bluuebunny/crossref_metadata_2025 huggingface dataset:
Visual Cycle: Dependence of Retinol Production and Removal on Photoproduct Decay and Cell Morphology
Authors: Ala-Laurila Petri , Kolesnikov Alexander V. , Crouch Rosalie K. , Tsina Efthymia , Shukolyukov Sergey A. , Govardovskii Victor I. , Koutalos Yiannis , Wiggert Barbara , Estevez Maureen E. , Cornwall M. Carter
Published: July 2006

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50–70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10–40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.
Mini-dystrophin Expression Down-regulates IP3-mediated Calcium Release Events in Resting Dystrophin-deficient Muscle Cells
Authors: Balghi Haouaria , Sebille Stéphane , Mondin Ludivine , Cantereau Anne , Constantin Bruno , Raymond Guy , Cognard Christian
Published: July 2006

We present here evidence for the enhancement, at rest, of an inositol 1,4,5-trisphosphate (IP3)–mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, the number of sites discharging calcium (release site density [RSD]) was quantified and found more elevated in SolC1(−) than in SolD(+) myotubes. Variations of membrane potential had no significant effect on this difference, and higher resting [Ca2+]i in SolC1(−) (Marchand, E., B. Constantin, H. Balghi, M.C. Claudepierre, A. Cantereau, C. Magaud, A. Mouzou, G. Raymond, S. Braun, and C. Cognard. 2004. Exp. Cell Res. 297:363–379) cannot explain alone higher RSD. The exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) and phospholipase C inhibitor (U73122) significantly reduced RSD in both cell types but with a stronger effect in dystrophin-deficient SolC1(−) myotubes. Immunocytochemistry allowed us to localize ryanodine receptors (RyRs) as well as IP3 receptors (IP3Rs), IP3R-1 and IP3R-2 isoforms, indicating the presence of both RyRs-dependent and IP3-dependent release systems in both cells. We previously reported evidence for the enhancement, through a Gi protein, of the IP3-mediated calcium signaling pathway in SolC1(−) as compared to SolD(+) myotubes during a high K+ stimulation (Balghi, H., S. Sebille, B. Constantin, S. Patri, V. Thoreau, L. Mondin, E. Mok, A. Kitzis, G. Raymond, and C. Cognard. 2006. J. Gen. Physiol. 127:171–182). Here we show that, at rest, these regulation mechanisms are also involved in the modulation of calcium release activities. The enhancement of resting release activity may participate in the calcium overload observed in dystrophin-deficient myotubes, and our findings support the hypothesis of the regulatory role of mini-dystrophin on intracellular signaling.
On the tips of their tongues: authors and their views on scholarly publishing
Authors: Nicholas David , Jamali Hamid R. , Rowlands Ian
Published: July 2006

The paper presents the results of an international questionnaire survey of the attitudes of senior authors towards scholarly publishing. It was commissioned by the Publishers Association (PA) and the International Association of Scientific, Technical and Medical Publishers (STM). A total of 5,513 authors participated. The questionnaire included 22 closed questions and one open‐ended question that 1,943 authors used to freely express their views. Using QSR N6 textual analysis software, their comments were processed and themes extracted. The main concerns of the authors were the peer‐review process, faster publishing, better access to journals, journal prices, and issues related to electronic publishing.
Ask and they shall deceive: how what we ask drives what they tell us, and why it's mostly useless
Authors: Anderson Kent
Published: July 2006

The author describes his experience of market research through surveys and focus groups. He proposes that both techniques are seriously flawed and offers a neurophysiological explanation of why that should be so.
Thought patterns of some editors of academic journals
Authors: Ju‐fang Shao , Hui‐yun Shen
Published: July 2006

Influenced by Chinese traditional thought, some contemporary editors of Chinese academic journals have holistic and authority‐based thought patterns and values. Editors with such ideas are easily misled in the refereeing of manuscriptss. This leads to some high‐quality manuscripts being turned down and others, of little academic value, being published. The editorial aim of publication of the best material is difficult to realize. If a journal wishes to develop and gain international acceptance, editors with such ideas must adjust and change their thought patterns and values.
Does body-volume constrain reproductive output in lizards?
Authors: Du Weiguo , Ji Xiang , Shine Richard
Published: July 2006

The numbers and sizes of eggs produced by adult females ultimately determine the viability of populations, as well as the evolutionary fitness of the females themselves. Despite an enormous amount of literature on the adaptive significance of fecundity variation within and among populations, simpler questions—such as the proximate mechanisms by which a female determines her clutch size—have attracted less attention. Our surgical manipulations show that the amount of space available to hold eggs within a female's abdomen influences her total reproductive allocation, enabling her to flexibly modify her reproductive output as she grows larger.
Linking protein structure and dynamics to catalysis: the role of hydrogen tunnelling
Authors: Klinman Judith P
Published: July 2006

Early studies of enzyme-catalysed hydride transfer reactions indicated kinetic anomalies that were initially interpreted in the context of a ‘tunnelling correction’. An alternate model for tunnelling emerged following studies of the hydrogen atom transfer catalysed by the enzyme soybean lipoxygenase. This invokes full tunnelling of all isotopes of hydrogen, with reaction barriers reflecting the heavy atom, environmental reorganization terms. Using the latter approach, we offer an integration of the aggregate data implicating hydrogen tunnelling in enzymes (i.e. deviations from Swain–Schaad relationships and the semi-classical temperature dependence of the hydrogen isotope effect). The impact of site-specific mutations of enzymes plays a critical role in our understanding of the factors that control tunnelling in enzyme reactions.
Proton-coupled electron transfer: the mechanistic underpinning for radical transport and catalysis in biology
Authors: Reece Steven Y , Hodgkiss Justin M , Stubbe JoAnne , Nocera Daniel G
Published: July 2006

