Luminal Face Research Articles

The gas bladder of Heterotis niloticus (Cuvier, 1829).

This work reports on the structural characteristics of the respiratory gas bladder of the osteoglossiform fish Heterotis niloticus. The bladder-vertebrae relationships are also analyzed. A slit-shaped orifice in the mediodorsal pharyngeal wall is surrounded by a muscle sphincter and serves as a glottis-like opening to the gas bladder. The dorsolateral internal surface of the gas bladder is lined by a parenchyma of highly vascularized trabeculae and septa displaying an alveolar-like structure. The trabeculae contain, in addition to vessels, numerous eosinophils probably involved in immune responses. The air spaces are endowed with a thin exchange barrier indicating a good potential for respiratory gas exchange. The ventral wall of the gas bladder is a well-vascularized membrane that exhibits an exchange barrier in the luminal face and an inner structure dominated by the presence of a layer of richly innervated smooth muscle. This is suggestive of an autonomous adjustability of the gas bladder ventral wall. The trunk vertebrae show large transverse processes (parapophyses) and numerous surface openings that lead into intravertebral spaces that become invaded by the bladder parenchyma. Curiously, the caudal vertebrae show a regular teleost morphology with neural and hemal arches, but have similar surface openings and intravertebral pneumatic spaces. The African Arowana hence rivals the freshwater butterfly fish Pantodon in its exceptional role of displaying postcranial skeletal pneumaticity outside of Archosauria. The possible significance of these findings is discussed.

Reduction in ABCG2 mRNA Expression in Human Immortalised Brain Microvascular Endothelial Cells by Ferric Ammonium Citrate is Mediated by Reactive Oxygen Species and Activation of ERK1/2 Signalling.

The ATP-binding cassette (ABC) transport protein ABCG2 (also known as breast cancer resistance protein (BCRP)) is expressed at the luminal face of the blood-brain barrier (BBB), where it limits the brain uptake of a number of therapeutic drugs. We recently reported that the ABC efflux transporter P-glycoprotein (P-gp) was downregulated in human immortalised brain endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC). The aim of the present study, therefore, was to assess whether BCRP expression is also affected by FAC and identify any signalling mechanisms involved. ABCG2 mRNA was assessed by RT-qPCR. Protein levels of BCRP, phosphorylated extracellular-regulated kinases 1 and 2 (p-ERK1/2) and total ERK 1/2 were assessed by Western blot. Reactive oxygen species (ROS) levels were determined using 2',7'-dichlorofluorescin diacetate. Treatment of hCMEC/D3 cells with FAC (250 µM, 72 h) significantly reduced ABCG2 mRNA levels (32.2 ± 3.7%) without a concomitant reduction in BCRP protein expression. ABCG2 mRNA levels were restored to control levels when co-treated with the antioxidant N-acetylcysteine (NAC), suggesting the effect of FAC was mediated by a ROS-sensitive pathway. We also found that FAC-treatment was associated with increased levels of p-ERK1/2, suggesting involvement of the ERK1/2 signalling pathway in the observed ABCG2 mRNA downregulation. The ERK1/2 signalling pathway inhibitor U0126 restored p-ERK1/2 levels and partially attenuated the FAC-induced reduction in ABCG2 mRNA. This study suggests that FAC-induced downregulation of ABCG2 mRNA isdriven byROS and ERK1/2 signalling, mechanisms which may be exploited to modulate BCRP expression at the BBB.

Glycosylphosphatidylinositol Anchor Biosynthesis Pathway-Related Protein GPI7 Is Required for the Vegetative Growth and Pathogenicity of Colletotrichum graminicola

