Cdk2 knockout mice are viable

BACKGROUND: Cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits control cell growth and division. Cdk2/cyclin E complexes are thought to be required because they phosphorylate the retinoblastoma protein and drive cells through the G1/S transition into the S phase of the cell cycle. In addition, Cdk2 associates with cyclin A, which itself is essential for cell proliferation during early embryonic development. RESULTS: In order to study the functions of Cdk2 in vivo, we generated Cdk2 knockout mice. Surprisingly, these mice are viable, and therefore Cdk2 is not an essential gene in the mouse. However, Cdk2 is required for germ cell development; both male and female Cdk2(-/-) mice are sterile. Immunoprecipitates of cyclin E1 complexes from Cdk2(-/-) spleen extracts displayed no activity toward histone H1. Cyclin A2 complexes were active in primary mouse embryonic fibroblasts (MEFs), embryo extracts and in spleen extracts from young animals. In contrast, there was little cyclin A2 kinase activity in immortalized MEFs and spleen extracts from adult animals. Cdk2(-/-) MEFs proliferate but enter delayed into S phase. Ectopic expression of Cdk2 in Cdk2(-/-) MEFs rescued the delayed entry into S phase. CONCLUSIONS: Although Cdk2 is not an essential gene in the mouse, it is required for germ cell development and meiosis. Loss of Cdk2 affects the timing of S phase, suggesting that Cdk2 is involved in regulating progression through the mitotic cell cycle.     

Ceramide increases mitochondrial free calcium levels via caspase 8 and Bid: role in initiation of cell death

We investigated how the mitochondrial phase of ceramide-mediated cell death is initiated in nerve growth factor (NGF)-differentiated PC12 cells. We distinguished three independent effects of ceramide: free radical production; a transient increase in cytosolic free calcium; and a long-lasting increase in mitochondrial free calcium. Only the latter led to cell death, which could be prevented by buffering of mitochondrial calcium with the calcium binding protein calbindin D-28K ectopically expressed in mitochondria. We showed that mitochondrial calcium did not increase as a result of the increase in cytosolic free calcium levels. Rather, it appears to derive from the endoplasmic reticulum (ER) since dantrolene, which inhibits release of calcium from ER into cytosol through ryanodine receptors, prevented the increase in cytosolic free calcium but potentiated the increase in mitochondrial free calcium. This suggests that a transfer of calcium occurs directly, or very locally, between the two organelles. This transfer implicated activation of caspase 8 and cleavage of its substrate Bid, a previously unknown function of these cell death intermediaries. The increase in mitochondrial free calcium was also responsible for the release of cytochrome c into the cytosol, underlining the critical role it plays in ceramide-mediated cell death.     

Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) shows a very restricted tropism for cells of the monocyte/macrophage lineage. It enters cells via receptor-mediated endocytosis. A monoclonal antibody (MAb) that is able to block PRRSV infection of porcine alveolar macrophages (PAM) and that recognizes a 210-kDa protein (p210) was described previously (MAb41D3) (X. Duan, H. Nauwynck, H. Favoreel, and M. Pensaert, J. Virol. 72:4520-4523, 1998). In the present study, the p210 protein was purified from PAM by immunoaffinity using MAb41D3 and was subjected to internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56 to 91% with mouse sialoadhesin, a macrophage-restricted receptor, were obtained with four p210 peptides. Using these peptide data, the full p210 cDNA sequence (5,193 bp) was subsequently determined. It shared 69 and 78% amino acid identity, respectively, with mouse and human sialoadhesins. Swine (PK-15) cells resistant to viral entry were transfected with the cloned p210 cDNA and inoculated with European or American PRRSV strains. Internalized virus particles were detected only in PK-15 cells expressing the recombinant sialoadhesin, demonstrating that this glycoprotein mediated uptake of both types of strains. However, nucleocapsid disintegration, like that observed in infected Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process. The ability of porcine sialoadhesin to mediate endocytosis was demonstrated by specific internalization of MAb41D3 into PAM. Altogether, these results show that sialoadhesin is involved in the entry process of PRRSV in PAM.     

sst2 Somatostatin receptor inhibits cell proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 activation

The G protein-coupled sst2 somatostatin receptor is a critical negative regulator of cell proliferation. sstII prevents growth factor-induced cell proliferation through activation of the tyrosine phosphatase SHP-1 leading to induction of the cyclin-dependent kinase inhibitor p27Kip1. Here, we investigate the signaling molecules linking sst2 to p27Kip1. In Chinese hamster ovary-DG-44 cells stably expressing sst2 (CHO/sst2), the somatostatin analogue RC-160 transiently stimulates ERK2 activity and potentiates insulin-stimulated ERK2 activity. RC-160 also stimulates ERK2 activity in pancreatic acini isolated from normal mice, which endogenously express sst2, but has no effect in pancreatic acini derived from sst2 knock-out mice. RC-160-induced p27Kip1 up-regulation and inhibition of insulin-dependent cell proliferation are both prevented by pretreatment of CHO/sst2 cells with the MEK1/2 inhibitor PD98059. In addition, using dominant negative mutants, we show that sst2-mediated ERK2 stimulation is dependent on the pertussis toxin-sensitive Gi/o protein, the tyrosine kinase Src, both small G proteins Ras and Rap1, and the MEK kinase B-Raf but is independent of Raf-1. Phosphatidylinositol 3-kinase (PI3K) and both tyrosine phosphatases, SHP-1 and SHP-2, are required upstream of Ras and Rap1. Taken together, our results identify a novel mechanism whereby a Gi/o protein-coupled receptor inhibits cell proliferation by stimulating ERK signaling via a SHP-1-SHP-2-PI3K/Ras-Rap1/B-Raf/MEK pathway.     

