bluuebunny/crossref_metadata_2025 huggingface dataset:
An autopsy case of obstructive jaundice due to hepatic multiple peribiliary cysts accompanying hepatolithiasis
Authors: Kai Keita , Eguchi Yuichiro , Kumagai Takafumi , Sugita Yasuo , Tokunaga Osamu
Published: July 2007

A 73‐year‐old Japanese male, who had a history of alcoholic cirrhosis, was admitted to Saga University Hospital to receive treatment for hepatocellular carcinoma. The patient was treated to maintain his liver function, however, the total bilirubin level continued to increase gradually. Endoscopic retrograde cholangiography demonstrated an obstruction of the bilateral intrahepatic ducts. Although endoscopic nasobiliary drainage was performed, the patient died two months after admission. At the autopsy, multiple peribiliary cysts were found to almost completely obstruct the bilateral intrahepatic bile ducts. In addition, hepatolithiasis was found in the right hepatic duct. To date, only a few such cases of multipleperibiliary cysts with obstructive jaundice have been reported, and no such case accompanied by hepatolithiasis. It is also important to note that all the reported cases resulted in a poor prognosis. We herein report a very rare autopsy case with obstructive jaundice due to multiple peribiliary cysts accompanying hepatolithiasis. Generally, multiple peribiliary cysts are considered to be clinically harmless, but once they cause obstructive jaundice, their presence suggests the occurrence of end‐stage cirrhosis and a poor prognosis.
Overexpression of CYP3A aggravates endotoxin‐induced liver injury in hypophysectomized female rats
Authors: Takemura Shigekazu , Minamiyama Yukiko , Toyokuni Shinya , Imaoka Susumu , Hai Seikan , Kubo Shoji , Hirohashi Kazuhiro , Funae Yoshihiko , Okada Shigeru
Published: July 2007

Aim: CYP3A2 is a male‐specific isoform of cytochrome P450 enzyme which is expressed abundantly in male rats but not in intact female rats. Having previously reported that hepatic CYP3A2 promotes lipopolysaccharide (LPS)‐induced liver injury in male rats, we further examined the impact of CYP3A on LPS‐induced liver injury by comparing intact and hypophysectomized female rats. In hypophysectomized female rats, phenobarbital (PB), a cytochrome P450 inducer, markedly increased the hepatic content and activity of CYP3A1/2, but did not do so in intact rats. CYP2B1 increased to similar levels in PB‐treated hypophysectomized and intact rats.

Methods: Rats were administered 10 mg/kg LPS intravenously and some were given PB for three days before LPS injection. Liver injury was analyzed 8 h after LPS injection.

Results: PB–LPS increased plasma alanine aminotransferase significantly more in hypophysectomized female rats than in intact female rats. Ketoconazole, a CYP3A inhibitor, inhibited the increase of liver injury. Hepatic 8‐hydroxydeoxyguanosine in nuclei and 4‐hydroxy‐2‐nonenal‐modified proteins, measured to evaluate oxidative stress by LPS treatment, increased markedly more in PB‐treated, hypophysectomized female rats, than in intact female rats.

Conclusion: Overexpression of CYP3A aggravated LPS‐induced liver injury in rats, apparently through the formation of reactive oxygen species.
Age‐related loss of cardiac vagal preganglionic neurones in spontaneously hypertensive rats
Authors: Corbett Eric K. A. , Mary David A. S. G. , McWilliam Peter N. , Batten Trevor F. C.
Published: July 2007

Despite the findings that impaired vagal control of the heart rate occurs in human hypertension, leading to greater cardiovascular risk, the mechanism of this impairment is as yet unknown. Observations in humans and experiments in the spontaneously hypertensive rat (SHR) suggested that such impairment may be related to an anomaly in central vagal neurones. We therefore set out to determine whether the numbers and distribution of cardiac‐projecting vagal preganglionic neurones in the medulla of adult (12 week) hypertensive SHR are different from those in young (4 week) prehypertensive SHR and in age‐matched Wistar‐Kyoto (WKY) rats of two age groups. The number of vagal neurones, identified by labelling with the fluorescent tracer DiI applied to the heart, was essentially similar in the three areas of the medulla analysed (dorsal vagal nucleus, nucleus ambiguus and intermediate reticular zone) in young SHR and young or adult WKY rats. In contrast, fewer vagal neurones were labelled in adult SHR compared with young SHR or WKY rats. This difference was due to highly significant reductions in vagal neurones in the dorsal vagal nucleus and nucleus ambiguus on the right side of the medulla. These observations suggest that a loss of parasympathetic preganglionic neurones supplying the heart with axons in the right vagus nerve, or a remodelling of their cardiac projections, may explain the known impairment of the baroreceptor reflex gain controlling heart rate in hypertension.
Cardiac vagal preganglionic neurones in the intermediate zone of the brainstem in anaesthetized cats
Authors: Kong Shuzhen , Liu Jian‐Hui , Ramage Andrew G. , Wang Yun
Published: July 2007

