bluuebunny/crossref_metadata_2025 huggingface dataset:
Early upsurge in anti‐HBs titer possibly caused by the immunomodulative, not by the mutagenetic effect of interferon and ribavirin
Authors: Yamazaki Kazuhide , Ohkoshi Shogo , Maruyama Masaki , Aoki Yo‐hei , Yano Masahiko , Kurita So , Suzuki Kenta , Matsuda Yasunobu , Sugimura Kazuhito , Aoyagi Yutaka
Published: May 2007

A patient with chronic hepatitis B and C undergoing treatment with interferon and ribavirin showed an upsurge in hepatitis B virus surface antibody (anti‐HBs) titer, accompanied by a decrease in hepatitis B virus surface antigen (HBsAg) during the early treatment phase. Simultaneously, elevation of alanine aminotransferase (ALT) was observed. Subsequently, the hepatitis B virus (HBV) DNA titer decreased and HBV e antigen (HBeAg) to anti‐HBe seroconversion occurred. The anti‐HBs titer gradually returned to the pretreatment level after cessation of ribavirin treatment and HBV‐DNA became undetectable. We found no nucleotide mutations in HBV‐DNA that could explain the sudden elevation in anti‐HBs titer. The appearance of anti‐HBs was considered to be a break in immune tolerance against some epitopes in HBsAg, possibly the r epitope, stimulated by interferon/ribavirin treatment. The immunomodulatory effect of ribavirin might have caused this unexpected early immune response to HBsAg that preceded seroconversion to anti‐HBe.
Parametric imaging of contrast ultrasound for the evaluation of neovascularization in liver tumors
Authors: Sugimoto Katsutoshi , Moriyasu Fuminori , Kamiyama Naohisa , Metoki Ryo , Iijima Hiroko
Published: May 2007

Aim:  To assess the efficacy of parametric imaging for the diagnosis of neovascularization in liver tumors.

Methods:  The subjects were 17 rabbits (five with normal liver and 12 with VX2 tumor implanted in the liver). The contrast agents used were SonoVue (Bracco, Milan, Italy). A diagnostic ultrasound system was used with a programmable replenishment sequence. The images obtained between the initial frame after the high mechanical index (MI) scan, which diminishes microbubbles in the scan volume, and the current frame were coded in color according to the arrival and peak times. After the experiment, the tumors were excised and sectioned. Sections were prepared for light microscopy with hematoxylin–eosin (HE) staining and CD31 staining to evaluate vascular density.

Results:  Arrival time imaging (ATI) delineated the fine blood vessels (100–200 μm in diameter) in all of the rabbits. Tortuous and meandering tumor vessels were visualized in the VX2 tumors. Differences of perfusion velocity between tumor tissue and tumor‐free areas were shown in peak time imaging (PTI). Vascularity evaluated on the ATI and perfusion speed recognized on the ATI and PTI were related to the vascular density measured by pathological investigation.

Conclusion:  Parametric imaging is a promising new method for the visualization of perfusion and the estimation of tumor blood vessels.
Possible inhibitory effect of oral zinc supplementation on hepatic fibrosis through downregulation of TIMP‐1: A pilot study
Authors: Takahashi Masahiko , Saito Hidetsugu , Higashimoto Makiko , Hibi Toshifumi
Published: May 2007

Aim:  To investigate the effect of oral zinc supplementation (polaprezinc) for 24 weeks (34 mg/day zinc) on liver fibrosis in patients with advanced chronic liver disease.

Methods:  Various markers of liver fibrosis, and fibrogenic and fibrolytic enzyme activities were measured before and after zinc supplementation in 17 patients with early cirrhosis.

Results:  Serum zinc levels were decreased in the patients as compared with healthy controls. No side‐effect was noted in any of the patients who received zinc supplementation. Serum levels of zinc increased by up to 156% over baseline levels in the group of patients who took oral zinc for 24 weeks. In patients whose serum zinc levels increased, the serum levels of type IV collagen and the activity of tissue inhibitors of metalloproteinase‐1 (TIMP‐1) were significantly reduced, but no such change was observed in the other groups of patients, and no other serum markers changed.

Conclusion:  These results suggest that oral zinc supplement therapy with polaprezinc is safe and may be a novel and useful strategy for antifibrosis therapy in patients with early liver cirrhosis.
Hepatoprotective action of prostaglandin A2 analogs under CCl4‐induced liver injury in vitro
Authors: Hubich Aksana I. , Bondar Alexey Y. , Kastsiuk Tatsiana U. , Kastsiuk Uladimir A. , Lakhvich Fyodar A. , Sholukh Mikhail V.
Published: May 2007

Aim:  The cytoprotective effects of six novel synthetic prostaglandin A2 analogs against carbon tetrachloride (CCl4) as a toxic agent were studied with isolated rat liver hepatocytes in vitro.

Results:  It was found that hepatocytes treatment with CCl4 induced: (i) a significant increase of lactic dehydrogenase (LDH) release from cytoplasm; (ii) leakage of glutamate dehydrogenase (GDH) and acid phosphatase from mitochondria and lysosomes, respectively; (iii) 10‐fold increase of trien conjugates formation; and (iv) a reduction of free SH‐groups by 50%. Prostanoids U‐26, U‐9 and U‐34 decreased cytotoxic index of CCl4 on average by 1.5–2.0 times and were more effective than PGI2, the well‐known hepatoprotector of prostanoids type. The protective action of the prostanoids was not a cAMP‐ or Ca2+‐dependent process. However, prostanoids U‐26, U‐9 and U‐34 normalized intracellular content of SH‐groups, reduced trien conjugates formation by 60–80% and strongly prevented enzyme leakage through cellular membranes. They were also able to inhibit CCl4 effects via decreasing cytochrome P4502E1 activity.

