Blockage of Voltage-Dependent Calcium Channels Affects Twitch Response of Rat Skeletal Muscle

Neurotransmitter release is controlled by calcium (Ca2+) entry into motor nerve terminals (MNTs) through voltage-dependent calcium channels (VDCCs). Upon Ca2+ influx, acetylcholine is released starting downstream processes causing muscle contraction. Co-release of acetylcholine and glutamate from MNTs has been reported. Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors on postsynaptic site is evident. In this study we aimed to observe nerve-evoked and directly-elicited twitch responses in the presence of VDCCs inhibitors without blocking NMDA receptors. We elicited contractions with nerve-evoked (0.1 Hz; 0.3 ms) and direct (0.1 Hz; 3 ms) electrical stimulations at rat hemidiaphragm preparations by using some VDCC toxins and drugs. We evaluated their effects at 15 minutes of application. P/Q-type VDCC blocker omega-conotoxin MVIIC (25 nM) suppressed the nerve-evoked and direct stimulation contractions relative to their initial value by 50 and 40%, respectively. N-type VDCC inhibitor omega-conotoxin GVIA (170 nM) had no effect on both stimulation contractions. P-type VDCC blocker omega-agatoxin IVA (10 nM) completely blocked the nerve-evoked stimulation contractions, whereas the direct stimulation contractions maintained the same values as baseline. L-type VDCC blockers verapamil (75 mu M) and diltiazem (75 mu M) depressed both nerve-evoked and direct stimulation contractions. Ethanol (650 mM) blocked nearly 100% of the contractions by nerve-evoked stimulation, meanwhile 45.5% of direct stimulation. Our finding may suggest the Ca2+ influx localization matters for decoding the neural information for the contractile force generation

CALCIUM SIGNALING: OVERVIEW AND RESEARCH DIRECTIONS OF A MOLECULAR COMMUNICATION PARADIGM

In the ongoing effort to build micro- and nanoscale machines, one of the key approaches is the bio-hybrid approach, which focuses on the use of biological constructs and engineered cells. As a natural extension of this concept to nanoscale communication, molecular communication is an umbrella term encompassing various communication systems that are built based on biological intra-and intercellular communication methods, most of which use molecules and molecular concentration as the information carrier. Compared to other proposed molecular communication systems such as diffusion-based communication and microtubular networks, calcium signaling is expected to provide a faster and more controllable system that is suitable for information dissemination and group behavior in nanoscale sensor networks. In this article, we give a general overview of calcium signaling, a novel communication paradigm that uses intercellular calcium waves in biology as a baseline, explain its capabilities, limitations, and some possible deployment scenarios. We also describe various open issues of this novel communication system and elaborate on some research directions for calcium signaling

Intracellular trafficking of diphtheria toxin and its mutated form, CRM197, in the endocytic pathway

WOS: 000434665900001PubMed ID: 30374472OBJECTIVE: Diphtheria toxin (DTx) is a well-characterized bacterial toxin. However, the endocytic pathway of the mutant of DTx, CRM197, which is used as an immunological adjuvant, has not yet been fully explained. The aim of this study was to investigate the intracellular trafficking of CRM197-loaded endosomes. METHODS: Human umbilical vein endothelial cells (HUVECs) were used in a cell culture. The effective incubation time was determined by transmission electron microscopy in toxin-treated cells. Density gradient centrifugation and ADP-ribosylation assay were used to isolate and detect toxin-loaded endosomal fractions. Endosomal fractions from CRM197-treated cells were elicited after 15 minutes of incubation and the presence of fragment A was demonstrated using Western blot. Immunofluorescence microscopy was used to identify endosomes in CRM197-treated endothelial cells. RESULTS: DTx-loaded endosomes were detected as enlarged vesicles in the perinuclear area with 15 minutes of toxin treatment. DTx-loaded endosomal fractions were determined by ADP-ribosyltransferase activity test and Western blot analysis. Enzymatic activity of the toxin-loaded endosomal fraction increased by 20% in actin cytoskeletal-damaged cells treated with cytochalasin D. The steps for the toxin treatment of HUVECs with DTx and obtaining endosomal fractions were repeated for CRM197. In the CRM197-loaded endosomal fraction, actin and Hsp90 were identified in addition to fragment A. Fluorescent images revealed that CRM197-loaded endosomes were co-localized with actin filaments and that Rab11, which signals the return to the plasma membrane, was more prominent than Rab7, the lysosomal pathway indicator. CONCLUSION: These results suggest that CRM197-loaded endosomes participate in the recycling pathway.Scientific Research Project Coordination Unit of Istanbul University [21270, 39536, 24735]This work was supported by the Scientific Research Project Coordination Unit of Istanbul University. Projects number: 21270, 39536 and 24735