Cardiac vagal preganglionic neurones (CVPNs) show respiratory modulation in the nucleus ambiguus but not in the dorsal vagal nucleus. Both types of neurones can be activated by pulmonary C fibre afferents. Another brainstem area that has been identified as containing CVPNs is the intermediate zone between the dorsal vagal nucleus and nucleus ambiguus. Experiments were carried out in α‐chloralose‐anaesthetized cats to determine the physiological properties of these CVPNs and their responses to pulmonary C fibre afferent activation. Seven CVPN axons in the right cardiac vagal branches were identified and found to be localized in the intermediate zone with a conduction velocity of between 1.2 and 1.6 m s−1, in the C fibre range. These seven CVPNs [either showing spontaneous activity (n= 1) or having activity induced by dl‐homocysteic acid applied ionophoretically (n= 3)] were neither respiratory modulated nor did they receive a baroreceptor input, thus being similar to those found in the dorsal vagal nucleus. Right atrial injections of phenylbiguanide excited all four CVPNs tested. In conclusion, CVPNs located in the intermediate zone have similar properties to those in the dorsal vagal nucleus but not the nucleus ambiguus.
Nonlinear Dynamic Analysis of an Unsymmetrical Generator-Bearing System
Authors: Liu Zhansheng , Huang Senlin , Su Jiexian
Published: July 2007

Considering both nonlinear oil film force and unsymmetrical stiffness, this paper presents a mechanical model of a generator-bearing system. The complex mode synthesis method is used to reduce the linear degrees of freedom of the high order model in the rotating coordinates, and one-order modal differential equations are obtained which may not be solved directly by Newmark-β method. To solve this problem, a modified Newmark-β method is presented to investigate dynamic effects of the asymmetry of rotor stiffness, the viscosity of oil, the rotor unbalance and the ratio of length to diameter of bearings. Three-dimension diagrams and unfiltered vibration curves are used as tools to examine the dynamic behavior of the system, and some insights into the dynamic behavior are given. Numerical results show that instability of the system may be improved by modifying these parameters.
Development of a Self-Organized Neuro-Fuzzy Model for System Identification
Authors: Yang S. M. , Chen C. J. , Chang Y. Y. , Tung Y. Z.
Published: July 2007

It has been known that it is difficult to establish a fuzzy logic model with effective fuzzy rules and the associated membership functions. Neural network with its learning capability has been incorporated to make the fuzzy model more adaptive and effective. A self-organized neuro-fuzzy model by integrating the Mamdani fuzzy model and the backpropagation neural network is developed in this paper for system identification. The five-layer network adaptively adjusts the membership functions and dynamically optimizes the fuzzy rules. A benchmark test is applied to validate the model accuracy in nonlinear system identification. Experimental verifications on the dynamics of a composite smart structure and on an acoustics system also demonstrate that the neuro-fuzzy model is superior to the neural network and to an adaptive filter in system identification. The model can be established systematically and is shown to be effective in engineering applications.
Forced Vibration Analysis of an Elevator Rope With Both Ends Moving
Authors: Kimura Hiroyuki , Ito Hiroaki , Fujita Yoshiaki , Nakagawa Toshiaki
Published: July 2007

An elevator rope for a high-rise building is forcibly excited by the displacement of the building caused by wind forces. Regarding the rope, there are two boundary conditions. In the first case, one end moves with time and the other end is fixed, while in the second case, both ends move with time. A theoretical solution to the forced vibration of a rope where one end is moving has been already obtained. In this paper, a theoretical solution to the forced vibration of a rope where both ends are moving is presented, based on the assumption that rope tension and movement velocity are constant, and that the damping coefficient of the rope is zero or small. The virtual sources of waves, which can be assigned to reflecting waves, are used to obtain the theoretical solution. Finite difference analyses of rope vibration are also performed to verify the validity of the theoretical solution. The calculated results of the finite difference analyses are in fairly good agreement with that of the theoretical solution. The effects of the changing rate of rope length and the damping factor on the maximum rope displacement are quantitatively clarified.
Dissimilatory Iodate Reduction by MarinePseudomonassp. Strain SCT
Authors: Amachi Seigo , Kawaguchi Nahito , Muramatsu Yasuyuki , Tsuchiya Satoshi , Watanabe Yuko , Shinoyama Hirofumi , Fujii Takaaki
Published: July 2007

Bacterial iodate (IO3−) reduction is poorly understood largely due to the limited number of available isolates as well as the paucity of information about key enzymes involved in the reaction. In this study, an iodate-reducing bacterium, designated strain SCT, was newly isolated from marine sediment slurry. SCT is phylogenetically closely related to the denitrifying bacteriumPseudomonas stutzeriand reduced 200 μM iodate to iodide (I−) within 12 h in an anaerobic culture containing 10 mM nitrate. The strain did not reduce iodate under the aerobic conditions. An anaerobic washed cell suspension of SCT reduced iodate when the cells were pregrown anaerobically with 10 mM nitrate and 200 μM iodate. However, cells pregrown without iodate did not reduce it. The cells in the former category showed methyl viologen-dependent iodate reductase activity (0.31 U mg−1), which was located predominantly in the periplasmic space. Furthermore, SCT was capable of anaerobic growth with 3 mM iodate as the sole electron acceptor, and the cells showed enhanced activity with respect to iodate reductase (2.46 U mg−1). These results suggest that SCT is a dissimilatory iodate-reducing bacterium and that its iodate reductase is induced by iodate under anaerobic growth conditions.
Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic
Vibrio parahaemolyticus
Bacteria in Oysters
Authors: Nordstrom Jessica L. , Vickery Michael C. L. , Blackstone George M. , Murray Shelley L. , DePaola Angelo
Published: July 2007

