In the central nervous system, angiotensin II (AngII) binds to angiotensin type 1 receptors (AT1R) to affect autonomic and endocrine functions as well as learning and memory. However, understanding the function of cells containing AT1Rs has been restricted by limited availability of specific antisera, difficulties discriminating AT1 receptor-immunoreactive cells in many brain regions and, the identification of AT1R-containing neurons for physiological and molecular studies. Here, we demonstrate that an Agtr1a bacterial artificial chromosome (BAC) transgenic mouse line that expresses type A AT1Rs (AT1aRs) identified by enhanced green fluorescent protein (EGFP) overcomes these shortcomings. Throughout the brain, AT1aR-EGFP was detected in the nuclei and cytoplasm of cells, most of which were neurons. EGFP often extended into dendritic processes and could be identified either natively or with immunolabeling of EGFP. The distribution of AT1aR-EGFP cells in brain closely corresponded to that reported for AngII binding and AT1aR protein and mRNA. In particular, AT1aR-EGFP cells were in autonomic regions (e.g., hypothalamic paraventricular nucleus, central nucleus of the amygdala, parabrachial nucleus, nuclei of the solitary tract and rostral ventrolateral medulla) and in regions involved in electrolyte and fluid balance (i.e., subfornical organ) and learning and memory (i.e., cerebral cortex and hippocampus). Additionally, dual label electron microscopic studies in select brain areas demonstrate that cells containing AT1aR-EGFP colocalize with AT1R-immunoreactivity. Assessment of AngII-induced free radical production in isolated EGFP cells demonstrated feasibility of studies investigating AT1aR signaling ex vivo. These findings support the utility of Agtr1a BAC transgenic reporter mice for future studies understanding the role of AT1 receptor containing cells in brain function.

Superoxide produced by the enzyme NADPH oxidase mediates crucial intracellular signaling cascades in the mNTS, a brain region populated by catecholaminergic neurons, as well as astroglia that play an important role in autonomic function. The mechanisms mediating NADPH oxidase (phox) activity in the neural regulation of cardiovascular processes are incompletely understood, however the subcellular localization of superoxide produced by the enzyme is likely to be an important regulatory factor. We used immunogold electron microscopy to determine the phenotypic and subcellular localization of the NADPH oxidase subunits p47 phox , gp91 phox, and p22 phox in the mNTS. The mNTS contains a large population of neurons that synthesize catecholamines. Significantly, catecholaminergic signaling can be modulated by redox reactions. Therefore, the relationship of NADPH oxidase subunit labeled neurons or glia with respect to catecholaminergic neurons was also determined by dual labeling for the superoxide producing enzyme and tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. In the mNTS, NADPH oxidase subunits were present primarily in somatodendritic processes and astrocytes, some of which also contained TH, or were contacted by TH labeled axons, respectively. Immunogold quantification of NADPH oxidase subunit localization showed that p47 phox and gp91 phox were present on the surface membrane, as well as vesicular organelles characteristic of calcium storing smooth endoplasmic reticula in dendritic and astroglial processes. These results indicate that NADPH oxidase assembly and consequent superoxide formation are likely to occur near the plasmalemma, as well as on vesicular organelles associated with intracellular calcium storage within mNTS neurons and glia. Thus, NADPH oxidase-derived superoxide may participate in intracellular signaling pathways linked to calcium regulation in diverse mNTS cell types. Moreover, NADPH oxidase-derived superoxide in neurons and glia may directly or indirectly modulate catecholaminergic neuron activity in the mNTS.

). The modulation by ATP of pre-synaptic ion channels (Zimmermann, 1994) via specific purinergic receptors, for example, could result in changes in subsequent release of neurotransmitters from the pre-synaptic site and, as a consequence, on the functional effect at the post-synaptic site.ATP is rapidly hydrolysed by ecto-nucleotidases (Gordon et al. 1989) even at pre-synaptic sites (Thirion et al. 1996) to adenosine 5‚-diphosphate (ADP), adenosine 5‚-monophosphate (AMP) and adenosine. Adenosine, the final hydrolysed metabolite of ATP (Gordon et al. 1989;Kennedy et al. 1996; see review by Rathbone et al. 1999), is another strong neuromodulator that may interact with

