Production of taxol from Phyllosticta dioscoreae, a leaf spot fungus isolated from Hibiscus rosa-sinensis

Taxol is a highly functionalized anticancer drug widely used in hospitals and clinics. The leaf spot fungus, Phyllosticta dioscoreae was isolated from diseased leaves of Hibiscus rosa-sinensis and screened for extracellular production of taxol in M1D (Modified liquid medium) and PDB (Potato dextrose broth) medium for the first time. The fungus was identified by its morphological and conidial features in the culture growth. The presence of taxol in the fungal culture filtrate was confirmed by different spectroscopic and chromatographic analyses. The amount of taxol produced was quantified by HPLC. The maximum amount of taxol produced was found to be 298 μg/L in M1D medium. Production rate was 5.96 × 10³ times faster than that found in culture broth of earlier reported fungus, Taxomyces andreanae. The extracted fungal taxol also showed strong cytotoxic activity in vitro in the cultures of human cancer cells tested by apoptotic assay. The results indicate that P. dioscoreae is an excellent source of taxol production, which suggests that the fungus has potential to undergo genetic engineering in order to improve its production level.     

Prediction of Candidate Primary Immunodeficiency Disease Genes Using a Support Vector Machine Learning Approach

Screening and early identification of primary immunodeficiency disease (PID) genes is a major challenge for physicians. Many resources have catalogued molecular alterations in known PID genes along with their associated clinical and immunological phenotypes. However, these resources do not assist in identifying candidate PID genes. We have recently developed a platform designated Resource of Asian PDIs, which hosts information pertaining to molecular alterations, protein–protein interaction networks, mouse studies and microarray gene expression profiling of all known PID genes. Using this resource as a discovery tool, we describe the development of an algorithm for prediction of candidate PID genes. Using a support vector machine learning approach, we have predicted 1442 candidate PID genes using 69 binary features of 148 known PID genes and 3162 non-PID genes as a training data set. The power of this approach is illustrated by the fact that six of the predicted genes have recently been experimentally confirmed to be PID genes. The remaining genes in this predicted data set represent attractive candidates for testing in patients where the etiology cannot be ascribed to any of the known PID genes.     

Yersinia yopQ mRNA encodes a bipartite type III secretion signal in the first 15 codons

The type III machinery of Yersinia transports Yop proteins across the bacterial envelope. The minimal secretion signal of yopQ is located in codons 1-10 that, when fused in frame to the neomycin phosphotransferase gene, is sufficient to promote type III secretion of YopQ(1-10)-Npt. Frame-shift mutations, generated by nucleotide insertions or deletions following the AUG start and suppressed at the fusion site with npt, abrogate signalling of yopQ(1-10) but not of yopQ(1-15). By generating transversions of every single nucleotide in yopQ(1-10), we identified 10 nucleotide positions in codons 2, 3, 5, 7, 9 and 10 that were each required for substrate recognition. One transversion that abolishes secretion, uridyl 9 to adenyl (U9A), is a synonymous codon 3 mutation that retains the original amino acid as confirmed by Edman degradation analysis, suggesting that the mRNA but not the amino acid sequence of yopQ(1-10) is involved in secretion signalling. Although transversion of U8A abrogates signalling of yopQ(1-10), fusion of yopQ codons 11-15 restores secretion. The nucleotides that are required for this suppression by yopQ(11-15) were identified and revealed both synonymous and non-synonymous mutations. Frame-shift mutations introduced into just this suppressor region (codons 11-15) did not abrogate its ability to suppress mutations in the minimal secretion signal (codons 1-10). Thus, elements downstream of the minimal secretion signal of YopQ increase the efficiency of YopQ secretion and suppress mutations elsewhere in the secretion signal.     

Low background,pulsatile, in vitro flow circuit for modeling coronary implant thrombosis

We have developed an in vitro method for creating pulsatile flows to mimic coronary type flow patterns on a beat-to-beat basis. The flow is created by accelerating fluid loops about an axis, inducing relative wall motion. Using this technique, a variety of oscillating flow patterns can be generated and modulated. Such flow generation offers the potential to monitor sensitive, flow-dependent, biological parameters like thrombosis while minimizing background disturbances from pump action and circuit effects. We examined this potential by measuring the loop occlusion time for loops stented with stainless steel 7-9 NIR stents and stentless control loops.     

