Identification, characterization, and molecular cloning of a novel transporter-like protein localized to the central nervous system.

During the course of large scale purification of the D1 dopamine receptor from rat brain, a protein of approximately 87,000 daltons (p87) was observed to copurify with the D1 receptor through four chromatographic steps. To characterize the nature of this protein, bovine and rat cDNA clones were isolated and sequenced. The bovine and rat clones were highly conserved (98.5% identity). Each clone possessed an open reading frame of 2226 base pairs encoding a protein of 742 amino acids (calculated MW of 82,500), containing three stretches of peptide sequence obtained from p87 sequence analysis. Comparison of the deduced peptide sequence of this protein with those found in available databanks revealed that it was a novel protein related to the family of nutrient transport proteins from eukaryotes and bacteria, including, the mammalian facilitated glucose transporters, the yeast transporters for maltose, lactose, and glucose, and the proton-driven bacterial transporters for arabinose, xylose, and citrate. In addition p87 also shares with these transporters a similar hydropathicity profile that suggests the presence of 12 transmembrane segments. The mRNA for p87 appears to be localized primarily, if not exclusively, to the central nervous system. Northern blot analysis reveals a message of approximately 4.8 kb in cortex, hippocampus, brain stem, and cerebellum, but no detectable signal in peripheral tissues such as spleen, liver, kidney, lung, heart, or skeletal muscle. Evidence form Western blot analysis and immunohistochemistry suggests that this protein may be expressed in intracellular organelles or the membrane of synaptosomes rather than plasma membrane. Based on its structure and properties, p87 appears to define a new class of transporter-like proteins.     

Improved RNA isolation from cells in tissue culture using a commercial nucleic acid extractor.

The Applied Biosystems 340A Nucleic Acid Extractor automates isolation of either DNA or RNA from tissue or cells in culture. We have found that several modifications to the manufacturer's recommended protocol greatly improve the quality of RNA that can be routinely isolated from cells in culture. These modifications include lysis of monolayer cells directly on plates, centrifuging samples after homogenization to remove precipitable RNase contaminants and purging the instrument's reagent lines with 0.1% diethyl pyrocarbonate. These simple modifications enhance both RNA quality and reproducibility of yield.     

Genetic and Molecular Organization of Ribosomal DNA (Rdna) Variants in Wild and Cultivated Barley

Twenty rDNA spacer-length variants (slvs) have been identified in barley. These slvs form a ladder in which each variant (with one exception) differs from its immediate neighbors by a 115-bp subrepeat. The 20 slvs are organized in two families, one forming an eight-step ladder (slvs 100-107) in the nucleolus organizer region (NOR) of chromosome 7 and the other a 12-step ladder (slvs 108a-118) in the NOR of chromosome 6. The eight shorter slvs (100-107) segregate and serve as markers of eight alleles of Mendelian locus Rrn2 and the 12 longer slvs (108a-118) segregate and serve as markers of 12 alleles of Mendelian locus Rrn1. Most barley plants (90%) are homozygous for two alleles, including one from each the 100-107 and the 108a-118 series. Two types of departures from this typical pattern of molecular and genetic organization were identified, one featuring compound alleles marked by two slvs of Rrn1 or of Rrn2, and the other featuring presence in Rrn1 of alleles normally found in Rrn2, and vice versa. The individual and joint effects on adaptedness of the rDNA alleles are discussed. It was concluded that selection acting on specific genotypes plays a major role in molding the strikingly different allelic and genotypic frequency distributions seen in populations of wild and cultivated barley from different ecogeographical regions.     

Connective tissue growth factor and cardiac fibrosis after myocardial infarction.

