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Biomedical subjects

T J Thomas

Publications and source records attributed to T J Thomas.

At least 55 records · Page 3Linked to original sources
PubMed1999

Interaction of pilocarpine with latanoprost in patients with glaucoma and ocular hypertension.

PURPOSE: To study any interaction between pilocarpine and latanoprost when administered together, and to determine the optimal timing of dosage to maximize reduction of intraocular pressure (IOP). METHODS: Nineteen adult patients with either primary open-angle glaucoma or ocular hypertension participated in a single-center, prospective case study with masked observer. After a baseline measurement of IOP during treatment with latanoprost was obtained, initial treatment with pilocarpine three times daily was added without bedtime administration. This was followed by three different dose regimens in which pilocarpine was administered four times daily, altering the bedtime pilocarpine dose to precede the latanoprost dose by 1 hour, or to follow it by 10 minutes or 1 hour. Intraocular pressure was measured at 8:00 AM and 75 minutes after administration of the morning dose of pilocarpine. RESULTS: Comparison of IOP at 8:00 AM with baseline showed no significant change when pilocarpine was taken three times daily, or when pilocarpine was taken four times daily when the bedtime dose preceded administration of latanoprost by 1 hour. There were significant decreases in IOP versus baseline when the bedtime dose of pilocarpine was taken simultaneously with or 1 hour after administration of latanoprost. Application of pilocarpine immediately after the 8:00 AM IOP measurement revealed a significant additional decrease in pressure. There were no significant differences between dosage schedules in the magnitude of the additional reduction in IOP. CONCLUSION: The order and timing of administration of pilocarpine and latanoprost can significantly alter their ocular hypotensive activity. Pilocarpine is most effective when administered four times daily, and when the bedtime dose is administered 1 hour after administration of latanoprost.

Aged↗
PubMed1998

Induction of p21 (CIP1/WAF1/SID1) by estradiol in a breast epithelial cell line transfected with the recombinant estrogen receptor gene: a possible mechanism for a negative regulatory role of estradiol.

Estrogens stimulate the growth of a majority of estrogen receptor (ER)-positive breast cancer cells. In contrast, estradiol exerted a 75% inhibition of DNA synthesis in the MCF-10AE(wt5) cell line, obtained by the transfection of the ER gene into a normal breast epithelial cell line, MCF-10A. The estradiol-mediated growth inhibitory effect was reversed by ICI 164384, a pure anti-estrogen. Analysis of cell cycle by flow cytometry showed a significant increase of G1 cells by estradiol treatment compared to controls. To understand the mechanism of action of estradiol on MCF-10AE(wt5) cells, we examined the level of a cyclin dependent kinase inhibitor (CKI), p21, by Western blot analysis. Our results showed a 5- to 10-fold increase in the level of p21 in estradiol-treated MCF-10AE(wt5) cells compared to controls. ICI 164384 reversed estradiol-mediated induction of p21. Northern blot analysis of p21 mRNA indicated that estradiol stimulated its message in MCF-10AE(wt5) cells. Analysis of a panel of 6 breast cancer cell lines showed the absence of p21 protein, whereas it was present at a very low level in MCF-10A cells. Comparison of p21 in MCF-10A and MCF-10AE(wt5) cells showed an abundance of p21 in the ER-transfected cells. However, this p21 appears to be inactive in the absence of estradiol. These results suggest a p21-mediated pathway as a possible mechanism for the growth inhibitory effects of estradiol on at least a subset of ER-transfected cell lines.

Breast↗
PubMed1997

Effects of natural and synthetic polyamines on the conformation of an oligodeoxyribonucleotide with the estrogen response element.

