bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel domain in the protein kinase SOS2 mediates interaction with the protein phosphatase 2C ABI2
Authors: Ohta Masaru , Guo Yan , Halfter Ursula , Zhu Jian-Kang
Published: October 2003

SOS2 (salt overly sensitive 2) is a serine/threonine protein kinase required for salt tolerance inArabidopsis thaliana. In this study, we identified the protein phosphatase 2C ABI2 (abscisic acid-insensitive 2) as a SOS2-interacting protein. Deletion analysis led to the discovery of a novel protein domain of 37 amino acid residues, designated as the protein phosphatase interaction (PPI) motif, of SOS2 that is necessary and sufficient for interaction with ABI2. The PPI motif is conserved in protein kinases of the SOS2 family (i.e., protein kinase S, PKS) and in the DNA damage repair and replication block checkpoint kinase, Chk1, from various organisms including humans. Mutations in the conserved amino acid residues in the PPI motif abolish the interaction of SOS2 with ABI2. We also identified a protein kinase interaction domain in ABI2 and examined the interaction specificity between PKS and the ABI phosphatases. We found that some PKSs interact strongly with ABI2 whereas others interact preferentially with ABI1. The interaction between SOS2 and ABI2 was disrupted by theabi2-1mutation, which causes increased tolerance to salt shock and abscisic acid insensitivity in plants. Our results establish the PPI motif and the protein kinase interaction domain as novel protein interaction domains that mediate the binding between the SOS2 family of protein kinases and the ABI1/2 family of protein phosphatases.
Expression of the type 2 receptor for cysteinyl leukotrienes (CysLT2R) by human mast cells: Functional distinction from CysLT1R
Authors: Mellor Elizabeth A. , Frank Nita , Soler Dulce , Hodge Martin R. , Lora Jose M. , Austen K. Frank , Boyce Joshua A.
Published: October 2003

Cysteinyl leukotrienes (cysLTs) mediate vascular leakage and bronchoconstriction through the smooth muscle-associated CysLT type 1 receptor (CysLT1R), one of at least two loosely homologous cysLT-binding G protein-coupled receptors. We previously reported that CysLT1R is expressed by cultured human mast cells (hMCs), and that priming these cells with IL-4 enhances their sensitivity to calcium flux and cytokine generation in response to cys-LTs and the nucleotide ligand, uridine diphosphate (UDP), without increasing their surface expression of CysLT1R. We now report that hMCs express the type 2 receptor for cysLTs (CysLT2R) as well, and that the amount of surface CysLT2R protein increases in response to priming with IL-4. The selective function of CysLT2R was evident based on uninhibited IL-8 secretion by IL-4-primed hMCs stimulated with cys-LTs or UDP in the presence of the selective CysLT1R antagonist MK571. MK571 did inhibit IL-5 generation, calcium flux, and phosphorylation of extracellular signal-regulated kinase. IL-8 secretion was inhibited by pertussis toxin and a selective p38 kinase inhibitor, SB203580. The CysLT2 response may permit the cys-LTs and nucleotides generated in infection and tissue injury to elicit IL-8 generation by hMCs, potentially leading to neutrophilic infiltration, a characteristic of aerosol challenge-induced late-phase responses and of sudden death associated with asthma.
Measuring stem cell frequency in epidermis: A quantitativein vivofunctional assay for long-term repopulating cells
Authors: Schneider T. E. , Barland C. , Alex A. M. , Mancianti M. L. , Lu Y. , Cleaver J. E. , Lawrence H. J. , Ghadially R.
Published: October 2003

Epidermal stem cells play a central role in tissue homeostasis, wound repair, tumor initiation, and gene therapy. A major impediment to the purification and molecular characterization of epidermal stem cells is the lack of a quantitative assay for cells capable of long-term repopulationin vivo, such as exists for hematopoietic cells. The tremendous strides made in the characterization and purification of hematopoietic stem cells have been critically dependent on the availability of competitive transplantation assays, because these assays permit the accurate quantitation of long-term repopulating cellsin vivo. We have developed an analogous functional assay for epidermal stem cells, and have measured the frequency of functional epidermal stem cells in interfollicular epidermis. These studies indicate that cells capable of long-term reconstitution of a squamous epithelium reside in the interfollicular epidermis. We find that the frequency of these long-term repopulating cells is 1 in 35,000 total epidermal cells, or in the order of 1 in 104basal epidermal cells, similar to that of hematopoietic stem cells in the bone marrow, and much lower than previously estimated in epidermis. Furthermore, these studies establish a novel functional assay that can be used to validate immunophenotypic markers and enrichment strategies for epidermal stem cells, and to quantify epidermal stem cells in various keratinocyte populations. Thus further studies using this type of assay for epidermis should aid in the progress of cutaneous stem cell-targeted gene therapy, and in more basic studies of epidermal stem cell regulation and differentiation.
Interference with T cell receptor–HLA-DR interactions by Epstein–Barr virus gp42 results in reduced T helper cell recognition
Authors: Ressing Maaike E. , van Leeuwen Daphne , Verreck Frank A. W. , Gomez Raquel , Heemskerk Bianca , Toebes Mireille , Mullen Maureen M. , Jardetzky Theodore S. , Longnecker Richard , Schilham Marco W. , Ottenhoff Tom H. M. , Neefjes Jacques , Schumacher Ton N. , Hutt-Fletcher Lindsey M. , Wiertz Emmanuel J. H. J.
Published: October 2003

