Glial-specific ablation of angiotensinogen lowers arterial pressure in renin and angiotensinogen transgenic mice

Angiotensinogen (AGT) is mainly expressed in glial cells in close proximity to renin-expressing neurons in the brain. We previously reported that glial-specific overexpression of ANG II results in mild hypertension. Here, we tested the hypothesis that glial-derived AGT plays an important role in blood pressure regulation in hypertensive mice carrying human renin (hREN) and human AGT transgenes under the control of their own endogenous promoters. To perform a glial-specific deletion of AGT, we used an AGT transgene containing loxP sites (hAGT(flox)), so the gene can be permanently ablated in the presence of cre-recombinase expression, driven by the glial fibrillary acidic protein (GFAP) promoter. Triple transgenic mice (RAC) containing a: 1) systemically expressed hREN transgene, 2) systemically expressed hAGT(flox) transgene, and 3) GFAP-cre-recombinase were generated and compared with double transgenic mice (RA) lacking cre-recombinase. Liver and kidney hAGT mRNA levels were unaltered in RAC and RA mice, as was the level of hAGT in the systemic circulation, consistent with the absence of cre-recombinase expression in those tissues. Whereas hAGT mRNA was present in the brain of RA mice (lacking cre-recombinase), it was absent from the brain of RAC mice expressing cre-recombinase, confirming brain-specific elimination of AGT. Immunohistochemistry revealed a loss of AGT immunostaining glial cells throughout the brain in RAC mice. Arterial pressure measured by radiotelemetry was significantly lower in RAC than RA mice and unchanged from nontransgenic control mice. These data suggest that there is a major contribution of glial-AGT to the hypertensive state in mice carrying systemically expressed hREN and hAGT genes and confirm the importance of a glial source of ANG II substrate in the brain.     

Characterization of the Bex gene family in humans, mice, and rats

To better understand the development of ventral mesencephalic dopamine neurons, we performed subtractive hybridization screens to find ventral mesencephalic genes expressed at rat embryonic day 10 when these neurons begin to differentiate. The most commonly identified genes in these screens were members of the Bex (Brain expressed X-linked) gene family, rat Bex1 (Rex3), and a novel gene, rat Bex4. After identifying these genes, we then sought to characterize the Bex gene family. Two additional novel Bex genes (human Bex5 and mouse Bex6) were discovered through genomic databases. Bex5 is present in humans and monkeys, but not rodents, while Bex6 exists in mice, but not humans. Bex4 and Bex5 are localized to the X chromosome, are expressed in brain, and are similar in sequence. Bex4 and Bex5 are 54% and 56% identical to human Bex3 (pHGR74, NADE). Mouse Bex6 is on chromosome 16 and is 67% identical to mouse Bex4. Human Bex gene expression was studied with tissue expression arrays probed with specific oligonucleotides. Human Bex1 and Bex2 have similar expression patterns in the central nervous system with high levels in pituitary, cerebellum, and temporal lobe, and Bex1 is widely expressed outside of the central nervous system with high expression in the liver. Human Bex4 is highly expressed in heart, skeletal muscle, and liver, while Bex3 and Bex5 are more widely expressed. The subcellular localization of the Bex proteins varies from nuclear (rat Bex1) to cytoplasmic (rat Bex3, human Bex5, and mouse Bex6) and to both nuclear and cytoplasmic (rat Bex2 and rat Bex4). Rat Bex3, rat Bex4, human Bex5, and mouse Bex6 are degraded by the proteasome, while rat Bex1 or Bex2 are not. Rat Bex3 protein can likely bind transition metals through a histidine-rich domain. Because this gene family was originally named Bex and because these genes are unified by sequence similarity and gene structure, we believe the Bex nomenclature should prevail over nomenclature based on function (NADE) that has not been extended to the other Bex genes. We conclude that the Bex gene family members are highly homologous but differ in their expression patterns, subcellular localization, and degradation by the proteasome.     

2-Cyano-4-fluoro-1-thiovalylpyrrolidine analogues as potent inhibitors of DPP-IV

We report the synthesis and biological activity of a series of 2-cyano-4-fluoro-1-thiovalylpyrrolidine inhibitors of DPP-IV. Within this series, compound 19 provided a potent, selective, and orally active DPP-IV inhibitor which demonstrated a very long duration of action in both rat and dog.     

Host effects of glandular-haired alfalfa on alfalfa weevil (Coleoptera: Curculionidae) and potato leafhopper (Homoptera: Cicadellidae) populations in Virginia

Cultivars of glandular-haired alfalfa, Medicago sativa L., such as '54H69', are currently available and marketed as being resistant to potato leafhopper, Empoasca fabae (Harris). Between 2000 and 2002, studies were conducted to compare the effects of '54H69' and a standard, nonglandular-haired alfalfa cultivar, 'Choice', on alfalfa weevil, Hypera postica (Gyllenhal), and potato leafhopper populations at Campbell and Montgomery counties, Virginia. '54H69' had no effect on alfalfa weevil populations. At each location, densities of alfalfa weevil in '54H69' and 'Choice' were similar, but pest pressure was higher at Campbell Co. than at Montgomery Co. and always exceeded the economic threshold before insecticide was applied. Densities of potato leafhopper also did not differ between '54H69' and 'Choice' in any year at the two locations. Insecticide treatment effectively reduced potato leafhopper densities in the two cultivars, although populations were below the economic threshold at both locations when the insecticides were applied. Overall, postinsecticide treatment comparisons showed that the densities of alfalfa weevil and potato leafhoppers were similar or higher in untreated '54H69' compared with insecticide-treated 'Choice'. In addition, there were no differences in seasonal dry yields between '54H69' and 'Choice' in any year at either location. Our results indicate that the glandular-haired alfalfa '54H69' does not provide acceptable resistance to potato leafhopper and also does not offer a yield advantage to growers in Virginia.     

