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D Ang

Publications and source records attributed to D Ang.

At least 19 recordsLinked to original sources
PubMed2006

Fluoroquinolones may delay the diagnosis of tuberculosis.

INTRODUCTION: Fluoroquinolones (FQs), frequently used for many common infections such as community-acquired pneumonia and urinary tract infection, are also effective against Mycobacterium tuberculosis. This study describes a series of patients in whom the empirical use of FQs for what appeared to be common community-acquired infections led to a delay in the diagnosis of tuberculosis (TB). METHODS: We reviewed the records of five patients with TB in whom the early use of FQs led to partial symptom resolution and a prolonged relapsing and remitting course. RESULTS: Of the five patients described, four presented with community-acquired pneumonia and one with urinary tract infection. All were given FQs and improved, though not completely. Their illnesses took a relapsing and remitting course. TB was eventually diagnosed, in four of them by culture and in one by characteristic histopathology (this patient required surgical resection of a lung abscess). CONCLUSION: FQs may lead to partial symptom resolution in TB. We highlight the problem of a delayed diagnosis, and voice our concern about inadvertent monotherapy of TB in such cases.

Adult↗
PubMed2000

Pseudo-T-even bacteriophage RB49 encodes CocO, a cochaperonin for GroEL, which can substitute for Escherichia coli's GroES and bacteriophage T4's Gp31.

Bacteriophage T4-encoded Gp31 is a functional ortholog of the Escherichia coli GroES cochaperonin protein. Both of these proteins form transient, productive complexes with the GroEL chaperonin, required for protein folding and other related functions in the cell. However, Gp31 is specifically required, in conjunction with GroEL, for the correct folding of Gp23, the major capsid protein of T4. To better understand the interaction between GroEL and its cochaperonin cognates, we determined whether the so-called "pseudo-T-even bacteriophages" are dependent on host GroEL function and whether they also encode their own cochaperonin. Here, we report the isolation of an allele-specific mutation of bacteriophage RB49, called epsilon22, which permits growth on the E. coli groEL44 mutant but not on the isogenic wild type host. RB49 epsilon22 was used in marker rescue experiments to identify the corresponding wild type gene, which we have named cocO (cochaperonin cognate). CocO has extremely limited identity to GroES but is 34% identical and 55% similar at the protein sequence level to T4 Gp31, sharing all of the structural features of Gp31 that distinguish it from GroES. CocO can substitute for Gp31 in T4 growth and also suppresses the temperature-sensitive phenotype of the E. coli groES42 mutant. CocO's predicted mobile loop is one residue longer than that of Gp31, with the epsilon22 mutation resulting in a Q36R substitution in this extra residue. Both the CocO wild type and epsilon22 proteins have been purified and shown in vitro to assist GroEL in the refolding of denatured citrate synthase.

Amino Acid Sequence↗
PubMed2000

Genetic analysis of bacteriophage-encoded cochaperonins.

Early genetic studies identified the Escherichia coli groES and groEL genes because mutations in them blocked the growth of bacteriophages lambda and T4. Subsequent genetic and biochemical analyses have shown that GroES and GroEL constitute a chaperonin machine, absolutely essential for E. coli growth, because it is needed for the correct folding of many of its proteins. In spite of very little sequence identity to GroES, the bacteriophage T4-encoded Gp31 protein and the bacteriophage RB49-encoded CocO protein are bona fide GroEL cochaperonins, even capable of substituting for GroES in E. coli growth. A major functional distinction is that only Gp31 and CocO can assist GroEL in the correct folding of Gp23, the major bacteriophage capsid protein. Conserved structural features between CocO and Gp31, which are absent from GroES, highlight their potential importance in specific cochaperonin function.

Bacterial Proteins↗
PubMed1999

Polypeptide flux through bacterial Hsp70: DnaK cooperates with trigger factor in chaperoning nascent chains.

