bluuebunny/crossref_metadata_2025 huggingface dataset:
Healthcare capacity measurement
Authors: Bamford David , Chatziaslan Elizabeth
Published: November 2009

Purpose

The purpose of this paper is to present findings from an action research intervention in the outpatient department of a National Health Service (NHS) Hospital. It investigates the perceived and actual problems of measuring capacity at a UK hospital.

Design/methodology/approach

An action research methodology was adopted: relevant literature on capacity measurement and performance was reviewed; the motivation behind its usage explored. Systems requiring improvement were identified and the adoption and implementation of new working methods initiated and explored. In addition to considering capacity measurement and performance issues, the authors examined the effects this may have on the long‐term potential of the organisation.

Findings

The research identified gaps in the capacity and activity measures used and in the dissemination of performance information. To address these problems and meet the changing needs of the department a new performance measurement and reporting tool was implemented.

Research limitations/implications

The methodology applied was appropriate, generating data to facilitate discussion and draw specific conclusions from. A perceived limitation is the single case approach, however, Remenyi argues this can be enough to add to the body of knowledge.

Practical implications

The paper gives details about the introduction of new performance measures and the development of a performance measurement tool for the Outpatient Department of the Hospital. The conclusions drawn add substantially to previous commentaries, and develop interesting questions for future research.

Originality/value

Specific recommendations for the implementation of a new performance tool for healthcare organisations are made.
Measuring and comparing the performance of Portuguese secondary schools
Authors: Sarrico Cláudia S. , Rosa Maria J.
Published: November 2009

Purpose

This paper aims to discuss methods for measuring and comparing the performance of Portuguese schools, confronting metric and practice benchmarking exercises.

Design/methodology/approach

Data on the schools was collected within the context of a programme for school evaluation launched by the Ministry of Education in 2007. The paper first uses a non‐parametric technique, data envelopment analysis, to assess the sample of schools using the data collected, taking a value‐added approach. The results obtained are compared with the results obtained by the panels of evaluators within the national evaluation programme of schools.

Findings

The paper concludes that the performance of the schools under the metric benchmarking exercise does vary substantially. However, the results do not correlate with the judgements made by the panels of assessors regarding schools' results. This might be because assessors find it difficult to take a value‐added approach and instead assess schools mostly on output measures, such as exam classifications and graduation rates, having difficulty in taking into account pupils' socio‐economic background and other variables not under the control of the school.

Research limitations/implications

The paper focuses on the Portuguese context, and its findings might not be directly applicable to other contexts. Also, if other quantitative and qualitative methods were used, other results might have been obtained.

Practical implications

Although the study is focused on the Portuguese context, contributing towards a better understanding of Portuguese secondary school performance, it is believed it will be helpful to inform the debate on school evaluation, performance improvement and policy setting in other contexts.

Originality/value

The paper concludes by arguing for metric benchmarking exercises of the type proposed, to inform schools, evaluators and policy decision‐makers, in combination with practice benchmarking exercises, which are better suited for qualitative aspects of performance.
Cross‐gender networking in the workplace: causes and consequences
Authors: Ng Ignace , Hau‐siu Chow Irene
Published: November 2009

Purpose

This study seeks to examine how individual and organizational characteristics as well as attitudinal factors can affect the network composition of female managers. Another of its objectives is to examine the effect of cross‐gender network on the quit intention of female managers.

Design/methodology/approach

A survey questionnaire was administered, seeking information on the personal characteristics and attitudes of the 91 managers, the characteristics of the organization for which the respondent works, and the network characteristics of the respondents in Hong Kong.

Findings

The results show that positive attitudes towards women's leadership qualities and higher ratio of females in top management positions are associated with a lower cross‐gender instrumental network for females. Perceived discrimination or being married encourages female managers to seek a cross‐gender network. Cross‐gender networks reduce the quit intentions of female managers.

Originality/value

The study offers a better understanding of how networks change involves an examination of both the characteristics of the network holder and the larger context in which the network holder is located. It contributes to the scant evidence on the consequences of cross‐gender networking for female managers in the Chinese context.
A more proactive approach to addressing gender‐related employment disparities in the United States
Authors: Graham Mary E. , Hotchkiss Julie L.
Published: November 2009

Purpose

The purpose of this paper is to propose a proactive public policy approach to complement relatively reactive existing policies addressing gender‐related employment disparities in the USA, and to provide an initial empirical illustration of the proposal.

Design/methodology/approach

The paper provides a conceptual application of theories of total quality management (TQM) to the topic of gender‐related employment disparities, followed by an empirical illustration using US Current Population Survey data and a gender equal employment opportunity (EEO) scorecard.

Findings

Using the TQM framework, company outliers were conceptualized on the EEO scorecard as “special” causes of economy‐wide equal employment variation and the industries in which companies are situated as “common” causes. The paper identifies two underperforming industries on gender‐related employment outcomes: Mining and Construction, and Transportation, Communication and Utilities.

