Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion.

In Pacific spiny dogfish (Squalus acanthias), plasma CO(2) reactions have access to plasma carbonic anhydrase (CA) and gill membrane-associated CA. The objectives of this study were to characterise the gill membrane-bound CA and investigate whether extracellular CA contributes significantly to CO(2) excretion in dogfish. A subcellular fraction containing membrane-associated CA activity was isolated from dogfish gills and incubated with phosphatidylinositol-specific phospholipase C. This treatment caused significant release of CA activity from its membrane association, a result consistent with identification of the dogfish gill membrane-bound CA as a type IV isozyme. Inhibition constants (K(i)) against acetazolamide and benzolamide were 4.2 and 3.5 nmol L(-1), respectively. Use of a low dose (1.3 mg kg(-1) or 13 micromol L(-1)) of benzolamide to selectively inhibit extracellular CA in vivo caused a significant 30%-60% reduction in the arterial-venous total CO(2) concentration difference, a significant increase in Pco(2) and an acidosis, without affecting blood flow or ventilation. No effect of benzolamide on any measure of CO(2) excretion was detected in rainbow trout (Oncorhynchus mykiss). These results indicate that extracellular CA contributes substantially to CO(2) excretion in the dogfish, an elasmobranch, and confirm that CA is not available to plasma CO(2) reactions in rainbow trout, a teleost.     

Genetic analysis of adiponectin and obesity in Hispanic families: the IRAS Family Study

Adiponectin, coded for by the APM1 gene, is a novel adipocyte-derived hormone implicated in energy homeostasis and obesity. Several genetic studies have observed evidence of association between APM1 gene polymorphisms and features of the metabolic syndrome, such as insulin resistance and obesity. As part of a comprehensive genetic analysis of the APM1 gene, we have screened 96 unrelated individuals for polymorphisms in the promoter, coding regions, and 3untranslated region (UTR). Three promoter single-nucleotide polymorphisms (SNPs), two rare coding SNPs (G113A and T1233C), and 13 SNPs in the 3UTR were identified. Eighteen SNPs were genotyped in 811 Hispanic individuals from 45 families in the IRAS Family Study (IRASFS). SNPs were tested for association with six obesity quantitative traits (body mass index, waist, waist:hip ratio, subcutaneous adipose tissue, visceral adipose tissue, and visceral:subcutaneous ratio). Significant evidence of association to at least one of the obesity traits was identified in seven of the 18 SNPs (<0.001–0.05). The promoter SNP INS CA-11156 was the most consistently associated SNP and was associated significantly with all measures of obesity, except the visceral:subcutaneous ratio (P-values 0.009–0.03). Haplotype analysis supported this evidence of association, with haplotypes containing an insertion of one CA repeat at position –11156 consistently being associated with lower obesity values (P-value <0.001–0.05). The adiponectin polymorphisms, in particular those in the promoter region, thus show significant association with obesity measures in the Hispanic population. Additional studies are needed to confirm our findings and determine which polymorphism causes the functional effect.     

Conditional MLL-CBP targets GMP and models therapy-related myeloproliferative disease

Chromosomal translocations that fuse the mixed lineage leukemia (MLL) gene with multiple partners typify acute leukemias of infancy as well as therapy-related leukemias. We utilized a conditional knockin strategy to bypass the embryonic lethality caused by MLL-CBP expression and to assess the immediate effects of induced MLL-CBP expression on hematopoiesis. Within days of activating MLL-CBP, the fusion protein selectively expanded granulocyte/macrophage progenitors (GMP) and enhanced their self-renewal/proliferation. MLL-CBP altered the gene expression program of GMP, upregulating a subset of genes including Hox a9. Inhibition of Hox a9 expression by RNA interference demonstrated that MLL-CBP required Hox a9 for its enhanced cell expansion. Following exposure to sublethal gamma-irradiation or N-ethyl-N-nitrosourea (ENU), MLL-CBP mice developed myelomonocytic hyperplasia and progressed to fatal myeloproliferative disorders. These represented the spectrum of therapy-induced acute myelomonocytic leukemia/chronic myelomonocytic leukemia/myelodysplastic/myeloproliferative disorder similar to that seen in humans possessing the t(11;16). This model of MLL-CBP therapy-related myeloproliferative disease demonstrates the selectivity of this MLL fusion for GMP cells and its ability to initiate leukemogenesis in conjunction with cooperating mutations.     