The temporal and spatial expression of transforming growth factor (TGF)-beta(1) and connective tissue growth factor (CTGF) was assessed in the left ventricle of a myocardial infarction (MI) model of injury with and without angiotensin-converting enzyme (ACE) inhibition. Coronary artery ligated rats were killed 1, 3, 7, 28, and 180 days after MI. TGF-beta(1), CTGF, and procollagen alpha1(I) mRNA were localized by in situ hybridization, and TGF-beta(1) and CTGF protein levels by immunohistochemistry. Collagen protein was measured using picrosirius red staining. In a separate group, rats were treated for 6 months with an ACE inhibitor. There were temporal and regional differences in the expression of TGF-beta(1), CTGF, and collagen after MI. Procollagen alpha1(I) mRNA expression increased in the border zone and scar peaking 1 week after MI, whereas collagen protein increased in all areas of the heart over the 180 days. Expression of TGF-beta(1) mRNA and protein showed major increases in the border zone and scar peaking 1 week after MI. The major increases in CTGF mRNA and protein occurred in the viable myocardium at 180 days after MI. Long-term ACE inhibition reduced left ventricular mass and decreased fibrosis in the viable myocardium, but had no effect on cardiac TGF-beta(1) or CTGF. TGF-beta(1) is involved in the initial, acute phase of inflammation and repair after MI, whereas CTGF is involved in the ongoing fibrosis of the heart. The antifibrotic benefits of captopril are not mediated through a reduction in CTGF.     

Assisted colonization as a climate change adaptation tool

Assisted colonization is a form of conservation translocation which introduces species at risk from extinction to new habitats, beyond their current range, in anticipation of more suitable conditions. Identifying which species, communities and ecosystems may benefit most from assisted colonization in coming decades is a key goal for conservation. Climate change is expected to lead to the loss or movement of suitable habitat for a range of species and anticipating which can be effectively conserved through assisted colonization is critical. Here, we identify a series of scenarios that may predispose terrestrial species to the need for assisted colonization in order to reduce extinction risk resulting from anthropogenic climate change and assemble a list of traits commonly associated with at‐risk species. These traits may help to provide broad‐scale guidance on how to select species to target for assisted colonization as a conservation management response to climate change. We also identify six key themes associated with successful conservation translocations including recipient site selection and preparation, a clear understanding of species biology and ecology, and taking lessons from invasive species research.     

Lack of dependence on p53 for DNA double strand break repair of episomal vectors in human lymphoblasts.

The p53 tumor suppressor gene has been shown to be involved in a variety of repair processes, and recent findings have suggested that p53 may be involved in DNA double strand break repair in irradiated cells. The role of p53 in DNA double strand break repair, however, has not been fully investigated. In this study, we have constructed a novel Epstein-Barr virus (EBV)-based shuttle vector, designated as pZEBNA, to explore the influence of p53 on DNA strand break repair in human lymphoblasts, since EBV-based vectors do not inactivate the p53 pathway. We have compared plasmid survival of irradiated, restriction enzyme linearized, and calf intestinal alkaline phosphatase (CIP)-treated pZEBNA with a Simian virus 40 (SV40)-based shuttle vector, pZ189, in TK6 (wild-type p53) and WTK1 (mutant p53) lymphoblasts and determined that p53 does not modulate DNA double strand break repair in these cell lines.     

Formation of Adeno-Associated Virus Circular Genomes Is Differentially Regulated by Adenovirus E4 ORF6 and E2a Gene Expression

A central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of both integrated and episomal genomes. However, the molecular processes associated with episomal long-term persistence of AAV genomes are only poorly understood. To investigate these mechanisms, we have utilized a recombinant AAV (rAAV) shuttle vector to identify circular AAV intermediates from transduced HeLa cells and primary fibroblasts. The unique structural features exhibited by these transduction intermediates included circularized monomer and dimer virus genomes in a head-to-tail array, with associated specific base pair alterations in the 5′ viral D sequence. In HeLa cells, the abundance and stability of AAV circular intermediates were augmented by adenovirus expressing the E2a gene product. In the absence of E2a, adenovirus expressing the E4 open reading frame 6 gene product decreased the abundance of AAV circular intermediates, favoring instead the linear replication form monomer (Rf_m) and dimer (Rf_d) structures. In summary, the formation of AAV circular intermediates appears to represent a new pathway for AAV genome conversion, which is consistent with the head-to-tail concatemerization associated with latent-phase persistence of rAAV. A better understanding of this pathway may increase the utility of rAAV vectors for gene therapy.