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Biomedical subjects

G C Fareed

Publications and source records attributed to G C Fareed.

At least 19 recordsLinked to original sources
PubMed1988

Novel antigenic markers of human tumor regression.

The development of tumor-specific antibodies was studied in a group of cancer patients undergoing active specific immunotherapy with irradiated human allogeneic and autochthonous (autologous) tumor cells injected by the intralymphatic route. Immunoblotting studies on extracts of various established tumor cell cultures and fresh tumor biopsies were performed using sera from these patients. Evaluable tumor regressions were associated with detection of antibodies against human tumor cell antigens of 22,000 daltons (22 kd), 38,000 daltons (38 kd), 43,000 daltons (43 kd), and 70,000 daltons (70 kd). Similar antigens of approximately 22, 43, and 70 kd have also been detected in fresh extracts of certain human tumor tissues when tested with antisera from patients responding to immunotherapy. Production of antibodies to these antigens may play a role in tumor regression with active specific immunotherapy. These human regression-associated antigens may, therefore, represent novel agents for cancer immunotherapy.

Adult↗
PubMed1985

Human apolipoprotein B: identification of cDNA clones and characterization of mRNA.

Apolipoprotein B (apoB) is a major protein component of low density and very low density lipoproteins. Because of its large size and heterogeneity, molecular studies of apoB have been difficult, and its structure and regulation remain poorly understood. We now report the identification of human apoB cDNA clones by antibody screening of hepatoma libraries in the expression vector lambda gt11. Both oligo(dT) primed and random primed libraries were constructed and screened with polyclonal antibodies to intact apoB, as well as with antibodies raised against a synthetic peptide based on the limited amino acid sequence available for apoB. The identity of the clones was unambiguously established by comparisons of the cloned cDNA sequences with apoB amino acid sequences. The clones hybridize to an exceptionally large 20 kb mRNA that is present in liver and intestine but not other tissues examined, consistent with the distribution expected from protein biosynthetic studies. The properties of the mRNA have implications for the biogenesis of the multiple apoB molecular weight forms secreted by liver and intestine.

Apolipoproteins B↗
PubMed1985

Subacute sclerosing panencephalitis: measles virus matrix protein nucleic acid sequences detected by in situ hybridization.

Subacute sclerosing panencephalitis (SSPE) is characterized by a hyperimmune state toward the polypeptides of measles virus except the matrix (M) protein. Using cloned (3H)-labeled complementary DNA probes for in situ hybridization, we found the M protein and nucleocapsid (NP) protein nucleotide sequences in glial cells and neurons of cryostat sections from two SSPE brains. In one SSPE brain, M protein was lacking, but the other measles polypeptides were present. IgG and IgM antibodies eluted from that brain lacked antibodies to M protein, but antibodies to other measles polypeptides were present. In SSPE brain, the viral M-protein defect is not a deletion of the M gene, but rather a block in gene expression.

Brain Chemistry↗
PubMed1979

Cloning the argF gene from Escherichia coli K-12 with simian virus 40.

We have inserted a gene coding for ornithine transcarbamylase (OTCase) from Escherichia coli K-12 into the late gene region of simian virus 40 (SV40) DNA and propagated the hybrid molecules as free episomes or by co-infection with an SV40 tsA helper virus. In the first case, the E. coli argF gene was inserted via the EcoRI and BamHI termini in the late gene region of SV40 and the recombinant molecules were used to transfect monkey kidney cells. The hybrid DNA, which was too large to be encapsidated, was replicated for a short time (14 days) but was eventually lost from the surviving cells. In order to allow the argF gene to be packaged into virions, we purified two SV40 vectors containing large deletions of late gene region sequences. One was a 3325 base pair segment from a HaeII + BamHI digest. The argF gene was joined to both vectors at the BamHI site and these linear molecules were used to transfect monkey cells in the presence of SV40 tsA58 DNA as helper. These hybrid DNAs were replicated and packaged into virions. Late in the lytic infection of monkey cells, polyadenylated, cytoplasmic argF transcripts were detected, but significant translation of these trancripts was not observed.

Cloning, Molecular↗
PubMed1979

Transformation of human embryonic kidney cells by human papovarirus BK.

