The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival

Cardiac contractility is regulated through the activity of various key Ca²⁺-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca²⁺ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca²⁺ by SR membranes during relaxation. Recently, the antiapoptotic HS-1–associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575–594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca²⁺ levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca²⁺ stores.     

Small leucine rich proteoglycan family regulates multiple signalling pathways in neural development and maintenance

The small leucine-rich repeat proteoglycan (SLRPs) family of proteins currently consists of five classes, based on their structural composition and chromosomal location. As biologically active components of the extracellular matrix (ECM), SLRPs were known to bind to various collagens, having a role in regulating fibril assembly, organization and degradation. More recently, as a function of their diverse proteins cores and glycosaminoglycan side chains, SLRPs have been shown to be able to bind various cell surface receptors, growth factors, cytokines and other ECM components resulting in the ability to influence various cellular functions. Their involvement in several signaling pathways such as Wnt, transforming growth factor-β and epidermal growth factor receptor also highlights their role as matricellular proteins. SLRP family members are expressed during neural development and in adult neural tissues, including ocular tissues. This review focuses on describing SLRP family members involvement in neural development with a brief summary of their role in non-neural ocular tissues and in response to neural injury.     

Mutational analysis and modeling reveal functionally critical residues in transmembrane segments 1 and 3 of the UapA transporter

Earlier, we identified mutations in the first transmembrane segment (TMS1) of UapA, a uric acid-xanthine transporter in Aspergillus nidulans, that affect its turnover and subcellular localization. Here, we use one of these mutations (H86D) and a novel mutation (I74D) as well as genetic suppressors of them, to show that TMS1 is a key domain for proper folding, trafficking and turnover. Kinetic analysis of mutants further revealed that partial misfolding and deficient trafficking of UapA does not affect its affinity for xanthine transport, but reduces that of uric acid and confers a degree of promiscuity towards the binding of other purines. This result strengthens the idea that subtle interactions among domains not directly involved in substrate binding refine the selectivity of UapA. Characterization of second-site suppressors of H86D revealed a genetic interaction of TMS1 with TMS3, the latter segment shown for the first time to be important for UapA function. Systematic mutational analysis of polar and conserved residues in TMS3 showed that Ser154 is crucial for UapA transport activity. Our results are in agreement with a topological model of UapA built on the recently published structure of UraA, a bacterial homolog of UapA.     

Expression and promoter methylation status of hMLH1, MGMT,APC, and CDH1 genes in patients with colon adenocarcinoma

Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. CRC development is the result of genetic and epigenetic alterations accumulation in the epithelial cells of colon mucosa. In the present study, DNA methylation, an epigenetic event, was evaluated in tumoral and matching normal epithelium in a cohort of 61 CRC patients. The results confirmed and expanded knowledge for the tumor suppressor genes hMLH1, MGMT, APC, and CDH1. Promoter methylation was observed for all the examined genes in different percentage. A total of 71% and 10% of the examined cases were found to be methylated in two or more and in all genes, respectively. mRNA and protein levels were also evaluated. Promoter methylation of hMLH1, MGMT, APC, and CDH1 genes was present at the early stages of tumor’s formation and it could also be detected in the normal mucosa. Correlations of the methylated genes with patient’s age and tumor’s clinicopathological characteristics were also observed. Our findings suggest that DNA methylation is a useful marker for tumor progression monitoring and that promoter methylation in certain genes is associated with more advanced tumor stage, poor differentiation, and metastasis.     

Non-β-cell progenitors of β-cells in pregnant mice

Pregnancy is a normal physiological condition in which the maternal β-cell mass increases rapidly about two-fold to adapt to new metabolic challenges. We have used a lineage tracing of β-cells to analyse the origin of new β-cells during this rapid expansion in pregnancy. Double transgenic mice bearing a tamoxifen-dependent Cre-recombinase construct under the control of a rat insulin promoter, together with a reporter Z/AP gene, were generated. Then, in response to a pulse of tamoxifen before pregnancy, β-cells in these animals were marked irreversibly and heritably with the human placental alkaline phosphatase (HP AP). First, we conclude that the lineage tracing system was highly specific for β-cells. Secondly, we scored the proportion of the β-cells marked with HP AP during a subsequent chase period in pregnant and non-pregnant females. We observed a dilution in this labeling index in pregnant animal pancreata, compared to nonpregnant controls, during a single pregnancy in the chase period. To extend these observations we also analysed the labeling index in pancreata of animals during the second of two pregnancies in the chase period. The combined data revealed statistically-significant dilution during pregnancy, indicating a contribution to new beta cells from a non-β-cell source. Thus for the first time in a normal physiological condition, we have demonstrated not only β-cell duplication, but also the activation of a non-β-cell progenitor population. Further, there was no transdifferentiation of β-cells to other cell types in a two and half month period following labeling, including the period of pregnancy.     

