Gene sequence, overproduction, purification and determination of the wild-type level of the Escherichia coli flagellar switch protein FliG.

The flagellar motor switch in Escherichia coli and Salmonella typhimurium controls swimming behavior by regulating the direction of flagellar rotation. The switch is a complex apparatus composed of at least three proteins--FliG, FliM and FliN. During chemotactic behavior, the switch responds to signals transduced by the chemotaxis sensory signaling system. CheY, the chemotaxis response regulator, is thought to act directly on the switch to induce tumbles in the swimming pattern, but physical interaction of CheY and switch proteins has not been shown. We have undertaken this work to develop the molecular tools to investigate CheY binding to switch proteins, as well as to understand more about the structure and function of the switch. We present here the sequences of the fliG gene and its protein product, the engineering and amplification of fliG by the polymerase chain reaction (PCR) and its subcloning, and the overproduction, purification and determination of the wild-type (wt) level of the FliG protein. The sequence data revealed a 91.8% amino acid (aa) identity between E. coli and S. typhimurium FliG. Engineering and amplifying fliG by PCR allowed convenient cloning into an efficient expression vector. FliG was successfully overproduced and purified to > 98% purity. Polyclonal antibodies (Ab) were generated against purified FliG and used in quantitative Western blots to determine that the wt expression level of fliG results in about 3700 FliG copies per cell. Purified FliG and anti-FliG Ab will be useful for direct biochemical analyses of CheY-switch protein interaction.

Nucleotide sequence analysis of the phosphomannose isomerase gene (pmi) of Pseudomonas aeruginosa and comparison with the corresponding Escherichia coli gene manA.

Phosphomannose isomerase (PMI) has been proposed to catalyze the first step of the alginic acid biosynthetic pathway in Pseudomonas aeruginosa. The nucleotide sequence of the P. aeruginosa pmi gene contained on a 2.0-kb BamHI-SstI DNA fragment has been determined. The gene was defined by the start and stop codons and by in vitro disruption of an open reading frame of 1440 bp corresponding to a polypeptide product with a predicted Mr of 52 860. This polypeptide displayed an apparent Mr of approx. 56 000 upon electrophoresis of a maxicell extract on sodium dodecyl sulfate-polyacrylamide gels. The codon utilization of the pmi gene was distinct in the wobble base preference and influenced by the high G + C content (66 mol%) of the P. aeruginosa DNA. Computer assisted matching analysis failed to demonstrate any significant homology at the nucleotide level between the P. aeruginosa pmi and Escherichia coli manA (pmi) genes. However, sequences homologous to the P. aeruginosa pmi gene were found in other Pseudomonas species, such as P. putida and P. mendocina, and in Azotobacter vinelandii, all capable of producing alginic acid.

Cloning and complete nucleotide sequence determination of the catB gene encoding cis,cis-muconate lactonizing enzyme.

The enzyme, cis,cis-muconate lactonizing enzyme I (MLEI; EC 5.5.1.1), has been proposed to play a key role in the beta-ketoadipate pathway of benzoate degradation. A 10.2-kb EcoRI fragment isolated from a Pseudomonas putida genomic library complemented a mutant deficient in this enzyme. The MLEI coding gene, catB, was localized to a 1.6-kb fragment which was sequenced by the dideoxy chain termination method. MLEI was purified 25-fold from crude extracts of benzoate-grown P. putida PRS2015 harboring the cloned catB gene. Purified MLEI was greater than 95% homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit Mr was 40,000 which was in close agreement with the nucleotide sequence data. N-terminal sequence analysis of purified MLEI protein agreed with the N terminus predicted by the nucleotide sequence. Comparison of the nucleotide and amino acid sequences for catB with the corresponding sequences of the clcB gene (K.L. Ngai, B.F., D.K. Chatterjee, L.N. Ornston, and A.M.C., unpublished), whose gene product catalyzes the analogous reaction in 3-chlorobenzoate degradation, showed significant homology. These results suggest that catB and clcB have diverged from a common ancestral gene.

Characterization and nucleotide sequence determination of a repeat element isolated from a 2,4,5-T degrading strain of Pseudomonas cepacia.

