bluuebunny/crossref_metadata_2025 huggingface dataset:
Glomerular Calcification Induced by Bolus Injection with Dibasic Sodium Phosphate Solution in Sprague—Dawley Rats
Authors: Tsuchiya Noriko , Matsushima Shuuichi , Takasu Nobuo , Kyokawa Yoshimasa , Torii Mikinori
Published: June 2004

To elucidate the nephrotoxicity of phosphate, dibasic sodium phosphate solution was given to Sprague—Dawley rats by daily bolus intravenous administration at concentrations of 0, 1, 25, 250, or 360 mM (0, 1, 28, 284, or 408 mg/kg Na2HPO4)for 14 days, and the kidneys were pathologically examined. There were no remarkable changes in blood chemistry values; however, urinalysis revealed mild to moderate proteinuria in the 250 and 360 mM groups. The kidneys from the 360 mM group were macroscopically pale. Histopathology revealed panglomerular deposition of basophilic dense granules, which were positive for von Kossa's staining, accompanied by dose-dependent degeneration of the glomerular epithelium and parietal epithelium in the 250 and 360 mM groups. Electron microscopic examination showed fusion of podocytes and increased microvilli, with large amounts of debris in the Bowman's space. Low-density lamellar structures were present not only in the glomerular epithelium, basement membrane, mesangial matrix and parietal epithelium but also within the Bowman's space depending on the severity of the glomerular lesion. Phosphorus and calcium were detected by X-ray microanalysis as fine particles admixed with lamellar structures. These results suggest that high-dose phosphate used in this study transiently overloads the glomerular epithelium during filtration through glomerular capillaries and produces insoluble calcium salt and glomerular lesions, resulting in proteinuria.
Results of Chronic Dietary Toxicity Studies of High Viscosity (P70H and P100H) White Mineral Oils in Fischer 344 Rats
Authors: Trimmer Gary W. , Freeman James J. , Priston R. A. J. , Urbanus Jan
Published: June 2004

Two-year dietary studies were conducted to determine the chronic toxicity and its reversibility, and the carcinogenicity of P70(H) and P100(H) white mineral oils in Fischer-344 rats (F-344). The studies were identical in design and followed the Organization for Economic Cooperation and Development, Guidelines for Testing Chemicals, Guideline 453, 1981. Additional endpoints evaluated were: (1) extent of mineral hydrocarbon deposition in liver, kidneys, mesenteric lymph nodes, and spleen of female rats at 3, 6, 12, 18 and 24 months, and (2) reversibility of effects following cessation of exposure. Dietary concentration were 60, 120, 240, and 1, 200 mg/kg/day, adjusted periodically to account for bodyweight changes. Study results were consistent with preceding subchronic studies. No treatment-related mortality, neoplastic lesions, or changes in clinical health, hematology, serum chemistry, or urine chemistry were evident in any group administered either white oil. Statistically significant higher food consumption was noted in the 1, 200 mg/kg group males and females exposed to either white oil and statistically significant higher body weights were noted in the 1, 200-mg/kg males during the latter portion of the P100(H) study. Higher mesenteric lymph node weights were accompanied by increased severity of infiltrating histiocytes. This occurred to a greater extent with the P70(H) than the P100(H) oil. No other histopathology of significance was observed. Mineral hydrocarbons were detected in the liver following exposure to either oil. Maximal concentrations of mineral hydrocarbons in the liver were similar with both oils but occurred more rapidly with the P70(H) oil. Liver mineral hydrocarbon content returned to near-background levels during the reversibility phase. In conclusion, lifetime exposer of F344 rats to P70(H) and P100(H) white oils resulted in only minimal findings and with no consequence to clinical health. Thus, under the conditions of these studies, the No Observable Adverse Effect Level (NOAEL) for these studies was considered to be 1, 200 mg/kg/day.
Relationships Between Organ Weight and Body/Brain Weight in the Rat: What Is the Best Analytical Endpoint?
Authors: Bailey Steven A. , Zidell Robert H. , Perry Richard W.
Published: June 2004

Analysis of organ weight in toxicology studies is an important endpoint for identification of potentially harmful effects of chemicals. Differences in organ weight between treatment groups are often accompanied by differences in body weight between these groups, making interpretation of organ weight differences more difficult. Using data from control rats that were part of 26 toxicity studies conducted under similar conditions, we have evaluated the relationship between organ weight and body/brain weight to determine which endpoint (organ weight, organ-to-body weight ratio, or organ-to-brain weight ratio) is likely to accurately detect target organ toxicity. This evaluation has shown that analysis of organ-to-body weight ratios is predictive for evaluating liver and thyroid gland weights, and organ-to-brain weight ratios is predictive for evaluating ovary and adrenal gland weights. Brain, heart, kidney, pituitary gland, and testes weights are not modeled well by any of the choices, and alternative analysis methods such as analysis of covariance should be utilized.
A 29.5 KDA Heat‐modifiable Major Outer Membrane Protein of Rickettsia Prowazekii, Putative Virulence Factor, is a Peptidyl‐Prolyl Cis/trans Isomerase
Authors: Emelyanov Victor V. , Loukianov Evgeny V.
Published: June 2004

