bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation of Antibiotic Biosynthesis by Specified Mutations in the
rpoB
Gene (Encoding the RNA Polymerase β Subunit) of
Streptomyces lividans
Authors: Hu Haifeng , Zhang Qin , Ochi Kozo 
Published: July 2002

ABSTRACT

We found that the biosynthesis of actinorhodin (Act), undecylprodigiosin (Red), and calcium-dependent antibiotic (CDA) are dramatically activated by introducing certain mutations into the
rpoB
gene that confer resistance to rifampin to
Streptomyces lividans
66, which produces less or no antibiotics under normal growth conditions. Activation of Act and/or Red biosynthesis by inducing mutations in the
rpoB
gene was shown to be dependent on the mutation's position and the amino acid species substituted in the β-subunit of the RNA polymerase. Mutation analysis identified 15 different kinds of point mutations, which are located in region I, II, or III of the
rpoB
gene and, in addition, two novel mutations (deletion of nucleotides 1287 to 1289 and a double substitution at nucleotides 1309 and 1310) were also found. Western blot analyses and S1 mapping analyses demonstrated that the expression of
act
II
-
ORF4 and
redD
, which are pathway-specific regulatory genes for Act and Red, respectively, was activated in the mutants able to produce Act and Red. The ActIV-ORF1 protein (an enzyme for Act biosynthesis) and the RedD protein were produced just after the upregulation of ActII-ORF4 and RedZ, respectively. These results indicate that the mutation in the
rpoB
gene of
S. lividans
, resulting in the activation of Act and/or Red biosynthesis, functions at the transcription level by activating directly or indirectly the key regulatory genes,
act
II
-
ORF4 and
redD
. We propose that the mutated RNA polymerase may function by mimicking the ppGpp-bound form in activating the onset of secondary metabolism in
Streptomyces
.
A Repressor Protein, PhaR, Regulates Polyhydroxyalkanoate (PHA) Synthesis via Its Direct Interaction with PHA
Authors: Maehara Akira , Taguchi Seiichi , Nishiyama Tatsuaki , Yamane Tsuneo , Doi Yoshiharu 
Published: July 2002

ABSTRACT

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the
phaR
gene, which is located downstream of
phaP
in
Paracoccus denitrificans
, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of
phaP
and
phaR
, suggesting that PhaR plays a role in the regulation of
phaP
expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant
Escherichia coli
was able to rebind specifically to poly[(
R
)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that
phaP
expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.
Role of the Murein Precursor UDP-
N
-Acetylmuramyl-
l
-Ala-γ-
d
-Glu-
meso
-Diaminopimelic Acid-
d
-Ala-
d
-Ala in Repression of β-Lactamase Induction in Cell Division Mutants
Authors: Uehara Tsuyoshi , Park James T. 
Published: July 2002

ABSTRACT

Certain β-lactam antibiotics induce the chromosomal
ampC
β-lactamase of many gram-negative bacteria. The natural inducer, though not yet unequivocally identified, is a cell wall breakdown product which enters the cell via the AmpG permease component of the murein recycling pathway. Surprisingly, it has been reported that β-lactamase is not induced by cefoxitin in the absence of FtsZ, which is required for cell division, or in the absence of penicillin-binding protein 2 (PBP2), which is required for cell elongation. Since these results remain unexplained, we examined an
ftsZ
mutant and other cell division mutants (
ftsA
,
ftsQ
, and
ftsI
) and a PBP2 mutant for induction of β-lactamase. In all mutants, β-lactamase was not induced by cefoxitin, which confirms the initial reports. The murein precursor, UDP-
N
-acetylmuramyl-
l
-Ala-γ-
d
-Glu-
meso
-diaminopimelic acid-
d
-Ala-
d
-Ala (UDP-MurNAc-pentapeptide), has been shown to serve as a corepressor with AmpR to repress β-lactamase expression in vitro. Our results suggest that β-lactamase is not induced because the
fts
mutants contain a greatly increased amount of corepressor which the inducer cannot displace. In the PBP2(Ts) mutant, in addition to accumulation of corepressor, cell wall turnover and recycling were greatly reduced so that little or no inducer was available. Hence, in both cases, a high ratio of repressor to inducer presumably prevents induction.
Detection and Differentiation of
Cryptosporidium
Parasites That Are Pathogenic for Humans by Real-Time PCR
Authors: Limor Josef R. , Lal Altaf A. , Xiao Lihua 
Published: July 2002

