Modulation of store-operated Ca2+ entry by cyclic-ADP-ribose

Store-operated Ca2+ entry plays an important role in Ca2+ homeostasis in cells but the mechanisms of control of these channels are not completely understood. We describe an investigation of the role of the CD38-cyclic-ADP-ribose (cADPR)-ryanodine-channel (RyR) signaling pathway in store-operated Ca2+ entry in human smooth muscle. We observed that human myometrial cells have a functional store-operated Ca2+ entry mechanism. Furthermore, we observed the presence of transient receptor potential 1, 3, 4, 5, and 6 ion channels in human myometrial cells. Store-operated Ca2+ transient was inhibited by at least 50-70% by several inhibitors of the RyR, including ryanodine (10 µM), dantrolene (10 µM), and ruthenium red (10 µM). Furthermore, the cell permeable inhibitor of the cADPR-system, 8-Br-cADPR (100 µM), is a potent inhibitor of the store-operated entry, decreasing the store operated entry by 80%. Pre-incubation of cells with 100 µM cADPR and the hydrolysis-resistant cADPR analog 3-deaza-cADPR (50 µM), but not with ADP-ribose (ADPR) leads to a 1.6-fold increase in the store-operated Ca2+ transient. In addition, we observed that nicotinamide (1-10 mM), an inhibitor of cADPR synthesis, also leads to inhibition of the store-operated Ca2+ transient by 50-80%. Finally, we observed that the transient receptor potential channels, RyR, and CD38 can be co-immunoprecipitated, indicating that they interact in vivo. Our observations clearly implicate the CD38-cADPR-ryanodine signaling pathway in the regulation of store-operated Ca2+ entry in human smooth muscle cells.

Anti-cyclic citrullinated peptide antibodies and rheumatoid factor in leprosy patients with articular involvement

The objective of the present research was to evaluate the usefulness of anti-cyclic citrullinated peptide (anti-CCP) antibodies and the IgM rheumatoid factor (IgM RF) test for the differential diagnosis of leprosy with articular involvement and rheumatoid arthritis (RA). Anti-CCP antibodies and IgM RF were measured in the sera of 158 leprosy patients (76 with and 82 without articular involvement), 69 RA patients and 89 healthy controls. Leprosy diagnosis was performed according to Ridley and Jopling classification criteria and clinical and demographic characteristics of leprosy patients were collected by a standard questionnaire. Leprosy patients with any concomitant rheumatic disease were excluded. Serum samples were obtained from all participants and frozen at _20°C. Measurement of anti-CCP antibodies and IgM RF were performed by ELISA, using a commercial second-generation kit, and the latex agglutination test, respectively. Anti-CCP antibodies and IgM RF were detected in low frequencies (2.6 and 1.3%, respectively) in leprosy patients and were not associated with articular involvement. Among healthy individuals both anti-CCP antibodies and IgM RF were each detected in 3.4% of the subjects. In contrast, in the RA group, anti-CCP antibodies were present in 81.2% and IgM RF in 62.3%. In the present study, both anti-CCP antibodies and IgM RF showed good positive predictive value for RA, helping to discriminate between RA and leprosy patients with articular involvement. However, anti-CCP antibodies were more specific for RA diagnosis in the population under study.

Effect of the human leukocyte antigen HLA-DRB1 and anti-cyclic citrullinated peptide on the outcome of rheumatoid arthritis patients

Our objective was to determine whether the presence of the human leukocyte antigen HLA-DRB1 locus is associated with production of anti-cyclic citrullinated peptide antibodies (anti-CCP Abs) and to what extent they are associated with increased susceptibility to and severity of rheumatoid arthritis (RA) in Egyptian patients. Twenty-nine RA patients gave informed consent to participate in a case-control study that was approved by the Ain Shams University Medical Ethics Committee. RA disease activity and severity were determined using the simplified disease activity index and Larsen scores, respectively. We used a wide scale national study on the pattern of HLA typing in normal Egyptians as a control study. Anti-CCP Abs and HLA-DRB1 typing were determined for all subjects. The alleles most strongly associated with RA were HLA-DRB1 [*01 , *04 and *06] (41.4%). RA patients with serum anti-CCP Ab titers above 60 U/mL had a significantly higher frequency of HLA-DRB1*01 (58.3%) and HLA-DRB1*04 alleles (83.3%). Significant positive correlations were found between serum and synovial anti-CCP Ab titer, RA disease activity, and severity (r = 0.87, 0.66 and 0.63, respectively; P < 0.05). HLA-DRB1 SE+ alleles [*01 and *04] were highly expressed among Egyptian RA patients. The presence of these alleles was associated with higher anti-CCP Ab titer, active and severe RA disease. Early determination of HLA-DRB1 SE+ alleles and serum anti-CCP Ab could facilitate the prediction of the clinical course and prognosis of RA when first evaluated leading to better disease control.