A role for DnaK, the major E. coli Hsp70, in chaperoning de novo protein folding has remained elusive. Here we show that under nonstress conditions DnaK transiently associates with a wide variety of nascent and newly synthesized polypeptides, with a preference for chains larger than 30 kDa. Deletion of the nonessential gene encoding trigger factor, a ribosome-associated chaperone, results in a doubling of the fraction of nascent polypeptides interacting with DnaK. Combined deletion of the trigger factor and DnaK genes is lethal under normal growth conditions. These findings indicate important, partially overlapping functions of DnaK and trigger factor in de novo protein folding and explain why the loss of either chaperone can be tolerated by E. coli.

Bacterial Proteins↗
PubMed1999

Diagnosis, etiology, and therapy of fibromyalgia.

Fibromyalgia is characterized by diffuse pain, multiple tender points, fatigue, and sleep disturbance. Its frequent concurrence with rheumatic diseases modifies the clinical picture of the "primary" disease. This article reviews new information about the etiopathogenesis and treatment of this syndrome.

Algorithms↗
PubMed1994

Isolation and characterization of point mutations in the Escherichia coli grpE heat shock gene.

The Escherichia coli grpE gene (along with dnaK, dnaJ, groEL, and groES) was originally identified as one of the host factors required for phage lambda growth. The classical grpE280 mutation was the only grpE mutation that resulted from the initial screen and shown to specifically block the initiation of lambda DNA replication. Here we report the isolation of several new grpE missense mutations, again using phage lambda resistance as a selection. All mutants fall into two groups based on their temperature-dependent phenotype for lambda growth. Members of the first group (I), including grpE17 and grpE280, which was obtained again, are resistant to lambda growth at both 30 and 42 degrees C. Members of the second group (II), including grpE25, grpE66, grpE103, grpE13a, grpE57b, and grpE61, are sensitive to lambda growth at 30 degrees C but resistant at 42 degrees C. All mutations are recessive, since an E. coli grpE null mutant strain carrying these mutant alleles on low-copy-number plasmids are sensitive to infection by the lambda grpE+ transducing phage. Both group I and group II mutants are temperature sensitive for E. coli growth above 42 degrees C. The nucleotide changes were identified by sequencing analyses and shown to be dispersed throughout the latter 75% of the grpE coding region. Most of the amino acid changes occur at conserved residues, as judged by sequence comparisons between E. coli and other bacterial and yeast GrpE homologs. The isolation of these new mutations is the first step toward a structure-function analysis of the GrpE protein.

Amino Acid Sequence↗
PubMed1994

Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes.

We characterized a 24-kDa protein associated with matrix hsp70 (mt-hsp70) of Neurospora crassa and Saccharomyces cerevisiae mitochondria. By using specific antibodies, the protein was identified as MGE, a mitochondrial homolog of the prokaryotic heat shock protein GrpE. MGE extracted from mitochondria was quantitatively bound to hsp70. It was efficiently released from hsp70 by the addition of Mg-ATP but not by nonhydrolyzable ATP analogs or high salt. A mutant mt-hsp70, which was impaired in release of bound precursor proteins, released MGE in an ATP-dependent manner, indicating that precursor proteins and MGE bind to different sites of hsp70. A preprotein accumulated in transit across the mitochondrial membranes was specifically coprecipitated by either antibodies directed against MGE or antibodies directed against mt-hsp70. The preprotein accumulated at the outer membrane was not coprecipitated by either antibody preparation. After being imported into the matrix, the preprotein could be coprecipitated only by antibodies against mt-hsp70. We propose that mt-hsp70 and MGE cooperate in membrane translocation of preproteins.

Ascomycota↗
PubMed1993

Function of the GrpE heat shock protein in bidirectional unwinding and replication from the origin of phage lambda.