Allelic genes from three Rickettsia prowazekii strains encoding parvulin‐like protein (Plp), a heat‐modifiable 29.5 kDa major outer membrane protein, were earlier cloned into expression vector pQE 30. In this work, recombinant proteins were overproduced in E. coli, purified, and found to exhibit an expected peptidyl‐prolyl cis/trans isomerase activity of a parvulin type in vitro with oligopeptide substrates. Native polypeptide of prototype virulent Breinl strain is known to differ by SDS ‐ PAGE mobility from those of both vaccine Madrid E and virulent EVir isolates. Being different in electrophoretic behavior, heat‐unmodified forms of the three strains were shown to migrate apart from lipopolysaccharides. A EVir Plp gene was sequenced, and deduced protein sequence was found to be identical to previously published Breinl and Madrid E. Present data indicate that unknown post‐translational modification(s) in rickettsiae are responsible for both interstrain difference and heat‐modifiability of Plp.
IUBMB Life, 56: 215‐219, 2004
Oxygen Free Radicals and the Systemic Inflammatory Response
Authors: Closa Daniel , Folch‐Puy Emma
Published: June 2004

The generation of oxygen free radicals is known to be involved in the development of the systemic inflammatory response syndrome. In addition to their actions as noxious mediators generated by inflammatory cells, these molecules play also a crucial role contributing to the onset and progression of inflammation in distant organs. In the early stages of the process, free radicals exert their actions via activation of nuclear factors, as NFκB or AP‐1, that induce the synthesis of cytokines. In later stages, endothelial cells are activated due to the synergy between free radicals and cytokines, promoting the synthesis of inflammatory mediators and adhesion molecules. Finally, free radicals exert their toxic effects at the site of inflammation by reacting with different cell components, inducing loss of function and cell death. This review focuses on progress in the understanding the different actions of free radicals at the sequential stages of the development of the systemic inflammatory response.
IUBMB Life, 56: 185‐191, 2004
Lis1 and doublecortin function with dynein to mediate coupling of the nucleus to the centrosome in neuronal migration
Authors: Tanaka Teruyuki , Serneo Finley F. , Higgins Christine , Gambello Michael J. , Wynshaw-Boris Anthony , Gleeson Joseph G.
Published: June 2004

Humans with mutations in either DCX or LIS1 display nearly identical neuronal migration defects, known as lissencephaly. To define subcellular mechanisms, we have combined in vitro neuronal migration assays with retroviral transduction. Overexpression of wild-type Dcx or Lis1, but not patient-related mutant versions, increased migration rates. Dcx overexpression rescued the migration defect in Lis1+/− neurons. Lis1 localized predominantly to the centrosome, and after disruption of microtubules, redistributed to the perinuclear region. Dcx outlined microtubules extending from the perinuclear “cage” to the centrosome. Lis1+/− neurons displayed increased and more variable separation between the nucleus and the preceding centrosome during migration. Dynein inhibition resulted in similar defects in both nucleus–centrosome (N-C) coupling and neuronal migration. These N-C coupling defects were rescued by Dcx overexpression, and Dcx was found to complex with dynein. These data indicate Lis1 and Dcx function with dynein to mediate N-C coupling during migration, and suggest defects in this coupling may contribute to migration defects in lissencephaly.
Dynamics of putative raft-associated proteins at the cell surface
Authors: Kenworthy Anne K. , Nichols Benjamin J. , Remmert Catha L. , Hendrix Glenn M. , Kumar Mukesh , Zimmerberg Joshua , Lippincott-Schwartz Jennifer
Published: June 2004

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (>4 μm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.
Extracellular matrix mineralization is regulated locally; different roles of two gla-containing proteins
Authors: Murshed Monzur , Schinke Thorsten , McKee Marc D. , Karsenty Gerard
Published: June 2004

Extracellular matrix mineralization (ECMM) is a physiologic process in the skeleton and in teeth and a pathologic one in other organs. The molecular mechanisms controlling ECMM are poorly understood. Inactivation of Matrix gla protein (Mgp) revealed that MGP is an inhibitor of ECMM. The fact that MGP is present in the general circulation raises the question of whether ECMM is regulated locally and/or systemically. Here, we show that restoration of Mgp expression in arteries rescues the arterial mineralization phenotype of Mgp−/− mice, whereas its expression in osteoblasts prevents bone mineralization. In contrast, raising the serum level of MGP does not affect mineralization of any ECM. In vivo mutagenesis experiments show that the anti-ECMM function of MGP requires four amino acids which are γ-carboxylated (gla residues). Surprisingly, another gla protein specific to bone and teeth (osteocalcin) does not display the anti-ECMM function of MGP. These results indicate that ECMM is regulated locally in animals and uncover a striking disparity of function between proteins sharing identical structural motifs.
Trafficking of Lyn through the Golgi caveolin involves the charged residues on αE and αI helices in the kinase domain
Authors: Kasahara Kousuke , Nakayama Yuji , Ikeda Kikuko , Fukushima Yuka , Matsuda Daisuke , Horimoto Shinya , Yamaguchi Naoto
Published: June 2004

Src-family kinases, known to participate in signaling pathways of a variety of surface receptors, are localized to the cytoplasmic side of the plasma membrane through lipid modification. We show here that Lyn, a member of the Src-family kinases, is biosynthetically transported to the plasma membrane via the Golgi pool of caveolin along the secretory pathway. The trafficking of Lyn from the Golgi apparatus to the plasma membrane is inhibited by deletion of the kinase domain or Csk-induced “closed conformation” but not by kinase inactivation. Four residues (Asp346 and Glu353 on αE helix, and Asp498 and Asp499 on αI helix) present in the C-lobe of the kinase domain, which can be exposed to the molecular surface through an “open conformation,” are identified as being involved in export of Lyn from the Golgi apparatus toward the plasma membrane but not targeting to the Golgi apparatus. Thus, the kinase domain of Lyn plays a role in Lyn trafficking besides catalysis of substrate phosphorylation.
Triggering p53 after cytokinesis failure
Authors: Stukenberg P. Todd
Published: June 2004

