Major Isoforms Research Articles

Fatal intracranial haemorrhage in acute promyelocytic leukemia patients with short isoform of PML-RARα: Review of molecular and radiological data

Three major PML-RARα fusion gene transcripts (long [bcr1], variant [bcr2], and short [bcr3]) are currently used in clinical laboratories for the diagnosis and treatment monitoring of APL patients. Despite highly improved outcome, relapse and intracranial haemorrhage that may lead to early death is still an unsolved complication in APL. We reviewed APL patients confirmed by qPCR for the presence of PML-RARα transcripts (n = 27) and studied their outcome in relation to the isoform expression at diagnosis and follow-up in King Fahad Medical City. Eight in twenty-seven patients showed bcr3 and nineteen patients with bcr1 as major isoforms at diagnosis. Half of the bcr3 patients (n = 4/8) showed early mortality, prolonged qPCR positivity, 4-fold higher neutrophil/lymphocyte ratio, higher creatinine levels, and significantly reduced relapse free and overall survival time compared with bcr1 patients. Radiological findings in bcr3 patients revealed CNS involvement in the form of intracranial haemorrhage and periventricular microangiopathy and no CNS involvement in bcr1 patients. In conclusion, PML-RARα isoform expression at diagnosis in selective patients influences disease course over time and may even lead to early mortality due to haemorrhage. Thus, timely reporting of the specific PML-RARα isoform by clinical laboratories and CNS assessment by radiology can prevent complications leading to death in some APL patients.

Differential glycosylation in mutant vitamin D-binding protein decimates the binding stability of vitamin D

Vitamin D (VD) is produced by the skin upon exposure to sunlight or is obtained from dietary sources. Several risk factors are associated with VD deficiency including mutations and post-translational modifications in its transport protein known as vitamin D binding protein (VDBP) or GC-globulin. The two common single nucleotide polymorphisms rs7041 and rs4588 create three major isoforms of VDBP, including GC-1F also called wild type, GC1S, and GC-2. The 3D models for both GC-1F and GC-2 were constructed in their glycosylated states to decipher the effect of these mutations on the overall conformational changes and VD-binding affinity. The binding affinities were estimated using the Molecular Mechanics Poison-Boltzmann surface area (MM-PBSA) method and conformational changes were investigated after free energy landscapes estimations. Total free energies suggest that GC-1F exhibits stronger affinity (ΔE = −116.09 kJ/mol) than GC-2 (ΔE = −95 kJ/mol) variant with VD. The GC-1F isoforms had more streamlined motion compared to GC-2 isoforms, predicting a trade-off between cross-talk residues that significantly impacts protein structural stability. The data suggest that glycation at Thr418 plays a vital role in the overall VDBP-VD affinity by stabilizing the N-T loop that holds the domain I (VD-pocket) and domain III intact. The loss of glycation in GC-2 has a pivotal role in the inter-domain conformational stability of VDBP, which may ultimately affect VD transportation and maturation. These findings describe a novel mechanism in how mutations distant from the VD-active site change the overall conformational of the VDBP and abrogate the VDBP-VD interaction. Communicated by Ramaswamy H. Sarma

Basement membrane proteins in extracellular matrix characterize NF1 neurofibroma development and response to MEK inhibitor.

Neurofibromatosis Type 1 (NF1) is one of the most common tumor-predisposing genetic disorders. Neurofibromas are NF1-associated benign tumors. A hallmark feature of neurofibromas is an abundant collagen-rich extracellular matrix (ECM) that constitutes >50% of the tumor dry weight. However, little is known about the mechanism underlying ECM deposition during neurofibroma development and treatment response. We performed a systematic investigation of ECM enrichment during plexiform neurofibroma (pNF) development, and identified basement membrane (BM) proteins, rather than major collagen isoforms, as the most upregulated ECM component. Following MEK inhibitor treatment, the ECM profile displayed an overall down-regulation signature, suggesting ECM reduction as a therapeutic benefit of MEK inhibition. Through these proteomic studies, TGF-β1 signaling was identified as playing a role in ECM dynamics. Indeed, TGF-β1 overexpression promoted pNF progression in vivo. Furthermore, by integrating single-cell RNA-sequencing, we found that immune cells including macrophages and T cells produce TGF-β1 to induce Schwann cells to produce and deposit BM proteins for ECM remodeling. Following Nf1 loss, neoplastic Schwann cells further increased BM protein deposition in response to TGF-β1. Our data delineate the regulation governing ECM dynamics in pNF and suggest that BM proteins could serve as markers for disease diagnosis and treatment response.