Glycosylphosphatidylinositol (GPI) anchoring is a common post-translational modification in eukaryotic cells and has been demonstrated to have a wide range of biological functions, such as signal transduction, cellular adhesion, protein transport, immune response, and maintaining cell wall integrity. More than 25 proteins have been proven to participate in the GPI anchor synthesis pathway which occurs in the cytoplasmic and the luminal face of the ER membrane. However, the essential proteins of the GPI anchor synthesis pathway are still less characterized in maize pathogen Colletotrichum graminicola. In the present study, we analyzed the biological function of the GPI anchor synthesis pathway-related gene, CgGPI7, that encodes an ethanolamine phosphate transferase, which is localized in ER. The vegetative growth and conidia development of the ΔCgGPI7 mutant was significantly impaired in C. graminicola. and qRT-PCR results showed that the transcriptional level of CgGPI7 was specifically induced in the initial infection stage and that the pathogenicity of ΔCgGPI7 mutant was also significantly decreased compared with the wild type. Furthermore, the ΔCgGPI7 mutant displayed more sensitivity to cell wall stresses, suggesting that CgGPI7 may play a role in the cell wall integrity of C. graminicola. Cell wall synthesis-associated genes were also quantified in the ΔCgGPI7 mutant, and the results showed that chitin and β-1,3-glucans synthesis genes were significantly up-regulated in ΔCgGPI7 mutants. Our results suggested that CgGPI7 is required for vegetative growth and pathogenicity and might depend on the cell wall integrity of C. graminicola.

Morphological and histochemical features of the digestive tract of Leiarius marmoratus (Gill, 1870).

Leiarius marmoratus, a freshwater catfish from Pimelodidae family, shows great biological and commercial relevance because of its geographic distribution and adaptation to fish-farm. The knowledge of the morphological characteristics of the digestive tract is fundamental to the understanding of fish physiology and nutrition, which helps in the planning of diets to provide better management and success in fish farming. Thus, this work described the morphology and histochemistry of the digestive tract of L. marmoratus adults. After euthanasia, the animals were dissected for analysis of the digestive tract. The oesophagus is a short and distensive organ with longitudinal folds that allow the passage of large food, e.g., other fishes. Oesophageal mucosa layer shows a stratified epithelium with goblet cells and club cells. The secretion of goblet cells is composed of neutral and acidic mucins that are anchored in the epithelium luminal face by epithelial cells fingerprint-like microridges, lubricating the surface to facilitate the food sliding. Club cells have protein secretion that can be involved in alarm signals when epithelium is damaged and in immunological defence. The saccular stomach is highly distensible to store large food. Gastric mucosa layer is composed of epithelial cells with intense secretion of neutral mucin to protect against self-digestion of gastric juice. Cardiac and fundic regions of stomach show well-developed gastric glands composed of oxynticopeptic cells. These cells have numerous mitochondria, highlighting their intense activity in the synthesis of acid and enzymes. The intestine is divided into three regions: anterior, middle and posterior. Although it is a short tube, intestine shows longitudinal folds and microvilli of enterocytes to increase the contact surface. These folds are higher in the anterior region of the intestine, highlighting their function in digestion and absorption. Intestinal goblet cells have acidic and neutral mucins that lubricate the epithelium and aid in digestive processes. These cells increase in number towards aboral, and they are related to the protection and lubrication to expulsion of faecal bolus.

Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody.

The role of glutamate in excitatory neurotransmission depends on its transport into synaptic vesicles by the vesicular glutamate transporters (VGLUTs). The three VGLUT isoforms exhibit a complementary distribution in the nervous system, and the knockout of each produces severe, pleiotropic neurological effects. However, the available pharmacology lacks sensitivity and specificity, limiting the analysis of both transport mechanism and physiological role. To develop new molecular probes for the VGLUTs, we raised six mouse monoclonal antibodies to VGLUT2. All six bind to a structured region of VGLUT2, five to the luminal face, and one to the cytosolic. Two are specific to VGLUT2, whereas the other four bind to both VGLUT1 and 2; none detect VGLUT3. Antibody 8E11 recognizes an epitope spanning the three extracellular loops in the C-domain that explains the recognition of both VGLUT1 and 2 but not VGLUT3. 8E11 also inhibits both glutamate transport and the VGLUT-associated chloride conductance. Since the antibody binds outside the substrate recognition site, it acts allosterically to inhibit function, presumably by restricting conformational changes. The isoform specificity also shows that allosteric inhibition provides a mechanism to distinguish between closely related transporters.

