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BBA - General Subjects (v.1810, #9)

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Rottlerin dissolves pre-formed protein amyloid: A study on hen egg white lysozyme by Nandini Sarkar; Manjeet Kumar; Vikash Kumar Dubey (pp. 809-814).

Deposition of protein fibrillar aggregates called amyloids in the tissue, is the principal cause of several degenerative diseases. Here, we have shown the disaggregation potential of rottlerin towards hen egg white lysozyme (HEWL) fibrils formed under alkaline conditions (pH-12.2).Several biophysical methods like Atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and fluorescence emission spectra were used for the study.Rottlerin exhibited instantaneous disaggregation effect on HEWL fibrils as monitored by Thioflavin T assay, anisotropy study and AFM imaging. Further we have monitored the conformational changes induced by rottlerin on the fibril in terms of surface hydrophobicity and secondary structure through 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence and FTIR study respectively. We have also attempted to elucidate the type of interaction between HEWL and rottlerin at pH-12.2 employing techniques like quenching study and FTIR.Rottlerin seems to have potential application as anti-amyloid compound.► Instantaneous disaggregation of HEWL amyloid by rottlerin. ► Disaggregation is followed by reduction in surface hydrophobicity. ► Addition of rottlerin to HEWL fibril reduces the β sheet content of HEWL. ► The interaction between HEWL and rottlerin is mainly governed by hydrogen bonding.

Keywords: Abbreviations; HEWL; Hen Egg White Lysozyme; ThT; Thioflavin T; DNSCl; Dansyl ChlorideAmyloid; Anisotropy; Thioflavin T; Fourier transform infrared spectroscopy

Mannose 6-phosphate receptor homology (MRH) domain-containing lectins in the secretory pathway by Alicia C. Castonguay; Linda J. Olson; Nancy M. Dahms (pp. 815-826).

The mannose 6-phosphate receptor homology (MRH) domain-containing family of proteins, which include recycling receptors (mannose 6-phosphate receptors, MPRs), resident endoplasmic reticulum (ER) proteins (glucosidase II β-subunit, XTP3-B, OS-9), and a Golgi glycosyltransferase (GlcNAc-phosphotransferase γ-subunit), are characterized by the presence of one or more MRH domains. Many MRH domains act as lectins and bind specific phosphorylated (MPRs) or non-phosphorylated (glucosidase II β-subunit, XTP3-B and OS-9) high mannose-type N-glycans. The MPRs are the only proteins known to bind mannose 6-phosphate (Man-6-P) residues via their MRH domains.Recent biochemical and structural studies that have provided valuable insight into the glycan specificity and mechanisms of carbohydrate recognition by this diverse group of MRH domain-containing proteins are highlighted.Currently, three-dimensional structures are known for ten MRH domains, revealing the conservation of a similar fold. OS-9 and the MPRs use the same four residues (Gln, Arg, Glu, and Tyr) to bind mannose.The MRH domain-containing proteins play key roles in the secretory pathway: glucosidase II, XTP3-B, and OS-9 are involved in the recognition of nascent glycoproteins, whereas the MPRs play an essential role in lysosome biogenesis by targeting Man-6-P-containing lysosomal enzymes to the lysosome.► Recycling receptors, ER proteins, and a glycosyltransferase contain MRH domain. ► Many MRH domains function as lectins and bind mannose-containing N-glycans. ► Structures are known for ten MRH domains, revealing conservation of a similar fold. ► OS-9 and the MPR use the same four residues (Gln, Arg, Glu, Tyr) to bind mannose.