Endothelial basement membrane laminins - new players in mouse and human myoendothelial junctions and shear stress communication

Basement membranes (BMs) are critical but frequently ignored components of the vascular system. Using high-resolution confocal imaging of whole-mount-stained mesenteric arteries, we identify integrins, vinculin, focal adhesion kinase (FAK) and several BM proteins including laminins as novel components of myoendothelial junctions (MEJs), anatomical microdomains that are emerging as regulators of cross-talk between endothelium and smooth muscle cells (SMCs). Electron microscopy revealed multiple layers of the endothelial BM that surround endothelial projections into the smooth muscle layer as structural characteristics of MEJs. The shear-responsive calcium channel TRPV4 is broadly distributed in endothelial cells and occurs in a proportion of MEJs where it localizes to the tips of the endothelial projections that are in contact with the underlying SMCs. In mice lacking the major endothelial laminin isoform, laminin 411 (Lama4−/−), which we have previously shown over-dilate in response to shear and exhibit a compensatory laminin 511 upregulation, localization of TRPV4 at the endothelial-SMC interface in MEJs was increased. Endothelial laminins do not affect TRPV4 expression, rather in vitro electrophysiology studies using human umbilical cord arterial endothelial cells revealed enhanced TRPV4 signalling upon culturing on an RGD-motif containing domain of laminin 511. Hence, integrin-mediated interactions with laminin 511 in MEJ structures unique to resistance arteries modulate TRPV4 localization at the endothelial-smooth muscle interface in MEJs and signalling over this shear-response molecule.

CNS-dominant human FMRP isoform rescues seizures, fear, and sleep abnormalities in Fmr1-KO mice.

Fragile X syndrome is a neurodevelopmental disorder caused by the absence of the mRNA-binding protein fragile X messenger ribonucleoprotein (FMRP). Because FMRP is a highly pleiotropic protein controlling the expression of hundreds of genes, viral vector-mediated gene replacement therapy is viewed as a potential viable treatment to correct the fundamental underlying molecular pathology inherent in the disorder. Here, we studied the safety profile and therapeutic effects of a clinically relevant dose of a self-complementary adeno-associated viral (AAV) vector containing a major human brain isoform of FMRP after intrathecal injection into wild-type and fragile X-KO mice. Analysis of the cellular transduction in the brain indicated primarily neuronal transduction with relatively sparse glial expression, similar to endogenous FMRP expression in untreated wild-type mice. AAV vector-treated KO mice showed recovery from epileptic seizures, normalization of fear conditioning, reversal of slow-wave deficits as measured via electroencephalographic recordings, and restoration of abnormal circadian motor activity and sleep. Further assessment of vector efficacy by tracking and analyzing individual responses demonstrated correlations between the level and distribution of brain transduction and drug response. These preclinical findings further demonstrate the validity of AAV vector-mediated gene therapy for treating the most common genetic cause of cognitive impairment and autism in children.

Retinal degeneration in rpgra mutant zebrafish.