The initiation of DNA replication by phage lambda depends on a specialized nucleoprotein structure that provides for the precise localization and activity of the Escherichia coli DnaB helicase at the lambda replication origin. Previous work has shown that the DnaJ and DnaK heat shock proteins function in the initiation pathway by releasing the DnaB helicase from the initiation complex to carry out localized unwinding of origin DNA. This DnaJ.DnaK pathway results in mainly unidirectional DNA unwinding and replication, whereas replication in vivo is mainly bidirectional. Based on recent replication work indicating an important role for the GrpE heat shock protein, we have used electron microscopy to study the action of GrpE in the DNA unwinding and replication reactions. We have found that GrpE acts with DnaJ and DnaK to facilitate the unwinding reaction at low concentrations of DnaK. In the presence of GrpE, bidirectional unwinding occurs in approximately half of the unwound DNA molecules. In addition, GrpE significantly increases the frequency of replication proceeding leftward from the origin. We suggest that reactions including GrpE result in more complete disassembly of the preinitiation nucleoprotein structure, thus allowing replication to proceed in both directions from the origin.

Bacterial Proteins↗
PubMed1992

The essential Escherichia coli msgB gene, a multicopy suppressor of a temperature-sensitive allele of the heat shock gene grpE, is identical to dapE.

The grpE gene product is one of three Escherichia coli heat shock proteins (DnaK, DnaJ, and GrpE) that are essential for both bacteriophage lambda DNA replication and bacterial growth at all temperatures. In an effort to determine the role of GrpE and to identify other factors that it may interact with, we isolated multicopy suppressors of the grpE280 point mutation, as judged by their ability to reverse the temperature-sensitive phenotype of grpE280. Here we report the characterization of one of them, designated msgB. The msgB gene maps at approximately 53 min on the E. coli chromosome. The minimal gene possesses an open reading frame that encodes a protein with a predicted size of 41,269 M(r). This open reading frame was confirmed the correct one by direct amino-terminal sequence analysis of the overproduced msgB gene product. Genetic experiments demonstrated that msgB is essential for E. coli growth in the temperature range of 22 to 37 degrees C. Through a sequence homology search, MsgB was shown to be identical to N-succinyl-L-diaminopimelic acid desuccinylase (the dapE gene product), which participates in the diaminopimelic acid-lysine pathway involved in cell wall biosynthesis. Consistent with this finding, the msgB null allele mutant is viable only when the growth medium is supplemented with diaminopimelic acid. These results suggest that GrpE may have a previously unsuspected function(s) in cell wall biosynthesis in E. coli.

Alleles↗
PubMed1992

Isolation and characterization of the Escherichia coli htrD gene, whose product is required for growth at high temperatures.

Those genes in Escherichia coli defined by mutations which result in an inability to grow at high temperatures are designated htr, indicating a high temperature requirement. A new htr mutant of E. coli was isolated and characterized and is designated htrD. The htrD gene has been mapped to 19.3 min on the E. coli chromosome. Insertional inactivation of htrD with a mini-Tn10 element resulted in a pleiotropic phenotype characterized by a severe inhibition of growth at 42 degrees C and decreased survival at 50 degrees C in rich media. Furthermore, htrD cells were sensitive to H2O2. Growth rate analysis revealed that htrD cells grow very slowly in minimal media supplemented with amino acids. This inhibitory effect has been traced to the presence of cysteine in the growth medium. Further studies indicated that the rate of cysteine transport is higher in htrD cells relative to the wild type. All of these results, taken together, indicate that the htrD gene product may be required for proper regulation of intracellular cysteine levels and that an increased rate of cysteine transport greatly affects the growth characteristics of E. coli.

Bacterial Proteins↗
PubMed1991

Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK.

