Minor Isoform Research Articles

Abstract P315: Vascular AT1 Angiotensin Receptors Do Not Contribute to Albuminuria or Hyperfiltration in Diabetes

Blockade of the renin angiotensin system (RAS) reduces albuminuria, attenuates hyperfiltration, and slows the progression of diabetic nephropathy (DN) by preventing vasoconstriction and subsequent increases in glomerular hydrostatic pressure. Since RAS blockade disrupts Ang II signaling in all tissues, the specific contribution of vascular actions of AT1 receptors in DN has been difficult to delineate. Therefore, we generated 129 SvEv mice with cell-specific loss of AT1A from VSMCs (SMKOs) using Cre-loxp . To eliminate AT1R from VSMCs, we crossed the SMKO mice with AT1BR -/- mice, lacking the minor AT1B isoform. To study the impact of vascular AT1R in DN, we crossed the AT1B- null SMKOs with mice having the Ins2 C96Y AKITA mutation, which develop DM1 early. To enhance kidney injury, mice underwent uninephrectomy (UNX) at 11wks. Blood glucose levels were elevated (~500mg/dL) and similar at 10, 16 and 24wks between the two groups. Prior to UNX, albuminuria was similar between Control AKITA and AT1B- null SMKO AKITA (62±10 Control AKITA versus 107±27 μg/24hrs SMKO AKITA, P=NS). Albuminuria increased with age in both Control Akita and AT1B- null SMKO AKITA but without significant differences between the groups at 16wks (307±106 vs 313±117 μg/24hrs; P=NS) or 24wks (494±236 versus 730±217 μg/24hrs; P=NS), despite a trend toward higher albuminuria in AT1B- null SMKO AKITAs. There was no significant difference in GFR (using FITC-inulin) between non-diabetic Control and AT1B- null SMKO (15.6±1.2 vs 14.8±0.8 μl/min/g BW), and hyperfiltration was observed in both Control AKITA (23.7±2.4 μl/min/g BW; P=0.003) and AT1B- null SMKO AKITA mice (20.7±1.7 μl/min/g BW; P=0.01) relative to their non-diabetic comparators. However, there was no significant difference in GFR between ControlAKITA and AT1B- null SMKO AKITA (P=NS). Finally we measured mRNA levels of putative kidney injury markers by RTqPCR and found no differences in levels of Col1A1 , NGAL , or TGFB1 mRNA between Control AKITA and AT1B null SMKO AKITA. Our studies indicate that the absence of vascular AT1R responses is not sufficient to reduce albuminuria and prevent hyperfiltration in a mouse model of DN. This suggests that blockade of AT1R in other cell lineages may contribute to beneficial actions of ARBs in DN.

Abstract 1911: ΔNp63α promotes TGFβ-induced metastasis by silencing a microRNA network restraining wound healing

Abstract Primary cancer cell dissemination is the key event in metastasis, yet context-specific determinants remain largely undefined. Multiple reports have suggested an anti-metastatic role for the p53 family member p63 linked to its minor epithelial isoform, TAp63. However, the role and contribution of the major p63 isoform, ΔNp63α, remain largely undefined. Here we report a distinct and TAp63-independent mechanism in which ΔNp63α-expressing cells, in a TGFβ-rich microenvironment, are positively selected for metastatic dissemination. We define the molecular mechanism of this effect, through ΔNp63α-mediated repression of miR-527 and miR-665 and the subsequent upregulation of two TGFβ pathway major effectors, SMAD4 and TβRII. Furthermore, we provide evidence that this mechanism reflects a fundamental role for ΔNp63α in the normal wound healing response. We show that ΔNp63α and miR-527/665 control the TGFβ-dependent signaling that switches off anti-migratory miR-198 by downregulating the regulatory factor KSRP. Collectively, these findings reveal a novel microRNA network orchestrated selectively by ΔNp63α that is involved in the physiological regulation of wound healing but is hijacked by tumors to promote metastatic dissemination. Citation Format: Lidia Rodriguez Calleja, camille Jacques, Francois Lamoureux, Marc Baud’huin, Thibault Quillard, Franck Verrecchia, Dominique Heymann, Leif Ellisen, Benjamin Ory. ΔNp63α promotes TGFβ-induced metastasis by silencing a microRNA network restraining wound healing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1911.