The T cell receptor gamma chain alternate reading frame protein (TARP), a prostate-specific protein localized in mitochondria

We previously showed that mRNA encoding TARP (T cell receptor gamma chain alternate reading frame protein) is exclusively expressed in the prostate in males and is up-regulated by androgen in LNCaP cells, an androgen-sensitive prostate cancer cell line. We have now developed an anti-TARP monoclonal antibody named TP1, and show that TARP protein is up-regulated by androgen in both LNCaP and MDA-PCa-2b cells. We used TP1 to determine the subcellular localization of TARP by Western blotting following subcellular fractionation and immunocytochemistry. Both methods showed that TARP is localized in the mitochondria of LNCaP cells, MDA-PCa-2b cells, and PC-3 cells transfected with a TARP-expressing plasmid. We also transfected a plasmid encoding TARP fused to green fluorescent protein into LNCaP, MDA-Pca-2b, and PC-3 cells and confirmed its specific mitochondrial localization in living cells. Fractionation of mitochondria shows that TARP is located in the outer mitochondrial membrane. Immunohistochemistry using a human prostate cancer sample showed that TP1 reacted in a dot-like cytoplasmic pattern consistent with the presence of TARP in mitochondria. These data demonstrate that TARP is the first prostate-specific protein localizing in mitochondria and indicate that TARP, an androgen-regulated protein, may act on mitochondria to carry out its biological functions.     

Tyrosine-to-cysteine modification of human alpha-synuclein enhances protein aggregation and cellular toxicity

The deposition of alpha-synuclein and other cellular proteins in Lewy bodies in midbrain dopamine neurons is a pathological hallmark of Parkinson's disease. Nitrative and oxidative stress can induce alpha-synuclein protein aggregation, possibly initiated by the formation of stable cross-linking dimers. To determine whether enhanced dimer formation can accelerate protein aggregation and increase cellular toxicity, we have substituted cysteine for tyrosine at positions 39, 125, 133, and 136 in human wild-type (WT) alpha-synuclein, and in A53T and A30P mutant alpha-synuclein. To reduce the likelihood of cross-linking, phenylalanine was substituted for tyrosine at the same sites. We have found that overexpression of Y39C or Y125C mutant proteins leads to increased intracellular inclusions and apoptosis in a rat dopaminergic cell line (N27 cells) and in human embryonic kidney 293 cells. Expression of Y133C, Y136C, and all four Tyr-to-Phe mutations were not more cytotoxic than WT control. Exposure to oxidative stress increased Y39C and Y125C alpha-synuclein aggregation and toxicity. Dimers and oligomers were found in Triton X-100-soluble fractions from adenovirus-mediated overexpression of Y39C and Y125C in N27 cells. In contrast, WT beta-synuclein and all four Tyr-to-Cys mutant beta-synucleins did not cause protein aggregation and cell death. We conclude that cysteine substitution at critical positions in the alpha-synuclein molecule can increase dimer formation and accelerate protein aggregation and cellular toxicity of alpha-synuclein.     

Phosphatized multicellular algae in the Neoproterozoic Doushantuo Formation, China, and the early evolution of florideophyte red algae

Phosphatic sediments of the Late Neoproterozoic (ca. 600 million years old [Myr]) Doushantuo Formation at Weng'an, South China, contain fossils of multicellular algae preserved in anatomical detail. As revealed by light microscopy and scanning electron microscopy, these fossils include both simple pseudoparenchymatous thalli with apical growth but no cortex-medulla differentiation and more complex thalli characterized by cortex-medulla differentiation and structures interpretable as carposporophytes, suggesting a multiphasic life cycle. Simple pseudoparenchymatous thalli, represented by Wengania, Gremiphyca, and Thallophycoides, are interpreted as stem group florideophytes. In contrast, complex pseudoparenchymatous thalli, such as Thallophyca and Paramecia, compare more closely to fossil and living corallinaleans than to other florideophyte orders, although they also differ in some important aspects (e.g., lack of biocalcification). These more complex thalli are interpreted as early stem group corallinaleans that diverged before Paleozoic stem groups such as Arenigiphyllum, Petrophyton, Graticula, and Archaeolithophyllum. This phylogenetic interpretation implies that (1) the phylogenetic divergence between the Florideophyceae and its sister group, the Bangiales, must have taken place before Doushantuo time-an inference supported by the occurrence of bangialean fossils in Mesoproterozoic rocks; (2) the initial diversification of the florideophytes occurred no later than the Doushantuo time; and (3) the corallinalean clade had a "soft" (uncalcified) evolutionary history in the Neoproterozoic before evolving biocalcification in the Paleozoic and undergoing crown group diversification in the Mesozoic.