Research limitations/implications

Further conceptual work on the application of TQM to gender disparities in employment is recommended. Also, the study considered broad industry categories; future research should refine these categories further.

Practical implications

It is recommended that US enforcement agencies incorporate industry considerations more explicitly into their activities. Employer insights may be beneficial to improving equal employment opportunity performance at the industry level.

Originality/value

The application of TQM theory to the topic of gender‐related employment disparities is a novel approach that may motivate new public policies.
The spouse of the female manager: role and influence on the woman's career
Authors: Välimäki Suvi , Lämsä Anna‐Maija , Hiillos Minna
Published: November 2009

Purpose

This paper aims to examine the role of the spouse, specifically the husband, for the woman manager's career by focusing on the gender role construction between spouses, and the relationship of these roles to the woman's career.

Design/methodology/approach

The topic was investigated within a Finnish context by analyzing the narratives of 29 female managers. A common feature among the women was their managerial position and extensive work experience. All the women had or had had one or more spouses in the course of their careers, and all but one were mothers, mostly of teenage or adult children.

Findings

A typology distinguishing five types of spouses was constructed: determining, supporting, instrumental, flexible, and counterproductive. The results suggest that fluidity in gender roles between spouses is associated with the woman manager's sense of success and satisfaction in her career compared with more conventional gender role construction. It seems that traditional gender roles between spouses can be one reason for women's difficulties in attaining (top) managerial positions in Finland.

Originality/value

The study contributes to the prior literature concerning the work‐family relationship by extending research into an area so far overlooked: namely, the role of the spouse in relation to the woman manager's career. The study calls into question the straightforward and unequivocal view of the family – so typical in discussions about work‐family issues – by showing the many different meanings that women managers attach to one of the family members.
Culture, job satisfaction and organizational commitment in India and the United States
Authors: Kwantes Catherine T.
Published: November 2009

Purpose

The purpose of this paper is to compare and contrast the roles of culture and job satisfaction as antecedents to organizational commitment in both a Western context (the US) and in India.

Design/methodology/approach

Responses come from a questionnaire distributed to engineers in India. Construct equivalence of measures is established, while hierarchical regression analysis is used to assess the extent to which each hypothesized antecedent is related to affective, continuance, and normative commitment. Responses from each national context are compared and contrasted.

Findings

Job satisfaction is found to relate to affective commitment in both the Indian and American samples. Moderate support is found for the hypothesized effect of collectivism on normative commitment in both samples, while the hypothesized antecedents to continuance commitment are not found in any sample. Different patterns of relationships emerge in the US and India.

Research limitations/implications

The results provide further cautionary evidence against uncritically applying organizational theories developed in a Western context to developing nations such as India. The sample in this research is restricted to engineers, future research should examine other occupations/professions as well as determining the applicability of these results to different levels in the organization.

Originality/value

This research examines theoretically suggested antecedents to organizational commitment, explicitly testing these relationships in two cultural contexts. The results presented in this paper suggest that context must be taken into account when developing organizational theories. Further, the results suggest specific activities that can be useful in the Indian context to increase both normative and affective commitment.
Myristoylated alanine‐rich C kinase substrate phosphorylation promotes cholangiocarcinoma cell migration and metastasis via the protein kinase C‐dependent pathway
Authors: Techasen Anchalee , Loilome Watcharin , Namwat Nisana , Takahashi Eri , Sugihara Eiji , Puapairoj Anucha , Miwa Masanao , Saya Hideyuki , Yongvanit Puangrat
Published: November 2009

(Cancer Sci 2010; 101: 658–665)

Myristoylated alanine‐rich C kinase substrate (MARCKS), a substrate of protein kinase C (PKC) has been suggested to be implicated in cell adhesion, secretion, and motility through the regulation of the actin cytoskeletal structure. The quantitative real‐time–polymerase chain reaction analysis revealed that MARCKS is significantly overexpressed in Opisthorchis viverrini‐associated cholangiocarcinoma (CCA) (P = 0.001) in a hamster model, which correlated with the results of mRNA in situ hybridization. An immunohistochemical analysis of 60 CCA patients revealed a significant increase of MARCKS expression. Moreover, the log–rank analysis indicated that CCA patients with a high MARCKS expression have significantly shorter survival times than those with a low MARCKS expression (P = 0.02). This study investigated whether MARCKS overexpression is associated with CCA metastasis. Using a confocal microscopic analysis of CCA cell lines that had been stimulated with the PKC activator, 12‐0‐tetradecanoyl phorbol‐13‐acetate (TPA), MARCKS was found to be translocated from the plasma membrane to the perinuclear area. In addition, phosphorylated MARCKS (pMARCKS) became highly concentrated in the perinuclear area. Moreover, an adhesion assay demonstrated that the exogenous overexpression of MARCKS remarkably promoted cell attachment. Interestingly, after TPA stimulation, the CCA cell line‐depleted MARCKS showed a decrease in migration and invasion activity. It can be concluded that in non‐stimulation, MARCKS promotes cell attachment to the extracellular matrix. After TPA stimulation, PKC phosphorylates MARCKS leading to cell migration or invasion. Taken together, the results of this study reveal a prominent role for MARCKS as one of the key players in the migration of CCA cells and suggest that cycling between MARCKS and pMARCKS can regulate the metastasis of biliary cancer cells.
Phase I study of irinotecan and doxifluridine for metastatic colorectal cancer focusing on the UGT1A1*28 polymorphism
Authors: Hazama Shoichi , Nagashima Atsushi , Kondo Hiroshi , Yoshida Shin , Shimizu Ryoichi , Araki Atsuhiro , Yoshino Shigefumi , Okayama Naoko , Hinoda Yuji , Oka Masaaki
Published: November 2009