Cells that fail to divide during cytokinesis often arrest in the next G1 phase by a mysterious mechanism that depends upon p53. What triggers this arrest is unclear. New studies, including a report in this issue (Uetake and Sluder, 2004) suggest that this arrest does not occur because cells are polyploid, are binucleate, have multiple centrosome, or have failed cytokinesis, making this phenomenon even more puzzling.
Surface μ Heavy Chain Signals Down-Regulation of the V(D)J-Recombinase Machinery in the Absence of Surrogate Light Chain Components
Authors: Galler Gunther R. , Mundt Cornelia , Parker Mathew , Pelanda Roberta , Mårtensson Inga-Lill , Winkler Thomas H.
Published: June 2004

Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre–B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible μHC transgene in Rag2−/− pro–B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-α. Furthermore, in wild-type mice and in mice lacking either λ5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in μHC+LC− pre–B cells. Surprisingly, μHC without LC is expressed on the surface of pro–/pre–B cells from λ5−/−, VpreB1−/−VpreB2−/−, and SLC−/− mice. Thus, SLC or LC is not required for μHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.
Distinct Time Effects of Vaccination on Long-Term Proliferative and IFN-γ–producing T Cell Memory to Smallpox in Humans
Authors: Combadiere Behazine , Boissonnas Alexandre , Carcelain Guislaine , Lefranc Evelyne , Samri Assia , Bricaire François , Debre Patrice , Autran Brigitte
Published: June 2004

Residual immunity to the smallpox virus raises key questions about the persistence of long-term immune memory in the absence of antigen, since vaccination ended in 1980. IFN-γ–producing effector–memory and proliferative memory T cells were compared in 79 vaccinees 13–25 yr after their last immunization and in unvaccinated individuals. Only 20% of the vaccinees displayed both immediate IFN-γ–producing effector–memory responses and proliferative memory responses at 6 d; 52.5% showed only proliferative responses; and 27.5% had no detectable vaccinia-specific responses at all. Both responses were mediated by CD4 and CD8 T cells. The vaccinia-specific IFN-γ–producing cells were composed mainly of CD4Pos CD45RANeg CD11aHi CD27Pos and CCR7Neg T cells. Their frequency was low but could be expanded in vitro within 7 d. Time since first immunization affected their persistence: they vanished 45 yr after priming, but proliferative responses remained detectable. The number of recalls did not affect the persistence of residual effector–memory T cells. Programmed revaccination boosted both IFN-γ and proliferative responses within 2 mo of recall, even in vaccinees with previously undetectable residual effector–memory cells. Such long-term maintenance of vaccinia-specific immune memory in the absence of smallpox virus modifies our understanding of the mechanism of persistence of long-term memory to poxviruses and challenges vaccination strategies.
CD25+ CD4+ T Cells, Expanded with Dendritic Cells Presenting a Single Autoantigenic Peptide, Suppress Autoimmune Diabetes
Authors: Tarbell Kristin V. , Yamazaki Sayuri , Olson Kara , Toy Priscilla , Steinman Ralph M.
Published: June 2004

In the nonobese diabetic (NOD) mouse model of type 1 diabetes, the immune system recognizes many autoantigens expressed in pancreatic islet β cells. To silence autoimmunity, we used dendritic cells (DCs) from NOD mice to expand CD25+ CD4+ suppressor T cells from BDC2.5 mice, which are specific for a single islet autoantigen. The expanded T cells were more suppressive in vitro than their freshly isolated counterparts, indicating that DCs from autoimmune mice can increase the number and function of antigen-specific, CD25+ CD4+ regulatory T cells. Importantly, only 5,000 expanded CD25+ CD4+ BDC2.5 T cells could block autoimmunity caused by diabetogenic T cells in NOD mice, whereas 105 polyclonal, CD25+ CD4+ T cells from NOD mice were inactive. When islets were examined in treated mice, insulitis development was blocked at early (3 wk) but not later (11 wk) time points. The expanded CD25+ CD4+ BDC2.5 T cells were effective even if administered 14 d after the diabetogenic T cells. Our data indicate that DCs can generate CD25+ CD4+ T cells that suppress autoimmune disease in vivo. This might be harnessed as a new avenue for immunotherapy, especially because CD25+ CD4+ regulatory cells responsive to a single autoantigen can inhibit diabetes mediated by reactivity to multiple antigens.
Negative Regulation of Immunoglobulin E–dependent Allergic Responses by Lyn Kinase
Authors: Odom Sandra , Gomez Gregorio , Kovarova Martina , Furumoto Yasuko , Ryan John J. , Wright Harry V. , Gonzalez-Espinosa Claudia , Hibbs Margaret L. , Harder Kenneth W. , Rivera Juan
Published: June 2004