DIFFERENTIAL EXPRESSION OF CD150/SLAMF1 IN NORMAL AND MALIGNANT B CELLS ON THE DIFFERENT STAGES OF MATURATION

Within B-cell lineage cell surface receptor CD150/SLAMF1 is broadly expressed starting from pre-B cells with upregulation toward plasma cells. However, expression of CD150 is rather limited on the surface of malignant B cells with the block of differentiation at the different stages of maturation. The aim of our work was to explore CD150 expression both on protein and mRNA levels with the emphasis on CD150 isoforms in malignant B-cell lines at the different stages of maturation in comparison with their normal B cell counterparts. Studies were performed on normal tonsillar B-cell subpopulations, B-lymphoblastoid cell lines, malignant B-cell lines of different origin, including pre-B acute lymphoblastic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, and multiple myeloma. Protein CD150 expression was assessed by western blot analysis and the expression level of CD150 isoforms was evaluated using qRT-PCR. Despite the similar CD150 expression both on mRNA and protein levels in normal B-cell subsets and B-lymphoblastoid cell lines, malignant B-cell lines demonstrated substantial heterogeneity in CD150 expression. Only Hodgkin's lymphoma cell lines, Burkitt's lymphoma cell lines BJAB and Raji, and also pre-B cell line BLIN-1 expressed CD150 protein. At the same time total CD150 and mCD150 mRNA was detected in all studied cell lines excluding pre-B cell line REH. The minor sCD150 isoform was found only in Hodgkin's lymphoma cell lines and Burkitt's lymphoma cell line Raji. The nCD150 isoform was broadly expressed in tested B cell lines with exception of REH and Daudi. Malignant B-cell lines at the different stages of maturation only partially resemble their normal counterparts by CD150 expression. In malignant B-cell lines, CD150 expression on mRNA level is much broader than on protein level. CD150 isoforms are differentially expressed in normal and malignant B cells with predominant expression of mCD150 isoform.

ΔNp63α Silences a miRNA Program to Aberrantly Initiate a Wound-Healing Program That Promotes TGFβ-Induced Metastasis.

Primary cancer cell dissemination is a key event during the metastatic cascade, but context-specific determinants of this process remain largely undefined. Multiple reports have suggested that the p53 (TP53) family member p63 (TP63) plays an antimetastatic role through its minor epithelial isoform containing the N-terminal transactivation domain (TAp63). However, the role and contribution of the major p63 isoform lacking this domain, ΔNp63α, remain largely undefined. Here, we report a distinct and TAp63-independent mechanism by which ΔNp63α-expressing cells within a TGFβ-rich microenvironment become positively selected for metastatic dissemination. Orthotopic transplantation of ΔNp63α-expressing human osteosarcoma cells into athymic mice resulted in larger and more frequent lung metastases than transplantation of control cells. Mechanistic investigations revealed that ΔNp63α repressed miR-527 and miR-665, leading to the upregulation of two TGFβ effectors, SMAD4 and TβRII (TGFBR2). Furthermore, we provide evidence that this mechanism reflects a fundamental role for ΔNp63α in the normal wound-healing response. We show that ΔNp63α-mediated repression of miR-527/665 controls a TGFβ-dependent signaling node that switches off antimigratory miR-198 by suppressing the expression of the regulatory factor, KSRP (KHSRP). Collectively, these findings reveal that a novel miRNA network involved in the regulation of physiologic wound-healing responses is hijacked and suppressed by tumor cells to promote metastatic dissemination. Cancer Res; 76(11); 3236-51. ©2016 AACR.