Proteomic analysis of astrocytic secretion in the mouse. Comparison with the cerebrospinal fluid proteome

Astrocytes, the most abundant cell type in the central nervous system, are intimately associated with synapses. They play a pivotal role in neuronal survival and the brain inflammatory response. Some astrocytic functions are mediated by the secretion of polypeptides. Using a proteomic approach, we have identified more than 30 proteins released by cultured astrocytes. These include proteases and protease inhibitors, carrier proteins, and antioxidant proteins. Exposing astrocytes to brefeldin A, which selectively blocks secretory vesicle assembly, suppressed the release of some of these proteins. This indicates that astrocytes secrete these proteins by a classic vesicular mechanism and others by an alternative pathway. Astrocytes isolated from different brain regions secreted a similar pattern of proteins. However, the secretion of some of them, including metalloproteinase inhibitors and apolipoprotein E, was region-specific. In addition, pro-inflammatory treatments modified the profile of astrocytic protein secretion. Finally, more than two thirds of the proteins identified in the astrocyte-conditioned medium were detectable in the mouse cerebrospinal fluid, suggesting that astrocytes contribute to the cerebrospinal fluid protein content. In conclusion, this study provides the first unbiased characterization of the major proteins released by astrocytes, which may play a crucial role in the modulation of neuronal survival and function.     

Slowed conduction and thin myelination of peripheral nerves associated with mutant rho Guanine-nucleotide exchange factor 10

Slowed nerve-conduction velocities (NCVs) are a biological endophenotype in the majority of the hereditary motor and sensory neuropathies (HMSN). Here, we identified a family with autosomal dominant segregation of slowed NCVs without the clinical phenotype of HMSN. Peripheral-nerve biopsy showed predominantly thinly myelinated axons. We identified a locus at 8p23 and a Thr109Ile mutation in ARHGEF10, encoding a guanine-nucleotide exchange factor (GEF) for the Rho family of GTPase proteins (RhoGTPases). Rho GEFs are implicated in neural morphogenesis and connectivity and regulate the activity of small RhoGTPases by catalyzing the exchange of bound GDP by GTP. Expression analysis of ARHGEF10, by use of its mouse orthologue Gef10, showed that it is highly expressed in the peripheral nervous system. Our data support a role for ARHGEF10 in developmental myelination of peripheral nerves.     

Males outcompete hermaphrodites for seed siring success in controlled crosses in the polygamous Fraxinus excelsior (Oleaceae)

Polygamy (including trioecy and subdioecy), the co-occurrence of males, hermaphrodites, and females in natural populations, is a rare and poorly studied breeding system expressed in Fraxinus excelsior L. (Oleaceae), a wind-pollinated tree. Here we investigate siring ability of pollen from male vs. hermaphrodite individuals to better understand this sex polymorphism. We conducted single-donor and two-donor pollination experiments and compared both fruit set and seed siring success, assessed with polymorphic microsatellite markers, of male and hermaphrodite individuals. Single pollen donor crosses allowed us to verify the male function of hermaphrodites. However, pollen from hermaphrodites was much less proficient than male pollen, with males siring 10 times as many fruits in single donor pollination treatments. This result was strengthened by the surprisingly low reproductive success of hermaphrodites in pollen competition conditions: of the 110 seedlings analyzed three were selfed and only one was sired by the hermaphrodite donor. The remaining 106 were sired by the male pollen donor. These results raise the question of the maintenance of male fertility in hermaphrodites in Fraxinus excelsior. Male function of hermaphrodites in this species now needs to be assessed under field conditions.     

The c-Cbl-associated protein and c-Cbl are two new partners of the SH2-containing inositol polyphosphate 5-phosphatase SHIP2

SHIP2 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase which contains motifs susceptible to mediate protein-protein interaction. Using yeast two-hybrid, GST-pulldown, and coimmunoprecipitation studies, we isolated the CAP cDNA as a specific partner of SHIP2 proline-rich domain and showed by GST-pulldown experiments that the interaction took place with the SH3C of CAP. The interaction was not modulated in COS-7 cells stimulated by EGF neither in CHO cells overexpressing the insulin receptor in the presence or absence of insulin stimulation. We also showed that SHIP2 was able to coimmunoprecipitate with endogenous c-Cbl protein in the absence of CAP and with the insulin receptor in CHO-IR cell extracts. The presence of SHIP2 in a complex around the insulin receptor could account for the very specific increase in insulin sensitivity of SHIP2 knock-out mice.