(Cancer Sci 2010; 101: 722–727)

Although individuals carrying the UGT1A1 allele *28 have an increased risk of severe toxicities associated with irinotecan, no phase I study has been conducted based on the polymorphism. This report presents the recommended doses of irinotecan for patients with the respective genotypes. Twenty‐seven patients with advanced colorectal cancer were enrolled in this study, and the UGT1A1*28 polymorphism was genotyped before chemotherapy. One course of chemotherapy consisted of irinotecan infused once every 2 weeks at 70, 100, 120, and 150 mg/m2 at dose levels 1, 2, 3, and 4, respectively, and doxifluridine was administered orally. This treatment continued for at least 12 weeks. The dose‐limiting toxicity was determined as grade 3 hematological and non‐hematological toxicities for the TA6/TA6 (6/6) and TA6/TA7 (6/7) genotypes. The pharmacokinetics of irinotecan, SN‐38, and SN‐38 glucuronide, was assessed at dose level 2. Eighteen and nine patients had the 6/6 and 6/7 genotypes, respectively. The maximum tolerated dose (MTD) was not observed up to dose level 4 in patients with the 6/6 genotype. In contrast, MTD was observed at dose level 2 (100 mg/m2) in patients with the 6/7 genotype. Patients with the 6/7 genotype had a significantly higher area under the plasma time–concentration curve 0‐∞ SN‐38 (P = 0.022) and biliary index (P = 0.030) than those with 6/6. The recommended starting doses of biweekly irinotecan for phase II/III were 150 mg/m2 for patients with the UGT1A1 6/6 genotype and 70 mg/m2 for those with the 6/7 genotype, respectively. The gene polymorphism should be considered when determining the precise recommended doses to be administered in phase I studies.
Up‐regulation of the type 3 ryanodine receptor is neuroprotective in the TgCRND8 mouse model of Alzheimer’s disease
Authors: Supnet Charlene , Noonan Charmaine , Richard Kelly , Bradley John , Mayne Michael
Published: November 2009

The cellular pathology of Alzheimer’s disease is progressive and protracted leading eventually to considerable neuronal death. The underlying mechanisms of the pathology are complex but changes in the control of intracellular Ca2+ are believed to contribute to the demise of neurons. In this study, we investigated the functional consequences of an increase in the expression of the type 3 isoform of the ryanodine receptor (RyR3). We found that although cortical neurons from TgCRND8 mice secreted significantly more amyloid beta protein and showed significantly increased RyR3 expression, they were no more sensitive to cell stress than non‐transgenic neurons. Furthermore, despite increased intracellular Ca2+ release in response to ryanodine, we found that basal Ca2+, K+‐evoked Ca2+ responses, and capacitative Ca2+ entry were no different in TgCRND8 neurons compared with non‐transgenic neurons. Therefore, as RyR3 up‐regulation did not affect neuronal health or global Ca2+ homeostasis, we investigated the effect of reducing RyR3 expression using small interfering RNA. Surprisingly, a reduction of RyR3 expression in TgCRND8, but not in non‐transgenic, neurons increased neuronal death. These data reveal a new role for RyR3 and indicate a novel potential therapeutic target to delay or prevent the progression of Alzheimer’s disease.
A Material History of Australia
Authors: Wood Richard , Lenzen Manfred , Foran Barney
Published: November 2009

This article presents an analysis of the material history of Australia in the period 1975–2005. The values of economy‐wide indicators of material flow roughly trebled since 1975, and we identify the drivers of this change through structural decomposition analysis. The purpose of this work is to delve beneath the top‐level trends in material flow growth to investigate the structural changes in the economy that have been driving this growth. The major positive drivers of this change were the level of exports, export mix, industrial structure, affluence, and population. Only improvements in material intensity offered retardation of growth in material flow. Other structural components had only small effects at the aggregate level. At a more detailed level, however, the importance of the mineral sectors became apparent. Improvements in mining techniques have reduced material requirements, but increased consumption within the economy and increased exports have offset these reductions. The full roll out of material flow accounting through Australian society and business and a systematic response to its implications will require change in the national growth focus of the last two generations, with serious consideration needed to reverse the current volume‐focused growth of the economy and also to recast neoliberal and globalized trade policies that have dominated the globe for the past decades.
Myocellular limitations of human performance and their modification through genome‐dependent responses at altitude
Authors: Flueck Martin
Published: November 2009