Conclusion:  The results obtained demonstrate that prostanoids provide cytoprotective effects on liver hepatocytes through the prevention of lipid peroxidation of the plasma and the cellular membranes and maintenance of their barrier function.
Fusion protein containing SH3 domain of c‐Abl induces hepatocarcinoma cells to apoptosis
Authors: Yin Ji Kai , Liang Ying Min , He Xian Li , Lu Jian Guo , Zhang Li , Bao Guo Qiang , Ma Qing Jiu
Published: May 2007

Aim:  Through a preliminary test on a novel protein containing an HIV1‐TAT domain and a SH3 domain of oncoprotein P210BCR‐ABL (we named it after PTD‐BCR/ABL SH3), we found that this protein shows inhibition activity of hepatocarcinoma cell HepG‐2. The purpose of the present study is to explore the biological behavior of PTD‐BCR/ABL SH3 fusion protein in hepatocarcinoma cells in vitro and in vivo.

Methods:  HepG‐2 cells were cocultured with the fusion protein for the indicated time and studied in vitro by immunocytochemistry staining to demonstrate the localization of the protein, light and electron microscope observation in morphology research, MTT assay to draw a growth curve and to analyze inhibition ratio, DNA ladder and TUNEL staining to study apoptosis. Nude mice bearing HepG‐2 tumors were used to test the antitumor activity of the fusion protein.

Results:  PTD‐BCR/ABL SH3 fusion protein successfully entered into HepG‐2 cells and localized in the nucleus. The protein had shown high cytotoxity through inducing HepG‐2 cells to apoptosis, and in vivo. The growth speed of tumors in the treatment group was distinctly slower than those in the control group, and the survival time of mice in the treatment group was longer than those in the control group. The growth of the tumors had been inhibited in the treatment group, while other tissues, such as heart, liver, lung and kidney displayed normal morphology.

Conclusion:  PTD‐BCR/ABL SH3 fusion protein displays significant inhibitory activity of inducing hepatocarcinoma HepG‐2 cells to apoptosis in vitro. It also showed therapeutic effects in vivo.
A prospective study of acute drug‐induced liver injury in patients suffering from non‐alcoholic fatty liver disease
Authors: Tarantino Giovanni , Conca Paolo , Basile Vincenzo , Gentile Antonio , Capone Domenico , Polichetti Giuliano , Leo Emilio
Published: May 2007

Aim:  Liver damage due to facultative hepatotoxins is scarcely foreseeable. We evaluated the prevalence of acute drug‐induced liver injury (DILI) in a specific setting, assessing eventual interactions with pre‐existing hepatic illnesses.

Methods:  The research was carried out in an Italian tertiary care hospital, by analyzing 248 patients with non‐advanced liver disease, divided into two well‐matched groups: 174 patients (median age 53, 94 females) with hepatitis C virus‐related chronic hepatitis; and 74 (median age 55, 39 females) with non‐alcoholic fatty liver disease (NAFLD).

Results:  Six patients (2.4% of the whole population) belonging to the NAFLD group (χ2‐test, P = 0.004) suffered from acute hepatoxicity related to the following drugs, that is antihypertensive, acting on platelet aggregation, antimicrobial, non‐steroidal anti‐inflammatory and proton pump inhibitor. The NAFLD presence was an independent risk factor in determining drug‐related acute hepatitis, with an odds ratio of 3.95 (95% confidence intervals: 11.48–1.35). Central obesity was relevant in every patient with acute toxicity. Alcohol consumption and drug association did not influence the acute drug‐induced liver damage.

Conclusion:  NAFLD conveys a nearly fourfold increase of DILI risk in obese middle‐aged patients. NAFLD, characterized by mitochondrial dysfunction, could predispose to drug‐induced hepatotoxicity that probably shares the same pathophysiological mechanism.
Uniaxial stretch‐induced regulation of mitogen‐activated protein kinase, Akt and p70 S6 kinase in the ageing Fischer 344 × Brown Norway rat aorta
Authors: Rice Kevin M. , Desai Devashish H. , Preston Deborah L. , Wehner Paulette S. , Blough Eric R.
Published: May 2007

The effects of ageing on the cardiovascular system contribute to substantial alterations in cellular morphology and function. The variables regulating these changes are unknown; however, one set of signalling molecules that may be of particular importance in mediating numerous cellular responses, including control of cell growth, differentiation and adaptation, are the proteins associated with the mitogen‐activated protein kinase (MAPK) signalling systems. The MAPKs, in conjunction with the p70 S6k signalling cascade, have emerged as critical components for regulating numerous mechanotransduction‐related cellular responses. Here we investigate the ability of uniaxial stretch to activate the MAPK and p70 S6k pathways in adult (6‐month‐old), aged (30‐month‐old) and very aged (36‐month‐old) Fischer 344/NNiaHSd × Brown Norway/BiNia (FBN) rats. Western blotting of the MAPK family proteins extracellular signal‐regulated kinase (Erk) 1/2, p38‐ and c‐Jun NH2‐terminal kinase (Jnk)‐MAPKs showed differential expression and activation between these proteins with age. An acute 15 min interval of 20% uniaxial stretch using an ex vivo aortic preparation demonstrated similar regulation of Erk1/2, p38‐ and Jnk‐MAPK. However, ageing altered uniaxial induced p70 S6k pathway signalling. These observations confirm previous data demonstrating that MAPK proteins are mechanically regulated and also suggest that p70 S6k signalling expression and activation are controlled differently with ageing. Taken together, these data may help to explain, in part, the age‐related changes in vascular morphology, function and response to injury.
Peripheral 5‐HT1Areceptors are not essential for increased ventilation evoked by systemic 8‐OH‐DPAT challenge in anaesthetized rats
Authors: Szereda‐Przestaszewska Małgorzata , Kaczyńska Katarzyna
Published: May 2007