Introduction: Pathogenic mutations in RPGR ORF15, one of two major human RPGR isoforms, were responsible for most X-linked retinitis pigmentosa cases. Previous studies have shown that RPGR plays a critical role in ciliary protein transport. However, the precise mechanisms of disease triggered by RPGR ORF15 mutations have yet to be clearly defined. There are two homologous genes in zebrafish, rpgra and rpgrb. Zebrafish rpgra has a single transcript homologous to human RPGR ORF15; rpgrb has two major transcripts: rpgrb ex1-17 and rpgrb ORF15, similar to human RPGR ex1-19 and RPGR ORF15, respectively. rpgrb knockdown in zebrafish resulted in both abnormal development and increased cell death in the dysplastic retina. However, the impact of knocking down rpgra in zebrafish remains undetermined. Here, we constructed a rpgra mutant zebrafish model to investigate the retina defect and related molecular mechanism. Methods: we utilized transcription activator-like effector nuclease (TALEN) to generate a rpgra mutant zebrafish. Western blot was used to determine protein expression. RT-PCR was used to quantify gene transcription levels. The visual function of embryonic zebrafish was detected by electroretinography. Immunohistochemistry was used to observe the pathological changes in the retina of mutant zebrafish and transmission electron microscope was employed to view subcellular structure of photoreceptor cells. Results: A homozygous rpgra mutant zebrafish with c.1675_1678delins21 mutation was successfully constructed. Despite the normal morphological development of the retina at 5 days post-fertilization, visual dysfunction was observed in the mutant zebrafish. Further histological and immunofluorescence assays indicated that rpgra mutant zebrafish retina photoreceptors progressively began to degenerate at 3-6 months. Additionally, the mislocalization of cone outer segment proteins (Opn1lw and Gnb3) and the accumulation of vacuole-like structures around the connecting cilium below the OSs were observed in mutant zebrafish. Furthermore, Rab8a, a key regulator of opsin-carrier vesicle trafficking, exhibited decreased expression and evident mislocalization in mutant zebrafish. Discussion: This study generated a novel rpgra mutant zebrafish model, which showed retinal degeneration. our data suggested Rpgra is necessary for the ciliary transport of cone-associated proteins, and further investigation is required to determine its function in rods. The rpgra mutant zebrafish constructed in this study may help us gain a better understanding of the molecular mechanism of retinal degeneration caused by RPGR ORF15 mutation and find some useful treatment in the future.

In-silico Identification and Analysis of Hub Proteins for Designing Novel First-line Anti-seizure Medications

Background: Epilepsy is a seizure-related disease with different symptoms and types, depending on the origin and propagation region of the brain. There are several marketed anti-seizure medications (ASMs) available for choice of treatment by clinicians but there is a huge paucity of ideal first-line ASMs. Objective: The present study was undertaken to identify and get an insight into the major target (hub) proteins, which can be comprehensively used as a platform for designing first-line ASMs. Method: Large-scale text mining was done to generate a data warehouse of available ASMs and their MOAs, followed by the identification of specific isoforms of target proteins for designing next-generation ASMs, using network biology and other in-silico approaches. Results: The study resulted in the identification of 3 major classes of target proteins of major ASMs and their specific isoforms, namely – GABA receptors (GABRA1, GABRB1, and GABARAP); VGSC (α- subunitSCN2A (Nav1.2)) and VGCC (α-subunitCACNA1G (Cav3.1)). The identified proteins were also observed to be concurrent with the target sites of majorly sold ASMs currently. Conclusion: The predicted hub protein families and their specific isoforms can be further validated and comprehensively used to design next-generation novel first-line ASM(s).

BE3 is the major branching enzyme isoform required for amylopectin synthesis in Chlamydomonas reinhardtii.

Starch-branching enzymes (BEs) are essential for starch synthesis in both plants and algae where they influence the architecture and physical properties of starch granules. Within Embryophytes, BEs are classified as type 1 and type 2 depending on their substrate preference. In this article, we report the characterization of the three BE isoforms encoded in the genome of the starch producing green algae Chlamydomonas reinhardtii: two type 2 BEs (BE2 and BE3) and a single type 1 BE (BE1). Using single mutant strains, we analyzed the consequences of the lack of each isoform on both transitory and storage starches. The transferred glucan substrate and the chain length specificities of each isoform were also determined. We show that only BE2 and BE3 isoforms are involved in starch synthesis and that, although both isoforms possess similar enzymatic properties, BE3 is critical for both transitory and storage starch metabolism. Finally, we propose putative explanations for the strong phenotype differences evidenced between the C. reinhardtii be2 and be3 mutants, including functional redundancy, enzymatic regulation or alterations in the composition of multimeric enzyme complexes.

Role of white matter hyperintensity in effects of apolipoprotein E on cognitive injury.