Charge transport and catalysis in enzymes often rely on amino acid radicals as intermediates. The generation and transport of these radicals are synonymous with proton-coupled electron transfer (PCET), which intrinsically is a quantum mechanical effect as both the electron and proton tunnel. The caveat to PCET is that proton transfer (PT) is fundamentally limited to short distances relative to electron transfer (ET). This predicament is resolved in biology by the evolution of enzymes to control PT and ET coordinates on highly different length scales. In doing so, the enzyme imparts exquisite thermodynamic and kinetic controls over radical transport and radical-based catalysis at cofactor active sites. This discussion will present model systems containing orthogonal ET and PT pathways, thereby allowing the proton and electron tunnelling events to be disentangled. Against this mechanistic backdrop, PCET catalysis of oxygen–oxygen bond activation by mono-oxygenases is captured at biomimetic porphyrin redox platforms. The discussion concludes with the case study of radical-based quantum catalysis in a natural biological enzyme, class I
Escherichia coli
ribonucleotide reductase. Studies are presented that show the enzyme utilizes both collinear and orthogonal PCET to transport charge from an assembled diiron-tyrosyl radical cofactor to the active site over 35 Å away via an amino acid radical-hopping pathway spanning two protein subunits.
Unusual origins of isotope effects in enzyme-catalysed reactions
Authors: Northrop Dexter B
Published: July 2006

High hydrostatic pressure is a neglected tool for probing the origins of isotope effects. In chemical reactions, normal primary deuterium isotope effects (DIEs) arising solely from differences in zero point energies are unaffected by pressure; but some anomalous isotope effects in which hydrogen tunnelling is suspected are partially suppressed. In some enzymatic reactions, high pressure completely suppresses the DIE. We have now measured the effects of high pressure on the parallel
13
C heavy atom isotope effect of yeast alcohol dehydrogenase and found that it is also suppressed by high pressure and, similarly, suppressed in its entirety. Moreover, the volume changes associated with the suppression of both deuterium and heavy atom isotope effects are virtually identical. The equivalent decrease in activation volumes for hydride transfer, when one mass unit is added to the carbon end of a scissile C–H bond as when one mass unit is added to the hydrogen end, suggests a common origin. Given that carbon is highly unlikely to undergo tunnelling, it follows that hydrogen is not doing so either. The origin of these isotope effects must lie elsewhere. We offer protein domain motions as a possibility.
Bacteraemic pneumonia caused by Neisseria lactamica with reduced susceptibility to penicillin and ciprofloxacin in an adult with liver cirrhosis
Authors: Wang Cheng-Yi , Chuang Yu-Min , Teng Lee-Jene , Lee Li-Na , Yang Pan-Chyr , Kuo Sow-Hsong , Hsueh Po-Ren
Published: July 2006

This report presents a case of bacteraemic pneumonia caused by Neisseria lactamica in an adult patient with liver cirrhosis who was successfully treated with ceftriaxone. The isolate was confirmed as N. lactamica by analysis of a partial sequence of the 16S rRNA gene; it had reduced susceptibilities to penicillin (MIC 0.75 μg ml−1) and ciprofloxacin (MIC ⩾0.5 mg l−1).
Epidemiological analysis of group A streptococci recovered from patients in China
Authors: Jing Hong-bo , Ning Boa-an , Hao Huai-jie , Zheng Yu-ling , Chang Dong , Jiang Wei , Jiang Yong-qiang
Published: July 2006

Since the mid-1980s, there has been a resurgence of severe forms of invasive group A streptococcal (GAS) disease in many countries and regions. However, there has not been any systemic epidemiologic analysis of GAS disease reported in mainland China. To analyse the molecular epidemiology of GAS disease, 86 strains from patients in different regions of mainland China were collected. The collection sites included blood, pus, wounds, the epipharynx and other sites. A total of 21 different emm types were identified in the isolates. In both invasive and non-invasive isolates, M1 (29.1 %) and M12 (23.3 %) were the most prevalent types, a different distribution to M type distributions reported in other countries. Furthermore, minor emm gene sequence alterations were noted for six types. Several important GAS virulence factors were detected by PCR using specific primers. The speB and slo genes were detected in all isolates and were species specific. Four superantigen genes, speA, speC, smeZ and ssa, were found in 52 % (45/86), 51 % (44/86), 82 % (71/86) and 23 % (27/86) of isolates, respectively. M1 isolates harboured more speA (84 %) and fewer speC genes (44 %), while M12 isolates had fewer speA (35 %) and more speC genes (100 %). There was also an association between some virulence genes and isolation sites, perhaps due to the correlation between the emm type distribution and virulence gene occurrence. For two important virulence genes related to necrotizing fasciitis, the sil gene was only carried by 11 of 86 isolates, and no sil gene contained the start codon ATA. The sla gene rarely occurred in GAS isolates, only four of 86 GAS strains being positive, including two isolates obtained from blood. In antimicrobial susceptibility tests, the overall rate of drug resistance in GAS isolates was higher than reported rates in other countries, and the resistance rates to erythromycin, tetracycline and clindamycin were 91.8, 93.4 and 80 %, respectively. This epidemiological study may help to understand the pathogenesis of GAS disease and aid in vaccine development.
Identification of a repetitive sequence belonging to a PPE gene of Mycobacterium tuberculosis and its use in diagnosis of tuberculosis
Authors: Srivastava Ranjana , Kumar D. , Waskar M. N. , Sharma Meera , Katoch V. M. , Srivastava Brahm S.
Published: July 2006