The respiratory effects resulting from stimulation of 5‐HT1Areceptors were studied in spontaneously breathing rats that were: (i) neurally intact and subsequently bilaterally vagotomized; (ii) subjected to bilateral midcervical vagotomy followed by supranodosal vagotomy; (iii) midcervically vagotomized and treated by carotid sinus/body denervation; or (iv) subjected to infra‐ and supranodosal vagotomy followed by pharmacological blockade of 5‐HT1Areceptors. An intravenous bolus of the 5‐HT1Areceptor agonist 8‐hydroxy‐dipropylaminotetralin (8‐OH‐DPAT, 10 μg kg−1) evoked increases in both breathing rate and tidal volume. After section of the midcervical and supranodosal vagi, 8‐OH‐DPAT challenge still increased the respiratory rate and tidal volume. Carotid sinus/body denervation did not reduce the augmentation of the tidal volume, but prevented the increase in breathing rate. Blockade of 5‐HT1Areceptors with intravenous doses of 1‐(2‐metoxyphenyl)‐4‐[4‐(2‐phthalimido) butyl] piperazine (NAN 190; 20 μg kg−1) abolished all respiratory effects of 8‐OH‐DPAT challenge. In all the neural states, 8‐OH‐DPAT evoked a significant fall in mean arterial blood pressure. Pretreatment with NAN 190 reduced baseline values of mean arterial pressure and prevented 8‐OH‐DPAT‐induced hypotension. These results indicate that: (i) 8‐OH‐DPAT‐evoked activation of 5‐HT1Areceptors increases breathing rate and tidal volume, which persists after section of the lung vagi and the nodose ganglia, but only the increase in breathing rate was abolished by carotid sinus/body denervation; and (ii) 8‐OH‐DPAT hyperventilatory and hypotensive responses result from the excitation of presumed 5‐HT1Acarotid receptors and the central 5‐HT1A‐expressing neurones.
Chronic ethanol increases fetal cerebral blood flow specific to the ethanol‐sensitive cerebellum under normoxaemic, hypercapnic and acidaemic conditions: ovine model
Authors: Parnell Scott E. , Ramadoss Jayanth , Delp Michael D. , Ramsey Michael W. , Chen Wei‐Jung A. , West James R. , Cudd Timothy A.
Published: May 2007

Cerebral hypoxia has been proposed as a mechanism by which prenatal ethanol exposure causes fetal alcohol spectrum disorder (FASD) in children, but no study had tested this hypothesis using a chronic exposure model that mimicks a common human exposure pattern. Pregnant sheep were exposed to ethanol, 0.75 or 1.75 g kg−1 (to create blood ethanol concentrations of 85 and 185 mg dl−1, respectively), or saline 3 days per week in succession (a ‘binge drinking’ model) from gestational day (GD) 109 until GD 132. Fetuses were instrumented on GD 119–120 and studied on GD 132. The 1.75 g kg−1 dose resulted in a significant increase in fetal biventricular output (measured by radiolabelled microsphere technique) and heart rate, and a reduction of mean arterial pressure and total peripheral resistance at 1 h, the end of ethanol infusion. The arterial partial pressure of CO2 was increased, arterial pH was decreased and arterial partial pressure of O2 did not change. Fetal whole‐brain blood flow increased by 37% compared with the control group at 1 h, resulting in increased cerebral oxygen delivery. The elevation in brain blood flow was region specific, occurring preferentially in the ethanol‐sensitive cerebellum, increasing by 44% compared with the control group at 1 h. There were no changes in the lower dose group. Assessment of regional differences in the teratogenic effects of ethanol by stereological cell‐counting technique showed a reduced number of cerebellar Purkinje cells in response to the 1.75 g kg−1 dose compared with the control brains. However, no such differences in neuronal numbers were observed in the hippocampus or the olfactory bulb. We conclude that repeated exposure to moderate doses of ethanol during the third trimester alters fetal cerebral vascular function and increases blood flow in brain regions that are vulnerable to ethanol in the presence of acidaemia and hypercapnia, and in the absence of hypoxia.
SB-431542, a Transforming Growth Factor β Inhibitor, ImpairsTrypanosoma cruziInfection in Cardiomyocytes and Parasite Cycle Completion
Authors: Waghabi Mariana C. , Keramidas Michelle , Calvet Claudia M. , Meuser Marcos , Soeiro Maria de Nazaré C. , Mendonça-Lima Leila , Araújo-Jorge Tania C. , Feige Jean-Jacques , Bailly Sabine
Published: May 2007

