Membrane lipids and ethanol tolerance in the mouse. The influence of dietary fatty acid composition

Mice of the TO Swiss strain received diets containing different amounts of saturated or unsaturated fat throughout life. These diets produced characteristic changes in cardiac phospholipid fatty acid composition, but produced no significant differences in fatty acid composition of phospholipids from a crude membrane fraction of brain. When littermates of these animals were exposed to ethanol vapour in an inhalation chamber it was observed that mice which had received a diet high in saturated fat lost the righting reflex at an estimated concentration of ethanol in blood higher than that required for mice receiving a control diet, or a diet rich in polyunsaturated fat. Analysis of the brain membrane fraction from those animals which had received ethanol revealed that mice receiving the highly saturated fat diet now had a significantly greater proportion of saturated fatty acids in brain membrane phospholipids. These results are discussed in relation to the hypothesis that brain membrane lipid composition may influence the behavioural response to ethanol.     

CD4+ T cell responses to SSX-4 in melanoma patients

Genes of the synovial sarcoma X breakpoint (SSX) family are expressed in different human tumors, including melanomas, but not in adult somatic tissues. Because of their specific expression at the tumor site, SSX-encoded Ags are potential targets for anticancer immunotherapy. In this study, we have analyzed CD4+ T cell responses directed against the Ag encoded by SSX-4. Upon in vitro stimulation of PBMC from four melanoma patients bearing Ag-expressing tumors with a pool of long peptides spanning the protein sequence, we detected and isolated SSX-4-specific CD4+ T cells recognizing several distinct antigenic sequences, mostly restricted by frequently expressed HLA class II alleles. The majority of the identified sequences were located within the Krüppel-associated box domain in the N-terminal region of the protein, indicating a high potential immunogenicity of this region. Together our data document the existence of CD4+ T cells specific for multiple SSX-4 derived sequences in circulating lymphocytes from melanoma patients and encourage further studies to assess the impact of SSX-4-specific T cell responses on disease evolution in cancer patients.     

Mouse model of split hand/foot malformation type I

Split hand/foot malformation type I (SHFM1) disease locus maps to chromosome 7q21.3-q22, a region that includes the distal-less-related (dll) genes DLX5 and DLX6. However, incomplete penetrance, variable expressivity, segregation distortion, and syndromic association with other anomalies have so far prevented the identification of the SHFM1 gene(s) in man. Here we show that the targeted double inactivation of Dlx5 and Dlx6 in the mouse causes in homozygous mutant animals bilateral ectrodactyly with a severe defect of the central ray of the hindlimbs, a malformation typical of SHFM1. This is the first evidence that the role of dll/Dlx genes in appendage development is conserved from insects to mammals and proves their involvement in SHFM1.     

Quantitative founder-effect analysis of French Canadian families identifies specific loci contributing to metabolic phenotypes of hypertension

The Saguenay-Lac St-Jean population of Quebec is relatively isolated and has genealogical records dating to the 17th-century French founders. In 120 extended families with at least one sib pair affected with early-onset hypertension and/or dyslipidemia, we analyzed the genetic determinants of hypertension and related cardiovascular and metabolic conditions. Variance-components linkage analysis revealed 46 loci after 100,000 permutations. The most prominent clusters of overlapping quantitative-trait loci were on chromosomes 1 and 3, a finding supported by principal-components and bivariate analyses. These genetic determinants were further tested by classifying families by use of LOD score density analysis for each measured phenotype at every 5 cM. Our study showed the founder effect over several generations and classes of living individuals. This quantitative genealogical approach supports the notion of the ancestral causality of traits uniquely present and inherited in distinct family classes. With the founder effect, traits determined within population subsets are measurably and quantitatively transmitted through generational lineage, with a precise component contributing to phenotypic variance. These methods should accelerate the uncovering of causal haplotypes in complex diseases such as hypertension and metabolic syndrome.     

