bluuebunny/crossref_metadata_2025 huggingface dataset:
Intramolecular Hydrogen Bond Interaction in Selected Diols
Authors: Plass Monika , Kolbe Alfred
Published: December 2003

Solute descriptors characterizing major interactions in solution are accessible based on quantitative structure-property relationships (QSPR). Parameters of such relationships should be additive for functional groups. Because added parameters of monools describing molecular interaction do not meet the experimentally found intermolecular interaction parameters of diols and triols, it is assumed that intramolecular hydrogen bonding is responsible for these deviations. In this paper the intramolecular interactions in several diols are illuminated by IR measurements. Particularly, the influence of intramolecular hydrogen bonding on the absorbances of the OH groups is subject of investigation. Two conclusions can be drawn from the results: The terminal OH groups, which underlie an OH–OH interaction, also change their absorbance intensity in comparison to the free OH band. Secondly, the intermolecular interaction potential is strongly affected by intramolecular hydrogen bonding. The first observation is tentatively quantified as well as the position of the equilibrium between intramolecularly bonded and free diols.
Particle Entropies and Entropy Quanta V. The P–t Uncertainty Relation
Authors: Zimmermann Herbert W.
Published: December 2003

We consider the temporal evolution of an isolated system of N particles from a non-equilibrium state of entropy S = S′ to the equilibrium state of maximum entropy, S = S
max, S′ ≤ S
max. The application of usual density matrix theory to the temporal development of S leads us to dS/dt = 0: The entropy S does not change in time t. Thereby it is irrelevant, whether we consider a non-equilibrium state or an equilibrium state. Consequently, the system cannot irreversibly change by entropy production dS/dt > 0 from S′ to S
max. This is a paradoxial result, which contradicts the experience. It can be traced back to the von Neumann equation, which in principle describes reversible processes and hence is unsuitable for calculating the irreversible evolution of the entropy S in time t. Each irreversible process is accompanied by a positive entropy production P = dS/dt ≥ 0 inside the system, which only vanishes in case of the equilibrium state. In order to overcome the above mentioned difficulties, we assign an operator
P
to the entropy production P, which is defined by the eigenvalue equation
P
|u〉 = P|u〉 with the state vector |u〉 of the N-particle system. There was an extensive discussion about the relation between the production of entropy P on one hand and the progression of time t on the other. Making use of this concept, we combine the operator of entropy production
P
with the time-development operator
U
(t, t
0) of the system and finally deduce the infinitesimal unitary operator
U
(t+τ,t) = 1 + (i/k)τ
P
by means of very general assumptions. Here
P
means the generator of the infinitesimal progression of time τ = t − t
0 and k the Boltzmann constant, representing the atomic entropy unit. Similarly to P we also treat the time t as an observable, defined by
t
|u〉 = t|u〉. We apply the infinitesimal time-evolution operator
U
(t+τ,t) to the operator of time
t
and finally obtain the
P
–
t
commutation relation i[
P
,
t
] = k, which is independent of τ. It shows that the operators
P
and
t
do not commute, and hence P and t are not sharply defined simultaneously. Instead we have uncertainties ΔP and Δt on measuring P and t, which are given by the P–t uncertainty relation ΔPΔt ≥ k/2. It readily allows a discussion of the evolution of the entropy S of the
isolated system in time t from S′ to S
max. Now, the irreversible steps are correctly described by the entropy production P = dS/dt > 0, while the thermal equilibrium is given by P = 0, ΔP = 0, and thus the lifetime of the equilibrium state Δt = ∞. According to the P–t uncertainty relation, the Boltzmann constant k is similarly important to the quantum thermodynamics of irreversible processes like Planck′s constant h to usual quantum mechanics.
Ultramicroelectrode Voltammetric Investigation of the Micellar Phase of Sodium Dodecyl Sulfate in Aqueous Solution
Authors: Yang Zhenyu , Zhao Jinsheng , Xie Yu , Bai Yamin , Du Zhankui , Yang Zhengyu
Published: December 2003

Ultramicroelectrode voltammetric measurements have been made on Sodium Dodecyl Sulfate (SDS) in aqueous solutions using K4Fe(CN)6 as the electroactive probe and H2SO4 as the supporting electrolyte. By extrapolation, the SDS micellar diffusion coefficient (D
m), hydrodynamic radius (R
m), aggregation number (n), and the molecular weight of SDS micellar in aqueous solutions at various surfactant concentrations are obtained. The partition coefficients, KD
, of the electroactive probe between micelle and water continuous phase are also estimated. The results suggest that the probe is sensitive to the surfactant concentration and reflect the changes of the structure and the hydrophobic nature of micelles.
Thermodynamic, Transport and Collision-Induced Light Scattering Data of Xenon Gas Simultaneously Fitted by an Isotropic Interatomic Potential
Authors: El-Kader M.S.A. , Taher B. M.
Published: December 2003

An approximate empirical isotropic interatomic potential for xenon interaction is developed by simultaneously fitting the Morse-Morse-Morse Spline van der Waals potential form to interaction second pressure virial coefficient, viscosity, thermal conductivity and depolarized interaction-induced light scattering data. Absolute zeroth and second moments of the two-body depolarized spectrum have been measured and compared with theoretical calculations using various models for the interatomic potential. In additions, vibrational energy levels, third pressure virial coefficient and self-diffusion calculated for the new potential are compared with experimental ones. The results show that it is the most accurate potentials yet reported for this system. The use of the new potential in lattice sum calculations yields good results for several properties of solid xenon.
Adsorption on Na-Montmorillonite of 1,10-Phenanthroline and Quinoline in Single and Binary Aqueous Solution
Authors: Ferreiro Eladio A. , de Bussetti Silvia G.
Published: December 2003