The antiinflammatory cytokine transforming growth factor β (TGF-β) plays an important role in Chagas disease, a parasitic infection caused by the protozoanTrypanosoma cruzi. In the present study, we show that SB-431542, an inhibitor of the TGF-β type I receptor (ALK5), inhibitsT. cruzi-induced activation of the TGF-β pathway in epithelial cells and in cardiomyocytes. Further, we demonstrate that addition of SB-431542 greatly reduces cardiomyocyte invasion byT. cruzi. Finally, SB-431542 treatment significantly reduces the number of parasites per infected cell and trypomastigote differentiation and release. Taken together, these data further confirm the major role of the TGF-β signaling pathway in bothT. cruziinfection andT. cruzicell cycle completion. Our present data demonstrate that small inhibitors of the TGF-β signaling pathway might be potential pharmacological tools for the treatment of Chagas disease.
Deoxyribonucleoside Kinases Activate Nucleoside Antibiotics in Severely Pathogenic Bacteria
Authors: Sandrini Michael P. B. , Shannon Oonagh , Clausen Anders R. , Björck Lars , Piškur Jure
Published: May 2007

Common bacterial pathogens are becoming progressively more resistant to traditional antibiotics, representing a major public-health crisis. Therefore, there is a need for a variety of antibiotics with alternative modes of action. In our study, several nucleoside analogs were tested against pathogenic staphylococci and streptococci. We show that pyrimidine-based nucleoside analogs, like 3′-azido-3′-deoxythymidine (AZT) and 2′,2′-difluoro-2′deoxycytidine (gemcitabine), are specifically activated by the endogenous bacterial deoxyribonucleoside kinases, leading to cell death. Deoxyribonucleoside kinase-deficient
Escherichia coli
strains become highly susceptible to nucleoside analogs when they express recombinant kinases from
Staphylococcus aureus
or
Streptococcus pyogenes
. We further demonstrate that recombinant
S. aureus
deoxyadenosine kinase efficiently phosphorylates the anticancer drug gemcitabine in vitro and is therefore the key enzyme in the activation pathway. When adult mice were infected intraperitoneally with a fatal dose of
S. pyogenes
strain AP1 and afterwards received gemcitabine, they failed to develop a systemic infection. Nucleoside analogs may therefore represent a promising alternative for combating pathogenic bacteria.
Chloramphenicol Is a Substrate for a Novel Nitroreductase Pathway in
Haemophilus influenzae
Authors: Smith Arnold L. , Erwin Alice L. , Kline Toni , Unrath William C. T. , Nelson Kevin , Weber Allan , Howald William N.
Published: May 2007

The
p
-nitroaromatic antibiotic chloramphenicol has been used extensively to treat life-threatening infections due to
Haemophilus influenzae
and
Neisseria meningitidis
; its mechanism of action is the inhibition of protein synthesis. We found that during incubation with
H. influenzae
cells and lysates, chloramphenicol is converted to a 4-aminophenyl allylic alcohol that lacks antibacterial activity. The allylic alcohol moiety undergoes facile re-addition of water to restore the 1,3-diol, as well as further dehydration driven by the aromatic amine to form the iminoquinone. Several
Neisseria
species and most chloramphenicol-susceptible
Haemophilus
species, but not
Escherichia coli
or other gram-negative or gram-positive bacteria we examined, were also found to metabolize chloramphenicol. The products of chloramphenicol metabolism by species other than
H. influenzae
have not yet been characterized. The strains reducing the antibiotic were chloramphenicol susceptible, indicating that the pathway does not appear to mediate chloramphenicol resistance. The role of this novel nitroreductase pathway in the physiology of
H. influenzae
and
Neisseria
species is unknown. Further understanding of the
H. influenzae
chloramphenicol reduction pathway will contribute to our knowledge of the diversity of prokaryotic nitroreductase mechanisms.
Sequencing and Comparative Genomic Analysis of pK29, a 269-Kilobase Conjugative Plasmid Encoding CMY-8 and CTX-M-3 β-Lactamases in
Klebsiella pneumoniae
Authors: Chen Ying-Tsong , Lauderdale Tsai-Ling , Liao Tsai-Lien , Shiau Yih-Ru , Shu Hung-Yu , Wu Keh-Ming , Yan Jing-Jou , Su Ih-Jen , Tsai Shih-Feng
Published: May 2007

A 269-kilobase conjugative plasmid, pK29, from a
Klebsiella pneumoniae
strain was sequenced. The plasmid harbors multiple antimicrobial resistance genes, including those encoding CMY-8 AmpC-type and CTX-M-3 extended-spectrum β-lactamases in the common backbone of IncHI2 plasmids. Mechanisms for dissemination of the resistance genes are highlighted in comparative genomic analyses.
Changes in
qnr
Prevalence and Fluoroquinolone Resistance in Clinical Isolates of
Klebsiella pneumoniae
and
Enterobacter
spp. Collected from 1990 to 2005
Authors: Strahilevitz Jacob , Engelstein Dalia , Adler Amos , Temper Violeta , Moses Allon E. , Block Colin , Robicsek Ari
Published: May 2007

Clinical isolates of
Klebsiella pneumoniae
and
Enterobacter
spp. collected from 1990 through 2005 at a tertiary care center were studied for
qnr
genes. Isolates bearing these genes emerged in the mid-1990s, coinciding with the time of a rapid increase in fluoroquinolone resistance. Sixty percent of these isolates were ciprofloxacin susceptible by CLSI breakpoints.
Rifaximin Does Not Induce Toxin Production or Phage-Mediated Lysis of Shiga Toxin-Producing
Escherichia coli
Authors: Ochoa Theresa J. , Chen Jane , Walker Christopher M. , Gonzales Elsa , Cleary Thomas G.
Published: May 2007