Epstein–Barr virus (EBV) persists lifelong in infected hosts despite the presence of antiviral immunity. Many viral antigens are expressed during lytic infection. Thus, for EBV to spread, it must have evolved effective ways to evade immune recognition. Here, we report that HLA class II-restricted antigen presentation to T helper cells is hampered in the presence of the lytic-phase protein gp42. This interference with T cell activation involves association of gp42 with class II peptide complexes. Using HLA-DR tetramers, we identify a block in T cell receptor (TCR)–class II interactions imposed by gp42 as the underlying mechanism. EBV gp42 sterically clashes with TCR Vα-domains as visualized by superimposing the crystal structures for gp42–HLA-DR1 and TCR–MHC class II complexes. Blocking TCR recognition provides a previously undescribed strategy for viral immune evasion.
Two CD95 tumor classes with different sensitivities to antitumor drugs
Authors: Algeciras-Schimnich Alicia , Pietras Eric M. , Barnhart Bryan C. , Legembre Patrick , Vijayan Shrijay , Holbeck Susan L. , Peter Marcus E.
Published: October 2003

CD95 type I and II cells differ in their dependence on mitochondria to execute apoptosis, because antiapoptotic members of the Bcl-2 family render only type II cells resistant to death receptor-induced apoptosis. They can also be distinguished by a more efficient formation of the death-inducing signaling complex in type I cells. We have identified a soluble form of CD95 ligand (S2) that is cytotoxic to type II cells but does not kill type I cells. By testing 58 tumor cell lines of the National Cancer Institute's anticancer drug-screening panel for apoptosis sensitivity to S2 and performing death-inducing signaling complex analyses, we determined that half of the CD95-sensitive cells are type I and half are type II. Most of the type I cell lines fall into a distinct class of tumor cells expressing mesenchymal-like genes, whereas the type II cell lines preferentially express epithelium-like markers. This suggests that type I and II tumor cells represent different stages of carcinogenesis that resemble the epithelial–mesenchymal transition. We then screened the National Cancer Institute database of >42,000 compounds for reagents with patterns of growth inhibition that correlated with either type I or type II cell lines and found that actin-binding compounds selectively inhibited growth of type I cells, whereas tubulin-interacting compounds inhibited growth of type II cells. Our analysis reveals fundamental differences in programs of gene expression between type I and type II cells and could impact the way actin- and microtubule-disrupting antitumor agents are used in tumor therapy.
A genomewide screen in
Saccharomyces cerevisiae
for genes that suppress the accumulation of mutations
Authors: Huang Meng-Er , Rio Anne-Gaëlle , Nicolas Alain , Kolodner Richard D.
Published: October 2003

A genomewide screen of a collection of 4,847 yeast gene deletion mutants was carried out to identify the genes required for suppressing mutations in the
CAN1
forward-mutation assay. The primary screens and subsequent analysis allowed (
i
) identification of 18 known mutator mutants, providing a solid means for checking the efficiency of the screen, and (
ii
) identification of a number of genes not known previously to be involved in suppressing mutations. Among the previously uncharacterized mutation-suppressing genes were six genes of unknown function including four (
CSM2
,
SHU2
,
SHU1
, and
YLR376c
) encoding proteins that interact with each other and promote resistance to killing by methyl methanesulfonate, one gene (
EGL1
) previously identified as suppressing Ty1 mobility and recombination between repeated sequences, and one gene (
YLR154c
) that was not associated with any known processes. In addition, five genes (
TSA1
,
SOD1
,
LYS7
,
SKN7
, and
YAP1
) implicated in the oxidative-stress responses were found to play a significant role in mutation suppression. Furthermore,
TSA1
, which encodes thioredoxin peroxidase, was found to strongly suppress gross chromosomal rearrangements. These results provide a global view of the nonessential genes involved in preventing mutagenesis. Study of such genes should provide useful clues in identification of human genes potentially involved in cancer predisposition and in understanding their mechanisms of action.
IL-3 receptor signaling is dispensable for BCR-ABL-induced myeloproliferative disease
Authors: Wong Stephane , McLaughlin Jami , Cheng Donghui , Shannon Kevin , Robb Lorraine , Witte Owen N.
Published: October 2003

BCR-ABL expression led to a dramatic up-regulation of the IL-3, IL-5, and granulocyte–macrophage colony-stimulating factor receptor β common (IL-3Rβc) and IL-3 receptor β (IL-3Rβ) chains in murine embryonic stem cell-derived hematopoietic cells coincident with an expansion of multipotent progenitors and myeloid elements. This up-regulation required BCR-ABL tyrosine kinase activity and led to IL-3Rβc/β chain tyrosine phosphorylation in the absence of detectable IL-3 production. These results suggested that cytokine-independent IL-3 receptor activation could be a dominant signaling component in BCR-ABL-induced leukemogenesis. To unambiguously define the significance of IL-3 receptor-dependent signaling in BCR-ABL-induced leukemogenesis, BCR-ABL-transduced bone marrow cells deficient in either IL-3Rβc chain or both IL-3Rβc/β chain expression were examined for their ability in generating myeloproliferative disease (MPD). These BCR-ABL-expressing knockout cells were capable of generating MPD similar to control cells, demonstrating that IL-3 receptor activation is not essential for BCR-ABL-induced MPD. However, the IL-3Rβc/β chain could act as a cofactor in BCR-ABL-induced leukemogenesis by activation of its many known oncogenic signaling pathways.
An early modern human from the Peştera cu Oase, Romania
Authors: Trinkaus Erik , Moldovan Oana , Milota ştefan , Bîlgăr Adrian , Sarcina Laurenţiu , Athreya Sheela , Bailey Shara E. , Rodrigo Ricardo , Mircea Gherase , Higham Thomas , Ramsey Christopher Bronk , van der Plicht Johannes
Published: October 2003

