Differential Effects of Tra2? Isoforms on HIV-1 RNA Processing and Expression

Balanced processing of HIV-1 RNA is critical to virus replication and is regulated by host factors. In this report, we demonstrate that overexpression of either Tra2α or Tra2β results in a marked reduction in HIV-1 Gag/ Env expression, an effect associated with changes in HIV-1 RNA accumulation, altered viral splice site usage, and a block to export of HIV-1 genomic RNA. A natural isoform of Tra2β (Tra2ß3), lacking the N-terminal RS domain, also suppressed HIV-1 expression but had different effects on viral RNA processing. The functional differences between the Tra2β isoforms were also observed in the context of another RNA substrate indicating that these factors have distinct functions within the cell. Finally, we demonstrate that Tra2ß depletion results in a selective reduction in HIV-1 Env expression as well as an increase in multiply spliced viral RNA. Together, the findings indicate that Tra2α/β can play important roles in regulating HIV-1 RNA metabolism and expression.     

Differential expression of TGF-β isoforms during postlactational mammary gland involution

After cessation of lactation, the mammary gland undergoes involution, which is characterized by a massive epithelial cell death and proteolytic degradation of the extracellular matrix. Whereas the expression patterns and also the function of TGF-beta isoforms during mammary gland branching morphogenesis and lactation are well understood, their expression during postlactational involution and therefore a possible role in this process is poorly known. In this study we show that TGF-beta3 expression is dramatically induced (>fivefold) during mouse mammary gland involution when compared to that of virgin mouse, reaching a maximal expression level at day 4 after weaning. In contrast, other TGF-beta isoforms do not display significant increase in expression during involution (TGF-beta1, 1.3-fold and TGF-beta2, <1.5-fold) when compared to that of virgin or lactating mice. During mammary gland involution, TGF-beta3 is expressed in the epithelial layer and particularly in myoepithelial cells. A comparison of the kinetics of TGF-beta3 expression to that of programmed cell death and degradation of the basement membrane suggests that TGF-beta3 functions in the remodeling events of the extracellular matrix during the second stage of involution.     

Analysis of isoform specific ERK signaling on the effects of interleukin-1β on COX-2 expression and PGE2 production in human chondrocytes

The MAPK/ERK pathway is involved in IL-1β-induced cyclooxygenase (COX-2) expression and prostaglandin E2 (PGE2) production; two factors that play important roles in OA pathogenesis. In the present study, we find that IL-1β induced COX-2 expression and PGE2 production in human chondrocytes via a process that required the activation of the MAPK/ERK pathway. To evaluate the respective roles and relationship of ERK1 and ERK2 on IL-1β induced COX-2 expression and PGE2 production, small interfering RNA was used to knockdown ERK1, ERK2 or both in human chondrocytes. COX-2 expression and PGE2 production were significantly suppressed to a similar degree by the silencing of ERK1 or ERK2 alone. Moreover, the combined knockdown displayed a synergistic effect. Simultaneously, Western blotting indicated that the knockdown of ERK1 or ERK2 down regulated phospho-ERK1 and ERK1 or phospho-ERK2 and ERK2 levels, respectively. No significant compensatory mechanism through the upregulation of the other phospho-ERK and ERK isoform was observed. The combined silencing suppressed both phospho-ERK1/2 and ERK1/2. In conclusion, each ERK isoform similarly influenced IL-1β-mediated COX-2 expression and PGE2 production in human chondrocytes, and ERK1 and ERK2 displayed synergistic effects. Although, inhibition of both ERK1 and ERK2 would be a more effective, each ERK isoform may sufficiently regulate these effects in human chondrocytes. ERK1 or ERK2 may be potential therapeutic target for the inflammatory process of OA.     

Differential expression of BK channel isoforms and β-subunits in rat neuro-vascular tissues

We investigated the expression of splice variants and β-subunits of the BK channel (big conductance Ca²⁺-activated K⁺ channel, Slo1, MaxiK, K_Ca1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An α-subunit splice variant X1_+ 24 was found expressed (RT-PCR) in nervous tissue only where also the SS4_+ 81 variant was dominating with little expression of the short form SS4₀. SS4_+ 81 was present in some cerebral vessels too. The SS2_+ 174 variant (STREX) was found in both blood vessels and in nervous tissue. In situ hybridization data supported the finding of SS4_+ 81 and SS2_+ 174 in vascular smooth muscle and trigeminal ganglion. β-subunits β2 and β4 showed high expression in brain and trigeminal ganglion and some in cerebral vessels while β1 showed highest expression in blood vessels. β3 was found only in testis and possibly brain. A novel splice variant X2_+ 92 was found, which generates a stop codon in the intracellular C-terminal part of the protein. This variant appears non-functional as a homomer but may modulate the function of other splice-variants when expressed in Xenopus oocytes. In conclusion a great number of splice variant and β-subunit combinations likely exist, being differentially expressed among nervous and vascular tissues.     

Regulatory effects of simvastatin and apoJ on APP processing and amyloid-β clearance in blood-brain barrier endothelial cells

Amyloid-β peptides (Aβ) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aβ metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aβ. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aβ metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aβ oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aβ clearance across the pBCEC monolayer. Treatment of pBCEC with Aβ_(1–40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3 × Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aβ oligomers and reduced Aβ uptake and cell-associated Aβ oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aβ processing and clearance at the BBB.     

