bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modified Pathway To Synthesize Ribulose 1,5-Bisphosphate in Methanogenic Archaea
Authors: Finn Michael W. , Tabita F. Robert 
Published: September 2004

ABSTRACT

Several sequencing projects unexpectedly uncovered the presence of genes that encode ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) in anaerobic archaea. RubisCO is the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway, a scheme that does not appear to contribute greatly, if at all, to net CO
2
assimilation in these organisms. Recombinant forms of the archaeal enzymes do, however, catalyze a bona fide RuBP-dependent CO
2
fixation reaction, and it was recently shown that
Methanocaldococcus
(
Methanococcus
)
jannaschii
and other anaerobic archaea synthesize catalytically active RubisCO in vivo. To complete the CBB pathway, there is a need for an enzyme, i.e., phosphoribulokinase (PRK), to catalyze the formation of RuBP, the substrate for the RubisCO reaction. Homology searches, as well as direct enzymatic assays with
M. jannaschii
, failed to reveal the presence of PRK. The apparent lack of PRK raised the possibility that either there is an alternative pathway to generate RuBP or RubisCO might use an alternative substrate in vivo. In the present study, direct enzymatic assays performed with alternative substrates and extracts of
M. jannsachii
provided evidence for a previously uncharacterized pathway for RuBP synthesis from 5-phospho-
d
-ribose-1-pyrophosphate (PRPP) in
M. jannaschii
and other methanogenic archaea. Proteins and genes involved in the catalytic conversion of PRPP to RuBP were identified in
M. jannaschii
(Mj0601) and
Methanosarcina acetivorans
(Ma2851), and recombinant Ma2851 was active in extracts of
Escherichia coli
. Thus, in this work we identified a novel means to synthesize the CO
2
acceptor and substrate for RubisCO in the absence of a detectable kinase, such as PRK. We suggest that the conversion of PRPP to RuBP might be an evolutional link between purine recycling pathways and the CBB scheme.
patS
Minigenes Inhibit Heterocyst Development of
Anabaena
sp. Strain PCC 7120
Authors: Wu Xiaoqiang , Liu Duan , Lee Martin H. , Golden James W. 
Published: September 2004

ABSTRACT

The
patS
gene encodes a small peptide that is required for normal heterocyst pattern formation in the cyanobacterium
Anabaena
sp. strain PCC 7120. PatS is proposed to control the heterocyst pattern by lateral inhibition.
patS
minigenes were constructed and expressed by different developmentally regulated promoters to gain further insight into PatS signaling.
patS
minigenes
patS4
to
patS8
encode PatS C-terminal 4 (GSGR) to 8 (CDERGSGR) oligopeptides. When expressed by P

petE

, P

patS

, or P

rbcL

promoters,
patS5
to
patS8
inhibited heterocyst formation but
patS4
did not. In contrast to the full-length
patS
gene, P

hepA
-patS5

failed to restore a wild-type pattern in a
patS
null mutant, indicating that PatS-5 cannot function in cell-to-cell signaling if it is expressed in proheterocysts. To establish the location of the PatS receptor, PatS-5 was confined within the cytoplasm as a
gfp-patS5
fusion. The green fluorescent protein GFP-PatS-5 fusion protein inhibited heterocyst formation. Similarly, full-length PatS with a C-terminal hexahistidine tag inhibited heterocyst formation. These data indicate that the PatS receptor is located in the cytoplasm, which is consistent with recently published data indicating that HetR is a PatS target. We speculated that overexpression of other
Anabaena
strain PCC 7120 RGSGR-encoding genes might show heterocyst inhibition activity. In addition to
patS
and
hetN
, open reading frame (ORF)
all3290
and an unannotated ORF,
orf77
, encode an RGSGR motif. Overexpression of
all3290
and
orf77
under the control of the
petE
promoter inhibited heterocyst formation, indicating that the RGSGR motif can inhibit heterocyst development in a variety of contexts.
Insecticidal Pilin Subunit from the Insect Pathogen
Xenorhabdus nematophila
Authors: Khandelwal Puneet , Choudhury Devapriya , Birah Ajanta , Reddy M. K. , Gupta Gorakh Prasad , Banerjee Nirupama 
Published: September 2004

