Biomedical subjects

Mark H Harpster

Publications and source records attributed to Mark H Harpster.

5 recordsLinked to original sources

PubMed2006

Earliest changes in the left ventricular transcriptome postmyocardial infarction.

We report a genome-wide survey of early responses of the mouse heart transcriptome to acute myocardial infarction (AMI). For three regions of the left ventricle (LV), namely, ischemic/infarcted tissue (IF), the surviving LV free wall (FW), and the interventricular septum (IVS), 36,899 transcripts were assayed at six time points from 15 min to 48 h post-AMI in both AMI and sham surgery mice. For each transcript, temporal expression patterns were systematically compared between AMI and sham groups, which identified 515 AMI-responsive genes in IF tissue, 35 in the FW, 7 in the IVS, with three genes induced in all three regions. Using the literature, we assigned functional annotations to all 519 nonredundant AMI-induced genes and present two testable models for central signaling pathways induced early post-AMI. First, the early induction of 15 genes involved in assembly and activation of the activator protein-1 (AP-1) family of transcription factors implicates AP-1 as a dominant regulator of earliest post-ischemic molecular events. Second, dramatic increases in transcripts for arginase 1 (ARG1), the enzymes of polyamine biosynthesis, and protein inhibitor of nitric oxide synthase (NOS) activity indicate that NO production may be regulated, in part, by inhibition of NOS and coordinate depletion of the NOS substrate, L: -arginine. ARG1: was the single-most highly induced transcript in the database (121-fold in IF region) and its induction in heart has not been previously reported.

Acute Disease↗

PubMed2003

Simultaneous transgenic suppression of LePG and LeExp1 influences rheological properties of juice and concentrates from a processing tomato variety.

Processing tomato lines suppressed in the accumulation of ripening-related polygalacturonase or expansin were generated by introduction of transgenes to silence expression of the LePG and LeExp1 genes, respectively. The rheological properties of juice and juice reconstituted from paste produced from lines suppressed in one of these genes, or in both, were compared with azygous controls. When assayed by measuring Bostwick consistency, paste produced from either suppressed LePG or suppressed LeExp1 lines and diluted to 5 degrees Brix was approximately 18% more viscous than that produced from controls. Simultaneous suppression of LePG and LeExp1 produced a small additional increase in viscosity of 4%. Rheometric flow analysis at 5 or 10 degrees Brix also showed substantial increases in the consistency index due to suppression of either LePG or LeExp1 alone, and a small additional increase when both genes were suppressed in the same transgenic line. Measurements by laser diffraction and [1H]NMR showed that suppression of LePG or LeExp1 accumulation altered the size distribution of insoluble particles and modified their surface properties. The data are consistent with suppression of LePG increasing serum viscosity, and suppression of either LePG or LeExp1 altering the properties of the insoluble particles and improving some aspect of particle-particle or particle-serum interaction, or both. However, relative to that caused by suppression of either gene alone, the additional increase in viscosity caused by simultaneous suppression of LePG and LeExp1 together was slight.

Beverages↗

PubMed2003

Inverted repeat of a heterologous 3'-untranslated region for high-efficiency, high-throughput gene silencing.

This report describes a method for the easy generation of inverted repeat constructs for the silencing of genes of unknown sequence which is applicable to high-throughput studies. This improved procedure for high-efficiency gene silencing is specific for a target gene, but does not require inverted repeat DNA of the target gene in the construct. The method employs an inverted repeat of the 3'-untranslated region (3'-UTR) of a heterologous gene, and has been demonstrated using the 3'-UTR region of the nopaline synthase (nos) gene from Agrobacterium tumefaciens, which is often used as the 3'-UTR for transgene constructs. In a population of independent tomato primary transformants harboring a stably integrated polygalacturonase (PG) transgene driven by a constitutive promoter and linked to an inverted repeat of the nos 3'-UTR, 51 of 56 primary transformants (91% of the population) showed highly effective post-transcriptional silencing of the PG gene, with PG mRNA abundance in ripe fruit reduced by 98% or more. The method was also effective in Arabidopsis, where two different, relatively uncharacterized plant transcription factors were also targeted effectively. This method has the advantage of ease and rapidity in preparation of the constructs, since a gene of interest can be inserted into a binary vector already containing the promoter and the inverted nos domain in a single-cloning step, and does not require any knowledge of the DNA sequence. The approach is suitable for high-throughput gene silencing studies, where it is necessary to investigate the function of hundreds to thousands of uncharacterized genes.

