bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of potassium ion as the endothelium‐derived hyperpolarizing factor (EDHF) in the bovine coronary artery
Authors: Nelli Silvia , Wilson William S , Laidlaw Hilary , Llano Andrea , Middleton Susan , Price Andrew G , Martin William
Published: July 2003

This study explored the role of the potassium ion in endothelium‐derived hyperpolarizing factor (EDHF)‐mediated vasodilatation in the bovine coronary artery.

Bradykinin‐induced, EDHF‐mediated vasodilatation was blocked by the Na+–K+ ATPase inhibitor, ouabain (1 μM), in a time‐dependent manner, with maximal blockade seen after 90 min. In contrast, the KIR channel inhibitor, Ba2+ (30 μM), had no effect.

When the potassium content of the bathing solution was increased in a single step from 5.9 to 7–19 mM, powerful vasodilatation (max. 75.9±3.6%) was observed. Vasodilatation was transient and, consequently, cumulative addition of potassium produced little vasodilatation, with vasoconstriction predominating at the higher concentrations.

The magnitude of potassium‐induced vasodilatation was similar in endothelium‐containing and endothelium‐denuded rings, and was unaffected by Ba2+ (30 μM), but abolished by ouabain (1 μM).

Ouabain (1 μM, 90 min) powerfully blocked bradykinin‐induced, nitric oxide‐mediated vasodilatation as well as that induced by the nitrovasodilator, glyceryl trinitrate, but that induced by the KATP channel opener, levcromakalim, was hardly affected.

Thus, activation of Na+–K+ ATPase is likely to be involved in the vasodilator responses of the bovine coronary artery to both nitric oxide and EDHF. These findings, together with the ability of potassium to induce powerful, ouabain‐ but not Ba2+‐sensitive, endothelium‐independent vasodilatation, are consistent with this ion contributing to the EDHF response in this tissue.

British Journal of Pharmacology (2003) 139, 982–988. doi:10.1038/sj.bjp.0705329
Blockade of HERG potassium currents by fluvoxamine: incomplete attenuation by S6 mutations at F656 or Y652
Authors: Milnes James T , Crociani Olivia , Arcangeli Annarosa , Hancox Jules C , Witchel Harry J
Published: July 2003

Pharmacological blockade of the Human ether‐a‐go‐go related gene (HERG) potassium channel is commonly linked with acquired long QT syndrome and associated proarrhythmia. The objectives of this study were (i) to identify and characterise any inhibitory action on HERG of the selective‐serotonin re‐uptake inhibitor fluvoxamine, (ii) to then determine whether fluvoxamine shared the consensus molecular determinants of HERG blockade of those drugs so far tested.

Heterologous HERG potassium current (IHERG) was measured at 37°C, using the whole‐cell patch‐clamp technique, from a mammalian cell line (Human embryonic kidney 293) expressing HERG channels. IHERG tails, following repolarisation from +20 to −40 mV, were blocked by fluvoxamine with an IC50 of 3.8 μM.

Blockade of wild‐type HERG was of extremely rapid onset (within 10 ms) and showed voltage dependence, with fluvoxamine also inducing a leftward shift in voltage‐dependent activation of IHERG. Characteristics of block were consistent with a component of closed channel (or extremely rapidly developing open channel) blockade and dependence on open and inactivated channel states. The attenuated‐inactivation mutation S631A partially reduced the blocking effect of fluvoxamine.

The S6 mutations, Y652A and F656A, and the pore helix mutant S631A only partially attenuated blockade by fluvoxamine at concentrations causing profound blockade of wild‐type HERG.

All HERG‐blocking pharmaceuticals studied to date have been shown to block F656 mutant channels with over 100‐fold reduced potency compared to their blockade of the wild‐type channel. Fluvoxamine is therefore quite distinct in this regard from previously studied agents.

British Journal of Pharmacology (2003) 139, 887–898. doi:10.1038/sj.bjp.0705335
Near‐field scanning optical microscopy probes: a comparison of pulled and double‐etched bent NSOM probes for fluorescence imaging of biological samples
Authors: Burgos P. , Lu Z. , Ianoul A. , Hnatovsky C. , Viriot M.‐L. , Johnston L. J. , Taylor R. S.
Published: July 2003

Bent near‐field optical probes for biological applications have been fabricated using a combination of a two‐step chemical etching method and focused ion beam milling to create a well‐defined aperture. The transmission efficiencies have been evaluated as a function of laser wavelength (λ) and aperture size (D) for both large and small core fibres. The probe transmission behaviour follows a (D/λ)3 relationship. The double‐etched probes are compared to pulled probes fabricated from highly GeO2‐doped dispersion compensating fibre and a standard single‐mode optical fibre. The transmission efficiencies of both types of pulled probes are approximately two orders of magnitude lower than double‐etched probes with similar aperture sizes. To demonstrate the utility of the various probes, their imaging performance has been evaluated for samples of polymer beads and phase‐separated phospholipid monolayers of dipalmitoylphosphatidylcholine or cholesterol/phosphatidylcholine/sphingomyelin mixtures. Both pulled and double‐etched probes are suitable for fluorescence imaging of polymer spheres. However, pulled probes are rapidly damaged at the higher input laser intensities required for fluorescence imaging of monolayer samples doped with < 1% of a fluorescent dye‐labelled lipid. The images obtained with the double‐etched probes show excellent spatial resolution and signal/noise, illustrating the potential of such probes for imaging of biological samples.
Polarization artefacts of an FTIR microscope and the consequences for intensity measurements on anisotropic materials
Authors: Coats A. M. , Hukins D. W. L. , Imrie C. T. , Aspden R. M.
Published: July 2003