The role of cyclic nucleotides in modulation of crayfish neuromuscular junctions by a neuropeptide /

DF2, a heptapeptide, is a member of the family of FMRFamide-like peptides and has been shown to increase the amount of transmitter released at neuromuscular junctions of the crayfish, Procambarus clarkit Recent evidence has shown that protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII) and the cAMPdependent protein kinase (PKA) play a role in the neuromodulatory pathway of DF2. The involvement of these kinases led to the prediction that a G-protein-coupled receptor (GPCR) is activated by DF2 due to the role that each kinase plays in traditional GPCR pathways seen in other organisms and in other cells. G-proteins can also act on an enzyme that generates cyclic guanosine monophosphate (cGMP) which mediates its effects through a cGMP-dependent protein kinase (PKG). This thesis addresses the question of whether or not DF2's effects on synaptic transmission in crayfish are mediated by the cyclic nucleotides cAMP and cGMP. The effects of DF2 on synaptic transmission were examined using deep abdominal extensor muscles of the crayfish Procambarus clarkii. An identified motor neuron was stimulated, and excitatory post-synaptic potentials (EPSPs) were recorded in abdominal extensor muscle LI . A number of activators and inhibitors were used to determine whether or not cAMP, PKA, cGMP and PKG mediate the effect of this peptide. Chemicals that are known to activate PKA (Sp-cAMPS) and/or PKG (8-pCPTcGMP) mimic and potentiate DF2's effect by increasing EPSP amplitude. Inhibitors of either PKA (Rp-cAMPS) or PKG (Rp-8-pCPT-cGMPS) block a portion of the increase in EPSP amplitude induced by the peptide. When both kinase inhibitors are applied simultaneously, the entire effect of DF2 on EPSPs is blocked. The PKG inhibitor blocks the effects of a PKG activator but does not alter the effect of a PKA activator on EPSP amplitude. Thus, the PKG inhibitor appears to be relatively specific for PKG. A trend in the data suggests that the PKA inhibitor blocks a portion of the response elicited by the PKG activator. Thus, the PKA inhibitor may be less specific for PKA. Phosphodiesterase inhibitors, which are known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of the peptide. These results support the hypothesis that cAMP and cGMP, acting through their respective protein kinase enzymes, mediate the ability of DFi to increase transmitter output.

Zero-sum problems in finite cyclic groups

The purpose of this thesis is to investigate some open problems in the area of combinatorial number theory referred to as zero-sum theory. A zero-sequence in a finite cyclic group G is said to have the basic property if it is equivalent under group automorphism to one which has sum precisely IGI when this sum is viewed as an integer. This thesis investigates two major problems, the first of which is referred to as the basic pair problem. This problem seeks to determine conditions for which every zero-sequence of a given length in a finite abelian group has the basic property. We resolve an open problem regarding basic pairs in cyclic groups by demonstrating that every sequence of length four in Zp has the basic property, and we conjecture on the complete solution of this problem. The second problem is a 1988 conjecture of Kleitman and Lemke, part of which claims that every sequence of length n in Zn has a subsequence with the basic property. If one considers the special case where n is an odd integer we believe this conjecture to hold true. We verify this is the case for all prime integers less than 40, and all odd integers less than 26. In addition, we resolve the Kleitman-Lemke conjecture for general n in the negative. That is, we demonstrate a sequence in any finite abelian group isomorphic to Z2p (for p ~ 11 a prime) containing no subsequence with the basic property. These results, as well as the results found along the way, contribute to many other problems in zero-sum theory.