Pseudomonas cepacia strain AC1100, capable of growth on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was mutated to the 2,4,5-T- strain PT88 by a ColE1::Tn5 chromosomal insertion. Using cloned DNA from the region flanking the insertion, a 1477-bp sequence (designated RS1100) was identified which was repeated several times on the wild-type chromosome and was also present on AC1100 plasmid DNA. Various chromosomal fragments containing this sequence were cloned and their nucleotide sequence was determined. Examination of RS1100 revealed the presence of 38-39-bp terminal inverted repeats immediately flanked by 8-bp direct repeats. The translated sequence of the single large open reading frame of RS1100 showed structural similarity to the phage Mu transposase and other DNA-binding proteins. Thus the AC1100 repeated sequence has several structural features in common with insertion sequence elements. Three copies of RS1100 were mapped near 2,4,5-t genes encoding degradation of 5-chloro-1,2,4-trihydroxybenzene, an intermediate in 2,4,5-T degradation. Neither RS1100 nor the 2,4,5-t genes hybridized to DNA isolated from Pseudomonas strains, including P. cepacia, suggesting that both gene fragments may be of foreign origin recruited in strain AC1100. The origin of these two DNA segments as well as the role played by RS1100 in the recruitment of 2,4,5-t genes in AC1100 are presently under investigation.

Design and synthesis of potent, selective, and orally bioavailable tetrasubstituted imidazole inhibitors of p38 mitogen-activated protein kinase.

Novel potent and selective diarylimidazole inhibitors of p38 MAP (mitogen-activated protein) kinase are described which have activity in both cell-based assays of tumor necrosis factor-alpha (TNF-alpha) release and an animal model of rheumatoid arthritis. The SAR leading to the development of selectivity against c-Raf and JNK2alpha1 kinases is presented, with key features being substitution of the 4-aryl ring with m-trifluoromethyl and substitution of the 5-heteroaryl ring with a 2-amino substituent. Cell-based activity was significantly enhanced by incorporation of a 4-piperidinyl moiety at the 2-position of the imidazole which also enhanced aqueous solubility. In general, oral bioavailability of this class of compounds was found to be poor unless the imidazole was methylated on nitrogen. This work led to identification of 48, a potent (p38 MAP kinase inhibition IC50 0.24 nM) and selective p38 MAP kinase inhibitor which inhibits lipopolysaccharide-stimulated release of TNF-alpha from human blood with an IC50 2.2 nM, shows good oral bioavailability in rat and rhesus monkey, and demonstrates significant improvement in measures of disease progression in a rat adjuvant-induced arthritis model.

Microbial degradation of synthetic recalcitrant compounds.

Synthetic compounds, particularly highly chlorinated aromatics, comprise the bulk of the environmental pollutants that somehow must be removed from the environment. Microbial degradation of such compounds is usually very slow, making them highly persistent in nature. Some synthetic compounds, with a lower degree of chlorination are, however, biodegradable; biochemical, genetic, and molecular studies demonstrate the evolution of new plasmid-encoded enzymatic activities specifically designed for the chlorinated substrates. Nucleotide sequences of many of the genes encoding such enzymatic activities demonstrate considerable homology either near the active sites or throughout the molecules with the chromosomal genes encoding enzymes catalyzing analogous reactions. In some cases, unique repeated sequences, reminiscent of prokaryotic insertion sequence elements, are present at or near the newly evolved genes. This suggests gene duplication and divergence as well as recombinational events mediated by transposable type elements as key ingredients in the evolution of new degradative functions. An understanding of such evolutionary processes is an essential feature for the development of genetically-improved bacteria capable of utilizing and thereby removing highly chlorinated environmental pollutants from our environment.

Real-time transmission of full-motion echocardiography over a high-speed data network: impact of data rate and network quality of service.

UNLABELLED: With high-resolution network transmission required for telemedicine, education, and guided-image acquisition, the impact of errors and transmission rates on image quality needs evaluation. METHODS: We transmitted clinical echocardiograms from 2 National Aeronautics and Space Administration (NASA) research centers with the use of Motion Picture Expert Group-2 (MPEG-2) encoding and asynchronous transmission mode (ATM) network protocol over the NASA Research and Education Network. Data rates and network quality (cell losses [CLR], errors [CER], and delay variability [CVD]) were altered and image quality was judged. RESULTS: At speeds of 3 to 5 megabits per second (Mbps), digital images were superior to those on videotape; at 2 Mbps, images were equivalent. Increasing CLR caused occasional, brief pauses. Extreme CER and CDV increases still yielded high-quality images. CONCLUSIONS: Real-time echocardiographic acquisition, guidance, and transmission is feasible with the use of MPEG-2 and ATM with broadcast quality seen above 3 Mbps, even with severe network quality degradation. These techniques can be applied to telemedicine and used for planned echocardiography aboard the International Space Station.