Keywords: Abbreviations; MRH; mannose 6-phosphate receptor homology; MPR; mannose 6-phosphate receptor; CD-MPR; cation-dependent MPR; CI-MPR; cation-independent MPR; Man-6-P; mannose 6-phosphate; ER; endoplasmic reticulum; ML-II; mucolipidosis II; LERP; lysosomal enzyme receptor protein; GlcNAc-phosphotransferase; UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase; Man-P-GlcNAc; mannose 6-phosphate N-acetylglucosamine ester; uncovering enzyme; N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase; TGN; trans; Golgi network; LSD; lysosomal storage disorder; LIF; leukemia inhibitory factor; TGF-β; transforming growth factor-β precursor; SPR; surface plasmon resonance; ERAD; endoplasmic reticulum associated degradation; IGF-II; insulin-like growth factor-II; GAA; acid α-glucosidaseLectin; Carbohydrate recognition; Protein targeting; Secretory pathway

Pre-screening and follow-up of childhood acute leukemia using biochemical infrared analysis of peripheral blood mononuclear cells by Udi Zelig; Shaul Mordechai; George Shubinsky; Ranjit Kumar Sahu; Mahmoud Huleihel; Eugene Leibovitz; Ilana Nathan; Joseph Kapelushnik (pp. 827-835).

Recent advances in chemotherapeutic treatment of childhood acute leukemia have improved remission rates to about 80%. With the development of novel drugs and treatment protocols adapted for specific individual patients, a simple diagnostic tool for following patients' responses on a daily basis is required. In the present clinical study, we have investigated the usefulness of Fourier transform infrared microscopy (FTIR-MSP) for pre-screening and follow-up of leukemia patients undergoing chemotherapy.Blood samples were collected from leukemia patients before and during treatment as well as from patients with high fever and healthy subjects which served as control groups. Peripheral blood mononuclear cells (PBMCs) were isolated and their spectra obtained using FTIR-MSP. The presence of blasts in bone marrow and other diagnostic and prognostic clinical parameters were determined during follow-up up to 1000days.Leukemia was efficiently indicated by a reduced lipids and elevated DNA absorption of PBMC together with additional characteristic spectral bands. These diagnostic markers were used for monitoring the biochemical changes in PBMCs during chemotherapy. The trends of several markers were found to be in agreement with blast percentage as determined by flow cytometry.Our findings reveal the utility of FTIR-MSP for leukemia pre-screening independently of symptoms common to leukemia. Furthermore, FTIR-MSP supplies precursor indication regarding patient response to treatment compared to current methods.This preliminary study shows a great potential of FTIR-MSP as a complementary tool for childhood leukemia pre-screening and follow-up which may allow faster response to critical problems arise during treatment.► A spectroscopic method for acute childhood leukemia pre-screening and follow up. ► Distinct spectral pattern for leukemia compared to sepsis and healthy subjects. ► Good correlation with flow cytometry. ► Faster identification of patient response to treatment. ► Optical method for fast pre-screening and long term daily follow up.

Keywords: Abbreviations; ALL; acute lymphoid leukemia; AML; acute myeloid leukemia; FTIR-MSP; Fourier transform infrared microscopy; PBMCs; peripheral blood mononuclear cells; PB; peripheral blood; BM; bone marrow; MRD; minimal residual disease; PCR; polymerase chain reaction; EDTA; ethylenediaminetetraacetic acid; ZnSe; zinc selenide; MCT; mercury-cadmium-telluride; FWHM; full width at half maximum; MAb; monoclonal antibodies; WBC; white blood cells; FACS; flow cytometry; LDH; lactate dehydrogenaseChildhood acute leukemia; Follow-up; FTIR-MSP; Flow cytometry

The induction of human CL-P1 expression in hypoxia/reoxygenation culture condition and rat CL-P1 after ischemic/reperfusion treatment by Satoshi Koyama; Katsuki Ohtani; Jun Fukuzawa; Naoyuki Yao; Mitsuko Fukuda; Seong-Jae Jang; Naoyuki Hasebe; Kenjiro Kikuchi; Hiroyuki Itabe; Itsuro Yoshida; Yasuhiko Suzuki; Nobutaka Wakamiya (pp. 836-842).