The 2002 discovery of a robust modern human mandible in the Peştera cu Oase, southwestern Romania, provides evidence of early modern humans in the lower Danubian Corridor. Directly accelerator mass spectrometry radiocarbon (
14
C)-dated to 34,000–36,000
14
C years B.P., the Oase 1 mandible is the oldest definite early modern human specimen in Europe and provides perspectives on the emergence and evolution of early modern humans in the northwestern Old World. The moderately long Oase 1 mandible exhibits a prominent tuber symphyseos and overall proportions that place it close to earlier Upper Paleolithic European specimens. Its symmetrical mandibular incisure, medially placed condyle, small superior medial pterygoid tubercle, mesial mental foramen, and narrow corpus place it closer to early modern humans among Late Pleistocene humans. However, its cross-sectional symphyseal orientation is intermediate between late archaic and early modern humans, the ramus is exceptionally wide, and the molars become progressively larger distally with exceptionally large third molars. The molar crowns lack derived Neandertal features but are otherwise morphologically undiagnostic. However, it has unilateral mandibular foramen lingular bridging, an apparently derived Neandertal feature. It therefore presents a mosaic of archaic, early modern human and possibly Neandertal morphological features, emphasizing both the complex population dynamics of modern human dispersal into Europe and the subsequent morphological evolution of European early modern humans.
Impact of the health and living conditions of migrant and non-migrant Senegalese adolescent girls on their nutritional status and growth
Authors: Garnier Denis , Simondon Kirsten B , Hoarau Thierry , Benefice Eric
Published: October 2003

Objective:

To describe the living conditions of Senegalese adolescent girls according to their migration status, and to define the main socio-economic and biological determinants of their nutritional and growth status.

Design:

Health and living conditions, sexual maturation, and nutritional and growth status of adolescent girls were determined within the framework of a longitudinal study on growth.

Settings:

The capital city of Senegal (Dakar) and a rural community (Niakhar), 120 km south-east of Dakar.

Subjects:

Three hundred and thirty-one girls, 14.5–16.6 years of age, were recruited from the same villages. Thirty-six per cent of the sample remained in the villages to attend school and/or to help with household subsistence tasks (non-migrants). The remaining (64%) migrated to cities to work as maids (migrants) and lived in two different socio-economic environments: at the home of a guardian during the night and in the house of the employer during the daytime.

Results:

Family rural environment and guardian and employer urban environments were socio-economically different (P < 0.001). Living conditions in urban areas were better than in rural areas and the employer's environment was socio-economically more favourable. Migrants had more advanced sexual maturation and higher body mass index (BMI), fat mass index (FMI) and mid-upper arm circumference than non-migrants. However, migrants were smaller than non-migrants. BMI, FMI and weight-for-age were related to socio-economic levels and duration of migration. Schooling was positively related to height and negatively related to BMI.

Conclusions:

Migrants enjoyed better living conditions than non-migrants. This could be partly due to the better socio-economic environment of the employer. It was associated with earlier puberty and better nutritional status, but not with a better growth.
A Belgian study on the reliability and relative validity of the Health Behaviour in School-Aged Children food-frequency questionnaire
Authors: Vereecken Carine A , Maes Lea
Published: October 2003

Objective:

In the Health Behaviour in School-Aged Children (HBSC) survey, the frequency of consumption of a limited number of food items – focusing on fibre, calcium and less healthy items – is queried using a 15-item food-frequency questionnaire (FFQ). The present study was conducted to assess the reliability and relative validity of the HBSC FFQ among school pupils in Belgium.

Design and subjects:

To assess the reliability of the FFQ, 207 pupils aged 11–12 years and 560 pupils aged 13–14 years completed the questionnaire twice, with a test–retest interval of 6 to 15 days. To assess the relative validity of the FFQ, in a first study data were collected as part of the Flemish HBSC 2000 survey: 7072 pupils (11–18 years) completed the FFQ and a 24-hour food behaviour checklist (FBC). In a second study, 101 pupils (11–12 years) completed the FFQ and a 7-day food diary (FD).

Results:

Reliability – weighted kappa values between test and retest ranged from 0.43 to 0.70, percentage agreement from 37 to 87%, and Spearman correlations from 0.52 to 0.82. Relative validity – comparison of the FBC with the percentage of respondents who should have consumed the food items on a random day, computed from the FFQ, showed good agreement between the FFQ and the FBC for most items. Only for cereals, diet soft drinks and other milk products were considerably higher food frequencies than expected found from the FBC. Comparison of the FFQ with the FD showed overestimation for all but three food items (cheese, soft drinks and chips). Spearman correlations ranged between 0.10 for crisps and 0.65 for semi-skimmed milk.

Conclusion:

The HBSC FFQ is a reliable questionnaire that can be used for ranking subjects for most food items, although one must consider the overestimation when the FFQ is used for estimating prevalences.
A conceptual framework for intelligence-based public health nutrition workforce development
Authors: Hughes Roger
Published: October 2003

Objective:

This paper describes a conceptual framework and associated intelligence requirements for problem-based workforce development for public health nutrition.

Methods:

A conceptual framework for public health nutrition workforce development was constructed based on a review of the literature and consideration of the intelligence needs to inform workforce development planning.

