Codon 249 mutation in exon 7 of p53 gene in plasma DNA:maybe a new early diagnostic marker of hepatocellular carcinoma in Qidong risk area,China

AIM: One of the characteristics of hepatocellular carcinoma (HCC) in Qidong area is the selective mutation resulting in a serine substitution at codon 249 of the p53 gene (1,20),and it has been identified as a 'hotspot' mutation in heptocellular carcinomas occurring in populations exposed to aflatoxin and with high prevalence of hepatitis B virus carriers (2, 3, 9, 10, 16, 24). We evaluated in this paper whether this 'hotspot' mutation could be detected in cellfree DNA circulating in plasma of patients with hepatocellular carcinoma and cirrhosis in Qidong, China, and tried to illustrate the significance of the detection of this molecular biomarker.METHODS: We collected blood samples from 25hepatocellular carcinoma patients, 20 cirrhotic patients and 30 healthy controls in Qidong area. DNA was extracted and purified from 200 μl of plasma from each sample. The 249ser p53 mutation was detected by restriction digestion analysis and direct sequencing of exon-7 PCR products.RESULTS: We found in exon 7 of p53 gene G→T transversion at the third base of codon 249 resulting 249Arg→249ser mutation in 10/25 (40%) hepatocellular carcinoma cases,4/20 (20%) cirrhotics, and 2/30 (7 %) healthy controls.The adjusted odds ratio for having the mutation was 22.1(95 % CI, 3.2～91.7) for HCC cases compared to controls.CONCLUSION: These data show that the 249ser p53mutation in plasma is strongly associated with hepatocellular carcinoma in Qidong patients. We found this mutation was also detected, although it was at a much lower frequency,in plasma DNA of Qidong cirrhotics and healthy controls;We consider that these findings, together with the usual method of HCC diagnosis, will give more information in early diagnosis of HCC, and 249ser p53 mutation should be developed to a new early diagnostic marker for HCC.

The in vitro reconstitution of nucleosome and its binding patterns with HMG1/2 and HMG14/17 proteins

Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociated from DNA at 1M NaCl. When the salt concentration was slowly reduced to 650 mMand 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic 'beads-on-a-string' structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution ofnucleosome,the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372to -194 bp) DNA sequences in the 5'flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG 14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene expression.

A Novel Method for Conversion of Valuable BDS Intermediates HPBS/Cx-HPBS from DBT/Cx-DBT

A simple recombinant PCR method was used to delete the dszB gene responsible for the slowest step of the Dsz pathway and allow the accumulation of hydroxyphenyl benzene sulfinate (HPBS) in the recombinant E. coli. Using GC/MS, HPBS accumulation was confirmed. The recombinant E. coli could also desulfurize Cx-DBT to corresponding Cx-HPBS. The result gave a new insight to BDS process and explored a new method to obtain valuable surfactants from cheap raw materials.

Somatic mutations of APC gene in carcinomas from hereditary non-polyposis colorectal cancer patients

AIM:To investigate the mutational features of adenomatous polyposis coil (APC) gene and its possible arising mechanism in hereditary non-polyposis colorectal cancers (HNPCC).METHODS: PCR-based In Vitro Synthesized Protein Test (IVSP) assay and sequencing analysis were used to confirm somatic mutations of whole APC gene in 19 HNPCC cases.RESULTS: Eleven cases with 13 mutations were determined to harbor APC mutations. The prevalence of APC mutation was 58%(11/19). The mutations consisted of 9 frameshift and 4 nonsense ones, indicating that there were more frameshift mutations (69%).The frameshift mutations all exhibited deletion or insertion of 1-2 bp and most of them (7/9) happened at simple nucleotide repeat sequences,particularly within (A)n tracts (5/9). All point mutations presented C-to-T transitions at CpG sites.CONCLUSION: Mutations of APC gene were detected in more than half of HNPCC, indicating that its mutation was a common molecular event and might play an important role in the tumorigenesis of HNPCC. Locations of frameshift mutations at simple nucleotide repeat sequences and point mutations at CpG sites suggested that many mutations probably derived from endogenous processes including mismatch repair (MMR) deficiency. Defective MMR might affect the nature of APC mutations in HNPCC and likely occur earlier than APC mutational inactivation in some patients.