ABSTRACT

Xenorhabdus nematophila
is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of
X. nematophila
. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of
X. nematophila
isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in
Escherichia coli
. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured
Helicoverpa armigera
larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest,
H. armigera
, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.
An Energy Taxis Transducer Promotes Root Colonization byAzospirillum brasilense
Authors: Greer-Phillips Suzanne E. , Stephens Bonnie B. , Alexandre Gladys 
Published: September 2004

ABSTRACTMotility responses triggered by changes in the electron transport system are collectively known as energy taxis. InAzospirillum brasilense, energy taxis was shown to be the principal form of locomotor control. In the present study, we have identified a novel chemoreceptor-like protein, named Tlp1, which serves as an energy taxis transducer. The Tlp1 protein is predicted to have an N-terminal periplasmic region and a cytoplasmic C-terminal signaling module homologous to those of other chemoreceptors. The predicted periplasmic region of Tlp1 comprises a conserved domain that is found in two types of microbial sensory receptors: chemotaxis transducers and histidine kinases. However, the function of this domain is currently unknown. We characterized the behavior of atlp1mutant by a series of spatial and temporal gradient assays. Thetlp1mutant is deficient in (i) chemotaxis to several rapidly oxidizable substrates, (ii) taxis to terminal electron acceptors (oxygen and nitrate), and (iii) redox taxis. Taken together, the data strongly suggest that Tlp1 mediates energy taxis inA. brasilense. Using qualitative and quantitative assays, we have also demonstrated that thetlp1mutant is impaired in colonization of plant roots. This finding supports the hypothesis that energy taxis and therefore bacterial metabolism might be key factors in determining host specificity inAzospirillum-grass associations.
Dynamic Cell Surface Hydrophobicity of
Lactobacillus
Strains with and without Surface Layer Proteins
Authors: Vadillo-Rodríguez Virginia , Busscher Henk J. , Norde Willem , de Vries Joop , van der Mei Henny C. 
Published: September 2004

ABSTRACT

Variations in surface hydrophobicity of six
Lactobacillus
strains with and without an S-layer upon changes in ionic strength are derived from contact angle measurements with low- and high-ionic-strength aqueous solutions. Cell surface hydrophobicity changed in response to changes in ionic strength in three out of the six strains, offering these strains a versatile mechanism to adhere to different surfaces. The dynamic behavior of the cell surface hydrophobicity could be confirmed for two selected strains by measuring the interaction force between hydrophobic and hydrophilic tips with use of atomic force microscopy.
Detection of Intrahepatic Hepatitis B Virus DNA and Correlation with Hepatic Necroinflammation and Fibrosis
Authors: Wong Danny Ka-Ho , Yuen Man-Fung , Tse Eric , Yuan HeJun , Sum Simon Siu-Man , Hui Chee-Kin , Lai Ching-Lung 
Published: September 2004