A quinoxaline-based charge-transfer compound for efficient deep-red organic light emitting diodes

Deep-red (DR) emitting materials with narrow band gap have important applications in optoelectronic field. However, limited by energy gap rules, the development of DR emitting materials with high luminous efficiency face great challenges. Herein, we present a new Quinoxaline-based compound 6,7-bis(4-(diphenylamino)phenyl)quinoxaline-2,3-dicarbonitrile (o-QxDC-DTPA) with D-A-D structure for deep-red organic light emitting diodes. Such a material features distinctive DR emitting property with strong charge transfer character. Highly efficient devices were achieved based on o-QxDC-DTPA with different doped concentrations. The doped device exhibited excellent electroluminescence efficiency, with a maximum EQE of 5.21% and DR emission with the emission peak at 680 nm.

Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes.

APOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs. Immunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies. Both endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants. APOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.

Microdomains form on the luminal face of neuronal extracellular vesicle membranes

Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been implicated in several pathologies including those of the central nervous system. They are released by all cell types, including neurons, and are highly heterogenous in size and composition. Yet much remains unknown regarding the biophysical characteristics of different EVs. Here, using cryo-electron microscopy (cryoEM), we analyzed the size distribution and morphology of EVs released from primary cortical neurons. We discovered massive macromolecular clusters on the luminal face of EV membranes. These clusters are predominantly found on medium-sized vesicles, suggesting that they may be specific to microvesicles as opposed to exosomes. We propose that these clusters serve as microdomains for EV signaling and play an important role in EV physiology.

Luminal addition of non-permeant Eu3+ interferes with luminal Ca2+ regulation of the cardiac ryanodine receptor

Dysregulation of the cardiac ryanodine receptor (RYR2) by luminal Ca2+ has been implicated in a life-threatening, stress-induced arrhythmogenic disease. The mechanism of luminal Ca2+-mediated RYR2 regulation is under debate, and it has been attributed to Ca2+ binding on the cytosolic face (the Ca2+ feedthrough mechanism) and/or the luminal face of the RYR2 channel (the true luminal mechanism). The molecular nature and location of the luminal Ca2+ site is unclear. At the single-channel level, we directly probed the RYR2 luminal face by Eu3+, considering the non-permeant nature of trivalent cations and their high binding affinities for Ca2+ sites. Without affecting essential determinants of the Ca2+ feedthrough mechanism, we found that luminal Eu3+ competitively antagonized the activation effect of luminal Ca2+ on RYR2 responsiveness to cytosolic caffeine, and no appreciable effect was observed for luminal Ba2+ (mimicking the absence of luminal Ca2+). Importantly, luminal Eu3+ caused no changes in RYR2 gating. Our results indicate that two distinct Ca2+ sites (available for luminal Ca2+ even when the channel is closed) are likely involved in the true luminal mechanism. One site facing the lumen regulates channel responsiveness to caffeine, while the other site, presumably positioned in the channel pore, governs the gating behavior.

RETRACTED ARTICLE:Deficiency in intestinal epithelial Reg4 ameliorates intestinal inflammation and alters the colonic bacterial composition

The regenerating islet-derived family member 4 (Reg4) in the gastrointestinal tract is up-regulated during intestinal inflammation. However, the physiological function of Reg4 in the inflammation is largely unknown. In the current study, the functional roles and involved mechanisms of intestinal epithelial Reg4 in intestinal inflammation were studied in healthy and inflamed states using human intestinal specimens, an intestinal conditional Reg4 knockout mouse (Reg4ΔIEC) model and dextran sulfate sodium (DSS)-induced colitis model. We showed that the elevated serum Reg4 in pediatric intestinal failure (IF) patients were positively correlated with the serum concentrations of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In inflamed intestine of IF patients, the crypt base Reg4 protein was increased and highly expressed towards the luminal face. The Reg4 was indicated as a novel target of activating transcription factor 2 (ATF2) that enhanced Reg4 expression during the intestinal inflammation. In vivo, the DSS-induced colitis was significantly ameliorated in Reg4ΔIEC mice. Reg4ΔIEC mice altered the colonic bacterial composition and reduced the bacteria adhere to the colonic epithelium. In vitro, Reg4 was showed to promote the growth of colonic organoids, and that this occurs through a mechanism involving activation of signal transducer and activator of transcription 3 (STAT3). In conclusion, our findings demonstrated intestinal-epithelial Reg4 deficiency protects against experimental colitis and mucosal injury via a mechanism involving alteration of bacterial homeostasis and STAT3 activation.