Superoxide-mediated activation of cardiac fibroblasts by serum factors in hypomagnesemia

Magnesium deficiency is known to produce myocardial fibrosis in different animal models, but the underlying mechanisms are unclear. However, circulating levels of pro-oxidant and mitogenic factors are reported to be elevated in a rodent model of acute magnesium deficiency, suggesting a role for humoral factors in the pathogenesis of the cardiovascular lesions. Probing the mechanism of cardiac fibrogenesis in magnesium deficiency, the present study furnished evidence that serum from magnesium-deficient rats has a more marked effect than serum from magnesium-sufficient rats on mitogenesis, net collagen production, and superoxide generation in cardiac fibroblasts from young adult rats. The enhanced mitogenic response was abolished by superoxide dismutase and N-acetyl cysteine, showing that it is mediated by superoxide anion. Further, a modest inhibitory effect of the neurokinin-1 receptor antagonist, spantide, suggested that factors acting via neurokinin-1 receptors may partly modulate cardiac fibroblast function in magnesium deficiency. The findings are consistent with the postulation that serum factors may activate cardiac fibroblasts via a superoxide-mediated mechanism and contribute to the fibrogenic response in the heart in magnesium deficiency.     

Structure and mechanism of ADP-ribose-1''-monophosphatase (Appr-1''-pase), a ubiquitous cellular processing enzyme

Appr-1'-pase, an important and ubiquitous cellular processing enzyme involved in the tRNA splicing pathway, catalyzes the conversion of ADP-ribose-1'monophosphate (Appr-1'-p) to ADP-ribose. The structures of the native enzyme from the yeast and its complex with ADP-ribose were determined to 1.9 A and 2.05 A, respectively. Analysis of the three-dimensional structure of this protein, selected as a target in a structural genomics project, reveals its putative function and provides clues to the catalytic mechanism. The structure of the 284-amino acid protein shows a two-domain architecture consisting of a three-layer alphabetaalpha sandwich N-terminal domain connected to a small C-terminal helical domain. The structure of Appr-1'-pase in complex with the product, ADP-ribose, reveals an active-site water molecule poised for nucleophilic attack on the terminal phosphate group. Loop-region residues Asn 80, Asp 90, and His 145 may form a catalytic triad.     

SAR of psilocybin analogs: discovery of a selective 5-HT 2C agonist

An SAR study of psilocybin and psilocin derivatives reveals that 1-methylpsilocin is a selective agonist at the h5-HT(2C) receptor. The corresponding phosphate derivative, 1-methylpsilocybin, shows efficacy in an animal model for obsessive-compulsive disorder, as does 4-fluoro-N,N-dimethyltryptamine. These results suggest a new area for development of novel 5-HT(2C) agonists with applications for drug discovery.     

Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement

The rapid evolution of sexual isolation in sympatry has long been associated with reinforcement (i.e., selection to avoid maladaptive hybridization). However, there are many species pairs in sympatry that have evolved rapid sexual isolation without known costs to hybridization. A major unresolved question is what evolutionary processes are involved in driving rapid speciation in such cases. Here, we focus on one such system; the Drosophila athabasca species complex, which is composed of three partially sympatric and interfertile semispecies: WN, EA, and EB. To study speciation in this species complex, we assayed sexual and genomic isolation within and between these semispecies in both sympatric and allopatric populations. First, we found no evidence of reproductive character displacement (RCD) in sympatric zones compared to distant allopatry. Instead, semispecies were virtually completely sexually isolated from each other across their entire ranges. Moreover, using spatial approaches and coalescent demographic simulations, we detected either zero or only weak heterospecific gene flow in sympatry. In contrast, within each semispecies we found only random mating and little population genetic structure, except between highly geographically distant populations. Finally, we determined that speciation in this system is at least an order of magnitude older than previously assumed, with WN diverging first, around 200K years ago, and EA and EB diverging 100K years ago. In total, these results suggest that these semispecies should be given full species status and we adopt new nomenclature: WN—D. athabasca, EA—D. mahican, and EB—D. lenape. While the lack of RCD in sympatry and interfertility do not support reinforcement, we discuss what additional evidence is needed to further decipher the mechanisms that caused rapid speciation in this species complex.