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Biomedical subjects

E Wickstrom

Publications and source records attributed to E Wickstrom.

At least 55 records · Page 3Linked to original sources
PubMed1991

Daily addition of an anti-c-myc DNA oligomer induces granulocytic differentiation of human promyelocytic leukemia HL-60 cells in both serum-containing and serum-free media.

Expression of the human protooncogene c-myc is necessary for replication, and may be inhibited in a sequence-specific, dose-dependent manner by an antisense oligodeoxynucleotide specific for the first five codons of c-myc mRNA. Antisense inhibition of c-myc inhibits the proliferation and enhances the differentiation of the HL-60 human promyelocytic leukemia cell line. In order to raise the efficacy of antisense oligomers, HL-60 cells were grown in a serum-free medium so as to minimize nuclease activity in the culture medium. Daily addition of anti-c-myc oligomer was then found to induce terminal granulocytic differentiation of 80% or more of HL-60 cells, and inhibit colony formation by greater than 50%, comparable to 1% Me2SO.

Blood↗
PubMed1990

Oligodeoxynucleoside phosphorothioate stability in subcellular extracts, culture media, sera and cerebrospinal fluid.

Degradation of a synthetic oligodeoxynucleoside phosphorothioate was studied in six systems used for antisense inhibition experiments. Oligodeoxynucleoside phosphorothioates were degraded very slowly at 37 degrees C in all of the systems studied. Measured half-lives of pentadecamers were 12 +/- 1 h in rabbit reticulocyte lysate, 7 +/- 1 h in HeLa cell postmitochondrial extract, 14 +/- 2 h in RPMI 1640 with 10% fetal bovine serum, 8 +/- 1 h in undiluted fetal bovine serum, 9 +/- 1 h in adult human serum, and 19 +/- 7 h in rat cerebrospinal fluid.

Animals↗
PubMed1990

Risk factors for morbidity and mortality in mitral valve replacement.

Risk factors of operative mortality and long term survival were identified in 219 patients who underwent mitral valve replacement (MVR) using Bjørk-Shiley mechanical prostheses. Early mortality was 7.3%. The accumulated follow-up time was 1134 patient-years, and the 5-year survival for the total cohort was 78 +/- 3%. Independent prognostic factors of early mortality were poor NYHA class, which carried a relative risk (RR) of 3.2, and ischaemic aetiology, with a RR of 2.2. Ischaemic aetiology was the sole predictor of heart pump failure requiring intra-aortic balloon pump support (RR = 2.7). Independent risk factors of total mortality (early and late) were male sex (RR = 2.3), NYHA class III-IV (RR = 2.4), presence of mitral regurgitation (RR = 3.2) and relative heart volume (RR = 1.6 for a 800 ml/m2 size compared to a heart of 550 ml/m2). Our results underline the importance of patient-related factors in MVR, and indicate that care is needed in comparing the quality of MVR from different institutions with respect to mortality and morbidity. The results of MVR are palliative rather than curative except in female patients with NYHA class II function and mitral stenosis, in whom cure was attained.

Adolescent↗
PubMed1990

Target dependence of antisense oligodeoxynucleotide inhibition of c-Ha-ras p21 expression and focus formation in T24-transformed NIH3T3 cells.

The relationship of antisense oligodeoxynucleotide inhibition to the predicted secondary structure of human c-Ha-ras oncogene mRNA was examined. Three antisense pentadecade-oxynucleotides complementary to the 5' cap region, a predicted loop in the 5' untranslated region, and the initiation codon region of c-Ha-ras mRNA were synthesized. T24-transformed NIH3T3 cells were treated for 24 hr with each anti-ras oligomer or control sequence. The levels of c-Ha-ras p21 antigen were then analyzed by radioimmunoprecipitation. Inhibition of p21 expression was sequence-specific and dose-dependent. The efficacy of the cap sequence in reducing p21 expression was greater than that of the initiation codon target, which in turn was more effective than the target upstream of the initiation codon. Antisense inhibition of p21 expression correlated with inhibition of focus formation.