Podocalyxin is required for maintaining blood–brain barrier function during acute inflammation

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele of Podxl in vascular endothelial cells (vECs) using Tie2Cre mice (PodxlΔTie2Cre). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood-brain barrier (BBB) in PodxlΔTie2Cre mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH2) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH2, LPS-primed PodxlΔTie2Cre mice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

The orientation of a decellularized uterine scaffold determines the tissue topology and architecture of the regenerated uterus in rats†.

A decellularized uterine scaffold (DUS) prepared from rats permits recellularization and regeneration of uterine tissues when placed onto a partially excised uterus and supports pregnancy in a fashion comparable to the intact uterus. The underlying extracellular matrix (ECM) together with an acellular, perfusable vascular architecture preserved in DUS is thought to be responsible for appropriate regeneration of the uterus. To investigate this concept, we examined the effect of the orientation of the DUS-preserving ECM and the vascular architecture on uterine regeneration through placement of a DUS onto a partially defective uterine area in the reversed orientation such that the luminal face of the DUS was outside and the serosal face was inside. We characterized the tissue structure and function of the regenerated uterus, comparing the outcome to that when the DUS was placed in the correct orientation. Histological analysis revealed that aberrant structures including ectopic location of glands and an abnormal lining of smooth muscle layers were observed significantly more frequently in the reversed group than in the correct group (70% vs. 30%, P<0.05). Despite the changes in tissue topology, the uteri regenerated with an incorrectly oriented DUS could achieve pregnancy in a way similar to uteri regenerated with a correctly oriented DUS. These results suggest that DUS-driven ECM orientation determines the regenerated uterus structure. Using DUS in the correct orientation is preferable when clinically applied. The disoriented DUS may deteriorate the tissue topology leading to structural disease of the uterus even though the fertility potential is not immediately affected.

Magnetic targeting of smooth muscle cells in vitro using a magnetic bacterial cellulose to improve cell retention in tissue-engineering vascular grafts

Tissue-engineered vascular grafts (TEVG) use biologically-active cells with or without supporting scaffolds to achieve tissue remodeling and regrowth of injured blood vessels. However, this process may take several weeks because the high hemodynamic shear stress at the damaged site causes cellular denudation and impairs tissue regrowth. We hypothesize that a material with magnetic properties can provide the force required to speed up re-endothelization at the vascular defect by facilitating high cell density coverage, especially during the first 24 h after implantation. To test our hypothesis, we designed a magnetic bacterial cellulose (MBC) to locally target cells in vitro under a pulsatile fluid flow (0.514 dynes cm−2). This strategy can potentially increase cell homing at TEVG, without the need of blood cessation. The MBC was synthesized by an in situ precipitation method of Fe3+ and Fe2+ iron salts into bacterial cellulose (BC) pellicles to form Fe3O4 nanoparticles along the BC’s fibrils, followed by the application of dextran coating to protect the embedded nanoparticles from oxidation. The iron salt concentration used in the synthesis of the MBC was tuned to balance the magnetic properties and cytocompatibility of the magnetic hydrogel. Our results showed a satisfactory MBC magnetization of up to 10 emu/g, which is above the value considered relevant for tissue engineering applications (0.05 emu/g). The MBC captured magnetically-functionalized cells under dynamic flow conditions in vitro. MBC magnetic properties and cytocompatibility indicated a dependence on the initial iron oxide nanoparticle concentration. Statement of SignificanceMagnetic hydrogels represent a new class of functional materials with great potential in TVEG because they offer a platform to (1) release drugs on demand, (2) speed up tissue regrowth, and (3) provide mechanical cues to cells by its deformability capabilities. Here, we showed that a magnetic hydrogel, the MBC, was able to capture and retain magnetically-functionalized smooth muscle cells under pulsatile flow conditions in vitro. A magnetic hydrogel with this feature can be used to obtain high-density cell coverage on sites that are aggressive for cell survival such as the luminal face of vascular grafts, whereas simultaneously can support the formation of a biologically-active cell layer that protects the material from restenosis and inflammation.

