Tra2-Mediated Recognition of HIV-1 5′ Splice Site D3 as a Key Factor in the Processing of vpr mRNA

Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3′ splice site (3′ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5′ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE_vpr) localized between exonic splicing silencer ESSV and 5′ss D3. The ESE_vpr sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE_vpr. Further analyses revealed that ESE_vpr supports the binding of U1 snRNA at 5′ss D3, allowing bridging interactions across the upstream exon with 3′ss A2. In line with this, an increase or decrease in the complementarity of 5′ss D3 to the 5′ end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3′ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5′ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3′ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.     

Tissue-Specific Expression of Neurexin-1α Isoforms in Rat Organs

Russian Journal of Bioorganic Chemistry - Neurexins are a family of synaptic adhesion proteins that play a key role in synapse formation and maintenance. Neurexins undergo extensive alternative...     

Effects of Bone Morphogenetic Protein-2 and Transforming Growth Factor-β1 on Gene Expression of Decorin and Biglycan by Cultured Osteoblastic Cells

The influence of bone morphogenetic protein-2 (BMP-2) and transforming growth factor (TGF-) on the expression of small proteoglycans, decorin and biglycan was investigated in a clonal rat osteoblastic cell line, ROS-C26 (C26) cells, which is a potential osteoblast precursor cell line and capable of differentiating into mature osteoblasts after treatment with recombinant BMP-2 (rhBMP-2). Following the culture of C26 cells for 3, 6, and 9 days in the presence or absence of rhBMP-2, alkaline phosphatase activity increased in the rhBMP-2 treated cells in direct proportion to their differentiation into more mature osteoblastic cells, whereas decorin mRNA decreased in the cells, when compared to control cells without rhBMP-2 treatment. These results were evident 6 days after treatment. However, rhBMP-2 treatment had no effect on biglycan mRNA expression in the cells. Subsequently, after removal of rhBMP-2 from the culture media, the cells were further cultured for 24h with graded concentrations of TGF-1 (0, 0.1, 1.0, 5.0, and 10ng/ml). TGF-1 decreased decorin mRNA expression in the cells dose dependently, but did not affect their biglycan mRNA expression. Furthermore, either removal of rhBMP-2 from the culture media or addition of TGF-1 significantly decreased alkaline phosphatase activity of rhBMP-2-induced cells. These results indicate that osteoblastic differentiation is accompanied by increased alkaline phosphatase activity and decreased expression of decorin mRNA, but continuous expression of biglycan mRNA. Both rhBMP-2 and TGF-1 inhibit decorin mRNA expression in osteoblasts at varying stages of differentiation, but their effects on biglycan mRNA expression and alkaline phosphatase are different.     

Differential Effects of IL-12 on Tregs and Non-Treg T Cells: Roles of IFN-γ, IL-2 and IL-2R

Complex interactions between effector T cells and Foxp3⁺ regulatory T cells (Treg) contribute to clinical outcomes in cancer, and autoimmune and infectious diseases. Previous work showed that IL-12 reversed Treg-mediated suppression of CD4⁺Foxp3⁻ T cell (Tconv) proliferation. We and others have also shown that Tregs express T-bet and IFN-γ at sites of Th1 inflammation and that IL-12 induces IFN-γ production by Tregs in vitro. To investigate whether loss of immunosuppression occurs when IFN-γ is expressed by Tregs we treated mouse lymphocyte cultures with IL-12. IFN-γ expression did not decrease the ability of Tregs to suppress Tconv proliferation. Rather, IL-12 treatment decreased Treg frequency and Foxp3 levels in Tregs. We further showed that IL-12 increased IL-2R expression on Tconv and CD8 T cells, diminished its expression on Tregs and decreased IL-2 production by Tconv and CD8 T cells. Together, these IL-12 mediated changes favored the outgrowth of non-Tregs. Additionally, we showed that treatment with a second cytokine, IL-27, decreased IL-2 expression without augmenting Tconv and CD8 T cell proliferation. Notably, IL-27 only slightly modified levels of IL-2R on non-Treg T cells. Together, these results show that IL-12 has multiple effects that modify the balance between Tregs and non-Tregs and support an important role for relative levels of IL-2R but not for IFN-γ expression in IL-12-mediated reversal of Treg immunosuppression.     