Vibrio parahaemolyticus
is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (
tlh
), thermostable direct hemolysin (
tdh
), and thermostable-related hemolysin (
trh
) genes of
V. parahaemolyticus
. The
tlh
gene is a species-specific marker, while the
tdh
and
trh
genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only
V. parahaemolyticus
strains possessing the appropriate target genes generated a fluorescent signal, except for a late
tdh
signal generated by three strains of
V. hollisae
. The multiplex assay detected <10 CFU/reaction of pathogenic
V. parahaemolyticus
in the presence of >10
4
CFU/reaction of total
V. parahaemolyticus
bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard
V. parahaemolyticus
isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for
V. parahaemolyticus
targets.
V. parahaemolyticus tlh
,
tdh
, and
trh
were detected in 44, 44, and 52% of the oyster samples, respectively.
V. parahaemolyticus
was isolated from 33% of the samples, and
tdh
+
and
trh
+
strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic
V. parahaemolyticus
.
Gliding Arc Discharge in the Potato PathogenErwinia carotovorasubsp.atroseptica: Mechanism of Lethal Action and Effect on Membrane-Associated Molecules
Authors: Moreau M. , Feuilloley M. G. J. , Veron W. , Meylheuc T. , Chevalier S. , Brisset J.-L. , Orange N.
Published: July 2007

Gliding arc (glidarc) discharge is a physicochemical technique for decontamination at atmospheric pressure and ambient temperature. It leads to the destruction of bacterial phytopathogens responsible for important losses in industrial agriculture, namely,Erwiniaspp., without the formation of resistant forms. We investigated the effect of a novel optimized prototype allowing bacterial killing without lag time. This prototype also decreases the required duration of treatment by 50%. The study of the time course effect of the process on bacterial morphology suggests that glidarc induces major alterations of the bacterial membrane. We showed that glidarc causes the release of bacterial genomic DNA. By contrast, an apparent decrease in the level of extractible lipopolysaccharide was observed; however, no changes in the electrophoretic pattern and cytotoxic activity of the macromolecule were noted. Analysis of extractible proteins from the outer membrane of the bacteria revealed that glidarc discharge induces the release of these proteins from the lipid environment, but may also be responsible for protein dimerization and/or aggregation. This effect was not observed in secreted enzymatic proteins, such as pectate lyase. Analysis of the data supports the hypothesis that the plasma generated by glidarc discharge is acting essentially through oxidative mechanisms. Furthermore, these results indicate that, in addition to effectively destroying bacteria, glidarc discharge should be used to improve the extraction of bacterial molecules.
Sensitivity of Nested PCR in the Detection of Low Numbers of
Giardia lamblia
Cysts
Authors: Miller Kathryn M. , Sterling Charles R.
Published: July 2007

Nested PCR was performed on individually isolated
Giardia lamblia
cysts in replicates of 50 for sets of 1, 2, 3, 4, 5, 7, and 10 cysts. Amplification ranged from 80% for 1 cyst to 100% for 10 cysts. The results suggest that nested PCR is well adapted for
G. lamblia
single-cyst detection.
Lactococcus lactis
Gene
yjgB
Encodes a γ-
d
-Glutaminyl-
l
-Lysyl- Endopeptidase Which Hydrolyzes Peptidoglycan
Authors: Redko Yulia , Courtin Pascal , Mézange Christine , Huard Carine , Chapot-Chartier Marie-Pierre
Published: July 2007

YjgB is one of five peptidoglycan hydrolases previously identified in
Lactococcus lactis
. Analysis of its amino acid sequence revealed that YjgB contains an NlpC/P60 domain, whereas no specific cell wall binding domain or motif could be identified. The NlpC/P60 family is characterized by three conserved residues, a cysteine, a histidine, and a polar residue. In agreement with the presence of a Cys residue in the catalytic site of YjgB, its enzymatic activity was enhanced in the presence of dithiothreitol. Peptidoglycan-hydrolyzing activity of YjgB was detected in growing cells of an
L. lactis
strain overexpressing YjgB, as revealed by the presence of disaccharide (DS)-dipeptide in the muropeptide composition of the overexpressing strain. YjgB hydrolyzes the peptide chains of
L. lactis
muropeptides between γ-
d
-Gln and
l
-Lys residues. Its hydrolytic activity was detected on DSs with tetra- and pentapeptide chains, whereas hydrolytic activity was very low on DS-tripeptides. Thus, we demonstrated that YjgB is an endopeptidase which cleaves γ-
d
-Gln-
l
-Lys bonds in peptide chains of
L. lactis
peptidoglycan.
Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters
Authors: Lee Sung Kuk , Chou Howard H. , Pfleger Brian F. , Newman Jack D. , Yoshikuni Yasuo , Keasling Jay D.
Published: July 2007

Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (P
BAD
) system that is more compatible with IPTG (isopropyl-β-
d
-1-thiogalactopyranoside) induction of a lactose-inducible (P
lac
) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.
Functional Analysis of the Fructooligosaccharide Utilization Operon in
Lactobacillus paracasei
1195
Authors: Goh Yong Jun , Lee Jong-Hwa , Hutkins Robert W.
Published: July 2007

