Baccatin III induces assembly of purified tubulin into long microtubules

Baccatin III is widely considered to be an inactive derivative of Taxol. We have reexamined its effect on in vitro assembly of tubulin under a variety of conditions. We found baccatin III to be active in all circumstances in which Taxol is active: it assembled GTP-tubulin, GDP-tubulin, and microtubule protein into normal microtubules and stabilized these polymers against cold-induced disassembly. The effect of baccatin III on in vitro microtubule assembly was quantitatively assessed through determination of critical concentrations, which can be used to obtain the apparent equilibrium constants for the addition of tubulin subunits to growing microtubules. The apparent equilibrium constants for the growth reaction for baccatin III-induced GTP-tubulin and GDP-tubulin assembly measured at 37 degrees C were 4.2-4.6-fold less than those measured for Taxol-induced GTP-tubulin and GDP-tubulin assembly. These data indicate that the entire Taxol side chain contributes only about -1 kcal/mol to the apparent standard free energy of microtubule growth at 37 degrees C regardless of the nature of the E site nucleotide. These data also support the idea that the majority of the interactions between Taxol and tubulin that affect this equilibrium occur between the baccatin portion of the molecule and the binding site. We have also observed a structural difference in microtubules formed using baccatin III and Taxol. Baccatin III-induced microtubules were routinely much longer than those assembled by Taxol, even when very high concentrations of baccatin III were employed. One interpretation of these data is that baccatin III and Taxol differ in their abilities to nucleate GTP-tubulin. This difference in activity may have bearing on the large disparity in cytotoxicity of the two molecules.     

Bio-production of Baccatin III,an Important Precursor of Paclitaxel by a Cost-Effective Approach

Natural production of anti-cancer drug taxol from Taxus has proved to be environmentally unsustainable and economically unfeasible. Currently, bioengineering the biosynthetic pathway of taxol is an attractive alternative production approach. 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT) was previously characterized as an acyltransferase, using 10-deacetylbaccatin III (10-DAB) and acetyl CoA as natural substrates, to form baccatin III in the taxol biosynthesis. Here, we report that other than the natural acetyl CoA (Ac-CoA) substrate, DBAT can also utilize vinyl acetate (VA), which is commercially available at very low cost, acylate quickly and irreversibly, as acetyl donor in the acyl transfer reaction to produce baccatin III. Furthermore, mutants were prepared via a semi-rational design in this work. A double mutant, I43S/D390R was constructed to combine the positive effects of the different single mutations on catalytic activity, and its catalytic efficiency towards 10-DAB and VA was successfully improved by 3.30-fold, compared to that of wild-type DBAT, while 2.99-fold higher than the catalytic efficiency of WT DBAT towards 10-DAB and Ac-CoA. These findings can provide a promising economically and environmentally friendly method for exploring novel acyl donors to engineer natural product pathways.     

Dynamics of Concomitant Field Populations of Hoplolaimus columbus and Meloidogyne incognita

From the fall of 1968 through the summer of 1973, a Georgia cotton field with a lengthy history of the Cotton Stunt Disease Complex was sampled for the presence of plant parasitic nematodes. Although Meloidogyne incognita was recovered on all sampling dates, concomitant populations of Hoplolaimus columbus were not recovered until the spring of 1970. During the succeeding four growing seasons, the population density and horizontal distribution of H. columbus increased, and H. columbus replaced M. incognita as the predominant phytopathogenie species. A second Georgia cotton field containing concomitant populations of H. columbus and M. incognita was observed from the fall of 1971 through the summer of 1973. In this case the horizontal distribution of both species remained relatively constant and the population density of H. columbus increased steadily. In both locations, the presence of either H. columbus or M. incognita significantly inhibited the presence of the concomitant species. In general, however, the initial spring or final fall population densities of H. columbus or M. incognita had no significant influence on the population density of the concomitant species, The data are also discussed in relation to the biological significance of H. columbus in the southeastern coastal plain.     

Fermentation and isolation of C10-deacetylase for the production of 10-deacetylbaccatin III from baccatin III

10-Deacetylabaccatin III (10 DAB), an important precursor for paclitaxel semisynthesis, is enhanced in yew extracts using C10-deacetylase and C13-deacylase enzymes.(4) C10-deacetylase is an intracellular enzyme produced by the fermentation of a soil microorganism, Nocardioides luteus (SC 13912). During the fermentation of Nocardioides luteus, the growth of cells reaches a maximum growth at 28 h. C10-deacetylase enzyme activity starts at 26 h and peaks at 38 h of the fermentation. The cells are recovered by centrifugation. The C10-deacetylase enzyme was purified from the Nocardioides luteus cells. The enzyme was purified 190-fold to near homogeneity. The purified enzyme appeared as a single band on 12.5% SDS-PAGE analysis with a molecular weight of 40,000 daltons. (c) 1995 John Wiley & Sons, Inc.     

