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V Shen

Publications and source records attributed to V Shen.

82 records · Page 5Linked to original sources
PubMed1981

Functional inactivation of lac alpha-peptide mRNA by a factor that purifies that Escherichia coli RNase III.

Using RNA-directed synthesis of the alpha-peptide of beta-galactosidase as an assay, a factor was purified that inactivated further function of the mRNA. In the presence of Ca2+ ions to inhibit most nuclease activity, inactivation of mRNA occurred during incubation with ribosomes or with a 1 M KCl wash of ribosomes. The inactivation activity required Mg2+ ions, and purified as a single factor which did not bind to DEAE-cellulose, but bound reversibly to phosphocellulose. The factor eluted from Sephadex G-150 with an apparent molecular weight of about 43,000. Purified 700-fold, it showed no detectable exonuclease activity, and little or no cleavage of a variety of single-stranded substrates, including full length lac operon mRNA; but repurified inactivated mRNA was still inactive for protein synthesis. The factor did not inhibit poly(U)-directed polyphenylalanine synthesis. When proteins isolated from the ribosomal wash were individually tested, highly purified RNase III, which purifies in the same way and has the same size, also inactivated lac mRNA. The ribosomal wash from an RNase III- strain showed little if any activity compared to that from an isogenic RNase III+ strain. The possibility of a site-specific inactivating cleavage of mRNA by RNase III at or near the 5' end is considered.

Calcium↗
PubMed1980

Monoclonal antibodies to Escherichia coli 50S ribosomes.

Hybridoma cell lines that produce monoclonal antibodies directed against 50S Ribosomal proteins have been isolated. Spleen cells (from BALB/c mice immunized with 50S ribosomal subunits extracted from Escherichia coli) were fused to mouse myeloma cell line SP2/O-Ag 14. The initial screening for antibody producing hybridomas was carried out by a double antibody sandwich method; hybridomas were subsequently cloned in soft agar. Antibodies were characterized by their specific binding to individual 50S ribsomal proteins separated on phosphocellulose columns and in two-dimensional polyacrylamide gels. The assignments were confirmed with purified single ribosomal proteins. Of four clones analyzed thus far, two are identical with specificity for r-protein L5. The other clones produce two different antibodies directed against r-protein L20. Each monoclonal antibody formed ribosome dimers visualizable in the electron microscope. Dimers could be reacted with a different second antibody to form chains containing 8 or more ribosomes, which may be useful for structural studies.

Animals↗
PubMed1980

Lactose operon transcription from wild-type and L8-UV5 lac promoters in Escherichia coli treated with chloramphenicol.

In cells treated with chloramphenicol and the inducer isopropyl-beta-D-thiogalacto-pyranoside, messenger ribonucleic acid transcription from the wild-type lac promoter was not detected. Transcription occurred from the mutant UV5-L8 promoter. The transcripts were of variable length; some included the whole Z gene. No major site of transcription arrest within the Z gene was apparent.

Chloramphenicol↗
PubMed1978

Coupling of lac mRNA transcription to translation in Escherichia coli cell extracts.

In an extract containing all the components for lac gene expression except washed ribosomes, lac mRNA formation was increased 4- to 6-fold by the addition of washed ribosomes. The formation of beta-galactosidase mRNA and enzyme showed very different dependency on added ribosomes. Enzyme was formed in proportion to the number of ribosomes added, whereas 10% of the standard level of ribosomes promoted full levels of transcription. Consistent with their action in vivo, chloramphenicol and erythromycin blocked the ribosome-dependent lac transcription. The same inhibition was seen with RNA pulse-labeled for 1 or 5 min, so that the effect was truly a blockage of formation rather than an increased hyperlability of nascent mRNA. The effect was specified for some RNA species, as it is in vivo: phage lambda N gene transcription was increased rather than inhibited in the presence of chloramphenicol. Chloramphenicol did not stop lac transcription as a result of its blockage of formation of the regulatory nucleotide tetraphosphate (ppGpp), because addition of the nucleotide did not restore mRNA formation in chloramphenicol-treated extracts. Rather, the data are consistent with the ideas that one or a few ribosomes moving closely behind RNA polymerase can prevent its arrest and that, when ribosome movement is blocked by chloramphenicol, the RNA polymerase is exposed to factors that provoke premature RNA chain termination.