3' Untranslated Regions↗

PubMed2002

Suppression of a ripening-related endo-1,4-beta-glucanase in transgenic pepper fruit does not prevent depolymerization of cell wall polysaccharides during ripening.

The function of the ripening-related endo-1,4-beta-D-glucanase (EGase) CaCel1 in fruit softening was investigated by suppression of CaCel1 gene expression in transgenic pepper (Capsicum annuum L.) plants using constitutive expression of a truncated sense CaCel1 transgene. In suppressed lines, immunodetectable CaCel1 protein and extractable CMCase activity were reduced to at or below the limit of detection in ripe mature red fruit, suggesting that in pepper ripening-related CMCase activity is the product of a single gene. However, the abundances of two mRNAs derived from the CaCel1 gene by differential transcription initiation were affected differently in suppressed lines. Accumulation of a 1.7 kb CaCel1 transcript was strongly suppressed, whereas the abundance of a 2.1 kb CaCel1 transcript was only partially reduced. This implies that the 1.7 kb mRNA is responsible for producing CaCel1 protein, while the 2.1 kb mRNA is translationally inactive, and as such is recalcitrant to co-suppression. Chelator-soluble polyuronides exhibited little or no depolymerization during ripening, but matrix glycans including xyloglucan were extensively depolymerized. Depolymerization of non-xyloglucan matrix glycans was the prominant cell wall change observed during pepper ripening. However, the lack of CaCel1 activity in suppressed fruit had no detectable effect on ripening-related matrix glycan depolymerization, which occurred at wild-type levels. Recombinant CaCel1 protein purified from a transgenic pepper line over-expressing functional CaCel1 was active against pepper matrix glycans in vitro, and showed greater activity against non-xyloglucan polysaccharides than against xyloglucan. Transgenic suppression of CaCel1 EGase activity has not identified the natural cell wall substrate for this enzyme, and shows that activities other than CaCel1 are responsible for the depolymerization of matrix glycans occurring during ripening in pepper.

Capsicum↗

PubMed2002

Constitutive overexpression of a ripening-related pepper endo-1,4-beta-glucanase in transgenic tomato fruit does not increase xyloglucan depolymerization or fruit softening.

The ripening-related pepper endo-1,4-beta-D-glucanase (EGase) CaCel1 was over-expressed in transgenic tomato plants under the control of the constitutive 35S promoter to investigate the effects on plant growth and fruit softening of high levels of a potential cell wall-degrading activity. In transgenic fruit, recombinant CaCel1 protein was associated with a high-salt putative cell wall fraction, and extractable CMCase activity was increased by up to 20-fold relative to controls. However, the effects of high levels of EGase activity on fruit cell wall metabolism were relatively small. The largest consequence observed was a decrease of up to 20% in the amount of matrix glycans in a 24% KOH-soluble fraction consisting of polysaccharides tightly bound to cellulose. This decrease was confined to polysaccharides other than xyloglucan, did not affect the size distribution of remaining molecules, and was not correlated with a corresponding increase in glycans in a 4% KOH-soluble fraction loosely bound to cellulose, suggesting that the missing polymers had been degraded to fragments small enough to be lost from the extracts. The amount of matrix glycans in the 4% KOH-soluble fraction was not substantially changed, but the size distribution showed a small relative increase in the amount of polymers in a peak eluting close to a linear dextran marker of 71 kDa. This could be due either to an increase in the amount of polymers of this size, or to a loss from the extract of other polymers present in peaks of higher molecular weight. Transgenic fruit were not softer than controls but appeared the same or slightly firmer at both green and red developmental stages, and no differences in plant vegetative growth were observed. CaCel1 did not cause depolymerization of tomato fruit xyloglucan in vivo, but differences in the amount or molecular weight profile of other matrix glycans were observed. The data suggest that degradation of a proportion of matrix glycans other than xyloglucan does not result in fruit softening, and that fruit softening is not limited by the amount of EGase activity present during ripening.

Amino Acid Sequence↗