The
fosABCDXE
operon encodes components of a putative fructose/mannose phosphoenolpyruvate-dependent phosphotransferase system and a β-fructosidase precursor (FosE) that are involved in the fructooligosaccharide (FOS) utilization pathway of
Lactobacillus paracasei
1195. The presence of an N-terminal signal peptide sequence and an LPQAG cell wall anchor motif in the C-terminal region of the deduced FosE precursor amino acid sequence predicted that the enzyme is cell wall associated, indicating that FOS may be hydrolyzed extracellularly. In this study, cell fractionation experiments demonstrated that the FOS hydrolysis activity was present exclusively in the cell wall extract of
L. paracasei
previously grown on FOS. In contrast, no measurable FOS hydrolysis activity was detected in the cell wall extract from the isogenic
fosE
mutant. Induction of β-fructosidase activity was observed when cells were grown on FOS, inulin, sucrose, or fructose but not when cells were grown on glucose. A diauxic growth pattern was observed when cells were grown on FOS in the presence of limiting glucose (0.1%). Analysis of the culture supernatant revealed that glucose was consumed first, followed by the longer-chain FOS species. Transcription analysis further showed that the
fos
operon was expressed only after glucose was depleted in the medium. Expression of
fosE
in a non-FOS-fermenting strain,
Lactobacillus rhamnosus
GG, enabled the recombinant strain to metabolize FOS, inulin, sucrose, and levan.
Water Buffaloes (
Bubalus bubalis
) Identified as an Important Reservoir of Shiga Toxin-Producing
Escherichia coli
in Brazil
Authors: Oliveira Murilo G. , Feitosa Brito José R. , Carvalho Roberta R. , Guth Beatriz E. C. , Gomes Tânia A. T. , Vieira Mônica A. M. , Kato Maria A. M. F. , Ramos Isabel I. , Vaz Tânia M. I. , Irino Kinue
Published: July 2007

The presence of Shiga toxin-producing
Escherichia coli
(STEC) in water buffaloes is reported for the first time in South America. The prevalence of STEC ranged from 0 to 64% depending on the farm. STEC isolates exhibiting the genetic profiles
stx
1
stx
2
ehxA iha saa
and
stx
2
ehxA iha saa
predominated. Of the 20 distinct serotypes identified, more than 50% corresponded to serotypes associated with human diseases.
New Genera of RNA Viruses in Subtropical Seawater, Inferred from Polymerase Gene Sequences
Authors: Culley Alexander I. , Steward Grieg F.
Published: July 2007

Viruses are an integral component of the marine food web, contributing to the disease and mortality of essentially every type of marine life, yet the diversity of viruses in the sea, especially those with RNA genomes, remains very poorly characterized. Isolates of RNA-containing viruses that infect marine plankton are still rare, and the only cultivation-independent surveys of RNA viral diversity reported so far were conducted for temperate coastal waters of British Columbia. Here, we report on our improvements to a previously used protocol to investigate the diversity of marine picorna-like viruses and our results from applying this protocol in subtropical waters. The original protocol was simplified by using direct filtration, rather than tangential flow filtration, to harvest viruses from seawater, and new degenerate primers were designed to amplify a fragment of the RNA-dependent RNA polymerase gene by reverse transcription-PCR from RNA extracted from the filters. Whereas the original protocol was unsuccessful in a preliminary test, the new protocol resulted in amplification of picorna-like virus sequences in every sample of subtropical and temperate coastal seawater assayed. These polymerase sequences formed a diverse, but monophyletic cluster along with other sequences amplified previously from seawater and sequences from isolates infecting marine protists. Phylogenetic analysis suggested that our sequences represent at least five new genera and 24 new species of RNA viruses. These results contribute to our understanding of RNA virus diversity and suggest that picorna-like viruses are a source of mortality for a wide variety of marine protists.
Potential Use of Toxic Thermolabile Proteins To Study Protein Quality Control Systems
Authors: Mizrahi Itzhak , Dagan Michael , Biran Dvora , Ron Eliora Z.
Published: July 2007

SulA is an
Escherichia coli
division inhibitor with a short half-life whose accumulation results in filamentation. Here, we show that SulA is thermally unstable and forms aggregates at elevated temperatures. This property enables the selection of isolates with mutated protein quality control systems.
Discovery of a Novel Styrene Monooxygenase Originating from the Metagenome
Authors: van Hellemond Erik W. , Janssen Dick B. , Fraaije Marco W.
Published: July 2007