New Tetrahydroisoquinoline Derivatives Overcome Pgp Activity in Brain-Blood Barrier and Glioblastoma Multiforme in Vitro.

P-glycoprotein (Pgp) determines resistance to a broad spectrum of drugs used against glioblastoma multiforme (GB). Indeed, Pgp is highly expressed in GB stem cells and in the brain-blood barrier (BBB), the peculiar endothelium surrounding the brain. Inhibiting Pgp activity in the BBB and GB is still an open challenge. Here, we tested the efficacy of a small library of tetrahydroisoquinoline derivatives with an EC50 for Pgp ≤ 50 nM, in primary human BBB cells and in patient-derived GB samples, from which we isolated differentiated/adherent cells (AC, i.e., Pgp-negative/doxorubicin-sensitive cells) and stem cells (neurospheres, NS, i.e., Pgp-positive/doxorubicin-resistant cells). Three compounds used at 1 nM increased the delivery of doxorubicin, a typical substrate of Pgp, across BBB monolayer, without altering the expression and activity of other transporters. The compounds increased the drug accumulation within NS, restoring doxorubicin-induced necrosis and apoptosis, and reducing cell viability. In co-culture systems, the compounds added to the luminal face of BBB increased the delivery of doxorubicin to NS growing under BBB and rescued the drug’s cytotoxicity. Our work identified new ligands of Pgp active at low nanomolar concentrations. These compounds reduce Pgp activity in BBB and GB and improve in vitro chemotherapy efficacy in this tumor.

In Vivo Imaging of Diacylglycerol at the Cytoplasmic Leaflet of Plant Membranes.

Diacylglycerol (DAG) is an important intermediate in lipid biosynthesis and plays key roles in cell signaling, either as a second messenger itself or as a precursor of phosphatidic acid. Methods to identify distinct DAG pools have proven difficult because biochemical fractionation affects the pools, and concentrations are limiting. Here, we validate the use of a genetically encoded DAG biosensor in living plant cells. The sensor is composed of a fusion between yellow fluorescent protein and the C1a domain of protein kinase C (YFP-C1aPKC) that specifically binds DAG, and was stably expressed in suspension-cultured tobacco BY-2 cells and whole Arabidopsis thaliana plants. Confocal imaging revealed that the majority of the YFP-C1aPKC fluorescence did not locate to membranes but was present in the cytosol and nucleus. Treatment with short-chain DAG or PMA (phorbol-12-myristate-13-acetate), a phorbol ester that binds the C1a domain of PKC, caused the recruitment of the biosensor to the plasma membrane. These results indicate that the biosensor works and that the basal DAG concentration in the cytoplasmic leaflet of membranes (i.e. accessible to the biosensor) is in general too low, and confirms that the known pools in plastids, the endoplasmic reticulum and mitochondria are located at the luminal face of these compartments (i.e. inaccessible to the biosensor). Nevertheless, detailed further analysis of different cells and tissues discovered four novel DAG pools, namely at: (i) the trans-Golgi network; (ii) the cell plate during cytokinesis; (iii) the plasma membrane of root epidermal cells in the transition zone, and (iv) the apex of growing root hairs. The results provide new insights into the spatiotemporal dynamics of DAG in plants and offer a new tool to monitor this in vivo.