A repetitive sequence specific toMycobacterium tuberculosiswas isolated from aλgt11 library ofM. tuberculosisby DNA–DNA hybridization using genomic DNA ofM. tuberculosisas probe followed by subtractive hybridization with a cocktail of other mycobacterial DNA. This led to identification of CD192, a 1291 bp fragment ofM. tuberculosiscontaining repetitive sequences, which produced positive hybridization signals withM. tuberculosisDNA within 30 min. Nucleotide sequencing revealed the presence of several direct and inverted repeats within the 1291 bp fragment that belonged to a PPE family gene (Rv0355) ofM. tuberculosis. The use of CD192 as a DNA probe for the identification ofM. tuberculosisin culture and clinical samples was investigated. The 1291 bp sequence was present inM. tuberculosis,Mycobacterium bovisandM. bovisBCG, but was not present in many of the other mycobacterial strains tested, includingM. tuberculosisH37Ra. More than 300 clinical isolates ofM. tuberculosiswere probed with CD192, and the presence of the 1291 bp sequence was observed in all the clinical strains, including those lacking IS6110. The sequence displayed RFLP among the clinical isolates. A PCR assay was developed which detectedM. tuberculosiswith 100 % specificity from specimens of sputum, cerebrospinal fluid and pleural effusion from clinically diagnosed cases of tuberculosis.
Rapid cost-effective subtyping of meticillin-resistant Staphylococcus aureus by denaturing HPLC
Authors: Jury F. , Al-Mahrous M. , Apostolou M. , Sandiford S. , Fox A. , Ollier W. , Upton M.
Published: July 2006

The importance of meticillin-resistant Staphylococcus aureus (MRSA) in hospital-acquired infection is widely acknowledged. The UK government has stated that MRSA bloodstream infection rates will have to be halved by 2008. Such radical improvements will require advances on several fronts. Screening for MRSA in high-risk patients on arrival at hospital allows isolation of carriers and reduces transmission to staff and other patients. Concurrent subtyping of MRSA could also inform outbreak investigations and long-term epidemiological studies. The variability within the staphylococcal protein A, or spaA, gene-repeat region can be used as a marker of short- and long-term genetic variation. A novel application is described of denaturing HPLC (DHPLC) for rapid, inexpensive characterization of spaA gene amplification products, without the need for DNA sequence determination. The method allowed rapid and precise sizing of spaA gene-repeat regions from 99 S. aureus strains and was combined with heteroduplex analysis, using reference PCR products, to indicate the spa type of the test isolate. The method allowed subtyping of strains in less than 5 h from receipt of a primary isolation plate. When applied to an outbreak that occurred during this study, the authors were able to demonstrate relatedness of the isolates more than 5 days before results were received from a reference laboratory. If combined with direct amplification from swabs, DHPLC analysis of spaA gene variation could prove extremely valuable in outbreak investigation and MRSA surveillance.
Genotyping of Campylobacter jejuni using seven single-nucleotide polymorphisms in combination with flaA short variable region sequencing
Authors: Price Erin P. , Thiruvenkataswamy Venugopal , Mickan Lance , Unicomb Leanne , Rios Rosa E. , Huygens Flavia , Giffard Philip M.
Published: July 2006

This investigation describes the development of a generally applicable, bioinformatics-driven, single-nucleotide polymorphism (SNP) genotyping assay for the common bacterial gastrointestinal pathogenCampylobacter jejuni. SNPs were identifiedin silicousing the program ‘Minimum SNPs’, which selects for polymorphisms providing the greatest resolution of bacterial populations based on Simpson's index of diversity (D). The high-DSNPs identified in this study were derived from the combinedC. jejuni/Campylobacter colimultilocus sequence typing (MLST) database. Seven SNPs were found that provided aDof 0.98 compared with full MLST characterization, based on 959 sequence types (STs). The seven high-DSNPs were interrogated using allele-specific real-time PCR (AS kinetic PCR), which negates the need for expensive labelled primers or probes and requires minimal assay optimization. The total turnaround time of the SNP typing assay was approximately 2 h. Concurrently, 69C. jejuniisolates were subjected to MLST and flagellin A short variable region (flaASVR) sequencing and combined with a population of 84C. jejuniandC. coliisolates previously characterized by these methods. Within this collection of 153 isolates, 19flaASVR types (D=0.857) were identified, compared with 40 different STs (D=0.939). When MLST andflaASVR sequencing were used in combination, the discriminatory power was increased to 0.959. In comparison, SNP typing of the 153 isolates alone provided aDof 0.920 and was unable to resolve a small number of unrelated isolates. However, addition of theflaASVR locus to the SNP typing procedure increased the resolving power to 0.952 and clustered isolates similarly to MLST/flaASVR. This investigation has shown that a seven-memberC. jejuniSNP typing assay, used in combination with sequencing of theflaASVR, efficiently discriminatesC. jejuniisolates.
Role of the ABC transporter TruMDR2 in terbinafine, 4-nitroquinoline N-oxide and ethidium bromide susceptibility in Trichophyton rubrum
Authors: Fachin Ana Lúcia , Ferreira-Nozawa Monica S. , Maccheroni Walter , Martinez-Rossi Nilce M.
Published: July 2006