We studied the effects of natural and synthetic polyamines on the conformation of an oligodeoxyribonucleotide (ODN1) harboring the estrogen response element (ERE) by circular dichroism (CD) spectroscopy and polyacrylamide gel electrophoresis. Putrescine and spermidine had no marked effect on the CD spectrum of ODN1. In contrast, spermine provoked and stabilized two characteristic changes in the CD spectrum. The first change was indicated by an increase in the intensity of the CD band at 280 nm at 0.5 mM spermine in Tris-HCl buffer containing 50 mM NaCl. This change appears to be related to changes in base tilt and conformational alterations similar to A-DNA. At 1-2 mM spermine, the CD spectrum was characterized by a loss of positive bands at 220 and 270 nm. This change might have contributions from polyamine-induced condensation/aggregation of DNA. Spectral measurements were also conducted in Tris-HCl buffer containing 150 mM NaCl to minimize contributions from condensation and aggregation of ODN1. Under these conditions, CD spectral changes were retained by (ODN1), although the magnitude of the change was diminished. In contrast, a control oligdeoxyribonucleotide (ODN2) having similar base composition did not show any significant change in the CD spectrum in the presence of 150 mM NaCl and 2 mM spermine. The changes in the CD spectrum of ODN1 were highly sensitive to polyamine structure, as evidenced by experiments using spermine analogs with altered number of -CH2- groups separating the amino and imino groups. Electrophoretic mobility shift analysis further showed ODN1 stabilization by spermine and its analogs. These data demonstrate the ability of an ODN containing ERE to undergo conformational transitions in the presence of polyamines and suggest a possible mechanism for polyamine-mediated alterations in the interaction of estrogen receptor with ERE.

Base Sequence↗
PubMed1997

Effects of chain length modification and bis(ethyl) substitution of spermine analogs on purine-purine-pyrimidine triplex DNA stabilization, aggregation, and conformational transitions.

The natural polyamines--putrescine, spermidine, and spermine--are known to stabilize pyrimidine-purine-pyrimidine and purine-purine-pyrimidine triplex DNA formation. We studied the ability of two tetramine and two pentamine analogs of spermine and their bis(ethyl) derivatives to stabilize triplex DNA formation between 5'-TG3TG4TG4TG3T-3' and its target duplex probe, consisting of the oligonucleotides 5'-TCGAAG3AG4AG4AG3A-3' and 5'-TCGATC3TC4TC4TC3T-3'. We used electrophoretic mobility shift assay (EMSA), melting temperature (Tm) measurements, and circular dichroism (CD) spectroscopy to evaluate the effects of these novel polyamine analogs on triplex DNA stability, dissociation constants, aggregation, and conformation. In general, pentamines were more efficacious than tetramines in stabilizing triplex DNA, although most of the polyamines with pendant free amino groups caused DNA aggregation below 50% conversion to triplex DNA. Ethyl substitution of these pendant amino groups lowered their efficacy approximately 2-fold in stabilizing triplex DNA; however, this effect was more than compensated for by the lack of DNA aggregation in the presence of bis(ethyl)polyamines. A concentration-dependent increase in the Tm of triplex DNA was observed in the presence of polyamines. CD spectral measurements showed distinct differences in the conformation of triplex DNA stabilized in the presence of polyamines compared to the CD spectra of the oligonucleotides alone. Temperature-dependent CD spectra of triplex DNA showed monophasic melting in the absence and presence of polyamines, suggesting duplex/triplex --> single-stranded DNA transition. These results indicate that structural modifications of polyamines is an effective strategy to develop triplex DNA-stabilizing ligands, with potential applications in antigene therapeutics.

Circular Dichroism↗
PubMed1997

Mechanism of action of a tyrphostin, 3,4-dihydroxy-alpha-cyanothiocinnamamide, in breast cancer cell growth inhibition involves the suppression of cyclin B1 and the functional activity of cyclin B1/p34cdc2 complex.