The Glycosylphosphatidylinositol Anchor Biosynthesis Genes GPI12, GAA1, and GPI8 Are Essential for Cell-Wall Integrity and Pathogenicity of the Maize Anthracnose Fungus Colletotrichum graminicola.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is one of the most common posttranslational modifications of proteins in eukaryotic cells and is important for associating proteins with the cell surface. In fungi, GPI-anchored proteins play essential roles in cross-linking of β-glucan cell-wall polymers and cell-wall rigidity. GPI-anchor synthesis is successively performed at the cytoplasmic and the luminal face of the ER membrane and involves approximately 25 proteins. While mutagenesis of auxiliary genes of this pathway suggested roles of GPI-anchored proteins in hyphal growth and virulence, essential genes of this pathway have not been characterized. Taking advantage of RNA interference (RNAi) we analyzed the function of the three essential genes GPI12, GAA1 and GPI8, encoding a cytoplasmic N-acetylglucosaminylphosphatidylinositol deacetylase, a metallo-peptide-synthetase and a cystein protease, the latter two representing catalytic components of the GPI transamidase complex. RNAi strains showed drastic cell-wall defects, resulting in exploding infection cells on the plant surface and severe distortion of in planta-differentiated infection hyphae, including formation of intrahyphal hyphae. Reduction of transcript abundance of the genes analyzed resulted in nonpathogenicity. We show here for the first time that the GPI synthesis genes GPI12, GAA1, and GPI8 are indispensable for vegetative development and pathogenicity of the causal agent of maize anthracnose, Colletotrichum graminicola.

Preceding bronchial cutting for exposure of the pulmonary artery buried in scar tissue after chemoradiotherapy.

It is often difficult to expose the pulmonary artery buried in a scar tissue, especially in lung cancer patients that responded well to neoadjuvant chemoradiotherapy. Difficulty to access pulmonary artery branches may lead to potentially unnecessary pneumonectomy. To complete lobectomy in such cases, a technique with preceding bronchial cutting for exposure of the pulmonary artery is presented. After dissecting the pulmonary vein, the lobar bronchus is cut from the opposite side of the pulmonary artery with scissors. The back wall of the lobar bronchus is cut using a surgical knife from the luminal face, which can expose the pulmonary artery behind the bronchial stump and then complete lobectomy. Fourteen patients have been treated using the present technique, enabling complete resection by lobectomy (including sleeve lobectomy in 3 patients) without major bleeding. The present procedure can expose pulmonary artery buried in scar tissue, resulting in making the lobectomy safer.

Cardiac ryanodine receptor: Selectivity for alkaline earth metal cations points to the EF-hand nature of luminal binding sites

A growing body of evidence suggests that the regulation of cardiac ryanodine receptor (RYR2) by luminal Ca2+ is mediated by luminal binding sites located on the RYR2 channel itself and/or its auxiliary protein, calsequestrin. The localization and structure of RYR2-resident binding sites are not known because of the lack of a high-resolution structure of RYR2 luminal regions. To obtain the first structural insight, we probed the RYR2 luminal face stripped of calsequestrin by alkaline earth metal divalents (M2+: Mg2+, Ca2+, Sr2+ or Ba2+). We show that the RYR2 response to caffeine at the single-channel level is significantly modified by the nature of luminal M2+. Moreover, we performed competition experiments by varying the concentration of luminal M2+ (Mg2+, Sr2+ or Ba2+) from 8mM to 53mM and investigated its ability to compete with 1mM luminal Ca2+. We demonstrate that all tested M2+ bind to exactly the same RYR2 luminal binding sites. Their affinities decrease in the order: Ca2+>Sr2+>Mg2+~Ba2+, showing a strong correlation with the M2+ affinity of the EF-hand motif. This indicates that the RYR2 luminal binding regions and the EF-hand motif likely share some structural similarities because the structure ties directly to the function.

