bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Characterization of
Thermotoga maritima
Endonuclease IV, a Thermostable Apurinic/Apyrimidinic Endonuclease and 3′-Repair Diesterase
Authors: Haas Brian J. , Sandigursky Margarita , Tainer John A. , Franklin William A. , Cunningham Richard P. 
Published: December 2019

ABSTRACT

An endonuclease IV homolog was identified as the product of a conceptual open reading frame in the genome of the hyperthermophilic bacterium
Thermotoga maritima
. The
T. maritima
endonuclease IV gene encodes a 287-amino-acid protein with 32% sequence identity to
Escherichia coli
endonuclease IV. The gene was cloned, and the expressed protein was purified and shown to have enzymatic activities that are characteristic of the endonuclease IV family of DNA repair enzymes, including apurinic/apyrimidinic endonuclease activity and repair activities on 3′-phosphates, 3′-phosphoglycolates, and 3′-
trans
-4-hydroxy-2-pentenal-5-phosphates. The
T. maritima
enzyme exhibits enzyme activity at both low and high temperatures. Circular dichroism spectroscopy indicates that
T. maritima
endonuclease IV has secondary structure similar to that of
E. coli
endonuclease IV and that the
T. maritima
endonuclease IV structure is more stable than
E. coli
endonuclease IV by almost 20°C, beginning to rapidly denature only at temperatures approaching 90°C. The presence of this enzyme, which is part of the DNA base excision repair pathway, suggests that thermophiles use a mechanism similar to that used by mesophiles to deal with the large number of abasic sites that arise in their chromosomes due to the increased rates of DNA damage at elevated temperatures.
YscP of
Yersinia pestis
Is a Secreted Component of the Yop Secretion System
Authors: Payne Patricia L. , Straley Susan C. 
Published: December 2019

ABSTRACT

The
Yersinia pestis
low-Ca
2+
response stimulon is responsible for the environmentally regulated expression and secretion of antihost proteins (V antigen and Yops). We have previously shown that
yscO
encodes a secreted core component of the Yop secretion (Ysc) mechanism. In this study, we constructed and characterized in-frame deletions in the adjacent gene,
yscP
, in the
yscN–yscU
operon. The ΔP1 mutation, which removed amino acids 246 to 333 of YscP, had no effect on Yop expression or secretion, and the mutant protein, YscP1, was secreted, as was YscP in the parent. In contrast, the ΔP2 strain expressed and secreted less of each Yop than did the parent under the inductive conditions of 37°C and the absence of Ca
2+
, with an exception being YopE, which was only minimally affected by the mutation. The YscP2 protein, missing amino acids 57 to 324 of YscP, was expressed but not secreted by the ΔP2 mutant. The effect of the ΔP2 mutation was at the level of Yop secretion because YopM and V antigen still showed limited secretion when overproduced in
trans
. Excess YscP also affected secretion: overexpression of YscP in the parent, in either
yscP
mutant, or in an
lcrG
mutant effectively shut off secretion. However, co-overexpression of YscO and YscP had no effect on secretion, and YscP overexpression in an
lcrE
mutant had little effect on Yop secretion, suggesting that YscP acts, in conjunction with YscO, at the level of secretion control of LcrE at the bacterial surface. These findings place YscP among the growing family of mobile Ysc components that both affect secretion and themselves are secreted by the Ysc.
Expression of Two Glutathione
S
-Transferase Genes in the Yeast
Issatchenkia orientalis
Is Induced by
o
-Dinitrobenzene during Cell Growth Arrest
Authors: Tamaki Hisanori , Yamamoto Kenji , Kumagai Hidehiko 
Published: December 2019

ABSTRACT

Glutathione
S
-transferases (GSTs) Y-1 and Y-2 from the yeast
Issatchenkia orientalis
were purified by passage through a glutathione-agarose column, and the cDNA for GST Y-1 was cloned and sequenced. The deduced amino acid sequence consisted of 188 residues with a total calculated molecular mass of 21,001 Da and showed 36.7% identity to that of GST Y-2, another GST isoenzyme expressed in this strain.
Escherichia coli
DH5α transformed with pUC119 harboring the GST Y-1 gene under the control of the
lac
promoter exhibited 29-fold-higher GST activity than the same strain with pUC119. Northern blot analysis revealed that both genes were highly expressed in cells cultured in the presence of 200 μM
o
-dinitrobenzene (DNB), one of the substrates of GST, while only the GST Y-1 gene was expressed, and only slightly, under normal (DNB-free) culture conditions. The DNB in the medium arrested cell growth until it was reduced by conjugation with reduced glutathione. Kinetic analysis of GST gene expression during detoxification of DNB revealed that the levels of expression of both genes were elevated within 3 h after the addition of DNB and that they further increased until 12 h postaddition. The levels of expression of both genes were decreased markedly when the DNB concentration in the culture medium was lowered. These results suggest that
I. orientalis
cells sense xenobiotics and arrest cell growth as a mechanism for preventing the induction of mutations by these compounds, while the levels of expression of the GST genes are up-regulated for detoxification.
The MobA-Linked Primase Is the Only Replication Protein of R1162 Required for Conjugal Mobilization
Authors: Henderson Dorian , Meyer Richard 
Published: December 2019