Diarrhea in children is often caused by enteropathogen infections that might benefit from early empirical antibiotic therapy. However, when the definition of the pathogen requires sophisticated laboratory studies, the etiology of enteritis is not known early in illness. Empirical therapy may be dangerous if the child is infected with a Shiga toxin-producing
Escherichia coli
(STEC) strain because antimicrobials may increase Shiga toxin (Stx) release, resulting in increased risk of microangiopathic hemolytic anemia with acute renal failure (hemolytic-uremic syndrome [HUS]) and death. There is a need for antimicrobials that would be effective against multiple bacterial enteropathogens yet not induce Stx release or increase the risk of HUS. Rifaximin has been evaluated in adults for treatment of bacterial enteritis and has a good record for safety and efficacy, but it has not been evaluated extensively in children with gastroenteritis. We therefore evaluated rifaximin's potential for phage induction, drug-induced bacteriolysis, and toxin release in 57 STEC strains (26 O157 and 31 non-O157 strains). Growth in ciprofloxacin, a known Stx phage inducer, caused bacteriolysis and release of toxin in 25/26 (96%) O157 strains and 15/31 (48%) non-O157 strains. In contrast, rifaximin did not induce phage replication or lysis in any strain. Toxin release in the presence of rifaximin was not different from release in the absence of antibiotic. Rifaximin, unlike many antibiotics used to treat pediatric gastroenteritis, does not induce phage-mediated bacteriolysis and Stx release.
Bacteriophage Therapy To Reduce
Salmonella
Colonization of Broiler Chickens
Authors: Atterbury R. J. , Van Bergen M. A. P. , Ortiz F. , Lovell M. A. , Harris J. A. , De Boer A. , Wagenaar J. A. , Allen V. M. , Barrow P. A.
Published: May 2007

Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing
Salmonella
colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which
Salmonella
colonization could be reduced. A total of 232
Salmonella
bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against
Salmonella enterica
serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific
Salmonella
host strains. The first phage reduced
S. enterica
serotype Enteritidis cecal colonization by ≥4.2 log
10
CFU within 24 h compared with controls. Administration of the second phage reduced
S. enterica
serotype Typhimurium by ≥2.19 log
10
CFU within 24 h. The third bacteriophage was ineffective at reducing
S. enterica
serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.
“Mark the Gene”: a Method for Nondestructive Introduction of Marker Sequences Inside the Gene Frame of Transgenes
Authors: Morono Yuki , Kitagawa Wataru , Kimura Nobutada , Noda Naohiro , Nakamura Kazunori , Kamagata Yoichi
Published: May 2007

A specific marking and detection technique is a fundamental requirement for the safer use of genetically modified (GM) organisms. Here we propose a simple and effective method for directly marking functional transgenes in GM organisms. For that purpose, we introduced nucleotide substitutions (NS), based on the degeneracy of codons as markers (NS markers), into the
bphC
(2,3-dihydroxybiphenyl dioxygenase) and
tomA3
(toluene-
ortho
-monooxygenase) gene frames using a PCR-based method. No change was observed in the enzyme activity of translated proteins, and alignments with homologous genes showed the uniqueness of the NS markers. Furthermore, we constructed
tomA3
variations harboring NS markers in different positions. Although the translational products were identical, the constructed variation genes could be distinguished through their marker patterns by multiplex PCR, showing that NS markers could serve as product-specific tags for identifying individual GM organisms. This direct method of marking the functional transgene provides a simple, low-risk, and robust marking method without causing the gene functions to deteriorate.
Comparative Sequence Analysis of Plasmids from
Lactobacillus delbrueckii
and Construction of a Shuttle Cloning Vector
Authors: Lee Ju-Hoon , Halgerson Jamie S. , Kim Jeong-Hwan , O'Sullivan Daniel J.
Published: May 2007

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with
Lactobacillus delbrueckii
, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of
Lactobacillus delbrueckii
subsp.
bulgaricus
was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic
ori
and encoded six open reading frames (ORFs), of which functions could be predicted for three—a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available
L. delbrueckii
plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for
L. delbrueckii
plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from
Streptococcus thermophilus
. This observation and the ability of the replicon to function in
S. thermophilus
support the idea that the origin of plasmids in
L. delbrueckii
was likely from
S. thermophilus
. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from
L. delbrueckii
has previously been constructed, an
Escherichia coli-L. delbrueckii
shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A
ori
, the chloramphenicol resistance gene of pCI372, and the
lacZ
polylinker from pUC18. This cloning vector was successfully introduced into
E. coli
,
L. delbrueckii
subsp.
bulgaricus
,
S. thermophilus
, and
Lactococcus lactis
. This shuttle cloning vector provides a new tool for molecular analysis of
Lactobacillus delbrueckii
and other lactic acid bacteria.
A General Framework for Designing and Validating Oligomer-Based DNA Microarrays and Its Application to
Clostridium acetobutylicum
Authors: Paredes Carlos J. , Senger Ryan S. , Spath Iwona S. , Borden Jacob R. , Sillers Ryan , Papoutsakis Eleftherios T.
Published: May 2007

