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Biomedical subjects

J R Gu

Publications and source records attributed to J R Gu.

36 records · Page 2Linked to original sources
PubMed1991

Strain analysis of hepatitis B virus on the basis of restriction endonuclease analysis of polymerase chain reaction products.

To assess the value of classifying hepatitis B virus (HBV) strains at the genomic level with products from the polymerase chain reaction (PCR), an HBV PCR DNA typing procedure was developed. The design of this method was based on the selective sensitivity of the PCR product to digestion with different restriction endonucleases and on the size of the fragment resulting from a specific nuclease digestion. On the basis of published nucleotide sequences of different HBV subtypes, a set of primers was selected within the preS-S region. One of the primers was 1679F containing 25 nucleotides (5'-GGGTGGAGCCCTCAGGCTCAGGGCA-3'), and the other was 2254R containing 24 nucleotides (5'-GAAGATGAGGCATAGCAGCAGGAT-3'). All of the reactive sera produced the same sized 575-bp identification band. The product was subjected to digestion by a selected panel of restriction endonucleases. By using prototype HBV of known subtype as a model, these restriction nuclease maps of the PCR product were subtype specific. Most adr subtype clinical samples produced predicted PCR DNA restriction nuclease fragmentation in accordance with the nucleotide sequence of the prototype virus. Occasionally, samples of either the adw or the ayw subtype gave discrepant results in which they appeared to be a different subtype or were resistant to the restriction nuclease digestion. This genetic alteration was consistent in the paired specimens from sexual transmission cases. These results show that the described procedures are applicable to HBV strain assessment.

Base Sequence↗
PubMed1991

Transthyretin (prealbumin) gene in human primary hepatic cancer.

From a subtracting cDNA library constructed from normal liver versus human primary hepatic cancer (PHC) a cDNA clone pG8 was isolated. Using it as a probe, RNA extracted from one human liver and 9 PHC samples were analyzed by Northern hybridization. As expected, its mRNA was highly expressed in liver; however, the expression was strikingly suppressed in PHC. Only weak signal was observed in 2 out of 9 PHC, while no signal was detectable in the other 7 samples. Utilizing pG8 as a probe, DNA from the same PHC specimens was analyzed after MspI digestion and Southern hybridization. Deletion of DNA fragment was observed in 4 out of 9 samples. In further study of cancer and non-cancerous liver from other 7 PHC patients, similar deletion of DNA fragments in cancer was observed in 4 out of 7 samples. After sequencing of the clone of 572 bp, it was unexpectedly found that pG8 was completely homologous to the coding sequence of transthyretin, TTR gene, as TTR (or prealbumin) gene has been known to be linked to a hereditary disorder, familial amyloidosis (FAP), and related to thyroxine transport and binding to retinol-RBP (the retinol binding protein) complex. This is the first report of a study on TTR in human primary hepatic cancer. Since TTR gene was strikingly suppressed in mRNA expression and possibly defective in its gene structure, it was strongly implicated that TTR might be an important gene marker or a candidate of anti-oncogene for human PHC. The biological activity of TTR gene is under study.

Base Sequence↗
PubMed1990

Enhanced expression of ganglioside GD3 in human and rat hepatocellular carcinoma cells and NIH 3T3 cells transfected with human tumor DNAs.

The gangliosides of human hepatoma biopsies, human hepatoma cell lines, and diethylnitrosamine-induced rat hepatomas were examined. These malignant tissues all expressed increased content of disialolactosylceramide (GD3) with respect to their normal counterparts. During the induction of rat hepatoma by diethylnitrosamine, an increase in GD3 levels appeared as early as 12 wk after initiation of diethylnitrosamine, concurrent with the appearance of precancerous hepatocytes. GD3 levels gradually increased to a peak of 4 times that of normal rat liver at 20 wk. CMP-NeuAc:GM3 sialyltransferase, the enzyme that synthesizes GD3 by transfer of sialic acid to GM3, also had tumor-associated elevation during the course of diethylnitrosamine-induction of rat hepatomas. To investigate the relationship of oncogene transformation and changes in ganglioside biosynthesis, NIH 3T3 cells transfected DNAs from human hepatoma or nasopharyngeal carcinoma were studied. The transfectants each expressed the same ganglioside composition, including a detectable level of GD3, as well as enhanced activity of CMP-NeuAc:GM3 sialyltransferase. A correlation between the tumor DNA transfection and the augmentation of GD3 in malignant cells is discussed. Because of the early appearance of GD3 in hepatoma and its possible relationship to oncogene activation, GD3 may be a potentially useful early tumor marker.

