Transcriptional analysis of pqqD and study of the regulation of pyrroloquinoline quinone biosynthesis in Methylobacterium extorquens AM1

Methanol dehydrogenase, the enzyme that oxidizes methanol to formaldehyde in gram-negative methylotrophs, contains the prosthetic group pyrroloquinoline quinone (PQQ). To begin to analyze how the synthesis of PQQ is coordinated with the production of other methanol dehydrogenase components, the transcription of one of the key PQQ synthesis genes has been studied. This gene (pqqD) encodes a 29-amino- acid peptide that is thought to be the precursor for PQQ biosynthesis. A unique transcription start site was mapped to a guanidine nucleotide 95 bp upstream of the pqqD initiator codon. RNA blot analysis identified two transcripts, a major one of 240 bases encoding pqqD and a minor one of 1,300 bases encoding pqqD and the gene immediately downstream, pqqG. Both transcripts are present at similar levels in cells grown on methanol and on succinate, but the levels of PQQ are about fivefold higher in cells grown on methanol than in cells grown on succinate. These results suggest that PQQ production is regulated at a level different from the transcription of pqqD. The genes mxbM, mxbD, mxcQ, mxcE, and mxaB are required for transcription of the genes encoding the methanol dehydrogenase subunits and were assessed for their role in PQQ production. PQQ levels were measured in mutants defective in each of these regulatory genes and compared with levels of pqqD transcription, measured with a transcriptional fusion between the pqqD promoter and xylE. The results showed that only a subset of these regulatory genes (mxbM, mxbD, and mxaB) is required for transcription of pqqD, and only mxbM and mxbD mutants affected the final levels of PQQ significantly

Genetics and Regulation of Pyrroloquinoline Quinone (PQQ) Biosynthesis in the Methylotrophic Bacterium Methylobacterium extorquens AM1

Pyrroloquinoline quinone (PQQ) has potential applications in the treatment of Alzheimers disease and jaundice. PQQ is produced and secreted in large amounts by methylotrophic bacteria, and they have potential for commercial PQQ production. PQQ is the prosthetic group in methanol dehydrogenase of the facultative methylotroph, Methylobacterium extorquens AM1. Previous work had shown that seven genes were required for PQQ production in M.extorquens AM1. Dr. Christina Morris in our lab had sequenced the first two genes, pqqD and pqqG. pqqD encoded a twenty-nine amino acid peptide containing tyrosine and glutamate separated by three amino acids. Tyrosine and glutamate had been shown to be the precursors of PQQ, and this peptide is believed to be the precursor of PQQ biosynthesis in vivo. This thesis was initiated by mapping the transcriptional start site in front of pqqD. A high abundance 240 base pair transcript containing pqqD and a low abundance 1300 base pair transcript containing both pqqD and the next gene pqqG were detected using RNA-DNA blots. This was supported by data with Tn5lac insertions. Studies were carried out to understand the regulation of PQQ biosynthesis in more detail. It was found that the products of the regulatory genes moxM, moxD and moxB were required for induction of pqqD on methanol plus methylamine compared to succinate, while the products of the regulatory genes moxQ and moxE were not required. The products of moxM and moxD were also required for high level transcription of pqqD. Measurements of PQQ in the culture supernatants of wild-type and mutants indicated that transcription of pqqD was not the rate limiting step in PQQ biosynthesis, and in addition that regulation of PQQ biosynthesis occurs at more than one step in the process. Using functional complementation it was found that pqqE of M.extorquens AM1 was equivalent to pqqF of K.pneumoniae. The middle portion of pqqE was sequenced and by aligning PqqE with PqqF of K.pneumoniae, the direction of transcription and size of pqqE was determined. PqqF shows similarity to proteases and this strongly suggests that a protease is involved in PQQ biosynthesis.</p

