Early-glycation of apolipoprotein E: effect on its binding to LDL receptor,scavenger receptor A and heparan sulfates

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) causes substantial morbidity afflicting approximately 10% of adult males. Treatment is often empirical and ineffective since the etiology is unknown. Other prostate and genitourinary diseases have genetic components suggesting that CP/CPPS may also be influenced by genetic predisposition. We recently reported a highly polymorphic short tandem repeat (STR) locus near the phosphoglycerate kinase gene within Xq11-13. Because this STR is in a region known to predispose towards other prostate diseases, we compared STR polymorphisms in 120 CP/CPPS patients and 300 control blood donors. Nine distinct allele sizes were detected, ranging from 8 to 15 repeats of the tetrameric STR plus a mutant allele (9.5) with a six base deletion in the flanking DNA sequence. The overall allele size distribution in the CP/CPPS patients differed from controls (Chi-square=19.252, df=8, P=0.0231). Frequencies of two specific alleles, 9.5 and 15, differed significantly in CP/CPPS vs. control subjects and allele 10 differed with marginal significance. Alleles 9.5 and 10 were both more common in CP/CPPS patients than controls while allele 15 was less common. These observations suggest that Xq11-13 may contain one or more genetic loci that predispose toward CP/CPPS. Further investigations involving family studies, larger patient populations, and other control groups may help elucidate this potential genetic predisposition in CP/CPPS.     

Oxidized low-density lipoprotein-induced apoptosis

Cultured cells are able to oxidize low-density lipoproteins (LDL) and oxidized LDL (oxLDL), which are present in atherosclerosis areas, exhibit a variety of biological properties potentially involved in atherogenesis. This review is focused on the toxicity of oxLDL, more precisely on the toxic compounds generated during LDL oxidation, the features and the mechanisms of cell death (apoptosis or necrosis) induced by oxLDL. After internalization, toxic oxidized lipids, namely lipid peroxides, oxysterols and aldehydes, induce modifications of cell proteins, elicit oxidative stress, lipid peroxidation and alter various signaling pathways and gene expression. These events may participate in the toxic effect, and converge to trigger an intense, delayed and sustained calcium peak which elicits either apoptosis or necrosis processes. OxLDL-induced apoptosis involves both mitochondrial and death-receptor (Fas/FasL) apoptotic pathways, thereby activating the classical caspase cascade and subsequent biochemical and morphological apoptotic features. When apoptosis is blocked by overexpression of Bcl-2, oxLDL trigger necrosis through a calcium-dependent pathway. Apoptosis occurring in atherosclerotic areas is potentially involved in endothelial cell lining defects, necrotic core formation and plaque rupture or erosion which may trigger atherothrombotic events. However, the precise role of oxLDL in apoptosis/necrosis occurring in vivo in atherosclerotic plaques remains to be clarified.     

A murine gene with circadian expression revealed by transposon insertion: self-sustained rhythmicity in the liver and the photoreceptors

We have previously identified in some mouse strains (e.g. BALB/c, DBA/2) a murine Intracisternal A-particle (IAP) transposable element specifically expressed in the liver. This IAP sequence is inserted within a gene, mCCR4/m. nocturnin, the sequence of which is related to the circadian Xenopus nocturnin gene. Here we show, using real-time quantitative RT-PCR, that both the IAP sequence and the m. nocturnin gene display strong circadian expression in the liver, with peak abundance after dusk. Circadian oscillations of m. nocturnin RNA are maintained in mice without the IAP insertion (e.g. CBA/J, 129/sv), are free-running under constant light and dark conditions, and persist upon food and water privation, demonstrating that m. nocturnin is a circadian gene. In situ hybridization analyses (in 129/sv mice) further show circadian expression of m. nocturnin also in the retina, precisely at the level of the photoreceptors, a result consistent with the previously described circadian expression of the Xenopus gene. These results strengthen the strong conservation of the nocturnin gene with the identification of a functional mouse ortholog of the Xenopus gene, and demonstrate the reciprocal influence of nearby genes on the expression of transposable elements via "position effects".     

Myostatin knockout in mice increases myogenesis and decreases adipogenesis

Growth differentiation factor-8 (GDF-8), or Myostatin, plays an important inhibitory role during muscle development. Since muscle and adipose tissue develop from the same mesenchymal stem cells, we hypothesized that Myostatin gene knockout may cause a switch between myogenesis and adipogenesis. Male and female wild type (WT) and Myostatin knockout (KO) mice were sacrificed at 4, 8, and 12 weeks of age. The gluteus muscle (GM) was larger in KO mice compared to WT mice at 8 (P < 0.01) and 12 (P < 0.001) weeks. At 12 weeks, KO mice had decreased fat depots (P < 0.01). Compared to 12-week-old WT mice, serum leptin concentration in KO mice was lower (P < 0.001) and leptin mRNA expression was decreased (P < 0.01) in inguinal adipose tissue. CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) levels in adipose tissue were significantly lower in KO mice compared to WT mice. Thus, increased muscle development in Myostatin knockout mice is associated with reduced adipogenesis and consequently, decreased leptin secretion.     

