Immunochemical approach to the mapping of an assembled epitope of human chorionic gonadotropin: Proximity of CTP-alpha to the receptor binding region of the beta-subunit

A single-step solid-phase RIA (SS-SPRIA) developed in our laboratory using hybridoma culture supernatants has been utilised for the quantitation of epitope-paratope interactions. Using SS-SPRIA as a quantitative tool for the assessment of epitope stability, it was found that several assembled epitopes of human chorionic gonadotropin (hCG) are differentially stable to proteolysis and chemical modification. Based on these observations an approach has been developed for identifying the amino acid residues constituting an epitopic region. This approach has now been used to map an assembled epitope at/near the receptor binding region of the hormone. The mapped site forms a part of the seat belt region and the cystine knot region (C34-C38-C88-C90-H106). The carboxy terminal region of the alpha-subunit forms a part of the epitope indicating its proximity to the receptor binding region. These results are in agreement with the reported receptor binding region identified through other approaches and the X-ray crystal structure of hCG.

Mapping of assembled epitopic regions of human chorionic gonadotropin reveals proximity of CTP alpha to the determinant loop beta 93-100

Three overlapping assembled epitopes of beta hCG have been mapped using MAb probes and a single step solid phase radioimmunoassay. These epitopes have been shown to be at receptor binding region comprising of the loop region beta Cys93-Cys100. Importance of disulphide bonds in maintaining integrity of these epitopes is assessed. Two MAbs (INN 58 and INN 22) interact with the beta region as well as the alpha C-terminal peptide, while the other MAb INN 24 interacts with only the beta region. Cross-reactivity pattern with beta hCG and hLH as web as the reported crystal structure of hCG substantiates the epitope identification. The results demonstrate utility of MAbs as probes in investigations on three-dimensional structure of gonadatropins.

The Role of UPF0157 in the Folding of M-tuberculosis Dephosphocoenzyme A Kinase and the Regulation of the Latter by CTP

Background: Targeting the biosynthetic pathway of Coenzyme A (CoA) for drug development will compromise multiple cellular functions of the tubercular pathogen simultaneously. Structural divergence in the organization of the penultimate and final enzymes of CoA biosynthesis in the host and pathogen and the differences in their regulation mark out the final enzyme, dephosphocoenzyme A kinase (CoaE) as a potential drug target. Methodology/Principal Findings: We report here a complete biochemical and biophysical characterization of the M. tuberculosis CoaE, an enzyme essential for the pathogen's survival, elucidating for the first time the interactions of a dephosphocoenzyme A kinase with its substrates, dephosphocoenzyme A and ATP; its product, CoA and an intrinsic yet novel inhibitor, CTP, which helps modulate the enzyme's kinetic capabilities providing interesting insights into the regulation of CoaE activity. We show that the mycobacterial enzyme is almost 21 times more catalytically proficient than its counterparts in other prokaryotes. ITC measurements illustrate that the enzyme follows an ordered mechanism of substrate addition with DCoA as the leading substrate and ATP following in tow. Kinetic and ITC experiments demonstrate that though CTP binds strongly to the enzyme, it is unable to participate in DCoA phosphorylation. We report that CTP actually inhibits the enzyme by decreasing its Vmax. Not surprisingly, a structural homology search for the modeled mycobacterial CoaE picks up cytidylmonophosphate kinases, deoxycytidine kinases, and cytidylate kinases as close homologs. Docking of DCoA and CTP to CoaE shows that both ligands bind at the same site, their interactions being stabilized by 26 and 28 hydrogen bonds respectively. We have also assigned a role for the universal Unknown Protein Family 0157 (UPF0157) domain in the mycobacterial CoaE in the proper folding of the full length enzyme. Conclusions/Significance: In view of the evidence presented, it is imperative to assign a greater role to the last enzyme of Coenzyme A biosynthesis in metabolite flow regulation through this critical biosynthetic pathway.

Thermal expansion studies of substituted CTP derivatives

Several new Na, Y and Zr substituted derivatives of Ca-0.5 Ti-2(PO4)(3) (CTP) have been synthesized. These derivatives retain the hexagonal structure of the parent (CTP) compound with minor changes in lattice parameters. Linear thermal expansion coefficients (alpha) have been obtained using a high sensitivity dilatometer.