Tyrphostins are a group of compounds specifically targeted for the inhibition of tyrosine phosphorylation in signal transduction pathways. We studied the effects of a tyrphostin, 3,4-dihydroxy-alpha-cyanothiocinnamamide (tyrphostin-47), on hormone-responsive MCF-7 and hormone-unresponsive MCF-7-5C cell growth by DNA analysis for a period of 10 days. The growth of both cell lines was inhibited by this drug at 50 and 100 microM concentrations. Flow cytometric analysis showed that tyrphostin treatment caused a significant delay in the progression of MCF-7 cells through G1 and S phases of the cell cycle. The level of cyclin B1, a component of the mitosis promoting factor (MPF), was reduced by 90% in the presence of 100 microM tyrphostin. The other component of MPF, p34cdc2 kinase, was not affected; however, its functional activity was dramatically reduced, as determined by histone H1 phosphorylation assay. In contrast, G1 cyclins (D1 and E) and tyrosine kinase activity were not markedly affected by tyrphostin-47, as determined by Western immunoblot detection with specific antibodies. Our results suggest that a possible mechanism of tyrphostin action in breast cancer cells might involve the suppression of cyclin B1 and inhibition of the functional activity of cyclin B1/p34cdc2 complex. Our data indicate that the cell cycle machinery might be a target for developing novel drugs for breast cancer.

Breast Neoplasms↗
PubMed1997

Immunoisolation of tumor cells: generation of antitumor immunity through indirect presentation of antigen.

In recent years, a number of cancer therapies have been developed based on the augmentation of the immune response in the tumor-bearing individual. We have developed a novel approach to such therapies, using a membrane-bound immunoisolation device containing tumor cells to enhance the immune response of mice to the adenocarcinoma, MCA-38. Use of an immunoisolation device for cancer immunotherapy has several advantages. First, safety is enhanced because the reintroduced tumor cells are sequestered within the device. Second, the immunoisolation device permits introduction and maintenance of live cells that can stimulate the immune system for extended periods. Finally, the cells can be quantitatively removed at the end of the treatment period. Cells derived from the murine adenocarcinoma cell line MCA-38 were implanted into mice within a membrane-enclosed (immunoisolation) device. After 3 weeks, the animals were challenged by injection of free tumor cells. All of the control animals (lacking implants or with empty devices) developed tumor at the challenge site, whereas all of the animals that received devices containing tumor cells remained tumor free. The animals also remained tumor free after a second challenge. It is our hypothesis that antigens shed from tumor cells within the device are taken up by antigen-presenting cells of the host and thus indirectly initiate the activation of the immune system.

Adenocarcinoma↗
PubMed1997

Yohimbine elimination in normal volunteers is characterized by both one- and two-compartment behavior.

We sought to determine the safety, pharmacodynamic response, and single- and multiple-dose pharmacokinetic profile of yohimbine hydrochloride. Thirty-two healthy volunteers received 6 days of yohimbine, 5.4 mg 3 times daily (t.i.d.), 10.8 mg t.i.d., 16.2 mg t.i.d., or 21.6 mg twice daily (b.i.d.), with determination of plasma catecholamine levels and mood/anxiety-inventory scores. The pharmacokinetic profile of yohimbine was determined after the first and last dose. Yohimbine exhibited one-compartment elimination in most subjects, with dose-dependent increases in maximal concentration (Cmax) and area under the curve (AUC) but no evidence of drug accumulation. At least two subjects in each cohort exhibited two-compartment elimination of yohimbine, with nonsignificant increases in day 7 AUC, Cmax, and terminal elimination half-life (t1/2beta). Plasma catecholamine levels increased significantly in relation to both average yohimbine AUC and Cmax, but there were no significant effects on heart rate, blood pressure, or anxiety/mood-inventory scores. The single- and multiple-dose pharmacokinetic profile of yohimbine exhibits a substantial degree of interpatient and intrapatient variability, possibly resulting from variability in first-pass and hepatic metabolism. There is a significant correlation between plasma norepinephrine levels and yohimbine AUC or Cmax. Further multiple-dose studies are warranted definitively to address the relation between yohimbine AUC or Cmax and pharmacologic effect.

Adult↗
PubMed1997

Pyrimidine-purine-pyrimidine triplex DNA stabilization in the presence of tetramine and pentamine analogues of spermine.