Provisional Patent Application: Hybrid System Modeling of Human Blood Clotting

The process of blood coagulation in mammals is complicated, and involves the interaction of more than a dozen coagulation factors as well as a number of proteins from the kininkallikrein system and protein inhibitors. Attempts to model coagulation mathematically have previously focused on a smaller subset of interactions, perhaps one of the pathways or just a portion of one of them (cf. [1],[2], [3],[4],[5], [6] and [7]) using continuous-time chemical rate differential equations. There are, however, at least two reasons why this methodology is inadequate for modeling the entire coagulation cascade. First, there is reason to believe [8] that certain events in the cascade are better modeled by punctuated phase changes, rather than as evolving continuously. For example, antithrombin III and α2-macroglobulin are only able to inhibit thrombin activation below a certain threshold of thrombin [9]. Similarly, concentrations of free zinc ions are thought to toggle the activation of several of the proteins of the contact activation portion of the clotting cascade [10],[11]. Second, the problem with modeling coagulation as a purely continuous-time phenomenon is that the process is too complicated (with thresholds and discontinous phase changes) to permit a precise description of the various parameters and their interactions in terms of differential equations. Our invention is a novel modeling framework and software program which constructs a robust, faithful, interactive, and graphical computer simulation of the entire coagulation process. The framework is faithful in that it accurately models mammalian blood clotting; robust in the sense of doing so over a wide range of parameter settings; interactive in that the system operation and parameter settings can be interactively changed while the software program is executing, and hypothetical “what-if ” simulations performed; and graphical in that the model is a formal graphical structure that supports visualization of the clotting process as well as exact quantitative analysis. Technically, the invention uses an emerging alternative modeling paradigm in which the coagulation cascade is viewed as a “hybri

WIPO Re:Search: Accelerating anthelmintic development through cross-sector partnerships

AbstractNeglected tropical diseases (NTDs), malaria, and tuberculosis have a devastating effect on an estimated 1.6billion people worldwide. The World Intellectual Property Organization (WIPO) Re:Search consortium accelerates the development of new drugs, vaccines, and diagnostics for these diseases by connecting the assets and resources of pharmaceutical companies, such as compound libraries and expertise, to academic or nonprofit researchers with novel product discovery or development ideas. As the WIPO Re:Search Partnership Hub Administrator, BIO Ventures for Global Health (BVGH) fields requests from researchers, identifies Member organizations able to fulfill these requests, and helps forge mutually beneficial collaborations. Since its inception in October 2011, WIPO Re:Search membership has expanded to more than 90 institutions, including leading pharmaceutical companies, universities, nonprofit research institutions, and product development partnerships from around the world. To date, WIPO Re:Search has facilitated over 70 research agreements between Consortium Members, including 11 collaborations focused on anthelmintic drug discovery

Fighting Plasmodium chloroquine resistance with acetylenic chloroquine analogues

Malaria is among the tropical diseases that cause the most deaths in Africa. Around 500,000 malaria deaths are reported yearly among African children under the age of five. Chloroquine (CQ) is a low-cost antimalarial used worldwide for the treatment of Plasmodium vivax malaria. Due to resistance mechanisms, CQ is no longer effective against most malaria cases caused by P. falciparum. The World Health Organization recommends artemisinin combination therapies for P. falciparum malaria, but resistance is emerging in Southeast Asia and some parts of Africa. Therefore, new medicines for treating malaria are urgently needed. Previously, our group identified the 4-aminoquinoline DAQ, a CQ analog containing an acetylenic bond in its side chain, which overcomes CQ resistance in K1 P. falciparum strains. In this work, the antiplasmodial profile, drug-like properties, and pharmacokinetics of DAQ were further investigated. DAQ showed no cross-resistance against standard CQ-resistant strains (e.g., Dd2, IPC 4912, RF12) nor against P. falciparum and P. vivax isolates from patients in the Brazilian Amazon. Using drug pressure assays, DAQ showed a low propensity to generate resistance. DAQ showed considerable solubility but low metabolic stability. The main metabolite was identified as a mono N-deethylated derivative (DAQM), which also showed significant inhibitory activity against CQ-resistant P. falciparum strains. Our findings indicated that the presence of a triple bond in CQ-analogues may represent a low-cost opportunity to overcome known mechanisms of resistance in the malaria parasite