While DNA microarray analysis is widely accepted as an essential tool for modern biology, its use still eludes many researchers for several reasons, especially when microarrays are not commercially available. In that case, the design, construction, and use of microarrays for a sequenced organism constitute substantial, time-consuming, and expensive tasks. Recently, it has become possible to construct custom microarrays using industrial manufacturing processes, which offer several advantages, including speed of manufacturing, quality control, no up-front setup costs, and need-based microarray ordering. Here, we describe a strategy for designing and validating DNA microarrays manufactured using a commercial process. The 22K microarrays for the solvent producer
Clostridium acetobutylicum
ATCC 824 are based on in situ-synthesized 60-mers employing the Agilent technology. The strategy involves designing a large library of possible oligomer probes for each target (i.e., gene or DNA sequence) and experimentally testing and selecting the best probes for each target. The degenerate
C. acetobutylicum
strain M5 lacking the pSOL1 megaplasmid (with 178 annotated open reading frames [genes]) was used to estimate the level of probe cross-hybridization in the new microarrays and to establish the minimum intensity for a gene to be considered expressed. Results obtained using this microarray design were consistent with previously reported results from spotted cDNA-based microarrays. The proposed strategy is applicable to any sequenced organism.
SIMPLE Approach for Isolating Mutants Expressing Fimbriae
Authors: Nuccio Sean-Paul , Chessa Daniela , Weening Eric H. , Raffatellu Manuela , Clegg Steven , Bäumler Andreas J.
Published: May 2007

Genomes of members of the family
Enterobacteriaceae
contain large repertoires of putative fimbrial operons. Since many of these operons are poorly expressed in vitro, a convenient method for inducing elaboration of the encoded fimbriae would greatly facilitate their functional characterization. Here we describe a new technique for identifying fimbriated bacteria from a library of transposon mutants by screening with immunomagnetic particles for ligand expression (SIMPLE). The SIMPLE method was applied to identify the T-POP mutants of
Salmonella enterica
serotype Typhimurium carrying on their surfaces filaments composed of PefA, the major subunit product of a fimbrial operon (
pef
) that is not expressed during growth in Luria-Bertani broth. Four such mutants were identified from a library of 24,000 mutants, each of which carried a T-POP insertion within the
hns
gene, which encodes a global silencer of horizontally acquired genes. Our data suggest that the SIMPLE method is an effective approach for isolating fimbriated bacteria, which can be readily applied to fimbrial operons identified by whole-genome sequencing.
Monophyly of
nodA
and
nifH
Genes across Texan and Costa Rican Populations of
Cupriavidus
Nodule Symbionts
Authors: Andam Cheryl P. , Mondo Stephen J. , Parker Matthew A.
Published: May 2007

nodA
and
nifH
phylogenies for
Cupriavidus
nodule bacteria from native legumes in Texas and Costa Rica grouped all strains into a single clade nested among neotropical
Burkholderia
strains. Thus,
Cupriavidus
symbiotic genes were not acquired independently in different regions and are derived from other
Betaproteobacteria
rather than from α-rhizobial donors.
Transcriptome Analysis Reveals that Multidrug Efflux Genes Are Upregulated To Protect
Pseudomonas aeruginosa
from Pentachlorophenol Stress
Authors: Muller Jocelyn Fraga , Stevens Ann M. , Craig Johanna , Love Nancy G.
Published: May 2007

Through chemical contamination of natural environments, microbial communities are exposed to many different types of chemical stressors; however, research on whole-genome responses to this contaminant stress is limited. This study examined the transcriptome response of a common soil bacterium,
Pseudomonas aeruginosa
, to the common environmental contaminant pentachlorophenol (PCP). Cells were grown in chemostats at a low growth rate to obtain substrate-limited, steady-state, balanced-growth conditions. The PCP stress was administered as a continuous increase in concentration, and samples taken over time were examined for physiological function changes with whole-cell acetate uptake rates (WAURs) and cell viability and for gene expression changes by Affymetrix GeneChip technology and real-time reverse transcriptase PCR. Cell viability, measured by heterotrophic plate counts, showed a moderately steady decrease after exposure to the stressor, but WAURs did not change in response to PCP. In contrast to the physiological data, the microarray data showed significant changes in the expression of several genes. In particular, genes coding for multidrug efflux pumps, including MexAB-OprM, were strongly upregulated. The upregulation of these efflux pumps protected the cells from the potentially toxic effects of PCP, allowing the physiological whole-cell function to remain constant.
Molecular Characterization of Potential Microcystin-Producing Cyanobacteria in Lake Ontario Embayments and Nearshore Waters
Authors: Hotto A. M. , Satchwell M. F. , Boyer G. L.
Published: May 2007

The distribution and genotypic variation of potential microcystin (MC) producers along the southern and eastern shores of Lake Ontario in 2001 and 2003 were examined using a suite of PCR primers. Cyanobacterial,
Microcystis
sp., and
Microcystis
-specific toxin primer sets identified shoreline distribution of cyanobacterial DNA (in 97% of the stations) and MC synthetase genes (in 50% of the stations). Sequence analysis of a partial
mcyA
amplicon targeting
Microcystis
,
Anabaena
, and
Planktothrix
species indicated that the
Microcystis
sp. genotype was the dominant MC genotype present and revealed a novel
Microcystis
-like sequence containing a 6-bp insert. Analysis of the same samples with genus-specific
mcyE
primers confirmed that the
Microcystis
sp. genotype was the dominant potential MC producer. Genotype compositions within embayments were relatively homogenous compared to those for shoreline and tributary samples. MC concentrations along the shoreline exhibited both temporal and spatial differences as evidenced by the protein phosphatase inhibition assay, at times exceeding the World Health Organization guideline value for drinking water of 1.0 μg MC-LR
eq
liter
−1
. MC genotypes are widespread along the New York State shoreline of Lake Ontario, appear to originate nearshore, and can be carried through the lake via wind and surface water current patterns.
Carvacrol Induces Heat Shock Protein 60 and Inhibits Synthesis of Flagellin in
Escherichia coli
O157:H7
Authors: Burt Sara A. , van der Zee Ruurd , Koets Ad P. , de Graaff Anko M. , van Knapen Frans , Gaastra Wim , Haagsman Henk P. , Veldhuizen Edwin J. A.
Published: May 2007