Magnetic Resonance Imaging (MRI) T2-weighted white matter hyperintensity (WMH) is a marker of small vessel cerebrovascular pathology and is of ischemic origin. The prevalence and severity of WMH is associated with cardiovascular risk factors, aging, and cognitive injury in mild cognitive impairment (MCI), vascular dementia, and Alzheimer's disease (AD). WMH especially affects executive function, with additional effects on memory and global cognition. Apolipoprotein E (apoE) plays a role in cholesterol metabolism and neuronal repair after injury. Human and animal studies support a role for apoE in maintaining white matter integrity. In humans, there are three major human apoE isoforms, E2, E3, and E4. Human apoE isoforms differ in risk to develop AD and in association with WMH. In this Mini Review, we propose an increased focus on the role of WMH in cognitive health and cognitive injury and the likely role of apoE and apoE isoform in modulating these effects. We hypothesize that apoE and apoE isoforms play a role in modulating WMH via apoE isoform-dependent effects on oxylipins and 7-ketocholesterol, as well as amyloid related vascular injury, as seen in cerebral amyloid angiopathy.

Evaluation of the Cytochrome P450 3A and P-glycoprotein Drug-Drug Interaction Potential of Futibatinib.

Futibatinib, a selective, irreversible fibroblast growth factor receptor 1-4 inhibitor, is being investigated for tumors harboring FGFR aberrations and was recently approved for the treatment of FGFR2 fusion/rearrangement-positive intrahepatic cholangiocarcinoma. In vitro studies identified cytochrome P450 (CYP) 3A as the major CYP isoform in futibatinib metabolism and indicated that futibatinib is likely a P-glycoprotein (P-gp) substrate and inhibitor. Futibatinib also showed time-dependent inhibition of CYP3A in vitro. Phase I studies investigated the drug-drug interactions of futibatinib with itraconazole (a dual P-gp and strong CYP3A inhibitor), rifampin (a dual P-gp and strong CYP3A inducer), or midazolam (a sensitive CYP3A substrate) in healthy adult participants. Compared with futibatinib alone, coadministration of futibatinib with itraconazole increased futibatinib mean peak plasma concentration and area under the plasma concentration-time curve by 51% and 41%, respectively, and coadministration of futibatinib with rifampin lowered futibatinib mean peak plasma concentration and area under the plasma concentration-time curve by 53% and 64%, respectively. Coadministration of midazolam with futibatinib had no effect on midazolam pharmacokinetics compared with midazolam administered alone. These findings suggest that concomitant use of dual P-gp and strong CYP3A inhibitors/inducers with futibatinib should be avoided, but futibatinib can be concomitantly administered with other drugs metabolized by CYP3A. Drug-drug interaction studies with P-gp-specific substrates and inhibitors are planned.

Calotropis gigantea stem bark extract activates HepG2 cell apoptosis through ROS and its effect on cytochrome P450

The 95% ethanolic extract of the dry powder of Calotropis gigantea (C. gigantea) stem bark was separated by fractionation with different solutions to yield 4 fractions: dichloromethane (CGDCM), ethyl acetate (CGEtOAc), and water (CGW). This research focused on CGDCM-induced apoptosis in HepG2 cells with IC50 and above-IC50 values, which provide useful information for future anticancer applications. CGDCM had lower cytotoxicity on normal lung fibroblast IMR-90 cells than on HepG2 cells. Apoptotic induction of CGDCM was mediated by decreased fatty acid and ATP synthesis while increasing reactive oxygen species production. The effects of the four extracts on the activity of the four major CYP450 isoforms (CYP1A2, CYP2C9, CYP2E1 and CYP3A4) were determined using the CYP-specific model activity of each isoform. All four fractions of the extract were shown to be poor inhibitors of CYP1A2 and CYP2E1 (IC50 > 1000 μg/mL) and moderate inhibitors of CYP3A4 (IC50 = 56.54–296.9 μg/mL). CGDCM and CGW exerted moderate inhibition activities on CYP2C9 (IC50 = 59.56 and 46.38 μg/mL, respectively), but CGEtOH and CGEtOAc exhibited strong inhibition activities (IC50 = 12.11 and 20.43 μg/mL, respectively). It is proposed that C. gigantea extracts at high doses have potential for further studies to develop alternative anticancer applications. Inhibiting CYP2C9 activity may also lead to drug-herb interactions.