A role for Lyn kinase as a positive regulator of immunoglobulin (Ig)E-dependent allergy has long been accepted. Contrary to this belief, Lyn kinase was found to have an important role as a negative regulator of the allergic response. This became apparent from the hyperresponsive degranulation of lyn−/− bone marrow–derived mast cells, which is driven by hyperactivation of Fyn kinase that occurs, in part, through the loss of negative regulation by COOH-terminal Src kinase (Csk) and the adaptor, Csk-binding protein. This phenotype is recapitulated in vivo as young lyn−/− mice showed an enhanced anaphylactic response. In vivo studies also demonstrated that as lyn−/− mice aged, their serum IgE increased as well as occupancy of the high affinity IgE receptor (FcεRI). This was mirrored by increased circulating histamine, increased mast cell numbers, increased cell surface expression of the high affinity IgE receptor (FcεRI), and eosinophilia. The increased IgE production was not a consequence of increased Fyn kinase activity in lyn−/− mice because both lyn−/− and lyn−/− fyn−/− mice showed high IgE levels. Thus, lyn−/− mice and mast cells thereof show multiple allergy-associated traits, causing reconsideration of the possible efficacy in therapeutic targeting of Lyn in allergic disease.
Nuclear Factor of Activated T Cells Balances Angiogenesis Activation and Inhibition
Authors: Zaichuk Tetiana A. , Shroff Emelyn H. , Emmanuel Rebekah , Filleur Stephanie , Nelius Thomas , Volpert Olga V.
Published: June 2004

It has been demonstrated that vascular endothelial cell growth factor (VEGF) induction of angiogenesis requires activation of the nuclear factor of activated T cells (NFAT). We show that NFATc2 is also activated by basic fibroblast growth factor and blocked by the inhibitor of angiogenesis pigment epithelial–derived factor (PEDF). This suggests a pivotal role for this transcription factor as a convergence point between stimulatory and inhibitory signals in the regulation of angiogenesis.

We identified c-Jun NH2-terminal kinases (JNKs) as essential upstream regulators of NFAT activity in angiogenesis. We distinguished JNK-2 as responsible for NFATc2 cytoplasmic retention by PEDF and JNK-1 and JNK-2 as mediators of PEDF-driven NFAT nuclear export.

We identified a novel NFAT target, caspase-8 inhibitor cellular Fas-associated death domain–like interleukin 1β–converting enzyme inhibitory protein (c-FLIP), whose expression was coregulated by VEGF and PEDF. Chromatin immunoprecipitation showed VEGF-dependent increase of NFATc2 binding to the c-FLIP promoter in vivo, which was attenuated by PEDF. We propose that one possible mechanism of concerted angiogenesis regulation by activators and inhibitors may be modulation of the endothelial cell apoptosis via c-FLIP controlled by NFAT and its upstream regulator JNK.
Targeting Plasma Cells in Autoimmune Diseases
Authors: Tarlinton David M. , Hodgkin Philip D.
Published: June 2004

Antibodies specific for self-antigens mediate life-threatening pathology in several autoimmune diseases. Clearly the ability to target the plasma cells (PCs) producing the autoantibodies would be of great clinical benefit. Current immunosuppressive therapies are based on the premise that autoreactive PCs are short-lived and replenished from ongoing immune responses. However, recent results question this assumption and suggest that optimizing the treatment of severe autoimmune conditions will require a significant investment in elucidating the details of PC biology.
The RCP–Rab11 Complex Regulates Endocytic Protein Sorting
Authors: Peden Andrew A. , Schonteich Eric , Chun John , Junutula Jagath R. , Scheller Richard H. , Prekeris Rytis
Published: June 2004

Rab 11 GTPase is an important regulator of endocytic membrane traffic. Recently, we and others have identified a novel family of Rab11 binding proteins, known as Rab11-family interacting proteins (FIPs). One of the family members, Rab coupling protein (RCP), was identified as a protein binding to both Rab4 and Rab11 GTPases. RCP was therefore suggested to serve a dual function as Rab4 and Rab11 binding protein. In this study, we characterized the cellular functions of RCP and mapped its interactions with Rab4 and Rab11. Our data show that RCP interacts only weakly with Rab4 in vitro and does not play the role of coupling Rab11 and Rab4 in vivo. Furthermore, our data indicate that the RCP–Rab11 complex regulates the sorting of transferrin receptors from the degradative to the recycling pathway. We therefore propose that RCP functions primarily as a Rab11 binding protein that regulates protein sorting in tubular endosomes.
Isolation and molecular identification of Candida dubliniensis from non-human immunodeficiency virus-infected patients in Kuwait
Authors: Ahmad Suhail , Khan Zaiba , Mokaddas Eiman , Khan Zia U.
Published: June 2004

Candida dubliniensisis an emerging pathogen capable of causing oropharyngeal, vaginal and bloodstream infections. AlthoughC. dubliniensisis similar toCandida albicansin several phenotypic characteristics, it differs from it with respect to epidemiology, certain virulence factors and the ability to develop resistance to fluconazole rapidly. In this study, the first seven isolations ofC. dubliniensisfrom Kuwait are described, all originating from non-human immunodeficiency virus (HIV)-infected patients. The isolates were initially identified by the Vitek 2 yeast identification system, positive germ tube test, production of rough colonies and chlamydospores on Staib agar and by their inability to assimilate xylose, trehalose or methyl α-d-glucoside. The species identity of the isolates was subsequently confirmed by specific amplification of rDNA targeting the internally transcribed spacer 2 (ITS2), restriction endonuclease digestion of the amplified DNA and direct DNA sequencing of the ITS2. Using the E-test method, the MICs ofC. dubliniensistest isolates were in the range 0.125–0.75 μg ml−1for fluconazole, 0.002–0.75 μg ml−1for itraconazole, 0.006–0.125 μg ml−1for ketoconazole, 0.002–0.5 μg ml−1for amphotericin B and 0.002–0.016 μg ml−1for voriconazole. Two of the isolates were resistant to 5-flucytosine (>32 μg ml−1), but none against fluconazole. The study reinforces the current view thatC. dubliniensishas a much wider geographical and epidemiological distribution.
Protection in a mouse peritonitis model mediated by iron-regulated outer-membrane protein of Salmonella typhi coupled to its Vi antigen
Authors: Chibber Sanjay , Bhardwaj Sonia B.
Published: June 2004

