Biomedical subjects

M L Tierney

Publications and source records attributed to M L Tierney.

28 records · Page 2Linked to original sources

PubMed1995

Rapid desensitization of alpha 1 beta 1 GABA A receptors expressed in Sf9 cells under optimized conditions.

alpha 1 and beta 1 subunits of human GABA A receptors were expressed in Sf9 cells using the Sf9-baculovirus system. Better expression was obtained by manipulating the system. Cell growth phase at the time of infection determined the practical range of virus titre, the period postinfection during which cells were useful for signal detection and the maximal current obtained. Cells in the early exponential phase were relatively insensitive to multiplicity of infection (MOI) whereas cells in the mid- to late-exponential phase were highly dependent on MOI and they responded with the largest Cl- current generated by GABA. Channels activated by GABA were chloride-selective. Half the maximum peak whole-cell current was obtained with 11 microM GABA. The time course of Cl- currents activated by saturating GABA concentrations in cells infected with alpha 1 beta 1-recombinant viruses was examined employing a rapid perfusion system which allowed whole-cell solution exchange in less than 1 msec. The current decay could be fitted by 3 to 4 exponentials for the first 8 sec. The initial fast current decrease had a time constant of about 23 msec. No voltage dependence of time constants was detected but the whole-cell IV relation showed outward rectification. Currents were depressed by bicuculline, penicillin and picrotoxin and potentiated by pentobarbitone.

Animals↗

PubMed1993

Deletion analysis and localization of SbPRP1, a soybean cell wall protein gene, in roots of transgenic tobacco and cowpea.

SbPRP1 is a member of the soybean (Glycine max L. Merr) proline-rich cell wall protein family and is expressed at high levels in root tissue. To characterize the sequences required for this expression, we have fused 1.1 kb of upstream flanking DNA sequence from an SbPRP1 genomic clone to a gene encoding beta-glucuronidase (GUS). This construct was introduced into tobacco using Agrobacterium tumefaciens-mediated transformation. Histochemical staining of GUS activity in transgenic tobacco indicated that SbPRP1 is expressed in the apical and elongating region of both primary and lateral roots, most strongly in the epidermis. A similar localization pattern was found in transformed hairy roots when this construct was introduced into cowpea (Vigna aconitifolia) using Agrobacterium rhizogenes-mediated transformation. Nested 5'-deletion analysis of the SbPRP1 promoter indicated that a minimal promoter for SbPRP1 expression in roots is located within the first 262 bases of upstream flanking DNA and that the region between -1080 and -262 is required for maximal expression of this gene. Gel retardation assays showed that nuclear factors can be detected in soybean roots which specifically bind to sequences located between -1080 and -623, a region which is needed for maximal expression of the SbPRP1 promoter. Northern hybridization analysis was also used to show that little SbPRP1 mRNA was present in roots during the first 24 h after imbibition. These studies indicate that SbPRP1 expression is localized to the actively growing region of the root and that this expression is temporally regulated during very early stages of seedling growth.

Base Sequence↗

PubMed1993

Expression of DcPRP1 is linked to carrot storage root formation and is induced by wounding and auxin treatment.

A carrot (Daucus carota, L.) genomic clone (DcPRP1) was isolated on the basis of its homology to previously described cDNAs encoding a wound-inducible, proline-rich cell wall protein. DNA sequence analysis showed that DcPRP1 contains a single open-reading frame encoding a 235-amino acid protein that is colinear with that predicted from the cDNA sequence with the exception of four amino acids at the N terminus and a 60-nucleotide insertion present within the genomic clone. Genomic Southern hybridization analysis showed that the cloned sequence hybridized with a single restriction enzyme fragment using several restriction enzymes. Primer extension and northern hybridization analysis indicated that the expression of DcPRP1 is developmentally regulated and linked to the formation of storage roots, where this gene is expressed at high levels after wounding. The level of DcPRP1 mRNA was greatest in tissue immediately adjacent to the wound site. Treatment of unwounded carrot storage roots with 10 microM 2,4-dichlorophenoxy-acetic acid, indoleacetic acid, or naphthalene-1-acetic acid also resulted in the accumulation of DcPRP1 transcripts to a level equal to that seen in wounded tissue.