The essential oils of oregano and thyme are active against a number of food-borne pathogens, such as
Escherichia coli
O157:H7. Carvacrol is one of the major antibacterial components of these oils, and
p
-cymene is thought to be its precursor in the plant. The effects of carvacrol and
p
-cymene on protein synthesis in
E. coli
O157:H7 ATCC 43895 cells were investigated. Bacteria were grown overnight in Mueller-Hinton broth with a sublethal concentration of carvacrol or
p
-cymene, and their protein compositions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by Western blotting. The presence of 1 mM carvacrol during overnight incubation caused
E. coli
O157:H7 to produce significant amounts of heat shock protein 60 (HSP60) (GroEL) (
P
< 0.05) and inhibited the synthesis of flagellin highly significantly (
P
< 0.001), causing cells to be aflagellate and therefore nonmotile. The amounts of HSP70 (DnaK) were not significantly affected.
p
-Cymene at 1 mM or 10 mM did not induce HSP60 or HSP70 in significant amounts and did not have a significant effect on flagellar synthesis. Neither carvacrol (0.3, 0.5, 0.8, or 1 mM) nor
p
-cymene (0.3, 0.5, or 0.8 mM) treatment of cells in the mid-exponential growth phase induced significant amounts of HSP60 or HSP70 within 3 h, although numerical increases of HSP60 were observed. Motility decreased with increasing concentrations of both compounds, but existing flagella were not shed. This study is the first to demonstrate that essential oil components induce HSP60 in bacteria and that overnight incubation with carvacrol prevents the development of flagella in
E. coli
O157:H7.
Nucleotide Sequence of Plasmid pCNB1 from
Comamonas
Strain CNB-1 Reveals Novel Genetic Organization and Evolution for 4-Chloronitrobenzene Degradation
Authors: Ma Ying-Fei , Wu Jian-Feng , Wang Sheng-Yue , Jiang Cheng-Ying , Zhang Yun , Qi Su-Wei , Liu Lei , Zhao Guo-Ping , Liu Shuang-Jiang
Published: May 2007

The nucleotide sequence of a new plasmid pCNB1 from
Comamonas
sp. strain CNB-1 that degrades 4-chloronitrobenzene (4CNB) was determined. pCNB1 belongs to the IncP-1β group and is 91,181 bp in length. A total of 95 open reading frames appear to be involved in (i) the replication, maintenance, and transfer of pCNB1; (ii) resistance to arsenate and chromate; and (iii) the degradation of 4CNB. The 4CNB degradative genes and arsenate resistance genes were located on an extraordinarily large transposon (44.5 kb), proposed as Tn
CNB1
. Tn
CNB1
was flanked by two IS
1071
elements and represents a new member of the composite I transposon family. The 4CNB degradative genes within Tn
CNB1
were separated by various truncated genes and genetic homologs from other DNA molecules. Genes for chromate resistance were located on another transposon that was similar to the Tn
21
transposon of the class II replicative family that is frequently responsible for the mobilization of mercury resistance genes. Resistance to arsenate and chromate were experimentally confirmed, and transcriptions of arsenate and chromate resistance genes were demonstrated by reverse transcription-PCR. These results described a new member of the IncP-1β plasmid family, and the findings suggest that gene deletion and acquisition as well as genetic rearrangement of DNA molecules happened during the evolution of the 4CNB degradation pathway on pCNB1.
Enteric Virus Survival during Household Laundering and Impact of Disinfection with Sodium Hypochlorite
Authors: Gerba Charles P. , Kennedy Denise
Published: May 2007

This study was conducted to determine whether enteric viruses (adenovirus, rotavirus, and hepatitis A virus) added to cotton cloth swatches survive the wash cycle, the rinse cycle, and a 28-min permanent press drying cycle as commonly practiced in households in the United States. Detergent with and without bleach (sodium hypochlorite) was added to washing machines containing sterile and virus-inoculated 58-cm
2
swatches, 3.2 kg of cotton T-shirts and underwear, and a soiled pillowcase designed to simulate the conditions (pH, organic load, etc.) encountered in soiled laundry. The most important factors for the reduction of virus in laundry were passage through the drying cycle and the addition of sodium hypochlorite. Washing with detergent alone was not found to be effective for the removal or inactivation of enteric viruses, as significant concentrations of virus were found on the swatches (reductions of 92 to 99%). It was also demonstrated that viruses are readily transferred from contaminated cloths to uncontaminated clothes. The use of sodium hypochlorite reduced the number of infectious viruses on the swatches after washing and drying by at least 99.99%. Laundering practices in common use in the United States do not eliminate enteric and respiratory viruses from clothes. The use of bleach can further reduce the numbers of enteric viruses in laundry.
Construction of a Low-Temperature Protein Expression System Using a Cold-Adapted Bacterium,
Shewanella
sp. Strain Ac10, as the Host
Authors: Miyake Ryoma , Kawamoto Jun , Wei Yun-Lin , Kitagawa Masanari , Kato Ikunoshin , Kurihara Tatsuo , Esaki Nobuyoshi
Published: May 2007