Human muscle operates along a continuum of power output, which is set through bioenergetic and anatomical principles. In turn, environmental and intrinsic factors during contractile work exert pronounced control over muscle performance by instructing muscle remodelling. This phenotypic control is specifically indicated with intense exercise at altitude, when extra strain is put on energy supply and the temperature‐dependent mechanical efficiency of contraction. While it is classically thought that chronic exposure to hypoxia is maladaptive, repeated short episodes of reduced oxygenation alone or in combination with intense endurance work is now understood to preserve exercise performance when atmospheric oxygen levels are low. Endurance training at moderate altitude exploits the temperature‐dependent malleability of energy supply that may maximize metabolic flux at altitude. The contribution of genomic mechanisms is important to the plasticity of metabolic pathways in exercised muscle. This is highlighted by the association of distinct gene polymorphisms in master governors of mitochondrial and vascular growth with exercise phenotypes. Feedforward control of human locomoter muscle by exercise involves the transient upregulation of transcript expression for metabolic processes. The response of the mitochondrial transcriptome to intense exercise is graded with respect to mitochondrial content and deoxygenation during muscle work and reflects exercise‐induced mitochondrial biogenesis. This supports the notion that genome‐mediated muscle malleability is under feedback control by design constraints of the pathway of oxygen. Thus, activity‐dependent and genetic mechanisms contribute to the interindividual difference in the metabolic bottlenecks in athletes performing in exceptional environmental conditions.
Influence of Sublethal Concentrations of Common Disinfectants on Expression of Virulence Genes in
Listeria monocytogenes
Authors: Kastbjerg Vicky G. , Larsen Marianne Halberg , Gram Lone , Ingmer Hanne
Published: November 2009

Listeria monocytogenes
is a food-borne human pathogen that causes listeriosis, a relatively rare infection with a high fatality rate. The regulation of virulence gene expression is influenced by several environmental factors, and the aim of the present study was to determine how disinfectants used routinely in the food industry affect the expression of different virulence genes in
L. monocytogenes
when added at sublethal concentrations. An agar-based assay was developed to screen the effect of disinfectants on virulence gene promoter expression and was validated at the transcriptional level by Northern blot analysis. Eleven disinfectants representing four different groups of active components were evaluated in this study. Disinfectants with the same active ingredients had a similar effect on gene expression. Peroxy and chlorine compounds reduced the expression of the virulence genes, and quaternary ammonium compounds (QAC) induced the expression of the virulence genes. In general, a disinfectant had similar effects on the expression of all four virulence genes examined. Northern blot analyses confirmed the downregulation of
prfA
and
inlA
expression by Incimaxx DES (a peroxy compound) and their upregulation by Triquart Super (a QAC) in
L. monocytogenes
EGD. Hence, sublethal concentrations of disinfectants routinely used in the food industry affect virulence gene expression in the human pathogen
L. monocytogenes
, and the effect depends on the active components of the disinfectant. From a practical perspective, the study underlines that disinfectants should be used at the lethal concentrations recommended by the manufacturers. Further studies are needed to elucidate whether the changes in virulence gene expression induced by the disinfectants have impact on virulence or other biological properties, such as antibiotic resistance.
Galleria mellonella
as a Model System for Studying
Listeria
Pathogenesis
Authors: Mukherjee Krishnendu , Altincicek Boran , Hain Torsten , Domann Eugen , Vilcinskas Andreas , Chakraborty Trinad
Published: November 2009

Essential aspects of the innate immune response to microbial infection are conserved between insects and mammals. This has generated interest in using insects as model organisms to study host-microbe interactions. We used the greater wax moth
Galleria mellonella
, which can be reared at 37°C, as a model host for examining the virulence potential of
Listeria
spp. Here we report that
Galleria
is an excellent surrogate model of listerial septic infection, capable of clearly distinguishing between pathogenic and nonpathogenic
Listeria
strains and even between virulent and attenuated
Listeria monocytogenes
strains. Virulence required listerial genes hitherto implicated in the mouse infection model and was linked to strong antimicrobial activities in both hemolymph and hemocytes of infected larvae. Following
Listeria
infection, the expression of immune defense genes such as those for lysozyme, galiomycin, gallerimycin, and insect metalloproteinase inhibitor (IMPI) was sequentially induced. Preinduction of antimicrobial activity by treatment of larvae with lipopolysaccharide (LPS) significantly improved survival against subsequent
L. monocytogenes
challenge and strong antilisterial activity was detected in the hemolymph of LPS pretreated larvae. We conclude that the severity of septic infection with
L. monocytogenes
is modulated primarily by innate immune responses, and we suggest the use of
Galleria
as a relatively simple, nonmammalian model system that can be used to assess the virulence of strains of
Listeria
spp. isolated from a wide variety of settings from both the clinic and the environment.
Isolation of Prasinoviruses of the Green Unicellular Algae
Ostreococcus
spp. on a Worldwide Geographical Scale
Authors: Bellec Laure , Grimsley Nigel , Desdevises Yves
Published: November 2009