A single-copy gene, designated TruMDR2, encoding an ATP-binding cassette (ABC) transporter was cloned and sequenced from the dermatophyte Trichophyton rubrum. The ORF of TruMDR2 was 4048 nt and the deduced amino acid sequence showed high homology with ABC transporters involved in drug efflux in other fungi. The encoded ABC protein predicted 12 transmembrane segments (TMSs) and two almost identical nucleotide-binding domains (NBDs) arranged in two halves in a (TMS6–NBD)2 configuration and could be classified as a member of the multidrug-resistance (MDR) class of ABC transporters. Northern blot analyses revealed an increased level of transcription of the TruMDR2 gene when mycelium was exposed to acriflavine, benomyl, ethidium bromide, ketoconazole, chloramphenicol, griseofulvin, fluconazole, imazalil, itraconazole, methotrexate, 4-nitroquinoline N-oxide (4NQO) or tioconazole. Disruption of the TruMDR2 gene rendered the mutant more sensitive to terbinafine, 4NQO and ethidium bromide than the control strain, suggesting that this transporter plays a role in modulating drug susceptibility in T. rubrum.
A microarray analysis of the murine macrophage response to infection with Francisella tularensis LVS
Authors: Andersson Henrik , Hartmanová Blanka , Rydén Patrik , Noppa Laila , Näslund Linda , Sjöstedt Anders
Published: July 2006

The response of cells of the mouse macrophage cell line J774 to infection with Francisella tularensis LVS was analysed by means of a DNA microarray representing approximately 18 500 genes (20 600 clones). The adaptive response was modest at all time points, and at most, 81 clones were differentially regulated from the time point of uptake of bacteria (0 min) up to 240 min later. For all five time points, 229 clones fulfilled the criteria of being differentially regulated, i.e. the ratio between infected versus non-infected cells was at least 1.7-fold up- or down-regulated and P <0.05. It was found that many of the differentially regulated genes are known to respond to stress in general and to oxidative stress specifically. However, at 120 min it was observed that genes that lead to depletion of glutathione were upregulated. Possibly, this was a result of mechanisms induced by F. tularensis. Generally, there was a conspicuous lack of inflammatory responses and, for example, although tumour necrosis factor alpha (TNF-α) was upregulated at 0 min, a significant down-regulation was noted at all subsequent time points. When cells were treated with an inhibitor of inducible nitric oxide synthase (iNOS) or the antioxidant N-acetylcysteine (NAC), the infection-induced cytopathogenic effect was significantly inhibited. Together, the results suggest that F. tularensis LVS infection confers an oxidative stress upon the target cells and that many of the host-defence mechanisms appear to be intended to counteract this stress. The infection is characterized by a very modest inflammatory response.
Emergence and spread of two distinct clonal groups of multidrug-resistant Escherichia coli in a veterinary teaching hospital in Australia
Authors: Sidjabat Hanna E. , Townsend Kirsty M. , Lorentzen Michael , Gobius Kari S. , Fegan Narelle , Chin James J.-C. , Bettelheim Karl A. , Hanson Nancy D. , Bensink John C. , Trott Darren J.
Published: July 2006

Multidrug-resistant Escherichia coli (MDREC) expressing AmpC β-lactamases have emerged as a cause of opportunistic infections in dogs. Following a cluster of extraintestinal infections caused by two distinct clonal groups (CGs) of bla
CMY-producing MDREC, a 12-month infection control study was undertaken at a veterinary teaching hospital in Brisbane, Australia. Swabs from the rectum of hospitalized dogs (n=780), hospital staff (n=16) and the hospital environment (n=220) were plated onto selective agar to obtain multidrug-resistant (MDR) coliforms. These were then tested by multiplex PCR for E. coli uspA, bla
CMY and the class 1 integron-associated dfrA17-aadA5 gene cassette for rapid identification of MDREC CG 1 (positive for all three genes) and CG 2 (positive for uspA and bla
CMY only). A total of 16.5 % of the dog rectal swabs and 4.1 % of the hospital environmental swabs yielded MDREC, and on the basis of multiplex PCR, PFGE and plasmid profiling, these were confirmed to belong to either CG 1 or CG 2. Both CG 1 and CG 2 isolates were obtained from clinical cases of extraintestinal infection and rectal swabs from hospitalized dogs over the same period of time, whereas only CG 1 isolates were obtained from the hospital environment. Both CGs were prevalent during the first 6 months, but only CG 2 was isolated during the second 6 months of the study. Two isolates obtained from rectal swabs of staff working in the hospital belonged to CG 2, with one of the isolates possessing the same REDP as nine isolates from dogs, including six isolates associated with cases of extraintestinal infection. CG 1 isolates belonged to E. coli serotypes O162 : H−, OR : H− or Ont : H−, whereas CG 2 isolates belonged to O153 : HR, OR : HR or OR : H34. These results confirm that in this particular outbreak, canine MDREC were highly clonal and CG 2 MDREC may colonize both humans and dogs.
Isolation and characterization of Streptococcus mutans in heart valve and dental plaque specimens from a patient with infective endocarditis
Authors: Nomura Ryota , Nakano Kazuhiko , Nemoto Hirotoshi , Fujita Kazuyo , Inagaki Satoko , Takahashi Toshiki , Taniguchi Kazuhiro , Takeda Munehiro , Yoshioka Hideo , Amano Atsuo , Ooshima Takashi
Published: July 2006