Oxygenases form an interesting class of biocatalysts, as they typically perform oxygenations with exquisite chemo-, regio-, and/or enantioselectivity. It has been observed that, once heterologously expressed in
Escherichia coli
, some oxygenases are able to form the blue pigment indigo. We have exploited this characteristic to screen a metagenomic library derived from loam soil and identified a novel oxygenase. This oxygenase shows 50% sequence identity with styrene monooxygenases from pseudomonads (StyA). Only a limited number of homologs can be found in the genome sequence database, indicating that this biocatalyst is a member of a relatively small family of bacterial monooxygenases. The newly identified monooxygenase catalyzes the epoxidation of styrene and styrene derivatives and forms the corresponding (
S
)-epoxides with excellent enantiomeric excess [e.g., (
S
)-styrene oxide is formed with >99% enantiomeric excess,
ee
] and therefore is named styrene monooxgenase subunit A (SmoA). SmoA shows high enantioselectivity towards aromatic sulfides [e.g., (
R
)-ethyl phenyl sulfoxide is formed with 92%
ee
]. This excellent enantioselectivity in combination with the moderate sequence identity forms a clear indication that SmoA from a metagenomic origin represents a new enzyme within the small family of styrene monooxygenases.
Spitzenkörper Localization and Intracellular Traffic of Green Fluorescent Protein-Labeled CHS-3 and CHS-6 Chitin Synthases in Living Hyphae of
Neurospora crassa
Authors: Riquelme Meritxell , Bartnicki-García Salomon , González-Prieto Juan Manuel , Sánchez-León Eddy , Verdín-Ramos Jorge A. , Beltrán-Aguilar Alejandro , Freitag Michael
Published: July 2007

The subcellular location and traffic of two selected chitin synthases (CHS) from
Neurospora crassa
, CHS-3 and CHS-6, labeled with green fluorescent protein (GFP), were studied by high-resolution confocal laser scanning microscopy. While we found some differences in the overall distribution patterns and appearances of CHS-3-GFP and CHS-6-GFP, most features were similar and were observed consistently. At the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). In distal regions (beyond 40 μm from the apex), CHS-GFP revealed a network of large endomembranous compartments that was predominantly comprised of irregular tubular shapes, while some compartments were distinctly spherical. In the distal subapex (20 to 40 μm from the apex), fluorescence was observed in globular bodies that appeared to disintegrate into vesicles as they advanced forward until reaching the proximal subapex (5 to 20 μm from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching suggested that the fluorescence of the Spk originated from the advancing population of microvesicles (chitosomes) in the subapex. The inability of brefeldin A to interfere with the traffic of CHS-containing microvesicles and the lack of colocalization of CHS-GFP with the endoplasmic reticulum (ER)-Golgi body fluorescent dyes lend support to the idea that CHS proteins are delivered to the cell surface via an alternative route distinct from the classical ER-Golgi body secretory pathway.
Peroxisomal Peripheral Membrane Protein YlInp1p Is Required for Peroxisome Inheritance and Influences the Dimorphic Transition in the Yeast
Yarrowia lipolytica
Authors: Chang Jinlan , Fagarasanu Andrei , Rachubinski Richard A.
Published: July 2007

Eukaryotic cells have evolved molecular mechanisms to ensure the faithful inheritance of organelles by daughter cells in order to maintain the benefits afforded by the compartmentalization of biochemical functions. Little is known about the inheritance of peroxisomes, organelles of lipid metabolism. We have analyzed peroxisome dynamics and inheritance in the dimorphic yeast
Yarrowia lipolytica
. Most peroxisomes are anchored at the periphery of cells of
Y. lipolytica
. In vivo video microscopy showed that at cell division, approximately half of the anchored peroxisomes in the mother cell are dislodged individually from their static positions and transported to the bud. Peroxisome motility is dependent on the actin cytoskeleton. YlInp1p is a peripheral peroxisomal membrane protein that affects the partitioning of peroxisomes between mother cell and bud in
Y. lipolytica
. In cells lacking YlInp1p, most peroxisomes were transferred to the bud, with only a few remaining in the mother cell, while in cells overexpressing YlInp1p, peroxisomes were preferentially retained in the mother cell, resulting in buds nearly devoid of peroxisomes. Our results are consistent with a role for YlInp1p in anchoring peroxisomes in cells. YlInp1p has a role in the dimorphic transition in
Y. lipolytica
, as cells lacking the Yl
INP1
gene more readily convert from the yeast to the mycelial form in oleic acid-containing medium, the metabolism of which requires peroxisomal activity, than does the wild-type strain. This study reports the first analysis of organelle inheritance in a true dimorphic yeast and identifies the first protein required for peroxisome inheritance in
Y. lipolytica
.
High-Affinity Interaction between the S-Layer Protein SbsC and the Secondary Cell Wall Polymer ofGeobacillus stearothermophilusATCC 12980 Determined by Surface Plasmon Resonance Technology
Authors: Ferner-Ortner Judith , Mader Christoph , Ilk Nicola , Sleytr Uwe B. , Egelseer Eva M.
Published: July 2007

Surface plasmon resonance studies using C-terminal truncation forms of the S-layer protein SbsC (recombinant SbsC consisting of amino acids 31 to 270 [rSbsC31-270] and rSbsC31-443) and the secondary cell wall polymer (SCWP) isolated fromGeobacillus stearothermophilusATCC 12980 confirmed the exclusive responsibility of the N-terminal region comprising amino acids 31 to 270 for SCWP binding. Quantitative analyses indicated binding behavior demonstrating low, medium, and high affinities.
The N-Terminal Propeptide of
Vibrio vulnificus
Extracellular Metalloprotease Is both an Inhibitor of and a Substrate for the Enzyme
Authors: Chang Alan K. , Park Jong Woo , Lee Eun Hee , Lee Jung Sup
Published: July 2007