Oxidative stress-induced endothelial dysfunction and oxidized low-density lipoprotein (LDL) might play a key role in the pathogenesis of atherosclerosis. We recently identified a vascular endothelial scavenger receptor, collectin placenta 1 (CL-P1), which acts as a receptor for oxidized LDL as well as for microbes.We demonstrate how hypoxic and oxidative stress induced CL-P1 expression and compared their effects with the expression of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), an endothelial scavenger receptor expressed by oxidative stress.Hypoxia/reoxygenation induced CL-P1 mRNA and protein expression in human umbilical vein endothelial cells (HUVECs). The expression of LOX-1 mRNA in these cells peaked slightly at 24h, while the expression of CL-P1 had an onset at 72h and was sustained for 120h after reoxygenation. Furthermore, the exposure of rat carotid artery endothelium to ischemia/reperfusion increased the maximal CL-P1 mRNA expression at 72h and expression of its protein peaked at 7days after this treatment. We demonstrate that CL-P1 up-regulation is induced in vitro and in vivo by oxidative stress.The inducible expression of CL-P1 by oxidative stress might play a crucial role in endothelial dysfunction or chronic activation leading to the pathogenesis of atherosclerosis.► We investigated how hypoxic and oxidative stress induced CL-P1 and LOX-1 expression. ► Hypoxia/reoxygenation induced CL-P1 mRNA and protein expression in HUVECs. ► The rat carotid artery after treatment of ischemia increased the CL-P1 expression. ► CL-P1 up-regulation was induced in vitro and in vivo oxidative stress.

Keywords: Scavenger receptor; Hypoxia; Ischemia; Reperfusion; LOX-1; Collectin

In vitro fertilization: Four decades of reflections and promises by Yulian Zhao; Paul Brezina; Chao-Chin Hsu; Jairo Garcia; Peter R. Brinsden; Edward Wallach (pp. 843-852).

In 2010, Robert Edwards was awarded the Nobel Prize in Medicine for his pioneering work in the development of in vitro fertilization, a field that has touched millions of lives across the globe. Edwards dedicated his career to helping couples overcome infertility. He first established principles of early embryo development that served as the foundation for his later work. In the 1960s, he achieved the first human fertilized oocyte in vitro while at the Johns Hopkins Hospital. He then continued his work at Cambridge University. In 1978, the world witnessed the birth of the first “test tube baby”. This achievement is a landmark not only in the reproductive sciences but also in the history of mankind's technological evolution.This article outlines the development and progression of IVF from its infancy to the refined and broadly utilized technology offered to patients today. We describe the evolution of the field and the current state of IVF, including its current technological and social challenges.We congratulate Professor Edwards for his well-deserved recognition as Nobel Laureate in Medicine.This article is a tribute to Edwards for his exceptional accomplishments in this specific and rewarding field of modern medicine.► Robert Edwards, Nobel Laureate in Medicine 2010, and the first human IVF; ► IVF technology refinement, advancement, and milestones; ► current IVF status including social, ethical, regulatory and financial aspects; ► future of IVF.

Keywords: Nobel Prize; Robert Edwards; In vitro; fertilization

Apelin-transgenic mice exhibit a resistance against diet-induced obesity by increasing vascular mass and mitochondrial biogenesis in skeletal muscle by Toshihiro Yamamoto; Yugo Habata; Yoshio Matsumoto; Yoshitaka Yasuhara; Tadatoshi Hashimoto; Hitomi Hamajyo; Hisashi Anayama; Ryo Fujii; Hiromitsu Fuse; Yasushi Shintani; Masaaki Mori (pp. 853-862).