Streptococcus mutans, known to be an aetiologic agent of dental caries, also causes infective endocarditis (IE), although a comparison of isolates from the oral cavity and infected heart valve of the same patient has not been reported. In the present study, infected heart valve and dental plaque samples from a patient with IE were analysed. Broad-range PCR with DNA sequencing revealed that 50 clones from the dental plaque isolates were composed of oral streptococci and periodontopathic bacteria, whereas onlyStreptococcus mutanswas detected in 50 clones from the heart valve. Eighteen strains ofStreptococcus mutanswere isolated from dental plaque and seven from the heart valve, and the biochemical properties of each were in accordance with those ofStreptococcus mutans. DNA fingerprinting analysis revealed that all the oral isolates ofStreptococcus mutanshad similar patterns, which were different from those of the isolates from the infected heart valve. Western blotting using glucosyltransferase (GTF)-specific antiserum showed that the seven strains from the heart valve lacked the three types of intact GTF. In addition, the sucrose-dependent adhesion rates of these isolates were significantly lower than those of the oral isolates (P<0.001). Furthermore, the isolates from the heart valve were less susceptible to erythromycin and kanamycin. These results indicate that the properties of theStreptococcus mutansstrains isolated from the infected valve were different from those of typical oral strains, which may be related to the effects of IE.
Morphological changes and proteome response of Corynebacterium glutamicum to a partial depletion of FtsI
Authors: Valbuena Noelia , Letek Michal , Ramos Angelina , Ayala Juan , Nakunst Diana , Kalinowski Joern , Mateos Luis M. , Gil José A.
Published: July 2006

In Corynebacterium glutamicum, as in many Gram-positive bacteria, the cell division gene ftsI is located at the beginning of the dcw cluster, which comprises cell division- and cell wall-related genes. Transcriptional analysis of the cluster revealed that ftsI is transcribed as part of a polycistronic mRNA, which includes at least mraZ, mraW, ftsL, ftsI and murE, from a promoter that is located upstream of mraZ. ftsI appears also to be expressed from a minor promoter that is located in the intergenic ftsL–ftsI region. It is an essential gene in C. glutamicum, and a reduced expression of ftsI leads to the formation of larger and filamentous cells. A translational GFP-FtsI fusion protein was found to be functional and localized to the mid-cell of a growing bacterium, providing evidence of its role in cell division in C. glutamicum. This study involving proteomic analysis (using 2D SDS-PAGE) of a C. glutamicum strain that has partially depleted levels of FtsI reveals that at least 20 different proteins were overexpressed in the organism. Eight of these overexpressed proteins, which include DivIVA, were identified by MALDI-TOF. Overexpression of DivIVA was confirmed by Western blotting using anti-DivIVA antibodies, and also by fluorescence microscopy analysis of a C. glutamicum RESF1 strain expressing a chromosomal copy of a divIVA-gfp transcriptional fusion. Overexpression of DivIVA was not observed when FtsI was inhibited by cephalexin treatment or by partial depletion of FtsZ.
Cryptococcus neoformans var. grubii isolates recovered from persons with AIDS demonstrate a wide range of virulence during murine meningoencephalitis that correlates with the expression of certain virulence factors
Authors: Clancy Cornelius J. , Nguyen M. Hong , Alandoerffer Ruth , Cheng Shaoji , Iczkowski Kenneth , Richardson Malcolm , Graybill John R.
Published: July 2006

Cryptococcus neoformansis a common cause of meningoencephalitis among AIDS patients. SeveralC. neoformansvirulence factors have been identified, but the relative importance of particular factors is unknown. This study examined the corrrelation of the virulence of 18C. neoformansvar.grubiiisolates from AIDS patients with the expression of several well-described virulence factors. The LD50at 15 days after intracranial inoculation of ICR mice was <100 c.f.u. for 22 % of isolates, 100–1000 for 28 %, 1000–10 000 for 11 % and >20 000 for 39 %. Higher cryptococcal concentrations in brains were noted for isolates with lower LD50(P=0.002). In survival studies, no immunocompetent BALB/c mice (nu/−) infected with 3×LD50of three virulent isolates (LD50=62, 99, 1280) survived beyond 23 days, whereas 100 %, 90 % and 90 % of mice infected with 20 000 c.f.u. of three hypovirulent isolates (LD50>20 000) survived for 60 days (P<0.0001). Even among BALB/c nude (nu/nu) mice, survival rates over 60 days were 100 %, 70 % and 50 %, respectively, for the hypovirulent isolates. Growth rate at 37 °C and capsule size within brains correlated with LD50by univariate (P=0.0001 and 0.028, respectively) and multivariate (P=0.017 and 0.016, respectively) analyses. There was no correlation between LD50and capsule sizein vitro, phospholipase activity, melanin formation, proteinase activity and fluconazole MIC. In conclusion, AIDS patients are susceptible to infection byC. neoformansisolates of wide-ranging virulence, including isolates that are markedly hypovirulent. The virulence of a given isolate reflects a composite of factors rather than the contribution of a dominant factor. Growth at 37 °C and capsule sizein vivomake particularly important contributions.
Global effects of homocysteine on transcription in Escherichia coli: induction of the gene for the major cold-shock protein, CspA
Authors: Fraser Katy R. , Tuite Nina L. , Bhagwat Arvind , O'Byrne Conor P.
Published: July 2006