Animals↗
PubMed1989

Escherichia coli initiation factor 3 protein binding to 30S ribosomal subunits alters the accessibility of nucleotides within the conserved central region of 16S rRNA.

Translational initiation factor 3 (IF3) is an RNA helix destabilizing protein which interacts with strongly conserved sequences in 16S rRNA, one at the 3' terminus and one in the central domain. It was therefore of interest to identify particular residues whose exposure changes upon IF3 binding. Chemical and enzymatic probing of central domain nucleotides of 16S rRNA in 30S ribosomal subunits was carried out in the presence and absence of IF3. Bases were probed with dimethyl sulfate (DMS), at A(N-1), C(N-3), and G(N-7), and with N-cyclohexyl-N'-[2-(N-methyl-4-morpholinio)ethyl] carbodiimide p-toluenesulfonate (CMCT), at G(N-1) and U(N-3). RNase T1 and nuclease S1 were used to probe unpaired nucleotides, and RNase V1 was used to monitor base-paired or stacked nucleotides. 30S subunits in physiological buffers were probed in the presence and absence of IF3. The sites of cleavage and modification were detected by primer extension. IF3 binding to 30S subunits was found to reduce the chemical reactivity and enzymatic accessibility of some sites and to enhance attack at other sites in the conserved central domain of 16S rRNA, residues 690-850. IF3 decreased CMCT attack at U701 and U793 and V1 attack at G722, G737, and C764; IF3 enhanced DMS attack at A814 and V1 attack at U697, G833, G847, and G849. Many of these central domain sites are strongly conserved and with the conserved 3'-terminal site define a binding domain for IF3 which correlates with a predicted cleft in two independent models of the 30S ribosomal subunit.

Bacterial Proteins↗
PubMed1989

Inducible high expression of the Escherichia coli infC gene subcloned behind a bacteriophage T7 promoter.

The gene for Escherichia coli translational initiation factor 3 (infC) has been inserted into an overexpression plasmid under the control of the bacteriophage T7 promoter. The infC plasmid was then used to transform a host with a chromosomal T7 RNA polymerase gene controlled by the lacUV5 promoter. Induction of T7 RNA polymerase expression in the host cells resulted in a 200-fold overexpression of infC mRNA and a 100-fold overproduction of initiation factor 3. Rapid batch purification of biologically active IF3 yielded predominantly the long form of IF3, implying that the short form is an artifact of purification by traditional methods.

Bacterial Proteins↗
PubMed1989

Anti-c-myc DNA increases differentiation and decreases colony formation by HL-60 cells.

The proto-oncogene c-myc, whose gene product has a role in replication, is overexpressed in the human promyelocytic leukemia HL-60 cell line. Treatment of HL-60 cells with an antisense oligodeoxyribonucleotide complementary to the start codon and the next four codons of c-myc mRNA has previously been observed to inhibit c-myc protein expression and cell proliferation in a sequence-specific, dose-dependent manner. Comparable effects are seen upon treatment of HL-60 cells with dimethylsulfoxide (Me2SO), which is also known to induce granulocytic differentiation of HL-60 cells. Hence, the effects of antisense oligomers on cellular differentiation were examined and compared with Me2SO. Differentiation of HL-60 cells into forms with granulocytic characteristics was found to be enhanced in a sequence-specific manner by the anti-c-myc oligomer. No synergism was observed between the anti-c-myc oligomer and Me2SO in stimulating cellular differentiation. In contrast, synergism did appear in the inhibition of cell proliferation. Finally, the anti-c-myc oligomer uniformly inhibited colony formation in semisolid medium. It is possible that further reduction in the level of c-myc expression by antisense oligomer inhibition may be sufficient to allow terminal granulocytic differentiation and reverse transformation.