Apelin is an endogenous ligand for the G-protein-coupled 7-transmembrane receptor, APJ. The administration of apelin-13, a truncated 13-amino acid apelin peptide, in diet-induced obese mice is reported to result in a decrease in adiposity due to the increase of energy expenditure with an increase in the expression of uncoupling proteins.We systematically compared the phenotype of human apelin-transgenic (apelin-Tg) mice fed standard or high-fat diets (HFD) with that of non-Tg control mice to clarify the effect of apelin on obesity. The beneficial effects of apelin were evaluated by multiple assay methods including indirect calorimetrical measurements, gene expression analysis, and immunohistochemical staining.Apelin-Tg mice inhibited HFD-induced obesity without altering food intake and exhibited increased oxygen consumption and body temperature compared to non-Tg controls. Interestingly, the mRNA expressions of angiopoietin-1 ( Ang1), a key molecule for vascular maturation, and its receptor, endothelium-specific receptor tyrosine kinase 2 ( Tie2), were significantly upregulated in the skeletal muscle of HFD-fed apelin-Tg mice, and the areas of anti-CD31 antibody-positive endothelial cells also increased. Furthermore, both the aerobic type-I muscle fibre ratio and the DNA copy number of mitochondrial NADH dehydrogenase subunit 1 increased 2.0- and 1.4-fold in skeletal muscle, respectively.These findings suggest that apelin stimulates energy expenditure via increase vascular mass and mitochondrial biogenesis in skeletal muscle.Apelin is a prerequisite factor for anti-obesity by stimulating energy expenditure via regulating homeostatic energy balance.► Human apelin-transgenic mice exhibited a resistance for diet-induced obesity. ► Apelin was found to increase type-I muscle ratio and mitochondria content in muscle. ► It was also supposed to be caused by increased vascular mass induced by apelin. ► A potential therapeutic strategy for obesity involves increasing energy expenditure. ► So apelin may have an important role in regulation of metabolism-related diseases.

Keywords: Apelin; APJ; Ang1; Tie2; Vascular mass; Mitochondrial biogenesis

A method to assess the migration properties of cell-derived microparticles within a living tissue by Thang Q. Hoang; Christine Rampon; Jean-Marie Freyssinet; Sophie Vriz; Danièle Kerbiriou-Nabias (pp. 863-866).

Cells undergoing activation or apoptosis exhibit plasma membrane changes, leading to the formation of shed vesicles (microparticles, MP). Although their effects on recipient cells in vitro, and their ability to support inflammatory or thrombotic events in the circulation have been studied, the spreading of such vesicles in tissues is still elusive. Our aim was to set up a method to examine the behavior of these vesicles in vivo.We examined the persistence of green-fluorescent microparticles (fMP), prepared after Ca²⁺ ionophore activation (iono-fMP) or apoptogenic treatment (eto-fMP) of human Jurkat T lymphoblastic or non-hematopoietic embryonic kidney (HEK) cell lines, following injection in zebrafish embryos 2h after egg fertilization.One hour post-injection, iono-fMP issued from both cell types formed a fluorescent dispersal in the intercellular space of embryos. In contrast, eto-fMP or MP deprived of sialic acid at their membrane, gathered together at the site of injection.We propose a method characterizing the abilities of MP to spread in the intercellular space. We showed that MP produced by apoptosis of T cells and those deprived of sialic acid at their membrane do not diffuse within the living cells. On the contrary, MP shed upon calcium induced activation of T and HEK cells, diffuse at a distance and spread in the intercellular space.The fate of injected MP relies on the type of induction rather than the cell species and results provide a model to test the ability of vesicles to interact locally or to spread outside of the site of production.► A method is given to examine, in vivo, the fate of cell-derived microparticles (MP) ► The MP can be visualized upon injection into developing zebrafish embryos. ► The fate of MP in the intercellular space relies on particle's phenotype. ► Calcium-induced MP diffuse within the living cells, but not apoptosis-derived MP.

Keywords: Abbreviations; MP; microparticles; fMP; green-fluorescent MP; iono-fMP; fMP produced after Ca; ²⁺; ionophore activation; eto-fMP; fMP produced after apoptogenic treatment with etoposide; GFPf; farnesylated enhanced green fluorescent proteinLymphocytes; Microparticles; Apoptosis; Calcium activation; Sialic acid; Zebrafish

The significance of chloride in the inhibitory action of disodium cromoglycate on immunologically-stimulated rat peritoneal mast cells by J.K.Y. Law; C.K. Yeung; S.P. Wan; S. Ingebrandt; H.Y.A. Lau; J.A. Rudd; M. Chan (pp. 867-874).