The infrared beam on both the main Nicolet Nexus bench and the attached Spectra‐Tech Continuum microscope has been shown to be partially polarized. The degree of polarization is ≈ 30%. Although the state of polarization of the infrared beam is of no consequence when measuring the spectra of isotropic materials (gases, liquids), there is a potential problem when considering the spectra of anisotropic materials. Single band intensities are particularly prone to error as small changes in sample thickness or orientation directly affect the intensity. Thickness effects can be overcome by measuring intensity ratios. However, because of the partially polarized nature of the infrared beam, even intensity ratios, illustrated here by the ratio amide I/II of collagen fibres, vary with sample orientation. If overlooked, this effect can be problematic when measuring infrared spectra with an FTIR microscope from samples that are anisotropic or contain anisotropic domains, even though they may appear isotropic on a macroscopic scale. Because dichroic ratios remain unaffected, the intensity ratio from two bands with different transition moments may be used to give a strong indication of the orientation of the sample. This work is illustrated by reference to the FTIR spectra of orientated polyethylene, collagen tape and human trabecular bone.
Variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM): realization and application of a compact illumination device
Authors: Stock K. , Sailer R. , Strauss W. S. L. , Lyttek M. , Steiner R. , Schneckenburger H.
Published: July 2003

A novel compact illumination device in variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) is described. This device replaces the standard condensor of an upright microscope. Light from different laser sources is delivered via a monomode fibre and focused onto identical parts of a sample under variable angles of total internal reflection. Thus, fluorophores in close proximity to a cell–substrate interface are excited by an evanescent wave with variable penetration depth, and localized with high (nanometre) axial resolution. In addition to quantitative measurements in solution, fluorescence markers of the cytoplasm and the plasma membrane, i.e. calcein and laurdan, were examined using cultivated endothelial cells. Distances between the glass substrate and the plasma membrane were determined using the mathematical algorithm of a four‐layer model, as well as a Gaussian‐shaped intensity profile of the illumination spot on the samples. Distances between 0 and 30 nm in focal contacts and between 100 and 300 nm in other parts of the cell were thus determined. In addition to measurements of cell–substrate topology, the illumination device appears appropriate for numerous applications in which high axial resolution is required, e.g. experiments on endocytosis or exocytosis, as well as measurements of ion concentrations proximal to the plasma membrane. The compact illumination device is also suitable for combining TIRFM with further innovative techniques, e.g. time‐resolved fluorescence spectroscopy, fluorescence lifetime imaging (FLIM) or fluorescence resonance energy transfer (FRET).
Dermatophytes isolated from domestic animals in Iran
Authors: Khosravi A. R. , Mahmoudi M.
Published: July 2003

Between 1994 and 1998, a total of 790 feather, hair and skin specimens from a variety of animals with suspected dermatophytoses were studied, of which 248 (31.4%) yielded dermatophytes. The most frequent dermatophytes isolated were Microsporum canis (38.3%), Trichophyton verrucosum (31.8%), T. mentagrophytes (13.3%) and M. gypseum (7.7%). There was a significantly higher proportion of positive cultures from cats (54.8%) than dogs (8.2%), and M. canis was the most common species isolated (87.2 and 50% respectively). Trichophyton verrucosum was the most frequent causative agent of dermatophytoses in ruminants, M. equinum in horses, M. gypseum in rabbits, M. gallinae in chickens and T. mentagrophytes in pet squirrels.
Use of Punica granatum as an antifungal agent against candidosis associated with denture stomatitis
Authors: César de Souza Vasconcelos Laurylene , Sampaio Maria Carméli Correia , Sampaio Fábio Correia , Higino Jane Sheila
Published: July 2003

The aim of this study was to evaluate the use of a gel containing the extract of Punica granatum as an antifungal agent against candidosis associated with denture stomatitis. Sixty patients with denture stomatitis confirmed by clinical and mycologic examination were selected. The patients were randomly allocated in two groups of 30 individuals each according to the medication prescribed: group A used miconazole (Daktarin® gel oral) and group B used a gel of P. granatum Linné (pomegranate). Both groups used the medicines three times per day for 15 days. Forty‐eight hours after finishing the treatment the patients were re‐examined and a second set of samples was collected for mycologic examination. The medicines were evaluated for their clinical response and negativity for Candida. The clinical results showed a satisfactory and regular response in 27 and 21 subjects of groups A and B, respectively. Negativity of yeasts was observed in 25 subjects of group A and 23 of group B. It can be concluded that the extract of P. granatum may be used as a topical antifungal agent for the treatment of candidosis associated with denture stomatitis.
Red information scientist: the information career of J.D. Bernal
Authors: Muddiman Dave
Published: July 2003

