Validated HPLC Method for the Determination of Paclitaxel-related Substances in an Intravenous Emulsion Loaded with a Paclitaxel-Cholesterol Complex.

A high-performance liquid chromatography method was developed for the determination of related substances in an intravenous emulsion loaded with a paclitaxel-cholesterol complex. The separation was achieved using Agilent Eclipse XDB-C18 column (150×4.6 mm, 3.5 µm), which was kept at 40°. The gradient mobile phase consisted of acetonitrile and water with a flow rate of 1.2 ml/min. The ultraviolet detection wavelength was set at 227 nm. The preparation of the sample solution began with the addition of anhydrous sodium sulphate to break the emulsion. Then, methanol and ethyl ether were added to pick up the drug and remove the accessories of the emulsion by extraction and centrifugation. Finally, paclitaxel was enriched by a nitrogen blow method and resolved with a mixture of methanol:glacial acetic acid (200:1). The method was proven to be selective, sensitive, robust, linear, repeatable, accurate and suitable for the determination of paclitaxel-related substances in the emulsion formulations, and the major degradation products in the potential pharmaceutical product were 7-epipaclitaxel and 10-deacetylpaclitaxel.

Kimura disease: review of the literature.

Kimura disease (KD) is a rare, chronic inflammatory disease of unknown cause and is characterized by painless s.c. swellings and lymphadenopathy commonly affecting the head and neck region. Much therapeutics has been used to treat KD, but is not satisfactory because of frequent relapse. Imatinib has been reported previously to be useful for treatment of hypereosinophilic syndrome and may work by selectively blocking protein-tyrosine kinases, such as platelet-derived growth factor receptor, and c-Kit. We carried out immunohistochemical examination of platelet-derived growth factor receptor-alpha and c-Kit in tissues from patients with KD. The results were positive and suggested that Imatinib might be an effective drug for the treatment of the disease. We have also briefly reviewed the epidemiology, aetiology, clinical manifestations, laboratory and pathological examinations, differential diagnoses, treatment and prognosis of KD in this manuscript.

Phenolic glycosides from leaves of Hopiciopsis lobata.

A new phenolic glycoside, 6'-[(E)-2''-hydroxymethyl, 2''-butenoyl] arbutin (1), and two known phenolic glycosides, 6'-[(E)-4''-hydroxycinnamoyl] arbutin (2) and 6'-[(E)-3'',4''-dihydroxycinnamoyl] arbutin (3), were isolated from the leaves of Heliciopsis lobata (Merr.) Sleum. Their structures were elucidated by various spectroscopic methods including 2D NMR spectroscopy.

RapA, a bacterial homolog of SWI2/SNF2, stimulates RNA polymerase recycling in transcription.

We report that RapA, an Escherichia coli RNA polymerase (RNAP)-associated homolog of SWI2/SNF2, is capable of dramatic activation of RNA synthesis. The RapA-mediated transcriptional activation in vitro depends on supercoiled DNA and high salt concentrations, a condition that is likely to render the DNA superhelix tightly compacted. Moreover, RapA activates transcription by stimulating RNAP recycling. Mutational analyses indicate that the ATPase activity of RapA is essential for its function as a transcriptional activator, and a rapA null mutant exhibits a growth defect on nutrient plates containing high salt concentrations in vivo. Thus, RapA acts as a general transcription factor and an integral component of the transcription machinery. The mode of action of RapA in remodeling posttranscription or posttermination complexes is discussed.

Growth phase and growth rate regulation of the rapA gene, encoding the RNA polymerase-associated protein RapA in Escherichia coli.

The Escherichia coli rapA gene encodes the RNA polymerase (RNAP)-associated protein RapA, which is a bacterial member of the SWI/SNF helicase-like protein family. We have studied the rapA promoter and its regulation in vivo and determined the interaction between RNAP and the promoter in vitro. We have found that the expression of rapA is growth phase dependent, peaking at the early log phase. The growth phase control of rapA is determined at least by one particular feature of the promoter: it uses CTP as the transcription-initiating nucleotide instead of a purine, which is used for most E. coli promoters. We also found that the rapA promoter is subject to growth rate regulation in vivo and that it forms intrinsic unstable initiation complexes with RNAP in vitro. Furthermore, we have shown that a GC-rich or discriminator sequence between the -10 and +1 positions of the rapA promoter is responsible for its growth rate control and the instability of its initiation complexes with RNAP.

Nucleoid proteins stimulate stringently controlled bacterial promoters: a link between the cAMP-CRP and the (p)ppGpp regulons in Escherichia coli.