The products of the Escherichia coli dnaK, dnaJ, and grpE heat shock genes have been previously shown to be essential for bacteriophage lambda DNA replication at all temperatures and for bacterial survival under certain conditions. DnaK, the bacterial heat shock protein hsp70 analogue and putative chaperonin, possesses a weak ATPase activity. Previous work has shown that ATP hydrolysis allows the release of various polypeptides complexed with DnaK. Here we demonstrate that the ATPase activity of DnaK can be greatly stimulated, up to 50-fold, in the simultaneous presence of the DnaJ and GrpE heat shock proteins. The presence of either DnaJ or GrpE alone results in a slight stimulation of the ATPase activity of DnaK. The action of the DnaJ and GrpE proteins may be sequential, since the presence of DnaJ alone leads to an acceleration in the rate of hydrolysis of the DnaK-bound ATP. The presence of GrpE alone increases the rate of release of bound ATP or ADP without affecting the rate of hydrolysis. The stimulation of the ATPase activity of DnaK may contribute to its more efficient recycling, and it helps explain why mutations in dnaK, dnaJ, or grpE genes often exhibit similar pleiotropic phenotypes.

Adenosine Triphosphatases↗
PubMed1990

Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication.

The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda. The complex is composed of both phage and host-coded proteins. The lambda O initiator protein binds specifically to ori lambda. The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure. The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex. The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork. In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda. Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase.

Bacterial Proteins↗
PubMed1990

Mutational analysis of the phage T4 morphogenetic 31 gene, whose product interacts with the Escherichia coli GroEL protein.

The phage T4 morphogenetic gene 31 has been sequenced. Its deduced gene product is a polypeptide of 111 aa, with a predicted Mr of 12064 and a pI of 4.88. The proof that the assigned open reading frame (ORF) encodes Gp31 rests on the sequencing of two known gene 31 amber mutations, amN54 and NG71, demonstrating that these mutations result in translational termination within the assigned ORF. Furthermore, the sequencing of four different T4 epsilon mutations, isolated on the basis of allowing the phage to propagate on Escherichia coli groEL- hosts, showed that they are either missense mutations or 3-bp deletions in the gene 31 reading frame. The sequencing of neighboring DNA revealed the presence of five other ORFs, one of which overlaps gene 31 substantially, but in the opposite orientation.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of dnaJ null mutants of Escherichia coli.

Bacteriophage lambda requires the lambda O and P proteins for its DNA replication. The rest of the replication proteins are provided by the Escherichia coli host. Some of these host proteins, such as DnaK, DnaJ, and GrpE, are heat shock proteins. Certain mutations in the dnaK, dnaJ, or grpE gene block lambda growth at all temperatures and E. coli growth above 43 degrees C. We have isolated bacterial mutants that were shown by Southern analysis to contain a defective, mini-Tn10 transposon inserted into either of two locations and in both orientations within the dnaJ gene. We have shown that these dnaJ-insertion mutants did not grow as well as the wild type at temperatures above 30 degrees C, although they blocked lambda DNA replication at all temperatures. The dnaJ-insertion mutants formed progressively smaller colonies at higher temperatures, up to 42 degrees C, and did not form colonies at 43 degrees C. The accumulation of frequent, uncharacterized suppressor mutations allowed these insertion mutants to grow better at all temperatures and to form colonies at 43 degrees C. None of these suppressor mutations restored the ability of the host to propagate phage lambda. Radioactive labeling of proteins synthesized in vivo followed by immunoprecipitation or immunoblotting with anti-DnaJ antibodies demonstrated that no DnaJ protein could be detected in these mutants. Labeling studies at different temperatures demonstrated that these dnaJ-insertion mutations resulted in altered kinetics of heat shock protein synthesis. An additional eight dnaJ mutant isolates, selected spontaneously on the basis of blocking phage lambda growth at 42 degrees C, were shown not to synthesize DnaJ protein as well. Three of these eight spontaneous mutants had gross DNA alterations in the dnaJ gene. Our data provide evidence that the DnaJ protein is not absolutely essential for E. coli growth at temperatures up to 42 degrees C under standard laboratory conditions but is essential for growth at 43 degrees C. However, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.

Bacterial Proteins↗
PubMed1990

The Escherichia coli groE chaperonins.

