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Biomedical subjects

C M Wei

Publications and source records attributed to C M Wei.

At least 55 records · Page 3Linked to original sources
PubMed1985

Expression of active human uterine tissue plasminogen activator in yeast.

Tissue-type plasminogen activator (tPA) cDNA derived from human uterine mRNA was inserted into different yeast expression vectors. All such expression plasmids carried a yeast acid phosphatase (PHO5) promoter, a 2-micron plasmid replication origin, transcription termination signals, and a selectable TRP1 gene. Plasmid pYBDT-10 contained the entire tPA coding region ("pre-pro-tPA"), pYBDT-10-PRO contained a sequence encoding the putative pro-tPA precusor, and pYBDT-6 contained only a mature tPA cDNA fused precisely in frame to the sequence encoding the entire signal peptide of acid phosphatase. All constructions directed the synthesis of single-chain tPA proteins that were readily precipitated with a specific antibody directed against human uterine tPA. Electrophoretic mobilities were approximately the same as those of the Bowes melanoma single-chain tPA and a 68-kD protein marker. Treatment of immunoprecipitates with endoglycosidase H resulted in increased electrophoretic mobilities, suggesting that these yeast products are glycosylated. Despite the use of either human or yeast signal sequences, however, tPA produced in yeast was not secreted into the culture medium, but rather was found only in cells following disruption with glass beads. Although this cellular tPA exhibited fibrinolytic activity, most of the activity was associated with large cellular debris.

Base Sequence↗
PubMed1985

Two-dimensional echocardiographic evaluation of right ventricular function during left heart bypass.

Right ventricular (RV) function in terms of hemodynamics and RV wall motion was studied in 14 mongrel dogs during left heart bypass (LHB) using a centrifugal blood pump. The wall motion was analyzed by two-dimensional echocardiography (2D-echo). Incremental changes in LHB flow ratios of 0% (controls), 25%, 50%, 75% and a maximum 85-100% were accompanied by decrements of segmental shortening of the interventricular septum (IVS) by 54 +/- 12%, 43 +/- 5%, 42 +/- 2%, 35 +/- 0% and 0%, respectively. In addition to akinesis of the IVS during maximum flow, a specific part of the RV free wall adjacent to the IVS also had marked depression of contractions and overall RV contraction was nearly dependent on the RV free wall opposite to the IVS. Maximum LHB flow induced complete depression of the left ventricular cavity, a marked increase in RV volume, and depression of the RV ejection fraction on 2D-echo. Excessive or prolonged LHB reduces the RV wall motion capability and may lead to right heart failure. Our results suggest that an LHB ratio of about 75% is optimum to maintain normal cardiac function, particularly that of the right heart.

Animals↗
PubMed1981

Construction and isolation of a transmissible retrovirus containing the src gene of Harvey murine sarcoma virus and the thymidine kinase gene of herpes simplex virus type 1.

We constructed lambda recombinants containing the Harvey murine sarcoma virus genome and the thymidine kinase (tk) gene of herpes simplex virus type 1 linked to each other. The tk gene was located in a position downstream from both the long terminal repeat and the src gene of Harvey murine sarcoma virus. The DNAs of the lambda recombinants were used to transfect NIH3T3 mouse fibroblasts in order to obtain Harvey murine sarcoma virus DNA-induced foci of transformed cells. The transformed foci were superinfected with a helper-independent retrovirus, and new individual retrovirus were isolated from the superinfected foci. The new viruses could induce focus formation on NIH3T3 cells and could convert NIH3T3(TK-) cells into TK+ cells by carrying the herpes simplex virus type 1 tk gene into the TK- cells. From virus-infected cells, we isolated nonproducer foci on NIH3T3 cells and TK+ transformants on NIH3T3(TK-) cells containing one such new viral genome coding for the dual properties. The new retroviral sequence in the nonproducer cells could be rescued into virus particles at high titers by superinfection with a helper-independent retrovirus. A hybridization analysis indicated that the recombinant virus contained both the Harvey murine sarcoma virus src sequence and the tk gene sequence in a single RNA species approximately 4.9 kilobases long. We concluded that retroviruses can be used as true vectors for genes other than genes that lead to oncogenesis.

Animals↗
PubMed1980

Mapping of transforming region of the Harvey murine sarcoma virus genome by using insertion-deletion mutants constructed in vitro.