Results:

A cyclical conceptual framework including five intelligence-linked components including public health nutrition problems and priorities, solutions and best buys, work needed, capacity to do the work and workforce development needs. This framework applied to the Australian situation illustrates its applications in workforce development research and planning. Although the existing availability of workforce development intelligence in each of these components varies, the framework does provide a systematic approach for workforce development research and planning directly related to public health nutrition problem resolution.

Conclusions:

This framework highlights deficiencies in the existing public health nutrition workforce development intelligence and the need for further research to inform workforce development strategy planning.
A comparison between the National Food Survey and the Family Expenditure Survey food expenditure data
Authors: Paterakis Sophia E , Nelson Michael
Published: October 2003

Objective:

Household budget surveys (HBSs) have been used to assess nutritional information for epidemiological purposes. The agreement between this information and other comparable data needs to be examined. The aim of this project was to compare household food expenditure data between two British HBSs: the National Food Survey (NFS) and the Family Expenditure Survey (FES).

Design:

Household food expenditure data were compared between the NFS and the FES for the years from 1982 to 1993. Differences in expenditure were assessed by year, by household composition, by income group and by region; for trends across time for all households and for regional, household composition and income group variations.

Setting:

Great Britain.

Subjects:

Approximately 88 000 NFS households and 85 000 FES households surveyed between 1982 and 1993 were used in this analysis.

Results:

Marked differences between the food expenditure data provided by the two surveys were observed. Furthermore, differences in time trends were substantial, which can lead to different conclusions regarding changes in consumption patterns.

Conclusions:

There is no obvious reason for the differences in household food expenditure between the NFS and the FES. Methodological differences between the two surveys cannot provide a full explanation for these discrepancies. The NFS and FES are now merged into a single survey (the Expenditure and Food Survey). If HBSs are to be used for epidemiological purposes their validity needs to be established.
Evaluation of the impact of an iodine supplementation programme on severely iodine-deficient schoolchildren with hypothyroidism
Authors: Salarkia Nahid , Hedayati Mehdi , Mirmiran Parvin , Kimiagar Masood , Azizi Fereidoun
Published: October 2003

Background:

Northern rural areas of Tehran have been shown to have severe iodine deficiency in our previous studies. In 1989 the inhabitants of these villages received an injection of iodised oil, followed by iodised salt distribution in 1993. The aim of the present study was to evaluate the effect of the iodine supplementation on iodine-deficient schoolchildren with hypothyroidism in these villages.

Methods:

In total, 571 students aged 6–14 years were studied. Goitre was graded according to the World Health Organization classification. Serum concentrations of thyroid hormones (thyroxine (T4) and triiodothyronine (T3)) and thyroid-stimulating hormone (TSH) were determined using commercial kits, and urinary iodine was measured using a digestion method. The results were compared with data from our previous study in 1989.

Results:

Total goitre rate decreased by 42% in 1999 compared with that in 1989. A significant decrease in Grade 2 goitre concomitant with an increase in Grade 1 goitre was seen (P < 0.001). Values of the variables studied before (1989) and 10 years after iodine supplementation (1999) were: median urinary iodine excretion, 2.0 vs. 19.0 μg dl−1 (P < 0.001); T4, 6.5 ± 2.0 vs. 8.4 ± 1.6 μg dl−1 (P < 0.001); T3, 177 ± 38.0 vs. 145 ± 29.0 ng dl−1 (P < 0.001); TSH, 10.8 ± 15.1 vs. 1.8 ± 0.8 μU ml−1 (P < 0.001). No correlation was found between thyroid hormones and TSH on the one hand, and goitre and urinary iodine, on the other. Serum T4, T3 and TSH concentrations were within normal ranges in all schoolchildren in 1999.

Conclusion:

This study showed that euthyroidism induced by administration of iodised oil in iodine-deficient schoolchildren with hypothyroidism is sustained following the consumption of iodised salt.
Definitions for public health nutrition: a developing consensus
Authors: Hughes Roger
Published: October 2003

Objectives:

To assess the level of agreement amongst a panel of public health nutrition leaders regarding the key descriptors used to define the field of public health nutrition.

Design:

Cross-sectional survey requiring quantitative and qualitative responses representing the first round of a consensus development Delphi technique.

Setting:

International.

Subjects:

Expert panel of 24 public health nutrition leaders from nine countries in the European Union, the USA and Australia.

Results:

All but one of the panel agreed it was important to have a consensus definition for public health nutrition to describe the field consistently. Opinion about the length and complexity of this definition tended to vary depending on the intended use of the definition. The large majority (18/24) supported the inclusion of specific reference to physical activity in a definition of public health nutrition, although there was not consensus (>83% agreement as criterion) on this point. Consensus descriptors regarded as important in a definition of public health nutrition included: population-based, focus on health promotion, food and nutrition systems focus, wellness maintenance, primary prevention, applies public health principles, education, environmental and political descriptors. Treatment as a descriptor was rated as unimportant by a majority (14/24) of panellists, delineating public health nutrition from clinical practice.

Conclusions:

There is strong international agreement amongst public health nutrition leaders in Europe, the USA and Australia about a range of descriptors that can be used to define public health nutrition. The limitations of using word-for-word definitions between and within countries may be overcome by explicit use of the consensus descriptors identified in this process.
The relationship between low income and household food expenditure patterns in Canada
Authors: Kirkpatrick Sharon , Tarasuk Valerie
Published: October 2003

Objectives:

To compare food expenditure patterns between low-income households and higher- income households in the Canadian population, and to examine the relationship between food expenditure patterns and the presence or absence of housing payments among low-income households.