John Desmond Bernal (1901‐1970) was one of the most eminent scientists of his generation; he also became, in mid‐twentieth century Britain, an important political figure – the leading public spokesperson of “red” science. One remarkable but hitherto underexplored aspect of his career is a lifelong interest in scientific communication, documentation and information science. Utilising records in the Bernal archive in Cambridge, UK, this paper assesses Bernal's information career. It explores Bernal's initial interest in scientific documentation in the 1930s and examines his blueprint for the reform of scientific communication in Britain, advanced in Bernal's 1939 work, The Social Function of Science. It details his subsequent role, in 1945‐1949, as figurehead of a co‐ordinated but unsuccessful left‐wing campaign to establish an Institute of Scientific Information in Britain. It analyses Bernal's later theoretical papers in information science, and describes his support, in the 1950s and 1960s, for an emerging information profession. Bernal, it concludes, can justifiably be regarded as a major influence on twentieth century information science, above all because of his pioneering focus on the social dimensions of the discipline.
Service please! Rethinking public library Web sites
Authors: Hildebrand Ian
Published: July 2003

A survey of Australian public library Web sites reveals that very few libraries have gone beyond establishing Web sites that provide information about their static library resources and services. Few attempts have been made to deliver a high level of interactive library service to online clients. This paper examines some of the issues that public libraries need to consider in order to rethink the way that the Web can be better used to provide interactive, real time online services. Issues include: commitment of staff and financial resources to online services; developing and maintaining Web technology skills; reclaiming the Web from IT departments; and individual effort versus collaboration.
Lifting parts: putting conceptual insights into practice
Authors: Benders Jos , Verlaar Sander
Published: July 2003

An organization concept is “a more or less coherent, prescriptive vision on (aspects of) organization design”. Practitioners are often faced with the problem of how to use insights from organization concepts. The redesign of a parts distribution centre is discussed to illustrate how two organization concepts and insights from other sources were drawn on and combined to formulate a new organization design. We derive four different reasons for such combinations, namely conceptual clarification, conceptual additions, technical instrumentalization and legitimation.
The role of market orientation on company performance through the development of sustainable competitive advantage: the Inditex‐Zara case
Authors: Mazaira Andrés , González E. , Avendaño Ruth
Published: July 2003

This paper has been developed as a part of research seeking to verify the effects of organisational culture in general, and market orientation in particular, on the behaviour and results of managerial organisations. The difference with other existing work on the same subject is that this work uses the case method to bring managerial reality into closer contact with the university environment. This report contains the first of the case studies carried out in the context of this research, and examines Zara, a strategic unit in the Inditex group, which is shown to be a paradigmatic example of the development of market orientation in a company, as a basis for the company’s performance and competitive advantages.
Rethinking the international marketing strategy: new dimensions in a competitive market
Authors: Rundh Bo
Published: July 2003

The internationalisation has been a slow process for most companies and it has often been a question of gaining experience of other countries step by step. However, during the last two decades, companies have developed their international activities more on a contingency basis. The international market behaviour has also been influenced by a need to take advantage of different market opportunities and by an increasing need to serve customers in the global market environment. Due to the competitive situation, it might also have been necessary to introduce products more quickly on different international markets. In other situations it has been of importance to acquire companies to be able to build up a market position in the local market The purpose with this paper has therefore been to study the need for changes in the international marketing strategy due to requirements from the customers in the local marketplace.
Trends in organisations and selection: an introduction
Authors: Kwiatkowski Richard
Published: July 2003

This article introduces the special issue “Beyond psychometrics: assessment for the new millennium” and further speculates on how a number of organisational trends may influence selection practice in the future. These trends include the continuing emphasis on delayering, of selecting “core” strategic staff, of organisational fit, the impact of technology, the devolution and outsourcing of selection, the rise of teleworking, the questioning of Western assumptions alongside globalisation, and diversity. Developments in selection responses to these factors are identified, such as the popularity of assessment centres, psychometric tests, personality instruments and genetic testing. Some suggestions are made for possible future developments including whole team (or board) selection, the greater use of depth interviews, clinical tests of neurological functioning, and of virtual simulations, and the notion of the possession by individuals of a portable and verifiable assessment portfolio.
Brainsex and occupation: the role of serendipity in the genesis of an idea
Authors: Govier Ernest
Published: July 2003

This paper sets out the events that led to the development of the hypothesis that brains are sexed and that this, at least in part, underlies the sex differentiation that is observed across the range of occupations. It is also suggested that an individual's brainsex is a powerful organising influence on his or her pattern of abilities, emotional life, motives, and interests. It therefore follows that choice of occupation is an indicator of an individual's essential psychological character. The roll played by chance factors in the evolution of the ideas presented here is highlighted.
Challenges and opportunities in mergers and acquisitions: three international case studies – Deutsche Bank‐Bankers Trust; British Petroleum‐Amoco; Ford‐Volvo
Authors: Salama Alzira , Holland Wayne , Vinten Gerald
Published: July 2003