固定化假单胞菌CTP-01细胞分解对硫磷的研究

用明胶-戊二醛(GGA)和聚丙烯酰胺(PAA)包埋的固定化Pseudomonas sp.CTP-01细胞具有降解对硫磷的特性。GGA固定化细胞水解对硫磷的活力比PAA固定化细胞高5.8倍。当保存在4℃时GGA和PAA固定化细胞分别可以保持活力31.3和70%。GGA和PAA包埋的细胞最适反应温度分别为50℃到70℃和60℃到70℃,然而整细胞在温度超过65℃时活力很快下降。GGA和PAA两种固定化细胞最适pH为8.0,当pH低于7.0时活力开始下降,pH4吋则完全失活。

有机磷农药在水生态系中生物净化机理研究——2.CTP-02降解对硝基酚的特性和动力学

从鸭儿湖氧化塘中分离出具有分解对硝基酚能力的细菌Pseudomonas sp.,代号CTP-02。在实验室条件下,细菌培养物降解对硝基酚的速度与时间之间的动力学方程为dc/dt=-K_1t-K_2,细菌降解对硝基酚的最适温度为35℃,最适pH为7.5。CTP-02菌降解对硝基酚过程中首先发生脱硝基作用。

有机磷农药在水生态系中生物净化机理研究---- ----4. Pseudomonas sp. CTP-01 的对硫磷水解酶诱导合成性质

Pseudomonas sp.CTP-01的对硫磷水解酶具有底物诱导合成性质。停滞生长期的细胞接触底物半小时即产生相应酶的合成,而指数生长期的细胞接触底物48小时后才发生酶的合成。甲基对硫磷及对硝基酚也具有诱导作用,可见合成对硫磷水解酶的诱导特异基团可能与对硝基酚及其苯环上的取代基有密切关系。

Thiophenecarboxamide derivatives activated by EthA kill mycobacterium tuberculosis by inhibiting the CTP synthetase PyrG

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análisis e implementación de estrategias de rutado dinámico basado en CTP para redes de sensores inalámbricas multi-salto

Una red de sensores inalámbrica es un conjunto de dispositivos electrónicos que se comunican entre sí sin la necesidad de una infraestructura, recogiendo información del entorno en el que han sido desplegados, procesándola y transmitiéndola hasta una estación base mediante saltos sucesivos entre los nodos de la red (multi-salto). Durante las dos últimas décadas, este campo ha sido muy desarrollado en la comunidad científica, debido a las ventajas que ofrece el despliegue de una red inalámbrica en un entorno con el fin de estudiarlo y/o controlarlo. La ausencia de una infraestructura, junto con el reducido tamaño de los nodos, permite este estudio sin que dicho entorno se vea significativamente afectado por factores externos como pueda ser la presencia humana, permitiendo además aumentar el número de nodos que componen la red o cambiar la posición de algunos de ellos sin que sea necesario reconfigurarla manualmente. El principal reto que presentan las redes de sensores inalámbricas es su autonomía. En general, se requiere que un nodo tenga la capacidad de funcionar durante largos períodos de tiempo (varios meses o incluso un año) antes de que su batería se agote. Esto hace de la gestión del consumo energético un aspecto crítico en el diseño de la red y sus nodos. En el presente trabajo se busca optimizar este consumo mediante la gestión del proceso de comunicación y enrutamiento de la red. Con este fin, se implementa el protocolo CTP (Collection Tree Protocol) en la plataforma Cookies desarrollada en el Centro de Electrónica Industrial (CEI) de la UPM. CTP es un protocolo de rutado centrado en los datos, que utiliza una topología en árbol, con el nodo coordinador o estación base como raíz del mismo, para la transmisión de la información desde los sensores hasta la estación base. Además, no utiliza direcciones predeterminadas, dotando a la red de la flexibilidad requerida para hacer frente a inconsistencias y/o variaciones en la densidad y tamaño de la red. La ruta escogida se basa en un gradiente de rutado decreciente, ETX (Expected Transmission Count), que representa la calidad de la conexión entre un nodo y su nodo padre. Este gradiente de enrutamiento se obtiene mediante una conversión directa a partir del LQI (Link Quality Indication) definido por el estándar IEEE 802.15.4. Esta conversión directa supone una aproximación utilizando valores umbral del LQI. Un nodo escogerá el siguiente salto que realizará el paquete a enviar seleccionando de entre sus vecinos a aquél que tenga el menor ETX, evitando de esta forma la aparición de bucles. Otro de los aspectos que supone un gran consumo es el proceso de mantenimiento de la estructura de la red, pues requiere el envío periódico de señales de control o beacons a lo largo de toda la red. El protocolo CTP aprovecha el algoritmo de goteo (Trickle Algorithm), para gestionar el mantenimiento: durante la formación de la red y cuando se detecte alguna inconsistencia, se incrementa la frecuencia de emisión de los beacons, permitiendo así una rápida propagación de las señales de control para crear o reparar las conexiones entre los nodos. En cambio, cuando la topología de la red es estable, esta frecuencia de emisión se reduce significativamente, limitándose a asegurar que la topología se mantiene estable y favoreciendo así el ahorro de energía.