ABSTRACT

Cryptosporidiosis is a significant cause of food-borne and waterborne outbreaks of diarrheal diseases. To better understand the route of transmission of
Cryptosporidium
parasites, a number of genotyping techniques have been developed, based on PCR-restriction fragment length polymorphism or sequencing analysis of antigen, structural, and housekeeping genes. In this study, a real-time assay for the detection of
Cryptosporidium
oocysts is described. This technique had a detection limit of five oocysts. By melting curve analysis of PCR products with fluorescence-labeled hybridization probes, this technique was able to differentiate five common
Cryptosporidium
parasites that are pathogenic for humans in a single PCR. We evaluated and validated the test using samples from presently known
Cryptosporidium
parasites that are pathogenic for humans. This technique provides an alternative molecular tool in epidemiologic studies of human cryptosporidiosis.
Rapid and Simple Approach for Identification of
Mycobacterium tuberculosis
Complex Isolates by PCR-Based Genomic Deletion Analysis
Authors: Parsons Linda M. , Brosch Roland , Cole Stewart T. , Somoskövi Ákos , Loder Arthur , Bretzel Gisela , van Soolingen Dick , Hale Yvonne M. , Salfinger Max 
Published: July 2002

ABSTRACT

Although the virulences and host ranges differ among members of the
Mycobacterium tuberculosis
complex (TBC;
M. tuberculosis
,
M. africanum
,
M. canettii
,
M. microti
,
M. bovis
, and
M. bovis
BCG), commercially available molecular assays cannot differentiate these organisms because of the genetic identities of their 16S rRNA gene sequences. Comparative genomic analyses with the complete DNA sequence of
M. tuberculosis
H37Rv has provided information on regions of difference (RD 1 to RD 16) deleted in members of the TBC other than
M. tuberculosis
. To determine whether deletion analysis could accurately differentiate members of TBC, we used PCR to assess the presence or absence of specific regions of the genome in 88 well-characterized isolates of
M. tuberculosis
,
M. africanum
,
M. microti
,
M. bovis
, and
M. bovis
BCG. The identifications obtained by use of the specific deletion profiles correlated 100% with the original identifications for all TBC members except
M. africanum
, but further characterization resulted in profiles specific for all members. Although six RD regions were used in the analyses with the original 88 isolates, it was found that the use of RD 1, RD 9, and RD 10 was sufficient for initial screenings, followed by the use of RD 3, RD 5, and RD 11 if the results for any of the first three regions were negative. When 605 sequential clinical isolates were screened, 578 (96%) were identified as
M. tuberculosis
, 6 (1%) were identified as
M. africanum
, 8 (1%) were identified as
M. bovis
, and 13 (2%) were identified as
M. bovis
BCG. Since PCR-based assays can be implemented in most clinical mycobacteriology laboratories, this approach provides a rapid and simple means for the differentiation of members of TBC, especially
M. bovis
and
M. tuberculosis
, when it is important to distinguish between zoonotic sources (i.e., cattle and unpasteurized dairy products) and human sources of tuberculosis disease.
Sensitivity of the Procleix HIV-1/HCV Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA in a High-Risk Population
Authors: Jackson J. B. , Smith K. , Knott C. , Korpela A. , Simmons A. , Piwowar-Manning E. , McDonough S. , Mimms L. , Vargo J. M. 
Published: July 2002

ABSTRACT
The Procleix HIV-1/HCV Assay is a high-throughput nucleic acid test for the simultaneous detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNA during blood donor screening. This study evaluated the clinical sensitivity of the Procleix assay and assessed the assay's ability to identify HIV-1- and HCV-infected individuals undetected by standard serologic tests. Plasma samples were obtained prospectively from 539 individuals at high risk for HIV-1 and HCV infection at seven clinics affiliated with Johns Hopkins University. Samples were tested in the Procleix HIV-1/HCV Assay and, if reactive, were then tested in the Procleix HIV-1 and HCV discriminatory assays to differentiate the source of viral RNA positivity. Of these 539 subjects, 287 (53.2%) tested reactive in the Procleix HIV-1/HCV Assay. In discriminatory assay testing, 12 of 287 subjects (4.2%) were reactive for HIV-1 RNA only, 260 (90.6%) were reactive for HCV RNA only, and 11 (3.8%) were coinfected with HIV-1 and HCV. The clinical sensitivity for samples tested neat was 100% for HIV-1 and 99.3% for HCV. Three subjects with Procleix HCV reactive/seronegative results seroconverted upon follow-up and were confirmed as Procleix HCV yield cases. The Procleix HIV-1/HCV Assay is a highly sensitive test that detects ongoing and early HIV-1 and HCV infection in a significant number of subjects at high risk for these diseases. Confirmation of Procleix yield cases upon follow-up demonstrated the ability of the Procleix HIV-1/HCV Assay to detect the presence of HIV-1 and HCV in blood earlier than standard serologic tests.
Seminested PCR for Diagnosis of Candidemia: Comparison with Culture, Antigen Detection, and Biochemical Methods for Species Identification
Authors: Ahmad Suhail , Khan Zaiba , Mustafa Abu S. , Khan Zia U. 
Published: July 2002