Transcription factors Mix1 and VegT, relocalization of vegt mRNA, and conserved endoderm and dorsal specification in frogs

Protein expression of the transcription factor genes mix1 and vegt characterized the presumptive endoderm in embryos of the frogs Engystomops randi, Epipedobates machalilla, Gastrotheca riobambae, and Eleutherodactylus coqui, as in Xenopus laevis embryos. Protein VegT was detected in the animal hemisphere of the early blastula in all frogs, and only the animal pole was VegT-negative. This finding stimulated a vegt mRNA analysis in X. laevis eggs and embryos. vegt mRNA was detected in the animal region of X. laevis eggs and early embryos, in agreement with the VegT localization observed in the analyzed frogs. Moreover, a dorso-animal relocalization of vegt mRNA occurred in the egg at fertilization. Thus, the comparative analysis indicated that vegt may participate in dorsal development besides its known roles in endoderm development, and germ-layer specification. Zygotic vegt (zvegt) mRNA was detected as a minor isoform besides the major maternal (mvegt) isoform of the X. laevis egg. In addition, α-amanitin-insensitive vegt transcripts were detected around vegetal nuclei of the blastula. Thus, accumulation of vegt mRNA around vegetal nuclei was caused by relocalization rather than new mRNA synthesis. The localization of vegt mRNA around vegetal nuclei may contribute to the identity of vegetal blastomeres. These and previously reportedly localization features of vegt mRNA and protein derive from the master role of vegt in the development of frogs. The comparative analysis indicated that the strategies for endoderm, and dorsal specification, involving vegt and mix1, have been evolutionary conserved in frogs.

The Drosophila Dicer-1 Partner Loquacious Enhances miRNA Processing from Hairpins with Unstable Structures at the Dicing Site.

In Drosophila, Dicer-1 binds Loquacious-PB (Loqs-PB) as its major co-factor. Previous analyses indicated that loqs mutants only partially impede miRNA processing, but the activity of minor isoforms or maternally deposited Loqs was not eliminated in these studies. We addressed this by generating a cell line from loqs-null embryos and found that only ∼40% of miRNAs showed clear Loqs dependence. Genome-wide comparison of the hairpin structure and Loqs dependence suggested that Loqs substrates are influenced by base-pairing status at the dicing site. Artificial alteration of base-pairing stability at this position in model miRNA hairpins resulted in predicted changes in Loqs dependence, providing evidence for this hypothesis. Finally, we found that evolutionarily young miRNA genes tended to be Loqs dependent. We propose that Loqs may have roles in assisting the de novo emergence of miRNA genes by facilitating dicing of suboptimal hairpin substrates.

SRSF3 represses the expression of PDCD4 protein by coordinated regulation of alternative splicing, export and translation

Gene expression is regulated at multiple steps, such as transcription, splicing, export, degradation and translation. Considering diverse roles of SR proteins, we determined whether the tumor-related splicing factor SRSF3 regulates the expression of the tumor-suppressor protein, PDCD4, at multiple steps. As we have reported previously, knockdown of SRSF3 increased the PDCD4 protein level in SW480 colon cancer cells. More interestingly, here we showed that the alternative splicing and the nuclear export of minor isoforms of pdcd4 mRNA were repressed by SRSF3, but the translation step was unaffected. In contrast, only the translation step of the major isoform of pdcd4 mRNA was repressed by SRSF3. Therefore, overexpression of SRSF3 might be relevant to the repression of all isoforms of PDCD4 protein levels in most types of cancer cell. We propose that SRSF3 could act as a coordinator of the expression of PDCD4 protein via two mechanisms on two alternatively spliced mRNA isoforms.