Design:

Secondary data analysis of the 1996 Family Food Expenditure Survey conducted by Statistics Canada.

Setting:

Sociodemographic data and 1-week food expenditure data for 9793 households were analysed.

Subjects:

Data were collected from a nationally representative sample drawn through stratified multistage sampling. Low-income households were identified using Statistics Canada's Low Income Measures.

Results:

Total food expenditures, expenditures at stores and expenditures in restaurants were lower among low-income households compared with other households. Despite allocating a slightly greater proportion of their food dollars to milk products, low-income households purchased significantly fewer servings of these foods. They also purchased fewer servings of fruits and vegetables than did higher-income households. The effect of low income on milk product purchases persisted when the sample was stratified by education and expenditure patterns were examined in relation to income within strata. Among low-income households, the purchase of milk products and meat and alternatives was significantly lower for households that had to pay rents or mortgages than for those without housing payments.

Conclusions:

Our findings indicate that, among Canadian households, access to milk products and fruits and vegetables may be constrained in the context of low incomes. This study highlights the need for greater attention to the affordability of nutritious foods for low-income groups.
Identification and characterization of a fibronectin-binding protein from Clostridium difficile
Authors: Hennequin Claire , Janoir Claire , Barc Marie-Claude , Collignon Anne , Karjalainen Tuomo
Published: October 2003

A 68 kDa fibronectin-binding protein (Fbp68) from Clostridium difficile displaying significant homology to several established or putative Fbps from other bacteria was identified. The one-copy gene is highly conserved in C. difficile isolates. Fbp68 was expressed in Escherichia coli in fusion with glutathione S-transferase; the fusion protein and the native Fbp68 were purified. Immunoblot analysis and cell fractionation experiments revealed that Fbp68 is present on the surface of the bacteria. Far-immuno dot-blotting demonstrated that Fbp68 was capable of fixing fibronectin. Indirect immunofluorescence and ELISA were employed to demonstrate that C. difficile could bind both soluble and immobilized fibronectin. With competitive adherence inhibition assays it was shown that antibodies raised against Fbp68 partially inhibited attachment of C. difficile to fibronectin and Vero cells. Furthermore, Vero cells could fix purified membrane-immobilized Fbp68. Thus Fbp68 appears to be one of the several adhesins identified to date in C. difficile.
Transcript map of the temperate Lactobacillus gasseri bacteriophage ϕadh
Authors: Altermann Eric , Henrich Bernhard
Published: October 2003

Temporal transcription of phage ϕadh was analysed during lytic reproduction. Based on Northern hybridizations the phage genome was divided into regions of early, middle and late transcription. Eight groups of overlapping transcripts, probably originating from common precursors, were distinguished. Early transcription of a 10·9 kb region adjacent to the lytic/lysogenic switch started within the first 10 min of infection and produced three groups of mRNAs mostly related to DNA replication. Four middle transcripts were observed 30 min after infection, corresponding to an 8·5 kb genomic region, which started at the replication origin (ori) and encompassed a DNA packaging function and the cos site. Three groups of late transcripts were first observed 50 min after infection, corresponding to a 21·1 kb region between the middle region and the attachment site (attP), encoding functions for capsid morphogenesis and host cell lysis. A fourth group of late-appearing mRNAs was divergently transcribed from the 3·2 kb section between attP and the lytic/lysogenic switch, including the repressor and integrase genes. Except for one set of early mRNAs, all the transcripts persisted until the end of the reproduction cycle. Two confirmed and two predicted promoters were assigned to transcript 5′ ends in the early region.
Degradation of alkanes and highly chlorinated benzenes, and production of biosurfactants, by a psychrophilic Rhodococcus sp. and genetic characterization of its chlorobenzene dioxygenase
Authors: Rapp Peter , Gabriel-Jürgens Lotte H. E.
Published: October 2003

Rhodococcus sp. strain MS11 was isolated from a mixed culture. It displays a diverse range of metabolic capabilities. During growth on 1,2,4-trichlorobenzene, 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB) and 3-chlorobenzoate stoichiometric amounts of chloride were released. It also utilized all three isomeric dichlorobenzenes and 1,2,3-trichlorobenzene as the sole carbon and energy source. Furthermore, the bacterium grew well on a great number of n-alkanes ranging from n-heptane to n-triacontane and on the branched alkane 2,6,10,14-tetramethylpentadecane (pristane) and slowly on n-hexane and n-pentatriacontane. It was able to grow at temperatures from 5 to 30 °C, with optimal growth at 20 °C, and could tolerate 6 % NaCl in mineral salts medium. Genes encoding the initial chlorobenzene dioxygenase were detected by using a primer pair that was designed against the α-subunit (TecA1) of the chlorobenzene dioxygenase of Ralstonia (formerly Burkholderia) sp. strain PS12. The amino acid sequence of the amplified part of the α-subunit of the chlorobenzene dioxygenase of Rhodococcus sp. strain MS11 showed >99 % identity to the α-subunit of the chlorobenzene dioxygenase from Ralstonia sp. strain PS12 and the parts of both α-subunits responsible for substrate specificity were identical. The subsequent enzymes dihydrodiol dehydrogenase and chlorocatechol 1,2-dioxygenase were induced in cells grown on 1,2,4,5-TeCB. During cultivation on medium-chain-length n-alkanes ranging from n-decane to n-heptadecane, including 1-hexadecene, and on the branched alkane pristane, strain MS11 produced biosurfactants lowering the surface tension of the cultures from 72 to ⩽29 mN m−1. Glycolipids were extracted from the supernatant of a culture grown on n-hexadecane and characterized by 1H- and 13C-NMR-spectroscopy and mass spectrometry. The two major components consisted of α,α-trehalose esterified at C-2 or C-4 with a succinic acid and at C-2′ with a decanoic acid. They differed from one another in that one 2,3,4,2′-trehalosetetraester, found in higher concentration, was esterified at C-2, C-3 or C-4 with one octanoic and one decanoic acid and the other one, of lower concentration, with two octanoic acids. The results demonstrate that Rhodococcus sp. strain MS11 may be well suited for bioremediation of soils and sediments contaminated for a long time with di-, tri- and tetrachlorobenzenes as well as alkanes.
A three-dimensional, stochastic simulation of biofilm growth and transport-related factors that affect structure
Authors: Chang Ivan , Gilbert Eric S. , Eliashberg Natalya , Keasling Jay D.
Published: October 2003