Homocysteine (Hcy) is a thiol-containing amino acid that is considered to be medically important because it is linked to the development of several life-threatening diseases in humans, including cardiovascular disease and stroke. It inhibits the growth ofEscherichia coliwhen supplied in the growth medium. Growth inhibition is believed to arise as a result of partial starvation for isoleucine, which occurs because Hcy perturbs the biosynthesis of this amino acid. This study attempted to further elucidate the inhibitory mode of action of Hcy by examining the impact of exogenously supplied Hcy on the transcriptome. Using gene macroarrays the transcript levels corresponding to 68 genes were found to be reproducibly altered in the presence of 0.5 mM Hcy. Of these genes, the biggest functional groups affected were those involved in translation (25 genes) and in amino acid metabolism (19 genes). Genes involved in protection against oxidative stress were repressed in Hcy-treated cells and this correlated with a decrease in catalase activity. The gene showing the strongest induction by Hcy wascspA, which encodes the major cold-shock protein CspA. RT-PCR and reporter fusion experiments confirmed thatcspAwas induced by Hcy. Induction ofcspAby Hcy was not caused by nutritional upshift, a stimulus known to induce CspA expression, nor was it dependent on the presence of a functional CspA protein. The induction ofcspAby Hcy was suppressed when isoleucine was included in the growth medium. These data suggest that the induction of CspA expression in the presence of Hcy occurs because of a limitation for isoleucine. The possibility that Hcy-inducedcspAexpression is triggered by translational stalling that occurs when the cells are limited for isoleucine is discussed.
Succinate dehydrogenase functioning by a reverse redox loop mechanism and fumarate reductase in sulphate-reducing bacteria
Authors: Zaunmüller Tanja , Kelly David J. , Glöckner Frank O. , Unden Gottfried
Published: July 2006

Sulphate- or sulphur-reducing bacteria with known or draft genome sequences (Desulfovibrio vulgaris, Desulfovibrio desulfuricans G20, Desulfobacterium autotrophicum [draft], Desulfotalea psychrophila and Geobacter sulfurreducens) all contain sdhCAB or frdCAB gene clusters encoding succinate : quinone oxidoreductases. frdD or sdhD genes are missing. The presence and function of succinate dehydrogenase versus fumarate reductase was studied. Desulfovibrio desulfuricans (strain Essex 6) grew by fumarate respiration or by fumarate disproportionation, and contained fumarate reductase activity. Desulfovibrio vulgaris lacked fumarate respiration and contained succinate dehydrogenase activity. Succinate oxidation by the menaquinone analogue 2,3-dimethyl-1,4-naphthoquinone depended on a proton potential, and the activity was lost after degradation of the proton potential. The membrane anchor SdhC contains four conserved His residues which are known as the ligands for two haem B residues. The properties are very similar to succinate dehydrogenase of the Gram-positive (menaquinone-containing) Bacillus subtilis, which uses a reverse redox loop mechanism in succinate : menaquinone reduction. It is concluded that succinate dehydrogenases from menaquinone-containing bacteria generally require a proton potential to drive the endergonic succinate oxidation. Sequence comparison shows that the SdhC subunit of this type lacks a Glu residue in transmembrane helix IV, which is part of the uncoupling E-pathway in most non-electrogenic FrdABC enzymes.
Interaction and localization studies of enteropathogenic Escherichia coli type IV bundle-forming pilus outer membrane components
Authors: Daniel Anu , Singh Aparna , Crowther Lynette J. , Fernandes Paula J. , Schreiber Wiebke , Donnenberg Michael S.
Published: July 2006

Typical enteropathogenicEscherichia colistrains express an established virulence factor belonging to the type IV pili family, called the bundle-forming pilus (BFP). BFP are present on the cell surface as bundled filamentous appendages, and are assembled and retracted by proteins encoded by thebfpoperon. These proteins assemble to form a molecular machine. The BFP machine may be conceptually divided into three components: the cytoplasmic membrane (CM) subassembly, which is composed of CM proteins and cytoplasmic nucleotide-binding proteins; the outer membrane (OM) subassembly and the pilus itself. The authors have previously characterized the CM subassembly and the pilus. In this study, a more complete characterization of the OM subassembly was carried out using a combination of biochemical, biophysical and genetic approaches. It is reported that targeting of BfpG to the OM was influenced by the secretin BfpB. BfpG and BfpU interacted with the amino terminus of BfpB. BfpU had a complex cellular distribution pattern and, along with BfpB and BfpG, was part of the OM subassembly.
A putative multicopper protein secreted by an atypical type II secretion system involved in the reduction of insoluble electron acceptors in Geobacter sulfurreducens
Authors: Mehta Teena , Childers Susan E. , Glaven Richard , Lovley Derek R. , Mester Tünde
Published: July 2006