Interleukin-1 beta effect on the endogenous ADP-ribosylation and phosphorylation of eukaryotic elongation factor 2

WOS: 000387773800039PubMed ID: 27510652Eukaryotic elongation factor 2 (eEF2) plays an important role in eukaryotic polypeptide chain elongation. Adenosine diphosphate (ADP)-ribosylation is a post-translational modification reaction that catalyzes the transfer of ADP-ribose group to eEF2 and this causes the inhibition of protein synthesis. Indeed, in the absence of diptheria toxin, endogenous ADP-ribosylation can occur. eEF2 is phosphorylated by eEF2 kinase which prevents binding to ribosomes thus inhibiting its activity. Increase in endogenous ADP-ribosylation level approximately 70-75 % was observed in IL-1 beta treated HUVECs. Moreover, a 70 % rise of phosphorylation of eEF2 was measured. Alteration of endogenous ADP-ribosylation of eEF2 activity was related with cellular mono-ADP-ribosyltransferases (ADPrT). Increment of endogenous ADP-ribosylation on eEF2 did not seem to occur as a direct effect of IL-1 beta; it arises from the activation of ADPrT. This 2.5 fold increase was abolished by ADPrT inhibitors. Due to these post-translational modifications, global protein synthesis is inhibited. After dephosphorylation of phospho-eEF2, around 20 % increase in protein synthesis was observed. In conclusion, systemic IL-1 beta has an important role in the regulation of global protein synthesis.Istanbul University [31384]This work was supported by the Research Fund of the Istanbul University (Grant 31384)

Cross-reacting material 197 (CRM197) affects actin cytoskeleton of endothelial cells

WOS: 000412155000003PubMed ID: 28653650CRM197, cross-reacting material 197, is a mutant of diphtheria toxin (DTx). CRM197 is used in pharmacology as a carrier protein. It has been recently shown that CRM197 causes breakdown in actin filaments. In order to show intracellular localization of CRM197 and visualize cell structure via actin cytoskeleton, endothelial cells were cultured and subjected to CRM197 in vitro. To address the interaction between CRM197 and actin both experimental and theoretical studies were carried out. Colocalization of CRM197 with actin filaments was determined by immunofluorescence microscopy. Following 24-hour incubation, the loss of cell-cell contact between cells was prominent. CRM197 was shown to bind to G-actin by gel filtration chromatography, and this binding was confirmed by Western blot analysis of eluted samples obtained following chromatography. Based on crystal structure, docked model of CRM197-actin complex was generated. Molecular dynamics simulation revealed that Lys42, Cys218, Cys233 of CRM197 interacts with Gly197, Arg62 and Ser60 of G-actin, respectively. CRM197 binding to G-actin, colocalization of CRM197 with actin filament, and actin cytoskeleton rearrangement resulting in the loss of cell-cell contact show that actin comes into sight as target molecule for CRM197.Scientific Research Project Coordination Unit of Istanbul University [21270, 51249]This work was supported by the Scientific Research Project Coordination Unit of Istanbul University. Projects number: 21270 and 51249. We are thankful to Gamze Kilic Berkmen for her editing assistance

Scientific research results of istanbul Faculty of Medicine in 31 years

The aim of this study was to present the 31 year scientific research of Istanbul Faculty of Medicine (IFM) and to evaluate its national and international scientific contribution. The IFM addressed original research articles published in the 31-year period (January 1980-December 2010) were determined by using search engines PubMed, ISI Web of Sciences and Google Scholar were evaluated. A total of 4,728 IFM addressed original articles were identified [yearly mean: 151.5 +/- 145.9 (range:14-479)]. The IFM articles were 3.5% of all medical articles originated from Turkey between 1981 and 2007. One hundred and fourteen IFM articles published between 2005 and 2009 were originated from thesis (6.3% of total IFM articles between the same periods). The number of original IFM articles in 1980 was 23 and increased to 443 in 2010. The highest numbers of articles were published in the field of Internal Medicine, followed by Surgery and Basic Sciences during the 31 year period. In the last 12 years, the highest number of articles per faculty was found in the Department of Medical Genetics (2.6/faculty/year) in the field of Internal Medicine, Department of Heart and Vascular Surgery (1.87/faculty/year) in the field of Surgery and Department of Medical Biology (1.05/faculty/year) in the field of Basic Science. The number of articles increased from 0.2 to 1 per faculty between the same periods. Until the end of 2011 the total citations were 28,750, with an impact of 6.08.The number of articles with higher than 50 citations were 412. Istanbul Faculty of Medicine with its 4,728 original articles between 1980 and 2010 contributed crucially to scientific reproduction of our country. With a scientific acceleration in the last decade, Istanbul Faculty of Medicine contributed notably for Istanbul University to become the 383rd in general and 156th in the field of medicine among the leading universities of the world