Infection of secondary human embryonic kidney (HEK) cells with human papovavirus BK (BKV) resulted in cellular lysis and degeneration within 7 days. After 30 days, multilayered colonies of transformed cells were found and subcultured for analyses. These BK-HEK cells uniformly expressed the BKV T-antigen but were only 1% V-antigen positive. They produced infectious virus and were resistant to superinfection by BKV. They reached a saturation density of 1.3 x 10(5) cells per cm2 in medium with 5% fetal calf serum, were able to grow in medium containing 2% serum, and did not form colonies in soft agar or tumors in nude mice. Nonintegrated, superhelical BKV DNA was detected in the noncloned cells as expected because they were persistently infected and contained RNA transcripts complementary to both early and late regions of the BKV genome. Analysis of T-antigen-positive clonal isolates of these BK-HEK cells by the Southern technique revealed an absence of free viral DNA and the presencce of integrated BKV DNA sequences corresponding to the early region of the BKV genome. These studies demonstrate the stable transformation of human cells by BKV. However, the transformed human cells which retain and express part of the BKV genome do not fully manifest the growth properties of other papovarirus-transformed cells.

Antigens, Neoplasm↗
PubMed1979

Persistent BK papovavirus infection of transformed human fetal brain cells. I. Episomal viral DNA in cloned lines deficient in T-antigen expression.

After infection of permissive human fetal brain cells by BK human papovavirus (BKV), the vast majority of the cells were killed by the virus, but rare survivors were recovered after frequent medium changes. These surviving cells grew and formed visible colonies after 5 to 6 weeks and were thereafter established as permanent cell lines. These cells, designated as BK-HFB cells, were persistently infected and shed BKV. Morphologically, they were small polygonal cells and had transformed growth properties. Their plating efficiency on solid substrates or in semisolid medium was high, and they were tumorigenic in athymic nude mice. Cloning experiments in medium containing BKV antiserum revealed that BKV did not persist in the cultures in a simple carrier state. All cloned cell lines were initially T-antigen negative and virus-free. However, every clone began to release BKV and again became persistently infected within 3 weeks after removal of BKV antiserum. After rigorous antibody treatment, four of seven clones still released virus spontaneously upon removal of antiserum; three clones have remained virus-free and are apparently cured. Although these cloned cell lines are T- and V-antigen negative when grown in antiserum-containing medium, they retain "free" or episomal BKV genomes; integrated viral DNA was not detected in any of the clones. These free genomes are indistinguishable from prototype BKV DNA and are found in much larger amounts in virus-shedding cell lines.

Antigens, Neoplasm↗
PubMed1978

Transduction of a bacterial gene into mammalian cells.

The transduction of an Escherichia coli gene into mammalian cells is described. A supressor tRNA gene was linked to a simian virus 40 (SV40) vector in vitro and the recombinant was used to transfect rat embryo cells and monkey kidney cells. The hybrid SV40 genome, SV40-su+ III, retained genetic information required for autonomous replication and cellular transformation and had a 1300-base-pair DNA segment in the late gene region (between the restriction endonuclease sits Hpa II at 0.735 and EcoRI at 0/1.0 on the SV40 genetic map) replaced by an 870-base-pair bacterial DNA segment containing the suppressor tRNA gene, su+ III (tRNATyrsu+III). The structure and fate of the SV40-su+III chimera were determined by DNA reassociation kinetic analysis and restriction enzyme cleavage of the total cellular DNA from transformed rat embryo cells and persistently infected monkey cells. Hybridization with radiolabeled probes specific for vector (SV40) or su+III DNA sequences revealed primarily nonintegrated or free hybrid genomes. In cloned lines of both cell types, the bacterial DNA segment was recovered intact, as judged by the length of the segment excised by restriction endonucleases and its ability to hybridize to the radiolabeled bacterial DNA probe and not to the SV40 probe.

Cell Line↗
PubMed1976

Transformation of cultured human vascular endothelium by SV40 DNA.

Primary cultures of human umbilical vein endothelial cells (HEC) developed extensive cytopathic changes and necrosis after high multiplicity infection with wild-type SV40 virus. Using the calcium co-precipitation technique, stable transformation was obtained with purified preparations of intact circular SV40 DNA and restriction endonuclease-derived linear DNA fragments containing the entire early gene region. Smooth muscle cells, isolated from the same blood vessels, showed neither cytopathic effects nor transformation after similar treatment with SV40 virus or DNA. The HEC cultures transformed by SV40 (SVHEC) expressed SV40-specific T (tumor) and Tr (transplantation) antigens, but not V (viral capsid) antigen. No evidence of infectious virus production was found upon co-cultivation with the CV-1 line of monkey kidney cells. Transformation resulted in markedly increased growth potential, loss of anchorage dependence and topoinhibition of growth, and a reduced serum requirement. Prolonged subcultivation was accompanied by chromosomal abnormalities and eventual "crisis". Transformed cells did not exhibit endothelial-specific organelles (Weibel-Palade bodies) or factor VIII antigen, but angiotensin-converting enzyme occasionally was detectable in SVHEC cultures. SV40-transformed human vascular endothelium, a nonfibroblast diploid cell type, may be useful in studies of oncogenesis and control of the differentiated state.

