Phosphatidylinositol 3,4,5-trisphosphate is a phospholipid signaling molecule involved in many cellular functions including growth factor receptor signaling, cytoskeletal organization, chemotaxis, apoptosis, and protein trafficking. Phosphorylation at the 3 position of the inositol ring is catalyzed by many different 3-kinases (classified as types I A , I B , II, and III), but the physiological roles played by each of the different 3-kinase isozymes during embryonic development and in homeostasis in animals is incompletely understood. Mammalian type I A kinase isozymes are heterodimers that are active at 37°C when the catalytic 110-kDa subunit interacts through an amino-terminal binding domain with a regulatory 85-or 55-kDa subunit. Using gene targeting in embryonic stem cells, we deleted this binding domain in the gene encoding the ␣ isoform of the 110-kDa catalytic subunit (Pik3ca) of the ␣ isozyme of the type I A kinases, leading to loss of expression of the p110 catalytic subunit. We show that Pik3ca del/del embryos are developmentally delayed at embryonic day (E) 9.5 and die between E9.5 and E10.5. E9.5 Pik3ca del/del embryos have a profound proliferative defect but no increase in apoptosis. A proliferative defect is supported by the observation that fibroblasts from Pik3ca del/del embryos fail to replicate in Dulbecco's modified Eagle's medium and fetal calf serum, even with supplemental growth factors.Phosphorylated phosphoinositides generated by phosphoinositide (PI) 1 3-kinases play crucial roles in signaling through receptors at the cell surface as well as in membrane trafficking mechanisms in Golgi and endosomal compartments, cytoskeletal organization, and regulation of apoptosis (1-9). The genes for many PI 3-kinases have been isolated from a wide range of tissues and organisms. They are divided into three major classes based on their amino acid sequence, the homology among their lipid-kinase domains, and substrate specificities (6, 9). Class I PI 3-kinases phosphorylate the 3Ј sites of PtdIns, PtdIns 4-phosphate, and PtdIns 4,5-diphosphate to form PtdIns 3-phosphate, PtdIns 3,4-diphosphate, and PtdIns 3,4,5-trisphosphate. Class I PI 3-kinases can be further divided into two subclasses. The class I A PI 3-kinases, which include at least three isozymes, ␣, ␤, and ␦, are heterodimers of a catalytic 110-kDa subunit (p110) and a regulatory subunit of 85 or 55 kDa (p85/p55) (10 -14). The interaction of p85/p55 and p110 via the inter-Src homology 2 domain on p85/p55 and the aminoterminal 123 amino acids of p110 is critical for achieving maximal activity of this class of PI 3-kinases in mammalian cells at 37°C (11,15,16).Despite a wealth of knowledge of the biochemistry and cell biological effects of phosphoinositides, very little information is available on the particular physiological roles played by these different enzymes and isozymes during mammalian development. Inhibitors of PI 3-kinases such as wortmannin (17) and LY294002 (18) are very useful for probing the functions of these enzymes but as with a...

The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked human genetic disorder characterized by mental retardation, congenital cataracts, and renal tubular dysfunction. The Lowe syndrome gene, OCRL1 , encodes a phosphatidylinositol 4,5-bisphosphate 5-phosphatase in the Golgi complex. The pathogenesis of Lowe syndrome due to deficiency of a phosphatidylinositol 4,5-bisphosphate 5-phosphatase in the Golgi complex is unknown. We have used targeted disruption in embryonic stem cells to make mice deficient in Ocrl1 , the mouse homologue for OCRL1 , as an animal model for the disease. Surprisingly, mice deficient in Ocrl1 do not develop the congenital cataracts, renal Fanconi syndrome, or neurological abnormalities seen in the human disorder. We hypothesized that Ocrl1 deficiency is complemented in mice by inositol polyphosphate 5-phosphatase ( Inpp5b ), an autosomal gene that encodes a phosphatidylinositol bisphosphate 5-phosphatase highly homologous to Ocrl1. We created mice deficient in Inpp5b ; the mice were viable and fertile without phenotype except for testicular degeneration in males beginning after sexual maturation. We crossed mice deficient in Ocrl1 to mice deficient in Inpp5b. No liveborn mice or embryos lacking both enzymes were found, demonstrating that Ocrl1 and Inpp5b have overlapping functions in mice and suggesting that the lack of phenotype in Ocrl1 -deficient mice may be due to compensating Inpp5b function. (

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α-Synuclein (SNCA) gene has been implicated in the development of rare forms of familial Parkinson disease (PD). Recently, it was shown that an increase in SNCA copy numbers leads to elevated levels of wild-type SNCA-mRNA and protein and is sufficient to cause early-onset, familial PD. A critical question concerning the molecular pathogenesis of PD is what contributory role, if any, is played by the SNCA gene in sporadic PD. The expansion of SNCA-Rep1, an upstream, polymorphic microsatellite of the SNCA gene, is associated with elevated risk for sporadic PD. However, whether SNCA-Rep1 is the causal variant and the underlying mechanism with which its effect is mediated by remained elusive. We report here the effects of three distinct SNCA-Rep1 variants in the brains of 72 mice transgenic for the entire human SNCA locus. Human SNCA-mRNA and protein levels were increased 1.7- and 1.25-fold, respectively, in homozygotes for the expanded, PD risk-conferring allele compared with homozygotes for the shorter, protective allele. When adjusting for the total SNCA-protein concentration (endogenous mouse and transgenic human) expressed in each brain, the expanded risk allele contributed 2.6-fold more to the SNCA steady-state than the shorter allele. Furthermore, targeted deletion of Rep1 resulted in the lowest human SNCA-mRNA and protein concentrations in murine brain. In contrast, the Rep1 effect was not observed in blood lysates from the same mice. These results demonstrate that Rep1 regulates human SNCA expression by enhancing its transcription in the adult nervous system and suggest that homozygosity for the expanded Rep1 allele may mimic locus multiplication, thereby elevating PD risk.

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Gut-associated lymphocytes were described in fish, but their involvement in immune responses is still unknown. In rainbow trout, intraepithelial lymphocytes (IELs) are scattered between gut epithelial cells, but neither Peyer’s patches nor mesenteric lymph nodes were identified. Rainbow trout IELs contain mainly T cells, because they expressed transcripts of T cell marker homologs of CD8, CD4, CD28, CD3ε, TCRζ, TCRγ, and TCRβ and lacked IgM. However, trout IELs did not show specific homing to the gut mucosa, which in mammals defines IELs as a distinctive mucosal population. A detailed analysis of the TCRβ repertoire of rainbow trout IELs was performed in both naive and virus-infected animals. TCRβ transcripts of rainbow trout IELs were highly diverse and polyclonal in adult naive individuals, in sharp contrast with the restricted diversity of IEL oligoclonal repertoires described in birds and mammals. Significant modifications of the trout IEL TCRβ repertoire were observed after a systemic infection with a fish rhabdovirus and were especially marked for Vβ4-bearing receptors as previously reported for spleen cells. Thus, we could not find any specific properties of the trout IEL TCRβ repertoire compared with the spleen and pronephros TCRβ repertoire, which questions the reality of a distinct IEL compartment in teleosts. Our findings suggest that a highly diversified αβ ΤCR repertoire is maintained in fish IELs in the absence of Peyer’s patches and mesenteric lymph nodes, whereas the restricted diversity of mouse αβ IELs is attributed to multiple cycles of activation and recirculation, allowing a progressive narrowing of the repertoire.