Vi polysaccharide and iron-regulated outer-membrane proteins (IROMPs) were extracted and purified from the standard strain of Salmonella typhi, Ty2. These were then conjugated by chemical coupling using the carbodimide method. Vi–IROMP conjugate was tested for its ability to protect against colonization by S. typhi in different organs. Mice immunized with 2.5 μg Vi–IROMP conjugate showed the most protection, as the least bacterial colonization of spleen, liver and Peyer's patches was observed. Peritoneal macrophages obtained from conjugate-treated mice phagocytosed bacteria efficiently. Circulating antibodies and the delayed type hypersensitivity response elucidated by mouse foot-pad swelling was significantly higher in conjugate-treated animals. These results clearly demonstrate that an IROMP and polysaccharide conjugate of S. typhi prepared from the same strain has the potential to protect animals against challenge.
Typing and characterization of carbapenem-resistant Acinetobacter calcoaceticus–baumannii complex in a Chinese hospital
Authors: Yu Yun-Song , Yang Qing , Xu Xiao-Wei , Kong Hai-Shen , Xu Gen-Yun , Zhong Bu-Yun
Published: June 2004

This study was designed to investigate the prevalence of carbapenem-resistantAcinetobacter calcoaceticus–baumanniicomplex (Acbcomplex) and to type carbapenemases. The relatedness of 45 isolates of carbapenem-resistantAcbcomplex collected from a clinical setting was analysed by PFGE. The carbapenemases produced by these isolates were typed by IEF, a three-dimensional test, 2-mercaptopropanoic acid inhibition assay, PCR and DNA cloning and sequencing. Results showed that all 45 isolates were resistant to multiple antibiotics including meropenem. The resistance rates to cefoperazone/sulbactam and ampicillin/sulbactam were 2.2 and 6.5 %, respectively. About 71.7–78.3 % of these isolates were intermediately resistant to cefepime, ceftazidime and cefotaxime. Forty-five isolates were classified into type A (98 %) and B (2 %) based on their PFGE patterns. Most of type A isolates were from the ICU. Type A was the dominant isolate, including subtypes A1 (22 %), A2 (71 %), A3 (2 %) and A4 (2 %). Only one isolate, from the haematology department, belonged to type B. Forty-three isolates (96 %) were positive for carbapenemase. One isolate had two bands by IEF, the pIs of which were 6.64 and 7.17. The band with the pI of 6.64 was OXA-23. The other 42 isolates produced two bands with pIs of 6.40 and 7.01 which could not be inhibited by clavulanic acid, cloxacillin or 2-mercaptopropanoic acid. It can be concluded that the prevalent carbapenem-resistantAcbcomplex isolates from this hospital all had similar β-lactamase patterns.
PCR-based assays for detection of Streptococcus pneumoniae serotypes 3, 14, 19F and 23F in respiratory specimens
Authors: Rubin Lorry G. , Rizvi Atqia
Published: June 2004

Current culture-based assays are insensitive for detection of simultaneous respiratory tract colonization by more than one pneumococcal serotype. Separate single-tube, nested PCR-based assays have been developed to detect Streptococcus pneumoniae serotypes 3, 14, 19F and 23F by amplifying unique DNA sequences in the capsular polysaccharide gene cluster of each serotype. Pairs of 27–32-base outer primers and 20–21-base inner primers and a 20–22-base probe were designed to amplify and detect a 200–221-base sequence by dot blotting using the labelled probe. Sensitivity of the assays was 0.01–10 fg using chromosomal DNA and ⩽ 1 viable cell using DNA extracted from exponential-phase bacteria. Each serotype-specific assay detected chromosomal DNA from all of five to ten clinical isolates of the homologous type and did not detect DNA sequences from any of 190–204 strains from 51–52 different serotypes or 28 non-pneumococcal bacterial strains. Sixteen throat swabs from children that had been cultured for S. pneumoniae were tested in PCR assays following DNA extraction. All of six that grew S. pneumoniae serotype 3, 14, 19F or 23F were positive in the PCR assay for the homologous serotype (and in a PCR assay for sequences in lytA, present in all pneumococci) and were negative in assays for other serotypes. Of eight culture-negative specimens in children not receiving antimicrobials, three were positive for both the lytA assay and an assay for one of the four serotypes, suggesting true positive results; in three others all five PCR assays were negative and, in the remaining two, the lytA assay was positive but each of the four assays for individual serotypes was negative, suggesting either false-positive results or presence of DNA sequences from an S. pneumoniae serotype other than 3, 14, 19F or 23F. These preliminary clinical data suggest that these PCR-based assays are sensitive and specific for detection of individual serotypes of pneumococci and may be used with respiratory tract specimens.
Characterization of Pseudomonas putida genes responsive to nutrient limitation
Authors: Syn Chris K. C. , Magnuson Jon K. , Kingsley Mark T. , Swarup Sanjay
Published: June 2004