Ostreococcus
spp. are extremely small unicellular eukaryotic green algae found worldwide in marine environments, and they are susceptible to attacks by a diverse group of large DNA viruses. Several biologically distinct species of
Ostreococcus
are known and differ in the ecological niches that they occupy: while
O. tauri
(representing clade C strains) is found in marine lagoons and coastal seas, strains belonging to clade A, exemplified by
O. lucimarinus
, are present in different oceans. We used laboratory cultures of clonal isolates of these two species to assay for the presence of viruses in seawater samples from diverse locations. In keeping with the distributions of their host strains, we found a decline in the abundance of
O. tauri
viruses from a lagoon in southwest France relative to the Mediterranean Sea, whereas in the ocean, no
O. tauri
viruses were detected. In contrast, viruses infecting
O. lucimarinus
were detected from distantly separated oceans. DNA sequencing, phylogenetic analyses using a conserved viral marker gene, and a Mantel test revealed no relationship between geographic and phylogenetic distances in viruses infecting
O. lucimarinus
.
Unique Ecophysiology among U(VI)-Reducing Bacteria as Revealed by Evaluation of Oxygen Metabolism in
Anaeromyxobacter dehalogenans
Strain 2CP-C
Authors: Thomas Sara H. , Sanford Robert A. , Amos Benjamin K. , Leigh Mary Beth , Cardenas Erick , Löffler Frank E.
Published: November 2009

Anaeromyxobacter
spp. respire soluble hexavalent uranium, U(VI), leading to the formation of insoluble U(IV), and are present at the uranium-contaminated Oak Ridge Integrated Field Research Challenge (IFC) site. Pilot-scale
in situ
bioreduction of U(VI) has been accomplished in area 3 of the Oak Ridge IFC site following biostimulation, but the susceptibility of the reduced material to oxidants (i.e., oxygen) compromises long-term U immobilization. Following oxygen intrusion, attached
Anaeromyxobacter dehalogenans
cells increased approximately 5-fold from 2.2 × 10
7
± 8.6 × 10
6
to 1.0 × 10
8
± 2.2 × 10
7
cells per g of sediment collected from well FW101-2. In the same samples, the numbers of cells of
Geobacter lovleyi
, a population native to area 3 and also capable of U(VI) reduction, decreased or did not change.
A. dehalogenans
cells captured via groundwater sampling (i.e., not attached to sediment) were present in much lower numbers (<1.3 × 10
4
± 1.1 × 10
4
cells per liter) than sediment-associated cells, suggesting that
A. dehalogenans
cells occur predominantly in association with soil particles. Laboratory studies confirmed aerobic growth of
A. dehalogenans
strain 2CP-C at initial oxygen partial pressures (pO
2
) at and below 0.18 atm. A negative linear correlation [μ = (−0.09 × pO
2
) + 0.051;
R
2
= 0.923] was observed between the instantaneous specific growth rate μ and pO
2
, indicating that this organism should be classified as a microaerophile. Quantification of cells during aerobic growth revealed that the fraction of electrons released in electron donor oxidation and used for biomass production (

f
s

) decreased from 0.52 at a pO
2
of 0.02 atm to 0.19 at a pO
2
of 0.18 atm. Hence, the apparent fraction of electrons utilized for energy generation (i.e., oxygen reduction) (

f
e

) increased from 0.48 to 0.81 with increasing pO
2
, suggesting that oxygen is consumed in a nonrespiratory process at a high pO
2
. The ability to tolerate high oxygen concentrations, perform microaerophilic oxygen respiration, and preferentially associate with soil particles represents an ecophysiology that distinguishes
A. dehalogenans
from other known U(VI)-reducing bacteria in area 3, and these features may play roles for stabilizing immobilized radionuclides
in situ
.
Morphological Response of the Halophilic Fungal Genus
Wallemia
to High Salinity
Authors: Kralj Kunčič Marjetka , Kogej Tina , Drobne Damjana , Gunde-Cimerman Nina
Published: November 2009