The microelectrode array (MEA) was used to investigate the pharmacological relevance of chloride (Cl⁻) ions in antigen-dependent mast cell activation and the inhibitory effect of disodium cromoglycate (DSCG) on mast cell activation.The movements of ions across the cellular membrane and the potential relationship between Cl⁻ channels and DSCG during immunological activation were investigated using the MEA. The results were then subsequently compared with the amount of histamine released from anti-IgE activated peritoneal mast cells.The inclusion of charybdotoxin (ChTX) in Cl⁻-free buffer showed that the measured field potentials during antigen-stimulated peritoneal mast cell were a combination of Cl⁻ influx and K⁺ efflux. The delayed onset time of Cl⁻ influx indicated the presence of a delayed outwardly-rectifying Cl⁻ current in the antigen-stimulated peritoneal mast cells. The use of 5-nitro-2-(3-phenylpropylamino) benzoic acid demonstrated that the activated mast cell membrane potential can be stabilised, thereby reducing the amount of histamine released from the anti-IgE activated mast cells. The correlation between the results of the histamine release assay and the electrophysiological measurements demonstrated the importance of Cl⁻ to anti-IgE dependent mast cell activation. The inhibitory effect of DSCG on anti-IgE activated cells, however, did not correlate with the presumed influx of Cl⁻.The MEA data suggest that Cl⁻ influx is crucial to IgE-dependent mast cell degranulation.While the MEA cannot yield information about single channel properties, it is convenient to use and can provide information on the global changes in electrophysiological responses of non-excitable cells.► Cl⁻ influx is crucial to IgE-dependent mast cell degranulation. ► The MEA is more convenient to use than the patch-clamp. ► The MEA provides a “physiological signature” for profiling or characterisation. ► The MEA provides information on the changes in electrophysiological responses.

Keywords: Abbreviations; MEA; microelectrode array; sodium cromoglycate; DSCG; delayed outwardly-rectifying chloride channel; I; _ORCC; calcium-release activated calcium channel; CRAC; charybdotoxin; ChTX; calcium-dependent potassium channel; K; _Ca; channelChloride; Anti-IgE; Rat peritoneal mast cells; Disodium cromoglycate; Histamine; Extracellular electrophysiology

Brief treatment with heparin-binding EGF-like growth factor, but not with EGF, is sufficient to accelerate epithelial wound healing by Michael A. Tolino; Ethan R. Block; Jes K. Klarlund (pp. 875-878).

Heparin-binding EGF-like growth factor (HB-EGF) contains, in contrast to EGF, a domain that binds to negatively charged glycans on cell surfaces and in extracellular matrix. We speculated that a short exposure to HB-EGF induces prolonged biological effects such as healing of wounds after immobilization in tissues.Epithelial cell sheets in tissue and corneas in organ culture were treated briefly with HB-EGF or EGF and binding of the growth factors, time course of activation of the EGF receptor, and healing of wounds were compared.Treating human corneal epithelial cells for 2min with HB-EGF resulted in 8h of detectable activation of the EGF receptor, but activation was much shorter after EGF treatment. A brief treatment with HB-EGF, but not with EGF, induced significant acceleration of healing in wounds in epithelial sheets in tissue and organ culture. Bound HB-EGF was detectable up to 16h after brief treatments. Neutralizing antibodies added after HB-EGF treatment blocked acceleration of healing, demonstrating the role of bound HB-EGF in accelerating healing.A brief exposure to HB-EGF, but not to EGF, is sufficient to induce prolonged activation of the EGF receptor and to enhance healing.Bound HB-EGF can serve as a pool that induces prolonged activation of the EGF receptor. EGF has been used experimentally to treat poorly healing wounds, but the frequent applications that are necessary have hampered its use clinically. The findings imply that HB-EGF may be a useful long-acting alternative to EGF.► Heparin-binding EGF-like growth factor (HB-EGF), but not EGF, binds to glycans on the cell surface. ► Bound HB-EGF induces prolonged EGF receptor activation and healing of epithelial wounds. ► A brief treatment with HB-EGF is sufficient to induce epithelial healing. ► EGF rapidly washed out from tissues, and is not generally useful for treating wounds. ► HB-EGF may be more useful than EGF as a therapeutic agent to treat wounds.