The low bioavailability of nutrients and oxygen in the soil environment has hampered successful expression of biodegradation and biocontrol genes that are driven by promoters highly active during routine laboratory conditions of high availability of nutrients and oxygen. Hence, in the present study, expression of thegus-tagged genes in 12 Tn5-gusmutants of the soil microbePseudomonas putidaPNL-MK25 were examined under various conditions chosen to mimic the soil environment: low carbon, phosphate, nitrate or oxygen, and in the rhizosphere. Based on their expression profiles, three nutrient-responsive mutant (NRM) strains, NRM5, NRM7 and NRM17, were selected for identification of the tagged genes. In strain NRM5, expression of the glutamate dehydrogenase (gdhA) gene was increased 4·9–26·4-fold under various low-nutrient conditions. In NRM7, expression of the novel NADPH : quinone oxidoreductase-like (nql) gene was consistently amongst the highest and was synergistically upregulated by low-nutrient and anoxic conditions. ThecyoDgene in NRM17, which encodes the fourth subunit of the cytochromeoubiquinol oxidase complex, had decreased expression in low-nutrient conditions but its absolute expression level was still amongst the highest. Additionally, it was independent of oxygen availability, in contrast to that inEscherichia coli.
The transcription of the cbb operon in Nitrosomonas europaea
Authors: Wei Xueming , Sayavedra-Soto Luis A. , Arp Daniel J.
Published: June 2004

Nitrosomonas europaeais an aerobic ammonia-oxidizing bacterium that participates in the C and N cycles.N. europaeautilizes CO2as its predominant carbon source, and is an obligate chemolithotroph, deriving all the reductant required for energy and biosynthesis from the oxidation of ammonia (NH3) to nitrite (). This bacterium fixes carbon via the Calvin–Benson–Bassham (CBB) cycle via a type I ribulose bisphosphate carboxylase/oxygenase (RubisCO). The RubisCO operon is composed of five genes,cbbLSQON. This gene organization is similar to that of the operon for ‘green-like’ type I RubisCOs in other organisms. ThecbbRgene encoding the putative regulatory protein for RubisCO transcription was identified upstream ofcbbL. This study showed that transcription ofcbbgenes was upregulated when the carbon source was limited, whileamo,haoand other energy-harvesting-related genes were downregulated.N. europaearesponds to carbon limitation by prioritizing resources towards key components for carbon assimilation. Unlike the situation foramogenes, NH3was not required for the transcription of thecbbgenes. All fivecbbgenes were only transcribed when an external energy source was provided. In actively growing cells, mRNAs from the five genes in the RubisCO operon were present at different levels, probably due to premature termination of transcription, rapid mRNA processing and mRNA degradation.
Oxidative and amphotericin B-mediated cell death in the opportunistic pathogen Aspergillus fumigatus is associated with an apoptotic-like phenotype
Authors: Mousavi S. Amin A. , Robson Geoffrey D.
Published: June 2004

When protoplasts of the opportunistic fungal pathogen Aspergillus fumigatus were treated with low but toxic levels of hydrogen peroxide (0·1 mM) or amphotericin B (0·5 μg ml−1), loss of cell viability and death were associated with a number of phenotypic changes characteristic of apoptosis. The percentage of protoplasts staining positive with annexin V-FITC, an indicator of the externalization of phosphatidylserine and an early marker of apoptosis, rose to ∼55 % within 1 h. This was followed by a similar increase in apoptotic DNA fragmentation detected by the TUNEL assay, and led to a loss of cell permeability and death in ∼90 % of protoplasts, as indicated by the uptake of propidium iodide. The development of an apoptotic phenotype was blocked when protoplasts were pre-treated with the protein synthesis inhibitor cycloheximide, indicating active participation of the cell in the process. However, no significant activity against synthetic caspase substrates was detected, and the inclusion of the cell-permeant broad-spectrum caspase inhibitor Z-VAD-fmk did not block the development of the apoptotic-like phenotype. Higher concentrations of H2O2 (1·8 mM) and amphotericin B (1 μg ml−1) caused protoplasts to die without inducing an apoptotic phenotype. As predicted, the fungistatic antifungal agent itraconazole, which inhibits growth without causing immediate cell death, did not induce an apoptotic-like phenotype.
Specific growth rate and not cell density controls the general stress response in Escherichia coli
Authors: Ihssen Julian , Egli Thomas
Published: June 2004

In batch cultures ofEscherichia coli, the intracellular concentration of the general stress response sigma factor RpoS typically increases during the transition from the exponential to the stationary growth phase. However, because this transition is accompanied by complex physico-chemical and biological changes, which signals predominantly elicit this induction is still the subject of debate. Careful design of the growth environment in chemostat and batch cultures allowed the separate study of individual factors affecting RpoS. Specific growth rate, and not cell density or the nature of the growth-limiting nutrient, controlled RpoS expression and RpoS-dependent hydroperoxidase activity. Furthermore, it was demonstrated that the standardE. coliminimal medium A (MMA) is not suitable for high-cell-density cultivation because it lacks trace elements. Previously reported cell-density effects in chemostat cultures ofE. colican be explained by a hidden, secondary nutrient limitation, which points to the importance of medium design and appropriate experimental set-up for studying cell-density effects.
AgmR controls transcription of a regulon with several operons essential for ethanol oxidation in Pseudomonas aeruginosa ATCC 17933
Authors: Gliese Nicole , Khodaverdi Viola , Schobert Max , Görisch Helmut
Published: June 2004