Site-directed mutagenesis of the basic N-terminal cluster of pancreatic bile salt-dependent lipase. Functional significance

Previous studies have postulated the presence of a heparin-binding site on the bile salt-dependent lipase (BSDL), whereas two bile salt-binding sites regulate the enzyme activity. One of these sites may overlap with the tentative heparin-binding site at the level of an N-terminal basic cluster consisting of positive residues Lys(32), Lys(56), Lys(61), Lys(62), and Arg(63). The present study uses specific site-directed mutagenesis to determine the functional significance of this basic cluster. Mutations in this sequence resulted in recombinant enzymes that were able to bind to immobilized and to cell-associated heparin before moving throughout intestinal cells. Recombinant BSDL was fully active on soluble substrate, but mutants were less active on micellar cholesteryl oleate in comparison with the wild-type enzyme. Activation studies by primary (sodium taurocholate) and by secondary (sodium taurodeoxycholate) bile salts revealed that the activation of BSDL by sodium taurocholate at concentrations below the critical micellar concentration, and not that evoked by micellar bile salts, was affected by substitutions, suggesting that this N-terminal basic cluster likely represents the specific bile salt-binding site of BSDL. Substitutions also affected the activation of the enzyme promoted by anionic phospholipids, extending the function of this site to that of a cationic regulatory site susceptible to accommodate anionic ligands.     

Pituitary adenylate cyclase-activating polypeptide stimulates nitric-oxide synthase type I expression and potentiates the cGMP response to gonadotropin-releasing hormone of rat pituitary gonadotrophs

Nitric-oxide synthase type I (NOS I) is expressed primarily in gonadotrophs and in folliculo-stellate cells of the anterior pituitary. In gonadotrophs, the expression and the activity of NOS I are stimulated by gonadotropin-releasing hormone (GnRH) under both experimental and physiological conditions. In the present study, we show that pituitary adenylate cyclase-activating polypeptide (PACAP) is twice as potent as GnRH at increasing NOS I levels in cultured rat anterior pituitary cells. The action of PACAP is detectable after 4-6 h and maximal at 24 h, this effect is mimicked by 8-bromo-cAMP and cholera toxin and suppressed by H89 suggesting a mediation through the cAMP pathway. Surprisingly, NADPH diaphorase staining revealed that these changes occurred in gonadotrophs exclusively although PACAP and cAMP, in contrast to GnRH, have the potential to target several types of pituitary cells including folliculo-stellate cells. There was no measurable alteration in NOS I mRNA levels after cAMP or PACAP induction. PACAP also stimulated cGMP synthesis, which was maximal within 15 min and independent of cAMP, however, only part resulted from NOS I/soluble guanylate cyclase activation implying that in contrast to GnRH, PACAP has a dual mechanism in cGMP production. Interestingly, induction of NOS I by PACAP markedly enhanced the capacity of gonadotrophs to produce cGMP in response to GnRH. The fact that PACAP may act on gonadotrophs to alter NOS I levels, generate cGMP, and potentiate the cGMP response to GnRH, suggests that cGMP could play important cellular functions.     

A role for DNA polymerase beta in mutagenic UV lesion bypass

We report here that DNA polymerase beta (pol beta), the base excision repair polymerase, is highly expressed in human melanoma tissues, known to be associated with UV radiation exposure. To investigate the potential role of pol beta in UV-induced genetic instability, we analyzed the cellular and molecular effects of excess pol beta. We firstly demonstrated that mammalian cells overexpressing pol beta are resistant and hypermutagenic after UV irradiation and that replicative extracts from these cells are able to catalyze complete translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). By using in vitro primer extension reactions with purified pol beta, we showed that CPD as well as, to a lesser extent, the thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct, were bypassed. pol beta mostly incorporates the correct dATP opposite the 3'-terminus of both CPD and the (6-4) photoproduct but can also misinsert dCTP at a frequency of 32 and 26%, respectively. In the case of CPD, efficient and error-prone extension of the correct dATP was found. These data support a biological role of pol beta in UV lesion bypass and suggest that deregulated pol beta may enhance UV-induced genetic instability.     

Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L-null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H-null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons.     

Dok-related protein negatively regulates T cell development via its RasGTPase-activating protein and Nck docking sites

Downstream of kinase (Dok)-related protein (DokR, also known as p56(dok)/FRIP/Dok-R) is implicated in cytokine and immunoreceptor signaling in myeloid and T cells. Tyrosine phosphorylation induces DokR to bind the signal relay molecules, RasGTPase-activating protein (RasGAP) and Nck. Here, we have examined the function of DokR during hematopoietic development and the requirement for RasGAP and Nck binding sites in its biological function. Retroviral-mediated expression of DokR in bone marrow cells dramatically inhibited their capacity to form colonies in vitro in response to the cytokines macrophage colony-stimulating factor and stem cell factor, whereas responses to interleukin-3 and granulocyte macrophage colony-stimulating factor were only weakly affected. When introduced into lethally irradiated mice, hematopoietic cells expressing DokR showed a drastically reduced capacity to repopulate lymphoid tissues. Most notably, DokR dramatically reduced repopulation of the thymus, in part by reducing the number of T cell precursors seeding in the thymus, but equally, through inhibiting the transition of CD4(-)CD8(-) to CD4(+)CD8(+) T cells. Consequently, the number of mature peripheral T cells was markedly reduced. In contrast, a minimal effect on B cell and myeloid lineage development was observed. Importantly, functional RasGAP and Nck binding sites were found to be essential for the biological effects of DokR in vitro and in vivo.