Explores the challenges and opportunities in integration processes, and the factors responsible for the success of cross‐boarder acquisitions within related industries. Emphasises the corporate strategies the three partnered companies used to maximise synergies, and to minimise the negative effects of the unavoidable, but necessary and complex, acculturation process. Evidence extracted from these case studies highlights that successful co‐operation between the firms resulted from the learning process developed by the partners. Knowledge acquisition and the subsequent organisational learning were the important desirable outcomes of the acquisition processes experienced by the organisations under study. The paper is structured as follows: a literature review on integration strategies and cultural impact on mergers and acquisitions precedes the three success stories. Accounts originating from semi‐structured interviews with top executives of Deutsche Bank – Bankers Trust; British Petroleum – Amoco; and Ford – Volvo are compared and contrasted with the literature. Outlines the results achieved in this self‐contained initial stage of an ongoing research project.
Strategy and strategic decision‐making in the smaller local authority
Authors: Dereli Cynthia
Published: July 2003

This paper is based on interviews with four chief executives of district/borough authorities in a two‐tier local government area. The interviews constitute a snapshot of opinion from a group of people ideally placed to relate to the issue under discussion, namely the importance of strategy and strategic partnering for smaller local authorities. The paper considers the extent to which the views of this group develop or interlink with the thinking around strategy, in the literature on strategy in the private sector, and with views of public sector strategic management, particularly in the work and in the critique of the operation of the modernising agenda in the NHS. It concludes that, far from strategy being an irrelevancy to small authorities, it may be that they are ideally placed to provide insights into some of the tensions evident in the strategy debate at all levels.
Supplier choice criteria of executing agencies in developing countries
Authors: Shahadat Khan
Published: July 2003

Procurement by executing agencies, statutory organisations or departments in developing countries responsible for procurement against aid‐funded projects, has received little attention from academic researchers. This study found that executing agencies’ buying decisions are primarily influenced by economic criteria, with most emphasis on price and timely delivery. The reliability of the supplier is the next most important aspect. Suppliers need to design their offers to these organisational buyers, with due consideration of these criteria. Along with these, the offer also needs to project the supplier's proven ability to supply quality products.
Point‐counterpoint: measuring workplace spirituality
Authors: Krahnke Keiko , Giacalone Robert A. , Jurkiewicz Carole L.
Published: July 2003

Presents a point‐counterpoint discussion on whether or not we can – or should – measure something as ineffable as spirituality in organizations. Topics covered include “Workplace spirituality: on the need for measurement” and “Towards wholeness in spirituality research: embracing other ways of knowing”
The training and retention of chefs
Authors: Pratten J.D.
Published: July 2003

The development of the British restaurant sector is described and the ages of those involved are investigated. The progress of a prospective entrant to the industry is traced. The high drop‐out rates are examined to see if any solutions can be offered. A college offering catering courses was visited, and staff interviewed. A total of ten restaurants, all mentioned in the Good Food Guide, were approached and asked about their staff. Whenever possible, the staff were interviewed, and their views noted. They were asked about colleagues who had left, their destinations, and the reasons for going. Some of these were met, and their opinions recorded. The results are often anecdotal, and figures have not been produced, as they may be misleading. However, there is a clear picture of grievances felt. Contemporary newspaper articles are used when they can augment the picture. Ideas about different types of training to develop inter‐personal and administrative skills are discussed, in the hope that they may assist the sector in retaining some of its departing staff.
Decision support system in oil spill cases (literature review)
Authors: Zahra Pourvakhshouri S. , Mansor Shattri
Published: July 2003

Oil pollution is one of the most important issues affecting the marine coastal environments all over the world. There are a large number of organizations to propose plans for managing the problems that coastal areas face, including various methods to combat the oil spills. This project proposes to achieve a decision support system (DSS) to assist the users/managers to choose the most suitable method for combating oil spills, according to the coastal area sensitivity. In this regard and in order to build an appropriate DSS model, some relevant documents regarding oil spills and spills management strategies, GIS‐based modeling, and DSS planning were reviewed, some of which are referred to here.
University student e‐tailing: a marketing study at the entrepreneurship interface
Authors: Teach Richard D. , Schwartz Robert G.
Published: July 2003

This paper is the third report on an enlarging worldwide study of university students’ attitudes and opinions related to e‐commerce. The data set is made up of over 600 business majors distributed between three US and one Australian university. The purpose of the study was to explore students’ attitudes and opinions related to e‐commerce and how those varied based on gender, technology adeptness, shopping intensity, and university affiliation. Differences did exist between the more and less technology adept shoppers. In addition, differences existed between those who had higher or lower shopping intensity. Gender and university affiliation appeared to play little role. These differences perhaps could be utilized by entrepreneurial e‐commerce firms to make their sites more efficient for shopping cart completions and, in this case, for student shoppers. E‐marketing at the entrepreneurship interface appeared similar for students in English speaking countries.
The impact of interpersonal factors on creativity
Authors: Walton André P.
Published: July 2003