Vibrio vulnificus
, a marine bacterium capable of causing wound infection and septicemia, secretes a 45-kDa metalloprotease (vEP) with many biological activities. The precursor of vEP consists of four regions: a signal peptide, an N-terminal propeptide (nPP), a C-terminal propeptide, and the mature protease. Two forms of vEP—vEP-45, which contains the mature protease plus the C-terminal propeptide, and vEP-34, which contains only the mature protease—were expressed in
Escherichia coli
and purified. vEP-45 and vEP-34 had similar activities with azocasein as a substrate, but vEP-34 had reduced activity toward insoluble proteins. The nPP of vEP was expressed as a His tag fusion protein, and its effect on vEP activity was investigated. nPP inhibited the activities of both vEP-45 and vEP-34 but not that of thermolysin, a different but related zinc-dependent protease. The inhibition of vEP by nPP was further examined using vEP-34 as a representative enzyme. The inhibition could be completely reversed under conditions of low enzyme and propeptide concentrations and with prolonged incubation, which resulted from the degradation of nPP by vEP. However, even at high nPP and vEP concentrations, inhibition of vEP by nPP at high temperatures was not effective, resulting in the degradation of both nPP and vEP. These results demonstrate that the nPP of vEP could bind to vEP and inhibit its activity, resulting in the degradation of the propeptide.
Mutations in Residues Involved in Zinc Binding in the Catalytic Site of
Escherichia coli
Threonyl-tRNA Synthetase Confer a Dominant Lethal Phenotype
Authors: Caillet Joël , Graffe Monique , Eyermann Flore , Romby Pascale , Springer Mathias
Published: July 2007

Escherichia coli
threonyl-tRNA synthetase is a homodimeric protein that acts as both an enzyme and a regulator of gene expression: the protein aminoacylates tRNA
Thr
isoacceptors and binds to its own mRNA, inhibiting its translation. The enzyme contains a zinc atom in its active site, which is essential for the recognition of threonine. Mutations in any of the three amino acids forming the zinc-binding site inactivate the enzyme and have a dominant negative effect on growth if the corresponding genes are placed on a multicopy plasmid. We show here that this particular property is not due to the formation of inactive heterodimers, the titration of tRNA
Thr
by an inactive enzyme, or its misaminoacylation but is, rather, due to the regulatory function of threonyl-tRNA synthetase. Overproduction of the inactive enzyme represses the expression of the wild-type chromosomal copy of the gene to an extent incompatible with bacterial growth.
Characterization and Comparison of Biofilm Development by Pathogenic and Commensal Isolates of
Histophilus somni
Authors: Sandal Indra , Hong Wenzhou , Swords W. Edward , Inzana Thomas J.
Published: July 2007

Histophilus somni
(
Haemophilus somnus
) is an obligate inhabitant of the mucosal surfaces of bovines and sheep and an opportunistic pathogen responsible for respiratory disease, meningoencephalitis, myocarditis, arthritis, and other systemic infections. The identification of an exopolysaccharide produced by
H. somni
prompted us to evaluate whether the bacterium was capable of forming a biofilm. After growth in polyvinyl chloride wells a biofilm was formed by all strains examined, although most isolates from systemic sites produced more biofilm than commensal isolates from the prepuce. Biofilms of pneumonia isolate strain 2336 and commensal isolate strain 129Pt were grown in flow cells, followed by analysis by confocal laser scanning microscopy and scanning electron microscopy. Both strains formed biofilms that went through stages of attachment, growth, maturation, and detachment. However, strain 2336 produced a mature biofilm that consisted of thick, homogenous mound-shaped microcolonies encased in an amorphous extracellular matrix with profound water channels. In contrast, strain 129Pt formed a biofilm of cell clusters that were tower-shaped or distinct filamentous structures intertwined with each other by strands of extracellular matrix. The biofilm of strain 2336 had a mass and thickness that was 5- to 10-fold greater than that of strain 129Pt and covered 75 to 82% of the surface area, whereas the biofilm of strain 129Pt covered 35 to 40% of the surface area. Since
H. somni
is an obligate inhabitant of the bovine and ovine host, the formation of a biofilm may be crucial to its persistence in vivo, and our in vitro evidence suggests that formation of a more robust biofilm may provide a selective advantage for strains that cause systemic disease.
Sulfite Reduction in Mycobacteria
Authors: Pinto Rachel , Harrison Joseph S. , Hsu Tsungda , Jacobs William R. , Leyh Thomas S.
Published: July 2007

Mycobacterium tuberculosis
places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in
Mycobacterium smegmatis
was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from
M. tuberculosis
were determined under strict anaerobic conditions [
k
cat
= 1.0 (±0.1) electron consumed per second, and K

m(SO
3
−2
)

= 27 (±1) μM], and the enzyme exhibits no detectible turnover of nitrite, which need not be the case in the sulfite/nitrite reductase family. Deletion of sulfite reductase (
sirA
, originally misannotated
nirA
) reveals that it is essential for growth on sulfate or sulfite as the sole sulfur source and, further, that the nitrite-reducing activities of the cell are incapable of reducing sulfite at a rate sufficient to allow growth. Like their nitrite reductase counterparts, sulfite reductases require a siroheme cofactor for catalysis. Rv2393 (renamed
che1
) resides in the sulfur reduction operon and is shown for the first time to encode a ferrochelatase, a catalyst that inserts Fe
2+
into siroheme. Deletion of
che1
causes cells to grow slowly on metabolites that require sulfite reductase activity. This slow-growth phenotype was ameliorated by optimizing growth conditions for nitrite assimilation, suggesting that nitrogen and sulfur assimilation overlap at the point of ferrochelatase synthesis and delivery.
SbcCD Regulation and Localization inEscherichia coli
Authors: Darmon Elise , Lopez-Vernaza Manuel A. , Helness Anne C. , Borking Amanda , Wilson Emily , Thacker Zubin , Wardrope Laura , Leach David R. F.
Published: July 2007