The adsorption of single aqueous solutions and binary mixtures of 1,10-phenanthroline (OP) and quinoline (Q) on Na-montmorillonite was studied. The isotherms were obtained at 28°C and pH = 11. 1,10-phenanthroline showed a higher values of adsorption on the clay (2.213mmol/g) than quinoline (1.577mmol/g). On the basis of these values a specific surface of 799.6m2/g was determined on the montmorillonite for OP adsorption and 555.6m2/g for Q. 1,10-phenanthroline shows a higher affinity for the clay surface than quinoline, both in pure solution and in binary mixture, especially at low equilibrium concentrations. In the mixtures with an initial concentration of 1:1, the isotherm of OP adsorption can be fitted to the Langmuir equation and reaches a maximum adsorption of 1.810mmol/g, whereas in the case of Q the adsorption isotherm fits the Freundlich equation. Based on the free area unoccupied by OP molecules, and assuming re-accommodation of the aromatic ring on the surface of the clay, the maximum adsorption of Q at this concentration was calculated to be 0.827mmol/g. The diminishing maximum adsorption of OP at other concentrations in the presence of a constant quantity of Q on the surface corresponds to a quadratic equation as a function of the quantity of quinoline adsorbed.The obtained data could not be fitted to the competitive Langmuir adsorption model or to the two- or multi-surface Langmuir models, since adsorption of Q in aqueous solutions with OP does not correspond to the classic Langmuir equation.
Linear Free-Energy Relationships in the Protonation Equilibria and Acid–Base Catalysed Reaction of 4-Substituted Benzohydroxamic Acids
Authors: Ghosh Kallol K. , Tamrakar Pankaj
Published: December 2003

As part of our investigations on the structure-reactivity relationships in protonation and hydrolysis of hydroxamic acids, we have now studied a series of 4-substituted benzohydroxamic acids (4-X-C6H4.CO.NH(OH), with X = H, Me, OMe, Cl, Br, F and NO2) in aqueous sulfuric acid. Good correlation was observed pointing out the validity of the Hammett equation. The protonation constant, ionization constant and rate constant for acid- and base-catalysed hydrolysis reactions seems are to be related in a linear free-energy (LEER) fashion.
Pressure and Temperature Dependence of the Decomposition Rate of Aliphatic tert-Amyl Peroxyesters
Authors: Buback Michael , Nelke Dorit , Vögele Hans-Peter
Published: December 2003

Thermal decomposition of several aliphatic tert-amyl (TA, 1,1-dimethylpropyl) peroxyesters, RC(O)OOTA, has been studied in dilute solution of n-heptane at pressures up to 2500bar and temperatures up to 195°C. The peroxides under investigation were: tert-amyl peroxyacetate (TAPA), tert-amyl peroxy-n-butanoate (TAPnB), tert-amyl peroxy-iso-butanoate (TAPiB, TA peroxy-2-methyl propionate), tert-amyl peroxy-2-ethylhexanoate (TAPO), and tert-amyl peroxypivalate (TAPP, TA peroxy-2,2-dimethyl propionate). The experiments were carried out in a tubular reactor at residence times up to 140s. Peroxide concentration was monitored under continuous flow conditions via quantitative FT-IR spectroscopic analysis in an optical high-pressure cell positioned behind the tubular reactor. First-order decomposition kinetics over several half-lives were observed. For each TA peroxyester, rate equations for the first-order rate coefficient, k
obs(p,T), are presented. k
obs values for TA peroxyester decomposition are by 23 ± 9 per cent above the corresponding numbers reported for associated tert-butyl (TB) peroxyesters, with the same R group and at identical p, T, and solvent environment. The carbon atom of the R moiety that is in α-position to the carbonyl group controls decomposition kinetics. k
obs is smallest in cases where this particular C-atom is a “primary” one and is largest when it is “tertiary”. The “primary” TA peroxyesters are associated with activation energies around 140 kJ mol-1 whereas the E
A′s of the “secondary” and “tertiary” TA peroxyesters are around 130 und 120 kJ mol-1, respectively. The activation volumes, ΔV
‡
obs, of the “secondary” and “tertiary” TA peroxyesters are in the narrow range of ΔV
‡
obs = 3.0 ± 1.5cm3mol-1. The ΔV
‡
obs′s of the “primary” TA peroxyesters are above 8cm3mol-1. Within the limits of experimental accuracy, the activation parameters of TA peroxyester decomposition are identical to the corresponding quantities reported for the associated TB peroxyesters. The activation parameters suggest that “primary” aliphatic peroxyesters decompose via single-bond scission whereas the “secondary” and “tertiary” TA peroxyesters undergo concerted two-bond scission or extremely fast successive scission of the two bonds which latter two processes can not be distinguished on the timescale of the experiments. The mode of primary bond dissociation, via single-bond or via concerted two-bond scission, largely affects initiator efficiency in free-radical polymerization. The peroxide decomposition rate data measured in dilute solution of compressed n-heptane are useful for simulation and optimization of technical high-pressure ethene polymerizations.
Qualities of memories for performed and imagined actions
Authors: Lampinen James M. , Odegard Timothy N. , Bullington Jennifer L.
Published: December 2003