This paper reviews current theories related to creativity, the techniques that have been used to measure it, and the implications of important, recent research on how creative actions might be affected by the relationship between the individual and the group. From this work, the possible ways in which creative actions may be influenced by organisational settings are examined, and a revised think‐tank model is suggested that is consistent with maximising individuals’ creative contributions.
Biofilm-Specific Cross-Species Induction of Antimicrobial Compounds in Bacilli
Authors: Yan Liming , Boyd Kenneth G. , Adams David R. , Burgess J. Grant
Published: July 2003

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When
Bacillus licheniformis
strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of
Bacillus subtilis
strain DSM10
T
and
Bacillus pumilus
strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in
B. licheniformis
isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10
T
and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.
Metabolic Engineering of the Carotenoid Biosynthetic Pathway in the Yeast
Xanthophyllomyces dendrorhous
(
Phaffia rhodozyma
)
Authors: Verdoes Jan C. , Sandmann Gerhard , Visser Hans , Diaz Maria , van Mossel Minca , van Ooyen Albert J. J.
Published: July 2003

The
crtYB
locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast
Xanthophyllomyces dendrorhous.
The
crtYB
gene of
X. dendrorhous
, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the
crtYB
gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a β-carotene-accumulating mutant of
X. dendrorhous.
In several transformants containing multiple copies of the
crtYB
gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the β-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (
crtI
) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered
X. dendrorhous
strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via β-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3′-4′-didehydro-β-ψ-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.
Prevalence of Pandemic Thermostable Direct Hemolysin-Producing
Vibrio parahaemolyticus
O3:K6 in Seafood and the Coastal Environment in Japan
Authors: Hara-Kudo Yukiko , Sugiyama Kanji , Nishibuchi Mitsuaki , Chowdhury Ashrafuzzaman , Yatsuyanagi Jun , Ohtomo Yoshimitsu , Saito Akinobu , Nagano Hidetoshi , Nishina Tokuhiro , Nakagawa Hiroshi , Konuma Hirotaka , Miyahara Michiko , Kumagai Susumu
Published: July 2003

Although thermostable direct hemolysin (TDH)-producing
Vibrio parahaemolyticus
has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated
tdh
gene-positive
V. parahaemolyticus
in Japanese seafoods with a
tdh-
specific PCR method, a chromogenic agar medium, and a most-probable-number method. The
tdh
gene was detected in 33 of 329 seafood samples (10.0%). The number of
tdh
-positive
V. parahaemolyticus
ranged from <3 to 93/10 g. The incidence of
tdh
-positive
V. parahaemolyticus
tended to be high in samples contaminated with relatively high levels of total
V. parahaemolyticus
. TDH-producing strains of
V. parahaemolyticus
were isolated from 11 of 33
tdh
-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of
V. parahaemolyticus
were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6
tdh
-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.
Formation of 4-Hydroxy-2,5-Dimethyl-3[2H]-Furanone by
Zygosaccharomyces rouxii
: Identification of an Intermediate
Authors: Hauck Tobias , Brühlmann Fredi , Schwab Wilfried
Published: July 2003

The formation of the important flavor compound 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDMF; Furaneol) from
d
-fructose-1,6-bisphosphate by the yeast
Zygosaccharomyces rouxii
was studied with regard to the identification of intermediates present in the culture medium. Addition of
o
-phenylenediamine, a trapping reagent for α-dicarbonyls, to the culture medium and subsequent analysis by high-pressure liquid chromatography with diode array detection revealed the formation of three quinoxaline derivatives derived from
d
-fructose-1,6-bisphosphate under the applied growth conditions (30°C; pH 4 to 5). Isolation and characterization of these compounds by tandem mass spectrometry and nuclear magnetic resonance spectroscopy led to the identification of phosphoric acid mono-(2,3,4-trihydroxy-4-quinoxaline-2-yl-butyl) ester (Q1), phosphoric acid mono-[2,3-dihydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q2), and phosphoric acid mono-[2-hydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q3). Q1 and Q2 were formed independently of
Z. rouxii
cells, whereas Q3 was detected only in incubation systems containing the yeast. Identification of Q2 demonstrated for the first time the chemical formation of 1-deoxy-2,3-hexodiulose-6-phosphate in the culture medium, a generally expected but never identified intermediate in the formation pathway of HDMF. Since HDMF was detected only in the presence of
Z. rouxii
cells, additional enzymatic steps were presumed. Incubation of periplasmic and cytosolic protein extracts obtained from yeast cells with
d
-fructose-1,6-bisphosphate led to the formation of HDMF, implying the presence of the required enzymes in both extracts.
Use of a Green Fluorescent Protein-Based Reporter Fusion for Detection of Nitric Oxide Produced by Denitrifiers
Authors: Yin Shixue , Fuangthong Mayuree , Laratta William P. , Shapleigh James P.
Published: July 2003