Trypanosoma brucei CTP synthetase: A target for the treatment of African sleeping sickness

The drugs in clinical use against African sleeping sickness are toxic, costly, or inefficient. We show that Trypanosoma brucei, which causes this disease, has very low levels of CTP, which are due to a limited capacity for de novo synthesis and the lack of salvage pathways. The CTP synthetase inhibitors 6-diazo-5-oxo-l-norleucine (DON) and α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) reduced the parasite CTP levels even further and inhibited trypanosome proliferation in vitro and in T. brucei-infected mice. In mammalian cells, DON mainly inhibits de novo purine biosynthesis, a pathway lacking in trypanosomes. We could rescue DON-treated human and mouse fibroblasts by the addition of the purine base hypoxanthine to the growth medium. For treatment of sleeping sickness, we propose the use of CTP synthetase inhibitors alone or in combination with appropriate nucleosides or bases.

AITPM national newsletter October 2008 president's report

It’s a pleasure for me to be penning my first President’s Message for the AITPM Newsletter. I am eagerly looking forward to serving the Institute and members over the coming couple of years. First though, I’d like to congratulate Andrew Hulse for steering the good ship AITPM over the past two years, bringing so many initiatives to the fore, including the Certified Transport Planner (CTP), stronger ties with other organisations and agencies such as IPENZ and Austroads, mutually beneficial sponsorship arrangements, and sharing his enthusiasm towards the Thunderbirds. Personally and largely thanks to my kids’ domination of the TV I’m a bit keener on the other great British sixties sci-fi classic, Doctor Who. Maybe we can generate a “favourite Doctor” dialogue in the Newsletter.

A nod to consent : in step with the UCPR

This article considers the decision of Robin DCJ in CTP Manager Limited v Ascent Pty Ltd [2011] QDC 74 and the likely impact of the decision on the practice in the court registries in similar circumstances.

Links between unlicensed and unregistered driving: Literature review. Report to Queensland Department of Transport and Main Roads

Vehicle registration represents an important component of the management of the road transport system in Queensland, with most vehicles required to be registered before they can be driven or parked on a public road (Department of Transport and Main Roads, 2010b). In addition to the collection of taxes for road construction and maintenance, the current registration system also: • Sets the safety standards required for vehicles to be allowed on public roads; • Allows driver behaviour to be managed by identifying vehicles, and the responsible owners of vehicles, for enforcement purposes; and • Facilitates the collection of insurance premiums for the Queensland Compulsory Third Party (CTP) insurance scheme.

Expression Profiling of Nucleotide Metabolism-Related Genes in Human Breast Cancer Cells After Treatment with 5-Fluorouracil

5-Fluorouracil (5-FU) is one of the most widely used drugs for treatment of cancers, including breast cancer that exhibits its anticancer activity by inhibiting DNA synthesis and also incorporated into DNA and RNA. The objective of this investigation was to find out the total nucleotide metabolism genes regulated by 5-FU in breast cancer cell line. The breast cancer cell line MCF-7 was treated with the drug 5-FU. To analyze the expression of genes, we have conducted the experiment using 1.7k and 19k human microarray slide and confirmed the expression of genes by semiquantitative reverse transcription-polymerase chain reaction. The expression of 44 genes involved in the nucleotide metabolism pathway was quantified. Of these 44 genes analyzed, transcription of 6 genes were upregulated and 9 genes were downregulated. Earlier studies revealed that the transcription of genes for key enzymes like thymidylate synthase, thymidinekinase, and dihydropyrimidine dehydrogenase are regulated by 5-FU. This study identified some novel genes like thioredoxin reductase, ectonucleotide triphosphate dephosphorylase, and CTP synthase are regulated by 5-FU. The data also reveal large-scale perturbation in transcription of genes not involved directly in the known mechanism of action of 5-FU.

Molecular pathogenesis of human parechovirus 1 infection

The Parechoviruses (HPEV) belong to the family Picornaviridae of positive-stranded RNA viruses. Although the parechovirus genome shares the general properties of other picornaviruses, the genus has several unique features when compared to other family members. We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein was found to be a RNA-binding protein that preferably binds to positive sense 3 UTR RNA. It was found to bind also duplex RNA containing 3 UTR(+)-3 UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Neither dATP nor other NTP:s were suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. The intracellular changes brought about by the individual HPEV1 non-structural proteins were studied through the expression of fusion proteins. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein, which could be found on the surface of lipid droplets but also on diverse intracellular membranes, was partly relocated to viral replication complexes in transfected, superinfected cells. Although Golgi to ER traffic was arrested in HPEV1-infected cells, none of the individually expressed non-structural proteins had any visible effect on the anterograde membrane traffic. Our results suggest that the HPEV1 replication strategy is different from that of many other picornaviruses. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.