Biofilm structural heterogeneity affects a broad range of microbially catalysed processes. Solute transport limitation and autoinhibitor production, two factors that contribute to heterogeneous biofilm development, were investigated using BacMIST, a computer simulation model. BacMIST combines a cellular automaton algorithm for biofilm growth with Brownian diffusion for solute transport. The simulation represented the growth of microbial unit cells in a three-dimensional domain modelled after a repeating section of a constant depth film fermenter. The simulation was implemented to analyse the effects of various levels of transport limitation on a growing single-species biofilm. In a system with rapid solute diffusion, cells throughout the biofilm grew at their maximum rate, and no solute gradient was formed over the biofilm thickness. In increasingly transport-limited systems, the rapidly growing fraction of the biofilm population decreased, and was found exclusively at the biofilm–liquid interface. Trans-biofilm growth substrate gradients also deepened with increasing transport limitation. Autoinhibitory biofilm growth was simulated for various rates of microbially produced inhibitor transport. Inhibitor transport rates affected both the biofilm population dynamics and the resulting biofilm structures. The formation of networks of void spaces in slow-growing regions of the biofilm and the development of columns in the fast-growing regions suggested a possible mechanism for the microscopically observed evolution of channels in biofilms.
Application of a bacterial two-hybrid system for the analysis of protein–protein interactions between FemABX family proteins
Authors: Rohrer Susanne , Berger-Bächi Brigitte
Published: October 2003

Protein–protein interactions play an important role in all cellular processes. The development of two-hybrid systems in yeast and bacteria allows for in vivo assessment of such interactions. Using a recently developed bacterial two-hybrid system, the interactions of the Staphylococcus aureus proteins FemA, FemB and FmhB, members of the FemABX protein family, which is involved in peptidoglycan biosynthesis and β-lactam resistance of numerous Gram-positive bacteria, were analysed. While FmhB is involved in the addition of glycine 1 of the pentaglycine interpeptide of S. aureus peptidoglycan, FemA and FemB are specific for glycines 2/3 and 4/5, respectively. FemA–FemA, FemA–FemB and FemB–FemB interactions were found, while FmhB exists solely as a monomer. Interactions detected by the bacterial two-hybrid system were confirmed using the glutathione S-transferase-pulldown assay and gel filtration.
A novel tannase from Aspergillus niger with β-glucosidase activity
Authors: Ramírez-Coronel M. Ascención , Viniegra-González Gustavo , Darvill Alan , Augur Christopher
Published: October 2003

An extracellular tannase was produced from solid-state cultures of Aspergillus niger. The enzyme was purified to homogeneity from the cell-free culture broth by preparative isoelectric focusing and by FPLC using anion-exchange and gel-filtration chromatography. SDS-PAGE analysis as well as gel localization studies of purified tannase indicated the presence of two enzyme forms, with molecular masses of 90 kDa and 180 kDa. The tannase had an isoelectric point of 3·8, a temperature optimum of 60–70 °C and a pH optimum of 6·0. The substrate specificity of the tannase was determined by HPLC analysis of tannin substrates and products. The enzyme was able to remove gallic acid from both condensed and hydrolysable tannins. Internal sequences were obtained from each of the gel-purified and trypsin-digested tannase forms. The peptide sequences obtained from both forms were identical to sequences within a β-glucosidase from Aspergillus
kawachii. The purified tannase was tested for β-glucosidase activity and was shown to hydrolyse cellobiose efficiently. However, no β-glucosidase activity was detected when the enzyme was assayed in the presence of tannic acid.
The Staphylococcus aureus surface protein SasG and its homologues promote bacterial adherence to human desquamated nasal epithelial cells
Authors: Roche Fiona M. , Meehan Mary , Foster Timothy J.
Published: October 2003