To determine if green fluorescent protein could be used as a reporter for detecting nitric oxide production,
gfp
was fused to
nnrS
from
Rhodobacter sphaeroides
2.4.3.
nnrS
was chosen because its expression requires nitric oxide. The presence of the fusion in
R. sphaeroides
2.4.3 resulted in a significant increase in fluorescent intensity of the cells, but only when nitrite reductase was active. Cells lacking nitrite reductase activity and consequently the ability to generate nitric oxide were only weakly fluorescent when grown under denitrification-inducing conditions. One of the
R. sphaeroides
strains unable to generate nitric oxide endogenously was used as a reporter to detect exogenously produced nitric oxide. Incubation of this strain with sodium nitroprusside, a nitric oxide generator, significantly increased its fluorescence intensity. Mixing of known denitrifiers with the reporter strain also led to significant increases in fluorescence intensity, although the level varied depending on the denitrifier used. The reporter was tested on unknown isolates capable of growing anaerobically in the presence of nitrate, and one of these was able to induce expression of the fusion. Analysis of the 16S rRNA gene sequence of this isolate placed it within the
Thauera aromatica
subgroup, which is known to contain denitrifiers. These experiments demonstrate that this green fluorescent protein-based assay provides a useful method for assessing the ability of bacteria to produce nitric oxide.
Transfection of
Diaporthe perjuncta
with
Diaporthe RNA Virus
Authors: Moleleki Ntsane , van Heerden Schalk W. , Wingfield Michael J. , Wingfield Brenda D. , Preisig Oliver
Published: July 2003

Diaporthe perjuncta
is a pathogen of grapevines worldwide. A positive-strand RNA virus,
Diaporthe RNA virus
(DaRV), occurs in hypovirulent isolates of this fungus. A virus-free isolate from a South African grapevine was transfected with in vitro
-
transcribed positive strands of DaRV. Based on reverse transcription-PCR and partial sequence analysis, the transfected virus was identified as DaRV. The in vitro-transcribed RNA transcripts used to transfect fungal spheroplasts contained parts of the vector at their distal ends. These vector sequences were separated from the DaRV genome during replication in the new host. The transfected isolate had morphological features that differed from those of the isogenic virus-free strain, including production of a yellow pigment, a decreased growth rate, and lack of sporulation. An apple-based pathogenicity test did not reveal any differences in virulence between the virus-free and DaRV-transfected isolates. This study showed that virus-free fungal hosts can be successfully transfected with viruses other than the
Cryphonectria parasitica
hypovirus.
Characterization of Genes Involved in the Metabolism of α-Galactosides by
Lactococcus raffinolactis
Authors: Boucher Isabelle , Vadeboncoeur Christian , Moineau Sylvain
Published: July 2003

Lactococcus raffinolactis
, unlike most lactococci, is able to ferment α-galactosides, such as melibiose and raffinose. More than 12 kb of chromosomal DNA from
L. raffinolactis
ATCC 43920 was sequenced, including the α-galactosidase gene and genes involved in the Leloir pathway of galactose metabolism. These genes are organized into an operon containing
aga
(α-galactosidase),
galK
(galactokinase), and
galT
(galactose 1-phosphate uridylyltransferase). Northern blotting experiments revealed that this operon was induced by galactosides, such as lactose, melibiose, raffinose, and, to a lesser extent, galactose. Similarly, α-galactosidase activity was higher in lactose-, melibiose-, and raffinose-grown cells than in galactose-grown cells. No α-galactosidase activity was detected in glucose-grown cells. The expression of the
aga-galKT
operon was modulated by a regulator encoded by the upstream gene
galR
. The product of
galR
belongs to the LacI/GalR family of transcriptional regulators. In
L. lactis
,
L. raffinolactis
GalR acted as a repressor of
aga
and lowered the enzyme activity by more than 20-fold. We suggest that the expression of the
aga
operon in lactococci is negatively controlled by GalR and induced by a metabolite derived from the metabolism of galactosides.
Effects of Furfural on the Respiratory Metabolism of
Saccharomyces cerevisiae
in Glucose-Limited Chemostats
Authors: Sárvári Horváth Ilona , Franzén Carl Johan , Taherzadeh Mohammad J. , Niklasson Claes , Lidén Gunnar
Published: July 2003

Effects of furfural on the aerobic metabolism of the yeast
Saccharomyces cerevisiae
were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments. Furfural, an important inhibitor present in lignocellulosic hydrolysates, was shown to have an inhibitory effect on yeast cells growing respiratively which was much greater than the inhibitory effect previously observed for anaerobically growing yeast cells. The residual furfural concentration in the bioreactor was close to zero at all steady states obtained, and it was found that furfural was exclusively converted to furoic acid during respiratory growth. A metabolic flux analysis showed that furfural affected fluxes involved in energy metabolism. There was a 50% increase in the specific respiratory activity at the highest steady-state furfural conversion rate. Higher furfural conversion rates, obtained during pulse additions of furfural, resulted in respirofermentative metabolism, a decrease in the biomass yield, and formation of furfuryl alcohol in addition to furoic acid. Under anaerobic conditions, reduction of furfural partially replaced glycerol formation as a way to regenerate NAD
+
. At concentrations above the inlet concentration of furfural, which resulted in complete replacement of glycerol formation by furfuryl alcohol production, washout occurred. Similarly, when the maximum rate of oxidative conversion of furfural to furoic acid was exceeded aerobically, washout occurred. Thus, during both aerobic growth and anaerobic growth, the ability to tolerate furfural appears to be directly coupled to the ability to convert furfural to less inhibitory compounds.
Plasmid Introduction in Metal-Stressed, Subsurface-Derived Microcosms: Plasmid Fate and Community Response
Authors: Smets Barth F. , Morrow Jayne B. , Arango Pinedo Catalina
Published: July 2003