Absence of AMPKα2 accelerates cellular senescence via p16 induction in mouse embryonic fibroblasts

Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK), an energy gauge and redox sensor, delays aging process. However, the molecular mechanisms by which AMPKα isoform regulates cellular senescence remain largely unknown. The aim of this study was to determine if AMPKα deletion contributes to the accelerated cell senescence by inducing p16INK4A (p16) expression thereby arresting cell cycle. The markers of cellular senescence, cell cycle proteins, and reactive oxygen species (ROS) were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J), AMPKα1, or AMPKα2 homozygous deficient (AMPKα1−/−, AMPKα2−/−) mice by Western blot and cellular immunofluorescence staining, as well as immunohistochemistry (IHC) in skin tissue of young and aged mice. Deletion of AMPKα2, the minor isoform of AMPKα, but not AMPKα1 in high-passaged MEFs led to spontaneous cell senescence demonstrated by accumulation of senescence-associated-β-galactosidase (SA-β-gal) staining and foci formation of heterochromatin protein 1 homolog gamma (HP1γ). It was shown here that AMPKα2 deletion upregulates cyclin-dependent kinase (CDK) inhibitor, p16, which arrests cell cycle. Furthermore, AMPKα2 null cells exhibited elevated ROS production. Interestingly, knockdown of HMG box-containing protein 1 (HBP1) partially blocked the cellular senescence of AMPKα2-deleted MEFs via the reduction of p16. Finally, dermal cells senescence, including fibroblasts senescence evidenced by the staining of p16, HBP1, and Ki-67, in the skin of aged AMPKα2−/− mice was enhanced when compared with that in wild type mice. Taken together, our results suggest that AMPKα2 isoform plays a fundamental role in anti-oxidant stress and anti-senescence.

A Loss-of-Function Variant in a Minor Isoform of ANK3 Protects Against Bipolar Disorder and Schizophrenia

BackgroundAnkyrin-3 (ANK3) was one of the first genes to reach significance in a bipolar disorder genome-wide association study. Many subsequent association studies confirmed this finding and implicated this gene in schizophrenia. However, the exact nature of the role of ANK3 in the pathophysiology remains elusive. In particular, the specific isoforms involved and the nature of the imbalance are unknown. MethodsWe genotyped a Norwegian sample of 402 patients with bipolar disorder, 293 patients with schizophrenia, and 330 healthy control subjects genome-wide with the Illumina Human Exome BeadChip. We performed allelic association tests at the genome-wide and gene levels and found a significantly associated single nucleotide polymorphism in a splice site of ANK3. We replicated this finding in two other samples and studied the functional effect of this single nucleotide polymorphism by performing quantitative polymerase chain reaction on the affected exon junction in complementary DNA from blood total RNA. ResultsThe splice site single nucleotide polymorphism (rs41283526) is located in an alternatively spliced exon of ANK3 and has a strong and significant protective effect against bipolar disorder (odds ratio = .31) and schizophrenia (odds ratio = .21). The minor allele of rs41283526 is a loss-of-function variant that disables the correct splicing of the transcript. Data from the BrainSpan human developmental transcriptome show that the exon bearing this variant is expressed only in a minor isoform of ANK3, the transcription of which is initiated in early adolescence. ConclusionsOur results suggest that an elevated expression of this transcript starting in adolescence may be an important factor in the pathophysiology of schizophrenia and bipolar disorder.

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins.

A SNP in the Immunoregulatory Molecule CTLA-4 Controls mRNA Splicing In Vivo but Does Not Alter Diabetes Susceptibility in the NOD Mouse.

CTLA-4 is a critical "checkpoint" regulator in autoimmunity. Variation in CTLA-4 isoform expression has been linked to type 1 diabetes development in human and NOD mouse studies. In the NOD mouse, a causative link between increased expression of the minor isoform ligand-independent CTLA-4 and a reduction in diabetes has become widely accepted. Altered splicing of CTLA-4 has been attributed to a single nucleotide polymorphism (SNP) in Ctla4 exon2 (e2_77A/G). To investigate this link, we have used NOD embryonic stem (ES) cells to generate a novel NOD transgenic line with the 77A/G SNP. This strain phenocopies the increase in splicing toward the liCTLA4 isoform seen in B10 Idd5.1 mice. Crucially, the SNP does not alter the spontaneous incidence of diabetes, the incidence of cyclophosphamide-induced diabetes, or the activation of diabetogenic T-cell receptor transgenic CD4(+) T cells after adoptive transfer. Our results show that one or more of the many other linked genetic variants between the B10 and NOD genome are required for the diabetes protection conferred by Idd5.1. With the NOD mouse model closely mimicking the human disease, our data demonstrate that knock-in transgenic mice on the NOD background can test causative mutations relevant in human diabetes.