The response regulator AgmR was identified to be involved in the regulation of the quinoprotein ethanol oxidation system of Pseudomonas aeruginosa ATCC 17933. Interruption of the agmR gene by insertion of a kanamycin-resistance cassette resulted in mutant NG3, unable to grow on ethanol. After complementation with the intact agmR gene, growth on ethanol was restored. Transcriptional lacZ fusions were used to identify four operons which are regulated by the AgmR protein: the exaA operon encodes the pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the exaBC operon encodes a soluble cytochrome c
550 and an aldehyde dehydrogenase, the pqqABCDE operon carries the PQQ biosynthetic genes, and operon exaDE encodes a two-component regulatory system which controls transcription of the exaA operon. Transcription of exaA was restored by transformation of NG3 with a pUCP20T derivative carrying the exaDE genes under lac-promoter control. These data indicate that the AgmR response regulator and the exaDE two-component regulatory system are organized in a hierarchical manner. Gene PA1977, which appears to form an operon with the agmR gene, was found to be non-essential for growth on ethanol.
Sequence typing reveals extensive strain diversity of the Lyme borreliosis agents Borrelia burgdorferi in North America and Borrelia afzelii in Europe
Authors: Bunikis Jonas , Garpmo Ulf , Tsao Jean , Berglund Johan , Fish Durland , Barbour Alan G.
Published: June 2004

The genetic polymorphism ofBorrelia burgdorferiandBorrelia afzelii, two species that cause Lyme borreliosis, was estimated by sequence typing of four loci: therrs–rrlAintergenic spacer (IGS) and the outer-membrane-protein genep66on the chromosome, and the outer-membrane-protein genesospAandospCon plasmids. The major sources of DNA for PCR amplification and sequencing were samples of theB. burgdorferitick vectorIxodes scapularis, collected at a field site in an endemic region of the north-eastern United States, and theB. afzeliivectorIxodes ricinus, collected at a similar site in southern Sweden. The sequences were compared with those of reference strains and skin biopsy isolates, as well as database sequences. ForB. burgdorferi, 10–13 alleles for each of the 4 loci, and a total of 9 distinct clonal lineages with linkage of all 4 loci, were found. ForB. afzelii, 2 loci,ospCand IGS, were examined, and 11 IGS genotypes, 12ospCalleles, and a total of 9 linkage groups were identified. The genetic variants ofB. burgdorferiandB. afzeliiamong samples from the field sites accounted for the greater part of the genetic diversity previously reported from larger areas of the north-eastern United States and central and northern Europe. AlthoughospCalleles of both species had higher nucleotide diversity than other loci, theospClocus showed evidence of intragenic recombination and was unsuitable for phylogenetic inference. In contrast, there was no detectable recombination at the IGS locus ofB. burgdorferi. Moreover, beyond the signature nucleotides that specified 10 IGS genotypes, there were additional nucleotide polymorphisms that defined a total of 24 subtypes. Maximum-likelihood and parsimony cladograms ofB. burgdorferialigned IGS sequences revealed the subtype sequences to be terminal branches of clades, and the existence of at least three monophyletic lineages withinB. burgdorferi. It is concluded thatB. burgdorferiandB. afzeliihave greater genetic diversity than had previously been estimated, and that the IGS locus alone is sufficient for strain typing and phylogenetic studies.
luxS mutants of Serratia defective in autoinducer-2-dependent ‘quorum sensing’ show strain-dependent impacts on virulence and production of carbapenem and prodigiosin
Authors: Coulthurst Sarah J. , Kurz C. Léopold , Salmond George P. C.
Published: June 2004

The enzyme LuxS is responsible for the production of autoinducer-2 (AI-2), a molecule that has been implicated in quorum sensing in many bacterial species. This study investigated whether there is aluxS-dependent signalling system in the Gram-negative bacteriaSerratiaspp.Serratia marcescensis a broad-host-range pathogen and an important cause of nosocomial infections. Production of AI-2 activity was detected inS. marcescensATCC 274 andSerratiaATCC 39006 and theirluxSgenes were sequenced.luxSmutants were constructed in these strains and were analysed to determine which phenotypes are regulated byluxSand therefore, potentially, by AI-2. The phenotypes of theluxSmutants included decreased carbapenem antibiotic production inSerratiaATCC 39006 and decreased prodigiosin and secreted haemolysin production inS. marcescensATCC 274. TheluxSmutant ofS. marcescensATCC 274 was also found to exhibit modestly reduced virulence in aCaenorhabditis elegansmodel. Finally, it was shown that the culture supernatant of a wild-type strain contains a signal, presumably AI-2, capable of complementing the prodigiosin defect of theluxSmutant of another strain, even when substantially diluted. It is concluded thatluxSmodulates virulence and antibiotic production inSerratia, in a strain-dependent manner, and that, for at least one phenotype, this regulation is via extracellular signalling.
The replication-related organization of bacterial genomes
Authors: Rocha Eduardo P. C.
Published: June 2004