The basidiomycetous genus
Wallemia
is an active inhabitant of hypersaline environments, and it has recently been described as comprising three halophilic and xerophilic species:
Wallemia ichthyophaga
,
Wallemia muriae
, and
Wallemia sebi
. Considering the important protective role the fungal cell wall has under fluctuating physicochemical environments, this study was focused on cell morphology changes, with particular emphasis on the structure of the cell wall, when these fungi were grown in media with low and high salinities. We compared the influence of salinity on the morphological characteristics of
Wallemia
spp. by light, transmission, and focused-ion-beam/scanning electron microscopy.
W. ichthyophaga
was the only species of this genus that was metabolically active at saturated NaCl concentrations.
W. ichthyophaga
grew in multicellular clumps and adapted to the high salinity with a significant increase in cell wall thickness. The other two species,
W. muriae
and
W. sebi
, also demonstrated adaptive responses to the high NaCl concentration, showing in particular an increased size of mycelial pellets at the high salinities, with an increase in cell wall thickness that was less pronounced. The comparison of all three of the
Wallemia
spp. supports previous findings relating to the extremely halophilic character of the phylogenetically distant
W. ichthyophaga
and demonstrates that, through morphological adaptations, the eukaryotic
Wallemia
spp. are representative of eukaryotic organisms that have successfully adapted to life in extremely saline environments.
A Chemical Genetic Screen for Modulators of Exocytic Transport Identifies Inhibitors of a Transport Mechanism Linked to
GTR2
Function
Authors: Zhang Lisha , Huang Min , Harsay Edina
Published: November 2009

Membrane and protein traffic to the cell surface is mediated by partially redundant pathways that are difficult to perturb in ways that yield a strong phenotype. Such robustness is expected in a fine-tuned process, regulated by environmental cues, that is required for controlled cell surface growth and cell proliferation. Synthetic genetic interaction screens are especially valuable for investigating complex processes involving partially redundant pathways or mechanisms. In a previous study, we used a triple-synthetic-lethal yeast mutant screen to identify a novel component of the late exocytic transport machinery, Avl9. In a chemical-genetic version of the successful mutant screen, we have now identified small molecules that cause a rapid (within 15 min) accumulation of secretory cargo and abnormal Golgi compartment-like membranes at low concentration (<2 μM), indicating that the compounds likely target the exocytic transport machinery at the Golgi. We screened for genes that, when overexpressed, suppress the drug effects, and found that the Ras-like small GTPase, Gtr2, but not its homolog and binding partner, Gtr1, efficiently suppresses the toxic effects of the compounds. Furthermore, assays for suppression of the secretory defect caused by the compounds suggest that Gtr proteins can regulate a pathway that is perturbed by the compounds. Because
avl9Δ
and
gtr
mutants share some phenotypes, our results indicate that the small molecules identified by our chemical-genetic strategy are promising tools for understanding Avl9 function and the mechanisms that control late exocytic transport.
Multisite Phosphorylation of the
Saccharomyces cerevisiae
Filamentous Growth Regulator Tec1 Is Required for its Recognition by the E3 Ubiquitin Ligase Adaptor Cdc4 and Its Subsequent Destruction In Vivo
Authors: Bao Marie Z. , Shock Teresa R. , Madhani Hiten D.
Published: November 2009

In
Saccharomyces cerevisiae
, the pheromone-induced ubiquitylation and degradation of the filamentation pathway-specific activator, Tec1, suppresses cross talk between the mating and filamentous growth mitogen-activated protein kinase (MAPK) pathways. The mating pathway MAPK, Fus3, phosphorylates Tec1, resulting in its recognition by the SCF (for Skp1, Cullin, F-box containing) E3 ubiquitin ligase complex, leading to its proteolysis. Previously, it was found that Tec1 destruction requires phosphorylation on threonine 273 (T273). T273 is embedded in the sequence LLpTP, which is identical to the canonical binding site for Cdc4, a conserved F-box substrate adaptor for the SCF complex. However, recent work on both Cdc4 and the human Cdc4 ortholog Fbw7 has shown that a second substrate phosphorylation can be required for optimal Cdc4 binding in vitro. We report here that high-affinity binding of recombinant Cdc4 to Tec1 phosphopeptides requires phosphorylation of not only T273 but also a second site, T276. Significantly, both phospho-sites on Tec1 and a conserved basic pocket on Cdc4 are critical for Tec1 proteolysis in response to pheromone treatment of cells, establishing a role for two-phosphate recognition by yeast Cdc4 in substrate targeting in vivo.
The Glycosylation Pathway of
Eimeria tenella
Is Upregulated during Gametocyte Development and May Play a Role in Oocyst Wall Formation
Authors: Walker Robert A. , Slapetova Iveta , Slapeta Jan , Miller Catherine M. , Smith Nicholas C.
Published: November 2009