Animals↗
PubMed1990

Subtraction hybridization cDNA libraries from colon carcinoma and hepatic cancer.

cDNA clones of differentially expressed mRNAs in a colon carcinoma and a hepatocellular carcinoma have been isolated by subtractive cDNA cloning. The subtracted material is at least 90 X enriched for differentially expressed sequences and can be used for construction of subtractive cDNA libraries and polymerase chain reaction (PCR) amplification to generate differential probes. Commercially available lambda ZAP II is used for construction of primary libraries since single-stranded phage bearing the cloned cDNA can be excised in vivo and because lambda libraries are convenient for subsequent screening and manipulations. Rare mRNAs (less than 0.01% abundance), which are differentially expressed, can be isolated utilizing this procedure.

Bacteriophage lambda↗
PubMed1988

Transforming genes in duck hepatic carcinoma--mht(raf) and Ha-ras.

The transfection of NIH 3T3 cells was performed with DNAs from 2 duck primary hepatic carcinomas (DHC 40K, 9K) and 1 tumor-adjacent liver tissue (TAL, 9N). Transfectants were found from 40K, 9K and 9N DNAs. The secondary transfectants were obtained after transfection of RAT-1 cells with DNAs from primary transfectants. After hybridization with Ha-ras, Ki-ras, N-ras and mht oncogenes, it was found that duck mht (5.2 and 3.2 kb EcoRI fragments) and duck Ha-ras (3.4 kb EcoRI fragments) were present in all these transformants. This is the first report on transforming genes in duck primary hepatic cancer as well as tumor-adjacent liver tissue.

Animals↗
PubMed1987

[Localization of oncoprotein P21ras in the human liver cancer].

Using the human liver cancer DNA transfected NIH/3T3 cell line, the human N-ras oncogene and the over expression of the oncoprotein P21ras was demonstrated, BALB/C mice were immunized. The spleen cells from the immunized mice were fused with SP2/0 myeloma cells. After the HAT medium selection and screening, two hybridoma cell lines, SCI-Oncogema 1 and 2, were established. In the immunoprecipitation test, the molecular weight of the protein reacting to Oncogema 1 was 21,000. This M.W 21,000 protein possessed the capability to bind with GTP, i.e. the character of P21ras. These data indicate that the Oncogema 1 is the monoclonal antibody against P21ras. Using Oncogema 1, specimens from 6 liver cancer patients were studied by immunopathology. With ABC stain, it was observed that the malignant cells in all the samples showed dark staining; the P21ras revealed over expression. Although the staining was heterogeneous, it implied that the ras oncogene was involved in the carcinogenesis of these six samples. No over expression was seen in the normal liver cells even in those around the cancerous lesion. However, dysplastic cells were moderately stained which means that the ras oncogene was activated and P21ras over expressed in these cells. The results suggest that the ras oncogene and P21ras play an important role in the early stage of liver cancer carcinogenesis.

Animals↗
PubMed1986

Oncogenes in human primary hepatic cancer.

Transfection assay of NIH 3T3 cells was performed with DNAs isolated from ten human PHC (primary hepatic cancer) specimens and a hepatoma 7402 cell line. Positive results were obtained in 7402 and in six out of ten PHC DNAs. Human N-ras gene was identified in transfectants from 7402 DNA and transformed cells from three PHC DNA samples, which had passed more than two cycles of transfection. The expression of N-ras was also remarkably enhanced in six out of nine poly(A)+RNA samples isolated from PHC tissues. P21 synthesis was elevated in 7402 cells as well as in transfectants derived from 7402 cells and PHC DNA. In analysis of PHC DNA, rearrangement and amplification of N-ras gene was observed in two PHC samples. The discrepancy of results of the transfection assay and mRNA expression was discussed. Furthermore, c-myc was also highly expressed in most PHC tissues. It implied that the cooperating activity of N-ras and c-myc might be responsible for the malignant phenotypic alteration in some or most cases in human primary liver cancer.

Autoradiography↗
PubMed1986

Transforming activity of human nasopharyngeal carcinoma DNA.