ABSTRACT
Cells newly transformed with plasmid R1162 DNA were used as donors in conjugal matings to determine if the plasmid replication genes are necessary for transfer. An intact system for vegetative replication is not required for transfer at normal frequency, but the plasmid primase, in the form linked to the nickase, must be present in donor cells.
First Isolation and Cultivation of
Borrelia burgdorferi
Sensu Lato from Missouri
Authors: Oliver J. H. , Kollars T. M. , Chandler F. W. , James A. M. , Masters E. J. , Lane R. S. , Huey L. O. 
Published: December 2019

ABSTRACT

Five
Borrelia burgdorferi
sensu lato isolates from Missouri are described. This represents the first report and characterization of such isolates from that state. The isolates were obtained from either
Ixodes dentatus
or
Amblyomma americanum
ticks that had been feeding on cottontail rabbits (
Sylvilagus floridanus
) from a farm in Bollinger County, Mo., where a human case of Lyme disease had been reported. All isolates were screened immunologically by indirect immunofluorescence by using monoclonal antibodies to
B. burgdorferi
-specific outer surface protein A (OspA) (antibodies H3TS and H5332),
B. burgdorferi
-specific OspB (antibody H6831),
Borrelia
(genus)-specific antiflagellin (antibody H9724), and
Borrelia hermsii
-specific antibody (antibody H9826). Analysis of the isolates also involved a comparison of their protein profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Finally, the isolates were analyzed by PCR with six pairs of primers known to amplify selected DNA target sequences specifically found in the reference strain
B. burgdorferi
B-31. Although some genetic variability was detected among the five isolates as well as between them and the B-31 strain, enough similarities were found to classify them as
B. burgdorferi
sensu lato.
Past and Present Hepatitis G Virus Infections in Areas Where Hepatitis C is Highly Endemic and Those Where It Is Not Endemic
Authors: Tanaka Eiji , Tacke Michael , Kobayashi Masakazu , Nakatsuji Yoshiyuki , Kiyosawa Kendo , Schmolke Susanne , Engel Alfred M. , Hess Georg , Alter Harvey J. 
Published: December 2019

ABSTRACT

We reported previously on an area in Japan where over 30% of the inhabitants were positive for hepatitis C virus (HCV) antibody. In the present study, clinical features of hepatitis G virus (HGV) infection in this area of high endemicity were compared to those in an area where HCV is not endemic. A total of 400 individuals were selected randomly from those who were medically screened for liver disease in 1993; 200 were from the high-endemicity area, and the other 200 were from the no-endemicity area. HGV RNA was measured by reverse transcription and PCR with primers in the 5′ noncoding region. Antibody to HGV envelope protein E2 was measured by an enzyme-linked immunosorbent assay. Prevalence of any HGV marker in the high-endemicity area (32%) was significantly (
P
< 0.0001) higher than that in the no-endemicity area (6%); similar differences, 32% versus 3% (
P
< 0.0001), had been observed for HCV markers (HCV RNA and HCV antibody). In areas of both high and no endemicity, HCV markers were significantly more prevalent in individuals with any HGV marker than in those without HGV markers, and age-specific prevalence of HGV markers was distributed similarly to that of any HCV marker. Among possible routes of HGV transmission that were analyzed, folk medicine was significant in the high-endemicity area, but blood transfusion was the major route in the no-endemicity area. The rate of accompanying viremia in HGV infection (15%) was significantly lower than that in HCV infection (78%) (
P
< 0.0001). In conclusion, HGV infection was highly prevalent in the area of high HCV endemicity and was closely associated with HCV infection. HGV seemed to be transmitted via the practice of folk medicine as well as blood transfusion. HGV resulted in a chronic carrier state less frequently than did HCV.
Enterobacterial Repetitive Intergenic Consensus Sequences as Molecular Targets for Typing of
Mycobacterium tuberculosis
Strains
Authors: Sechi Leonardo A. , Zanetti Stefania , Dupré Ilaria , Delogu Giovanni , Fadda Giovanni 
Published: December 2019