The formation and stability of triplex DNA were investigated in the presence of a number of tetramine (+4) and pentamine (+5) derivatives of spermine with altered spacing between the positive charges and bis(ethyl) substitution of pendant amino groups. Thermal denaturation profiles were measured for the duplex and triplex forms of poly[d(TC)].poly[d(GA)] and poly(dA).poly(dT); in both cases the pentamines were more effective than the tetramines in increasing the melting temperature (Tm) of the triplexes. Some structural effects were evident, although bisethylation of the polyamines had only a minor effect on the Tm of pyrimidine-purine-pyrimidine triplexes. Relative association constants to poly(dT).poly(dA).poly(dT) and poly[d(AT)] were measured by an ethidium competition assay. These results demonstrated tighter binding of the pentamines by a factor of up to 10-fold, but bisethylation consistently decreased the relative association constants to the triplex. A third assay involving transmolecular triplex formation between separated pyrimidine-purine tracts in plasmid DNA was also employed. Again the pentamines promoted triplex formation at lower concentrations than the tetramines but structural effects were very important in determining the degree of triplex formation. These results may be important for the design of suitable ligands to stabilize triplex DNA in antigene therapeutics and to elucidate the mechanism of action of polyamine analogues as antitumor drugs.

Binding, Competitive↗
PubMed1996

Structural specificity effects of trivalent polyamine analogues on the stabilization and conformational plasticity of triplex DNA.

Natural polyamines, i.e. putrescine, spermidine and spermine, are excellent promoters of triplex DNA. Using melting temperature (Tm) measurements and CD spectroscopy, we found that structural alterations on spermidine backbone, including methylation, or acetylation at the N1-, N4- and/or N8-positions had a profound influence on the stability and conformation of poly(dA).2poly(dT) triplex. The conformation of the polynucleotide complex underwent sequential changes from B-DNA to triplex DNA as the concentration of spermidine increased from 0 to 50 microM in a buffer containing 10 mM sodium cacodylate and 1 mM EDTA (pH 7.2). At 60 microM spermidine, the CD spectrum of triplex DNA was comparable with that of psi-DNA, with a strong positive band centred around 260 nm. A negative band was also found at 295 nm. At higher concentrations of spermidine, however, the intensity of the positive band progressively decreased and the peak intensity was found at a 1:0.3 molar ratio of DNA phosphate:spermidine. Temperature-dependent CD analysis showed that the psi-DNA structure melted to single-stranded DNA at temperatures above the Tm determined from the absorbance versus temperature profile. Comparable effects were exerted on the conformation of triplex DNA by Co(NH3)6(3+), an inorganic trivalent cation. Substitution of the N4-hydrogen of spermidine by a cyclohexyl ring or the fusion of the N4-nitrogen in a cyclic ring system, as in piperidine, enhanced the ability of spermidine analogues to stabilize triplex and psi-DNA forms over a wider concentration range compared with spermidine. These data demonstrate a differential effect of trivalent cations in stabilizing triplex DNA and provoking unusual conformations such as psi-DNA. Synthetic homologues of spermidine that stabilize triplex DNA over a wider range of concentrations than that stabilized by spermidine itself might have potential therapeutic applications in the development of an anti-gene strategy against several diseases, including cancer and AIDS.

DNA↗
PubMed1996

Effects of yohimbine on autonomic measures are determined by individual values for area under the concentration-time curve.