Abstract 317: Adipose Tissue Phosphatidylcholine Remodeling Modulates Insulin Signaling

Phosphatidylcholines (PCs) are major components of cell membranes. Previous studies indicated that membrane PC composition is regulated by lysophosphatidylcholine acyltransferase (LPCAT) and an alteration in cell membrane saturation has been implicated in a variety of metabolic disorders, including obesity, insulin resistance, and cardiovascular diseases. LPCAT3 is the major isoform of LPCAT in adipose tissues. To study the impact of LPCAT3 on adipose tissue lipid metabolism and its metabolic consequences, we prepared adipocyte-specific Lpcat3 gene knockout (KO) mice. Based on the analysis of bulk RNA sequencing, we gained insights to investigate the role of LPCAT3 in regulating adipose tissue insulin signaling. We revealed that LPCAT3 deficiency significantly reduced polyunsaturated PCs in the plasma membrane of adipocytes, resulting in increased insulin sensitivity. Mechanistically, we demonstrated that inhibiting LPCAT3 in adipose tissue promoted insulin receptor and AKT activation by influencing membrane lipid rafts and attenuated diet-induced insulin resistance. Conversely, adipose tissues from ob/ob, db/db, and high fat diet-fed mice had significantly higher LPCAT3 activity which blunts insulin signaling. Moreover, the treatment of polyunsaturated PCs on human or mouse mature adipocytes significantly worsened cellular insulin signaling. Our findings suggest that targeting LPCAT3 in adipose tissues to manipulate membrane phospholipid saturation would be a new strategy to treat insulin resistance.

FKBP12 binds to the cardiac ryanodine receptor with negative cooperativity: implications for heart muscle physiology in health and disease.

Cardiac ryanodine receptors (RyR2) release the Ca2+ from intracellular stores that is essential for cardiac myocyte contraction. The ion channel opening is tightly regulated by intracellular factors, including the FK506 binding proteins, FKBP12 and FKBP12.6. The impact of these proteins on RyR2 activity and cardiac contraction is debated, with often apparently contradictory experimental results, particularly for FKBP12. The isoform that regulates RyR2 has generally been considered to be FKBP12.6, despite the fact that FKBP12 is the major isoform associated with RyR2 in some species and is bound in similar proportions to FKBP12.6 in others, including sheep and humans. Here, we show time- and concentration-dependent effects of adding FKBP12 to RyR2 channels that were partly depleted of FKBP12/12.6 during isolation. The added FKBP12 displaced most remaining endogenous FKBP12/12.6. The results suggest that FKBP12 activates RyR2 with high affinity and inhibits RyR2 with lower affinity, consistent with a model of negative cooperativity in FKBP12 binding to each of the four subunits in the RyR tetramer. The easy dissociation of some FKBP12/12.6 could dynamically alter RyR2 activity in response to changes in in vivo regulatory factors, indicating a significant role for FKBP12/12.6 in Ca2+ signalling and cardiac function in healthy and diseased hearts. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Analysis of 200 unrelated individuals with a constitutional NF1 deep intronic pathogenic variant reveals that variants flanking the alternatively spliced NF1 exon 31 [23a] cause a classical neurofibromatosis type 1 phenotype while altering predominantly NF1 isoform type II

Neurofibromatosis type 1 results from loss-of-function NF1 pathogenic variants (PVs). Up to 30% of all NF1 PVs disrupt mRNA splicing, including deep intronic variants. Here, we retrospectively investigated the spectrum of NF1 deep intronic PVs in a cohort of 8,090 unrelated individuals from the University of Alabama at Birmingham (UAB) dataset with a molecularly confirmed neurofibromatosis type 1. All variants were identified through their effect on the NF1 transcript, followed by variant characterization at the DNA-level. A total of 68 distinct variants, which were ≥ 20 nucleotides away from the closest exon–intron junction, were identified in 2.5% unrelated individuals with NF1 (200/8,090). Nine different pathogenic splice variants, identified in 20 probands, led to exonization of different parts of intron 30 [23.2] or 31 [23a]. The two major NF1 transcript isoforms, distinguished by the absence (type I) or presence (type II) of the alternatively spliced cassette exon 31 [23a], are equally expressed in blood in control individuals without NF1 or NF1-affected individuals carrying their PV not in the introns flanking exon 31 [23a]. By fragment and cloning analysis we demonstrated that the exonization of intron 31 [23a] sequences due to deep intronic PV predominantly affects the NF1 isoform II. Seven additional (likely) pathogenic NF1 deep intronic variants not observed in the UAB dataset were found by classification of 36 variants identified by a literature search. Hence, the unique list of these 75 deep intronic (likely) PVs should be included in any comprehensive NF1 testing strategy.