ABSTRACT

The presence of enterobacterial repetitive intergenic consensus (ERIC) sequences was demonstrated for the first time in the genome of
Mycobacterium tuberculosis
; these sequences have been found in transcribed regions of the chromosomes of gram-negative bacteria. In this study genetic diversity among clinical isolates of
M. tuberculosis
was determined by PCR with ERIC primers (ERIC-PCR). The study isolates comprised 71 clinical isolates collected from Sardinia, Italy. ERIC-PCR was able to identify 59 distinct profiles. The results obtained were compared with IS
6110
and PCR-GTG fingerprinting. We found that the level of differentiation obtained by ERIC-PCR is greater than that obtained by IS
6110
fingerprinting and comparable to that obtained by PCR-GTG. This method of fingerprinting is rapid and sensitive and can be applied to the study of the epidemiology of
M. tuberculosis
infections, especially when IS
6110
fingerprinting is not of any help.
Detection of
Porphyromonas gingivalis
from Saliva by PCR by Using a Simple Sample-Processing Method
Authors: Mättö Jaana , Saarela Maria , Alaluusua Satu , Oja Virva , Jousimies-Somer Hannele , Asikainen Sirkka 
Published: December 2019

ABSTRACT

Simple sample-processing methods for PCR detection of
Porphyromonas gingivalis
, a major pathogen causing adult periodontitis, from saliva were studied. The ability to detect
P. gingivalis
from 118 salivary samples by PCR after boiling and Chelex 100 processing was compared with bacterial culture.
P. gingivalis
was detected three times more often by PCR than by culture. Chelex 100 processing of saliva proved to be effective in preventing PCR inhibition and was applied to determine the occurrence of
P. gingivalis
in saliva samples from 263 Finnish subjects between 5 and 80 years of age. The occurrence of
P. gingivalis
increased with age, and it was detected by PCR in the saliva of 5.0% of subjects between 5 and 10 years of age, 13.8% of subjects between 11 and 20 years of age, 13.4% of subjects between 21 and 30 years of age, and 63.3% of subjects between 31 and 80 years of age. The results indicate that
P. gingivalis
is a rare finding in saliva from periodontally healthy children and young adults but a frequent one in saliva from adult periodontitis patients.
An Internal Control for Routine Diagnostic PCR: Design, Properties, and Effect on Clinical Performance
Authors: Rosenstraus Maurice , Wang Zhuang , Chang Sheng-Yung , DeBonville David , Spadoro Joanne P. 
Published: December 2019

ABSTRACT

We constructed internal controls (ICs) to provide assurance that clinical specimens are successfully amplified and detected. The IC nucleic acids contain primer binding regions identical to those of the target sequence and contain a unique probe binding region that differentiates the IC from amplified target nucleic acid. Because only 20 copies of the IC are introduced into each test sample, a positive IC signal indicates that amplification was sufficient to generate a positive signal from targets present at the limit of test sensitivity. The COBAS AMPLICOR
Chlamydia trachomatis
,
Neisseria gonorrhoeae
,
Mycobacterium tuberculosis
, and human hepatitis C virus tests exhibited inhibition rates ranging from 5 to 9%. Approximately 64% of these inhibitory specimens were not inhibitory when a second aliquot was tested. Because repeatedly inhibitory specimens were not reported as false negative and because additional infected specimens were detected during retesting, test sensitivities were 1 to 6% greater than they would have been if the IC had not been used.
In Vitro Activity of the New Triazole Voriconazole (UK-109,496) against Opportunistic Filamentous and Dimorphic Fungi and Common and Emerging Yeast Pathogens
Authors: Espinel-Ingroff Ana 
Published: December 2019