ABSTRACT

Assessment of intrahepatic hepatitis B virus (HBV) DNA levels in patients with chronic hepatitis B is important in understanding the natural history of the disease and designing antiviral therapy regimens. However, there is no standardized method for the measurement of intrahepatic HBV DNA levels. We describe a convenient novel method for the measurement of intrahepatic HBV DNA levels based on a modified COBAS Amplicor HBV Monitor test for HBV DNA measurement and real-time PCR β-actin gene detection for human genomic DNA (hgDNA) quantitation. Fifteen hepatitis B e antigen (HBeAg)-positive patients, 26 patients positive for antibody to HBeAg (anti-HBe), and 8 control patients were recruited. The mean between-run coefficient of variation for the β-actin real-time PCR assay was 15.4%. All eight control patients had undetectable intrahepatic and serum HBV DNA levels. All chronic hepatitis B patients had detectable intrahepatic HBV DNA levels, and all but one anti-HBe-positive patient had detectable serum HBV DNA levels. HBeAg-positive patients had higher median intrahepatic and serum HBV DNA levels than anti-HBe-positive patients (6,950 versus 676 HBV DNA copies/ng of hgDNA, respectively [
P
< 0.001] and 184 × 10
6
versus 6.65 × 10
6
copies/ml, respectively [
P
< 0.001]). The intrahepatic HBV DNA levels correlated strongly with the serum HBV DNA levels (
r
= 0.842;
P
< 0.001) and with the degree of fibrosis (
P
= 0.014). We conclude that the method that we describe is reliable and convenient for the measurement of intrahepatic HBV DNA levels and has potential clinical significance.
rpoB
Gene Sequencing for Identification of
Corynebacterium
Species
Authors: Khamis Atieh , Raoult Didier , La Scola Bernard 
Published: September 2004

ABSTRACT

The genus
Corynebacterium
is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete
rpoB
sequences of 56
Corynebacterium
species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete
rpoB
sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of
Corynebacterium
species.
Long-Term Follow-Up Study of Chinese Patients with YMDD Mutations: Significance of Hepatitis B Virus Genotypes and Characteristics of Biochemical Flares
Authors: Yuen Man-Fung , Yuan He-Jun , Sablon Erwin , Wong Danny Ka-Ho , Chan Annie On-On , Wong Benjamin Chun-Yu , Lai Ching-Lung 
Published: September 2004

ABSTRACT

We sought to examine the role of hepatitis B virus (HBV) genotypes in virological breakthroughs and biochemical flares in patients with YMDD mutations during lamivudine therapy. Virologic breakthroughs (i.e., the reappearance of HBV DNA as determined by bDNA assay) and biochemical flares (mild flares = alanine aminotransferase [ALT] between 2 and 10 times the upper limit of normal [ULN]; severe flares = ALT >10 times ULN) were monitored in 154 hepatitis B e antigen-positive patients receiving long-term lamivudine. The HBV genotypes and YMDD mutations were determined. Forty-three patients had virological breakthroughs with YMDD mutations (median follow-up of 29.6 months [range, 22.3 to 61.4]). Twenty patients (47%) patients had mild biochemical flares; seven (16%) had severe flares. Two patients showed an elevation of bilirubin level that is >2 times the ULN. All patients recovered spontaneously. The cumulative risks for biochemical flares were 28, 47, and 58% for the first 3 years, respectively. Patients with biochemical flares compared to those without flares had a significantly higher median pretreatment ALT level (61 U/liter versus 34.5 U/liter [
P
= 0.012]). There were no differences in the cumulative risk of virological breakthroughs, risk, and severity of biochemical flares between patients with genotypes B (
n
= 11) and C (
n
= 32). There was an increase in the percentage of patients with single YMDD mutant at last follow-up compared to that at the time of virological breakthroughs (74% [
n
= 32] versus 47% [
n
= 20], respectively;
P
= 0.015). The chances of YMDD mutations with virological breakthroughs and biochemical flares were similar in patients with genotypes B and C. Biochemical flares were common, with 16% being severe in nature. High pretreatment ALT levels were associated with a higher chance of biochemical flares.
Characteristics of
Streptococcus pneumoniae
,
Haemophilus influenzae
, and
Moraxella catarrhalis
Isolated from the Nasopharynges of Asymptomatic Children and Molecular Analysis of
S. pneumoniae
and
H. influenzae
Strain Replacement in the Nasopharynx
Authors: Sulikowska Agnieszka , Grzesiowski Paweł , Sadowy Ewa , Fiett Janusz , Hryniewicz Waleria 
Published: September 2004