When people imagine performing simple actions they can sometimes become confused about whether they actually performed the actions or only imagined doing so. In the present study participants performed, imagined, and heard groups of actions. During a second session participants imagined some of the earlier actions as well as some entirely new actions. On a later memory test participants sometimes falsely indicated that they had performed actions that they had only imagined and this tendency was a positive function of number of imaginings. We also found that true and false memories differed in terms of degree of perceptual detail, associated thoughts, emotions, contextual information, and kinesthetic detail. Differences between true and false memories were smaller when the action was imagined five times but were not entirely eliminated. These findings suggest that false memories produced in this paradigm can be both compelling and yet subtly different from true memories. Copyright © 2003 John Wiley & Sons, Ltd.
Classroom experiments on the effects of different noise sources and sound levels on long‐term recall and recognition in children
Authors: Hygge Staffan
Published: December 2003

A total of 1358 children aged 12–14 years participated in ten noise experiments in their ordinary classrooms and were tested for recall and recognition of a text exactly one week later. Single and combined noise sources were presented for 15 min at 66 dBA Leq (equivalent noise level). Single source presentations of aircraft and road traffic noise were also presented at 55 dBA Leq. Data were analysed between subjects since the first within‐subjects analysis revealed a noise after‐effect or a asymmetric transfer effect. Overall, there was a strong noise effect on recall, and a smaller, but significant effect on recognition. In the single‐source studies, aircraft and road traffic noise impaired recall at both noise levels. Train noise and verbal noise did not affect recognition or recall. Some of the pairwise combinations of aircraft noise with train or road traffic, with one or the other as the dominant source, interfered with recall and recognition. Item difficulty, item position and ability did not interact with the noise effect. Arousal, distraction, perceived effort, and perceived difficulty in reading and learning did not mediate the effects on recall and recognition. Copyright © 2003 John Wiley & Sons, Ltd.
Acoustic, semantic and phonetic influences in spoken warning signal words
Authors: Edworthy Judy , Hellier Elizabeth , Walters Kathryn , Clift‐Mathews Wendy , Crowther Mark
Published: December 2003

Three experiments are reported which explore the relationship between semantic, acoustic and phonetic variables in the judgement of eight warning signal words. Experiment 1 shows that listeners can distinguish very clearly between urgent and non‐urgent versions of the words when spoken by real speakers, and that some signal words such as ‘deadly’ and ‘danger’ score more highly than words such as ‘attention’ and ‘don't’. It also shows that the three dimensions of perceived urgency, appropriateness and believability of these words are highly correlated. Experiment 2 replicates Experiment 1 using synthesized voices where acoustic variables are controlled. The semantic effects are replicated, and to some extent appropriateness and believability are found to function differently from that of perceived urgency. Experiment 3 compares the same set of eight signal words with a set of phonetically similar neutral words, showing that warning signal words are rated significantly higher, and largely maintain their previous rank ordering. Copyright © 2003 John Wiley & Sons, Ltd.
A comparison of preschoolers' recall of experienced versus non‐experienced events across multiple interviews
Authors: Powell Martine B. , Jones Carolyn H. , Campbell Claire
Published: December 2003

Preschool children's recall of both experienced and non‐experienced activities was examined across three interviews. One hundred and six children aged 4 to 5 years (58 males, 48 females) from both low and high socioeconomic status areas participated in an event called the Deakin Activities, which consisted of two experienced activities. One or two days later, the children were asked to recall what happened in the two activities and an activity they had not experienced which was suggested to have occurred along with the experienced activities. Next, children were given false suggestions about one of the experienced (true‐biased) activities and the non‐experienced (false) activity. For the remaining experienced (true‐unbiased) activity, no questions were asked. Three and eight days after the activities were presented, children were again required to recall all three activities in their own words while a variety of suggestive techniques were used as encouragers. The results revealed that irrespective of the SES group, assent rates across the true and false activities became more similar after the first interview. Furthermore, children's narratives about the false activity became more similar in detail, structure and quality to their narratives about the true activities across interviews. However, the rate of fantastic/improbable details was higher for the false activity compared to the true activities, children reported more interviewer suggestions about the false activity than the true‐biased activity, and there were fewer confabulation errors reported about the false activity compared to the true activities. The implications of the results are discussed. Copyright © 2003 John Wiley & Sons, Ltd.
Awareness of the origin of knowledge predicts child witnesses' recall of alleged sexual and physical abuse
Authors: Thierry Karen L. , Lamb Michael E. , Orbach Yael
Published: December 2003

Children who understand that knowledge may have different origins produce more information in their free‐recall accounts than children who are less aware of source. We examined whether the tendency to make knowledge attributions was related to the number and proportion of details elicited from child witnesses using open‐ended invitations and whether this relationship varied depending on the number of incidents of abuse reported. The tendency to make knowledge attributions was measured in the presubstantive portion of protocol‐guided interviews with 3‐ to 11‐year‐old alleged victims of abuse. Hierarchical regression analyses revealed that the production of knowledge attribution details was positively related to the proportion of substantive episodic details produced by 3‐ to 6‐year‐olds recalling a single incident and by 3‐ to 11‐year‐olds recalling multiple incidents of abuse. Presubstantive source details were also positively correlated with the amount of source information recalled about incidents of abuse. These findings remained significant after controlling for the children's verbosity, the relative prominence of open‐ended invitations, and the children's ages. © 2003 US Government work.
Characteristics of eyewitness identification that predict the outcome of real lineups
Authors: Valentine Tim , Pickering Alan , Darling Stephen
Published: December 2003