Iodmetric and spectrophotometric assays for cephradine after its hydrolysis with a beta-lactamase.

Iodmetric and spectrophotometric methods were developed for the analysis of the new antibiotic cephradine. The procedures are modifications of known methods but are novel in employing a specific beta-lactamase for hydrolysis of the beta-lactam ring of the cephalosporin molecule. The iodometric method is rapid, precise, and accurate, but it requires fairly large amounts of cephradine. The spectrophotometric method, using differential UV absorption at 260 nm, is more rapid and more sensitive than the iodometric method but somewhat less accurate. Both methods proved useful for the routine assay of cephradine in certain formulations.

Tissue interactions with dentin specimens after demineralization using tetracycline.

Greater cell attachment to demineralized dentin has been associated with subsequent development of a fiber attachment system. The purpose of this study was to evaluate tissue interactions to dentin demineralized with different concentrations of tetracycline solution. Dentin specimens were obtained from beneath root surfaces covered by periodontal ligament. Each rectangular specimen had a face of root surface dentin and an opposite surface of pulpal dentin. Experimental specimens were treated with a tetracycline solution of either 200 mg/cc or 100 mg/cc for five minutes. The remaining group of specimens served as untreated controls. Specimens were implanted transcutaneously into incisional wounds on the dorsal surface of rats with one end protruding through the skin. Four specimens in each group were available for examination one and ten days after implantation. Histologic and histometric analysis of both root and pulpal surface of implants included counts of adhering cells, assessment of implant length within the connective tissue, and evaluation of connective tissue fiber relationships. In each group, specimens became severely extruded between days one and ten, the number of attached cells decreased, and a fiber attachment system did not develop. Tetracycline-treated surfaces had greater numbers of attached cells at both time points compared to untreated controls. No differences were discernible relating to different tetracycline concentrations. It was concluded that tetracycline-demineralized dentin provided a substrate that increased cell attachment; however, this enhanced response did not result in a connective tissue attachment.

Associations between bleeding and visual signs of interdental gingival inflammation.

Interdental gingival tissues are designated inflamed on the basis of their color and bleeding after stimulation. Gingival bleeding was previously shown in histological studies to indicate the presence of inflammatory lesions. The present study was undertaken to determine associations between bleeding and visual signs of interdental gingival inflammation. Each interdental site in 82 males, aged 18 to 30, was evaluated for the presence or absence of visual signs of inflammation. The interdental sites on one side of the mouth were evaluated for bleeding tendency using the Papilla Bleeding Index (PBI), while the other half was evaluated using the Eastman Interdental Bleeding Index (EIBI). The percentage of inflamed areas detected with the EIBI and visual method was similar and significantly higher than with the PBI. When the visually noninflamed sites were examined, 38.5% of these areas bled, indicating that interdental inflammatory lesions existed in the absence of visual signs of inflammation. Of the sites that bled but were visually noninflamed, 33.1% were detected using the PBI, while 66.9% were detected using the EIBI. The Eastman Interdental Bleeding Index was a more reliable clinical indicator for detecting interdental inflammatory lesions than the Papilla Bleeding Index.

Guided tissue regeneration in interproximal defects in the monkey.

The potential for guided tissue regeneration was evaluated in one-walled interproximal sites in Macaca fascicularis. Histologic differences were evaluated at 1 and 3 months. Within the experimental (barrier) group, 100% of the root surfaces with potential for regeneration were covered with new cementum, whereas the control specimens had 20% or less new cementum. The amount of regeneration was determined by the position of the barrier membrane; the more coronal the barrier, the greater the regeneration. Observations indicated that the optimal time for barrier removal is between 1 and 3 months.

DNA distortion accompanies transcriptional activation by the metal-responsive gene-regulatory protein MerR.

Transcriptional regulation of the bacterial mercuric ion resistance operon (mer) in response to nanomolar concentrations of mercuric ion is achieved by the allosterically modulated transcriptional activator protein MerR. We now show that mercuric ion modification of MerR activates transcription, facilitating the conversion of an RNA polymerase complex with the mer promoter from the closed conformation to the strand-separated, transcriptionally competent open complex. An Hg-MerR-induced structural alteration at the center of the promoter has been detected in the presence or absence of RNA polymerase by use of chemical nucleases sensitive to variations in DNA secondary structure. This hypersensitivity correlates directly with transcriptional activation, lending further support to a previous proposal that a protein-induced distortion in local DNA structure can be the key step in an allosterically modulated transcription activation mechanism.