Amino Acid Sequence↗

PubMed1992

A combination of human alpha 1 and beta 1 subunits is required for formation of detectable GABA-activated chloride channels in Sf9 cells.

The baculovirus expression system was used to produce alpha 1 and beta 1 subunits of the human GABAA receptor in Sf9 cells. In cells infected with both alpha 1 and beta 1 recombinant viruses, GABA elicited an outwardly rectifying chloride current that was blocked by bicuculline and potentiated by pentobarbitone. GABA did not produce detectable currents in cells infected with either alpha 1 or beta 1 recombinant viruses alone. In these cells, and in control (non-infected) Sf9 cells, pentobarbitone depressed the leakage current (Ki = 55 microM). Fluorescently labelled monoclonal antibodies to the alpha 1 subunit showed greater amounts of the alpha 1 subunit in cells infected with only the alpha 1 recombinant virus than in cells co-infected with the alpha 1 and beta 1 recombinant viruses. Fluorescence of the plasma membrane was seen in cells co-infected with the alpha 1 and beta 1 recombinant viruses, but was absent in cells infected with only the alpha 1 recombinant virus. It was concluded that the alpha 1 subunit normally interacts with the beta 1 subunit to be transported to the plasma membrane in Sf9 cells.

Animals↗

PubMed1992

Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression.

Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are plant lipid derivatives that resemble mammalian eicosanoids in structure and biosynthesis. These compounds are proposed to play a role in plant wound and pathogen responses. Here we report the quantitative determination of JA/MeJA in planta by a procedure based on the use of [13C,2H3]MeJA as an internal standard. Wounded soybean (Glycine max [L] Merr. cv. Williams) stems rapidly accumulated MeJA and JA. Addition of MeJA to soybean suspension cultures also increased mRNA levels for three wound-responsive genes (chalcone synthase, vegetative storage protein, and proline-rich cell wall protein) suggesting a role for MeJA/JA in the mediation of several changes in gene expression associated with the plants' response to wounding.

Acyltransferases↗

PubMed1990

Isolation and characterization of a proline-rich cell wall protein from soybean seedlings.

We have purified a cell wall protein from extracts of soybean (Glycine max) that was previously shown to be immunologically related to p33, a wound-induced carrot cell wall protein (Tierney ML, Wiechert J, Pluymers D [1988] Mol Gen Genet 211: 393-399). Amino acid composition analysis reveals that the protein is 36% proline and hydroxyproline and its amino-terminus was determined to be AsnTyrGluAsnProHypValTyrLysProHypThrGluLysProHypValTyr. In vivo [(3)H]proline labeling of soybean hypocotyl tissues and Western blot analysis indicate that the apical hook, a region characterized by active growth, is a predominant site for synthesis of this protein and that wounding does not significantly affect its synthesis or accumulation. Antibodies raised against the hook protein also cross-reacted with a higher molecular weight protein in cell wall extracts of wounded hook tissue. These data indicate that there are at least two related, proline-rich cell wall proteins in the hook region of soybean seedlings, one that appears to be developmentally regulated and another that is wound-inducible. RNA encoding the developmentally regulated seedling cell wall protein was detected in both the hook and elongating region of the hypocotyl. The presence of this RNA in the elongating region in the absence of detectable levels of the protein indicates either that the expression of this cell wall protein is subject to posttranscriptional control or that the rate of insolubilization of this protein increases in more mature regions of the hypocotyl.

Journal Article↗

PubMed1990

Physicochemical and biological characterization of recombinant human inhibin A.

