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Biomedical subjects

I Winicov

Publications and source records attributed to I Winicov.

At least 19 recordsLinked to original sources
PubMed2000

Alfin1 transcription factor overexpression enhances plant root growth under normal and saline conditions and improves salt tolerance in alfalfa.

Plant root development is an essential determinant of plant growth and crop yield that could be enhanced by induced changes in the expression of root-specific regulatory factors. We reported previously that Alfin1 binds DNA in a sequence-specific manner and that Alfin1 overexpression in transgenic alfalfa (Medicago sativa L.) enhances expression of the salt-inducible MsPRP2 gene in roots, suggesting that Alfin1 functions to regulate gene expression in roots. Here we show that Alfin1 is an essential gene for root growth and that its overexpression in transgenic plants confers a many-fold increase in root growth under normal and saline conditions. Alfin1-binding sites occur in promoters of genes expressed in roots of a wide variety of plant species and we propose that it is a general root growth regulator. Even though Alfin1 overexpression was under the control of the CaMV 35S promoter, plant shoot growth was not adversely affected. We show further that introduction of the Alfin1 transgene in plants confers a dominant characteristic that significantly increases plant growth and salt tolerance.

Caulimovirus↗
PubMed1999

Transgenic overexpression of the transcription factor alfin1 enhances expression of the endogenous MsPRP2 gene in alfalfa and improves salinity tolerance of the plants

Alfin1 cDNA encodes a putative transcription factor associated with NaCl tolerance in alfalfa (Medicago sativa L.). The recombinant protein binds DNA in a sequence-specific manner, including promoter fragments of the NaCl-inducible gene MsPRP2. Alfin1 function was tested in transgenic alfalfa under the control of the 35S promoter in the sense and antisense orientations with the endogenous MsPRP2 as a reporter gene. Calli overexpressing Alfin1 were more resistant to growth inhibition by 171 mM NaCl than vector-transformed controls, whereas calli expressing Alfin1 in the antisense orientation were more sensitive to NaCl inhibition. Transgenic plants overexpressing Alfin1 in the sense orientation grew well. In contrast, the antisense transgenic plants grew poorly in soil, demonstrating that Alfin1 expression is essential for normal plant development. Transgenic calli and plant roots overexpressing Alfin1 showed enhanced levels of endogenous MsPRP2 mRNA accumulation. However, MsPRP2 mRNA accumulation was also regulated in a tissue-specific manner, as shown in leaves of transgenic plants overexpressing Alfin1. These results suggest that Alfin1 acts as a transcriptional regulator in plants and regulates MsPRP2 expression in alfalfa. Alfin1 overexpressing transgenic plants showed salinity tolerance comparable to one of our NaCl-tolerant plants, indicating that Alfin1 also functions in gene regulation in NaCl tolerance.

Journal Article↗
PubMed1998

Alfin1, a novel zinc-finger protein in alfalfa roots that binds to promoter elements in the salt-inducible MsPRP2 gene.

Alfin1 cDNA, obtained by differential screening of a poly(A)+ library from salt-tolerant alfalfa cells, encodes a novel protein with a Cys4 and His/Cys3 putative zinc-binding domain that suggests a possible role for this protein in transcriptional regulation. We have expressed the cDNA in Escherichia coli and show that the recombinant Alfin1 protein binds DNA in a sequence-specific manner. The DNA recognition sequence was determined from individual clones isolated after four rounds of random oligonucleotide selection in gel retardation assays, coupled with PCR amplification of the selected sequences. The consensus binding site for Alfin1 is shown to contain two to five G-rich triplets with the conserved core of GNGGTG or GTGGNG in clones showing high-efficiency binding. DNA binding of the recombinant Alfin1 was inhibited by EDTA. Alfin1 mRNA was found predominantly in alfalfa roots. Growth of salt-sensitive Medicago sativa L on 171 mM NaCl led to a slight decrease in Alfin1 mRNA, while the salt-tolerant plants showed no decrease in Alfin1 mRNA levels. Interestingly, recombinant Alfin1 binds efficiently to three fragments of the MsPRP2 promoter, each containing consensus sequences identified by the random oligonucleotide selection. Since MsPRP2 transcripts were shown to be root-specific and accumulated in alfalfa roots in a salt-inducible manner, Alfin1 may play a role in the regulated expression of MsPRP2 in alfalfa roots and contribute to salt tolerance in these plants.

Amino Acid Sequence↗
PubMed1996

MNU affects mouse erythroleukemia cell differentiation at sub-cytotoxic doses.

