Mathematical models of diffusion-constrained polymerase chain reactions: basis of high-throughput nucleic acid assays and simple self-organizing systems

DNA templates amplified by polymerase chain reaction in thin polyacrylamide gels form diffusion-constrained amplicons called "polonies" (polymerase colonies) that have been used to phase DNA haplotypes over long distances, to analyse RNA splice variants, and to assay other phenomena of biological interest. We present two sets of mathematical models, one for single polony growth (SPGM) and one for two polony interaction (TPIM), that will be used to optimize polony technology. The models provide detailed predictions of polony yield, concentration profiles, growth of isolated polonies, and the interaction of neighboring polonies. The TPIM explains an experimental observation that nearby polonies deform against each other rather than interpenetrate, an effect important for optimizing polony protocols. However, the TPIM also predicts that polonies may invade each other with a complex geometry when sufficiently close. Polonies are also of interest as simple abiotic systems that exhibit lifelike properties of self-organization, growth, and development, and the models may also apply to biological phenomena involving propagation through tethering and diffusion. Our polony modeling software is available at our web site.     

Sphingosine 1-phosphate phosphatase 2 is induced during inflammatory responses

Sphingosine 1-phosphate (S1P) levels in cells and, consequently, its bioactivity as a signalling molecule are controlled by the action of enzymes responsible for its synthesis and degradation. In the present report, we examined alterations in expression patterns of enzymes involved in S1P-metabolism (sphingosine kinases including their splice variants, sphingosine 1-phosphate phosphatases, and sphingosine 1-phosphate lyase) under certain inflammatory conditions. We found that sphingosine kinase type 1 (SPHK1) mRNA could be triggered in a cell type-specific manner; individual SPHK1 splice variants were induced with similar kinetics. Remarkably, expression and activity of S1P phosphatase 2 (SPP2) was found to be highly upregulated by inflammatory stimuli in a variety of cells (e.g., neutrophils, endothelial cells). Bandshift analysis using oligonucleotides spanning predicted NFkappaB sites within the SPP2 promoter and silencing of NFkappaB/RelA via RelA-directed siRNA demonstrated that SPP2 is an NFkappaB-dependent gene. Silencing of SPP2 expression in endothelial cells, in turn, led to a marked reduction of TNF-alpha-induced IL-1beta mRNA and protein and to a partial reduction of induced IL-8, suggesting a pro-inflammatory role of SPP2. Notably, up-regulation of SPP2 was detected in samples of lesional skin of patients with psoriasis, an inflammatory skin disease. This study provides detailed insights into the regulation of SPP2 gene expression and suggests that SPP2 might be a novel player in pro-inflammatory signalling.     

Sulf2 gene is alternatively spliced in mammalian developing and tumour tissues with functional implications

SULF2 enzyme regulates the activities of a number of signalling pathways that in many tissues are up-regulated during development and disease. As we recently showed for avian Sulf1, the present study demonstrates that mammalian Sulf2 gene can also generate functionally distinct splice variants that would regulate normal development and tumour growth differentially. It is thus important to distinguish SULF1/SULF2 isoforms in mammalian tissues to understand their functional and clinical relevance to disease. This study demonstrates that unlike normal adult lung with little or no SULF2 expression, this enzyme is expressed at high levels in most lung tumours showing differential cellular distribution of full length and shorter SULF2 variants in such tumours. Furthermore, we show that the short SULF2 splice variants are associated with those signalling pathways that are inhibited by full length SULF1/SULF2 variants and therefore could promote growth in such lung tumours.     

Expression profiles of BDNF splice variants in cultured DRG neurons stimulated with NGF

Expression of brain-derived neurotrophic factor (BDNF) mRNA is increased in the dorsal root ganglion (DRG) in response to peripheral inflammation. Nerve growth factor (NGF) from inflammatory tissue is thought to induce expression of BDNF. Recently, it was reported that the BDNF gene has eight non-coding exons that are transcribed independently into several splice variants. Expression of these splice variants in DRG neurons stimulated with NGF has not been studied. We examined changes in expression of BDNF splice variants in a rat model of peripheral inflammation and in cultured DRG neurons exposed to NGF. Total BDNF mRNA was increased by inflammation in vivo and by NGF in vitro. Among all splice variants, exon 1-9 showed the greatest increase in expression in both experiments. Our results indicate that exon 1-9 contributes to changes in total BDNF levels and may play an important role in the acute response of DRG to NGF.     