Properties and algorithms of the (n, k)-arrangement graphs

The (n, k)-arrangement interconnection topology was first introduced in 1992. The (n, k )-arrangement graph is a class of generalized star graphs. Compared with the well known n-star, the (n, k )-arrangement graph is more flexible in degree and diameter. However, there are few algorithms designed for the (n, k)-arrangement graph up to present. In this thesis, we will focus on finding graph theoretical properties of the (n, k)- arrangement graph and developing parallel algorithms that run on this network. The topological properties of the arrangement graph are first studied. They include the cyclic properties. We then study the problems of communication: broadcasting and routing. Embedding problems are also studied later on. These are very useful to develop efficient algorithms on this network. We then study the (n, k )-arrangement network from the algorithmic point of view. Specifically, we will investigate both fundamental and application algorithms such as prefix sums computation, sorting, merging and basic geometry computation: finding convex hull on the (n, k )-arrangement graph. A literature review of the state-of-the-art in relation to the (n, k)-arrangement network is also provided, as well as some open problems in this area.

SYNTHESIS OF CYCLIC AZOBENZENE ANALOGUES FOR INCORPORATION INTO OLIGONUCLEOTIDES, PEPTIDES AND POLYMERS

(A) In recent years, considerable amount of effort has contributed towards enhancing our understanding of the new photoswitch, cyclic azobenzene, particularly from the theoretical point of view. However, the challenging part with this system was poor efficiency of its synthesis from 2,2’- dinitrodibenzyl and lack of effective methods for further modification which would be useful to incorporate this system into biomolecules as a photoswitch. We report the synthesis of cyclic azobenzene and analogues from 2,2’-dinitrodibenzyl, which would allow for further incorporation of this cyclic azobenzene into biomolecules. Reaction of 2,2’-dinitrodibenzyl with zinc metal powder in the presence of triethylammonium formate buffer (pH-9.5) gave a cyclic azoxybenzene, 11,12-dihydrodibenzo[c,g][1,2]diazocine-5-oxide. The latter compound was converted into cyclic azobenzene analogues (bromo-, chloro-, cyano-, and carboxyl) through subsequent transformations. The carboxylic acid analogue was reacted with D-threoninol to give the corresponding amide, which readily undergoes photo-isomerization upon illumination with light. Upon illumination with light at 400 nm, approximately 70% of cis- isomer of amide was isomerized to trans- isomer. It was observed that cis- to trans- isomerization reached the maximum steady state of light transmission after approximately 40 min, whereas the trans- to cis- isomerization approximately acquired in 2 h to regain full recovery of light transmission. Cyclic azobenzene phosphoramidite was synthesized from DMT-protected D-threoninol linked cyclic azobenzene. (B) In recent years, there has been considerable interest invested towards the synthesis of azobenzene analogues for incorporation into proteins. Among the many azobenzene analogues, the synthesis of bi-functional cyclic azobenzene analogues for the incorporation into proteins is relatively new. In this thesis, we report the synthesis of a cyclic azobenzene biscarboxylic acid from 4-(bromomethyl)benzonitrile. (C) Azobenzene has been widely used in the field of polymer science to study the surface morphology and surface properties of polymers. In this thesis, we report the incorporation of cyclic azobenzene into a commercial polymer 2- (hydroxyethyl)methacrylate. Samples collected after 24 h from the reaction solution showed approximately 9% of incorporation of cyclic azobenzene into polymer compared to samples collected after 10 h, which showed approximately 6% incorporation.

Synthesis of 3', 5'-cyclic diguanylic acid (c-di-GMP) analogues

In the past few years, interest in signaling networks involving 3ʹ, 5ʹ -cyclic diguanylic acid (c-di-GMP) has increased dramatically. Evidence started to emerge that connects c-di-GMP to the regulation of a range of biological processes in bacteria, such as bacterial biofilm formation, virulence, extracellular polysaccharide synthesis, however, much remains to be explored in the signaling pathways that involve this secondary messenger. This molecule has also been shown to be a very powerful immunostimulating agent and potent mucosal vaccine adjuvant.