Recombinant human inhibin A was isolated from recombinant mammalian cell line culture media. Two forms of inhibin were identified with Mr of 34 and 31 Kd composed of subunits (alpha, beta) of 24 and 15 Kd and 21 and 15 Kd respectively. Both forms are bioactive in an inhibin in vitro bioassay and immunoactive with potencies comparable to or higher than purified bovine inhibin. Amino acid analyses and NH2-terminal sequences of each of the subunits are consistent with those predicted from their cDNA structures. The inhibin alpha- but not beta-subunit is glycosylated based on its binding to the lectins concanavalin A and wheat germ lectin. The difference in molecular weight of 31 and 34 Kd inhibin is attributed to variation in glycosylation of the alpha-subunit. The 31+34 Kd inhibin is heterogeneous on isoelectric focusing gels consisting of four isoforms in the pH range 6.2-7.6. Inhibition also exhibits in vivo biological activity by suppressing serum FSH but not LH in castrate male rats. These physicochemical and biological characteristics of recombinant human inhibin are similar to those described for native inhibin isolated from a variety of other species.

Amino Acid Sequence↗

PubMed1989

Immunisation against the amino-terminal peptide (alpha N) of the alpha 43 subunit of inhibin impairs fertility in sheep.

Processing of the 58 kDa to the 31 kDa form of inhibin (Inh) involves cleavage of the amino-terminal peptide (alpha N) from the alpha 43-subunit. We show that active immunisation of female sheep against a recombinant bovine alpha N impairs their fertility. In Exp 1, 5 treated (Group 1; 300 micrograms alpha N) and 6 control ewes (Group 2; adjuvant only) were immunized (Day 1) and given boosters on Days 22 and 56. In Group 1, mean +/- SEM binding of 125I-31 kDa Inh was less than 0.5% on Days 33 and 44, whereas binding of 125I-58 kDa Inh was 4.9 +/- 0.7 and 6.2 +/- 0.6%, respectively. In Group 2 binding of both tracers was less than 0.5%. The corpora lutea (CL)/ewe in Group 1 on Days 44 and 82 were 1.8 +/- 0.2 and 2.8 +/- 0.9, respectively, and were not different from those in Group 2 (1.7 +/- 0.3 and 1.5 +/- 0.2, respectively). One ewe in Group 1 versus 5/6 ewes in Group 2 were diagnosed pregnant. In Exp 2, 18 treated and 16 controls were immunized as in Exp 1. The binding of 125I-58 kDa Inh in treated ewes (2.4 +/- 0.3%) was greater than in controls (less than 0.5%) on Day 56. The CL/ewe in treated ewes (1.8 +/- 0.2) was similar to that in controls (2.0 +/- 0.1) on Day 76. All 16 control ewes but only 7/17 treated ewes were subsequently diagnosed pregnant. The plasma progesterone concentrations were similar in treated ewes which did (7.6 +/- 1.2 nmol/L) and did not (7.0 +/- 0.7) become pregnant. Neither basal nor GnRH-stimulated concentrations of LH, nor basal concentrations of Inh differed between treated and controls in Exp 2. Similarly, there were no differences in FSH, except that basal concentrations were higher in the luteal phase of treated ewes. We conclude that immunisation of ewes against alpha N results in a significant reduction in fertility.

Animals↗

PubMed1987

The extensins.

The plant cell was includes a matrix which is species specific and which chages in composition during growth and development. Characterization of the protein component of the wall matrix has resulted in the purification of extensin and the genes which encode it. Analysis of the protein sequences for the extensins has provided clues about the types of interactions which may occur as the chemistry and architecture of the cell wall accommodate growth and development.

Journal Article↗

PubMed1987

Developmental Regulation of beta-Conglycinin in Soybean Axes and Cotyledons.

Analysis of the expression of genes encoding the beta-conglycinin seed storage proteins in soybean has been used to extend our understanding of developmental gene expression in plants. The alpha, alpha', and beta subunits of beta-conglycinin are encoded by a multigene family which is organ-specific in its expression. In this study we report the differentially programmed accumulation of the alpha, alpha', and beta subunits of beta-conglycinin. Multiple isomeric forms of each subunit are present in the dry seed, but the timing of their accumulation is unique for each subunit. The previously reported variation in amount of alpha' and alpha subunits in axis and cotyledons is also reflected in the amount of subunit specific mRNA which is present in each tissue. The beta subunit, previously undetected in soybean axes, is found to be synthesized but rapidly degraded. These differences in beta-conglycinin protein accumulation may be reflected by the morphological differences observed in protein bodies between these two tissues.

Journal Article↗