ABSTRACT

The rapid detection and identification of
Candida
species in clinical laboratories are extremely important for the management of patients with hematogenous candidiasis. The presently available culture and biochemical methods for detection and species identification of
Candida
are time-consuming and lack the required sensitivity and specificity. In this study, we have established a seminested PCR (snPCR) using universal and species-specific primers for detection of
Candida
species in serum specimens. The universal outer primers amplified the 3′ end of 5.8S ribosomal DNA (rDNA) and the 5′ end of 28S rDNA, including the internally transcribed spacer 2 (ITS2), generating 350- to 410-bp fragments from the four commonly encountered
Candida
species, viz.,
C. albicans
,
C. tropicalis
,
C. glabrata
, and
C. parapsilosis.
The species-specific primers, complementary to unique sequences within the ITS2 of each test species, amplified species-specific DNA in the reamplification step of the snPCR. The sensitivity of
Candida
detection by snPCR in spiked serum specimens was close to 1 organism/ml. Evaluation of snPCR for specific identification of
Candida
species with 76 clinical
Candida
isolates showed 99% concordant results with the Vitek and/or ID32C yeast identification system. Further evaluation of snPCR for detection of
Candida
species in sera from culture-proven (
n
= 12), suspected (
n
= 16), and superficially colonized (
n
= 10) patients and healthy subjects (
n
= 12) showed that snPCR results were consistently negative with sera from healthy individuals and colonized patients. In culture-proven candidemia patients, the snPCR results were in full agreement with blood culture results with respect to both positivity and species identity. In addition, snPCR detected candidemia due to two
Candida
species in five patients, compared to three by blood culture. In the category of suspected candidemia with negative blood cultures for
Candida
, nine patients (56%) were positive by snPCR; two of them had dual infection with
C. albicans
and either
C. tropicalis
or
C. glabrata
. In conclusion, the snPCR developed in this study is specific and more sensitive than culture for the detection of
Candida
species in serum specimens. Moreover, the improved detection of cases of candidemia caused by more than one
Candida
species is an additional advantage.
Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing
Escherichia coli
Authors: Reischl Udo , Youssef Mohammad T. , Kilwinski Jochen , Lehn Norbert , Zhang Wen Lan , Karch Helge , Strockbine Nancy A. 
Published: July 2002

ABSTRACT

PCR assays have proved useful for detecting and characterizing Shiga toxin-producing
Escherichia coli
(STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (
stx
1
and
stx
2
) and another that simultaneously detects the genes for intimin (
eae
) and enterohemolysin (E-
hly
). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of
E. coli
negative for
stx
genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the
stx
1
,
eae
, and E-
hly
genes and 96 and 100%, respectively, for the
stx
2
gene. No
stx
2
genes were detected from 10
stx
2f
-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains.
Differences between
Staphylococcus aureus
Isolates from Medical and Nonmedical Hospital Personnel
Authors: Cespedes Christian , Miller Maureen , Quagliarello Bianca , Vavagiakis Peter , Klein Robert S. , Lowy Franklin D. 
Published: July 2002

ABSTRACT

It is unclear whether the levels of
Staphylococcus aureus
colonization of hospital personnel with patient exposure are increased or whether personnel become colonized with more antibiotic-resistant strains. Differences in nasal and hand carriage of
S. aureus
between medical and nonmedical hospital personnel were examined. No differences in nasal carriage between the two groups were found; however, there was a trend that suggested differences in the rates of hand carriage of
S
.
aureus
(18% of nonmedical personnel and 10% of medical personnel). Medical personnel were colonized with more antibiotic-resistant isolates than nonmedical personnel (mean, 2.8 versus 2.1 isolates [
P
< 0.03]), and the strain profiles indicated that they tended to be more clonal in origin, suggesting that exposure to hospital isolates alters the colonization profile.
Transmission of
Campylobacter hyointestinalis
from a Pig to a Human
Authors: Gorkiewicz Gregor , Feierl Gebhard , Zechner Rudolf , Zechner Ellen L. 
Published: July 2002