Cell-Free System↗
PubMed1977

Chloramphenicol-induced changes in the synthesis of ribosomal, transfer, and messenger ribonucleic acids in Escherichia coli B/r.

The synthesis of ribosomal ribonucleic acid (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA) was measured in Escherichia coli B/r after the addition of 100 mug of chloramphenicol (CAM) per ml to cultures growing either in one of three minimal media (succinate, glycerol, or glucose) or in one of the same three media supplemented with 20 amino acids. (i) During CAM treatment, rRNA and tRNA were synthesized in the same relative proportions (85:15) as during exponential growth. The faster accumulation of tRNA relative to rRNA in CAM was due to a decreased stability of rRNA that is synthesized in the presence of or immediately before the addition of CAM. (ii) CAM stimulated the synthesis of rRNA and tRNA two- to eightfold. The results fell into two groups; one group was from studies done in minimal media and the other was from amino acid-supplemented media. In each group the stimulation decreased with increasing growth rate of the culture during exponential growth before the addition of CAM; however, the stimulation in minimal media was lower than that in amino acid-supplemented media. (iii) CAM caused an increase in the proportion of rRNA and tRNA synthesis and a corresponding decrease in the proportion of mRNA synthesis. In minimal media, the residual proportion of mRNA synthesis after CAM treatment was 10 to 15% of total RNA synthesis; in amino acid-supplemented media this proportion was 0 to 10%. In either case, the residual proportion of mRNA synthesis was independent of the proportions observed during exponential growth in these media. (iv) The absolute rate of mRNA synthesis decreased severalfold with the addition of CAM; i.e., the rate of synthesis of rRNA and tRNA was increased at the expense of mRNA synthesis. (v) During exponential growth, the fraction of the instantaneous rate of total RNA synthesis that corresponds to mRNA is a function of both the growth rate and the presence or absence of amino acids in the growth medium: in the absence of amino acids, this fraction decreased with increasing growth rate; in the presence of amino acids, the fraction increased slightly with growth rate. These results are consistent with a regulation of rRNA and tRNA synthesis at the transcriptional level, e.g., with a CAM-induced increase in the affinity of RNA polymerase for the rRNA and tRNA promoters. The results also suggest the occurrence of a regulation of RNA polymerase enzyme activity, i.e., of an activation of RNA polymerase that is inactive during exponential growth. A distinction between these alternatives requires measurements of the rRNA chain growth rates during CAM treatment.

Chloramphenicol↗
PubMed1977

Rate of ribosomal ribonucleic acid chain elongation in Escherichia coli B/r during chloramphenicol treatment.

In Escherichia coli B/r growing in glucose-amino acids medium, the radioactive labeling of 5S ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) was measured after the simultaneous addition to the bacteria of chloramphenicol (CAM) (100 mug/ml), rifampin (200 mug/ml), and radioactive uracil. Accumulation of 5S rRNA ceased 85 s after the addition of rifampin, independent of the presence or absence of CAM; this indicates that CAM did not affect the rRNA chain growth rate. Together with previous measurements of the synthesis of rRNA and messenger RNA under these conditions, the results imply that CAM caused a redistribution of RNA polymerase which greatly favored stable RNA synthesis (77 to 97% of total functioning RNA polymerase engaged in synthesis of rRNA and tRNA). Further, it is inferred that RNA polymerase molecules were activated that were inactive during exponential growth. The labeling of tRNA observed under these conditions suggests the existence of clusters of tRNA genes at the 3' end of long transcripts that resemble the rRNA precursor in length and response to CAM and may be parts of rRNA transcripts.