(A) Photoregulation of DNA Functions by Cyclic Azobenzene-tethered Oligonucleotides (B) Site-specific Fluorescent Labeling of DNA using Inverse Electron Demand Diels-Alder Reaction between trans-Cyclooctene Derivatives and BODIPY-Tetrazine Adducts

(A) Most azobenzene-based photoswitches require UV light for photoisomerization, which limit their applications in biological systems due to possible photodamage. Cyclic azobenzene derivatives, on the other hand, can undergo cis-trans isomerization when exposed to visible light. A shortened synthetic scheme was developed for the preparation of a building block containing cyclic azobenzene and D-threoninol (cAB-Thr). trans-Cyclic azobenzene was found to thermally isomerize back to the cis-form in a temperature-dependent manner. cAB-Thr was transformed into the corresponding phosphoramidite and subsequently incorporated into oligonucleotides by solid phase synthesis. Melting temperature measurement suggested that incorporation of cis-cAB into oligonucleotides destabilizes DNA duplexes, these findings corroborate with circular dichroism measurement. Finally, Fluorescent Energy Resonance Transfer experiments indicated that trans-cAB can be accommodated in DNA duplexes. (B) Inverse Electron Demand Diels-Alder reactions (IEDDA) between trans-olefins and tetrazines provide a powerful alternative to existing ligation chemistries due to its fast reaction rate, bioorthogonality and mutual orthogonality with other click reactions. In this project, an attempt was pursued to synthesize trans-cyclooctene building blocks for oligonucleotide labeling by reacting with BODIPY-tetrazine. Rel-(1R-4E-pR)-cyclooct-4-enol and rel-(1R,8S,9S,4E)-Bicyclo[6.1.0]non-4-ene-9-ylmethanol were synthesized and then transformed into the corresponding propargyl ether. Subsequent Sonogashira reactions between these propargylated compounds with DMT-protected 5-iododeoxyuridine failed to give the desired products. Finally a methodology was pursued for the synthesis of BODIPY-tetrazine conjugates that will be used in future IEDDA reactions with trans-cyclooctene modified oligonucleotides.

Some problems in set topology relating group of homeomorphisms and order

In this thesis we investigate some problems in set theoretical topology related to the concepts of the group of homeomorphisms and order. Many problems considered are directly or indirectly related to the concept of the group of homeomorphisms of a topological space onto itself. Order theoretic methods are used extensively. Chapter-l deals with the group of homeomorphisms. This concept has been investigated by several authors for many years from different angles. It was observed that nonhomeomorphic topological spaces can have isomorphic groups of homeomorphisms. Many problems relating the topological properties of a space and the algebraic properties of its group of homeomorphisms were investigated. The group of isomorphisms of several algebraic, geometric, order theoretic and topological structures had also been investigated. A related concept of the semigroup of continuous functions of a topological space also received attention

Some Lattice Problems In Fuzzy Set Theory And Fuzzy Topology

It is believed that every fuzzy generalization should be formulated in such a way that it contain the ordinary set theoretic notion as a special case. Therefore the definition of fuzzy topology in the line of C.L.CHANG E9] with an arbitrary complete and distributive lattice as the membership set is taken. Almost all the results proved and presented in this thesis can, in a sense, be called generalizations of corresponding results in ordinary set theory and set topology. However the tools and the methods have to be in many of the cases, new. Here an attempt is made to solve the problem of complementation in the lattice of fuzzy topologies on a set. It is proved that in general, the lattice of fuzzy topologies is not complemented. Complements of some fuzzy topologies are found out. It is observed that (L,X) is not uniquely complemented. However, a complete analysis of the problem of complementation in the lattice of fuzzy topologies is yet to be found out

Some Problems In Algebra And Topology A Study Of Fuzzy Convexity With Special Reference To Separation Properties

This thesis is a study of abstract fuzzy convexity spaces and fuzzy topology fuzzy convexity spaces No attempt seems to have been made to develop a fuzzy convexity theoryin abstract situations. The purpose of this thesis is to introduce fuzzy convexity theory in abstract situations