B23 is a downstream target of polyamine- modulated CK2

Our previous studies have shown that the overexpression of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, increases the enzymatic activity of the polyamine-responsive enzyme casein kinase 2 (CK2). Because CK2 is known to preferentially associate with the nuclear matrix in response to other trophic stimuli, we investigated the effects of ODC overexpression on CK2 localisation and on the CK2-mediated phosphorylation of a known CK2 substrate, the nucleolar phosphoprotein B23. Immunofluorescence analysis of CK2 and B23 in primary keratinocytes revealed that ODC overexpression resulted in the colocalisation of CK2 with B23 at the nucleolar borders. ODC overexpression also increased CK2 kinase activity 2-fold at the nuclear matrix, a response which could be abrogated by treatment of K6/ODC transgenic keratinocytes with the ODC inhibitor α-difluoromethylornithine (DFMO). Levels of B23 protein were also elevated in ODC-overexpressing cells compared to normal cells or transgenic cells treated with DFMO. This increase in protein level was neither due to an increase in steady-state mRNA levels, nor was it due to increased stability of B23 protein. Phosphorylation of B23 was also increased in ODC-overexpressing cells, and this increased phosphorylation could be blocked by treatment of the cells with the CK2 kinase inhibitors apigenin or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). These data suggest that B23 may be a downstream effector of polyamines via phosphorylation by the protein kinase CK2.     

GeneCruiser: a web service for the annotation of microarray data

SUMMARY: GeneCruiser is a web service allowing users to annotate their genomic data by mapping microarray feature identifiers to gene identifiers from databases, such as UniGene, while providing links to web resources, such as the UCSC Genome Browser. It relies on a regularly updated database that retrieves and indexes the mappings between microarray probes and genomic databases. Genes are identified using the Life Sciences Identifier standard. AVAILABILITY: GeneCruiser is freely available in the following forms: Web service and Web application, http://www.genecruiser.org; GenePattern, GeneCruiser access has been integrated into our microarray analysis platform, GenePattern. http://www.genepattern.org.     

Evidence that nervy, the Drosophila homolog of ETO/MTG8, promotes mechanosensory organ development by enhancing Notch signaling

In the imaginal tissue of developing fruit flies, achaete (ac) and scute (sc) expression defines a group of neurally-competent cells called the proneural cluster (PNC). From the PNC, a single cell, the sensory organ precursor (SOP), is selected as the adult mechanosensory organ precursor. The SOP expresses high levels of ac and sc and sends a strong Delta (Dl) signal, which activates the Notch (N) receptor in neighboring cells, preventing them from also adopting a neural fate. Previous work has determined how ac and sc expression in the PNC and SOP is regulated, but less is known about SOP-specific factors that promote SOP fate. Here, we describe the role of nervy (nvy), the Drosophila homolog of the mammalian proto-oncogene ETO, in mechanosensory organ formation. Nvy is specifically expressed in the SOP, where it interacts with the Ac and Sc DNA binding partner Daughterless (Da) and affects the expression of Ac and Sc targets. nvy loss- and gain-of-function experiments suggest that nvy reinforces, but is not absolutely required for, the SOP fate. We propose a model in which nvy acts downstream of ac and sc to promote the SOP fate by transiently strengthening the Dl signal emanating from the SOP.     

Heterologous expression of hexaheme fragments of a multidomain cytochrome from Geobacter sulfurreducens representing a novel class of cytochromes c

Multiheme cytochromes c are of great interest for researchers for a variety of reasons but difficult to obtain in quantities sufficient for the majority of studies. The genome of delta-proteobacterium Geobacter sulfurreducens contains more than a hundred genes coding for c-type cytochromes. Three of them represent a new class of multiheme cytochromes characterized by a mixed type of heme coordination and multidomain organization. We cloned and expressed in Escherichia coli three hexaheme fragments corresponding to two-domain fragments of one such protein containing 12 heme binding motifs and believed to consist of four triheme domains. Despite high sequence similarity among the fragments, expression levels varied significantly. Expression was optimized either by host strain variation or by reducing the rate of apoprotein synthesis. All three fragments were purified by cation exchange followed by gel filtration and were shown to contain six covalently attached hemes as confirmed by mass spectrometry. Their visible spectra are typical of c-type cytochromes. One of the fragments was crystallized and its preliminary X-ray structure shows two separate domains.