Circular DNA intermediates of Harvey murine sarcoma virus (Ha-MuSV) have been cloned in lambda gtWES . lambda B and shown to be capable of transforming mouse NIH 3T3 cells [Hager, G. L., Chang, E. H., Chan, H. W., Garon, C. F., Israel, M. A., Martin, M. A., Scolnick, E. M. & Lowy, D. R. (1979) J. Virol. 31, 795-809]. By using the cloned Ha-MuSV DNA insert as a parental genome, we have constructed a series of insertion-deletion mutants by inserting an octomer containing the Sal I linker sequence (G-G-T-C-G-A-C-C) into various regions of the Ha-MuSV genome after partial digestion with Hae III. After ligation into lambda gtWES . lambda B-Sal I vector molecules, the mutant Ha-MuSV DNAs were cloned. Fourteen insertion-deletion mutants have been mapped by restriction enzyme digestion, and their biological activities have been correlated with the locations of mutations. The mutants whose lesion mapped within 3.0 kilobases (kb) frm the 3'-end of the Ha-MuSV genome retained full transforming ability. The mutants containing the Sal I linker insertion at 0.4 or 1.5 kb from the 5'-end also retained transforming ability, but the number of foci induced by the DNAs in transfection assays was greatly reduced. However, a mutant containing a deletion of 1.5 kb at the 5'-end and a mutant with a deletion of the sequences between 1.0 and 1.5 kb from the 5'-end completely lost their transforming potential. A model for the transforming region of Ha-MuSV is discussed. Furthermore, because Ha-MuSV sequences can be rescued from the mouse cells transformed by these mutants using Moloney murine leukemia virus as a helper virus, it implies that the in vitro modified DNAs may be converted into genuine mutant viruses.

Cell Transformation, Viral↗
PubMed1980

Functional organization of the Harvey murine sarcoma virus genome.

The comparative infectivity of Harvey murine sarcoma virus (Ha-MuSV) DNA for NIH 3T3 cells was determined for supercoiled Ha-MuSV DNA molecularly cloned in lambda phage and pBR322 at its unique EcoRI site (which is located near the middle of the 6-kilobase pair [kbp] unintegrated linear viral DNA) and for two cloned subgenomic fragments: one was 3.8 kbp and lacked about 1 kbp from each side of the EcoRI site, and the second did not contain the 3 kbp of the unintegrated linear viral DNA located on the 3' side of the EcoRI site. Each subgenomic DNA induced foci of transformed cells, but with a lower relative efficiency then genomic DNA. Transfection with intact vector Ha-MuSV DNA yielded results similar to those obtained after separation of Ha-MuSV DNA from vector DNA. Cells lines were then derived from individual foci transformed with each type of viral DNA. Focus-forming virus was recovered from transformed cells after superinfection with a helper-independent virus, but the efficiency varied by several orders of magnitude. For several transformed lines, the efficiency of recovery of focus-forming virus was correlated with the structure of the Ha-MuSV DNA in the cells before superinfection. When 32P-labeled Ha-MuSV DNA probes specific for sequences on either the 3' or 5' side of the EcoRI site were used to analyze the viral RNA in the transformed cell lines, all lines were found to hybridize with the 5' probe, but some lines did not hybridize with the 3' probe. The transformed lines contained high levels of the Ha-MuSV-coded p21 or its associated GDP-binding activity. We conclude that the transforming region and the sequences that code for the viral p21 protein are both located within the 2 kilobases closest to the 5' end of the Ha-MuSV genome.

Animals↗
PubMed1977

Nucleotide sequences at the N6-methyladenosine sites of HeLa cell messenger ribonucleic acid.

Borate gel chromatography was used to separate internal oligonucleotides containing N6-methyladenosine (m6A) from methylated 5'-terminal oligonucleotides of HeLa cell polyadenylylated mRNA. N6-Methyladenosine occurs primarily in two sequences, -G-m6A-C (70%) and -A-m6A-C-(30%). The nucleoside immediately following cytidine may be uridine, cytidine, or adenosine, while guanosine as well as other nucleosides occupy subsequent positions. Each of the four positions preceding the -(G or A)-m6A-C- sequence may be occupied by a pyrimidine or a purine ribonucleoside. Since on a random basis all possible sequences containing -(G or A)-A-C-(U or C or A)- could occur once per 43 nucleotides whereas there is only one m6A residue per thousand nucleotides, then either (1) not all potential sites are methylated, (2) there are multiple unique sequences perhaps methylated by several different enzymes, or (3) there are other unrecognized discriminating factors. The possibility that methylation of adenosine occurs exclusively in the region close to the 5' terminus of the mRNA was considered. However, such a localization was excluded since the majority of m6A residues were not found in 4 to 6S 5'-terminal fragments isolated by borate gel chromatography.

Adenosine↗
PubMed1976

5'-Terminal and internal methylated nucleotide sequences in HeLa cell mRNA.