Effects of Concomitant Development on Reproduction of Meloidogyne incognita and Rotylenchulus reniformis on Sweet Potato

The influence of various factors on reproduction of concomitant Meloidogyne incognita (Mi) and Rotylenchulus reniformis (Rr) on sweet potato were studied in the greenhouse. Reproduction of Rr was reduced by Mi at all inoculum levels and experiment durations used, while Mi reproduction was not inhibited. Both species failed to affect each other when inoculated simultaneously onto root systems developed in separate pots from different nodes of the same plant. Reproduction of each species was not significantly greater when inoculation of the second species was delayed 1-2 weeks compared to simultaneous inoculation. After shoot excision, Rr increased in the soil but Mi decreased. Fibrous root weights of plants inoculated with Rr + Mi in some tests were higher than those inoculated with Mi alone, indicating an early suppression of Mi and/or root stintulation by Rr. Drought stress delayed Rr egg hatching and movement of larvae into the soil, but had little effect on Mi reproduction.     

Enzymatic acetylation of 10-deacetylbaccatin III to baccatin III by C-10 deacetylase from Nocardioides luteus SC 13913

Baccatin III is a polycyclic diterpene which can be used for the semi-synthesis of paclitaxel and analogs. An enzymatic process was developed for the conversion of 10-deacetylbaccatin III (10-DAB) to baccatin III without requiring protection of the 7-hydroxyl group. A C-10 deacetylase from Nocardioides luteus SC 13912 was immobilized on diethylaminoethyl cellulose (DEAE-Cellulose) and the immobilized enzyme was used in the biotransformation process. The reaction was catalyzed using vinyl acetate as acyl donor at ambient temperature and at pH 7. A reaction yield of 51% was obtained.     

Infection of Seedlings of Alfalfa and Red Clover by Concomitant Populations of Meloidogyne incognita and Pratylenchus penetrans

Invasion of 2-day-old seedlings of ''Buffalo'' alfalfa and ''Kenland'' red clover by larvae of M. incognita and adults of P. penetrans, during 1-3 day periods of incubation at 24 C, was investigated in 50-mm petri dishes on 1% agar. Penetration by both nematodes increased arithmetically with increased numbers in inocula. P. penetrans invaded alfalfa more readily than red cover, but M. incognita invaded red clover more readily than alfalfa. Both nematodes inhibited root-elongation of alfalfa more than that of red clover. In combinations of 10 and 50 of both nematodes, invasion of both plants by both nematodes was the same as for each nematode alone. Penetration by M. incognita into alfalfa, but not into red clover, was significantly reduced when combinations of 50 M. incognita and 200 P. penetrans were inoculated simultaneously. In the presence of large numbers of entrant P. penetrans in both plants, penetration by M. incognita was highly significantly reduced. Penetration by P. penetrans was unaffected in the reciprocal situations.     

Trichomonas Transmembrane Cyclases Result from Massive Gene Duplication and Concomitant Development of Pseudogenes

Background

Trichomonas vaginalis has an unusually large genome (∼160 Mb) encoding ∼60,000 proteins. With the goal of beginning to understand why some Trichomonas genes are present in so many copies, we characterized here a family of ∼123 Trichomonas genes that encode transmembrane adenylyl cyclases (TMACs).

Methodology/Principal Findings

The large family of TMACs genes is the result of recent duplications of a small set of ancestral genes that appear to be unique to trichomonads. Duplicated TMAC genes are not closely associated with repetitive elements, and duplications of flanking sequences are rare. However, there is evidence for TMAC gene replacements by homologous recombination. A high percentage of TMAC genes (∼46%) are pseudogenes, as they contain stop codons and/or frame shifts, or the genes are truncated. Numerous stop codons present in the genome project G3 strain are not present in orthologous genes of two other Trichomonas strains (S1 and B7RC2). Each TMAC is composed of a series of N-terminal transmembrane helices and a single C-terminal cyclase domain that has adenylyl cyclase activity. Multiple TMAC genes are transcribed by Trichomonas cloned by limiting dilution.

Conclusions/Significance

We conclude that one reason for the unusually large genome of Trichomonas is the presence of unstable families of genes such as those encoding TMACs that are undergoing massive gene duplication and concomitant development of pseudogenes.     