The nonconjugal IncQ plasmids pMOL187 and pMOL222, which contain the metal resistance-encoding genes
czc
and
ncc
, were introduced by using
Escherichia coli
as a transitory delivery strain into microcosms containing subsurface-derived parent materials. The microcosms were semicontinuously dosed with an artificial groundwater to set a low-carbon flux and a target metal stress (0, 10, 100, and 1,000 μM CdCl
2
), permitting long-term community monitoring. The broad-host-range IncPα plasmid RP4 was also transitorily introduced into a subset of microcosms. No novel community phenotype was detected after plasmid delivery, due to the high background resistances to Cd and Ni. At fixed Cd doses, however, small but consistent increases in Cd
r
or Ni
r
density were measured due to the introduction of a single pMOL plasmid, and this effect was enhanced by the joint introduction of RP4; the effects were most significant at the highest Cd doses. The pMOL plasmids introduced could, however, be monitored via
czc-
and
ncc
-targeted infinite-dilution PCR (ID-PCR) methods, because these genes were absent from the indigenous community: long-term presence of
czc
(after 14 or 27 weeks) was contingent on the joint introduction of RP4, although RP4 cointroduction was not yet required to ensure retention of
ncc
after 8 weeks. Plasmids isolated from Ni
r
transconjugants further confirmed the presence and retention of a pMOL222-sized plasmid. ID-PCR targeting the RP4-specific
trafA
gene revealed retention of RP4 for at least 8 weeks. Our findings confirm plasmid transfer and long-term retention in low-carbon-flux, metal-stressed subsurface communities but indicate that the subsurface community examined has limited mobilization potential for the IncQ plasmids employed.
Activity of Free and Clay-Bound Insecticidal Proteins from
Bacillus thuringiensis
subsp.
israelensis
against the Mosquito
Culex pipiens
Authors: Lee LanNa , Saxena Deepak , Stotzky G.
Published: July 2003

Bacillus thuringiensis
subsp.
israelensis
produces parasporal insecticidal crystal proteins (ICPs) that have larvicidal activity against some members of the order Diptera, such as blackflies and mosquitoes. Hydrolysis of the ICPs in the larval gut results in four major proteins with a molecular mass of 27, 65, 128, and 135 kDa. Toxicity is caused by synergistic interaction between the 25-kDa protein (proteolytic product of the 27-kDa protein) and one or more of the higher-molecular-mass proteins. Equilibrium adsorption of the proteins on the clay minerals montmorillonite and kaolinite, which are homoionic to various cations, was rapid (<30 min for maximal adsorption), increased with protein concentration and then reached a plateau (68 to 96% of the proteins was adsorbed), was significantly lower on kaolinite than on montmorillonite, and was not significantly affected by the valence of the cation to which the clays were homoionic. Binding of the toxins decreased as the pH was increased from 6 to 11, and there was 35 to 66% more binding in phosphate buffer at pH 6 than in distilled water at pH 6 or 7.2. Only 2 to 12% of the adsorbed proteins was desorbed by two washes with water; additional washings desorbed no more toxins, indicating that they were tightly bound. Formation of clay-toxin complexes did not alter the structure of the proteins, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the equilibrium supernatants and desorption washes and by dot blot enzyme-linked immunosorbent assay of the complexes, which was confirmed by enhanced chemiluminescence Western blot analysis. Free and clay-bound toxins resulted in 85 to 100% mortality of the mosquito
Culex pipiens
. Persistence of the bound toxins in nonsterile water after 45 days was significantly greater (mortality of 63% ± 12.7%) than that of the free toxins (mortality of 25% ± 12.5%).
Thermal Inactivation of Susceptible and Multiantimicrobial-Resistant
Salmonella
Strains Grown in the Absence or Presence of Glucose
Authors: Bacon R. T. , Ransom J. R. , Sofos J. N. , Kendall P. A. , Belk K. E. , Smith G. C.
Published: July 2003