MNU is a potent carcinogen and mutagen to various tissues. Molecular events during differentiation show particular sensitivity to MNU exposure. We have investigated the mouse erythroleukemia (MEL) cell differentiation in response to DMSO and the influence of subcytotoxic doses of MNU on this process to assess the role of MNU on the course of differentiation and specific gene expression in a single cell type. Differentiation was followed by determining the extent of hemoglobinization and beta-globin gene expression, which are representative measures of red cell maturation. In this study we have shown a delay and decrease in the extent of MEL cell differentiation by MNU exposure at the time of induction to differentiate, even at sub-lethal MNU concentrations. Once the differentiation process was initiated, exposure to MNU at sub-lethal doses showed a significantly smaller effect on the molecular course of events. Pre-treatment of MEL cells with MNU before DMSO induction did not affect differentiation. The MNU-induced delay in differentiation was reflected in the delayed appearance of beta-globin transcripts during the first 12 h post induction. However, transcription could not account for reduced hemoglobinization of the MNU-treated cells at 48 and 72 h post induction.

Animals↗
PubMed1995

Nuclear proteins that interact with the beta maj globin promoter start to accumulate in MEL cells within 12 hours of induction and RNA copies of the promoter successfully compete their binding in vitro.

The induction of differentiation in mouse erythroleukemia (MEL) cells by dimethylsulfoxide (DMSO) is characterized by increased transcription of globin genes. We have determined that DMSO treated cells increase the levels of nuclear factors capable of overall interactions with the beta maj globin promoter during the initial 24 h post induction, as measured by gel mobility analysis. Two unprocessed beta maj globin mRNA precursors, which are present in MEL cell nuclei early in differentiation, were previously shown to contain the 5' promoter flanking region, and thereby provided the nucleus with a pool of regulatory sequences in multiple RNA copies. We have studied the effect of RNA copies of the promoter region on binding interactions between DNA sequences of the beta maj globin promoter and nuclear factors that interact with these sequences. The promoter region RNA transcripts competed effectively for DNA binding proteins in vitro, while the antisense RNA from the same region did not. The most pronounced competition was observed with proteins from 12 h after DMSO induction, when the concentration of the DNA binding proteins was still increasing. Since the 'upstream' transcripts predominate at 12 h after DMSO induction, these results indicate that the promoter region transcripts may influence the equilibrium of binding between the beta maj globin promoter and the nuclear factors that bind to this region during DMSO induction.

Animals↗
PubMed1995

Post-transcriptional regulation of a salt-inducible alfalfa gene encoding a putative chimeric proline-rich cell wall protein.

A cDNA previously shown to identify a salt-inducible root-specific transcript in Medicago sativa was used to screen an alfalfa library for the corresponding genomic sequence. One positive clone was recovered. The nucleotide sequence of a subclone contained a 329 bp 5' region upstream of the first ATG codon, a 1143 bp coding segment, and a 447 bp 3'-untranslated region interrupted by a single 475 bp intron. Translation of the coding segment, which was designated MsPRP2, suggested it encodes a chimeric 40,569 Da cell wall protein with an amino-terminal signal sequence, a repetitive proline-rich sequence, and a cysteine-rich carboxyl-terminal sequence homologous to nonspecific lipid transfer proteins. The 3'-untranslated region of MsPRP2 contained a sequence similar to one found to destabilize mRNAs transcribed from the elicitor-regulated proline-rich protein gene PvPRP1. Transcription run-on experiments using nuclei from salt-sensitive and salt-tolerant alfalfa callus suggested that the accumulation of MsPRP2 transcripts in salt-tolerant alfalfa cells grown in the presence of salt is due primarily to increased mRNA stability. The MsPRP2 gene thus may be a useful model for studying post-transcriptional salt-regulated expression of cell wall proteins.

Amino Acid Sequence↗
PubMed1994

Two 10-bp regions are critical for phytochrome regulation of a Lemna gibba Lhcb gene promoter.