The 5'-terminal oligonucleotides m7G(5')ppp(5')NmpNp and m7G(5')ppp(5')NmpNmpNp were isolated by DEAE-cellulose column chromatography after enzymatic digestion of 32P- or methyl-3H-labeled poly(A)" HeLa cell mRNA. The recovery of such oligonucleotides indicated that a high percentage of mRNA has blocked termini. The dimethylated nucleoside, N6, O2'-dimethyladenosine (m6Am), as well as the four common 2'-O-methylribonucleosides (Gm, Am, Um, Cm) were present in the second position linked through the triphosphate bridge to 7-methylguanosine (m7G) whereas little m6Am was in the third position. The only internal methylated nucleoside, N6-methyladenosine (m6A), was found exclusively as m6ApC and Apm6ApC after digestion with RNase A, T1, and alkaline phosphatase. Digestion with RNase A and alkaline phat pyrimidines are present in much smaller amounts or absent from this position. These results imply a considerable sequence specificity since there are thousands of different mRNA species in HeLa cells. Our studies are consistent with the following model of HeLa cell mRNA in which Nm may be m6Am, Gm, Cm, Um, or Am and one or more m6A residues are present at an unspecified internal location: m7G(5')ppp(5')Nm-(Nm)---(G or A)-m6A-C---(A)100-200A.

Alkaline Phosphatase↗
PubMed1975

Methylated nucleotides block 5' terminus of HeLa cell messenger RNA.

Polyadenylylated [poly(A)+] mRNA from HeLa cells that were labeled with [3H-methyl]-methionine and 14C-uridine was isolated by poly(U)-Sepharose chromatography. The presence of approximately two methyl groups per 1000 nucleotides of poly(A)+ RNA was calculated from the 3H/14C ratios and known degrees of methylation of 18S and 28S ribosomal RNAs. All four 2'-O-methylribonucleosides, but only two base-methylated derivatives, 7-methylguanosine (7MeG) and 6-methyladenosine (6MeA), were identified. 6MeA was the major component accounting for approximately 50% of the total methyl-labeled ribonucleosides. 7MeG, comprising about 10% of the total, was present exclusively at the 5' terminus of the poly(A)+ RNA and could be removed by periodate oxidation and beta elimination. Evidence for a 5' to 5' linkage of 7MeG to adjacent 2'-O-methylribonucleosides through at least two and probably three phosphates to give structures of the type 7MeG5'ppp5pNMep- and 7MeG5'ppp5'NMepNmep- was presented. The previous finding of similar sequences of methylated nucleotides in mRNA synthesized in vitro by enzymes associated with virus cores indicates that blocked 5' termini may be a characteristic feature of mRNAs that function in eucaryotic cells.

Adenosine↗
PubMed1975

Methylated nucleotides block 5'-terminus of vaccinia virus messenger RNA.

Studies on the nature and location of the methylated nucleotides in mRNA synthesized in vitro by vaccinia virus particles revealed an unusual 5'-terminal structure. Evidence that the pyrophosphate group is blocked by 7-methylguanosine and that both 2'-O-methyl-adenosine and 2'-O-methylguanosine occupy penultimate positions was presented. According to this model, the 5'-termini of vaccinia virus mRNAs are: 7MeG-5'ppp-5'GMepNp and 7MeG-5'AMepNp.

Adenosine↗
PubMed1974

Mechanism of action of the mycotoxin trichodermin, a 12,13-epoxytrichothecene.

Trichodermin is a member of a group of closely related compounds-the 12,13-epoxytrichothecenes-that form a medically and economically important class of mycotoxins produced by fungi that spoil fruit and grain. Our studies show that trichodermin is a very potent inhibitor of protein synthesis in mammalian cells. Since ribosomes remain in polyribosomes in inhibited cells, trichodermin inhibits the elongation and/or termination processes of protein synthesis. In vitro, trichodermin is a potent inhibitor of the peptidyl transferase activity required for elongation and/or termination. An in vitro comparison of the effects of three peptidyl transferase inhibitors on elongation and termination indicates that anisomycin acts primarily on elongation while trichodermin and sparsomycin act primarily on termination. A new in vivo test to distinguish elongation inhibitors from termination inhibitors confirms that trichodermin inhibits primarily the termination process. Thus trichodermin inhibits protein synthesis by blocking the activity of peptidyl transferase required for termination. These studies suggest that the toxicosis caused by one of the 12,13-epoxytrichothecenes is due to its action as a protein synthesis inhibitor involving the peptidyl transferase activity of the eukaryotic ribosomes.

Amino Acids↗