ABSTRACT

We report on a case of human gastroenteritis caused by the pathogen
Campylobacter hyointestinalis
. Recurrent watery diarrhea and intermittent vomiting were the most significant symptoms of the previously healthy patient. Whole-cell protein electrophoresis and 16S rRNA gene sequencing were used to identify this
Campylobacter
species. Investigation of the patient's surroundings led to the recovery of a second
C. hyointestinalis
strain originating from porcine feces. Subsequent typing of the human and the porcine isolates by pulsed-field gel electrophoresis revealed similar macrorestriction profiles, indicating transmission of this pathogen.
Large-Scale Outbreak of Infection with
Mycobacterium chelonae
subsp.
abscessus
after Penicillin Injection
Authors: Zhibang Yang , BiXia Zhang , Qishan Lu , Lihao Chen , Xiangquan Liu , Huaping Li 
Published: July 2002

ABSTRACT

An outbreak of infection with
Mycobacterium chelonae
subsp.
abscessus
after the injection of penicillin in 86 patients attending a factory hospital is reported. The bacterium was isolated both from lids and from the soil where the drug was stored. Molecular analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and plasmids revealed a pattern identical to that of the strains isolated from the wounds. The source of the infections was soil contamination of the vial lids and was caused by improper use and sterilization of penicillin vials.
Septicemia Due to
Acinetobacter junii
Authors: Linde Hans-Jörg , Hahn Joachim , Holler Ernst , Reischl Udo , Lehn Norbert 
Published: July 2002

ABSTRACT

Acinetobacter
spp. are considered to be emerging nosocomial pathogens.
Acinetobacter junii
is a rare cause of disease in humans and was associated mainly with bacteremia in preterm infants and pediatric oncologic patients. In this report we describe a case of catheter-related infection by
A. junii
in an adult oncologic patient. Application of molecular methods for precise species identification of
Acinetobacter
spp. will help to further clarify their role as human pathogens.
Human Deep Tissue Infection with an EntomopathogenicBeauveriaSpecies
Authors: Henke Markus Oliver , de Hoog G. Sybren , Gross Uwe , Zimmermann Gisbert , Kraemer Doris , Weig Michael 
Published: July 2002

ABSTRACTBeauveriaspp. are ubiquitous fungal entomopathogens that are commercially distributed as biological insecticides worldwide. In this paper we describe the clinical manifestation, diagnosis, and therapy of the first documented human deep tissue infection with an entomopathogenicBeauveriaspecies in a patient receiving immunosuppressive therapy and describe the morphological and molecular characterization of the mold.
AIDS Vaccination Studies Using an Ex Vivo Feline Immunodeficiency Virus Model: Failure To Protect and Possible Enhancement of Challenge Infection by Four Cell-Based Vaccines Prepared with Autologous Lymphoblasts
Authors: Giannecchini Simone , Isola Patrizia , Sichi Olimpia , Matteucci Donatella , Pistello Mauro , Zaccaro Lucia , Del Mauro Daniela , Bendinelli Mauro 
Published: July 2002

ABSTRACT
Immunogenicity and protective activity of four cell-based feline immunodeficiency virus (FIV) vaccines prepared with autologous lymphoblasts were investigated. One vaccine was composed of FIV-infected cells that were paraformaldehyde fixed at the peak of viral expression. The other vaccines were attempts to maximize the expression of protective epitopes that might become exposed as a result of virion binding to cells and essentially consisted of cells mildly fixed after saturation of their surface with adsorbed, internally inactivated FIV particles. The levels of FIV-specific lymphoproliferation exhibited by the vaccinees were comparable to the ones previously observed in vaccine-protected cats, but antibodies were largely directed to cell-derived constituents rather than to truly viral epitopes and had very poor FIV-neutralizing activity. Moreover, under one condition of testing, some vaccine sera enhanced FIV replication in vitro. As a further limit, the vaccines proved inefficient at priming animals for anamnestic immune responses. Two months after completion of primary immunization, the animals were challenged with a low dose of homologous ex vivo FIV. Collectively, 8 of 20 vaccinees developed infection versus one of nine animals mock immunized with fixed uninfected autologous lymphoblasts. After a boosting and rechallenge with a higher virus dose, all remaining animals became infected, thus confirming their lack of protection.
Engineering of Adenovirus Vectors Containing Heterologous Peptide Sequences in the C Terminus of Capsid Protein IX
Authors: Dmitriev Igor P. , Kashentseva Elena A. , Curiel David T. 
Published: July 2002

ABSTRACT
The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.