A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium,
Shewanella
sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4°C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce β-lactamase in
Shewanella
sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4°C and 139 mg/liter of culture at 18°C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium,
Desulfotalea psychrophila
DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the
Escherichia coli
T7 promoter system.
Insertional Mutagenesis To Generate Lantibiotic Resistance in
Lactococcus lactis
Authors: Guinane Caitriona M. , Cotter Paul D. , Lawton Elaine M. , Hill Colin , Ross R. Paul
Published: May 2007

While the potential emergence of food spoilage and pathogenic bacteria with resistance to lantibiotics is a concern, the creation of derivatives of starter cultures and adjuncts that can grow in the presence of these antimicrobials may have applications in food fermentations. Here a bank of
Lactococcus lactis
IL1403 mutants was created and screened, and a number of novel genetic loci involved in lantibiotic resistance were identified.
Mobile Genetic Elements Provide Evidence for a Bovine Origin of Clonal Complex 17 of
Streptococcus agalactiae
Authors: Héry-Arnaud Geneviève , Bruant Guillaume , Lanotte Philippe , Brun Stella , Picard Bertrand , Rosenau Agnès , van der Mee-Marquet Nathalie , Rainard Pascal , Quentin Roland , Mereghetti Laurent
Published: May 2007

We sought an explanation for epidemiological changes in
Streptococcus agalactiae
infections by investigating the link between ecological niches of the bacterium by determining the prevalence of 11 mobile genetic elements. The prevalence of nine of these elements differed significantly according to the human or bovine origin of the isolate. Correlating this distribution with the phylogeny obtained by multilocus sequence analysis, we observed that human isolates harboring GBSi1, a clear marker of the bovine niche, clustered in clonal complex 17. Our results are thus consistent with the emergence of this virulent human clone from a bovine ancestor.
Potential Interactions of Particle-Associated Anammox Bacteria with Bacterial and Archaeal Partners in the Namibian Upwelling System
Authors: Woebken Dagmar , Fuchs Bernhard M. , Kuypers Marcel M. M. , Amann Rudolf
Published: May 2007

Recent studies have shown that the anaerobic oxidation of ammonium by anammox bacteria plays an important role in catalyzing the loss of nitrogen from marine oxygen minimum zones (OMZ). However, in situ oxygen concentrations of up to 25 μM and ammonium concentrations close to or below the detection limit in the layer of anammox activity are hard to reconcile with the current knowledge of the physiology of anammox bacteria. We therefore investigated samples from the Namibian OMZ by comparative 16S rRNA gene analysis and fluorescence in situ hybridization. Our results showed that “
Candidatus
Scalindua” spp., the typical marine anammox bacteria, colonized microscopic particles that were likely the remains of either macroscopic marine snow particles or resuspended particles. These particles were slightly but significantly (
P
< 0.01) enriched in
Gammaproteobacteria
(11.8% ± 5.0%) compared to the free-water phase (8.1% ± 1.8%). No preference for the attachment to particles could be observed for members of the
Alphaproteobacteria
and
Bacteroidetes
, which were abundant (12 to 17%) in both habitats. The alphaproteobacterial SAR11 clade, the
Euryarchaeota
, and group I
Crenarchaeota
, were all significantly depleted in particles compared to their presence in the free-water phase (16.5% ± 3.5% versus 2.6% ± 1.7%, 2.7% ± 1.9% versus <1%, and 14.9% ± 4.6% versus 2.2% ± 1.8%, respectively, all
P
< 0.001). Sequence analysis of the crenarchaeotal 16S rRNA genes showed a 99% sequence identity to the nitrifying “
Nitrosopumilus maritimus
.” Even though we could not observe conspicuous consortium-like structures of anammox bacteria with particle-enriched bacterioplankton groups, we hypothesize that members of
Gammaproteobacteria
,
Alphaproteobacteria
, and
Bacteroidetes
play a critical role in extending the anammox reaction to nutrient-depleted suboxic water layers in the Namibian upwelling system by creating anoxic, nutrient-enriched microniches.
New Insights into the Colonization and Release Processes of
Xenorhabdus nematophila
and the Morphology and Ultrastructure of the Bacterial Receptacle of Its Nematode Host,
Steinernema carpocapsae
Authors: Snyder Holly , Stock S. Patricia , Kim Sam-Kyu , Flores-Lara Yolanda , Forst Steven
Published: May 2007

We present results from epifluorescence, differential interference contrast, and transmission electron microscopy showing that
Xenorhabdus nematophila
colonizes a receptacle in the anterior intestine of the infective juvenile (IJ) stage of
Steinernema carpocapsae
. This region is connected to the esophagus at the esophagointestinal junction. The process by which
X. nematophila
leaves this bacterial receptacle had not been analyzed previously. In this study we monitored the movement of green fluorescent protein-labeled bacteria during the release process. Our observations revealed that
Xenorhabdus
colonizes the distal region of the receptacle and that exposure to insect hemolymph stimulated forward movement of the bacteria to the esophagointestinal junction. Continued exposure to hemolymph caused a narrow passage in the distal receptacle to widen, allowing movement of
Xenorhabdus
down the intestine and out the anus. Efficient release of both the wild type and a nonmotile strain was evident in most of the IJs incubated in hemolymph, whereas only a few IJs incubated in nutrient-rich broth released bacterial cells. Incubation of IJs in hemolymph treated with agents that induce nematode paralysis dramatically inhibited the release process. These results suggest that bacterial motility is not required for movement out of the distal region of the receptacle and that hemolymph-induced esophageal pumping provides a force for the release of
X. nematophila
out of the receptacle and into the intestinal lumen.