bluuebunny/crossref_metadata_2025 huggingface dataset:
Beyond food and nutrition: How can cities be made healthy?
Authors: Gross Rainer
Published: December 2004

Urbanization is one of the predominant demographic trendsof the late 20th century, with populations in developing countriesmoving in ever greater numbers from the rural countryside to citiesof all sizes. The nutrition community is logically mostly fixatedon issues of intake of foods and determination by nutritional status,and see urbanization and urban populations through that prism. Asidefrom assorted positive implications for better nutrition and healthof urbanization, urban residence can have negative trade‐offs including:social deterioration with drugs, crime and homelessness; sedentarism;and chemical, radiation, noise and illumination pollution. Pollutioninteracts with diet to the extent that there is balancing to beaccomplished between the free‐radical damage of pollutants and dietaryantioxidants. Environmental lead contamination continues to be a problemin Jakarta, Indonesia. Sedentarism and lack of solar exposure incities has negative implications for bone health, and risks forosteoporosis. Moreover, the processes in rural areas to produceagricultural products for urban populations can produce airbornepollutants that flow to cities, affecting the quality of urban life. Giventhese considerations, pollution and environmental factors shouldbe added to the alimentary and health‐related determinants oftenplaced into the causal models for malnutrition used for planningpolicy and programs by international agencies.
Systematic review of European Nimravinae (Mammalia, Carnivora, Nimravidae) and the phylogenetic relationships of Palaeogene Nimravidae
Authors: Peigné Stéphane
Published: December 2004

Peigné, S. (2003). Systematic review of European Nimravinae (Mammalia, Carnivora, Nimravidae) and the phylogenetic relationships of Palaeogene Nimravidae. —Zoologica Scripta, 32, 199–229.

The Nimravidae is a family of sabre‐tooth predators which are well represented in North America and Europe. Most of the previous studies have concentrated on the North American taxa which are the best known. However, the relationships between the European and North American genera have never been carefully investigated. This study presents the results of the first phylogenetic systematic review of Palaeogene Nimravidae. The first part of this work presents the systematic revision of several European taxa (Nimravus intermedius, Dinailurictis bonali and Quercylurus major) and also includes the description of new material from the Early Oligocene of western Europe. The second part deals with the phylogenetic relationships of the Palaeogene Nimravidae. Thirty‐three cranial, mandibular and dental characters were analysed, including a precise analysis of the sabre‐tooth adaptation. The resulting data matrix was analysed through a cladistic analysis which included 19 taxa, including a hypothetical ancestor (outgroup), a primitive skull assigned to cf. Eofelis, and the genera Nimravus (two species), Dinaelurus, Eofelis, Dinailurictis, Quercylurus, Dinictis, Pogonodon, Nanosmilus, Hoplophoneus (four species) and Eusmilus (four species). The analysis of the main core of the character matrix (only 15 taxa + hypothetical ancestor) using the program hennig86 resulted in three most parsimonious trees [length 75; consistency index (CI) = 0.82; retention index (RI) = 0.89]; the strict consensus tree (length 76; CI = 0.81; RI = 0.89) is presented. The main results of the analysis are the strong support for the monophyly of the Nimravine; the confirmation of the basal divergence between Nimravini (Nimravus and Dinaelurus) and all other genera; the sister group relationship of Dinictis to Pogonodon; the plesiomorphic morphology of the European genera Eofelis, Dinailurictis and Quercylurus; the strong support of the clade Nanosmilus/Hoplophoneus/Eusmilus; the unresolved relationships of Hoplophoneus species; and the unequivocal assignment of the species sicarius to Eusmilus. Palaeogeographical and evolutionary implications for the Nimravidae are discussed.
Addressing barriers to change: an RCT of practice‐based education to improve the management of hypertension in the elderly.
Authors: Bhrolchain Cliona Ni
Published: December 2004

Method A randomised, single‐blind, controlled trial of incorporating an exploration of barriers to changing practice into an educational strategy for GPs was conducted in 18 practices. Both groups of practices received visits during which they received the results of a previous audit on the management of hypertension in the elderly. The nine intervention practices were encouraged to explore barriers that would prevent them from implementing the pertinent research findings, in addition to an exploration of the issues and creation of a practice action plan. Control groups received the education without the element of exploring barriers to change. The outcome measures were changes in response to a questionnaire before and after the educational intervention.

Results There were statistically significant differences between the two groups after the educational visit. Those practices who were encouraged to explore barriers to change were more likely to show change in behaviour compared to the control group. All the intervention practices produced an action plan for improving the performance, whereas none of the controls did so. The intervention practices also showed more concordance in management protocols after the intervention.