Abstract 4363: Selective modulation of IRES-mediated translation in malignant cells

Abstract There are two distinct modes of protein synthesis in the cell. The conventional mechanism of cap-dependent ribosomal scanning is utilized for general protein synthesis, whereas internal ribosome entry site (IRES)-mediated translation appears to be reserved for synthesis of those gene products most critical for cell proliferation and survival, providing a fail-safe or emergency system to ensure that such essential polypeptides can be manufactured when needed, particularly under conditions of stress when general protein synthesis is compromised. To date, there has been no means, experimental or therapeutic, by which to selectively modulate IRES function; rather, IRES-mediated translation has typically been studied by inhibiting general protein synthesis and attributing the remaining translational activity to the IRES. To address this problem, a cell-based high-throughput screen of 135,000 compounds was performed to identify small molecules capable of selectively inhibiting translation mediated through the IGF1R IRES. The identification of these compounds allows us, for the first time, to selectively perturb IRES-mediated translation in its native context, and assess the consequences. Data obtained using the bicistronic reporter system demonstrate that the lead compounds and their structural analogs selectively inhibit second cistron translation (mediated through the IRES). Western analyses show that, at sufficient concentration (≥ 5 μg/ml), the IRES inhibitors completely block de novo IGF1R protein synthesis in genetically-unmodified breast tumor cells, confirming activity against the endogenous IRES. The IRES inhibitors function in a manner which is essentially reciprocal to rapamycin, and can be clearly distinguished from cycloheximide. Spectrum of activity of the small molecule IRES inhibitors extends beyond IGF1R to a subset of potential IRES targets, including c-myc. The IRES inhibitors differentially modulate synthesis of the two c-Myc protein isoforms, bringing about a dramatic decrease in p64, the dominant (oncogenic) isoform, while transiently stimulating synthesis of p67, the minor (growth-inhibitory) isoform. RNase protection analyses show no discernible changes in c-myc mRNA, confirming that the effects of the IRES inhibitor are exerted at the translational level. Sustained IRES inhibition has profound, detrimental effects on human tumor cells, inducing massive (>99%) cell death and complete loss of clonogenic survival in tumor models as disparate as triple negative breast carcinoma, glioblastoma, and acute myeloid leukemia. Beyond a critical threshold of dose and duration of exposure, there is no evidence of recovery, even with prolonged incubation in absence of the compound. Together, these findings support the conclusion that IRES-mediated translation is of fundamental importance to the survival of malignant cells, and may represent a critical point of vulnerability which could be exploited therapeutically. Citation Format: Christos Vaklavas, Zheng Meng, Hyoungsoo Choi, William E. Grizzle, Kurt R. Zinn, Scott W. Blume. Selective modulation of IRES-mediated translation in malignant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4363. doi:10.1158/1538-7445.AM2015-4363