Data were analysed from 640 attempts by eyewitnesses to identify the alleged culprit in 314 lineups organized by the Metropolitan Police in London. Characteristics of the witness, the suspect, the witness's opportunity to view the culprit, the crime and the lineup were recorded. Data analysis, using mixed effects multinomial logistic regression, revealed that the suspect was more likely to be identified if the witness is younger than 30, the suspect is a white European (rather than African‐Caribbean), the witness gave a detailed description, viewed the culprit for over a minute and made a fast decision at the lineup. None of the explanatory variables were significantly associated with a mistaken identification of a foil. No independent, statistically reliable effects of weapon focus, cross‐race identification, or of the delay before the identification attempt were observed. Copyright © 2003 John Wiley & Sons, Ltd.
Thematically‐induced arousal in naturally‐occurring emotional memories
Authors: Laney Cara , Heuer Friderike , Reisberg Daniel
Published: December 2003

A number of studies have examined participants' recollection of previously‐experienced emotional events. In these studies, emotion has generally been evoked by means of a specific attention‐grabbing visual stimulus (the sight of a bloody face or a weapon, views of surgery, and the like). We argue, however, that emotion outside of the laboratory is typically induced not by a particular visual stimulus, but by involvement and empathy with an unfolding event. We refer to this as ‘thematically‐induced arousal,’ as opposed to the ‘visually‐induced’ reactions involved in previous studies. In the present paper, we discuss the potential importance of this distinction, and report two studies that analysed sets of memories for emotional events that occurred outside the laboratory. The data make it clear that naturally‐occurring emotional events tend to be thematically‐arousing, not visually‐arousing, and provide an initial characterization of how these two types of arousal might shape memory. We discuss the implications of this finding, but we also emphasize the need for further data in this largely‐unexplored domain. Copyright © 2003 John Wiley & Sons, Ltd.
Tetraploid embryos rescue the early defects of tw5/tw5 mouse embryos
Authors: Sugimoto Michihiko , Karashima Yuko , Abe Kuniya , Tan Seong‐Seng , Takagi Nobuo
Published: December 2003

Summary: tclw5 is a t‐complex recessive lethal mutation of the tw5‐haplotype. Since tw5/tw5 embryos die soon after implantation, the tclw5 gene is thought to play an important role in early embryogenesis. Previous histological studies have demonstrated that tw5 homozygotes do not survive past the gastrulation stage due to extensive death of the embryonic ectoderm, whereas the extraembryonic tissues were less affected. In the present study, we demonstrate that tw5/tw5 embryos may be distinguished from wildtype littermates at embryonic (E) day 5.5. At this stage, the visceral endoderm of tw5/tw5 embryos appeared to be different, possessing smaller and fewer vacuoles compared to normal littermates. This led us to hypothesize that the visceral endoderm may be affected by tclw5. Confirmation was provided by the rescue of tw5/tw5 embryos following aggregation with tetraploid embryos. However, rescued embryos did not survive past E9.0 and displayed an underdeveloped posterior region. This would indicate that the actions of tclw5 extend beyond the midgestation stage. genesis 37:162–171, 2003. © 2003 Wiley‐Liss, Inc.
De novo DNA methylation at the CpG island of the zebrafish no tail gene
Authors: Yamakoshi Kimi , Shimoda Nobuyoshi
Published: December 2003

The zebrafish no tail gene (ntl) is indispensable for the formation of the notochord and the tail structure. Here we showed that de novo DNA methylation occurred at the CpG island of ntl. The methylation started at the segmentation stage and continued after the larval stage. However, it occurred predominantly between 14 and 48 h postfertilization, which overlaps the period in which ntl expression disappears in the notochord and the tailbud. This inverse correlation, together with the methylation‐associated formation of an inaccessible chromatin structure at the ntl CpG island region, suggested the involvement of the de novo methylation in ntl repression. Since no changes in methylation patterns were observed at the CpG islands of four other zebrafish genes, there must be a mechanism in zebrafish for specific methylation of the ntl CpG island. genesis 37:195–202, 2003. © 2003 Wiley‐Liss, Inc.
Analysing the primacy of distance in the utilization of health services in the Ahafo‐Ano South district, Ghana
Authors: Buor Daniel
Published: December 2003

Although the distance factor has been identified as key in the utilization of health services in rural areas of developing countries, it has been analysed without recourse to related factors of travel time and transport cost. Also, the influence of distance on vulnerable groups in utilization has not been an object of survey by researchers.

This paper addresses the impact of distance on utilization, and how distance compares with travel time and transport cost that are related to it in the utilization of health services in the Ahafo‐Ano South (rural) district in Ghana. The study, a cross‐sectional survey, also identifies the position of distance among other important factors of utilization.

A sample of 400, drawn through systematic random technique, was used for the survey. Data were analysed using the regression model and some graphic techniques. The main instruments used in data collection were formal (face‐by‐face) interview and a questionnaire.