We report that the H-NS nucleoid protein plays a positive role in the expression of stringently regulated genes in Escherichia coli. Bacteria lacking both H-NS and the paralog StpA show reduced growth rate. Colonies displaying an increased growth rate were isolated, and mapping of a suppressor mutation revealed a base pair substitution in the spoT gene. The spoT(A404E) mutant showed low ppGpp synthesizing ability. The crp gene, which encodes the global regulator CRP, was subject to negative stringent regulation. The stable RNA/protein ratio in an hns, stpA strain was decreased, whereas it was restored in the suppressor strain. Our findings provide evidence of a direct link between the cAMP-CRP modulon and the stringent response.

Interaction between RNA polymerase and RapA, a bacterial homolog of the SWI/SNF protein family.

Recently, we identified a novel Escherichia coli RNA polymerase (RNAP)-associated protein, an ATPase, called RapA (Sukhodolets, M. V. , and Jin, D. J. (1998) J. Biol. Chem. 273, 7018-7023). RapA is a bacterial homolog of SWI2/SNF2. We showed that RapA forms a stable complex with RNAP holoenzyme and that binding to RNAP holoenzyme stimulates the ATPase activity of RapA. We have further analyzed the interactions between purified RapA and the two forms of RNAP: core RNAP and RNAP holoenzyme. We found that RapA interacts with either form of RNAP. However, RapA exhibits higher affinity for core RNAP than for RNAP holoenzyme. Chemical cross-linking of the RNAP-RapA complex indicated that the RapA-binding sites are located at the interface between the alpha and beta' subunits of RNAP. Contrary to previously reported results (Muzzin, O., Campbell, E., A., Xia, L., Severinova, E., Darst, S. A., and Severinov, K. (1998) J. Biol. Chem. 273, 15157-15161), our in vivo analysis of a rapA null mutant suggested that RapA is not likely to be directly involved in DNA repair.

The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like "stringent" RNA polymerases in Escherichia coli.

In Escherichia coli, stringently controlled genes are highly transcribed during rapid growth, but "turned off" under nutrient limiting conditions, a process called the stringent response. To understand how transcriptional initiation at these promoters is coordinately regulated, we analyzed the interactions between RNA polymerase (RNAP) (both wild type and mutants) and four stringently controlled promoters. Our results show that the interactions between RNAP and stringently controlled promoters are intrinsically unstable and can alternate between relatively stable and metastable states. The mutant RNAPs appear to specifically further weaken interactions with these promoters in vitro and behave like "stringent" RNAPs in the absence of the stringent response in vivo, constituting a novel class of mutant RNAPs. Consistently, these mutant RNAPs also activate the expression of other genes that normally require the response. We propose that the stability of initiation complexes is coupled to the transcription of stringently controlled promoters, and this unique feature coordinates the expression of genes positively and negatively regulated by the stringent response.

RapA, a novel RNA polymerase-associated protein, is a bacterial homolog of SWI2/SNF2.

We have identified a novel Escherichia coli RNA polymerase (RNAP)-associated protein, an ATPase named RapA. Almost all of this 110-kDa protein in the cell copurifies with RNAP holoenzyme as a 1:1 complex. Purified to homogeneity, RapA also forms a stable complex with RNAP, as if it were a subunit of RNAP. The ATPase activity of RapA is stimulated by binding to RNAP, and thus, RapA and RNAP interact physically as well as functionally. Interestingly, RapA is a homolog of the SWI/SNF family of eukaryotic proteins whose members are involved in transcription activation, nucleosome remodeling, and DNA repair.

Multiple regions on the Escherichia coli heat shock transcription factor sigma32 determine core RNA polymerase binding specificity.

We have analyzed the core RNA polymerase (RNAP) binding activity of the purified products of nine defective alleles of the rpoH gene, which encodes sigma32 in Escherichia coli. All mutations studied here lie outside of the putative core RNAP binding regions 2.1 and 2.2. Based on the estimated K(s)s for the mutant sigma and core RNAP interaction determined by in vitro transcription and by glycerol gradient sedimentation, we have divided the mutants into three classes. The class III mutants showed greatly decreased affinity for core RNAP, whereas the class II mutants' effect on core RNAP interaction was only clearly seen in the presence of sigma70 competitor. The class I mutant behaved nearly identically to the wild type in core RNAP binding. Two point mutations in class III altered residues that were distant from one another. One was found in conserved region 4.2, and the other was in a region conserved only among heat shock sigma factors. These data suggest that there is more than one core RNAP binding region in sigma32 and that differences in contact sites probably exist among sigma factors.

RNA polymerase beta mutations have reduced sigma70 synthesis leading to a hyper-temperature-sensitive phenotype of a sigma70 mutant.