The replication of the chromosome is among the most essential functions of the bacterial cell and influences many other cellular mechanisms, from gene expression to cell division. Yet the way it impacts on the bacterial chromosome was not fully acknowledged until the availability of complete genomes allowed one to look upon genomes as more than bags of genes. Chromosomal replication includes a set of asymmetric mechanisms, among which are a division in a lagging and a leading strand and a gradient between early and late replicating regions. These differences are the causes of many of the organizational features observed in bacterial genomes, in terms of both gene distribution and sequence composition along the chromosome. When asymmetries or gradients increase in some genomes, e.g. due to a different composition of the DNA polymerase or to a higher growth rate, so do the corresponding biases. As some of the features of the chromosome structure seem to be under strong selection, understanding such biases is important for the understanding of chromosome organization and adaptation. Inversely, understanding chromosome organization may shed further light on questions relating to replication and cell division. Ultimately, the understanding of the interplay between these different elements will allow a better understanding of bacterial genetics and evolution.
Characterization of the Streptomyces lavendulae IMRU 3455 linear plasmid pSLV45
Authors: Hosted Thomas J. , Wang Tim , Horan Ann C.
Published: June 2004

Streptomyces lavendulae IMRU 3455 contains two large linear plasmids designated pSLV45 (45 kb) and pSLV195 (195 kb). A cosmid, pSPRX604, containing 42 kb from pSLV45 was cloned and sequenced. pSLV45 was tagged with a hygromycin-resistance marker by homologous recombination to generate the derivatives pSLV45.680 and pSLV45.681. An apramycin-resistance marker was introduced into S. lavendulae IMRU 467 using the pSPR910 integration vector to yield the recipient strain SPW910. The self-transmissible nature of pSLV45 was determined by transfer of pSLV45.680 and pSLV45.681 from the donor strains SPW680 and SPW681 into the recipient strain SPW910. Southern analysis indicated the presence of hygromycin- and pSLV45-hybridizing sequences within SPW910 exconjugants. PFGE analysis confirmed pSLV45.680 and pSLV45.681 were transferred intact and formed freely replicating linear plasmids. Sequence analysis of pSPRX604 revealed genes predicted to be involved in plasmid transfer, partitioning and regulation. The transfer of the linear plasmid pSLV45 from S. lavendulae IMRU 3455 into S. lavendulae IMRU 467 may allow the development of pSLV45 as an actinomycete-to-actinomycete conjugative shuttle vector.
FpvB, an alternative type I ferripyoverdine receptor of Pseudomonas aeruginosa
Authors: Ghysels Bart , Dieu Bui Thi Min , Beatson Scott A. , Pirnay Jean-Paul , Ochsner Urs A. , Vasil Michael L. , Cornelis Pierre
Published: June 2004

Under conditions of iron limitation, Pseudomonas aeruginosa secretes a high-affinity siderophore pyoverdine to scavenge Fe(III) in the extracellular environment and shuttle it into the cell. Uptake of the pyoverdine–Fe(III) complex is mediated by a specific outer-membrane receptor protein, FpvA (ferripyoverdine receptor). Three P. aeruginosa siderovars can be distinguished, each producing a different pyoverdine (type I–III) and a cognate FpvA receptor. Growth of an fpvA mutant of P. aeruginosa PAO1 (type I) under iron-limiting conditions can still be stimulated by its cognate pyoverdine, suggesting the presence of an alternative uptake route for type I ferripyoverdine. In silico analysis of the PAO1 genome revealed that the product of gene PA4168 has a high similarity with FpvA. Inactivation of PA4168 (termed fpvB) in an fpvA mutant totally abolished the capacity to utilize type I pyoverdine. The expression of fpvB is induced by iron limitation in Casamino acids (CAA) and in M9-glucose medium, but, unlike fpvA, not in a complex deferrated medium containing glycerol as carbon source. The fpvB gene was also detected in other P. aeruginosa isolates, including strains producing type II and type III pyoverdines. Inactivation of the fpvB homologues in these strains impaired their capacity to utilize type I ferripyoverdine as a source of iron. Accordingly, introduction of fpvB
in trans restored the capacity to utilize type I ferripyoverdine.
Branched swarming patterns on a synthetic medium formed by wild-type Bacillus subtilis strain 3610: detection of different cellular morphologies and constellations of cells as the complex architecture develops
Authors: Julkowska Daria , Obuchowski Michal , Holland I. Barry , Séror Simone J.
Published: June 2004

After optimizing the conditions, including nutrients and temperature, swarming of Bacillus subtilis 3610 was obtained on a synthetic, fully defined medium. The swarms formed highly branched (dendritic) patterns, generated by successive waves of moving cells. A detailed microscopic in situ analysis of swarms 1 and 2 revealed varied cell morphologies and a remarkable series of events, with cells assembling into different ‘structures’, as the architecture of the swarm developed. Long filamentous cells begin to form before the onset of the first swarming (11 h) and are again observed at later stages in the interior of individual mature dendrites. Swarm 2, detected at 18–22 h, is accompanied by the rapid movement of a wave of dispersed (non-filamentous) cells. Subsequently at the forward edge of this swarm, individual cells begin to cluster together, gradually forming de novo the shape of a dendrite tip with progressive lengthening of this new structure ‘backwards' towards the swarm centre. In both swarms 1 and 2, after the initial clustering of cells, there is the progressive appearance of a spreading monolayer of rafts (4–5 non-filamented cells, neatly aligned). The alternative possible roles of the rafts in the development of the swarm are discussed.