The survey finds that distance is the most important factor that influences the utilization of health services in the Ahafo‐Ano South district. Other key factors are income, service cost and education. The effect of travel time on utilization reflects that of distance and utilization. Recommendations to reduce distance coverage, improve formal education and reduce poverty have been made. Copyright © 2003 John Wiley & Sons, Ltd.
Working in a decentralized system: a threat to health workers' respect and survival in Uganda
Authors: Kyaddondo David , Whyte Susan Reynolds
Published: December 2003

This article contributes to the sparse empirical material on the position of health workers within health sector reform. Using qualitative data gathered in 1999, it shows how staff at rural health units in Tororo and Busia Districts experienced the reforms during the first 5 years of decentralization in Uganda. The analysis builds on a framework proposed by Franco et al. to examine the relation between health sector reform and health worker motivation. However, it diverges from their objective description of the factors determining motivation, giving more emphasis to the subjective perspective of the health workers. The categorical distinction between organizational and cultural/community factors was less relevant for them as actors. Two themes cross‐cut their lives inside and outside the health facilities: professional identity, which entailed recognition by both the organization and members of the community; and ‘survival strategies’, which were necessitated by the desire to maintain a status and lifestyle befitting a professional. Reform weakened workers' positions as professionals and hindered facility‐based ‘survival strategies’ that helped them get by on poor salaries. With an overall fall in remuneration, they were more motivated than ever to establish supplementary sources of income outside the formal government health care system. Copyright © 2003 John Wiley & Sons, Ltd.
Determinants of health status and the influence of primary health care services in Latin America, 1990–98
Authors: Moore David , Castillo Eliana , Richardson Chris , Reid Robert J.
Published: December 2003

Primary health care (PHC) services have been advocated as a means by which less developed countries may improve the health of their populations even in the face of poverty, low levels of literacy, poor nutrition and other factors that negatively influence health status. Using aggregated data from the World Bank and UNICEF this study examined which factors, both within the health care system and outside of it, are associated with under‐5 mortality rates in 22 countries of Latin America and the Caribbean during the 1990s. In a multivariate analysis using generalized estimating equations for repeated measures, five factors were found to be independent predictors of lower under‐5 mortality rates (U5MRs). These were vaccination levels, female literacy, the use of oral rehydration therapy, access to safe water and GNP per capita. When the magnitude of these associations were assessed, higher levels of GNP per capita was found to be very weakly associated with lower U5MRs, compared with female literacy and vaccination rates. These findings suggest that government policies which focus only on promoting economic growth, while not making important investments in PHC services, female education and access to safe water are unlikely to see large improvements in health status. Copyright © 2003 John Wiley & Sons, Ltd.
The importance of quality, access and price to health care consumers in Bulgaria: a self‐explicated approach
Authors: Pavlova Milena , Groot Wim , van Merode Godefridus
Published: December 2003

One approach to the problem of low patient satisfaction in Bulgaria is to identify attributes of health care services that the consumers value most and to focus on their improvement. Based on data from a household survey, this paper examines the importance that health care consumers attach to quality, access and price.

The survey was conducted in 2000 among the population of the region of Varna (the third largest city in Bulgaria). The elicitation of attribute importance was based on a self‐explicated method. To analyse the data, an ordered logit regression was performed.

The analysis shows that clinical quality is the most valued characteristic by Bulgarian health care consumers compared with social quality, access and price. Given the poor quality of health care provision in Bulgaria, the allocation of revenues to its improvement appears to be essential in order to raise patient satisfaction and to enhance social efficiency. Copyright © 2003 John Wiley & Sons, Ltd.
Health policy and its implementation in small island developing states in the British West Indies
Authors: McNaught Allan
Published: December 2003

Health policy and its implementation in small island developing states (SIDS) is a neglected area of study, and, seemingly, of little interest. The existing literature is generally characterized by descriptions of failure or incompetence, with little attempt to understand the nature and workings of the policy process in these small, yet complex, societies.

The research undertaken in this article was carried out over 6 years in Anguilla (pop. 9000) and the British Virgin Islands (pop. 20 000), two British Overseas Territories in the North East Caribbean. The purpose of the research was to determine to what extent policy theory and the tools of policy analysis could be used to explain the nature and the outcomes of the health policy and implementation process.

In trying to analyse and understand the policy process in these small islands it was necessary to understand their socio‐politic character. In addition, the development of a model of the public policy and public administration system in the English‐speaking Caribbean was an essential part of this process.

It was found that the conduct and outcome of the policy and implementation process in these islands varied significantly. It is postulated that the variations are anchored in the nature of the local context, the working practices and ideologies of politicians, senior public servants and the local policy elite. Copyright © 2003 John Wiley & Sons, Ltd.
Novel macrolide‐adjustable bidirectional expression modules for coordinated expression of two different transgenes in mice
Authors: Fux Cornelia , Weber Wilfried , Daoud‐El Baba Marie , Heinzen Christoph , Aubel Dominique , Fussenegger Martin
Published: December 2003

Background

Precise control of transgene expression is essential for a variety of applications ranging from gene‐function analysis, biopharmaceutical manufacturing to next‐generation molecular interventions in gene therapy and tissue engineering. The regulation of gene expression is currently a key issue for clinical implementation of gene‐therapy‐based treatments since desired transgene expression may need to be maintained within a narrow therapeutic window for successful treatment of a particular human disease.