The SbcCD complex and its homologues play important roles in DNA repair and in the maintenance of genome stability. InEscherichia coli, the in vitro functions of SbcCD have been well characterized, but its exact cellular role remains elusive. This work investigates the regulation of thesbcDCoperon and the cellular localization of the SbcC and SbcD proteins. Transcription of thesbcDCoperon is shown to be dependent on starvation and RpoS protein. Overexpressed SbcC protein forms foci that colocalize with the replication factory, while overexpressed SbcD protein is distributed through the cytoplasm.
Global Analysis of mRNA Decay in
Halobacterium salinarum
NRC-1 at Single-Gene Resolution Using DNA Microarrays
Authors: Hundt Sonja , Zaigler Alexander , Lange Christian , Soppa Jörg , Klug Gabriele
Published: July 2007

RNA degradation is an important factor in the regulation of gene expression. It allows organisms to quickly respond to changing environmental conditions by adapting the expression of individual genes. The stability of individual mRNAs within an organism varies considerably, contributing to differential amounts of proteins expressed. In this study we used DNA microarrays to analyze mRNA degradation in exponentially growing cultures of the extremely halophilic euryarchaeon
Halobacterium salinarum
NRC-1 on a global level. We determined mRNA half-lives for 1,717 open reading frames, 620 of which are part of known or predicted operons. Under the tested conditions transcript stabilities ranged from 5 min to more than 18 min, with 79% of the evaluated mRNAs showing half-lives between 8 and 12 min. The overall mean half-life was 10 min, which is considerably longer than the ones found in the other prokaryotes investigated thus far. As previously observed in
Escherichia coli
and
Saccharomyces cerevisiae
, we could not detect a significant correlation between transcript length and transcript stability, but there was a relationship between gene function and transcript stability. Genes that are known or predicted to be transcribed in operons exhibited similar mRNA half-lives. These results provide initial insights into mRNA turnover in a euryarchaeon. Moreover, our model organism,
H. salinarum
NRC-1, is one of just two archaea sequenced to date that are missing the core subunits of the archaeal exosome. This complex orthologous to the RNA degrading exosome of eukarya is found in all other archaeal genomes sequenced thus far.
Outer Membrane Proteins ofFibrobacter succinogeneswith Potential Roles in Adhesion to Cellulose and in Cellulose Digestion
Authors: Jun Hyun-Sik , Qi Meng , Gong Joshua , Egbosimba Emmanuel E. , Forsberg Cecil W.
Published: July 2007

Comparative analysis of binding of intact glucose-grownFibrobacter succinogenesstrain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One- and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grownF. succinogeneswere identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system ofF. succinogenes.
Origins of Flagellar Gene Operons and Secondary Flagellar Systems
Authors: Liu Renyi , Ochman Howard
Published: July 2007

Forty-one flagellated species representing 11 bacterial phyla were used to investigate the origin of secondary flagellar systems and the structure and formation of flagellar gene operons over the course of bacterial evolution. Secondary (i.e., lateral) flagellar systems, which are harbored by five of the proteobacterial species considered, originated twice, once in the alphaproteobacterial lineage and again in the common ancestor of theBeta- andGammaproteobacteria. The order and organization of flagellar genes have undergone extensive shuffling and rearrangement among lineages, and based on the phylogenetic distributions of flagellar gene complexes, the flagellar gene operons existed as small, usually two-gene units in the ancestor ofBacteriaand have expanded through the recruitment of new genes and fusion of gene units. In contrast to the evolutionary trend towards larger flagellar gene complexes, operon structures have been highly disrupted through gene disassociation and rearrangements in theEpsilon- andAlphaproteobacteria. These results demonstrate that the genetic basis of this ancient and structurally conserved organelle has been subject to many lineage-specific modifications.
Mycothiol Import by
Mycobacterium smegmatis
and Function as a Resource for Metabolic Precursors and Energy Production
Authors: Bzymek Krzysztof P. , Newton Gerald L. , Ta Philong , Fahey Robert C.
Published: July 2007

