from corresponding homozygous normal patterns; whether the profiles for two amplicons containing heterozygous RET sequence were distinct from homozygous cases was equivocal. To investigate the usefulness of this method for detecting mutations in tumor DNAs, each of the phosphatase and tensin homologue deleted on chromosome ten gene (PTEN) exons were examined for mutations in 63 malignant gliomas. Seventeen PTEN PCR products from this series of brain tumors showed elution profiles indicating sample heterozygosity and in each instance conventional sequencing confirmed the presence of a mutation. PTEN amplicons containing exons 1, 3 and 5 were sequenced for each of the 63 tumor DNAs to determine whether any mutations may have escaped DHPLC detection, and this analysis identified one such alteration in addition to the eight mutations that DHPLC had revealed. In total, DHPLC identified 37 of 40 (92.5%) PCR products containing defined sequence variation and no alterations were indicated among 196 amplicons containing homozygous normal sequence.     

Use of LPC antagonist, choline, in the management of bronchial asthma

BACKGROUND: In the Taiwanese literature, few articles describe the pertinent features of aortic stenosis (AS). This study explores the features of AS in Chinese children. METHODS: 3808 children with congenital heart diseases have undergone cardiac catheterization at our institution over the past 19 years. Among them, 51 (1.3%) cases were AS. The clinical, electrocardiographic, echocardiographic and catheterization findings, the methods of treatment and outcomes were reviewed. RESULTS: Valvular AS occurred in 39 children (76.5%), subvalvular AS in 5 (9.8%), and supravalvular AS in 7(13.7%). Male was predominant (M/F ratio, 2.6) except in supravalvular type. Forty-three patients had associated cardiovascular defects. Aortic regurgitation (AR) was the most common one. Most patients (56.9%) were asymptomatic. Classic symptoms included exertional dyspnea (17.6%), syncope (9.8%), and chest pain (7.8%), etc. Left ventricular hypertrophy was noted in 31.2% of cases. The mean duration of follow-up was 3.9 +/- 3.4 years. Ten patients received open-heart surgery and 2 received balloon dilation. The pressure gradients across the stenotic area dropped from 95.3 +/- 29.3 to 51.4 +/- 35.8 and 53.1 +/- 12.3 mm Hg in early and late Doppler follow-up studies, respectively (p < 0.05). The average gradient increased from 36.9 +/- 25.3 to 40.8 +/- 32.6 mm Hg in nonsurgical patients. The result was insignificant. No mortality occurred following open-heart surgery. One child expired due to heart failure after the ligation of the patent ductus arteriosus and dilation of the stenotic aortic valve on the surgical table under general anesthesia. Autopsy revealed valvular rupture. In the nonsurgical group, no mortality occurred, but one patient was brought home by parents in critical condition and later died. CONCLUSIONS: We found that some clinical features of AS in Chinese children were different from those in occidental populations. (1) The incidence of AS was relatively low. (2) Subvalvular AS was the least common type in contrast to supravalvular AS in western studies. (3) Male predominance was not present in the supravalvular type, which lacked sexual proclivity. (4) Williams syndrome was a more frequently associated anomaly. Turner syndrome was not present in our study. (5) Isolated AS was less frequent. (6) The unusual finding such as right ventricular hypertrophy on EKG was present due to associated cardiac anomalies. Open-heart surgery is effective and safe, but the efficacy of balloon dilation requires further investigation.     

Problems of ecology. Technology of submerged biological treatment of sewage

A technological process is described for the submerged biological treatment of household and industrial sewage and in particular pharmaceutical industry and pooled sewage. The submerged biological treatment of the sewage to the maximum permissible concentrations adopted for the water in fish farming is achieved by the use of submerged and immobilized microorganisms in a multistage process under anaerobic and aerobic conditions and the use of chemically active and inert materials as carriers of the microorganisms inducing certain biological processes. The data on the ecological estimation of the treated sewage by the stages of the technological process are presented.     

Study of suspected plague cases for isolation and identification of Yersinia pestis

A total of 62 suspected patients of plague were investigated for evidence of Yersinia pestis, by blood culture, lymph node aspirate culture, sputum culture, animal inoculation and serology for f1 antibodies against f1 antigen of Yersinia pestis. None of the samples was positive by direct smear examination and culture for Yersinia pestis, as well as for serology. The non positivity of the cultures is discussed.     

The O-specific polysaccharide chain of Campylobacter fetus serotype B lipopolysaccharide is a D-rhamnan terminated with 3-O-methyl-D-rhamnose (D-acofriose)

An O-specific polysaccharide was liberated from Campylobacter fetus subsp. fetus serotype B lipopolysaccharide by mild acid hydrolysis followed by gel chromatography. This polysaccharide was found to contain D-rhamnose and 3-O-methyl-D-rhamnose (D-Rha3Me, D-acofriose) in a ratio of approximately 24:1, as well as lipopolysaccharide core constituents. The structure of the polysaccharide was studied by 1H-NMR and 13C-NMR spectroscopy, which included two-dimensional COSY, rotating-frame NOE spectroscopy (ROESY), and computer-assisted analysis of the 13C-NMR spectrum. Methylation analysis using [2H3]methyl iodide and Smith degradation followed by GLC/MS of the derived acetylated oligosaccharide-alditols was used to determine the location of D-acofriose. The O-specific polysaccharide is linear, consists on average of 12 disaccharide repeating units, and is terminated by a residue of D-acofriose. The following structure of the D-rhamnan chain was established: [sequence: see text]     

High-speed rotational atherectomy: six-month serial quantitative coronary angiographic follow-up

One hundred twenty-three patients treated with high-speed rotational atherectomy (HSRA) were restudied 6.9 +/- 1.2 months later. At the follow-up, the number of focal concentric lesions increased from 32.2 percent to 63.0 percent, p<0.01, with decrease of type C lesions from 54.8 percent to 30.8 percent, p<0.05. Comparison of the degree of the net gain (NG) showed more severe baseline lesions in the high-gain group (NG >20 percent) compared with the moderate-gain group (20 percent > NG > 0 percent) and to the loss group (minimal luminal diameter [MLD] 0.8 +/- 0.4 mm vs 1.0 +/ 0.4 mm, p<0.05; and 1.2 +/- 0.5 mm; p<0.01, respectively). Highest initial gain (36.5 percent +/- 26.2 percent vs 24.5 percent +/- 18.1 percent; p<0.015; and 19.0 percent +/- 23.2 percent; p<0.001) as well as lowest late loss (1.8 percent +/- 21.7 percent vs 14.0 percent +/-18.4 percent; p<0.01 and 28.1 percent +/- 25.0 percent; p<0.01) were found in the high NG group. A higher interaction between burr and atheroma resulted in the lowest restenosis rate of 6 percent.