Methods

We have designed a novel bidirectional expression module that enables adjustable coregulation of two different transgenes in response to clinical doses of macrolide antibiotics. A bidirectional macrolide‐responsive promoter consisting of a central operator module (ETR) specific for the macrolide‐dependent transactivator (ET1) is flanked by two minimal promoters (PhCMVmin; Phsp70min) which drive expression of two divergently oriented transgenes. Macrolide antibiotics modulate the binding affinity of ET1 to ETR and adjust expression of both transgenes to desired levels.

Results

Bidirectional expression configurations enabled excellent macrolide‐adjustable coregulation profiles of two secreted reporter genes or one‐vector‐based autoregulated fine‐tuning of a single transgene in various transgenic rodent and human cell lines. Following implantation of microencapsulated CHO‐K1 cell derivatives transgenic for macrolide‐controlled bidirectional expression of erythropoietin (EPO) and the human secreted alkaline phosphatase (SEAP) intraperitoneally into mice, serum EPO and SEAP levels could be coadjusted to desired levels by administration of different erythromycin doses.

Conclusions

Based on their in vivo compatibility, the versatile bidirectional and macrolide‐responsive expression modules represent an important advancement on the way to implementing targeted and conditional molecular interventions into a clinical reality. Copyright © 2003 John Wiley & Sons, Ltd.
Increased in vitro and in vivo transgene expression levels mediated through cis‐acting elements
Authors: Johansen Jens , Tornøe Jens , Møller Arne , Johansen Teit E.
Published: December 2003

Background

Gene therapy for neurodegenerative diseases depends critically on the vector system to direct sustained and stable expression of the transgene. It is, however, a commonly observed phenomenon that transgene expression from currently available vectors is down‐regulated following ex vivo gene transfer to the central nervous system (CNS). In an attempt to circumvent this problem, we have systematically evaluated the potential of different cis‐acting elements to increase and stabilize transgene expression in vitro and after grafting of engineered cell lines to the CNS.

Methods

Plasmid vector constructs incorporating Woodchuck hepatitis post‐transcriptional regulatory element (WPRE), cHS4 insulator elements and/or the translational enhancer element SP163 were produced. Stable, polyclonal cultures of HiB5 cells were generated by transfection with reporter constructs, and in vitro transgene mRNA and protein levels were determined. Finally, HiB5 clones engineered to express the enhanced green fluorescent protein (EGFP) were grafted to the rat striatum and expression levels were evaluated.

Results

Inserting the WPRE element downstream of the open reading frame (ORF) of a reporter gene and flanking the transcriptional unit with cHS4 insulator elements significantly increased protein and mRNA expression levels. Surprisingly, the SP163 element, previously reported to be a translational enhancer, apparently did not promote any translational enhancing activity. Furthermore, the SP163 element exerted a negative effect on transcription. The ability of cHS4 and WPRE elements to stabilize in vivo transgene expression was demonstrated by transplantation of HiB5 clones containing expression constructs into the rat striatum.

Conclusion

The data suggest that incorporating cis‐acting elements in gene therapy vectors may result in improvements to currently available therapeutic vectors. Copyright © 2003 John Wiley & Sons, Ltd.
Enhancement of ultrasound‐mediated gene transfection by membrane modification
Authors: Nozaki Tetsuo , Ogawa Ryohei , Feril Loreto B. , Kagiya Go , Fuse Hideki , Kondo Takashi
Published: December 2003

Background

Ultrasound‐mediated gene transfection (USMGT) with an echo contrast agent could be a new promising physical method of triggering localized gene delivery, but the effect is still modest. The aim of this study is to devise a method to improve efficiency of USMGT. We examined the effect of lidocaine and different temperatures on USMGT, each of which is a known membrane modifier, since the plasma membrane can be considered a site of action in USMGT.

Methods

We observed the effect of lidocaine (0.01, 0.1 or 1.0 mM) and different temperatures (7, 20, 37, 42 or 44 °C) on USMGT (1 MHz, 3.6 W/cm2 (ISATA) and 20 s exposure) in the presence of Levovist (10 mg/ml). At 20 h after sonication, transfection efficiency was evaluated by luciferase assay. Membrane fluidity was examined by fluorescence polarization measurement. Cavitational activity was measured by ESR spin trapping with 5,5‐dimethyl‐1‐pyrroline N‐oxide. The number of cells transfected with the GFP gene was counted under a fluorescence microscope.

Results

Lidocaine (1 mM) and heat (42–44 °C) significantly increased luciferase expression approximately 18‐fold and 19‐fold higher than Levovist only. Both treatments were shown to increase membrane fluidity; in addition, heat enhanced a cavitational effect. It was confirmed by an experiment using the GFP gene that increase in luciferase expression was due to the increase in number of cells.