Sexual-stage glycoproteins of
Eimeria
are important components of the oocyst wall, a structure that ensures the efficient transmission of these and related parasites. In this study, the primary enzyme in the glycosylation pathway of
Eimeria tenella
, glucosamine:fructose-6-phosphate aminotransferase (EtGFAT), has been characterized as a macrogamete-specific protein. Although the transcription of Et
GFAT
was observed early in macrogamete development, protein expression was restricted to mature macrogametes, prior to their conversion into unsporulated oocysts. Genes coding for three other enzymes required for
N
-acetylgalactosamine (GalNAc) synthesis were also transcribed during
E. tenella
macrogamete development. Gene transcription of the enzyme responsible for the O-linked transfer of GalNAc to proteins, EtGalNAc-T, was upregulated primarily in unsporulated oocyst stages, and accordingly, a significant increase in GalNAc levels was observed in
E. tenella
gametocytes and oocysts. Gam56 and Gam82, two well-characterized glycoproteins of
Eimeria
macrogametes and the oocyst wall, contain high levels of GalNAc and represent probable targets of GalNAc O linkage. It appears that the glycosylation pathway, specifically relating to the formation of GalNAc O links, is dramatically upregulated in
E. tenella
sexual stages and may play a role in directing a number of macrogamete proteins to the developing oocyst wall.
Functional Characterization and Localization of
Pneumocystis carinii
Lanosterol Synthase
Authors: Joffrion Tiffany M. , Collins Margaret S. , Sesterhenn Thomas , Cushion Melanie T.
Published: November 2009

Organisms in the genus
Pneumocystis
are ubiquitous, opportunistic pathogenic fungi capable of causing a lethal pneumonia in immunocompromised mammalian hosts.
Pneumocystis
spp. are unique members of the fungal kingdom due to the absence of ergosterol in their cellular membranes. Although these organisms were thought to obtain cholesterol by scavenging, transcriptional analyses indicate that
Pneumocystis carinii
encodes gene homologs involved in sterol biosynthesis. To better understand the sterol pathway in these uncultivable fungi, yeast deletion strains were used to interrogate the function and localization of
P. carinii
lanosterol synthase (
ERG7
). The expression of PcErg7p in an
ERG7
-null mutant of the yeast
Saccharomyces cerevisiae
did not alter its growth rate and produced a functional lanosterol synthase, as evidenced by the presence of lanosterol detected by gas chromatographic analysis in levels comparable to that produced by the yeast enzyme. Western blotting and fluorescence microscopy revealed that, like the
S. cerevisiae
Erg7p, the PcErg7p localized to lipid particles in yeast. Using fluorescence microscopy, we show for the first time the presence of apparent lipid particles in
P. carinii
and the localization of PcErg7p to lipid particles in
P. carinii
. The detection of lipid particles in
P. carinii
and their association with PcErg7p therein provide strong evidence that the enzyme serves a similar function in
P. carinii
. Moreover, the yeast heterologous system should be a useful tool for further analysis of the
P. carinii
sterol pathway.
Differential Roles of the Glycogen-Binding Domains of β Subunits in Regulation of the Snf1 Kinase Complex
Authors: Mangat Simmanjeet , Chandrashekarappa Dakshayini , McCartney Rhonda R. , Elbing Karin , Schmidt Martin C.
Published: November 2009

Members of the AMP-activated protein kinase family, including the Snf1 kinase of
Saccharomyces cerevisiae
, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic α subunit and regulatory β and γ subunits. In this study, the role of the β subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (α), Snf4 (γ), and one of three alternative β subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three β subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the β subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.
Identification and Characterization of EctR1, a New Transcriptional Regulator of the Ectoine Biosynthesis Genes in the Halotolerant Methanotroph
Methylomicrobium alcaliphilum
20Z
Authors: Mustakhimov Ildar I. , Reshetnikov Alexander S. , Glukhov Anatoly S. , Khmelenina Valentina N. , Kalyuzhnaya Marina G. , Trotsenko Yuri A.
Published: November 2009

Genes encoding key enzymes of the ectoine biosynthesis pathway in the halotolerant obligate methanotroph
Methylomicrobium alcaliphilum
20Z have been shown to be organized into an
ectABC
-
ask
operon. Transcription of the
ect
operon is initiated from two promoters,
ectAp
1
and
ectAp
2
(
ectAp
1
p
2
), similar to the σ
70
-dependent promoters of
Escherichia coli
. Upstream of the gene cluster, an open reading frame (
ectR1
) encoding a MarR-like transcriptional regulator was identified. Investigation of the influence of EctR1 on the activity of the
ectAp
1
p
2
promoters in wild-type
M. alcaliphilum
20Z and
ectR1
mutant strains suggested that EctR1 is a negative regulator of the
ectABC
-
ask
operon. Purified recombinant EctR1-His
6
specifically binds as a homodimer to the putative −10 motif of the
ectAp
1
promoter. The EctR1 binding site contains a pseudopalindromic sequence (TATTTAGT-GT-ACTATATA) composed of 8-bp half-sites separated by 2 bp. Transcription of the
ectR1
gene is initiated from a single σ
70
-like promoter. The location of the EctR1 binding site between the transcriptional and translational start sites of the
ectR1
gene suggests that EctR1 may regulate its own expression. The data presented suggest that in
Methylomicrobium alcaliphilum
20Z, EctR1-mediated control of the transcription of the
ect
genes is not the single mechanism for the regulation of ectoine biosynthesis.
The Mtr Respiratory Pathway Is Essential for Reducing Flavins and Electrodes inShewanella oneidensis
Authors: Coursolle Dan , Baron Daniel B. , Bond Daniel R. , Gralnick Jeffrey A.
Published: November 2009