Organization and nucleotide sequence determination of a gene cluster involved in 3-chlorocatechol degradation.

Three critical enzymes catechol oxygenase II (chlorocatechol dioxygenase), muconate cycloisomerase II, and dienelactone hydrolase, are involved in the degradation of chlorocatechols, which are obligatory intermediates in the catabolism of chlorinated aromatic compounds. The organization and complete nucleotide sequence of the genes for these enzymes have been determined on a 4.2-kilobase-pair (kbp) Bgl II fragment cloned from the plasmid pAC27, based on the agreement of open reading frame lengths with apparent mobilities of polypeptides expressed in Escherichia coli maxicells, predicted N-terminal amino acid sequences with those of the purified proteins, and predicted total amino acid compositions with those of the purified proteins. The 4.2-kbp fragment contains the three genes and ribosome binding sites for those genes but no promoter. When placed downstream of the tac promoter in the broad-host-range plasmid pMMB22, this fragment directs the synthesis of all three enzymes in both E. coli and Pseudomonas putida only on induction with isopropyl beta-D-thiogalactopyranoside, suggesting that the gene cluster is regulated as a single unit under the control of a single promoter. Endogenous transcription initiation of the gene cluster on pAC27, however, occurs from a site present within a 386-bp Bgl II fragment upstream of the 4.2-kbp fragment, and sequences 5' to that site are similar to the sequences of other positively controlled Pseudomonas promoters occurring on the TOL and NAH plasmids.

Flagellar transcriptional activators FlbB and FlaI: gene sequences and 5' consensus sequences of operons under FlbB and FlaI control.

The regulation of the expression of the operons in the flagella-chemotaxis regulon in Escherichia coli has been shown to be a highly ordered cascade which closely parallels the assembly of the flagellar structure and the chemotaxis machinery (T. Iino, Annu. Rev. Genet. 11:161-182, 1977; Y. Komeda, J. Bacteriol. 168: 1315-1318). The master operon, flbB, has been sequenced, and one of its gene products (FlaI) has been identified. On the basis of the deduced amino acid sequence, the FlbB protein has similarity to an alternate sigma factor which is responsible for expression of flagella in Bacillus subtilis. In addition, we have sequenced the 5' regions of a number of flagellar operons and compared these sequences with the 5' region of flagellar operons directly and indirectly under FlbB and FlaI control. We found both a consensus sequence which has been shown to be in all other flagellar operons (J. D. Helmann and M. J. Chamberlin, Proc. Natl. Acad. Sci. USA 84:6422-6424) and a derivative consensus sequence, which is found only in the 5' region of operons directly under FlbB and FlaI control.

Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13.

The clcD structural gene encodes dienelactone hydrolase (EC 3.1.1.45), an enzyme that catalyzes the conversion of dienelactones to maleylacetate. The gene is part of the clc gene cluster involved in the utilization of chlorocatechol and is carried on a 4.3-kilobase-pair BglII fragment subcloned from the Pseudomonas degradative plasmid pAC27. A 1.9-kilobase-pair PstI-EcoRI segment subcloned from the BglII fragment was shown to carry the clcD gene, which was expressed inducibly under the tac promoter at levels similar to those found in 3-chlorobenzoate-grown Pseudomonas cells carrying the plasmid pAC27. In this study, we present the complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence. The NH2-terminal amino acid sequence encoded by the clcD gene from plasmid pAC27 corresponds to a 33-residue sequence established for dienelactone hydrolase encoded by the Pseudomonas sp. strain B13 plasmid pWR1. A possible relationship between the clcD gene and pcaD, a Pseudomonas putida chromosomal gene encoding enol-lactone hydrolase (EC 3.1.1.24) is suggested by the fact that the gene products contain an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites; the cysteinyl residue occupies position 60 in the predicted amino acid sequence of dienelactone hydrolase.

Electroresection followed by neodymium-YAG laser photocoagulation of the dog prostate for establishment of safety parameters.

Electroresection of the prostate followed by Nd-YAG laser coagulation of residual prostate was carried out in eight dogs. All dogs voided spontaneously without significant bleeding in the immediate postoperative period. At the time of sacrifice in 6 to 8 weeks, no damage to adjacent tissue was seen on microscopic examination. Re-epithelization had occurred. This study suggests that the addition of neodymium-YAG coagulation to residual tissue after TURP in humans can be done safely.