Cell Differentiation↗
PubMed1988

alpha-Oligodeoxynucleotide stability in serum, subcellular extracts and culture media.

Degradation of a synthetic alpha-oligodeoxynucleotide was studied in order to compare its survival with naturally occurring beta-oligodeoxynucleotides in five systems used for antisense hybridization arrest experiments. In contrast to beta-oligodeoxynucleotides, alpha-oligodeoxynucleotides were not detectably degraded over 24 h at 37 degrees C in HeLa cell postmitochondrial cytoplasmic extract or RPMI 1640 with 10% fetal bovine serum, and showed significant survival after 24 h at 37 degrees C in rabbit reticulocyte lysate, fetal bovine serum and human serum.

Base Sequence↗
PubMed1988

Human promyelocytic leukemia HL-60 cell proliferation and c-myc protein expression are inhibited by an antisense pentadecadeoxynucleotide targeted against c-myc mRNA.

The human promyelocytic leukemia cell line HL-60 overexpresses the c-myc protooncogene. A calculated secondary structure for c-myc mRNA placed the initiation codon in a bulge of a weakly base-paired region. Treatment of HL-60 cells with 5' d(AACGTTGAGGGGCAT) 3', complementary to the initiation codon and the next four codons of c-myc mRNA, inhibited c-myc protein expression in a dose-dependent manner. However, treatment of HL-60 cells with 5' d(TTGGGATAACACTTA) 3', complementary to nucleotides 17-31 of vesicular stomatitis virus matrix protein mRNA, displayed no such effects. These results agree with analogous studies of normal human T lymphocytes [Heikkila, R., Schwab, G., Wickstrom, E., Loke, S. L., Pluznik, D. H., Watt, R. & Neckers, L. M. (1987) Nature (London) 328, 445-449], except that only one-third as much oligomer was needed for a comparable effect. Proliferation of HL-60 cells in culture was inhibited in a sequence-specific, dose-dependent manner by the c-myc-complementary oligomer, but neither the oligomer complementary to vesicular stomatitis virus matrix protein mRNA nor 5' d(CATTTCTTGCTCTCC) 3', complementary to nucleotides 5399-5413 of human immunodeficiency virus tat gene mRNA, inhibited proliferation. It thus appears that antisense oligodeoxynucleotides added to myc-transformed cells via culture medium are capable of eliciting sequence-specific, dose-dependent inhibition of c-myc protein expression and cell proliferation.

Base Sequence↗
PubMed1986

Circular dichroism and 500-MHz proton magnetic resonance studies of the interaction of Escherichia coli translational initiation factor 3 protein with the 16S ribosomal RNA 3' cloacin fragment.

The RNA helix destabilizing properties of Escherichia coli initiation factor 3 protein (IF3), and its affinity for an evolutionarily conserved sequence at the 3' end of 16S rRNA, led us to examine the details of the protein-nucleic acid interactions upon IF3 binding to the 49-nucleotide 3'-terminal cloacin DF13 fragment of 16S rRNA by studying the circular dichroism (CD) and proton magnetic resonance spectra of the RNA, the protein, and their complex. In a physiological tris(hydroxymethyl)aminomethane buffer, where the interaction is primarily nonionic and sequence specific, addition of IF3 decreases the RNA 268-nm CD peak hyperbolically by 19% to an end point of about one IF3 per RNA strand. The titration curve is best fit by an association constant of (1.80 +/- 0.05) X 10(7) M-1, within the range estimated by a nuclease mapping study of the same system [Wickstrom, E. (1983) Nucleic Acids Res. 11, 2035-2052]. In a low-salt phosphate buffer without Mg2+, where the interaction is primarily ionic and nonspecific, titration with IF3 decreases the peak CD sigmoidally by 35% to an end point of two IF3 per strand. The titration curve is best fit by an intrinsic association constant of (1.7 +/- 0.7) X 10(6) M-1 for each IF3 and a cooperativity constant of 33 +/- 6. In a physiological phosphate buffer lacking Mg2+, the dispersion of aromatic proton magnetic resonance peaks and upfield-shifted methyl proton resonances indicates a high degree of secondary and tertiary structure in the protein. In an equimolar mixture of IF3 and RNA cloacin fragment, several changes in identifiable IF3 and RNA resonances are observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriocins↗
PubMed1986

Oligodeoxynucleotide stability in subcellular extracts and culture media.