Extracellular electron transfer onto Fe(III) oxides in Geobacter sulfurreducens is considered to require proteins that must be exported to the outer surface of the cell. In order to investigate this, the putative gene for OxpG, the pseudopilin involved in a type II general secretion pathway of Gram-negative bacteria, was deleted. The mutant was unable to grow with insoluble Fe(III) oxide as the electron acceptor. Growth on soluble Fe(III) was not affected. An analysis of proteins that accumulated in the periplasm of the oxpG mutant, but not in the wild-type, led to the identification of a secreted protein, OmpB. OmpB is predicted to be a multicopper protein, with highest homology to the manganese oxidase, MofA, from Leptothrix discophora. OmpB contains a potential Fe(III)-binding site and a fibronectin type III domain, suggesting a possible role for this protein in accessing Fe(III) oxides. OmpB was localized to the membrane fraction of G. sulfurreducens and in the supernatant of growing cultures, consistent with the type II secretion system exporting OmpB. A mutant in which ompB was deleted had the same phenotype as the oxpG mutant, suggesting that the failure to export OmpB was responsible for the inability of the oxpG-deficient mutant to reduce Fe(III) oxide. This is the first report that proposes a role for a multicopper oxidase-like protein in an anaerobic organism. These results further emphasize the importance of outer-membrane proteins in Fe(III) oxide reduction and suggest that outer-membrane proteins other than c-type cytochromes are required for Fe(III) oxide reduction in Geobacter species.
Transcriptional regulation of the fad regulon genes of Escherichia coli by ArcA
Authors: Cho Byung-Kwan , Knight Eric M. , Palsson Bernhard Ø.
Published: July 2006

ArcA is a global transcription factor required for optimal growth ofEscherichia coliduring anaerobic growth. In this study, the role of ArcA on the transcriptional regulatory subnetwork of thefadregulon was investigated. Gene expression profiles of deletion mutants (ΔarcA, ΔfadRand ΔarcA/ΔfadR) indicated that (i) ArcA is a major transcription factor for the transcriptional regulation of fatty acid metabolism in the absence of oxygen, and (ii) ArcA and FadR cooperatively regulate thefadregulon under anaerobic conditions. To determine the direct interaction between ArcA and the promoters of thefadregulon genes, chromatin immunoprecipitation (ChIP) analysis was performed. ChIP analysis suggested that ArcA directly binds to the promoter regions of thefadregulon genesin vivo. An ArcA-binding motif was identified from known binding sequences and predicted putative binding sites in the promoter regions of thefadregulon genes. These results indicate that ArcA directly represses the expression offadregulon genes during anaerobic growth.
Characterization and a role of Pseudomonas aeruginosa spermidine dehydrogenase in polyamine catabolism
Authors: Dasu Veeranki Venkata , Nakada Yuji , Ohnishi-Kameyama Mayumi , Kimura Keitarou , Itoh Yoshifumi
Published: July 2006

Pseudomonas aeruginosaPAO1 has two possible catabolic pathways of spermidine and spermine; one includes thespuAandspuBproducts with unknown functions and the other involves spermidine dehydrogenase (SpdH; EC 1.5.99.6) encoded by an unknown gene. The properties of SpdH inP. aeruginosaPAO1 were characterized and the correspondingspdHgene in this strain identified. The deduced SpdH (620 residues, calculatedMrof 68 861) had a signal sequence of 28 amino acids at the amino terminal and a potential transmembrane segment between residues 76 and 92, in accordance with membrane location of the enzyme. Purified SpdH oxidatively cleaved spermidine into 1,3-diaminopropane and 4-aminobutyraldehyde with a specific activity of 37 units (mg protein)−1and aKmvalue of 36 μM. The enzyme also hydrolysed spermine into spermidine and 3-aminopropanaldehyde with a specific activity of 25 units (mg protein)−1and aKmof 18 μM. Knockout ofspdHhad no apparent effect on the utilization of both polyamines, suggesting that this gene is minimally involved in polyamine catabolism. However, whenspdHwas fused to the polyamine-inducible promoter ofspuA, it fully restored the ability of aspuAmutant to utilize spermidine. It is concluded that SpdH can perform a catabolic rolein vivo, butP. aeruginosaPAO1 does not produce sufficient amounts of the enzyme to execute this function.
Functional characterization of a fatty acyl-CoA-binding protein (ACBP) from the apicomplexan Cryptosporidium parvum
Authors: Zeng Bin , Cai Xiaomin , Zhu Guan
Published: July 2006

In this paper, the identification and functional analysis of a fatty acyl-CoA-binding protein (ACBP) gene from the opportunistic protist Cryptosporidium parvum are described. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ACBPs (i.e. ∼90 aa) of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (∼90 aa) and a C-terminal ankyrin repeat sequence (∼170 aa). The entire CpACBP1 ORF was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane.
Differential turnover of the multiple processed transcripts of the Escherichia coli focA-pflB operon
Authors: Sawers R. Gary
Published: July 2006