Two small regions of the promoter of an Lhcb gene encoding a light-harvesting chlorophyll a/b protein were identified as essential in conferring phytochrome responsiveness by using a transient expression assay. Initially, 5' deletion analysis of cabAB19, an Lhcb2 gene of Lemna, showed that sequences within the region from -174 to -104 relative to the start of transcription were necessary for phytochrome regulation. Internal deletion and substitution mutants were used to demonstrate that no additional phytochrome-responsive regions exist between -1600 and -174 in this promoter. A 171-bp fragment of the promoter extending from -239 to -69 was sufficient to impart phytochrome responsiveness to a minimal ubiquitin promoter that was not itself regulated by light. Specific binding of Lemna proteins to the region necessary for phytochrome responsiveness was demonstrated using in vitro polyacrylamide gel mobility shift assays and 1,10-phenanthroline copper ion footprinting. Further analysis of the region from -174 to -104 demonstrated that mutations in two separate 10-bp sequences, from -134 to -125 and from -114 to -105, could abolish phytochrome responsiveness; thus, there are two unique regions that are necessary for phytochrome regulation of this gene. One of these regions contains a CCAAT motif and the other a GATA motif. These motifs are conserved in the promoters of many Lhcb genes and may be important elements in the phytochrome responsiveness of this gene family.

Base Sequence↗
PubMed1990

Dynamic histone acetylation in alfalfa cells. Butyrate interference with acetate labeling.

Dynamic histone acetylation of alfalfa (Medicago sativa) was studied in suspension cultures by short-term labeling with radioactive acetate. The relative labeling rates for the acetylated histones were in order of decreasing incorporation; H3.2 greater than H3.1 greater than H4 greater than H2B.1 greater than H2A.3. Histone H3 showed at least seven sites of acetylation, histone H2B.1 had six sites and histone H4 had five sites. Low numbers of acetylation sites were observed for histone H2B.2 and all histone H2A variants. The mass ratio, steady state acetylation and dynamic acetylation between major variant H3.1 and minor variant H3.2 were approx. 2:1, 1:2 and 2:5, respectively. Treatment of alfalfa cells with 50 mM n-butyrate did not lead to histone hyperacetylation, but instead interfered with histone acetylation labeling by acetate. The extent of apparent inhibition increased with time and concentration of butyrate. It is likely that the conversion of butyrate to acetylCoA results in dilution of the specific radioactivity of [3H]acetate in the acetylCoA pool thereby inhibiting the labeling reaction. This interpretation is supported by 14C-labeling of alfalfa acetylated histones by [1-14C]butyrate.

Acetates↗
PubMed1990

Protein synthesis during acquisition of long-term facilitation is needed for the persistent loss of regulatory subunits of the Aplysia cAMP-dependent protein kinase.

Depending on the number or the length of exposure, application of serotonin can produce either short-term or long-term presynaptic facilitation of Aplysia sensory-to-motor synapses. The cAMP-dependent protein kinase, a heterodimer of two regulatory and two catalytic subunits, has been shown to become stably activated only during long-term facilitation. Both acquisition of long-term facilitation and persistent activation of the kinase is blocked by anisomycin, an effective, reversible, and specific inhibitor of protein synthesis in Aplysia. We report here that 2-hr exposure of pleural sensory cells to serotonin lowers the concentration of regulatory subunits but does not change the concentration of catalytic subunits, as assayed 24 hr later; 5-min exposure to serotonin has no effect on either type of subunit. Increasing intracellular cAMP with a permeable analog of cAMP together with the phosphodiesterase inhibitor isobutyl methylxanthine also decreased regulatory subunits, suggesting that cAMP is the second messenger mediating serotonin action. Anisomycin blocked the loss of regulatory subunits only when applied with serotonin; application after the 2-hr treatment with serotonin had no effect. In the Aplysia accessory radula contractor muscle, prolonged exposure to serotonin or to the peptide transmitter small cardioactive peptide B, both of which produce large increases in intracellular cAMP, does not decrease regulatory subunits. This mechanism of regulating the cAMP-dependent protein kinase therefore may be specific to the nervous system. We conclude that during long-term facilitation, new protein is synthesized in response to the facilitatory stimulus, which changes the ratio of subunits of the cAMP-dependent protein kinase. This alteration in ratio could persistently activate the kinase and produce the persistent phosphorylation seen in long-term facilitated sensory cells.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Sphingosine interacts directly with the receptor complex to inhibit thyrotropin-releasing hormone binding.