Molecular mechanisms of the effect of herpesvirus saimiri protein StpC on the signaling pathway leading to NF-kappaB activation

Herpesvirus saimiri (Saimiriine herpesvirus-2) causes lethal T lymphoproliferative diseases in the susceptible species and transforms T lymphocytes to continuous growth in vitro. H. saimiri-induced transformation of T cells is becoming an important experimental tool of biomedical research. Two proteins of H. saimiri subgroup C, Tip and StpC, are essential for T cell transformation by this virus. It has been shown previously that StpC transforms fibroblasts, activates NF-kappaB, and binds to tumor necrosis factor (TNF)-receptor-associated factor (TRAF) proteins, but the molecular mechanism of its action remains insufficiently understood. This study further characterized the effect of StpC on NF-kappaB. First, StpC activates NF-kappaB via the consensus pathway involving activation of I-kappaB kinase and subsequent phosphorylation and degradation of I-kappaB in both T lymphoid and epithelial cells. Second, triggering of this pathway by StpC in both T lymphoid and epithelial cells is dependent on the presence of functional NF-kappaB-inducing kinase (NIK). Third, StpC physically interacts with TRAF in epithelial cells, and the effect of StpC on NF-kappaB activity in these cells requires the presence of functional TRAF. Finally the effect of StpC is completely independent of TNF-alpha, a well described stimulus of NF-kappaB activity. Moreover it appears that StpC uncouples stimulation of NF-kappaB activity from TNF-alpha stimulation. Overall these results argue that the effect of StpC on NF-kappaB is similar to the effects of other viral proteins, "usurping" the TRAF/NIK/I-kappaB kinase pathway, and reinforce the notion that the role of StpC in cell transformation by H. saimiri may be mediated by signaling that results in NF-kappaB activation.     

Stabilization of Mdm2 via decreased ubiquitination is mediated by protein kinase B/Akt-dependent phosphorylation

The tumor suppressor p53 is commonly inhibited under conditions in which the phosphatidylinositide 3'-OH kinase/protein kinase B (PKB)Akt pathway is activated. Intracellular levels of p53 are controlled by the E3 ubiquitin ligase Mdm2. Here we show that PKB inhibits Mdm2 self-ubiquitination via phosphorylation of Mdm2 on Ser(166) and Ser(188). Stimulation of human embryonic kidney 293 cells with insulin-like growth factor-1 increased Mdm2 phosphorylation on Ser(166) and Ser(188) in a phosphatidylinositide 3'-OH kinase-dependent manner, and the treatment of both human embryonic kidney 293 and COS-1 cells with phosphatidylinositide 3'-OH kinase inhibitor LY-294002 led to proteasome-mediated Mdm2 degradation. Introduction of a constitutively active form of PKB together with Mdm2 into cells induced phosphorylation of Mdm2 at Ser(166) and Ser(188) and stabilized Mdm2 protein. Moreover, mouse embryonic fibroblasts lacking PKBalpha displayed reduced Mdm2 protein levels with a concomitant increase of p53 and p21(Cip1), resulting in strongly elevated apoptosis after UV irradiation. In addition, activation of PKB correlated with Mdm2 phosphorylation and stability in a variety of human tumor cells. These findings suggest that PKB plays a critical role in controlling of the Mdm2.p53 signaling pathway by regulating Mdm2 stability.     

Bilateral enucleation and captivity influence the reproductive cycle of male Viscacha (Lagostomus maximus maximus)

The viscacha (Lagostomus maximus maximus) is a seasonal rodent living in the Southern Hemisphere. The adult males exhibit an annual reproductive cycle characterized by a gonadal regression period during winter. In this study, we investigated the effects of bilateral enucleation and captivity on their annual reproductive cycle. Testicular volume relative to body weight was recorded monthly in intact and bilaterally enucleated animals placed under natural photoperiod, water, and food ad lib. and constant temperature. Testes and accessory organs were evaluated by qualitative and quantitative light microscopic studies. The intact animals showed an annual reproductive cycle with complete gonadal atrophy in the first year. In the second year, testicular regression was observed but attenuated in regard to that recorded in the first winter period, indicating that adaptive changes might be involved. Bilateral enucleation in the viscacha dampened and extended the period of its annual reproductive cycle. The results suggest that both conditions, constant captivity and enucleation, produced stimulatory effects on the reproductive system of this rodent. Furthermore, local control mechanisms might be responsible for the morphological differences observed in testes, epididymis, and seminal vesicles from both groups, which exhibited similar levels of serum testosterone. Finally, an intact retinohypothalamic-pineal axis and/or photoperiodic input would be necessary to maintain the reproductive cycle amplitude and timing in viscacha.