ABSTRACT

Nasopharyngeal carriage of
Streptococcus pneumoniae
,
Haemophilus influenzae
, and
Moraxella catarrhalis
in 226 children in different settings (in a crèche [day care center], in an orphanage, and at home) during two seasons (winter and spring) was studied. The rates of carriage of
S. pneumoniae
and
H. influenzae
were markedly higher in the crèche and in the orphanage than in the home setting (e.g., 56.5, 63.3, and 25.9%, respectively, for
S. pneumoniae
in winter). Approximately 80% of the
S. pneumoniae
isolates identified in the crèche and in the orphanage belonged to the serotypes represented in the seven-valent pneumococcal vaccine, and 4.4% of the children were colonized by
H. influenzae
type b. Almost all
H. influenzae
isolates were fully susceptible to the antimicrobial agents tested, and only five (3.6%) produced β-lactamase; in contrast, 100% of the
M. catarrhalis
isolates were β-lactamase positive. Among
S. pneumoniae
isolates, 36.2% were nonsusceptible to penicillin (PNSP) and 11.8% were fully resistant to penicillin (PRP). All PNSP isolates were obtained from children at the crèche and at the orphanage but not among children brought up at home, and all PRP isolates showed a multiresistant phenotype. Colonization by PRP isolates correlated well with prior treatment with β-lactams. For the majority of children colonized at both sampling times, strain replacement of
S. pneumoniae
and
H. influenzae
was observed; long-term colonization by a single strain was rare.
Mutation in a Lordsdale Norovirus Epidemic Strain as a Potential Indicator of Transmission Routes
Authors: Dingle Kate E. 
Published: September 2004

ABSTRACT
An increase in norovirus outbreaks was reported internationally during 2002 and 2003 and was also observed in Oxfordshire (United Kingdom) hospitals. To understand their epidemiological relationships, viruses from 22 outbreaks (15 from one hospital) were subjected to nucleotide sequencing. The 3′-terminal 3,255 nt or complete genomes were determined for 49 viruses. All outbreaks were caused by a genogroup II norovirus related to the Lordsdale virus (GII 4), common in healthcare settings. The norovirus mutation rate was sufficiently high that the 3,255-nucleotide sequences allowed separate and potentially connected outbreaks to be identified, since all outbreaks with identical sequences were temporally or geographically linked. The high mutation rate was further indicated by four mutations and three microheterogeneities in 3,255 nucleotides during 17 days of norovirus shedding by an immunocompromised patient. The data suggested that multiple virus introductions from the community, occasional transmission among wards, and one instance of ongoing environmental contamination had occurred. The accumulation, or lack, of mutations within an outbreak was also used to indicate the predominant transmission route. In an outbreak where person-to-person spread was thought to predominate, six mutations were detected throughout the genome, whereas one mutation was detected when point source infection was suspected. This norovirus epidemic strain differed from its closest previously described relative by 11.4 to 13.6% in the outer P2 domain of the capsid, which also had a single-amino-acid insertion. Alterations to the capsid structure compared to previous noroviruses may explain the increased number of outbreaks during 2002 and 2003.
The Species
Mycobacterium africanum
in the Light of New Molecular Markers
Authors: Niemann S. , Kubica T. , Bange F. C. , Adjei O. , Browne E. N. , Chinbuah M. A. , Diel R. , Gyapong J. , Horstmann R. D. , Joloba M. L. , Meyer C. G. , Mugerwa R. D. , Okwera A. , Osei I. , Owusu-Darbo E. , Schwander S. K. , Rüsch-Gerdes S. 
Published: September 2004