Mycothiol ([MSH] AcCys-GlcN-Ins, where Ac is acetyl) is the major thiol produced by
Mycobacterium smegmatis
and other actinomycetes. Mutants deficient in MshA (strain 49) or MshC (transposon mutant Tn
1
) of MSH biosynthesis produce no MSH. However, when stationary phase cultures of these mutants were incubated in medium containing MSH, they actively transported it to generate cellular levels of MSH comparable to or greater than the normal content of the wild-type strain. When these MSH-loaded mutants were transferred to MSH-free preconditioned medium, the cellular MSH was catabolized to generate GlcN-Ins and AcCys. The latter was rapidly converted to Cys by a high deacetylase activity assayed in extracts. The Cys could be converted to pyruvate by a cysteine desulfhydrase or used to regenerate MSH in cells with active MshC. Using MSH labeled with [U-
14
C]cysteine or with [6-
3
H]GlcN, it was shown that these residues are catabolized to generate radiolabeled products that are ultimately lost from the cell, indicating extensive catabolism via the glycolytic and Krebs cycle pathways. These findings, coupled with the fact the
myo
-inositol can serve as a sole carbon source for growth of
M. smegmatis
, indicate that MSH functions not only as a protective cofactor but also as a reservoir of readily available biosynthetic precursors and energy-generating metabolites potentially important under stress conditions. The half-life of MSH was determined in stationary phase cells to be ∼50 h in strains with active MshC and 16 ± 3 h in the MshC-deficient mutant, suggesting that MSH biosynthesis may be a suitable target for drugs to treat dormant tuberculosis.
Investigation of Transcription Repression and Small-Molecule Responsiveness by TetR-Like Transcription Factors Using a HeterologousEscherichia coli-Based Assay
Authors: Ahn Sang Kyun , Tahlan Kapil , Yu Zhou , Nodwell Justin
Published: July 2007

The SCO7222 protein and ActR are two of ∼150 TetR-like transcription factors encoded in theStreptomyces coelicolorgenome. Using bioluminescence as a readout, we have developedEscherichia coli-based biosensors that accurately report the regulatory activity of these proteins and used it to investigate their interactions with DNA and small-molecule ligands. We found that the SCO7222 protein and ActR repress the expression of their putative target genes, SCO7223 andactII-ORF2 (actA), respectively, by interacting with operator sequence in the promoters. The operators recognized by the two proteins are related such thatO7223(an operator for SCO7223) could be bound by both the SCO7222 protein and ActR with similar affinities. In contrast,Oact(an operator foractII-ORF2) was bound tightly by ActR and more weakly by the SCO7222 protein. We demonstrated ligand specificity of these proteins by showing that while TetR (but not ActR or the SCO7222 protein) interacts with tetracyclines, ActR (but not TetR or the SCO7222 protein) interacts with actinorhodin and related molecules. Through operator-targeted mutagenesis, we found that at least two nucleotide changes inO7223were required to disrupt its interaction with SCO7222 protein, while ActR was more sensitive to changes onOact. Most importantly, we found that the interaction of each protein with wild-type and mutant operator sequences in vivo and in vitro correlated perfectly. Our data suggest thatE. coli-based biosensors of this type should be broadly applicable to TetR-like transcription factors.
Specificity and Regulation of Interaction between the P
II
and AmtB
1
Proteins in
Rhodospirillum rubrum
Authors: Wolfe David M. , Zhang Yaoping , Roberts Gary P.
Published: July 2007

The nitrogen regulatory protein P
II
and the ammonia gas channel AmtB are both found in most prokaryotes. Interaction between these two proteins has been observed in several organisms and may regulate the activities of both proteins. The regulation of their interaction is only partially understood, and we show that in
Rhodospirillum rubrum
one P
II
homolog, GlnJ, has higher affinity for an AmtB
1
-containing membrane than the other two P
II
homologs, GlnB and GlnK. This interaction strongly favors the nonuridylylated form of GlnJ and is disrupted by high levels of 2-ketoglutarate (2-KG) in the absence of ATP or low levels of 2-KG in the presence of ATP. ADP inhibits the destabilization of the GlnJ-AmtB
1
complex in the presence of ATP and 2-KG, supporting a role for P
II
as an energy sensor measuring the ratio of ATP to ADP. In the presence of saturating levels of ATP, the estimated

K
d

of 2-KG for GlnJ bound to AmtB
1
is 340 μM, which is higher than that required for uridylylation of GlnJ in vitro, about 5 μM. This supports a model where multiple 2-KG and ATP molecules must bind a P
II
trimer to stimulate release of P
II
from AmtB
1
, in contrast to the lower 2-KG requirement for productive uridylylation of P
II
by GlnD.
MukEF Is Required for Stable Association of MukB with the Chromosome
Authors: She Weifeng , Wang Qinhong , Mordukhova Elena A. , Rybenkov Valentin V.
Published: July 2007

MukB is a bacterial SMC(structural maintenance of chromosome) protein required for correct folding of theEscherichia colichromosome. MukB acts in complex with the two non-SMC proteins, MukE and MukF. The role of MukEF is unclear. MukEF disrupts MukB-DNA interactions in vitro. In vivo, however, MukEF stimulates MukB-induced DNA condensation and is required for the assembly of MukB clusters at the quarter positions of the cell length. We report here that MukEF is essential for stable association of MukB with the chromosome. We found that MukBEF forms a stable complex with the chromosome that copurifies with nucleoids following gentle cell lysis. Little MukB could be found with the nucleoids in the absence or upon overproduction of MukEF. Similarly, overproduced MukEF recruited MukB-green fluorescent protein (GFP) from its quarter positions, indicating that formation of MukB-GFP clusters and stable association with the chromosome could be mechanistically related. Finally, we report that MukE-GFP forms foci at the quarter positions of the cell length but not in cells that lack MukB or overproduce MukEF, suggesting that the clusters are formed by MukBEF and not by its individual subunits. These data support the view that MukBEF acts as a macromolecular assembly, a scaffold, in chromosome organization and that MukEF is essential for the assembly of this scaffold.