Differential Membrane Localization and Intermolecular Associations of α-Dystrobrevin Isoforms in Skeletal Muscle

α-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex. Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length α-dystrobrevin-1 (84 kD), and COOH-terminal truncated α-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that α-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds. α-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts. In contrast, α-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin. In yeast two-hybrid experiments and coimmunoprecipitation of in vitro–translated proteins, α-dystrobrevin-2 binds dystrophin, whereas α-dystrobrevin-1 binds both dystrophin and utrophin. α-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin. In contrast, α-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants. Thus, the distinct distributions of α-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved. These results show that alternative splicing confers distinct properties of association on the α-dystrobrevins.     

Everything’s Relative? Relative Differences in Processing Fluency and the Effects on Liking

Explanations of aesthetic pleasure based on processing fluency have shown that ease-of-processing fosters liking. What is less clear, however, is how processing fluency arises. Does it arise from a relative comparison among the stimuli presented in the experiment? Or does it arise from a comparison to an internal reference or standard? To address these questions, we conducted two experiments in which two ease-of-processing manipulations were applied: either (1) within-participants, where relative comparisons among stimuli varying in processing ease were possible, or (2) between-participants, where no relative comparisons were possible. In total, 97 participants viewed simple line drawings with high or low visual clarity, presented at four different presentation durations, and rated for felt fluency, liking, and certainty. Our results show that the manipulation of visual clarity led to differences in felt fluency and certainty regardless of being manipulated within- or between-participants. However, liking ratings were only affected when ease-of-processing was manipulated within-participants. Thus, feelings of fluency do not depend on the nature of the reference. On the other hand, participants liked fluent stimuli more only when there were other stimuli varying in ease-of-processing. Thus, relative differences in fluency seem to be crucial for liking judgments.     

Evaluation of Lovastatin Effects on Expression of Anti-apoptotic Nrf2 and PGC-1α Genes in Neural Stem Cells Treated with Hydrogen Peroxide

Reactive oxygen species and oxidative stress are associated with various cell processes, including cell survival and apoptosis. Oxidative stress has been implicated in the pathogenesis of several neurological disorders including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and multiple sclerosis (MS). In the present study, we evaluated the effects of lovastatin chemoprotection against hydrogen peroxide-induced oxidative stress in bone marrow stromal cell-derived neural stem cells (BMSC-derived NSCs) and whether it has protective effects. BMSC-derived NSCs were pretreated with different doses of lovastatin for 48 h and then exposed to 125 μM H₂O₂ for 30 min. Using MTT, TUNEL assay, and real-time RT-PCR, we evaluated the effects of lovastatin on cell survival, apoptosis, and PGC-1α and Nrf2 expression rates in pretreated BMSC-derived NSCs compared to control groups. Results showed that apoptosis rate in the lovastatin-pretreated BMSC-derived NSCs was significantly decreased compared to the control group. Our findings suggest that lovastatin protects NSCs against oxidative stress-induced cell death, and therefore, it may be used to promote the survival rate of NSCs and can be a candidate for treatment of oxidative stress-mediated neurological diseases.     