Sphingosine inhibition of [3H] [N3-Me-His] TRH (MeTRH) binding, previously shown to be independent of its effects on protein kinase-C, has been further characterized in GH3 cell membranes and in a partially purified, digitonin-solubilized receptor preparation. In membranes, as in intact cells, sphingosine inhibited [3H]MeTRH binding by decreasing receptor affinity, but, in contrast to its effect in intact cells, did not affect the number of available binding sites. The inhibition of binding was linear up to 75 microM sphingosine (in the presence of 100 microM BSA at 0.1 mg membrane protein/ml), yielding an apparent Ki of 51 microM. Since GTP decreases the affinity for MeTRH binding in GH3 cell membranes, we studied interactions between GTP and sphingosine. While the effects of low concentrations of GTP gamma S and sphingosine were additive, sphingosine inhibition of MeTRH binding surpassed and was not affected by the addition of maximally inhibitory concentrations of GTP gamma S. Also, sphingosine (75 microM) did not affect the ability of a maximally effective dose of TRH to stimulate the low Km GTPase (vehicle, +35 +/- 5%; sphingosine, +32 +/- 10%); there was a 25% decrease in total GTPase activity in the presence of sphingosine. MeTRH binding to digitonin-solubilized receptors, which had properties similar to those described previously by others, including no effect of GTP on binding, was inhibited by sphingosine. In solubilized receptors, as in membranes, sphingosine caused a decrease in apparent affinity without changes in the number of binding sites. These data suggest that sphingosine interacts directly with the TRH receptor [or an associated factor(s) in the receptor complex] to decrease affinity by a mechanism that does not involve uncoupling of G-proteins.

Digitonin↗
PubMed1989

Beta globin gene transcripts originating in the promoter region during early hexamethylene bisacetamide and dimethylsulfoxide induction of Friend erythroleukemia cells.

The beta-globin transcripts which are induced by dimethylsulfoxide (DMSO) and hexamethylene bisacetamide (HMBA) have been characterized in order to assess potential differences in their mechanisms of induction. Transcripts which initiate in the 5' flanking promoter region are likely indicators of promoter accessibility and were therefore characterized during the time course of induction with each inducer in Friend Erythroleukemia cells. S1 analysis with probes labeled at - 12 or +82 relative to the (+1) cap site showed no major differences between 5' ends of the upstream initiated transcripts in cells induced by DMSO or HMBA. We detected several upstream bands with each inducer corresponding to beta-globin transcripts with 5' ends between - 190 and -55 relative to the cap site and found that cells induced with DMSO and HMBA show a similar transcription response as measured by initiation in the 5' flanking region of the beta-globin gene. Interestingly, the upstream initiated transcripts reach their peak concentration levels much earlier in the time course of induction than do the mRNA transcripts with 5' ends at the major (+1) cap site. Northern blot analysis detected the upstream initiated transcripts as early as 16 hours after induction with DMSO, primarily in unprocessed large transcripts. We find that the promoter region containing transcripts constitute a higher percent of total beta-globin transcripts at the start of the induction and may therefore have an early function in the multistep induction process.

Acetamides↗
PubMed1989

Receptor density determines secretory response patterns mediate by inositol lipid-derived second messengers. Comparison of thyrotropin-releasing hormone and carbamylcholine actions in thyroid-stimulating hormone-secreting mouse pituitary tumor cells.

Signal transduction by thyrotropin-releasing hormone (TRH) and carbamylcholine (CCH) in some cells is mediated by inositol lipid hydrolysis forming the second messengers, inositol 1,4,5-trisphosphate (I-1,4,5-P3) and 1,2-diacylglycerol, and causing elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i). In mouse thyrotropic tumor (TtT) cells, maximally effective doses of TRH caused biphasic stimulation of thyroid-stimulating hormone (TSH) secretion, whereas CCH stimulated monophasic sustained TSH secretion without a burst phase. TRH, at maximally effective doses, stimulated a rapid marked increase in I-1,4,5-P3 which was associated with a rapid elevation of [Ca2+]i to approximately 1000 nM, whereas maximally effective doses of CCH caused little increase in I-1,4,5-P3 and no burst elevation of [Ca2+]i. Both TRH and CCH caused sustained modest (to 210-280 nM) elevations of [Ca2+]i which were inhibited by voltage-sensitive channel-blocking agents and stimulated sustained hydrolysis of inositol lipids. CCH-like responses were observed when TtT cells were stimulated by low doses of TRH. In TtT cells prepared from five tumors, the ratio of the number of TRH receptors to muscarinic receptors ranged from 10 to 40:1. Lastly, CCH-like responses were observed with maximally effective doses of TRH when the TRH receptor number was down-regulated to a level similar to that of muscarinic receptors. These data suggest that the kinetic pattern of stimulated TSH secretion caused by secretagogues that use the inositol lipid signal transduction pathway is determined by the density of receptors. In particular, there appears to be a minimal number of receptor-ligand complexes which is required to generate rapidly sufficient I-1,4,5-P3 to release intracellular Ca2+ and cause a secretory burst.