Evaluation of metabolism and cytochrome P450 mediated interaction liabilities of naringenin

Naringenin is one of the major components of grapefruit juice. It has a broad spectrum of pharmacological activities, and many studies report that grapefruit juice inhibits cytochrome P450 (CYP) 3A4 leading to drug interactions. Naringenin was profiled through various in vitro studies like metabolic stability and glucuronidation in rat and human liver microsomes while, CYP inhibition using human liver microsomes. In addition, pharmacokinetic profiling was conducted upon intravenous (i.v.) and oral administration in rats. Naringenin undergoes both phase I and phase II metabolism in rat liver microsomes, and in human liver microsomes, it is predominantly metabolized by phase II. Glucuronidation which is addition (conjugation) of glucuronic acid to various functional groups is one of the major metabolic pathways of Naringenin. Naringenin, at 1.0 μM and 10.0 μM, did not elicit any appreciable inhibition of the 5 major CYP isoforms (CYP1A2, CYP3A4, CYP2C9, CYP2C19, and CYP2D6). Oral pharmacokinetic studies at 100, 300,and 1000 mg/kg dose and intravenous pharmacokinetic studies at 1 mg/kg dose were performed in male SD rats. Naringenin exhibited very short half-life (0.27 h) and rapid elimination (Clearance=110.65 mL/min/kg) after i.v. administration. There was saturation in Cmax and exposure beyond 100 mg/kg, and the absolute bioavailability was found to be ≤ 5% at the tested oral doses. This present experiment suggests that naringenin does not substantially inhibit CYP3A4 (or any of the tested five isoforms) isoforms per se. Given the minimal involvement of CYP enzymes in the metabolism of naringenin and minimal inhibition of CYP enzymes (IC50> 10 μM), the potential for drug-drug interactions involving CYP substrates and inhibitors is very minimal in humans.

NOX4 as a critical effector mediating neuroinflammatory cytokines, myeloperoxidase and osteopontin, specifically in astrocytes in the hippocampus in Parkinson's disease

Oxidative stress and mitochondrial dysfunction have been believed to play an important role in the pathogenesis of aging and neurodegenerative diseases, including Parkinson's disease (PD). The excess of reactive oxygen species (ROS) increases with age and causes a redox imbalance, which contributes to the neurotoxicity of PD. Accumulating evidence suggests that NADPH oxidase (NOX)-derived ROS, especially NOX4, belong to the NOX family and is one of the major isoforms expressed in the central nervous system (CNS), associated with the progression of PD. We have previously shown that NOX4 activation regulates ferroptosis via astrocytic mitochondrial dysfunction. We have previously shown that activation of NOX4 regulates ferroptosis through mitochondrial dysfunction in astrocytes. However, it remains unclear why an increase in NOX4 in neurodegenerative diseases leads to astrocyte cell death by certain mediators. Therefore, this study was designed to evaluate how NOX4 in the hippocampus is involved in PD by comparing an MPTP-induced PD mouse model compared to human PD patients. We could detect that the hippocampus was dominantly associated with elevated levels of NOX4 and α-synuclein during PD and the neuroinflammatory cytokines, myeloperoxidase (MPO) and osteopontin (OPN), were upregulated particularly in astrocytes. Intriguingly, NOX4 suggested a direct intercorrelation with MPO and OPN in the hippocampus. Upregulation of MPO and OPN induces mitochondrial dysfunction by suppressing five protein complexes in the mitochondrial electron transport system (ETC) and increases the level of 4-HNE leading to ferroptosis in human astrocytes. Overall, our findings indicate that the elevation of NOX4 cooperated with the MPO and OPN inflammatory cytokines through mitochondrial aberration in hippocampal astrocytes during PD.

VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals.

Vascular endothelial growth factor A (VEGF-A) plays pivotal roles in regulating tumor angiogenesis as well as physiological vascular function. The major VEGF-A isoforms, VEGF-A121 and VEGF-A165, in serum, plasma, and platelets have not been exactly evaluated due to the lack of the appropriate assay system. Antibodies against human VEGF-A121 and VEGF-A165 (hVEGF-A121 and hVEGF-A165) were successfully produced and Enzyme-Linked ImmunoSorbent Assay (ELISA) for hVEGF-A121 and hVEGF-A165 were separately created by these monoclonal antibodies. The measurement of recombinant hVEGF-A121 and hVEGF-A165 by the created ELISA showed no cross-reaction between hVEGF-A121 and hVEGF-A165 in conditioned media from HEK293 cells transfected with either hVEGF-A121 or hVEGF-A165 expression vector. The levels of VEGF-A121 and VEGF-A165 in serum, plasma, and platelets from 59 healthy volunteers proved that VEGF-A121 level was higher than VEGF-A165 in both plasma and serum in all the cases. VEGF-A121 or VEGF-A165 in serum represented higher level than that in plasma. In contrast, the level of VEGF-A165 was higher than VEGF-A121 in platelets. The newly developed ELISAs for hVEGF-A121 and hVEGF-A165 revealed different ratios of VEGF isoforms in serum, plasma, and platelets. Measuring these isoforms in combination provides useful information as biomarkers for diseases involving VEGF-A121 and VEGF-A165.

Abstract 2559: Loss of AKT2 correlates with activation of β-catenin signaling in immortalized mouse hepatocytes

Abstract Background: Primary liver cancer is the second most common cause of cancer-related mortality. Abnormal activation of the phosphatidylinositol 3-kinase (PI3K)/AKT and Wnt/β-catenin signaling pathways, which act as key regulators in cell proliferation and growth, is frequently observed in liver cancer. This study investigated the role of AKT2, the major AKT isoform expressed in the liver, on the activation of β-catenin signaling in immortalized mouse hepatocytes. Methods: Regulation of β-catenin signaling by AKT2 was examined in hepatocytes lacking the negative regulator of PI3K/AKT signaling, PTEN (phosphatase and tensin homolog deleted on chromosome 10). Immortalized hepatocytes were established from mice with liver specific deletions of Pten alone (PtenloxP/loxP; albumin [Alb]-Cre+) or in combination with Akt2 (PtenloxP/loxP; Akt2-/-; Alb-Cre+). Control mice were PtenloxP/loxP; Alb-Cre-. Immunoblotting was used to assess protein expression of AKT and β-catenin. Gene expression of β-catenin targets cyclin D1, CD44, and Jag1 was determined with quantitative PCR. Cell growth was evaluated with hemacytometer count. Results: Pharmacological inhibition of PI3K using LY294002 resulted in decreased phosphorylation levels of AKT (Ser473 and Thr308) and β-catenin (Ser552), suggesting crosstalk between the PI3K/AKT and β-catenin pathways. When compared to wild-type hepatocytes, Pten null and Pten;Akt2 double mutant (DM) expressed significantly higher protein levels of β-catenin (p≤0.0327), with no difference observed in Pten null vs. Pten;Akt2 double mutant groups (p>0.05). Phosphorylation of β-catenin at Ser552, a residue that has been previously shown to be phosphorylated by AKT, was also significantly enhanced in both Pten null and Pten;Akt2 double mutant groups, when compared to wild-type cells. Enhanced phosphorylation of β-catenin in these groups correlated with increased expression of β-catenin transcriptional targets CD44 and Jag1 (p≤0.0041), with no significant difference in mRNA levels of these targets observed in Pten null vs. DM group (p>0.05). Activation of the β-catenin signaling pathway in Pten null and DM groups correlated with enhanced cell growth and significantly increased mRNA levels of cyclin D1 (p<0.0001). Interestingly, when compared to the control group, DM group showed enhanced expression of Akt1 and total phosphorylated AKT. Conclusions: Pten loss alone or accompanied with Akt2 deletion resulted in enhanced activation of β-catenin signaling and cell proliferation. These changes correlated with increased expression of AKT1 and total phosphorylated AKT in the Pten;Akt2 DM group. These findings suggest the need to further evaluate the role of AKT isoforms in the PI3K/AKT/β-catenin signaling axis and a promise for evaluation of isoform-specific inhibitors targeted at AKT to block tumor growth. Citation Format: Ielyzaveta Slarve, Bangyan Stiles, Lina He. Loss of AKT2 correlates with activation of β-catenin signaling in immortalized mouse hepatocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2559.