Influence of Concomitant Pratylenchus brachyurus and Meloidogyne spp. on Root Penetration and Population Dynamics

Populations of Pratylenchus brachyurus on cotton were increased significantly in the presence of either Meloidogyne incognita or M. arenaria.This occurred with either simultaneous inoculation or prior invasion by M. incognita. P. brachyurus penetrated cotton roots previously invaded by, or simultaneously inoculated with, M. incognita, as well as, or better than, in the absence of M. incognita. Prior invasion by M. incognita, however, suppressed P. brachyurus populations on tomato, while it had no effect on alfalfa and tobacco. Populations of M. incognita on cotton were generally inhibited by the presence of P. brachyurus. Simultaneous inoculation with, or previous invasion by, P. brachyurus also inhibited root penetration by M. incognita. These findings emphasize the importance of host susceptibility in the study of concomitant nematode populations.     

Dynamics of Concomitant Populations of Hoplolaimus columbus,Scutellonema brachyurum,and Meloidogyne incognita on Cotton

Cotton seedlings grown in a greenhouse and a growth chamber were inoculated with Scutellonema brachyurum, Hoplolaimus columbus, and Meloidogyne incognita, singly and in all possible combinations, at two initial population (Pi) levels (100 and 300/100 cm³). S. brachyurum alone was not pathogenic to cotton at these population levels. It fed primarily as an ectoparasite but matured and reproduced within the root when it penetrated. Populations of S. brachyurum increased in the presence of H. columbus but were suppressed by M. incognita. H. columbus suppressed dry shoot weights of cotton (P = 0.05) at a Pi of 300/100 cm³ soil. Simultaneous inoculation of H. columbus with either M. incognita or S. brachyurum increased H. columbus populations over treatments with H. columbus alone, both at 60 and 90 d after inoculation. M. incognita suppressed cotton shoot weights significantly (P = 0.05) at both Pi levels. Inoculation with S. brachyurum increased M. incognita populations 60 d after inoculation, while H. columbus suppressed populations of M. incognita. Most larvae of M. incognita did not develop to maturity in the presence of H. columbus. Giant cells aborted and were necrotic 20-25 d after inoculation. Since M. incognita and H. columbus feed on different tissues, the inhibition of M. incognita may have resulted from a physiological effect of H. columbus on the host.     

Nucleosomal DNA is digested to repeats of 10 bases by exonuclease III

Nucleosomes were treated with increasing concentrations of exonuclease III (Exo III) from E. coli. At low levels of Exo III, the heterogeneous distribution of monomers (with associated DNA fragments ranging in size between 140 and 170 bp) is "trimmed" down to a discrete core of 140 bp. The "trimming" of monomers to 140 bp results from a 3' exonucleolytic digestion accompanied by a 5' clipping activity which is specific for the conformation of internucleosomal DNA. At higher concentrations of Exo III, the enzyme digests the 140 bp "trimmed" nucleosome core from both 3' ends without associated 5' nuclease activity. Most striking is the observation that the fragments produced during such a digestion display discrete single-stranded lengths that are integer multiples of 10 bases. For some dimer nucleosomes, Exo III can digest as many as 200 bases from at least one 3' end and produce a 10 base interval ladder from about 400 bases down to 180 bases. This suggests that the enzyme can traverse the length of an entire nucleosome without destroying whatever structural features are necessary to produce a 10 base DNA ladder.     

Identification of Novel Type III Secretion Chaperone-Substrate Complexes of Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of humans that uses a type III secretion (T3S) system to manipulate host cells through the delivery of effector proteins into their cytosol and membranes. The function of T3S systems depends on small bacterial cytosolic chaperone-like proteins, which bind T3S substrates and ensure their appropriate secretion. To find novel T3S chaperone-substrate complexes of C. trachomatis we first searched its genome for genes encoding proteins with features of T3S chaperones. We then systematically tested for interactions between candidate chaperones and chlamydial T3S substrates by bacterial two-hybrid. This revealed interactions between Slc1 (a known T3S chaperone) or CT584 and several T3S substrates. Co-immunoprecipation after protein expression in Yersinia enterocolitica and protein overlay binding assays indicated that Slc1 interacted with the N-terminal region of the known T3S substrates Tarp (a previously described substrate of Slc1), CT694, and CT695, and that CT584 interacted with a central region of CT082, which we identified as a C. trachomatis T3S substrate using Y. enterocolitica as a heterologous system. Further T3S assays in Yersinia indicated that Slc1 or CT584 increased the amount of secreted Tarp, CT694, and CT695, or CT082, respectively. Expression of CT584 increased the intra-bacterial stability of CT082, while Slc1 did not affect the stability of its substrates. Overall, this indicated that in C. trachomatis Slc1 is a chaperone of multiple T3S substrates and that CT584 is a chaperone of the newly identified T3S substrate CT082.