NIH 3T3 cells were transfected with the DNAs from biopsy specimens of human nasopharyngeal carcinoma (NPC, EBV DNA positive) using calcium phosphate precipitation method. The malignant, transformed foci of NIH 3T3 cells have been observed and cloned. The hybridization of transfectant DNA digested by EcoRI with total human leukocyte DNA as probe was performed. The strong signal of smear comparing with NIH 3T3 DNA as control was observed. It was implied that the putative human transforming sequences had been integrated into transformed cells. Employing soft agar culture, the transformed cells can grow and form cell colonies. Following transfer, the foci were able to grow and adhere to a glass wall. These cells were easily agglutinated by con A. The cloned foci have been inoculated into nude mice with the formation of highly malignant sarcomas. In preliminary experiments for characterizing the transforming sequences, Ha-ras and Blym 1 were found in transfectants derived from one of the NPC DNA samples. It is implicated that these two oncogenes might be responsible for the acquisition of malignant phenotypic character of some human NPC. The further identification of oncogenes in NPC is currently in progress.

Animals↗
PubMed1986

Identification of human N-ras as the common oncogene in NIH 3T3 cells transformed with DNAs from human primary hepatic cancer and hepatoma 7402 line.

DNA was extracted from NIH 3T3 cells transformed with DNAs from human primary hepatic cancer (PHC) and Hepatoma 7402 cell line. The transformant DNA was analyzed by Southern transfer and hybridization with 32P-labeled probes of various oncogenes. The EcoRI 7.2 and 9.0 kb bands characteristic of human N-ras gene were identified in transformed NIH 3T3 cells derived both from PHC and 7402 DNA. The BamHI 6.6 kb band characteristic of human c-Ha-ras I was present only in 7402 transformants, but not in PHC transformants. Using 35S-methionine incorporation, immunoprecipitation with anti-p21 monoclonal antibodies, SDS-PAGE and autoradiography, it was demonstrated that p21 synthesis was remarkably enhanced in 7402 cells as well as in transformed cells derived from both 7402 and PHC DNA. Taking the data together, it strongly implies that N-ras is one of the transforming genes for human liver cancer.

Animals↗
PubMed1986

Expression of N-ras gene in human primary hepatocarcinoma.

Poly(A)+ RNA was isolated from 9 specimens of human primary hepatic carcinoma, 1 non-tumorous liver tissue adjacent to cancer and 1 normal liver tissue samples. The Oligo-dT cellulose-purified poly(A)+ RNAs were subjected to formaldehyde agarose gel electrophoresis, Northern transfer and hybridization with various oncogene probes. Two RNA species, 5.6 kb and 2.2 kb were identified by N-ras gene hybridization in 6 out of 9 mRNA samples from primary hepatic carcinoma specimen. N-ras specific mRNA was not detectable in mRNA samples from normal human liver and tumor surrounding cirrhotic tissue. No detectable hybridization of mRNA from hepatoma and normal liver with Ki-ras or Ha-ras was observed. As human N-ras gene has been identified in DNA of mouse transfectants transformed with PHC DNA, it strongly suggests that N-ras gene might be responsible for the transforming activity of part of cases of human liver cancer.

Carcinoma, Hepatocellular↗
PubMed1985

State of hepatitis B virus DNA in leucocytes of hepatitis B patients.

Hepatitis B virus (HBV) DNA in leucocytes from 50 hepatitis patients with various patterns of HBV serological markers and serum HBV DNA and 13 normal controls were examined by Southern blot hybridization with 32P-labeled 3.2 Kb HBV DNA. A free form of HBV DNA was observed in leucocytes of 8 patients, 7 of whom were positive for serum HBeAg, and in 6 patients an integrated form of HBV DNA was identified. HBV DNA was not identified in leucocytes from 13 normal controls. The free form of HBV DNA in leucocytes existed as a heterogeneous smear from 2.0 to 3.2 Kb, similar to the pattern in liver and hepatocellular carcinoma cells but different from serum HBV DNA in which the 3.2 Kb band was absent. The banding pattern of the integrated form of HBV DNA in leucocytes varied among different patients. During preparation of white blood cells and purification of HBV DNA probes, it was important to remove plasma contamination and traces of pBR322, respectively. The presence of extrachromosomal DNA sequences partially homologous to pBR322 could cause false results. The presence of a free and integrated form of HBV DNA in leucocytes is important for explaining the biology of HBV, the harbouring and replication sites of extrahepatic origin, the mechanism of recurrent infection, and the rationale of the treatment of hepatitis B.

Carrier State↗
PubMed1981

DNA binding proteins in chromatin structure and function.

The binding of chromosomal proteins to cloned mouse and human globin genes can be determined by transferring proteins from one dimensional electrophoretic separations on to nitrocellulose paper and exposing them to nick translated cloned globin DNAs. The usefulness of this technique is assessed in the case of RNA polymerases isolated from mouse Ascites tumor cells where a specific initiation site for transcription of mouse beta globin DNA can be demonstrated.

Animals↗