“Building an Architecture for the Wanderer”: A Conversation with Nathalie Handal

12 WLT MAY–AUGUST 2016 photo : rachel eliza griffiths Building an Architecture for the Wanderer” A Conversation with Nathalie Handal by Rachel Morgenstern-Clarren “ Q&A WORLDLITERATURETODAY.ORG 13 Rachel Morgenstern-Clarren: Your books are written in English but laced with Arabic , French, Spanish, Italian, Creole, and even Russian and Sanskrit words. Given your fluency in so many languages, why did you decide to write primarily in English ? How do you think that impacts what and how you write? Nathalie Handal: I didn’t choose it. English just came to me, and I welcomed it. It doesn’t impact what I write. My work would be about a search for connectedness no matter what language I wrote in. The more languages you know, the more unique and complex your own language becomes. RMC: What languages did you speak growing up? Did you ever consciously learn a language, or do you consider all of them to be your hyphenated “mother tongue”? NH: I’ve often said I don’t have a mother tongue. In fact, they are all hyphenated mother tongues. Together they create a symphony, a new language, and the multiplicity of that new language reflects my identity and life. RMC: Has your work been translated into other languages? Which language would you most like to be translated into in the future? NH: My work has been translated into fifteen languages. There isn’t a language that I’d most want to be translated into—they all intrigue me, and it’s a gift to expand the conversation. But I would like to be retranslated into all the languages I’ve previously been translated into. I now have a deeper understanding of the fundamental importance of translating culture alongside the words themselves. I would like to work with translators who have an understanding of the multiplicity of cultures present in my work—the nuances and localisms, rhythms and beats, histories and idiosyncrasies —in order to better translate it into whatever the equivalent would be in their own language and culture. Still, I’m aware that at certain levels translation is impossible , and equivalents don’t exist. But a proximity—textually, musically, and emotionally —would be wonderful. RMC: Did you always know that you were a poet? When did you decide to pursue poetry “professionally”? NH: Poetry is a mystical and magical experience—it doesn’t come from what we know. The only time I feel whole is while writing. Along the way, I met mentors who helped me clarify to myself that wanting to spend a life with the page wasn’t such a crazy idea—they had the same impulse. Derek Walcott told me in my early twenties , and his words resonated with my nomadic spirit, “Do whatever you have to do to write; don’t settle in a job.” Lucille Clifton made me understand the generous power of poetry. She also taught me how to put a poetry book together when I took a workshop with her in London . I still remember her luminous face as she placed my poems on the floor and asked me how they spoke to one another. Gerald Stern, at a Poetry Society of America event on black iconic poets of the twentieth century that we both participated in, said, “Clifton is our prophet.” I agree. The first poems I ever published appeared in the British magazine Ambit, with the enthusiastic support of John Burnside. It took me nearly twenty years to cross paths with John and tell him that his support meant so much to me that it nearly eliminated the bloody rejections that writers are all too familiar with. I’ve always been dedicated to my craft. Earlier on, I was only thinking about the page, not the rent, the endless bills and headaches that slowly tiptoed behind me and for which I had no solution. So if your question is, when did I connect poetry in any way with income, I would say, when I moved to the United States in my early G roundbreaking poet, playwright, and editor Nathalie Handal is one of our most diverse contemporary writers, and as the Washington Independent Review of Books writes, it’s with “startling...

High-Resolution Scanning Electron Microscopy and Immuno-Gold Labeling of the Nuclear Lamina and Nuclear Pore Complex.

Scanning electron microscopy (SEM) is a technique used to image surfaces. Field emission SEMs (feSEMs) can resolve structures that are ~0.5-1.5 nm apart. FeSEM, therefore is a useful technique for imaging molecular structures that exist at surfaces such as membranes. The nuclear envelope consists of four membrane surfaces, all of which may be accessible for imaging. Imaging of the cytoplasmic face of the outer membrane gives information about ribosomes and cytoskeletal attachments, as well as details of the cytoplasmic peripheral components of the nuclear pore complex, and is the most easily accessed surface. The nucleoplasmic face of the inner membrane is easily accessible in some cells, such as amphibian oocytes, giving valuable details about the organization of the nuclear lamina and how it interacts with the nuclear pore complexes. The luminal faces of both membranes are difficult to access, but may be exposed by various fracturing techniques. Protocols are presented here for the preparation, labeling, and feSEM imaging of Xenopus laevis oocyte nuclear envelopes.