A study was conducted to examine tolerability and pharmacodynamics of single doses of yohimbine in healthy volunteers using measures of mood, heart rate, blood pressure, and serum catecholamine levels. Participants were given single oral doses of yohimbine hydrochloride as high as 21.6 mg. Plasma concentrations of yohimbine, epinephrine, norepinephrine, and MHPG (3-methoxy-4-hydroxyphenylethylene-glycol) were quantified by means of high-performance liquid chromatography with electrochemical detection. Mood was assessed by visual analogue scale (VAS), the Profile of Mood States, and the Spielberger State Anxiety Index. Yohimbine was well tolerated and rapidly absorbed and eliminated. Dose-related increases in area under the concentration-time curve (AUC) were observed. Administration of yohimbine in the presence of a high fat meal diminished both the rate and extent of drug absorption. Significant intersubject variability in the pharmacokinetic parameters of yohimbine was observed, with some individuals exhibiting greatly increased oral bioavailability of yohimbine. Increases in blood pressure, respiratory rate, plasma catecholamine levels, and total VAS score were observed in participants with elevated AUC values. The AUC of yohimbine had the largest effect on total VAS score. The results indicate that higher doses of yohimbine are both well tolerated and produce dose-related increases in AUC, which are associated with more pronounced autonomic effects. Increases in respiratory rate and plasma MHPG appear to be the most reliable pharmacodynamic measures for single oral doses of yohimbine. Individual differences in the pharmacokinetics of yohimbine are important in determining pharmacodynamic effects and should be considered in evaluations of its clinical effectiveness.

Adrenergic alpha-Antagonists↗
PubMed1996

Structure-activity relations of S-adenosylmethionine decarboxylase inhibitors on the growth of MCF-7 breast cancer cells.

SAMDC is a key enzyme in the biosynthesis of spermidine and spermine, 2 polyamines that are essential for cell proliferation. Inhibition of polyamine biosynthesis is often targeted as a therapeutic strategy to suppress cancer cell growth as these cells contain elevated levels of polyamines. We examined the effect of a new group of SAMDC inhibitors, CGP33829, CGP35753, CGP36958, CGP39937, and CGP48664, (obtained from Ciba-Geigy, Basel, Switzerland), and their parent compound, MGBG, on the proliferation of MCF-7 breast cancer cells. MGBG had minimal effects on the proliferation of MCF-7 cells up to 6 microM concentration. In contrast, CGP48664 and CGP39937, containing 2 aromatic rings that delocalize the pi electron system of the backbone of MGBG, were potent inhibitors with 50% growth inhibition at 0.5 microM concentration. Other CGP compounds were less effective in inhibiting cell growth. The ability of CGP48664 to inhibit MCF-7 cell proliferation was related to its ability to inhibit SAMDC and to consequently deplete spermidine and spermine levels in the cell. Exogenous spermidine and spermine could reverse the growth inhibitory effects of this compound. CGP compounds also increased the activity of ODC, another enzyme involved in polyamine biosynthesis. Northern blot analysis of mRNA from MCF-7 cells progressing in cell cycle after G1 synchronization did not show an increase in ODC mRNA level by CGP48664. These data demonstrate structure-activity relationships of a series of MGBG derivatives on cell growth, enzyme activities, and polyamine biosynthesis in a hormone-responsive breast cancer cell line and suggest potential application of SAMDC inhibitors as therapeutic agents.

Acetyltransferases↗
PubMed1995

Defective signal-transduction pathways in T-cells from autoimmune MRL-lpr/lpr mice are associated with increased polyamine concentrations.