Conclusions

This enhancement could be useful for ultrasound‐mediated gene therapy in the future since both treatments for membrane modification could be directly applied to the living body. Copyright © 2003 John Wiley & Sons, Ltd.
Comparison of wild‐type and class I integrase mutant‐FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epithelium
Authors: Loewen Nils , Leske David A. , Chen Yi , Teo Wu‐Lin , Saenz Dyana T. , Peretz Mary , Holmes Jonathan M. , Poeschla Eric M.
Published: December 2003

Background

In neonatal and adult rodent retina, substantial lentiviral vector expression has been detected primarily in retinal pigment epithelium (RPE), except in very young animals (2–5 days post‐natal). In non‐retinal tissues, studies of lentiviral vectors have utilized various controls. Among the most stringent are class I integrase mutants, which selectively block the integration reaction while leaving all other gag/pol‐encoded functions intact. For HIV‐1 vectors injected into brain, these have been used to simultaneously control for pseudotransduction and verify that long‐term expression requires integration. Such experiments compare particles that differ only in a single amino acid within a single enzyme that forms a very small molar fraction of the virion. Class I integrase mutants have not been described for feline immunodeficiency virus (FIV) integrase, or tested in the eye for any lentiviral vector.

Methods

We compared subretinally and intravitreally injected FIV vectors and followed animals for up to 7 months, a duration that exceeds prior studies. We also compared the wild‐type (WT) vector with one incorporating a single class I amino acid mutation in FIV integrase (D66V). A mock vector (packaging construct absent) was an alternative control. All vectors were vesicular stomatitis virus glycoprotein G (VSV‐G)‐pseudotyped and were injected on day 7 of life. One group of animals received either subretinal or intravitreal injections of WT vector in the right eyes. Control left eyes were injected with mock vector. These animals were sacrificed at 2 or 7 days post‐injection. A second group received subretinal injections of either WT vector or equivalent D66V vector (reverse transcriptase‐normalized to WT), and were analyzed after 2, 3 and 7 months. All eyes were scored for marker gene (β‐galactosidase) expression by an observer blinded to vector assignments.

Results

Subretinal FIV vector injections were much more effective than intravitreal injections. The RPE was the principal retinal layer transduced by the WT vector, and at least 50% of the area of the retina expressed the marker gene at 3 and 7 months. Occasional cells in inner retinal layers also expressed β‐galactosidase at these time points. The sustained retinal expression produced by subretinally injected vector was blocked by the D66V mutation.

Conclusions

These results show that class I integrase mutant FIV vectors are useful control vectors, and that VSV‐G‐pseudotyped FIV vectors produce extensive retinal expression for at least 215 days, the longest duration yet reported for lentiviral vectors in retina. Transgene expression is mostly restricted to RPE after post‐natal day 7 in rats, suggesting that FIV vectors could be used to target RPE for gene therapy. Copyright © 2003 John Wiley & Sons, Ltd.
Engineering of technetium‐99m‐binding artificial receptors for imaging gene expression
Authors: Simonova Maria , Shtanko Olena , Sergeyev Nikolai , Weissleder Ralph , Bogdanov Alexei
Published: December 2003

Background

Optimization of gene therapy protocols requires accurate and non‐invasive quantification of vector delivery and gene expression. To facilitate non‐invasive imaging of gene expression, we have genetically engineered ‘artificial receptors’, i.e. membrane proteins that bind 99mTc‐oxotechnetate (99mTcOT) via transchelation from a complex with glucoheptonate. The latter is a component of a widely used clinical imaging kit.

Methods

The engineered marker proteins were designed as type I and II membrane proteins and consisted of (1) an 99mTcOT‐binding domain, metallothionein (MT), and (2) a membrane‐anchoring domain. Engineered constructs were used for transfection of COS‐1 and 293 cells; the expression of mRNA was verified by RT‐PCR.

Results

Immunofluorescent analysis, cell fractionation and immunoblotting revealed expression of marker proteins on plasma membrane. Transfection of cells resulted in strong positive staining of plasma membrane with anti‐His‐tag antibodies. Scintigraphic imaging in vitro confirmed the ability of transfected cells to bind 99mTcOT. The fraction of bound radioactivity reached a peak (3.53%) when 0.93 MBq 99mTcOT was added to transfected COS‐1 cells. The experiment‐to‐control signal ratio was equal to 32 at the same added dose.

Conclusions

(1) Both types of engineered ‘artificial receptors’ were expressed on the surface of eukaryotic cells; (2) marker proteins were functional in binding 99mTcOT; and (3) type II membrane proteins were more efficient in binding 99mTcOT than type I proteins. We anticipate that the developed approach could be useful for ‘tagging’ transfected cells with 99mTcOT enabling imaging of tracking in vivo transduced cells or cell therapies. Copyright © 2003 John Wiley & Sons, Ltd.
In vivo knockdown of gene expression in brain cancer with intravenous RNAi in adult rats
Authors: Zhang Yun , Boado Ruben J. , Pardridge William M.
Published: December 2003

Background

Gene silencing in mammalian cells is possible with RNA interference (RNAi) with expression plasmids that encode for short hairpin RNAs (shRNA) that hybridize to a specific sequence within a target mRNA. The limiting factor in developing RNAi therapeutics in mammals is the gene delivery system.

Methods

The present studies describe the production of anti‐luciferase shRNA expression plasmids, which are encapsulated in the interior of 85 nm pegylated immunoliposomes (PILs). C6 rat glioma cells, permanently transfected with the luciferase gene, are implanted in the caudate‐putamen nucleus of adult rats, which produces luciferase‐expressing intracranial brain cancer. The PILs are targeted across the blood‐brain barrier and across the tumor cell membrane in vivo with a monoclonal antibody (MAb) to the rat transferrin receptor (TfR). The TfRMAb is tethered to the tips of 1–2% of the poly(ethylene glycol) strands conjugated to the surface of the liposome.