Differential Regulation of Amyloid-β Endocytic Trafficking and Lysosomal Degradation by Apolipoprotein E Isoforms

Aggregation of amyloid-β (Aβ) peptides leads to synaptic disruption and neurodegeneration in Alzheimer disease (AD). A major Aβ clearance pathway in the brain is cellular uptake and degradation. However, how Aβ traffics through the endocytic pathway and how AD risk factors regulate this event is unclear. Here we show that the majority of endocytosed Aβ in neurons traffics through early and late endosomes to the lysosomes for degradation. Overexpression of Rab5 or Rab7, small GTPases that function in vesicle fusion for early and late endosomes, respectively, significantly accelerates Aβ endocytic trafficking to the lysosomes. We also found that a portion of endocytosed Aβ traffics through Rab11-positive recycling vesicles. A blockage of this Aβ recycling pathway with a constitutively active Rab11 mutant significantly accelerates cellular Aβ accumulation. Inhibition of lysosomal enzymes results in Aβ accumulation and aggregation. Importantly, apolipoprotein E (apoE) accelerates neuronal Aβ uptake, lysosomal trafficking, and degradation in an isoform-dependent manner with apoE3 more efficiently facilitating Aβ trafficking and degradation than apoE4, a risk factor for AD. Taken together, our results demonstrate that Aβ endocytic trafficking to lysosomes for degradation is a major Aβ clearance pathway that is differentially regulated by apoE isoforms. A disturbance of this pathway can lead to accumulation and aggregation of cellular Aβ capable of causing neurotoxicity and seeding amyloid.     

Differential Association of Phosphodiesterase 4D Isoforms with ��2-Adrenoceptor in Cardiac Myocytes

cAMP and protein kinase A (PKA) activation represents a key signaling mechanism upon β-adrenergic stimulation under stress. Both β₁- and β₂-adrenoreceptor (ARs) subtypes induce cAMP accumulation, yet play distinct roles in cardiac contraction and myocyte apoptosis. Differences in controlling cAMP/PKA activities through the assembly of complexes between the receptors and cAMP-specific phosphodiesterases contribute to the distinct biological outcomes. Here, we demonstrate that β₂ARs form signaling complexes with a set of PDE4D isoforms expressed in cardiac myocytes. PDE4D9 and PDE4D8 bind to the β₂AR at resting conditions; however, agonist stimulation induces dissociation of PDE4D9 from the receptor but recruitment of PDE4D8 to the receptor. Agonist stimulation also induces recruitment of PDE4D5 to the β₂AR. Moreover, the receptor-associated PDE4D isoforms play distinct roles in controlling cAMP activities and regulating the PKA phosphorylation of the receptor and myocyte contraction rate responses. Knockdown of PDE4D9 with short hairpin RNA enhances the β₂AR-induced cAMP signaling, whereas knockdown of PDE4D8 only slightly prolongs the receptor-induced cAMP signaling in myocytes. Inhibition of PDE4D9 and PDE4D5 enhances the base-line levels of contraction rates, whereas inhibition of PDE4D9 and PDE4D8 enhances the maximal contraction rate increases upon activation of β₂AR. Our data underscore the complex regulation of intracellular cAMP by β₂AR-associated phosphodiesterase enzymes to enforce the specificity of the receptor signaling for physiological responses.     

The Promotive Effects of Thymosin β4 on Neuronal Survival and Neurite Outgrowth by Upregulating L1 Expression