The heat resistance of susceptible and multiantimicrobial-resistant
Salmonella
strains grown to stationary phase in glucose-free tryptic soy broth supplemented with 0.6% yeast extract (TSBYE−G; nonadapted), in regular (0.25% glucose) TSBYE, or in TSBYE−G with 1.00% added glucose (TSBYE+G; acid adapted) was determined at 55, 57, 59, and 61°C. Cultures were heated in sterile 0.1% buffered peptone water (50 μl) in heat-sealed capillary tubes immersed in a thermostatically controlled circulating-water bath. Decimal reduction times (
D
values) were calculated from survival curves having
r
2
values of >0.90 as a means of comparing thermal tolerance among variables.
D
59°C
values increased (
P
< 0.05) from 0.50 to 0.58 to 0.66 min for TSBYE−G, TSBYE, and TSBYE+G cultures, respectively.
D
61°C
values of antimicrobial-susceptible
Salmonella
strains increased (
P
< 0.05) from 0.14 to 0.19 as the glucose concentration increased from 0.00 to 1.00%, respectively, while
D
61°C
values of multiantimicrobial-resistant
Salmonella
strains did not differ (
P
> 0.05) between TSBYE−G and TSBYE+G cultures. When averaged across glucose levels and temperatures, there were no differences (
P
> 0.05) between the
D
values of susceptible and multiantimicrobial-resistant inocula. Collectively,
D
values ranged from 4.23 to 5.39, 1.47 to 1.81, 0.50 to 0.66, and 0.16 to 0.20 min for
Salmonella
strains inactivated at 55, 57, 59, and 61°C, respectively.

z
D

values were 1.20, 1.48, and 1.49°C for
Salmonella
strains grown in TSBYE+G, TSBYE, and TSBYE−G, respectively, while the corresponding activation energies of inactivation were 497, 493, and 494 kJ/mol. Study results suggested a cross-protective effect of acid adaptation on thermal inactivation but no association between antimicrobial susceptibility and the ability of salmonellae to survive heat stress.
Contrasting Patterns of Phytoplankton Community Pigment Composition in Two Salt Marsh Estuaries in Southeastern United States
Authors: Noble Peter A. , Tymowski Raphael G. , Fletcher Madilyn , Morris James T. , Lewitus Alan J.
Published: July 2003

Phytoplankton community pigment composition and water quality were measured seasonally along salinity gradients in two minimally urbanized salt marsh estuaries in South Carolina in order to examine their spatial and temporal distributions. The North Inlet estuary has a relatively small watershed with minimal fresh water input, while the Ashepoo, Combahee, and Edisto (ACE) Basin is characterized by a relatively greater influence of riverine drainage. Sampling stations were located in regions of the estuaries experiencing frequent diurnal tidal mixing and had similar salinity and temperature regimens. Phytoplankton community pigment composition was assessed by using high-performance liquid chromatography (HPLC) and multivariate statistical analyses. Shannon diversity index, principal-component, and cluster analyses revealed that phytoplankton community pigments in both estuaries were seasonally variable, with similar diversities but different compositions. The temporal pigment patterns indicated that there was a relatively weak correlation between the pigments in ACE Basin and the relative persistence of photopigment groups in North Inlet. The differences were presumably a consequence of the unpredictability and relatively greater influence of river discharge in the ACE Basin, in contrast to the greater environmental predictability of the more tidally influenced North Inlet. Furthermore, the timing, magnitude, and pigment composition of the annual phytoplankton bloom were different in the two estuaries. The bloom properties in North Inlet reflected the predominance of autochthonous ecological control (e.g., regenerated nutrients, grazing), and those in ACE Basin suggested that there was greater influence of allochthonous environmental factors (e.g., nutrient loading, changes in turbidity). These interestuarine differences in phytoplankton community structure and control provide insight into the organization of phytoplankton in estuaries.
A Modified
Saccharomyces cerevisiae
Strain That Consumes
l
-Arabinose and Produces Ethanol
Authors: Becker Jessica , Boles Eckhard
Published: July 2003

Metabolic engineering is a powerful method to improve, redirect, or generate new metabolic reactions or whole pathways in microorganisms. Here we describe the engineering of a
Saccharomyces cerevisiae
strain able to utilize the pentose sugar
l
-arabinose for growth and to ferment it to ethanol. Expanding the substrate fermentation range of
S. cerevisiae
to include pentoses is important for the utilization of this yeast in economically feasible biomass-to-ethanol fermentation processes. After overexpression of a bacterial
l
-arabinose utilization pathway consisting of
Bacillus subtilis
AraA and
Escherichia coli
AraB and AraD and simultaneous overexpression of the
l
-arabinose-transporting yeast galactose permease, we were able to select an
l
-arabinose-utilizing yeast strain by sequential transfer in
l
-arabinose media. Molecular analysis of this strain, including DNA microarrays, revealed that the crucial prerequisite for efficient utilization of
l
-arabinose is a lowered activity of
l
-ribulokinase. Moreover, high
l
-arabinose uptake rates and enhanced transaldolase activities favor utilization of
l
-arabinose. With a doubling time of about 7.9 h in a medium with
l
-arabinose as the sole carbon source, an ethanol production rate of 0.06 to 0.08 g of ethanol per g (dry weight) · h
−1
under oxygen-limiting conditions, and high ethanol yields, this yeast strain should be useful for efficient fermentation of hexoses and pentoses in cellulosic biomass hydrolysates.