Results

The TfRMAb‐targeted PILs inhibit luciferase gene expression in the brain cancer by 90%, and this effect persists for at least 5 days after a single intravenous injection of 10 µg/rat of plasmid DNA. RNAi therapy directed against the luciferase gene caused no change in expression of tumor γ‐glutamyl transpeptidase. Targeting the empty expression plasmid with the TfRMAb‐PIL resulted in no change in luciferase activity in the brain cancer in vivo.

Conclusions

In vivo RNAi is enabled with a new form of gene delivery system that encapsulates expression plasmids in PILs, which are targeted to distant sites based on the specificity of a receptor‐specific monoclonal antibody. The combined application of the PIL gene delivery system and RNAi expression plasmids enables gene silencing in remote sites such as brain cancer in mammals after intravenous administration. Copyright © 2003 John Wiley & Sons, Ltd.
Ganciclovir nucleotides accumulate in mitochondria of rat liver cells expressing the herpes simplex virus thymidine kinase gene
Authors: van der Eb Marjolijn M. , Geutskens Sacha B. , van Kuilenburg André B. P. , van Lenthe Henk , van Dierendonck Jan‐Hein , Kuppen Peter J. K. , van Ormondt Hans , van de Velde Cornelis J. H. , Wanders Ronald J. A. , van Gennip Albert H. , Hoeben Rob C.
Published: December 2003

Background

Ganciclovir exhibits broad‐spectrum activity against DNA viruses such as cytomegaloviruses, herpes simplex viruses, varicella‐zoster virus, Epstein‐Barr virus and human herpes virus‐6. Ganciclovir is widely applied for anti‐herpetic treatment, cytomegalovirus prophylaxis after organ transplantation, and, more recently, in experimental gene therapy to eradicate cycling cells that express the herpes simplex virus thymidine kinase gene. Although ganciclovir supposedly acts as a chain terminator, there is compelling evidence demonstrating the presence of ganciclovir, but not of acyclovir, incorporated internally into DNA, leaving the precise mechanism by which ganciclovir inhibits DNA synthesis enigmatic.

Methods

To study the potential involvement of mitochondria in the ganciclovir nucleotide cytotoxicity, we used adenovirus‐mediated gene transfer to express herpes simplex virus thymidine kinase in rat liver and administered ganciclovir 2 days post‐infection. The integrity and function of mitochondria in the rat liver cells were evaluated by several techniques. In addition, we analyzed the nucleotide pools in cellular extracts and in isolated mitochondria.

Results

We show that ganciclovir nucleotides are abundantly present in the mitochondria of rat livers that express the HSVtk gene. Already 48 h after administration, 10–30% of the total mitochondrial nucleotide pool consists of ganciclovir nucleotides. Their presence is correlated with a lower amount of mitochondrial DNA, a reduced mitochondrial‐membrane potential, morphological abnormalities, and liver dysfunction.

Conclusions

These data provide evidence for the involvement of mitochondria in the hepatotoxicity of the HStk/ganciclovir combination. This may explain the toxicity of the HSVtk/gancilovir combination in some metabolically active but non‐proliferating cells, such as liver cells. This toxicity limits the applicability of this enzyme/prodrug combination. Copyright © 2003 John Wiley & Sons, Ltd.
Mesenchymal stem cells home to injured tissues when co‐infused with hematopoietic cells to treat a radiation‐induced multi‐organ failure syndrome
Authors: Chapel Alain , Bertho Jean Marc , Bensidhoum Morad , Fouillard Loic , Young Randell G. , Frick Johanna , Demarquay Christelle , Cuvelier Frédérique , Mathieu Emilie , Trompier François , Dudoignon Nicolas , Germain Claire , Mazurier Christelle , Aigueperse Jocelyne , Borneman Jade , Gorin Norbert Claude , Gourmelon Patrick , Thierry Dominique
Published: December 2003

Background

Recent studies have suggested that ex vivo expansion of autologous hematopoietic cells could be a therapy of choice for the treatment of bone marrow failure. We investigated the potential of a combined infusion of autologous ex vivo expanded hematopoietic cells with mesenchymal (MSCs) for the treatment of multi‐organ failure syndrome following irradiation in a non‐human primate model.

Methods

Hematopoietic cells and MSCs were expanded from bone marrow aspirates. MSCs were transduced with the gene encoding for the green fluorescent protein (e‐GFP), in order to track them following infusion. Twelve animals were studied. Nine animals received total‐body irradiation at 8 Gy from a neutron/gamma source thus resulting in heterogeneous exposure; three animals were sham‐irradiated. The animals were treated with expanded hematopoietic stem cells and MSCs, expanded hematopoietic stem cells alone, or MSCs alone. Unmanipulated bone marrow cell transplants were used as controls.

Results

Depending on the neutron/gamma ratio, an acute radiation sickness of varying severity but of similar nature resulted. GFP‐labeled cells were found in the injured muscle, skin, bone marrow and gut of the treated animals via PCR up to 82 days post‐infusion.

Conclusions

This is the first evidence of expanded MSCs homing in numerous tissues following a severe multi‐organ injury in primates. Localization of the transduced MSCs correlated to the severity and geometry of irradiation. A repair process was observed in various tissues. The plasticity potential of the MSCs and their contribution to the repair process in vivo remains to be studied. Copyright © 2003 John Wiley & Sons, Ltd.