We previously reported that difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, exerted significant beneficial effects on the lifespan and disease expression of MRL-lpr/lpr mice, which spontaneously develop a lupus-like syndrome. Polyamine levels in splenic T-cells of MRL-lpr/lpr mice were significantly higher than those of Balb/c mice. In the present investigation, we examined the role of endogenous polyamines in transmembrane Ca2+ influx, generation of InsP3 and tyrosine phosphorylation of the p56lck protein in concanavalin A-stimulated splenic T-cells. Cytosolic free calcium concentrations ([Ca2+]i) in concanavalin A-stimulated T-cells of MRL-lpr/lpr and Balb/c mice were 250 +/- 25 and 450 +/- 42 nM respectively. Treatment of MRL-lpr/lpr mice with DFMO increased [Ca2+]i to 360 +/- 30 nM (P < 0.05). InsP3 levels of concanavalin A-stimulated MRL-lpr/lpr splenic T-cells were only 20% higher than those of unstimulated controls, whereas those of Balb/c T-cells were 90% higher. DFMO treatment increased InsP3 levels in concanavalin A-treated MRL-lpr/lpr T-cells to 67%. Western-blot analysis showed a 7-fold higher level of p56lck phosphorylation of MRL-lpr/lpr splenic T-cells than that of Balb/c mice. DFMO treatment reduced tyrosine phosphorylation of p56lck of MRL-lpr/lpr mice significantly (P < 0.001). Two-colour flow-cytometric analysis revealed no significant difference in the CD4+/CD8+ ratio in splenic T-cells of MRL-lpr/lpr mice after DFMO treatment. Polyamine levels in splenocytes were significantly reduced by DFMO treatment. These data show that DFMO treatment could alter signal-transduction pathways of splenic T-cells of MRL-lpr/lpr mice. Increased levels of polyamines in T-cells of untreated lpr mice contribute to defective signal-transduction pathways and the pathogenesis of lupus-like symptoms.

Animals↗
PubMed1995

Triplex-DNA stabilization by hydralazine and the presence of anti-(triplex DNA) antibodies in patients treated with hydralazine.

Hydralazine is an antihypertensive drug that elicits andti-nuclear antibodies in patients as an adverse effect. We investigated the ability of hydralazine to promote/stabilize the triplex DNA form of poly(dA).2poly(dT). Under conditions of low ionic strength, the polynucleotide melted as a double helix with a melting temperature (Tm) of 55.3 degrees C. Hydralazine destabilized this duplex form by reducing its Tm to 52.5 degrees C. Spermidine (2.5 microM), a natural polyamine, provoked the triplex form of poly(dA)-.2poly(dT) with two melting transitions, Tm1 of 42.8 degrees C corresponding to triplex-->duplex+single-stranded DNA and Tm2 of 65.4 degrees C, corresponding to duplex melting. Triplex DNA thus formed in the presence of spermidine was further stabilized by hydralazine (250 microM) with a Tm1 of 53.6 degrees C. A similar stabilization effect of hydralazine was found on triplex DNA formed in the presence of 5 mM Mg2+. CD spectra revealed conformational perturbations of DNA in the presence of spermidine and hydralazine. These results support the hypothesis that hydralazine is capable of stabilizing unusual forms of DNA. In contrast with the weak immunogenicity of DNA in its right-handed B-DNA conformation, these unusual forms are immunogenic and have the potential to elicit anti-DNA antibodies. To test this possibility, we analysed sera from a panel of 25 hydralazine-treated patients for anti-(triplex DNA) antibodies using an ELISA. Our results showed that 72% of sera from hydralazine-treated patients contained antibodies reacting toward the triplex DNA. In contrast, there was no significant binding of normal human sera to triplex DNA. Taken together our data indicate that hydralazine and related drugs might exert their action by interacting with DNA and stabilizing higher-order structures such as the triplex DNA.

Adolescent↗
PubMed1995

Suppression of c-myc oncogene expression by a polyamine-complexed triplex forming oligonucleotide in MCF-7 breast cancer cells.

Polyamines are excellent stabilizers of triplex DNA. Recent studies in our laboratory revealed a remarkable structural specificity of polyamines in the induction and stabilization of triplex DNA. 1,3-Diaminopropane (DAP) showed optimum efficacy amongst a series of synthetic diamines in stabilizing triplex DNA. To utilize the potential of this finding in developing an anti-gene strategy for breast cancer, we treated MCF-7 cells with a 37mer oligonucleotide to form triplex DNA in the up-stream regulatory region of the c-myc oncogene in the presence of DAP. As individual agents, the oligonucleotide and DAP did not downregulate c-myc mRNA in the presence of estradiol. Complexation of the oligonucleotide with 2 mM DAP reduced c-myc mRNA signal by 65% at 10 microM oligonucleotide concentration. In contrast, a control oligonucleotide had no significant effect on c-myc mRNA. The expression of c-fos oncogene was not significantly altered by the triplex forming oligonucleotide (TFO). DAP was internalized within 1 h of treatment; however, it had no significant effect on the level of natural polyamines. These data indicate that selective utilization of synthetic polyamines and TFOs might be an important strategy to develop anti-gene-based therapeutic modalities for breast cancer.