The Bacillus bacteriocin thurincin H exhibits a wide inhibitory spectrum of activity against various foodborne pathogens, such as Listeria monocytogenes, and dairy spoilage bacteria, especially different Bacillus species commonly existing in dairy products. Previously, we constructed 3 plasmids to express native thurincin H homologously in an engineered natural producer, Bacillus thuringiensis SF361thnH(-). This host is deficient in thurincin H production because of an in-frame deletion of structural genes thnA1, thnA2, and thnA3 from the chromosome of the natural producer B. thuringiensis SF361. The previously constructed expression vectors were constructed by cloning the native thurincin H promoter, 3 (or 1) copies of structural genes, and the native (or Cry protein) terminator into an Escherichia coli-B. thuringiensis shuttle vector pHT315. In this study, 3 corresponding expression vectors (pGW134, pGW135, and pGW136) were constructed to express recombinant thurincin H-His6 in the same host, in which a 6-histidine tag was fused to the C terminus of each structural gene. The resulting low level of bacteriocin production indicated that the His tag might negatively interfere with subsequent posttranslational modification or exportation processes after the thurincin H-His6 prepeptide was translated. Additionally, in order to overexpress native thurincin H, 2 additional plasmids (pGW137 and pGW138) were constructed, consisting of the sporulation-dependent Cry protein dual promoter BtI and BtII, the thnA1 structural gene, and the thurincin H native or Cry protein terminator. However, production was low on Luria broth plates and absent on sporulation plates. It is possible that the resulting thurincin H prepeptide was not correctly modified or exported to the extracellular environment, due to the undesired biochemical and physiological changes during the sporulation phase.

No abstract

11034 Background: Skp2, a known oncogene, is overexpressed in several types of tumors and is associated with worse recurrence rate and overall survival in primary melanoma patients. Moreover, the anti-proliferative effects of Skp2 siRNA on various tumor cell lines have prompted the preclinical testing of Skp2 small molecule inhibitors. In this study, we assessed the clinical relevance and molecular mechanism(s) underlying Skp2 overexpression in metastatic melanoma patients. Methods: Skp2 protein levels were measured in 122 metastatic melanoma specimens using immunohistochemistry (IHC), and the association between Skp2 overexpression and post-recurrence survival was examined. Moreover, 22 cell lines (2 normal primary melanocytes, 2 primary immortal melanocytes, 4 primary melanoma cell lines, and 18 metastatic melanoma cell lines) were evaluated for Skp2 genomic amplification using Single Nucleotide Polymorphism (SNP) arrays (Affymetrix 6.0) and Skp2 gene expression using mRNA arrays (Affymetrix U133A 2.0) and quantitative RT-PCR. We also screened 18 cell lines for Skp2 mutation by sequencing. Results: Skp2 overexpression, defined as >25% tumor cells, was associated with shorter 3-yr post-recurrence survival (37%) compared to Skp2 expression ≤25% (55%) (HR=1.89, 95%, CI= 1.04, 3.42, p=0.04). Skp2 overexpression was significantly associated with the site of melanoma metastasis: visceral (n= 12; 89%), lymph node (n=49; 36%), brain (n=15; 14%), and soft-tissue (n=36; 6%) (p<0.001). SNP array revealed genomic amplification at the Skp2 locus in 6 (33%) metastatic cell lines and one primary melanoma cell line. Skp2 genomic amplification was associated with increased transcript expression. No Skp2 mutations were identified. Conclusions: Skp2 protein overexpression is associated with worse prognosis in metastasis in melanoma. Our results also support that gene amplification, rather than a Skp2 gene mutation, may be the major mechanism responsible for Skp2 aberrant expression in metastatic melanoma. No significant financial relationships to disclose.

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Heterologous expression of bacteriocin genetic determinants (or operons) has long been a research interest for the functional analysis of genes involved in bacteriocin biosynthesis, regulation, modification, and immunity. Previously, construction of genomic libraries of the bacteriocin producer strains was usually required to identify new bacteriocin operons, a method that is tedious and time consuming. For the first time, we directly amplified an 8.14-kb bioinformatically identified thurincin H gene cluster using a one-step PCR method with 100% accuracy. This amplified gene cluster was cloned into plasmid pHT315, resulting in plasmid pGW139, and subsequently transformed to Bacillus thuringiensis EG10368, a strain naturally sensitive to thurincin H. Heterologous expression of the gene cluster makes the sensitive B. thuringiensis EG10368 produce thurincin H at a higher level compared with the wild-type producer, B. thuringiensis SF361. Moreover, B. thuringiensis EG10368pGW139 acquired complete immunity to thurincin H. The results indicated that one-step PCR is a promising tool to accurately amplify long bacteriocin gene clusters used in bacteriocin functional analysis studies and it is an effective way to produce bacteriocins at a higher level, without the need to clone large chromosomal fragments.