Conclusion Addressing the barriers preventing practitioners from implementing research findings is effective in implementing change.
On Fisher's exact test for detecting gametic disequilibrium between DNA polymorphisms
Authors: ZAPATA C. , ALVAREZ G.
Published: December 2004

Nonrandom associations between DNA polymorphisms are commonly tested by Fisher's exact test in spite of it is seriously conservative. Theoretical statistical studies have shown that the exact test with Tocher's correction does not present this problem but Tocher's correction is never used by experimentalists for detecting gametic associations. We have examined the practical consequences of using these two alternative tests for the detection of nonrandom associations in populations. A total of 1566 pairs of RFLPs of eleven gene regions and sixteen populations from previously published human and Drosophila disequilibrium data were examined. The analysis reveals remarkable differences between the two tests for detecting gametic associations. In some gene regions, the exact test with Tocher's correction detects a percentage of significant associations between RFLPS which is twice or three times higher than that detected by the exact test without correction. Therefore, the widely used exact test can be seriously underestimating disequilibrium in populations. In addition, the study shows that the standard chi‐square test for independence in 2 × 2 tables detects a similar percentage of significant associations between RFLPs than Tocher's test.
Fine structure physical mapping of a 1·9 Mb region of chromosome 13q12
Authors: STILL I. H. , ROBERTS T. , COWELL J. K.
Published: December 2004

Through linkage analysis and the identification of structural chromosome rearrangements, a number of disease genes have been mapped to the pericentromeric region of the long arm of chromosome 13. Structural rearrangements, or deletions, of the 13q12 region have been implicated in a range of myeloproliferative neoplasms, and other haematopoietic malignancies. In particular, seven cases of a t(8;13) (p11; q12·1) rearrangement have been noted in patients with an atypical myeloproliferative disorder associated with T‐cell leukemia and eosinophilia. We have previously identified a CEPH megaYAC, 943E4, which crosses the translocation breakpoint in archival tumour samples from two patients with this t(8;13) translocation. As an initial step in the characterisation of this translocation breakpoint, we have generated a fine structure physical map of this 1·9 Mb YAC. We have used the method of YAC fragmentation to generate a series of deletion constructs of known size, which provide discreet physical landmarks convenient for mapping genetic markers along the 943E4 YAC. Analysis of these deletion constructs defined the order of ESTs and microsatellite markers in 943E4 as: cen‐NIB1257‐(ATP1AL1/D13S283)‐ D13S179E‐(D13S504E/D13S505E)‐D13S824E‐D13S182E‐D13S221‐tel. These markers have also been assigned to physically defined regions relative to the fragmented YAC endpoints and a derived NotI restriction map.
The functional α‐adrenoceptor in dog caudal vesical arteries is mainly an α1A subtype
Authors: Nakazawa M. , Taguchi Y. , Yang X.‐P. , Chiba S.
Published: December 2004

1 The present study attempted to pharmacologically characterize the subtypes of α‐adrenoceptors mediating the vasoconstriction in the isolated and perfused canine vesical artery.

2 Noradrenaline (NA) and phenylephrine (PE, an α1‐adrenoceptor agonist) induced a dose‐dependent vasoconstriction, whereas xylazine (an α2‐agonist) did not induce any clear vascular constrictor response.

3 Prazosin at 0.01 μm and rauwolscine at 0.1 μm failed to affect the NA‐induced vasoconstriction. Prazosin at 0.1 μm antagonized the vasoconstrictor responses to NA, with pKB value of 7.8.

4 WB 4101 at 0.01–0.1 μm dose‐dependently inhibited the responses to NA, with a pKB value of 8.9. The vasoconstrictor responses to NA were not significantly affected by chloroethylclonidine (10–30 μm) or BMY 7378 (0.1 μm).

5 The present results indicate that the canine vesical arteries dominantly contain α1‐adrenoceptors but have no α2‐adrenoceptors, and the functional subtype of α1‐adrenoceptor is characterized as an α1A‐adrenoceptor subtype.
Psychiatric epidemiology of old age: the H70 study – the NAPE Lecture 2003
Authors: Skoog I.
Published: December 2004

Objective: To describe methodological issues and possibilities in the epidemiology of old age psychiatry using data from the H70 study in Göteborg, Sweden.

Method: A representative sample born during 1901–02 was examined at 70, 75, 79, 81, 83, 85, 87, 90, 92, 95, 97, 99 and 100 years of age, another during 1906–07 was examined at 70 and 79 years of age, and samples born between 1922 and 1930 were examined at 70 years of age. The study includes psychiatric examinations and key informant interviews performed by psychiatrists, physical examinations performed by geriatricians, psychometric testings, blood sampling, computerized tomographies of the brain, cerebrospinal fluid analyses, anthropometric measurements, and psychosocial background factors.

Results: Mental disorders are found in approximately 30% of the elderly, but is seldom detected or properly treated. Incidence of depression and dementia increases with age. The relationship between blood pressure and Alzheimer's disease is an example of how cross‐sectional and longitudinal studies yield completely different results. Brain imaging is an important tool in epidemiologic studies of the elderly to detect silent cerebrovascular disease and other structural brain changes. The high prevalence of psychotic symptoms is an example of the importance to use several sources of information to detect these symptoms. Dementia should be diagnosed in all types of studies in the elderly, as it influences several outcomes such as mortality, blood pressure, and rates of depression. Suicidal feelings are rare in the elderly and are strongly related to mental disorders.