Oligodeoxynucleotide degradation was studied in four systems in order to assess the importance of degradation in hybridization arrest experiments dependent on oligodeoxynucleotides complementary to mRNA sequences. Oligodeoxynucleotides were not detectably degraded over 2 h at 37 degrees C in rabbit reticulocyte lysate or Dulbecco's modified essential medium with 5% fetal calf serum, but were degraded over 2 h in HeLa cell postmitochondrial cytoplasmic extract, and were degraded within 15 min in bovine calf serum.

Autoradiography↗
PubMed1983

Nuclease mapping of the secondary structure of the 49-nucleotide 3' terminal cloacin fragment of Escherichia coli 16s RNA and its interactions with initiation factor 3.

Escherichia coli translational initiation factor 3 (IF3) may be crosslinked to the 3' end of 16S RNA in 30S ribosomal subunits. In order to determine the sequence to which IF3 may bind in vivo, samples of 5'-32P labelled 3' terminal 49-nucleotide fragment of 16S RNA were incubated 5 min. at 37 degrees in 40 mM Tris-HOAc, pH 7.4, 100 mM NaCl, 1 mM Mg (OAc)2, 1 mM ZnSO4, with or without IF3, then reacted a further 5 min with nuclease S1, RNase T1, or RNase A. Base pairing between the 5' and 3' legs of the fragment occurs in the absence of IF3, but is disrupted by IF3 binding. IF3 appears to protect some residues near the 5' end of the fragment (U1495, A1499, A1500, A1502, and A1503) from nuclease S1, and potentiates S1 attack on others (G1494, G1497, C1501, G1504, G1505, U1506, G1517, G1529, G1530, and C1533). A series of equimolar reactions at increasing dilution imply an association constant range of 1.4-7.0 X 10(7) M-1.

Bacteriocins↗
PubMed1983

Crosslinking of Escherichia coli 50S ribosomal subunits with chlorambucilyl oligoprolyl phenylalanyl-tRNA molecular rulers.

A series of P-site probes, chlorambucilyl-(Pro)n-Phe-tRNAPhe, were prepared and reacted with poly(U)-directed Escherichia coli MRE 600 ribosomes. Upon binding of the probes to ribosomes, 90% of the cpm bound were not released following subsequent interaction with puromycin. In the absence of poly(U) or in the presence of poly(C), binding was limited to the amount of cpm bound if ribosomes were incubated in the presence of puromycin before adding modified tRNA and poly(U). AcPhe-tRNAPhe was a competitive inhibitor of chlorambucilyl Phe-tRNAPhe. Binding to 50S subunits was strongly stimulated by poly(U), while binding to 30S subunits was not. Crosslinked 50S proteins were analyzed by two-dimensional gel electrophoresis. Crosslinking with molecular rulers containing zero prolines led to poly(U)-dependent labeling of L1 and L27. With rulers containing five prolines, L6, L25, L28, and the group L18,23,24 were labeled. Analysis of crosslinked ribosomal RNA on sucrose density gradients revealed almost no cpm in the 16S or 23S peaks, but only in the 5S peaks. This was observed with molecular rulers containing either zero or five proline residues.

Binding, Competitive↗
PubMed1983

Veratrum alkaloids in sneezing-powder a potential danger.

Sneezing-powders containing pulverized root of veratrum album (white hellebore) have recently been marketed in the Scandinavian countries. The powder, imported from the Federal Republic of Germany, has caused severe intoxications.

Child↗