The Mtr respiratory pathway ofShewanella oneidensisstrain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboflavin and flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. OtherShewanellaspecies tested accumulated flavins in supernatants to an extent similar to that of MR-1, suggesting that flavin secretion is a general trait of the species. External flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled flavin concentrations. While mutants lacking MtrC were able to reduce flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.
Biochemical and Domain Analyses of FSUAxe6B, a Modular Acetyl Xylan Esterase, Identify a Unique Carbohydrate Binding Module in
Fibrobacter succinogenes
S85
Authors: Yoshida Shosuke , Mackie Roderick I. , Cann Isaac K. O.
Published: November 2009

Acetyl xylan esterase (EC 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of β-1,4-linked xylopyranoside residues decorated with acetyl side groups.
Fibrobacter succinogenes
S85 Axe6B (FSUAxe6B) is an acetyl xylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Sequences that are homologous to the region of unknown function are paralogously distributed, thus far, only in
F. succinogenes
. Therefore, the sequences were designated
Fibrobacter succinogenes
-specific paralogous module 1 (FPm-1). The FPm-1s are associated with at least 24 polypeptides in the genome of
F. succinogenes
S85. A bioinformatics search showed that most of the FPm-1-appended polypeptides are putative carbohydrate-active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies of highly conserved active-site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser
44
, His
273
, Glu
194
, and Asp
270
, with both Glu
194
and Asp
270
functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis.
Identification of Enhancer Binding Proteins Important for
Myxococcus xanthus
Development
Authors: Giglio Krista M. , Eisenstatt Jessica , Garza Anthony G.
Published: November 2009

Enhancer binding proteins (EBPs) control the temporal expression of fruiting body development-associated genes in
Myxococcus xanthus
. Eleven previously uncharacterized EBP genes were inactivated. Six EBP gene mutations produced minor but reproducible defects in fruiting body development. One EBP gene mutation that affected A-motility produced strong developmental defects.
Identification and Characterization of a Novel Member of the Radical AdoMet Enzyme Superfamily and Implications for the Biosynthesis of the Hmd Hydrogenase Active Site Cofactor
Authors: McGlynn Shawn E. , Boyd Eric S. , Shepard Eric M. , Lange Rachel K. , Gerlach Robin , Broderick Joan B. , Peters John W.
Published: November 2009

The genetic context, phylogeny, and biochemistry of a gene flanking the H
2
-forming methylene-H
4
-methanopterin dehydrogenase gene (
hmdA
), here designated
hmdB
, indicate that it is a new member of the radical
S
-adenosylmethionine enzyme superfamily. In contrast to the characteristic CX
3
CX
2
C or CX
2
CX
4
C motif defining this family, HmdB contains a unique CX
5
CX
2
C motif.
The
Citrobacter rodentium
Genome Sequence Reveals Convergent Evolution with Human Pathogenic
Escherichia coli
Authors: Petty Nicola K. , Bulgin Richard , Crepin Valerie F. , Cerdeño-Tárraga Ana M. , Schroeder Gunnar N. , Quail Michael A. , Lennard Nicola , Corton Craig , Barron Andrew , Clark Louise , Toribio Ana L. , Parkhill Julian , Dougan Gordon , Frankel Gad , Thomson Nicholas R.
Published: November 2009

Citrobacter rodentium
(formally
C
itrobacter
freundii
biotype 4280) is a highly infectious pathogen that causes colitis and transmissible colonic hyperplasia in mice. In common with enteropathogenic and enterohemorrhagic
Escherichia coli
(EPEC and EHEC, respectively),
C. rodentium
exploits a type III secretion system (T3SS) to induce attaching and effacing (A/E) lesions that are essential for virulence. Here, we report the fully annotated genome sequence of the 5.3-Mb chromosome and four plasmids harbored by
C. rodentium
strain ICC168. The genome sequence revealed key information about the phylogeny of
C. rodentium
and identified 1,585
C. rodentium
-specific (without orthologues in EPEC or EHEC) coding sequences, 10 prophage-like regions, and 17 genomic islands, including the locus for enterocyte effacement (LEE) region, which encodes a T3SS and effector proteins. Among the 29 T3SS effectors found in
C. rodentium
are all 22 of the core effectors of EPEC strain E2348/69. In addition, we identified a novel
C. rodentium
effector, named EspS.
C. rodentium
harbors two type VI secretion systems (T6SS) (CTS1 and CTS2), while EHEC contains only one T6SS (EHS). Our analysis suggests that
C. rodentium
and EPEC/EHEC have converged on a common host infection strategy through access to a common pool of mobile DNA and that
C. rodentium
has lost gene functions associated with a previous pathogenic niche.