Staphylococcus aureusbinds to human desquamated nasal epithelial cells, a phenomenon likely to be important in nasal colonization. ClfB was identified previously as one staphylococcal adhesin that promoted binding to nasal epithelia. In this study, it is shown that theS. aureussurface protein SasG, identified previously byin silicoanalysis of genome sequences, and two homologous proteins, Pls ofS. aureusand AAP ofStaphylococcus epidermidis, also promote bacterial adherence to nasal epithelial cells. Conditions forin vitroexpression of SasG byS. aureuswere not found. Adherence assays were therefore performed withS. aureusandLactococcus lactisexpressing SasG from an expression plasmid. These studies showed that SasG did not bind several ligands typically bound byS. aureus. Significantly, SasG and Pls did promote bacterial adherence to nasal epithelial cells. Furthermore, pre-incubation of epithelial cells with purified recombinant proteins revealed that the N-terminal A regions of SasG, Pls and AAP, but not the B repeats of SasG, inhibited adherence ofL. lactisexpressing SasG in a dose-dependent fashion. These results suggest that SasG, Pls and AAP bind to the same as-yet-unidentified receptor on the surface of nasal epithelial cells. In addition, expression of SasG, like Pls, reduced adherence ofS. aureusto fibronectin and fibrinogen.
Identification of sporulation genes by genome-wide analysis of the σ
E regulon of Bacillus subtilis
Authors: Feucht Andrea , Evans Louise , Errington Jeff
Published: October 2003

Differentiation in the spore-forming bacterium Bacillus subtilis is governed by the sequential activation of five sporulation-specific transcription factors. The early mother-cell-specific transcription factor, σ
E, directs the transcription of many genes that contribute to the formation of mature, dormant spores. In this study, DNA microarrays were used to identify genes belonging to the σ
E regulon. In total, 171 genes were found to be under the control of σ
E. Of these, 101 genes had not previously been described as being σ
E dependent. Disruption of some of the previously unknown genes (ydcC, yhaL, yhbH, yjaV and yqfD) resulted in a defect in sporulation.
A cryptic plasmid of Yersinia enterocolitica encodes a conjugative transfer system related to the regions of CloDF13 Mob and IncX Pil
Authors: Strauch Eckhard , Goelz Greta , Knabner Dorothea , Konietzny Antje , Lanka Erich , Appel Bernd
Published: October 2003

Yersinia enterocolitica 29930 (biotype 1A; O : 7,8), the producing strain of the phage-tail-like bacteriocin enterocoliticin, possesses a plasmid-encoded conjugative type IV transfer system. The genes of the conjugative system were found by screening of a cosmid library constructed from total DNA of strain 29930. The cosmid Cos100 consists of the vector SuperCos1 and an insert DNA of 40 303 bp derived from a cryptic plasmid of strain 29930. The conjugative transfer system consists of genes encoding a DNA transfer and replication system (Dtr) with close relationship to the mob region of the mobilizable plasmid CloDF13 and a gene cluster encoding a mating pair formation system (Mpf) closely related to the Mpf system of the IncX plasmid R6K. However, a gene encoding a homologue of TaxB, the coupling protein of the IncX system, is missing. The whole transfer region has a size of approximately 17 kb. The recombinant plasmid Cos100 was shown to be transferable between Escherichia coli and Yersinia with transfer frequencies up to 0·1 transconjugants per donor. Mutations generated by inserting a tetracycline cassette into putative tri genes yielded a transfer-deficient phenotype. Conjugative transfer of the cryptic plasmid could not be demonstrated in the original host Y. enterocolitica 29930. However, a kanamycin-resistance-conferring derivative of the plasmid was successfully introduced into E. coli K-12 by transformation and was shown to be self-transmissible. Furthermore, Southern blot hybridization and PCR experiments were carried out to elucidate the distribution of the conjugative transfer system in Yersinia. In total, six Y. enterocolitica biotype 1A strains harbouring closely related systems on endogenous plasmids were identified.
Aggregation of yeast cells: direct measurement of discrete lectin–carbohydrate interactions
Authors: Touhami Ahmed , Hoffmann Barbara , Vasella Andrea , Denis Frédéric A. , Dufrêne Yves F.
Published: October 2003

Aggregation of microbial cells mediated by specific interactions plays a pivotal role in the natural environment, in medicine and in biotechnological processes. Here we used atomic force microscopy (AFM) to measure individual lectin–carbohydrate interactions involved in the flocculation of yeast cells, an aggregation event of crucial importance in fermentation technology. AFM probes functionalized with oligoglucose carbohydrates were used to record force-distance curves on living yeast cells at a rate of 0·5 μm s−1. Flocculating cells showed adhesion forces of 121±53 pN, reflecting the specific interaction between individual cell-surface lectins and glucose residues. Similar adhesion forces, 117±41 pN, were measured using probes functionalized with the lectin concanavalin A and attributed to specific binding to cell-surface mannose residues. By contrast, specific interaction forces were not observed in non-flocculating conditions, i.e. in the presence of mannose or when using non-flocculating cells, pointing to their involvement in yeast flocculation. The single molecule force spectroscopy measurements presented here provide a means to study a variety of cellular interactions at the molecular level, such as the adhesion of bacteria to animal and plant tissues.
Bacillus subtilis spoVIF (yjcC) gene, involved in coat assembly and spore resistance
Authors: Kuwana Ritsuko , Yamamura Satoko , Ikejiri Hiromi , Kobayashi Kazuo , Ogasawara Naotake , Asai Kei , Sadaie Yoshito , Takamatsu Hiromu , Watabe Kazuhito
Published: October 2003