Antigens↗
PubMed1976

Propagation of a segment of bacteriophage lamda-DNA in monkey cells after covalent linkage to a defective simian virus 40 genome.

A 520 base pair DNA segment was excised from the bacteriophage lamda-genome by cleavage with the bacterial restriction endonuclease, endo R. Hindll. This segment was covalently joined in vitro to an 880 base pair simian virus 40 (SV40) DNA segment which contains the initation site for SV40 DNA replication. The latter segment was derived from the genome of a defective reiteration mutant of SV40 also by endo R. Hindlll cleavage. When the recombinant molecule, together with wild-type SV40 DNA as helper, was introduced into monkey cells by DNA infection, replication of the lamda-DNA sequences was observed, and hybrid genomes were encapsidated into progeny SV40 virions. The structure of the lamda-DNA segment after serial passage in monkey cells was examined by use of restriction endonucleases and electron microscopic heteroduplex analysis.

Cell Line↗
PubMed1976

Construction and propagation of a defective simian virus 40 genome bearing an operator from bacteriophage lambda.

A 2400 base pair DNA segment containing the leftward operator (OL) of phage lambda was covalently joined in vitro to a fragment of simian virus 40 (SV40) DNA harboring the SV40 replication origin. The recombinant molecule was propagated in the presence of helper wild-type SV40 DNA in monkey kidney cells and partially cloned by an infectious center procedure. After propagation in monkey cells and purification, the hybrid DNA could be distinguished from wild-type SV40 DNA by its shortened length (about 80% that of SV40), specific hybridization to denatured lambda DNA immobilized on filters, specific affinity for lambda repressor, and preservation of a large part (about 2300 base pairs) of the lambda immunity region as determined by restriction nuclease cleavage patterns and electron microscopic heteroduplex analysis. These results indicate that defective SV40 replicons can serve as vectors for propagating foreign DNA in mammalian cells.

Cell Line↗
PubMed1976

Structure of a defective simian virus 40 genome bearing an operator from bacteriophage lambda.

We examined further the physical structure of the simian virus 40 (SV40) and bacteriophage lambda DNA sequences in an SV40-lambda hybrid that had been propagated in monkey kidney cells. The SV40 vector portion of the hybrid, which was a small fragment isolated from a reiteration mutant of SV40, contained the site for initiation of SV40 DNA replication. Electron microscope heteroduplex and restriction endonuclease analyses revealed a tandem duplication of the SV40 vector segment linked to a 2,300-base pair portion (lambda map units 71 to 76) of the lambda immunity region. The defective hybrid genome thus harbors two origins for SV40 DNA replication in addition to the leftward operator and the N gene of lambda.

Coliphages↗
PubMed1975

Formation of reiterated simian virus 40 DNA.

We have described studies on the biological fate of a minicircular DNA molecule that is a specific, complex deletion mutant of SV40. When the minicircular DNA alone was used to infect monkey cells, its replication was not detected. However, after infection with the minicircles and SV40 DNA together, incorporation of (3H)thymidine into both species of viral DNA was demonstrated. This finding suggests that circular, duplex viral DNA segments, much smaller than SV40 DNA, are able to be replicated in vivo. Furthermore, 26% of the (3H)thymidine-labeled, superhelical DNA sedimented more rapidly than SV40 DNA I (21S) in neutral sucrose gradients (22S-32S). A similar amount of this rapidly sedimenting DNA was also detected when intact DAR DNA containing the triplication mutant was tested. Cleavage of the purified, rapidly sedimenting DNA with R.EcoRI produced 10.4S segments (one-third the size of unit-length SV40) in addition to full-length linears (14.5S) and a new cleavage product (16.7S). Cleavage of the 21S DNA I molecules also produced 10.4S DNA. These results indicate that the minicircular molecules are amplified in vivo, yielding not only the original triplication mutant but also a heterogeneous population of oligomers in which the 10.4S segment has been reiterated as many as 6 to 9 times. Our studies support the model proposed by Khoury et al. (1974) for the generation of the original DAR triplication mutant. In our experiments, cells were infected with a minicircular DNA molecule formed in vitro, which then served as a precursor in vivo in the formation of trimers and higher oligomers, as predicted by the proposed model. The DAR triplication mutant first appeared after the third passage in primary monkey kidney cells and rapidly became the predominant species in later passages (Fareed et al. 1974)...

Cell Line↗