Pathogenicity of C-terminal mutations in CDKL5

It was with great interest that we read the report by Martinez et al. [1] entitled “CDKL5 in different atypical Rett syndrome variants: Description of the first eight patients from Spain”, and in particular of a late C-terminal missense mutation in CDKL5. The mutation described, p.Pro976Leu (c.2927C>T) was identified in a female patient with Rett syndrome (RTT) with regression of late onset. The patient had very mild symptoms in comparison with the others in the report and most strikingly, had never had any seizure episodes. This is in contrast to the general description of patients with CDKL5 mutations [2–4], characterized by early-onset seizures, usually difficult to control. The mutation in this patient was de novo and X-chromosome inactivation (XCI) in the patient’s lymphocytes showed a random pattern of inactivation. We propose that the effect of this missense mutation is minimal due to its position along the CDKL5 gene. Recently, we reported an alternate CDKL5 protein isoform, which differs from the recognized isoform at the C-terminal end at exon 18 (GenBank reference sequence NM_003159.1: c.2713) onwards [5]. The C-terminus of the newly-identified isoform is encoded by an alternatively-spliced exon 18, which extends into intron 18 by at least 170 bases. The presence of an in-frame termination codon in this sequence means that translation beyond exon 18 does not occur in this isoform. As the missense mutation p.Pro976Leu is located on exon 20, the mutation would only affect the previouslyrecognized isoform (GenBank reference sequences NM_001037343 and NM_003159). Expression analysis in different human tissues indicates that the initially recognized isoform is the minor isoform [5], and most abundantly expressed in testis. In contrast, the newlyidentified isoform is expressed in all tissues, highest in parts of the brain, and is likely to be of greater physiological importance. To the best of our knowledge, only three other C-terminal CDKL5 variations in exons 19, 20 or 21 have been reported in patients with RTT or a related disorder. One of these is also a missense variation, p.Val999Met (c.2995G>A), reported as a polymorphism by Intusoma et al. [6] and has a heterozygosity of 0.118 (NCBI dbSNP, rs35693326). The other two variations are nonsensemutations, p.Arg952X and p.Arg970X. The latter mutation (p.Arg970X, c.2908C>T)was reported in a female patient with a Rett-like phenotype, and late-onset seizures at 17 mo [7]. Parental screening was limited only to the mother, and XCI studies were not carried out. The other nonsense mutation p.Arg952X (c.2854C>T) was identified in a female with severe mental retardation without RTT features, and seizures starting from 11 mo [6]. Familial screening found the same mutation in the grandmother, mother and half-sister of the patient. A difference in phenotypes could not be attributed to skewed XCI. Furthermore, population screening revealed an allele frequency of 0.8%, suggesting that although rare, the truncating variation is not likely to be pathogenic. In all, the existing data indicate that C-terminal mutations in CDKL5 should be interpreted with care. *Corresponding author: John Christodoulou, Disciplines of Pediatrics and Child Health and Genetic Medicine, Sydney Medical School, University of Sydney, Sydney, Australia. Tel.: +61 2 9845 3452; Fax: +61 2 9845 1864; E-mail: john.christodoulou@health.nsw.gov.au. Journal of Pediatric Epilepsy 1 (2012) 185–186 DOI 10.3233/PEP-2012-029 IOS Press 185

High expression of survivin and its splice variants survivin ΔEx3 and survivin 2 B in oral cancers

We have previously reported inactivation of p53 in 46% of Indian patients with oral cancer. Survivin, a p53 target gene and an inhibitor of apoptosis protein (IAP), is overexpressed in several cancers, including oral cancers. Studies assessing the role of survivin and its splice variants in oral cancers are, however, rare. The expression of 6 survivin isoforms in 4 oral cancer cell lines (AW8507, AW13516, UPCI-SCC040, UPCI-SCC029 B), a dysplastic oral cell line (DOK), 75 paired oral tumor and adjacent normal tissues, and 12 normal oral tissue samples from healthy individuals was analyzed by real-time PCR. The expression was correlated with clinicopathologic parameters, which included age, sex, tumor-node-metastasis (TNM) staging, tobacco and/or alcohol consumption, site, and differentiation status of tumor. This is the first study to find overexpression of the 6 characterized survivin isoforms in oral cancers compared with normal tissues (P < .05). Additionally, a significant (P < .05) correlation among the fold changes of all 6 survivin isoforms was observed. Survivin wild type (wt) was the predominantly expressed isoform in oral cell lines and tumor tissues versus normal tissues (P < .05). Among the minor isoforms, survivin ΔEx3 and survivin 2 B were dominantly expressed, whereas survivin 2 α and survivin 3 α overexpression was found for the first time. Further high survivin 3 B expression exhibited a significant association (P < .05) with poorly differentiated tumors. Interestingly the combined expression of the antiapoptotic survivin isoforms, survivin wt, survivin ΔEx3, and survivin 3 B, exhibited a significant association with TNM staging of the tumor. Our studies thus indicate that oral cancers overexpress the antiapoptotic survivin variants, which exhibit an association with advanced tumor stage, implying a role for these variants in oral tumorigenesis.

Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms.

The Wilms’ tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3’ end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms.

Analysis of transthyretin in human serum by capillary zone electrophoresis electrospray ionization time-of-flight mass spectrometry. Application to familial amyloidotic polyneuropathy type I.

Transthyretin (TTR) is known to misfold and aggregate, causing different types of amyloidosis. Familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, is associated with a TTR variant that presents a single amino acid substitution of valine for methionine at position 30 (Met 30). To screen for TTR-related amyloidosis rapidly and reliably, we have developed a novel procedure based on the analysis of monomers from the homotetrameric protein (∼56 kDa). First, we established a CZE-ESI-TOF-MS method to detect wild-type (normal) TTR with or without several PTMs, as well as an extra minor isoform in TTR standard solutions. Later, a sample pretreatment based on immunoprecipitation (IP) and centrifugal filtration was optimized to analyze serum samples from healthy controls and FAP-I patients (including an asymptomatic patient, a symptomatic patient, a liver-transplanted patient with the specific mutation, and a patient originally without the mutation who received a liver transplant from an FAP-I patient (iatrogenic FAP-I)). The mutant TTR (Met 30) variant with a relative molecular mass 32.07 higher than the wild-type TTR was found in the asymptomatic, the symptomatic and the iatrogenic FAP-I patients, who interestingly also presented the same concentration ratio between both variants of TTR (abnormal and normal). In contrast, as in the healthy controls, the abnormal TTR variant was not detected in the liver-transplanted patient with the specific mutation, which confirms the effectiveness of the treatment. The proposed procedure could be regarded as a suitable screening system for individuals with suspected TTR amyloidosis, and to gain insight into TTR structure, to understand the mechanism underlying the disease.

Arrhythmogenic Biophysical Phenotype for SCN5A Mutation S1787N Depends upon Splice Variant Background and Intracellular Acidosis.

Background SCN5A is a susceptibility gene for type 3 long QT syndrome, Brugada syndrome, and sudden infant death syndrome. I Na dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in 1 of 295 healthy white controls. Here, we determined the in vitro biophysical phenotype of SCN5A-S1787N in an effort to further assess its possible pathogenicity.Methods and ResultsWe engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position 1077 (Q1077del) and the minor isoform containing Q1077, and expressed these two engineered constructs in HEK293 cells for electrophysiological study. Macroscopic voltage-gated I Na was measured 24 hours after transfection with standard whole-cell patch clamp techniques. We applied intracellular solutions with pH7.4 or pH6.7. S1787N in the Q1077 background had WT-like I Na including peak I Na density, activation and inactivation parameters, and late I Na amplitude in both pH 7.4 and pH 6.7. However, with S1787N in the Q1077del background, the percentages of I Na late/peak were increased by 2.1 fold in pH 7.4 and by 2.9 fold in pH 6.7 when compared to WT.ConclusionThe LQT3-like biophysical phenotype for S1787N depends on both the SCN5A splice variant and on the intracellular pH. These findings provide further evidence that the splice variant and environmental factors affect the molecular phenotype of cardiac SCN5A-encoded sodium channel (Nav1.5), has implications for the clinical phenotype, and may provide insight into acidosis-induced arrhythmia mechanisms.

Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

Background/Aims: Sterol regulatory element-binding protein (SREBP) 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specifc and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specifc knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression.

Integrating evolutionary and functional tests of adaptive hypotheses: a case study of altitudinal differentiation in hemoglobin function in an Andean Sparrow, Zonotrichia capensis.

In air-breathing vertebrates, the physiologically optimal blood-O2 affinity is jointly determined by the prevailing partial pressure of atmospheric O2, the efficacy of pulmonary O2 transfer, and internal metabolic demands. Consequently, genetic variation in the oxygenation properties of hemoglobin (Hb) may be subject to spatially varying selection in species with broad elevational distributions. Here we report the results of a combined functional and evolutionary analysis of Hb polymorphism in the rufous-collared sparrow (Zonotrichia capensis), a species that is continuously distributed across a steep elevational gradient on the Pacific slope of the Peruvian Andes. We integrated a population genomic analysis that included all postnatally expressed Hb genes with functional studies of naturally occurring Hb variants, as well as recombinant Hb (rHb) mutants that were engineered through site-directed mutagenesis. We identified three clinally varying amino acid polymorphisms: Two in the α(A)-globin gene, which encodes the α-chain subunits of the major HbA isoform, and one in the α(D)-globin gene, which encodes the α-chain subunits of the minor HbD isoform. We then constructed and experimentally tested single- and double-mutant rHbs representing each of the alternative α(A)-globin genotypes that predominate at different elevations. Although the locus-specific patterns of altitudinal differentiation suggested a history of spatially varying selection acting on Hb polymorphism, the experimental tests demonstrated that the observed amino acid mutations have no discernible effect on respiratory properties of the HbA or HbD isoforms. These results highlight the importance of experimentally validating the hypothesized effects of genetic changes in protein function to avoid the pitfalls of adaptive storytelling.

Rear actomyosin contractility-driven directional cell migration in three-dimensional matrices: a mechano-chemical coupling mechanism

Cell migration is of vital importance in many biological processes, including organismal development, immune response and development of vascular diseases. For instance, migration of vascular smooth muscle cells from the media to intima is an essential part of the development of atherosclerosis and restenosis after stent deployment. While it is well characterized that cells use actin polymerization at the leading edge to propel themselves to move on two-dimensional substrates, the migration modes of cells in three-dimensional matrices relevant to in vivo environments remain unclear. Intracellular tension, which is created by myosin II activity, fulfils a vital role in regulating cell migration. We note that there is compelling evidence from theoretical and experimental work that myosin II accumulates at the cell rear, either isoform-dependent or -independent, leading to three-dimensional migration modes driven by posterior myosin II tension. The scenario is not limited to amoeboid migration, and it is also seen in mesenchymal migration in which a two-dimensional-like migration mode based on front protrusions is often expected, suggesting that there may exist universal underlying mechanisms. In this review, we aim to shed some light on how anisotropic myosin II localization induces cell motility in three-dimensional environments from a biomechanical view. We demonstrate an interesting mechanism where an interplay between mechanical myosin II recruitment and biochemical myosin II activation triggers directional migration in three-dimensional matrices. In the case of amoeboid three-dimensional migration, myosin II first accumulates at the cell rear to induce a slight polarization displayed as a uropod-like structure under the action of a tension-dependent mechanism. Subsequent biochemical signalling pathways initiate actomyosin contractility, producing traction forces on the adhesion system or creating prominent motile forces through blebbing activity, to drive cells to move. In mesenchymal three-dimensional migration, cells can also take advantage of the elastic properties of three-dimensional matrices to move. A minor myosin isoform, myosin IIB, is retained by relatively stiff three-dimensional matrices at the posterior side, then activated by signalling cascades, facilitating prominent cell polarization by establishing front-back polarity and creating cell rear. Myosin IIB initiates cell polarization and coordinates with the major isoform myosin IIA-assembled stress fibres, to power the directional migration of cells in the three-dimensional matrix.