This work describes a mutational analysis of the interaction between the beta and sigma subunits of Escherichia coli RNA polymerase. The rpoD800 mutant has a temperature-sensitive growth phenotype because the mutant sigma70 polypeptide is not stable at a high temperature. Some rpoB mutations, including rpoB114, enhanced the temperature sensitivity of the rpoD800 mutant. We determined the mechanism by which the rpoB114 rpoD800 double mutant becomes hyper-temperature sensitive for growth. We found that the levels of the mutant sigma70 in the rpoB114 rpoD800 mutant were dramatically reduced compared to that in the rpoD800 mutant after temperature shift-up. The rate of synthesis of the sigma70 polypeptide was reduced in the rpoB114 rpoD800 double mutant compared to the rpoD800 mutant, whereas the half-life of the mutant sigma70 polypeptide after temperature shift-up was the same in both strains. We conclude that because of the reduction of expression of rpoD800 by rpoB114, in concert with the intrinsic instability of the mutant sigma70 polypeptide, the amount of holoenzyme containing sigma70 becomes limiting upon temperature shift-up. This results in the hyper-temperature sensitivity of the rpoB114 rpoD800 double mutant. Furthermore, the effect of rpoB114 on the expression of sigma70 is independent of the rpoD800 allele and is at the transcriptional level. In vitro transcription assays showed that the mutant RNA polymerase RpoB114 was defective in transcribing the two major promoters of the rpoD operon specifically. The effects of these rpoB mutations on gene expression are discussed.

Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2.

Escherichia coli 397c is temperature sensitive for growth at 43.5 degrees C and unable to plate bacteriophage P2 at 33 degrees C. The mutation conferring these phenotypes was mapped to the rpoC gene. RNA synthesis is temperature sensitive in the mutant strain, and the beta' subunit of RNA polymerase isolated from this strain exhibits increased electrophoretic mobility. DNA sequence analysis revealed that the mutation is a deletion of 16 bp, resulting in a frameshift that leads to truncation of the beta' subunit at the carboxy terminus.

Transcripts that increase the processivity and elongation rate of RNA polymerase.

Transcripts encoded by the cis-acting antitermination sites (put sites) of lambdoid phage HK022 promote readthrough of downstream transcription terminators. Proper conformation of the transcripts is essential for activity, since put mutations that prevent the formation of predicted RNA stems prevented antitermination, and suppressor mutations that restore the stems restored antitermination. Antitermination does not appear to require proteins other than RNA polymerase, since put-dependent readthrough of multiple sequential terminators was observed in a purified transcription system consisting of template, polymerase, substrates, and buffer. Transcription of put also increased the elongation rate of polymerase, very likely by suppressing pausing. A mutation that alters the zinc-finger region of the beta' subunit of polymerase specifically prevented the put-dependent increases in terminator readthrough and elongation rate. The simplicity of HK022 antitermination contrasts with that of other known antitermination pathways. We propose that the central effector is a transcript that directly alters the elongation properties of RNA polymerase.

A mechanism of rifamycin inhibition and resistance in Pseudomonas aeruginosa.

We sought to study the nature of rifampicin resistance in Pseudomonas aeruginosa. We hypothesized that the rifamycin regions of RNA polymerase are conserved in P. aeruginosa and that rifampicin resistance is mediated by a mutation in the rpoB gene encoding the beta subunit of RNA polymerase. Transcription assays showed that 50 nM of rifampicin inhibited transcription > 99% in a clinical isolate (MIC = 32 mg/L) and only < 40% in the rifampicin resistant mutant (MIC = 1000 mg/L). DNA sequencing revealed that the rifampicin regions are conserved in P. aeruginosa and the rifampicin regions of the rifampicin-resistant strain contained a mutation. Sodium hexametaphosphate lowered rifamycin MIC in a rifamycin-resistant mutant four-fold and in the clinical isolate 32-fold, suggesting that P. aeruginosa has a natural membrane barrier to rifamycins.

The mechanism of early transcription termination by Rho026.

We previously found that nusD-type mutations in Escherichia coli transcription termination factor Rho enhance in vitro transcription termination at four points within the lambdacro gene. Here we show that the early termination points are part of one Rho-dependent termination site, tRE, with properties like those of previously characterized Rho-dependent sites lamda tR1 and trpt'. The early termination points are all RNA polymerase pause sites, and by deletion analysis and oligonucleotide blocking experiments, a common 5' Rho entry site for the early termination points (rutE) is identified. We show that both Rho026 and Rho+ can use rutE as an entry point for termination, but that Rho026 is more efficient in releasing the nascent RNA at tRE. The RNA-dependent ATPase activities of wild-type and mutant Rhos are similar, as are their abilities to bind free RNA and to use (rC)10 oligomers for ATPase activation. We therefore suggest that Rho-RNA polymerase interactions that define the site of RNA 3' end formation are altered in NusD Rho mutants. NusD Rho mutants are less dependent on, but still responsive to, the transcription termination factor NusG. However, addition of NusG to in vitro termination assays allows Rho+ to terminate more efficiently at tRE. These results suggest that NusG aids in the 3' end formation process. The decreased dependence on NusG for termination by the mutant Rhos in vitro provides an explanation for poorer lambda growth in rho(nusD) cells by interference with lamdaN-mediated antitermination at Rho-dependent sites.