ABSTRACT

The findings of recent studies addressing the molecular characteristics of
Mycobacterium tuberculosis
complex isolates have initiated a discussion on the classification of
M. africanum
, especially of those isolates originating from East Africa (cluster F, subtype II) and displaying phenotypic and biochemical characteristics more similar to those of
M. tuberculosis
. To further address this question, we analyzed a representative collection of 63
M. tuberculosis
complex strains comprising 30
M. africanum
subtype I strains, 20
M. africanum
subtype II strains, 10 randomly chosen
M. tuberculosis
isolates, and type strains of
M. tuberculosis
,
M. bovis
, and
M. africanum
for the following biochemical and molecular characteristics: single-nucleotide polymorphisms (SNPs) in
gyrB
and
narGHJI
and the presence or absence of RD1, RD9, and RD12. For all molecular markers analyzed, subtype II strains were identical to the
M. tuberculosis
strains tested. In contrast, the subtype I strains as well as the
M. africanum
type strain showed unique combinations of SNPs in
gyrB
and genomic deletions (the absence of RD9 and the presence of RD12), which proves their independence from
M. tuberculosis
and
M. bovis
. Accordingly, all subtype I strains displayed main biochemical characteristics included in the original species description of
M. africanum
. We conclude that the isolates from West Africa were proved to be
M. africanum
with respect to the phenotypic and genetic markers analyzed, while the isolates from East Africa must be regarded as phenotypic variants of
M. tuberculosis
(genotype Uganda). We propose the addition of the molecular characteristics defined here to the species description of
M. africanum
, which will allow clearer species differentiation in the future.
Comprehensive Detection and Serotyping of Human Adenoviruses by PCR and Sequencing
Authors: Sarantis Helen , Johnson Grant , Brown Martha , Petric Martin , Tellier Raymond 
Published: September 2004

ABSTRACT
Human adenoviruses are common pathogens associated with many diseases, including respiratory, gastrointestinal, and ocular infections. Because they are now being increasingly recognized as agents of life-threatening disseminated infection in immunocompromised patients, robust and sensitive laboratory detection methods are needed for their rapid diagnosis. We describe here a PCR assay using a single primer pair, targeting a region of the hexon gene containing hypervariable region 7, that can detect all known human adenovirus serotypes and allows for serotype determination through the analysis of the nucleotide sequence. This comprehensive assay has proven effective for diagnosing adenoviruses at the serotype level in a broad range of patient specimens, including conjunctival, nasopharyngeal, stool, blood, and urine specimens.
Identification of Emerging Human-Pathogenic
Pythium insidiosum
by Serological and Molecular Assay-Based Methods
Authors: Vanittanakom Nongnuch , Supabandhu Jitwadee , Khamwan Chantana , Praparattanapan Jutarut , Thirach Sophit , Prasertwitayakij Narawudt , Louthrenoo Worawit , Chiewchanvit Siri , Tananuvat Napaporn 
Published: September 2004

ABSTRACT

Pythium insidiosum
is a pathogen that causes disease in both animals and humans. Human infection is rare; however, when it does occur, most patients, especially those having underlying hemoglobinopathy syndromes, such as thalassemia, exhibit a severe form. We identified four isolates of
P. insidiosum
. Two were recovered from tissue biopsy specimens from thalassemic and leukemic patients, one was derived from brain tissue from a thalassemic patient, and another was isolated from a corneal ulcer from a fourth patient. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed with a serum sample derived from one thalassemic patient. The methods used to identify the
P. insidiosum
isolates were based on morphology, nucleic acid sequencing, and a PCR assay. To confirm the identification, portions of the 18S rRNA genes of these four isolates were sequenced. The sequences were shown to be homologous to previously described
P. insidiosum
DNA sequences. In addition, PCR amplification of the internal transcribed spacer region specific for
P. insidiosum
was positive for all four isolates. The ELISA with the serum sample from the thalassemic patient gave a positive result from a serum dilution of 1:800. Finally, Western immunoblotting with this serum sample showed positive immunoglobulin G recognition for proteins of 110, 73, 56, 42 to 35, 30 to 28, 26, and 23 kDa. The results of this study show that both molecularly based diagnostic and serodiagnostic techniques are useful for the rapid identification of human pythiosis. The predominant antigens recognized by Western blotting may be useful in the development of a more sensitive and specific diagnostic tool for this disease.