Differential effects of calcium on PI3K-Akt and HIF-1α survival pathways

Calcium signaling participates in the regulation of numberless cellular functions including cell cycle progression and cellular migration, important processes for cancer expansion. Cancer cell growth, migration, and invasion are typically supported by PI3K/Akt activation, while a hypoxic environment is critical in cancer development. Accordingly, in the present study, we aimed at investigating whether perturbations in calcium homeostasis induce alterations of HIF-1α and activate Akt levels in epithelial A549 and A431 cells. Survival was drastically reduced in the presence of calcium chelator BAPTA-AM and thapsigargin, a SERCA inhibitor inducing store-operated calcium entry, to a lesser extent. Calcium chelation provoked a transient but strong upregulation of HIF-1α protein levels and accumulation in the nucleus, whereas in the presence of thapsigargin, HIF-1α levels were rapidly abolished before reaching and exceeding control levels. Despite cell death, calcium chelation merely inhibited Akt, which was significantly activated in the presence of thapsigargin. Moreover, when store-operated calcium entry was simulated by reintroducing calcium ions in cell suspensions, Akt was rapidly activated in the absence of any growth factor. These data further underscore the growing importance of calcium entry and directly link this elementary event of calcium homeostasis to the Akt pathway, which is commonly deregulated in cancer.     

How does precursor RNA structure influence RNA processing and gene expression?

RNA is a fundamental biomolecule that has many purposes within cells. Due to its single-stranded and flexible nature, RNA naturally folds into complex and dynamic structures. Recent technological and computational advances have produced an explosion of RNA structural data. Many RNA structures have regulatory and functional properties. Studying the structure of nascent RNAs is particularly challenging due to their low abundance and long length, but their structures are important because they can influence RNA processing. Precursor RNA processing is a nexus of pathways that determines mature isoform composition and that controls gene expression. In this review, we examine what is known about human nascent RNA structure and the influence of RNA structure on processing of precursor RNAs. These known structures provide examples of how other nascent RNAs may be structured and show how novel RNA structures may influence RNA processing including splicing and polyadenylation. RNA structures can be targeted therapeutically to treat disease.     

Differential expression of secreted phosphoprotein 1 in response to estradiol-17β and in ovarian tumors in chickens

Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.     

The protective effects of resveratrol on ulcerative colitis via changing the profile of Nrf2 and IL-1β protein

Molecular Biology Reports - Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) with increasing incidence and prevalence in developed countries. The presence of inflammatory cytokines is...     

Apoptosis and expression of bcl-2 α, β mRNA isoforms and protein in neuroblastoma

We studied apoptosis in 36 neuroblastomas by DNA ladder assay. Expression of bcl2- and mRNA isoforms and protein were detected by RT-PCR and by immunohistochemistry, respectively. Internucleosomal DNA fragmentation was found in 20/36 (56%) tumor tissues collected both at onset and relapse of disease. Bcl-2 and mRNAs and protein were found in almost all examined tumors irrespective of DNA ladder, thus showing lack of correlation with the clinical stage. BCL-2 protein was observed to be expressed at various levels in undifferentiated and in more differentiated neuroblasts, while the stroma and the fibrovascular tissue were negative. Our results show that apoptosis is present in neuroblastoma at all stages and that bcl-2 gene is widely expressed in tumor tissue. In our series of neuroblastomas, bcl-2 expression is not correlated with unfavorable prognosis.     

Differential effects of short-term β agonist and growth hormone treatments on expression of myosin heavy chain IIB and associated metabolic genes in sheep muscle

Growth hormone (GH) and β agonists increase muscle mass, but the mechanisms for this response are unclear and the magnitude of response is thought to vary with age of animal. To investigate the mechanisms driving the muscle response to these agents, we examined the effects of short-term (6 day) administration of GH or cimaterol (a β2-adrenergic agonist, BA) on skeletal muscle phenotype in both young (day 60) and mature (day 120) lambs. Expression of myosin heavy chain (MyHC) isoforms were measured in Longissimus dorsi (LD), Semitendinosus (ST) and Supraspinatus (SS) muscles as markers of fibre type and metabolic enzyme activities were measured in LD. To investigate potential mechanisms regulating the changes in fibre type/metabolism, expression or activity of a number of signalling molecules were examined in LD. There were no effects of GH administration on MyHC isoform expression at either the mRNA or protein level in any of the muscles. However, BA treatment induced a proportional change in MyHC mRNA expression at both ages, with the %MyHCI and/or IIA mRNA being significantly decreased in all three muscles and %MyHCIIX/IIB mRNA significantly increased in the LD and ST. BA treatment induced de novo expression of MyHCIIB mRNA in LD, the fastest isoform not normally expressed in sheep LD, as well as increasing expression in the other two muscles. In the LD, the increased expression of the fastest MyHC isoforms (IIX and IIB) was associated with a decrease in isocitrate dehydrogenase activity, but no change in lactate dehydrogenase activity, indicating a reduced capacity for oxidative metabolism. In both young and mature lambs, changes in expression of metabolic regulatory factors were observed that might induce these changes in muscle metabolism/fibre type. In particular, BA treatment decreased PPAR-γ coactivator-1β mRNA and increased receptor-interacting protein 140 mRNA. The results suggest that the two agents work via different mechanisms or over different timescales, with only BA inducing changes in muscle mass and transitions to a faster, less oxidative fibre type after a 6-day treatment.