Keywords: Abbreviations; HB-EGF; heparin-binding EGF-like growth factor; EGFR; EGF receptor; HCLE; human corneal limbal epithelial; NI; non-immuneEGF; Heparin-binding EGF-like growth factor; Wound healing; Cornea

Ligand-induced fit affects binding modes and provokes changes in crystal packing of aldose reductase by Cornelia Koch; Andreas Heine; Gerhard Klebe (pp. 879-887).

Flexibility is a common feature of proteins. For human aldose reductase, a variety of conformers have been observed in crystalline complexes with different inhibitors.A study of crystal structures and isothermal titration calorimetry was performed on wild type and mutated aldose reductase.Though the interaction to the mutated residue Thr113 does not directly alter the binding mode of zopolrestat to aldose reductase, a shift of its basic scaffold is induced which affects the interaction with a flexible loop and introduces disorder. With the related inhibitor IDD393, two distinct binding site conformations result in two different crystal forms: While a backbone flip of the same residues as for zopolrestat is present in both crystal forms, a considerable side-chain movement of a phenylalanine is observed for only one crystal form. In consequence, residual mobility of adjacent amino acids is increased and some crystal contacts are prevented which reinforces different crystal packing. The structure of a benzothiazepine reveals a protein conformer, where this phenylalanine is further relocated resulting in the same altered crystal packing. Differences in the thermodynamic signature recorded for the various complexes relate to the structural differences.Crystal structures are accepted as “gold standard” for the interpretation of protein geometry, however, they are only one possible structure and can be influenced by crystal packing. In reverse, ligand binding can affect protein conformation and determine crystal packing. The phenomenon of such “polymorphic forms” is well appreciated, however rarely understood at the molecular level.► Structural and energetic consequences of the mutational exchange of a residue in the speciﬁcity pocket of aldose reductase. ► Study by high resolution crystallography and isothermal titration calorimetry. ► Three carboxylate-type inhibitors investigated, one with speciﬁcity pocket in closed, two in opened state. ► Inhibitor induced-ﬁt adaptations provoke the transition into another crystal form. ► New crystal form avoids packing contacts to the crucially affected residues.

Keywords: Protein flexibility; Aldose reductase; Protein–ligand interaction; Thermodynamic signature; Different crystal forms

Biological targets of isothiocyanates by Kristin K. Brown; Mark B. Hampton (pp. 888-894).

Isothiocyanates are phytochemicals with a broad array of effects in biological systems. Bioactivity includes the stimulation of cellular antioxidant systems, induction of apoptosis and interference with cytokine production and activity. Epidemiological evidence and experimental studies indicate that naturally occurring isothiocyanates and synthetic derivatives have anti-cancer and anti-inflammatory properties.This review focuses on the molecular targets of isothiocyanates, and how target modification translates into a biological response.Isothiocyanates may mediate their effects via direct protein modification or indirectly by disruption of redox homeostasis and increased thiol oxidation. Some target proteins have been identified, but in-depth searches with new techniques are needed to reveal novel targets. Site-directed mutagenesis and isothiocyanate structure-activity relationships will assist in determining the biological significance of specific modifications.Target identification is important for rational drug design and exploiting the therapeutic potential of isothiocyanates. It also provides insight into the diverse pathways that these compounds regulate.► Plant-derived isothiocyanates modify biological processes. ► Protein microenvironment promotes selective isothiocyanate reactivity. ► Probes are revealing previously unidentified targets.