Thymosin β₄ (Tβ4) is a major actin-sequestering peptide widely distributed in mammalian tissues including the nervous system. The presence of this peptide in the nervous system likely plays a role in synaptogensis, axon growth, cell migration, and plastic changes in dendritic spine. However, the effects of Tβ4 on the survival of neurons and axonal outgrowth have still not been fully understood. So far it is not clear if the effects of Tβ4 are associated with L1 functions. In the present study, we hypothesized that Tβ4-induced up-regulation of L1 synthesis could be involved in the survival and axon outgrowth of cultured spinal cord neurons. To test this hypothesis, primarily cultured neurons were prepared from the mouse spinal cord and treated with various concentrations of Tβ4 ranging from 0.1 to 10 μg/ml. The analysis of L1 mRNA expression and protein synthesis in neurons was then carried out using RT-PCR and western blot assays, respectively. After the addition of Tβ4 to cultures, cells were then treated with antibodies against distinct domains of L1-Fc. Subsequently, β-tubulin III and L1 double-labeled indirect immunofluorescence was carried out. Meanwhile, L1 immunofluorescent reactivity was analyzed and compared in cells treated with Tβ4. Furthermore, the number of β-tubulin III-positive cells and neurite lengths were measured. We found that Tβ4 enhanced L1 expression in a dose-dependent manner, and the highest L1 mRNA and protein synthesis in cells increased by more than 2.1- and 2.3-fold in the presence of Tβ4 at identical concentrations, respectively. Moreover, it also dose dependently enhanced neurite outgrowth and neuronal survival. Compared to conditions without Tβ4, the length of neurite and neuronal survival increased markedly in presence of 0.5, 1, and 5 μg/ml Tβ4, respectively, whereas the effects of Tβ4 were significantly attenuated or inhibited in the process of L1-Fc antibodies treatment. These above results indicate that the promotive effect of Tβ4 on the survival and neurite outgrowth of cultured spinal cord neurons might be mediated, at least in part via a stimulation of the production of L1 in the neurons.     

Stability and conformation of the dimeric HIV-1 genomic RNA 5′UTR

During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5′ untranslated region (5′UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5′UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation. Two 5′UTR dimer conformations, the kissing dimer (KD) and the extended dimer (ED), have been identified in vitro, which differ in the extent of intermolecular basepairing. Whether 5′UTRs from different HIV-1 strains with distinct sequences have access to the same dimer conformations has not been determined. Here, we applied fluorescence cross-correlation spectroscopy and single-molecule Förster resonance energy transfer imaging to demonstrate that 5′UTRs from two different HIV-1 subtypes form (KDs) with divergent stabilities. We further show that both 5′UTRs convert to a stable dimer in the presence of the viral NC protein, adopting a conformation consistent with extensive intermolecular contacts. These results support a unified model in which the genomes of diverse HIV-1 strains adopt an ED conformation.     

γ-Secretase Processing and Effects of γ-Secretase Inhibitors and Modulators on Long Aβ Peptides in Cells

Understanding how different species of Aβ are generated by γ-secretase cleavage has broad therapeutic implications, because shifts in γ-secretase processing that increase the relative production of Aβx-42/43 can initiate a pathological cascade, resulting in Alzheimer disease. We have explored the sequential stepwise γ-secretase cleavage model in cells. Eighteen BRI2-Aβ fusion protein expression constructs designed to generate peptides from Aβ1–38 to Aβ1–55 and C99 (CTFβ) were transfected into cells, and Aβ production was assessed. Secreted and cell-associated Aβ were detected using ELISA and immunoprecipitation MALDI-TOF mass spectrometry. Aβ peptides from 1–38 to 1–55 were readily detected in the cells and as soluble full-length Aβ proteins in the media. Aβ peptides longer than Aβ1–48 were efficiently cleaved by γ-secretase and produced varying ratios of Aβ1–40:Aβ1–42. γ-Secretase cleavage of Aβ1–51 resulted in much higher levels of Aβ1–42 than any other long Aβ peptides, but the processing of Aβ1–51 was heterogeneous with significant amounts of shorter Aβs, including Aβ1–40, produced. Two PSEN1 variants altered Aβ1–42 production from Aβ1–51 but not Aβ1–49. Unexpectedly, long Aβ peptide substrates such as Aβ1–49 showed reduced sensitivity to inhibition by γ-secretase inhibitors. In contrast, long Aβ substrates showed little differential sensitivity to multiple γ-secretase modulators. Although these studies further support the sequential γ-secretase cleavage model, they confirm that in cells the initial γ-secretase cleavage does not precisely define subsequent product lines. These studies also raise interesting issues about the solubility and detection of long Aβ, as well as the use of truncated substrates for assessing relative potency of γ-secretase inhibitors.