ABSTRACTThe in vitro antifungal activity of a new triazole derivative, voriconazole, was compared with those of itraconazole and amphotericin B against 67 isolates ofAspergillus flavus,Aspergillus fumigatus,Bipolarisspp.,Fusarium oxysporum,Fusarium solani,Pseudallescheria boydii,Rhizopus arrhizus,Blastomyces dermatitidis,Histoplasma capsulatum, andSporothrix schenckii. The in vitro activities of voriconazole were also compared with those of amphotericin B, fluconazole, and itraconazole against 189 isolates of emerging and common yeast pathogens ofBlastoschizomyces capitatus,Candida(13 species),Cryptococcus neoformans,Hansenula anomala,Rhodotorula rubra,Saccharomyces cerevisiae,Sporobolomyces salmonicolor, andTrichosporon beigelii. MICs were determined according to a procedure under evaluation by the National Committee for Clinical Laboratory Standards (NCCLS) for broth microdilution testing of filamentous fungi and by the NCCLS M27-A broth microdilution method for yeasts. The in vitro activities of voriconazole were similar to or better than those of itraconazole and amphotericin B againstAspergillusspp.,Fusariumspp., andP. boydiias well as againstB. dermatitidisandH. capsulatum. The activities of voriconazole were also comparable to or better than those of amphotericin B, fluconazole, and itraconazole against most species of yeasts tested. Exceptions were certain isolates ofR. rubraandS. salmonicolor. These results suggest that voriconazole has a wide spectrum of activity in vitro; its effectiveness in the treatment of human mycoses is under evaluation in clinical trials.
Characterization of
rpoB
Mutations in Rifampin-Resistant Clinical
Mycobacterium tuberculosis
Isolates from Greece
Authors: Matsiota-Bernard Peggy , Vrioni Georgia , Marinis Evangelos 
Published: December 2019

ABSTRACT

There is a geographic distribution of
Mycobacterium tuberculosis
strains with various
rpoB
gene mutations that account for rifampin resistance. We studied 17 rifampin-resistant clinical isolates from patients in Greece to identify
rpoB
mutations. The aim of our study was the evaluation of a commercially available line probe assay kit (INNO-LiPA Rif. TB) to detect
rpoB
mutations and rifampin resistance. The results obtained with the commercially available assay were compared to those obtained by automated DNA sequence analysis of amplified PCR products. Randomly amplified polymorphic DNA (RAPD) analyses of the isolates were also performed. The overall concordance of the line probe assay with phenotypic rifampin susceptibility test was 94%. Three distinct
rpoB
mutations in codons Ser
531
, His
526
, and Asp
516
were correctly identified with the kit, but mutations in external regions and insertions were detected only by automated DNA sequence analysis. The changes in codons Ser
531
and His
526
accounted for the majority of rifampin resistance, as previously described for isolates from other geographic areas. The results obtained by RAPD analyses of the isolates suggested that clonally related
M. tuberculosis
strains can have subclones bearing distinct mutant
rpoB
alleles. We conclude that this line probe assay kit, which is fast and with which tests are easy to perform, can be used for the rapid detection of rifampin resistance in
M. tuberculosis
before the availability of results by conventional methods and for epidemiological studies but that negative results obtained by this method do not rule out rifampin resistance.
Antitoxin-in-Membrane and Antitoxin-in-Well Assays for Detection of Toxigenic
Corynebacterium diphtheriae
Authors: Reinhardt D. J. , Lee A. , Popovic T. 
Published: December 2019

ABSTRACT

The Elek culture plate precipitin test is routinely used for the detection of exotoxin from toxigenic strains of
Corynebacterium diphtheriae
. Recently, the World Health Organization standardized this test to ensure accuracy, reliability, and reproducibility. In this study, we further modified the standard Elek test by using the antitoxin-in-membrane (AIM) and antitoxin-in-well (AIW) approaches. In the AIM tests, each strain was stabbed and streaked backwards and away from a point approximately 7 mm from the edge of a sterile cellulose acetate-cellulose nitrate filter membrane disk (pore size, 0.45 μm; diameter, 25 mm) containing 25 IU of diphtheria antitoxin. For AIW tests, a central well (diameter, 5 mm) containing 9 μl of antitoxin (4.5 IU) was surrounded by eight equidistant stab-streaks of each strain placed 10 mm from the well. In both methods, precipitin bands of identity typically were noted after 24- and 48-h incubations at 37°C. Both toxigenic and weak toxigenic strains gave clear and reproducible results. Compared with the standard Elek test, the AIM and AIW tests each use 50% less medium and 75 and 87% less antitoxin, respectively. AIM has the potential to test up to 14 isolates and AIW has the potential to test up to 24 isolates on the same plate. Furthermore, clearer positives were noted with weak toxigenic strains. In a blinded test of 209 verified
C. diphtheriae
isolates, a 99.5% agreement with the standard Elek test was obtained overall. Both modifications conserve reagents and medium, permit the simultaneous testing of a larger number of strains, and may be particularly suitable for reference laboratories or hospitals involved in diphtheria epidemic settings.
Repetitive Sequence-Based PCR versus Pulsed-Field Gel Electrophoresis for Typing of
Enterococcus faecalis
at the Subspecies Level
Authors: Malathum Kumthorn , Singh Kavindra V. , Weinstock George M. , Murray Barbara E. 
Published: December 2019