Revisiting and corrections to the annotated SRSF3 (SRp20) gene structure and RefSeq sequences from the human and mouse genomes.

SRSF3 (SRp20) is the smallest member of the serine/arginine (SR)-rich protein family. We found the annotated human SRSF3 and mouse Srsf3 RefSeq sequences are much larger than the detected SRSF3/Srsf3 RNA size by Northern blot. Mapping of RNA-seq reads from various human and mouse cell lines to the annotated SRSF3/Srsf3 gene illustrated only a partial coverage of its terminal exon 7. By 5' RACE and 3' RACE, we determined that SRSF3 gene spanning over 8422 bases and Srsf3 gene spanning over 9423 bases. SRSF3/Srsf3 gene has seven exons with exon 7 bearing two alternative polyadenylation signals (PAS). Through alternative PAS selection and exon 4 exclusion/inclusion by alternative RNA splicing, SRSF3/Srsf3 gene expresses four RNA isoforms. The major SRSF3 mRNA isoform with exon 4 exclusion by using a favorable distal PAS to encode a full-length protein is 1411nt long (not annotated 4228nt) and the same major mouse Srsf3 mRNA isoform is only 1295nt (not annotated 2585nt). The difference from the redefined RNA size of SRSF3/Srsf3 to the corresponding RefSeq sequence is at the 3' UTR region. Collectively, the redefined SRSF3/Srsf3 gene structure and expression will allow better understanding of SRSF3 functions and its regulations in health and diseases.

Primer Raynaud fenomeni olgularında ATP-bağımlı potasyum kanal proteinlerini kodlayan genlerde rastlanan varyasyonların belirlenmesi

Purpose: Primary Raynaud’s phenomenon (PRP) is a vascular disorder characterized by recurrent vasospastic response of the fingers and toes to cold or stress. ATP-sensitive potassium (KATP) channels are widely distributed in vasculatures, and play an important role in the vascular tone regulation. The major vascular isoform of KATP channels is composed of Kir6.1/SUR2 (KCNJ8/ABCC9). It would be important to determine whether variations of KATP genes related to PRP is thought to be associated with vasospasm. It is believed that the studies describing mechanisms involved in the pathogenesis of inherited vascular disorders offers the best opportunity for investigation of the early stages of pathogenicity and diagnosis of PRP and associated other diseases. In this study we aim to investigate possible association between genetic variations observed in KATP channel coding genes and vasospasm associated with PRP.
 Materials and Methods: In our study; the cases with PRP, the relation between the variation in the KCNJ8/ABCC9 genes (S422L/V734I or rs72554071/rs61688134) was examined. 50 subjects who were diagnosed with PRP (patient group) and 50 healthy subjects (control group) were included in the study. Variations were determined using the Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction (T-ARMS PCR) method. 
 Results: Of the individuals in the patient and control group included in the study, 21 were male and 29 were female. The mean age of the patients was 25.7±3.36 years, and the mean age of the control group was 25.9±3.44 years. No significant relationship was found between PRP disease and genotype and allele distribution of KCNJ8/ABCC9 genes.
 Conclusion: This study presented the first findings about KCNJ8/ABCC9 gene variations in the Turkish population and may lead to future studies. Studies involving a higher number of cases and more mutations will be able to show whether there is a relationship between KATP channels and PRP and contribute to the elucidation of PRP pathogenesis in terms of genetic factors.