In systematic screening four sporulation-specific genes, yjcA, yjcB, yjcZ and yjcC, of unknown function were found in Bacillus subtilis. These genes are located just upstream of the cotVWXYZ gene cluster oriented in the opposite direction. Northern blot analysis showed that yjcA was transcribed by the SigE RNA polymerase beginning 2 h (t
2) after the onset of sporulation, and yjcB, yjcZ and yjcC were transcribed by the SigK RNA polymerase beginning at t
4 of sporulation. The transcription of yjcZ was dependent on SigK and GerE. The consensus sequences of the appropriate sigma factors were found upstream of each gene. There were putative GerE-binding sites upstream of yjcZ. Insertional inactivation of the yjcC gene resulted in a reduction in resistance of the mutant spores to lysozyme and heat. Transmission electron microscopic examination of yjcC spores revealed a defect of sporulation at stage VI, resulting in loss of spore coats. These results suggest that YjcC is involved in assembly of spore coat proteins that have roles in lysozyme resistance. It is proposed that yjcC should be renamed as spoVIF.
Characterization of Enterococcus faecium mutants resistant to mundticin KS, a class IIa bacteriocin
Authors: Sakayori Youko , Muramatsu Mizuho , Hanada Satoshi , Kamagata Yoichi , Kawamoto Shinichi , Shima Jun
Published: October 2003

The emergence and spread of mutants resistant to bacteriocins would threaten the safety of using bacteriocins as food preservatives. To determine the physiological characteristics of resistant mutants, mutants ofEnterococcus faeciumresistant to mundticin KS, a class IIa bacteriocin, were isolated. Two types of mutant were found that had different sensitivities to other antimicrobial agents such as nisin (class I) and kanamycin. Both mutants were resistant to mundticin KS even in the absence of Mg2+ions. The composition of unsaturated fatty acids in the resistant mutants was significantly increased in the presence of mundticin KS. The composition of the phospholipids in the two resistant mutants also differed from those in the wild-type strain. The putative zwitterionic amino-containing phospholipid in both mutants significantly increased, whereas amounts of phosphatidylglycerol and cardiolipin decreased. These changes in membrane structure may influence resistance of enterococci to class IIa and class I bacteriocins.
Multiple methionine sulfoxide reductase genes in Staphylococcus aureus: expression of activity and roles in tolerance of oxidative stress
Authors: Singh Vineet K. , Moskovitz Jackob
Published: October 2003

Staphylococcus aureus contains three genes encoding MsrA-specific methionine sulfoxide reductase (Msr) activity (msrA1, msrA2 and msrA3) and an additional gene that encodes MsrB-specific Msr activity. Data presented here suggest that MsrA1 is the major contributor of the MsrA activity in S. aureus. In mutational analysis, while the total Msr activity in msrA2 mutant was comparable to that of the parent, Msr activity was significantly up-regulated in the msrA1 or msrA1
msrA2 double mutant. Assessment of substrate specificity together with increased reactivity of the cell-free protein extracts of the msrA1 mutants to anti-MsrB polyclonal antibodies in Western analysis provided evidence that increased Msr activity was due to elevated synthesis of MsrB in the MsrA1 mutants. Previously, it was reported that oxacillin treatment of S. aureus cells led to induced synthesis of MsrA1 and a mutation in msrA1 increased the susceptibility of the organism to H2O2. A mutation in the msrA2 gene, however, was not significant for the bacterial oxidative stress response. In complementation assays, while the msrA2 gene was unable to complement the msrA1
msrA2 double mutant for H2O2 resistance, the same gene restored H2O2 tolerance in the double mutant when placed under the control of the msrA1 promoter. However, msrA1 which was able to complement the oxidative stress response in msrA1 mutants could not restore the tolerance of the msrA1
msrA2 mutants to H2O2 when placed under the control of the msrA2 promoter. Additionally, although the oxacillin minimum inhibitory concentration of the msrA1 mutant was comparable to that of the wild-type parent, in shaking liquid culture, the msrA1 mutant responded more efficiently to sublethal doses of oxacillin. The data suggest complex regulation of Msr proteins and a more significant physiological role for msrA1/msrB in S. aureus.
Alterations in the formation of lipopolysaccharide and membrane vesicles on the surface of Pseudomonas aeruginosa PAO1 under oxygen stress conditions
Authors: Sabra W. , Lünsdorf H. , Zeng A.-P.
Published: October 2003

It has been postulated that phenotypic variation in the relative expression of two chemically distinct types of lipopolysaccharide (LPS), a serotype-specific LPS (B-band) and a common antigen LPS (A-band) in Pseudomonas aeruginosa is an important mechanism enabling this opportunistic pathogen to alter its surface characteristics to mediate adhesion and to survive under extreme conditions. To further investigate this, the relative expression levels of the two distinct types of LPS in P. aeruginosa PAO1 were investigated with cells grown in a chemostat at different dissolved oxygen tensions (pO2). The A-band LPS was constitutively expressed as pO2 was increased from nearly zero to 350 % of air saturation. In contrast, the B-band LPS showed a remarkable increase with increased pO2. Almost no B-band LPS was found in cells grown at a pO2 of less than 3 % of air saturation. Electron microscopic examination of cells revealed increased formation of membrane vesicles (MVs) on the surface of P. aeruginosa PAO1 under oxygen stress conditions. The toxicity of the supernatant of P. aeruginosa cultures to the growth of a hybridoma cell line significantly increased in samples taken from oxygen-stressed steady-state cultures. Furthermore, studies of adhesion in a continuous-flow biofilm culture revealed an increased adhesiveness for hydrophilic surfaces in P. aeruginosa PAO1 grown at a higher pO2. The oxygen-dependent alterations of cell-surface components and properties observed in this work provide a possible explanation for the emergence of P. aeruginosa lacking the B-band LPS in chronically infected cystic fibrosis patients. The results are also useful for understanding the processes involved in the formation of MVs in P. aeruginosa.