Kinetics of Toll-like receptor-4 splice variants expression in lipopolysaccharide-stimulated antigen presenting cells of healthy donors and patients with cystic fibrosis

Toll-like receptors (TLR) are key components of innate immune system. As TLR activation could induce potentially harmful inflammatory response, activation of TLR signaling pathways has to be under tight control. Besides other control mechanisms, an inhibitory function of murine TLR4 splice variants was recently demonstrated. In this study we investigated expression of four TLR4 splice variants in human antigen presenting cells (APC). Furthermore, we studied modification in TLR4 splice variants expression in APC in cystic fibrosis (CF) patients chronically infected by Gram-negative bacteria. We developed a novel reliable real-time PCR detection system that allowed monitoring of individual TLR4 splice variants expression. In APC from healthy donors we detected a characteristic transient increase of two out of four splice variants after lipopolysaccharide (LPS) stimulation. Similarly to murine TLR4, one of these variants, NM 003266, might translate to a potentially inhibitory protein. In contrast to controls, CF monocytes had significantly changed LPS-induced expression of TLR4 gene and its variants including reduced ability to up-regulate the expression of the potentially inhibitory variant upon stimulation. In accordance with this observation, monocytes from CF patients produced significantly more tumor necrosis factor after LPS stimulation than healthy controls. Our results thus describe the kinetics of TLR4 splicing variants expression after LPS stimulation and indicate a possible alteration of its regulation in CF patients.     

K-RAS Mutant Pancreatic Tumors Show Higher Sensitivity to MEK than to PI3K Inhibition In Vivo

Activating K-RAS mutations occur at a frequency of 90% in pancreatic cancer, and to date no therapies exist targeting this oncogene. K-RAS signals via downstream effector pathways such as the MAPK and the PI3K signaling pathways, and much effort has been focused on developing drugs targeting components of these pathways. To better understand the requirements for K-RAS and its downstream signaling pathways MAPK and PI3K in pancreatic tumor maintenance, we established an inducible K-RAS knock down system that allowed us to ablate K-RAS in established tumors. Knock down of K-RAS resulted in impaired tumor growth in all pancreatic xenograft models tested, demonstrating that K-RAS expression is indeed required for tumor maintenance of K-RAS mutant pancreatic tumors. We further examined signaling downstream of K-RAS, and detected a robust reduction of pERK levels upon K-RAS knock down. In contrast, no effect on pAKT levels could be observed due to almost undetectable basal expression levels. To investigate the requirement of the MAPK and the PI3K pathways on tumor maintenance, three selected pancreatic xenograft models were tested for their response to MEK or PI3K inhibition. Tumors of all three models regressed upon MEK inhibition, but showed less pronounced response to PI3K inhibition. The effect of MEK inhibition on pancreatic xenografts could be enhanced further by combined application of a PI3K inhibitor. These data provide further rationale for testing combinations of MEK and PI3K inhibitors in clinical trials comprising a patient population with pancreatic cancer harboring mutations in K-RAS.     

Expression of mRNAs Encoding Subunits of the NMDA Receptor in Developing Rat Brain

Abstract: Developmental changes in the levels of N -methyl- d -aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.     

Osteopontin Splice Variants Differentially Exert Clinicopathological Features and Biological Functions in Gastric Cancer

Purpose: Gastric cancer (GC) remains a leading cause of death worldwide, and an elevated expression of osteopontin (OPN) may correlate with its poor survival. Alternative splicing of OPN can result in three isoforms, OPN-a, OPN-b and OPN-c. The aim of our current study is to examine the expression pattern and biological functions of OPN splice variants in GC.Methods: Firstly, we evaluated the expression of OPN splice variants in 7 gastric cell lines, 101 pairs of GC tissues and their adjacent non-tumor tissues by Quantative real-time PCR (QT-PCR). Gain-of-function experiments were subsequently performed to determine their diverse roles in malignant behaviors of GC. Besides, their differential effects on the regulation of crucial downstream molecules were further explored in the anti-apoptotic and pro-metastatic process.Results: We found that OPN-b is the dominant kind of OPN isoform in GC cell lines. Although the expression levels of three variants were all elevated in GC tissues, increased OPN-b or OPN-c expression could correlate with clinicopathological features. Functional analyses further showed that OPN-b most strongly promoted GC cell survival possibly by regulation of Bcl-2 family proteins and CD44v expressions. Moreover, OPN-c most effectively stimulated GC metastatic activity by increasing secretion of MMP-2, uPa, and IL-8.Conclusions: Our results suggest that OPN splice variants differentially exert clinicopathological features and biological functions in GC. Therefore, focusing on specific OPN isoform could be a novel direction for developing diagnostic and therapeutic approaches in GC.     