The E.coli groES and groEL genes have been shown to form an operon, to be essential for E. coli viability, and to belong to the so-called heat-shock class of genes whose expression is regulated by the intracellular levels of sigma factor sigma 32. Both groE chaperonin proteins possess a seven-fold axis of symmetry, groES being composed of seven identical subunits of 97 amino acids each, and groEL of fourteen identical subunits of 548 amino acids each. The two groE chaperonins interact intimately as judged by both genetic and biochemical criteria. This interaction has been shown to be required for both bacteriophage morphogenesis and bacterial growth. The groEL chaperonin has been shown to bind to a number of incomplete or unfolded polypeptides in vitro. Such binding may prevent misfolding and promote rapid intra- or intermolecular folding of polypeptides in vivo. The proposed role of the groES chaperonin is to displace the polypeptides bound to groEL, thus effectively promoting the recycling of groEL.

Bacterial Proteins↗
PubMed1989

Initiation of lambda DNA replication with purified host- and bacteriophage-encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins.

Based on previous in vivo genetic analysis of bacteriophage lambda growth, we have developed two in vitro lambda DNA replication systems composed entirely of purified proteins. One is termed 'grpE-independent' and consists of supercoiled lambda dv plasmid DNA, the lambda O and lambda P proteins, as well as the Escherichia coli dnaK, dnaJ, dnaB, dnaG, ssb, DNA gyrase and DNA polymerase III holoenzyme proteins. The second system includes the E.coli grpE protein and is termed 'grpE-dependent'. Both systems are specific for plasmid molecules carrying the ori lambda DNA initiation site. The major difference in the two systems is that the 'grpE-independent' system requires at least a 10-fold higher level of dnaK protein compared with the grpE-dependent one. The lambda DNA replication process may be divided into several discernible steps, some of which are defined by the isolation of stable intermediates. The first is the formation of a stable ori lambda-lambda O structure. The second is the assembly of a stable ori lambda-lambda O-lambda P-dnaB complex. The addition of dnaJ to this complex also results in an isolatable intermediate. The dnaK, dnaJ and grpE proteins destabilize the lambda P-dnaB interaction, thus liberating dnaB's helicase activity, resulting in unwinding of the DNA template. At this stage, a stable DNA replication intermediate can be isolated, provided that the grpE protein has acted and/or is present. Following this, the dnaG primase enzyme recognizes the single-stranded DNA-dnaB complex and synthesizes RNA primers. Subsequently, the RNA primers are extended into DNA by DNA polymerase III holoenzyme. The proposed model of the molecular series of events taking place at ori lambda is substantiated by the many demonstrable protein-protein interactions among the various participants.

Bacterial Proteins↗
PubMed1989

The heat-shock-regulated grpE gene of Escherichia coli is required for bacterial growth at all temperatures but is dispensable in certain mutant backgrounds.

Previous work has established that the grpE+ gene product is a heat shock protein that is essential for bacteriophage lambda growth at all temperatures and for Escherichia coli growth at temperatures above 43 degrees C. Here it is shown that the grpE+ gene product is essential for bacterial viability at all temperatures. The strategy required constructing a grpE deletion derivative carrying a selectable chloramphenicol drug resistance marker provided by an omega insertion and showing that this deletion construct can be crossed into the bacterial chromosome if and only if a functional grpE+ gene is present elsewhere in the same cell. As a control, the same omega insertion could be placed immediately downstream of the grpE+ coding sequence without any observable effects on host growth. This result demonstrates that the inability to construct a grpE-deleted E. coli strain is not simply due to a lethal polar effect on neighboring gene expression. Unexpectedly, it was found that the grpE deletion derivative could be crossed into the bacterial chromosome in a strain that was defective in DnaK function. Further analysis showed that it was not the lack of DnaK function per se that allowed E. coli to tolerate a deletion in the grpE+ gene. Rather, it was the presence of unknown extragenic suppressors of a dnaK mutation that somehow compensated for the deficiency in both DnaK and GrpE function.

Blotting, Western↗