Inflammatory factors produced by sensitized guinea-pig peripheral blood lymphocytes

Summary The supernatants obtained from stimulated tuberculin-sensitive guinea-pig peripheral blood lymphocytes contain factors that induce a cutaneous inflammatory response in normal guinea-pigs similar to the tuberculin reaction and inhibit the migration of normal guinea-pigs peritoneal exudate cells. There appears to be a correlation between the presence of in vitro migration inhibitory activity and inflammatory activity in vivo.     

Erythrocyte 2,3-diphosphoglycerate in anaemic sheep

Summary Experimental anaemia resulted in an increase of red cell 2,3-DPG from 0.11 M/gHb to 0.99 M in haemoglobin A sheep and from 0.21 M to 1.5 M in haemoglobin B sheep. Production of haemoglobin C as a result of anaemia was confined to haemoglobin A only. The results, therefore, appear to suggest that the rise in 2,3-DPG in the red blood cells of different haemoglobin types is independent of haemoglobin C.This work was supported by grants from the National Health and Medical Research Council, The Clive and Vera Ramaciotti New South Wales Foundations and Helena Rubinstein Foundation, Inc. We wish to thank Mr J. Sheedy for his expert technical assistance, and Professor G. J. Brewer for his constructive criticism.     

Essential roles of PI(3)K-p110beta in cell growth, metabolism and tumorigenesis

On activation by receptors, the ubiquitously expressed class IA isoforms (p110alpha and p110beta) of phosphatidylinositol-3-OH kinase (PI(3)K) generate lipid second messengers, which initiate multiple signal transduction cascades. Recent studies have demonstrated specific functions for p110alpha in growth factor and insulin signalling. To probe for distinct functions of p110beta, we constructed conditional knockout mice. Here we show that ablation of p110beta in the livers of the resulting mice leads to impaired insulin sensitivity and glucose homeostasis, while having little effect on phosphorylation of Akt, suggesting the involvement of a kinase-independent role of p110beta in insulin metabolic action. Using established mouse embryonic fibroblasts, we found that removal of p110beta also had little effect on Akt phosphorylation in response to stimulation by insulin and epidermal growth factor, but resulted in retarded cell proliferation. Reconstitution of p110beta-null cells with a wild-type or kinase-dead allele of p110beta demonstrated that p110beta possesses kinase-independent functions in regulating cell proliferation and trafficking. However, the kinase activity of p110beta was required for G-protein-coupled receptor signalling triggered by lysophosphatidic acid and had a function in oncogenic transformation. Most strikingly, in an animal model of prostate tumour formation induced by Pten loss, ablation of p110beta (also known as Pik3cb), but not that of p110alpha (also known as Pik3ca), impeded tumorigenesis with a concomitant diminution of Akt phosphorylation. Taken together, our findings demonstrate both kinase-dependent and kinase-independent functions for p110beta, and strongly indicate the kinase-dependent functions of p110beta as a promising target in cancer therapy.     

SAP-controlled T-B cell interactions underlie germinal centre formation

Generation of long-term antibody-mediated immunity depends on the germinal centre reaction, which requires cooperation between antigen-specific T and B lymphocytes. In human X-linked lymphoproliferative disease and its gene-targeted mouse model, loss-of-function mutations in signalling lymphocyte activation molecule-associated protein (SAP, encoded by SH2D1a) cause a profound defect in germinal centre formation by an as yet unknown mechanism. Here, using two-photon intravital imaging, we show that SAP deficiency selectively impairs the ability of CD4(+) T cells to stably interact with cognate B cells but not antigen-presenting dendritic cells. This selective defect results in a failure of antigen-specific B cells to receive adequate levels of contact-dependent T-cell help to expand normally, despite Sap(-/-) T cells exhibiting the known characteristics of otherwise competent helper T cells. Furthermore, the lack of stable interactions with B cells renders Sap(-/-) T cells unable to be efficiently recruited to and retained in a nascent germinal centre to sustain the germinal centre reaction. These results offer an explanation for the germinal centre defect due to SAP deficiency and provide new insights into the bi-directional communication between cognate T and B cells in vivo.     

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.     

Towards a proteome-scale map of the human protein-protein interaction network

Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.     

Natural variation in cardiac metabolism and gene expression in Fundulus heteroclitus

Individual variation in gene expression is important for evolutionary adaptation and susceptibility to diseases and pathologies. In this study, we address the functional importance of this variation by comparing cardiac metabolism to patterns of mRNA expression using microarrays. There is extensive variation in both cardiac metabolism and the expression of metabolic genes among individuals of the teleost fish Fundulus heteroclitus from natural outbred populations raised in a common environment: metabolism differed among individuals by a factor of more than 2, and expression levels of 94% of genes were significantly different (P < 0.01) between individuals in a population. This unexpectedly high variation in metabolic gene expression explains much of the variation in metabolism, suggesting that it is biologically relevant. The patterns of gene expression that are most important in explaining cardiac metabolism differ between groups of individuals. Apparently, the variation in metabolism seems to be related to different patterns of gene expression in the different groups of individuals. The magnitude of differences in gene expression in these groups is not important; large changes in expression have no greater predictive value than small changes. These data suggest that variation in physiological performance is related to the subtle variation in gene expression and that this relationship differs among individuals.     

Deficiency of glutaredoxin 5 reveals Fe-S clusters are required for vertebrate haem synthesis

Iron is required to produce haem and iron-sulphur (Fe-S) clusters, processes thought to occur independently. Here we show that the hypochromic anaemia in shiraz (sir) zebrafish mutants is caused by deficiency of glutaredoxin 5 (grx5), a gene required in yeast for Fe-S cluster assembly. We found that grx5 was expressed in erythroid cells of zebrafish and mice. Zebrafish grx5 rescued the assembly of grx5 yeast Fe-S, showing that the biochemical function of grx5 is evolutionarily conserved. In contrast to yeast, vertebrates use iron regulatory protein 1 (IRP1) to sense intracellular iron and regulate mRNA stability or the translation of iron metabolism genes. We found that loss of Fe-S cluster assembly in sir animals activated IRP1 and blocked haem biosynthesis catalysed by aminolaevulinate synthase 2 (ALAS2). Overexpression of ALAS2 RNA without the 5' iron response element that binds IRP1 rescued sir embryos, whereas overexpression of ALAS2 including the iron response element did not. Further, antisense knockdown of IRP1 restored sir embryo haemoglobin synthesis. These findings uncover a connection between haem biosynthesis and Fe-S clusters, indicating that haemoglobin production in the differentiating red cell is regulated through Fe-S cluster assembly.