Chloramphenicol↗
PubMed1975

Stability of ribosomal and transfer ribonucleic acid in Escherichia coli B/r after treatment with ethylenedinitrilotetraacetic acid and rifampicin.

A short treatment with ethylenedinitrilotetraacetic acid to permeabilize bacteria for various antibiotics or treatment with the ribonucleic acid (RNA) synthesis inhibitor rifampin causes a slow degradation of 50S and 30S ribosomal particles and of the corresponding 23S and 16S ribosomal RNA species (about 25 percent in 1 h). The effects are additive such that the decay is about 50 percent/h if rifampin is employed after permeabilization by ethylenedinitrilotetraacetic acid. The 5S ribosomal RNA and transfer RNA are essentially stable under these conditions.

Drug Stability↗
PubMedDate not supplied

Feasibility study of IL-11 and granulocyte colony-stimulating factor after myelosuppressive chemotherapy to mobilize peripheral blood stem cells from heavily pretreated patients.

PURPOSE: Pediatric patients with solid tumors treated with prolonged dose-intensive chemoradiotherapy are poor mobilizers of peripheral blood stem cells (PBSC). We have conducted a pilot study to mobilize PBSC in eight pediatric patients with relapsed solid tumors using ifosfamide, carboplatin, and etoposide (ICE) followed-up by IL-11 plus granulocyte colony-stimulating factor (G-CSF). PATIENTS AND METHODS: Patients received ifosfamide 1.8 g/m2 per day for 5 days, carboplatin 400 mg/m2 per day for 2 days, and etoposide 100 mg/m2 per day for 5 days. After completion of ICE chemotherapy, patients received daily subcutaneous injections of G-CSF (5 microg/kg per day) and IL-11 (50-100 microg/kg per day) until peripheral stem cell apheresis. RESULTS: The median age was 11 years. Diagnosis included three relapsed Hodgkin disease, three relapsed central nervous system tumors, one relapsed Wilms tumor, and one relapsed rhabdomyosarcoma. The median number of apheresis procedures required to obtain 5 x 10(6) CD34+ cells/kg was one. The mean +/- standard error of mean (SEM) total CD34+ cells collected was 14.0+/-2.7 x 10(6)/kg. The mean +/- SEM total CD34+/CD41+ cells collected was 4.6+/-1.9 x 10(6)/kg. Seven of the eight patients have subsequently undergone myeloablative chemotherapy with autologous PBSC transplantation and have reconstituted hematopoiesis with a median time to neutrophil recovery of 10 days and platelet recovery of 15.5 days. CONCLUSIONS: We conclude that the regimen of ICE/IL-11 plus G-CSF is successful in mobilizing large numbers of CD34+ PBSC cells with a limited number (one) of apheresis collections in patients that have previously been heavily pretreated with chemotherapy/radiotherapy.

Antineoplastic Combined Chemotherapy Protocols↗
PubMedDate not supplied

Uterine cervical extrarenal Wilms tumor managed without hysterectomy.

This report describes an unusual case of uterine cervical Wilms tumor treated successfully without hysterectomy or radiation therapy. The 12-year-old white girl developed a persistent vaginal discharge. Her pelvic examination revealed a large mass involving the entire upper vagina, obscuring the cervix. Biopsy of the mass was consistent with Wilms tumor with favorable histology. The tumor was not initially resected because the resection would involve hysterectomy and partial resection of the bladder wall. The patient was treated with preexcisional chemotherapy consisted of alternating vincristine, doxorubicin, cyclophosphamide and carboplatin/etoposide. Repeat magnetic resonance imaging after 5 weeks of chemotherapy demonstrated marked reduction of the tumor size. The tumor was easily removed by transsection of the stalk followed by cold-knife conization of the cervix. The patient received four more cycles of chemotherapy and remained in complete remission 12 months after completion of chemotherapy. This report suggests that in selected cases, chemotherapy can reduce tumor size sufficiently in patients with bulky cervical Wilms tumor to allow local resection and avoid hysterectomy.

Antineoplastic Combined Chemotherapy Protocols↗