Antineoplastic Agents↗
PubMed1995

Polyamine-mediated conformational perturbations in DNA alter the binding of estrogen receptor to poly(dG-m5dC).poly(dG-m5dC) and a plasmid containing the estrogen response element.

The binding estrogen receptor (ER) to the upstream regions of estrogen-responsive genes, the estrogen response elements (ERE), is of fundamental importance in the regulation of gene expression by estradiol. Multiple cell-specific factors affect ER-ERE binding and modulate the responses of estradiol. We studied the role of polyamines in the recognition of ER, a ligand-activated transcription factor, with a left-handed Z-DNA forming polynucleotide as well as with a plasmid containing ERE. Polyamines are cellular organic cations with multiple functions in cell growth and differentiation. Polyamines induce Z-DNA conformation in alternating purine-pyrimidine sequences. To understand the role of polyamine-induced DNA conformational transition in ER-DNA interaction, we studied the binding of partially purified rabbit uterine ER to poly(dG-m5dC).poly(dG-m5dC). The induction of Z-DNA in the polynucleotide was monitored by circular dichroism and ultraviolet spectroscopic measurements. Binding of ER to poly(dG-m5dC).poly(dG-m5dC) increased from 15% to approx. 50-60% in the presence of 7.5 mM putrescine, 0.5 mM spermidine or 0.25 mM spermine. Maximal binding of ER to the polynucleotide was observed near the midpoint of the B-DNA to Z-DNA transition of the polynucleotide. N1-acetyl spermidine and N1-acetyl spermine facilitated the B-DNA to Z-DNA transition and the binding of ER although they were less effective than the unacetylated analog. Co(NH3)6(3+), a trivalent inorganic cation, also provoked the B-DNA to Z-DNA transition of the polynucleotide and increased its binding to ER. At higher polyamine concentrations, there was an inhibition of ER binding to the polynucleotide. In the presence of polyamines, the binding of ER to a plasmid containing ERE was 2-3-fold higher than that to a control plasmid devoid of ERE. Polyamine-induced facilitation of ER-ERE binding was also confirmed by gel mobility shift assay. Our data indicate that conformational perturbations, similar to that of the early stages of B-DNA to Z-DNA transition, are important in the recognition of ER and ERE.

Animals↗
PubMed1995

Cyclins and autoimmunity: cyclin B1 gene expression and restriction fragment length polymorphism in lupus-prone mice.

Cyclin B1 is the major component of M-phase promoting factor that plays a major role in the G2/M transition of cell cycle. We examined the expression of cyclin B1 at the protein and mRNA levels in the thymus of 12-week-old autoimmune and normal mice. We found an abundance of cyclin B1 protein (58 kDa) in the thymus of lupus-prone MRL-lpr/lpr mice, whereas the level of this protein was negligible in other strains. The level of the predominant cyclin B1 mRNA (1.7 kb) species was not markedly different in these strains, suggesting post transcriptional modification of cyclin B1 in the thymus of MRL-lpr/lpr mice. Southern blot analysis of cyclin B1 gene showed multiple forms of cyclin B1-related sequences in various murine genomes. Flow cytometry showed a significantly higher level of cells in the G2/M phase and a significantly lower level in the S phase in thymocytes of MRL-lpr/lpr compared to that in normal BALB/c mice, indicating an alteration of cell cycle machinery in thymocytes of MRL-lpr/lpr mice. Taken together, these data show that an upregulation of cyclin B1 protein and accumulation of thymocytes at the G2/M phase in MRL-lpr/lpr mice might play an important role in the aberrant development of T cells in these mice.

Animals↗