Amino acid substitutions in the two largest subunits of Escherichia coli RNA polymerase that suppress a defective Rho termination factor affect different parts of the transcription complex.

Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201. These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all rho-dependent and rho-independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (betaN1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (beta'R352C). Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons. The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for betaN1072H. In contrast, beta'R352C and two adjacent substitutions (beta'G351S and beta'S350F), but not betaN1072H, formed open complexes of greatly reduced stability. The sequence in this region of beta' modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals. Core determinants affecting open complex formation do not reside exclusively in beta', however, since the Rifr mutation rpoB2 in beta also dramatically destabilized open complexes. We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of beta region H in nucleoside triphosphate-binding and nucleotide addition and of beta' region C in contacts to the DNA strands that could be important for translocation. Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites.

A mutant RNA polymerase reveals a kinetic mechanisms for the switch between nonproductive stuttering synthesis and productive initiation during promoter clearance.

During transcription initiation from galP2, one of the two promoters of the Escherichia coli galactose operon with an initially transcribed sequence of pppAUUUC, RNA polymerase (RNAP) is known to engage nonproductive stuttering synthesis, which is sensitive to the concentration of UTP. This study examines the effect of this nonproductive synthesis on promoter clearance and determines other parameters that might affect stuttering synthesis by analyzing a mutant RNAP, RpoB3449, that has altered its function at this process at galP2. RpoB3449 has dramatically diminished stuttering synthesis, and consequently, it has increased the rate of productive initiation due to its enhanced rate of promoter clearance of galP2 compared with wild-type RNAP. Thus, a direct linkage between promoter clearance and productive transcription is demonstrated. The mechanism by which the mutant RNAP has altered the switch between nonproductive stuttering synthesis and productive initiation during promoter clearance is studied. Apparently, RpoB3449 has increased its efficiency in incorporating CTP at the +5 position of the galP2 transcript leading to its reduced stuttering synthesis, indicating that the rate of an RNAP incorporating the CTP after a stretch of uridine residues is important for promoter clearance at galP2. Because RpoB3449 demonstrates "wild-type" stuttering synthesis at the mutant galP2 promoter, which contains the 6 residue at the +5 position, it indicates that the mutant RNAP has altered in binding CTP at this context. Further experiments indicate that it is the +5 position per se of the galP2 sequence rather than a particular nucleotide at that position that is critical in determining the switch between the two alternate pathways during transcription initiation. A checkpoint model for the switch between nonproductive and productive initiations during promoter clearance is discussed.

A zinc-binding region in the beta' subunit of RNA polymerase is involved in antitermination of early transcription of phage HK022.

Antitermination of early transcription in phage HK022 requires no virus-encoded proteins and thus differs from antitermination by other lambdoid phages. It does require cis-acting phage sequences, which may be analogous to the lambdoid nut sites. To identify host proteins involved in antitermination, we isolated 14 Escherichia coli mutants that are specifically blocked in HK022 growth. The mutations are located in the rpoC gene, which encodes the beta' subunit of RNA polymerase. Each mutation alters one of three amino acid residues located within a cluster of four completely conserved cysteine residues that are believed to bind zinc. We examined the effect of one mutation on HK022 antitermination in vivo. rpoCY75N greatly reduced readthrough of a strong rho-independent transcription terminator placed downstream of the HK022 PL promoter and nutL analog, but did not decrease promoter activity. Purified enzyme had a similar effect on PL-directed transcription in vitro: wild-type but not mutant polymerase read through a strong rho-independent terminator located immediately downstream of the nutL analog with high efficiency. We suggest that interaction of the putative zinc-binding domain of the RNA polymerase beta' subunit with the HK022 antitermination sites suppresses transcription termination, and that this interaction can occur in the absence of other proteins.

Determination of intrinsic transcription termination efficiency by RNA polymerase elongation rate.

Transcription terminators recognized by several RNA polymerases include a DNA segment encoding uridine-rich RNA and, for bacterial RNA polymerase, a hairpin loop located immediately upstream. Here, mutationally altered Escherichia coli RNA polymerase enzymes that have different termination efficiencies were used to show that the extent of transcription through the uridine-rich encoding segment is controlled by the substrate concentration of nucleoside triphosphate. This result implies that the rate of elongation determines the probability of transcript release. Moreover, the position of release sites suggests an important spatial relation between the RNA hairpin and the boundary of the terminator.