Developmentally regulated alternative splicing of densin modulates protein-protein interaction and subcellular localization

Densin is a member of the leucine-rich repeat (LRR) and PDZ domain (LAP) protein family that binds several signaling molecules via its C-terminal domains, including calcium/calmodulin-dependent protein kinase II (CaMKII). In this study, we identify several novel mRNA splice variants of densin that are differentially expressed during development. The novel variants share the LRR domain but are either prematurely truncated or contain internal deletions relative to mature variants of the protein (180 kDa), thus removing key protein–protein interaction domains. For example, CaMKIIα coimmunoprecipitates with densin splice variants containing an intact C-terminal domain from lysates of transfected HEK293 cells, but not with variants that only contain N-terminal domains. Immunoblot analyses using antibodies to peptide epitopes in the N- and C- terminal domains of densin are consistent with developmental regulation of splice variant expression in brain. Moreover, putative splice variants display different subcellular fractionation patterns in brain extracts. Expression of green fluorescent protein (GFP)-fused densin splice variants in HEK293 cells shows that the LRR domain can target densin to a plasma membrane-associated compartment, but that the splice variants are differentially localized and have potentially distinct effects on cell morphology. In combination, these data show that densin splice variants have distinct functional characteristics suggesting multiple roles during neuronal development.     

Interaction of human GTP cyclohydrolase I with its splice variants

Tetrahydrobiopterin is an essential cofactor for aromatic amino acid hydroxylases, ether lipid oxidase and nitric oxide synthases. Its biosynthesis in mammals is regulated by the activity of the homodecameric enzyme GCH (GTP cyclohydrolase I; EC 3.5.4.16). In previous work, catalytically inactive human GCH splice variants differing from the wild-type enzyme within the last 20 C-terminal amino acids were identified. In the present study, we searched for a possible role of these splice variants. Gel filtration profiles of purified recombinant proteins showed that variant GCHs form high-molecular-mass oligomers similar to the wild-type enzyme. Co-expression of splice variants together with wild-type GCH in mammalian cells revealed that GCH levels were reduced in the presence of splice variants. Commensurate with these findings, the GCH activity obtained for wild-type enzyme was reduced 2.5-fold through co-expression with GCH splice variants. Western blots of native gels suggest that splice variants form decamers despite C-terminal truncation. Therefore one possible explanation for the effect of GCH splice variants could be that inactive variants are incorporated into GCH heterodecamers, decreasing the enzyme stability and activity.     

ENaC alpha-subunit variants are expressed in lung epithelial cells and are suppressed by oxidative stress

Amiloride-sensitive epithelial sodium channel (ENaC) is a major sodium channel in the lung facilitating fluid absorption. ENaC is composed of alpha-, beta-, and gamma-subunits, and the alpha-subunit is indispensable for ENaC function in the lung. In human lungs, the alpha-subunit is expressed as various splice variants. Among them, alpha(1)- and alpha(2)-subunits are two major variants with different upstream regulatory sequences that possess similar channel characteristics when tested in Xenopus oocytes. Despite the importance of alpha-ENaC, little was known about the relative abundance of its variants in lung epithelial cells. Furthermore, lung infection and inflammation are often accompanied by reduced alpha-ENaC expression, oxidative stress, and pulmonary edema. However, it was not clear how oxidative stress affects expression of alpha-ENaC variants. In this study, we examined relative expression levels of alpha-subunit variants in four human lung epithelial cell lines. We also tested the hypothesis that oxidative stress inhibits alpha-ENaC expression. Our results show that both alpha(1)- and alpha(2)-ENaC variants are expressed in the cells we tested, but relative abundance varies. In the two monolayer-forming cell lines, H441 and Calu-3, alpha(2)-ENaC is the predominant variant. We also show that H(2)O(2) specifically suppresses alpha(1)- and alpha(2)-ENaC variant expression in H441 and Calu-3 cells in a dose-dependent fashion. This suppression is achieved by inhibition of their promoters and is attenuated by dexamethasone. These data demonstrate the importance of the alpha(2)-subunit variant and suggest that glucocorticoids and antioxidants may be useful in correcting infection/inflammation-induced lung fluid imbalance.