ABSTRACT

Repetitive sequence-based PCR was compared to pulsed-field gel electrophoresis (PFGE) for the ability to discriminate
Enterococcus faecalis
isolates at the subspecies level. The BOXA2R primer, derived from repetitive sequences in
Streptococcus pneumoniae
, was applied to 41 isolates of
E. faecalis
collected from various sources. The REP1R-Dt and REP2-Dt primers, derived from the gram-negative repetitive extragenic palindromic element, were also applied to 18 selected isolates. Of the 41 isolates examined, 7 were β-lactamase producing and 8 were vancomycin resistant. By PFGE, 17 isolates had distinct patterns; the other 24 were classified into eight different clonal groups. By PCR using the BOXA2R primer, 16 isolates generated distinct patterns; the other 25 were classified into nine different clonal groups. There were only minor differences in the PCR results obtained by using the BOXA2R primer and the REP1R-Dt and REP2-Dt primers. Two isolates among vancomycin-resistant enterococci from the greater Houston, Tex., area were related by PFGE, distinct by PCR with the BOXA2R primer, and related by PCR with the REP1R-Dt and REP2-Dt primers. Clonal relationships among the remaining 39 isolates were similar by both PFGE and PCR. PCR reliably discriminated all epidemiologically unrelated isolates. Although PCR is less time consuming than PFGE, PCR results were more difficult to interpret than PFGE results, perhaps because fewer bands were generated by PCR than by PFGE and some PCR products were inconsistently seen.
Hepatitis C Virus Heteroduplex Tracking Assay for Genotype Determination Reveals Diverging Genotype 2 Isolates in Italian Hemodialysis Patients
Authors: Calvo Pier Luigi , Kansopon Joe , Sra Kuldip , Quan Stella , DiNello Robert , Guaschino Roberto , Calabrese Giovanni , Danielle Franca , Brunetto Mauizia Rossana , Bonino Ferruccio , Massaro Anna Lucia , Polito Alan , Houghton Michael , Weiner Amy J. 
Published: December 2019

ABSTRACT
A heteroduplex tracking assay (HTA) was developed for genetic analyses of the hepatitis C virus (HCV) using single-stranded probes from the core (C)/E1 region. Nucleotide sequencing of reverse transcriptase (RT)-PCR products from 15 Italian dialysis patients confirmed the specificity and accuracy of the HTA genotyping method, which identified 5 of 15 (33.3%) 1b, 7 of 15 (46.7%) 3a, and 3 of 15 (20%) type 2 infections. The genotypes of an additional 12 HCV antibody-positive blood donors from different geographical locations were also in agreement with the genotypes determined by the Inno-LiPA HCV II kit (Innogenetics) and/or restriction fragment length polymorphism (RFLP). Isolates which had between 35 to 40% nucleotide divergence from control subtype 1a, 1b, 2a, 2b, or 3a standards could be typed. Surprisingly, HTA detected one 1b-2 coinfection which was missed by DNA sequencing. Three samples that were designated non-2a or 2b type 2 by HTA were found to be type 2a by both RFLP and direct nucleotide sequencing of the 5′ untranslated region. The genetic distance between patient type 2 and control 2a, 2b, and 2c isolates indicated that a new subtype was present in the population being studied. Serotyping (RIBA serotyping strip immunoblot assay kit) of 23 dialysis patients showed that the genotype could be determined in 6 of 8 (75%) C/E1 RT-PCR-negative and 15 of 23 (65.2%) RT-PCR-positive samples, indicating that the two tests complement each other.
Comparison of Six Sets of PCR Primers from Two Different Genomic Regions for Amplification of GB Virus C/Hepatitis G Virus RNA
Authors: Andonov Anton , Sauder Connie , Jacobsen Heather , Chaudhary Rabindra 
Published: December 2019

ABSTRACT
Forty-four clinical samples positive for GB virus C (GBV-C)/hepatitis G virus (HGV) were tested with six primer sets, four from the 5′ untranslated region (5′-UTR) and two from the NS5a genomic region. Two of the 5′-UTR primer sets, when used in a single-round 60-cycle PCR, detected between 86.4 and 97.7% of the positive samples, while two different sets from the same area, when used in a nested PCR, amplified between 97.7 and 100% of the positive specimens. Both sets from the NS5a region, when used in a single-round PCR, detected 95.5% of the GBV-C/HGV-positive samples. Parallel testing with two PCR sets, one from the 5′-UTR and a second from NS5a, may eliminate false-negative results attributable to the genetic heterogeneity of the virus.