Expression of the anaerobically induciblefocA-pflBoperon ofEscherichia coliis subject to complex transcriptional and post-transcriptional control, which generates eight transcripts whose 5′ ends span ∼1.2 kb. All eight transcripts have the same 3′ end. The 5′ ends of three of the transcripts, termed 6, 6a and 7, are located upstream of the operon. The promoters generating transcripts 6 and 7 are anaerobically regulated by FNR and ArcA∼P, while promoter 6a is constitutively active. The 5′ ends of the other five transcripts are all located within the operon. Most of the 5′ ends of these operon-internal transcripts result from RNA polymerase-dependent processing of the three longer primary transcripts, 6, 6a and 7. Here, it is demonstrated that subsequent to, and distinct from, these processing events, post-transcriptional modification of these transcripts also occurs through the action of the endoribonuclease RNase E. Transcripts 6 and 7 exhibit differential stability with half-lives of 1 and 5 min, respectively. Transcript 7, which has the longer half-life, is the longest transcript of the operon and has a ∼340 base untranslated leader. Two of the operon-internal transcripts, 4 and 5, also have comparatively short half-lives in the wild-type, which are significantly increased in a mutant with impaired RNase E activity. A precursor-product relationship is observed between the longer transcripts 3–7 and transcripts 1 and 2. The 5′ ends of transcripts 1 and 2 are closest to thepflBgene and have half-lives of approximately 7–8 min. The consequence of this regulation is an accumulation of full-lengthpflBtranscript and comparably low levels of dicistronic transcript. This ensures different levels of synthesis of the formate transporter FocA and pyruvate formate-lyase during anaerobic growth, while maintaining coordinate regulation. Transcript analysis throughout the growth phase revealed that maximal anaerobic expression of thefocA-pflBoperon was restricted to exponentially growing cells. Expression of transcript 7 peaked in early to mid-exponential phase, while the levels of transcript 6 steadily accumulated toward the late-exponential phase of growth. Taken together, these findings indicate that although subject to common positive control by ArcA∼P and FNR, the transcripts generated by promoters 6 and 7 are subject to differential temporal and post-transcriptional regulation.
A survey of all 11 ABC transporters in fission yeast: two novel ABC transporters are required for red pigment accumulation in a Schizosaccharomyces pombe adenine biosynthetic mutant
Authors: Iwaki Tomoko , Giga-Hama Yuko , Takegawa Kaoru
Published: July 2006

ATP-binding cassette (ABC) proteins transport a wide variety of substrates, including sugars, amino acids, metal ions, lipids, peptides and proteins, across membranes, and most ABC proteins contain transmembrane domains (ABC transporters). Sequencing of theSchizosaccharomyces pombegenome has allowed identification of all genes encoding ABC transporters in fission yeast. To date, six such genes have been characterized, and an additional five genes encoding ABC transporters were identified from the genome sequence. In an attempt to characterize all of the ABC transporters in fission yeast, all 11 genes were disrupted. While all the genes were found to be dispensable for cell viability, some disruptants lacked apparent phenotypes. GFP-tagged ABC transporters were localized to membranes as follows: plasma membrane (2), vacuolar membrane (4), mitochondrial membrane (2), endoplasmic reticulum membrane (2), and endosome and Golgi membranes (1). Two Cluster II. 1 proteins, Abc2p (SPAC3F10.11c) and Abc4p (SPAC30.04c), were found to be localized to vacuolar membranes, and to be responsible for accumulation of a characteristic red pigment in the vacuole of an adenine biosynthetic mutant. The doubly disrupted mutantabc2Δabc4Δ exhibited drug sensitivity, and a decreased accumulation of monochlorobimane, suggesting that both of the proteins encoded by these genes are involved in detoxification of xenobiotics, and vacuolar sequestration of glutathioneS-conjugates.
Candida albicans Als3p is required for wild-type biofilm formation on silicone elastomer surfaces
Authors: Zhao Xiaomin , Daniels Karla J. , Oh Soon-Hwan , Green Clayton B. , Yeater Kathleen M. , Soll David R. , Hoyer Lois L.
Published: July 2006

Candida albicans ALS3encodes a large cell-surface glycoprotein that has adhesive properties. Immunostaining of culturedC. albicansgerm tubes showed that Als3p is distributed diffusely across the germ tube surface. Two-photon laser scanning microscopy of model catheter biofilms grown using a PALS3-green fluorescent protein (GFP) reporter strain showed GFP production in hyphae throughout the biofilm structure while biofilms grown using a PTPI1-GFP reporter strain showed GFP in both hyphae and yeast-form cells. Model catheter biofilms formed by anals3Δ/als3Δ strain were weakened structurally and had approximately half the biomass of a wild-type biofilm. Reintegration of a wild-typeALS3allele restored biofilm mass and wild-type biofilm structure. Production of an Als3p–Agα1p fusion protein under control of theALS3promoter in theals3Δ/als3Δ strain restored some of the wild-type biofilm structural features, but not the wild-type biofilm mass. Despite its inability to restore wild-type biofilm mass, the Als3p–Agα1p fusion protein mediated adhesion of theals3Δ/als3ΔC. albicansstrain to human buccal epithelial cells (BECs). The adhesive role of the Als3p N-terminal domain was further demonstrated by blocking adhesion ofC. albicansto BECs with immunoglobulin reactive against the Als3p N-terminal sequences. Together, these data suggest that portions of Als3p that are important for biofilm formation may be different from those that are important in BEC adhesion, and that Als3p may have multiple functions in biofilm formation. Overexpression ofALS3in anefg1Δ/efg1Δ strain that was deficient for filamentous growth and biofilm formation resulted in growth of elongatedC. albicanscells, even under culture conditions that do not favour filamentation. In the catheter biofilm model, theALS3overexpression strain formed biofilm with a mass similar to that of a wild-type control. However,C. albicanscells in the biofilm had yeast-like morphology. This result uncouples the effect of cellular morphology from biofilm formation and underscores the importance of Als3p in biofilm development on silicone elastomer surfaces.