Remote Ischemic Preconditioning (RIPC) Modifies the Plasma Proteome in Children Undergoing Repair of Tetralogy of Fallot: A Randomized Controlled Trial

Background

Remote ischemic preconditioning (RIPC) has been applied in paediatric cardiac surgery. We have demonstrated that RIPC induces a proteomic response in plasma of healthy volunteers. We tested the hypothesis that RIPC modifies the proteomic response in children undergoing Tetralogy of Fallot (TOF) repair.

Methods and Results

Children (n=40) were randomized to RIPC and control groups. Blood was sampled at baseline, after cardiopulmonary bypass (CPB) and 6, 12 and 24h post-CPB. Plasma was analysed by liquid chromatography mass spectrometry (LC-MS) in an untargeted approach. Peptides demonstrating differential expression (p<0.01) were subjected to tandem LC-MS/MS and protein identification. Corresponding proteins were identified using the NCBI protein database. There was no difference in age (7.3±3.5vs6.8±3.6 months)(p=0.89), weight (7.7±1.8vs7.5±1.9 kg)(p=0.71), CPB time (104±7vs94±7 min)(p=0.98) or aortic cross-clamp time (83±22vs75±20 min)(p=0.36). No peptides were differentially expressed at baseline or immediately after CPB. There were 48 peptides with higher expression in the RIPC group 6h post-CPB. This was no longer evident at 12 or 24h, with one peptide down-regulated in the RIPC group. The proteins identified were: inter-alpha globulin inhibitor (42.0±11.8 vs 820.8±181.1, p=0.006), fibrinogen preproprotein (59.3±11.2 vs 1192.6±278.3, p=0.007), complement-C3 precursor (391.2±160.9 vs 5385.1±689.4, p=0.0005), complement C4B (151.5±17.8 vs 4587.8±799.2, p=0.003), apolipoprotein B100 (53.4±8.3 vs 1364.5±278.2, p=0.005) and urinary proteinase inhibitor (358.6±74.9 vs 5758.1±1343.1, p=0.009). These proteins are involved in metabolism, haemostasis, immunity and inflammation.

Conclusions

We provided the first comprehensive analysis of RIPC-induced proteomic changes in children undergoing surgery. The proteomic changes peak 6h post-CPB and return to baseline within 24h of surgery.

Trial Registration

ACTR.org.au ACTRN12610000496011     

Transforming growth factor-β modulates pancreatic cancer associated fibroblasts cell shape,stiffness and invasion

Background

Tumor microenvironment consists of the extracellular matrix (ECM), stromal cells, such as fibroblasts (FBs) and cancer associated fibroblasts (CAFs), and a myriad of soluble factors. In many tumor types, including pancreatic tumors, the interplay between stromal cells and the other tumor microenvironment components leads to desmoplasia, a cancer-specific type of fibrosis that hinders treatment. Transforming growth factor beta (TGF-β) and CAFs are thought to play a crucial role in this tumor desmoplastic reaction, although the involved mechanisms are unknown.

Study of changes in physicochemical and microbiological characteristics of shrimps (Melicertus kerathurus) stored under modified atmosphere packaging

Fresh minimally processed shrimps were stored under modified atmosphere packaging (60% CO₂:40% N₂ for MAP A and 92.9% N₂:5.1% CO₂:2% O₂ for MAP B) for 5 days at 3 °C. Total mesophiles, H₂S forming bacteria, Pseudomonas spp., Brochothrix thermosphacta, firmness, color and sensory parameters were investigated throughout the whole time of the experiment. During storage period samples stored under MAP B managed to retain firmness values close to the initial values. All microbial populations growth was suppressed by the presence of MAP A. Samples stored under MAP B managed to maintain their firmness values close to the initial ones while MAP A samples were significantly less firm (p < 0.05).     

A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability

Background

The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34⁺ cord blood using non-viral, non-integrating methods.

Methodology/Principal Findings

We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34⁺ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64–89%) of cardiac troponin I⁺ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs.

Conclusion/Significance

This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.     

Assembly of an APC-Cdh1-substrate complex is stimulated by engagement of a destruction box

The anaphase-promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators. Cdh1p is an APC coactivator that directly binds APC substrates. A genetic screen in budding yeast identified residues within Cdh1p critical for its function. Cdh1p proteins containing mutations within the "C box" or the "IR" motif could bind substrate, but not the APC, whereas mutants that only bound the APC were not identified, suggesting an ordered assembly of the ternary APC-Cdh1p-substrate complex. Supporting this hypothesis, we found that substrate binding to wild-type Cdh1p enhanced its association with the APC in yeast cells. We used peptide competition assays to demonstrate that Cdh1p interacts directly with the D box and the KEN box, two motifs within APC substrates known to be required for APC-mediated degradation. Moreover, an intact D box domain within a substrate was required to stimulate the association between the Cdh1p-substrate complex and the APC.