Keywords: Isothiocyanate; Electrophile; Redox signalling; Oxidative stress; Post-translational modification

Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na⁺/K⁺-ATPase by Alessandra Linardi; Thomaz A.A. Rocha e Silva; Elen H. Miyabara; Carla F. Franco-Penteado; Kiara C. Cardoso; Patrícia A. Boer; Anselmo S. Moriscot; José A.R. Gontijo; Paulo P. Joazeiro; Carla B. Collares-Buzato; Stephen Hyslop (pp. 895-906).

Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na⁺/K⁺-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na⁺/K⁺-ATPase expression and activity in rats injected with Bothrops alternatus snake venom.Male Wistar rats were injected with venom (0.8mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72h and 7days post-venom. The rats were then killed and renal Na⁺/K⁺-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively.Venom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na⁺/K⁺-ATPase α₁ subunit were increased 6h post-venom, whereas Na⁺/K⁺-ATPase activity increased 6h and 24h post-venom. Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na⁺/K⁺-ATPase expression and activity in the early phase of renal damage.Enhanced Na⁺/K⁺-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage. ► Bothrops alternatus snake venom causes renal dysfunction in rats. ► This dysfunction is accompanied by marked renal structural alterations. ► Na+/K+-ATPase expression and activity are enhanced during early renal damage. ► Enhanced Na+/K+-ATPase activity may attenuate renal dysfunction after envenoming.

Keywords: Acute renal failure; Bothrops alternatus; Collagen deposition; F-actin; Na; ⁺; /K; ⁺; -ATPase; Renal function

Monocyte-endothelium-smooth muscle cell interaction in co-culture: Proliferation and cytokine productions in response to advanced glycation end products by Mi-Hyun Nam; Hyun-Sun Lee; Young Seomun; Yanhouy Lee; Kwang-Won Lee (pp. 907-912).

During hyperglycemia, reducing sugars react with the amino groups to form Amadori products which then form advanced glycation end-products (AGEs). Studies have shown that the AGEs and the receptor binding generated reactive oxygen species, and triggered secretion of cytokines contributing to the local regulations of proliferation and inflammation in cells. Interaction of vessel wall cells and monocytes may trigger the processes leading to atherosclerosis. We evaluated the effects of AGEs on smooth muscle cell (SMC) proliferation and cytokine synthesis in co-cultures of human monocytes (THP-1), endothelial cells (HUVEC) and aortic vascular smooth muscle cell (SMC) to clarify the effects of AGEs on vascular cells and to investigate the mechanisms of arteriosclerosis.Glycolaldehyde-induced AGEs (glycol-AGEs) was prepared. The THP-1 and HUVEC were cultured with SMC in transwell plates with 100μg/ml of glycol-AGEs for 24 to 48h.The proliferation of SMC was induced by glycol-AGEs in the co-culture system. Moreover, SMC treated with glycol-AGEs also expressed interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), and the level of cytokines expression was significantly elevated in the co-culture system of HUVEC and THP-1 when treated with glycol-AGEs.These results suggest that employing a co-culture system is necessary to investigate the synergistic effects of AGEs on intercellular cellular interactions and it creates a more in vivo-like environment for AGEs implicated atherosclerosis research.All three cell types are required to be investigated together to understand the effects of AGEs on intercellular interactions occurring among these cells.► We establish a co-culture system to clarify the effects of vascular cells by AGEs. ► Monocytes, vein endothelial cells and aortic vascular smooth muscle cells are co-cultured. ► Proliferation of vascular smooth muscle cells (SMC) is induced by AGEs in co-culture system. ► SMC treated with AGEs express cytokines, IL-6 and MCP-1. ► Cytokines expression is elevated in the co-culture system of HUVEC and THP-1 when treated with AGEs.

Keywords: Glycation; Co-culture; Atherosclerosis; Proliferation

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