Animals↗
PubMed1989

Differential Histone Acetylation in Alfalfa (Medicago sativa) Due to Growth in NaCl : Responses in Salt Stressed and Salt Tolerant Callus Cultures.

The steady state distribution of histone variant proteins and their modifications by acetylation were characterized in wild type and salinity stress adapted alfalfa (Medicago sativa). Isotopic labeling detected dynamic acetylation at four sites in the histone H3 variants and five sites in histones H4 and H2B. Histone variant H3.2 was the most highly acetylated histone with 25% higher steady state acetylation and a two- to threefold higher acetylation labeling than histone H3.1. Histone phosphorylation was limited to histone variants H1.A, H1.B, and H1.C and to histone H2A.3, which was also acetylated. Histone variant composition was unaffected by cellular exposure to NaCl. Histone acetylation was qualitatively similar in salt-tolerant and salt-sensitive cells under normal growth conditions. However, short term salt stress in salt sensitive cells or continued growth at 1% NaCl in salt tolerant cells led to major increases in the multiacetylated forms of histone H4 and the two variants of histone H3. These changes were more pronounced in the diploid than in the tetraploid alfalfa strains. The increase in multiacetylation of core histones serves as an in vivo reporter suggesting an altered intranuclear ionic environment in the presence of salt. It may also represent an adaptive response in chromatin structure to permit chromatin function in a more saline intranuclear environment.

Journal Article↗
PubMed1988

Sphingosine inhibits thyrotropin-releasing hormone binding to pituitary cells by a mechanism independent of protein kinase C.

Sphingosine inhibited [3H]methylhistidine-thyrotropin-releasing hormone (MeTRH) binding to intact GH3 cells and to GH3 membranes. This inhibition was dependent on the concentration of sphingosine and on the ratio of sphingosine to cell number (or membrane protein) and was partly reversed by washing. In intact cells, the IC50 was 63 microM (1.8 X 10(6) cells/ml; 2 nM MeTRH), and 100 microM sphingosine was found, by Scatchard analysis, to increase the apparent dissociation constant (Kd) from 1.1 +/- 0.3 to 6.5 +/- 2.3 nM and to decrease the maximal binding capacity (Bmax) to 41 +/- 9.5% of control. Kinetic analysis showed that the major effect of sphingosine on Kd was due to a marked decrease in the apparent association rate constant for MeTRH from 2.5 +/- 0.4 X 10(5) M-1 s-1 to 0.10 +/- 0.015 X 10(5) M-1 s-1. At 100 microM, sterylamine was as effective as sphingosine in inhibiting MeTRH binding, whereas sphinganine was less effective, and psychosine and steroylsphingosine were without effect. The following observations show that sphingosine inhibition of MeTRH binding did not involve protein kinase C. The IC50 for sphingosine inhibition of MeTRH binding was the same in GH3 cells that had been incubated with 1 microM phorbol 12-myristate 13-acetate for 16 h, to "down-regulate" protein kinase C, as in control cells. Sphingosine inhibited MeTRH binding to membranes isolated from GH3 cells that contain very little protein kinase C activity. In GH3 membranes, 100 microM sphingosine increased the Kd for MeTRH from 3.4 +/- 0.1 to 13 +/- 3.1 nM but did not significantly decrease Bmax (12 +/- 5.0% of control, p greater than 0.05). And, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, an inhibitor of protein kinase C, failed to decrease MeTRH binding to intact GH3 cells or to membranes, and did not interfere with the effects of sphingosine. These data show that sphingosine and its analogs have complex actions to inhibit MeTRH binding to GH3 cells, at least some of which are independent of protein kinase C, and thereby demonstrate that sphingolipids cannot be used as specific inhibitors of protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

RNA processing of beta-globin transcripts containing 5' flanking and structural gene sequences.

RNA species have been identified in murine erythroid cells which contain both 5' flanking and structural gene sequences from the beta maj globin gene. Two nonpolyadenylated RNA transcripts, average 3700 and 1800-1900 nucleotides long, were identified by denaturing agarose gel electrophoresis and were found to hybridize to both 5' and 3' beta maj globin flanking sequences. This finding suggests that transcription initiated in the 5' flanking region does proceed past the polyadenylation site. The apparent higher concentration of the 5' flanking sequences in precursor RNA molecules, as compared to mature polyadenylated globin mRNA, suggests increased precursor stability of beta globin transcripts initiated in the 5' flanking region.

Animals↗