Conclusion: Modern epidemiologic studies in population samples should be longitudinal and include assessments of psychosocial risk factors as well as comprehensive sets of biologic markers, such as brain imaging, neurochemical analyses, and genetic information to maximize the contribution that epidemiology can provide to increase our knowledge about the etiology of mental disorders.
Kinematical analysis of handwriting movements in depressed patients
Authors: Mergl R. , Juckel G. , Rihl J. , Henkel V. , Karner M. , Tigges P. , Schröter A. , Hegerl U.
Published: December 2004

Objective:  Motor disturbances are a relevant aspect of depression. Kinematical analysis of movements can be applied to explore which type of motor dysfunction is associated with depression. We hypothesized that depressed patients draw and write significantly slower than controls and that motor disturbances become more pronounced under bi‐manual demands.

Method:  We examined 37 depressed patients and 37 healthy controls using a digitizing graphic tablet and subsequent kinematical analysis of handwriting and rapid drawing movements.

Results:  Depressed patients performed drawing with significantly less regular velocity than controls (P < 0.001), but normal velocity. Motor differences between patients and controls did not increase under bi‐manual demands. Handwriting of patients was abnormally slow (P = 0.04).

Conclusion:  Irregular patterns of velocity peaks in depressed patients point to basal ganglia dysfunction and/or deficient activity of the sensorimotor cortex and the supplementary motor area as a possible substrate of hand‐motor disturbances in depression.
Manifestation of noncentrality in the EPR spectrum of Fe3+ in polycrystalline substances
Authors: Vasyukov V. N. , Chabanenko V. V. , Kochkanyan R. O. , Nechitaı̆lov M. M. , Nabyalek A. , Piechota S. , Szymczak H.
Published: December 2004

The EPR spectrum of an impurity Fe3+ ion in three organic substances is investigated in the temperature range 4.2–295 K: bromocresol green (C21H14Br4O5S), crystal violet (C25H30ClN3), and a compound of crystal violet with fullarene ((C25H30ClN3)+C60). An unusual change in the shape of the EPR spectrum as a function of temperature is observed in all substances investigated. At 4.2 K the spectrum is a superposition of three contributions: two resonance lines with effective g factors g1=4.39 and g2=2.03 and a nonresonance contribution. As the temperature changes, the intensities become redistributed between these three contributions. As temperature increases, the total intensity of line 1 and the nonresonance contribution decrease and the intensity of line 2 increases. The observed properties of the EPR spectra are characteristic for noncentral ions which possess a multiple-minimum potential. The redistribution of the populations of vibronic states of a noncentral Fe3+ ion determines the temperature variation of the EPR spectrum.
Observation of charged excimer complexes radiating in the VUV range in Xe–Ne cryoalloys
Authors: Belov A. G. , Bludov M. A. , Bondarenko E. A. , Doronin Yu. S. , Samovarov V. N. , Yurtaeva E. M.
Published: December 2004

Comparative measurements are performed of the cathodoluminescence of Xe–Ne solid alloys and free Xe clusters. A nonelementary band is observed on the low-energy side of the well-known transition in the neutral excimer complex Xe2* (7.1 eV). The structure and intensity of this band depend on Xe concentration. It is concluded on the basis of an analysis of the experimental data for cryocrystals and ionized clusters that this new band is a superposition of the luminescence of homo- and heteronuclear charged excimer complexes. It is shown that the neon matrix can serve as an effective reservoir for accumulation of hole centers and localized electrons.
Vortex dynamics in starting square water jets
Authors: Ai Jiao Jian , Yu S. C. M. , Law Adrian Wing-Keung , Chua L. P.
Published: December 2004

This paper presents the results of an experimental study on starting square jets at three Reynolds numbers (Re=U0De∕υ=2358,3528,4716) utilizing planar laser induced fluorescence. Starting circular jets under the same initial Re were also investigated as a basis for comparison. Observations showed that for both circular and square jets, the rate of penetration is almost constant within the first four diameters from the exit plane. Beyond that, the rate reaches an asymptotic behavior that is inversely proportional to the square root of time (t1∕2). It was also found that the jet front travels slower in square jets. The interaction and deformation between the spanwise and streamwise vortices in square jets lead to an enhanced entrainment. The phenomena of axis switching, vortex pinch-off, vortices leapfrogging and coalescence were all observed in the experiments and their formation mechanisms are discussed in detail. The normalized value for the pinch-off time obtained is found to be about 7 and the expansion rate of the head vortex core is proportional to t1.5.
320-channel dual phase lock-in optical spectrometer
Authors: Fodor P. S. , Rothenberger S. , Jevy J.
Published: December 2004

The development of a multiple-channel lock-in optical spectrometer (LIOS) is presented, which enables parallel phase-sensitive detection at the output of an optical spectrometer. The light intensity from a spectrally broad source is modulated at the reference frequency, and focused into a high-resolution imaging spectrometer. The height at which the light enters the spectrometer is controlled by an acousto-optic deflector, and the height information is preserved at the output focal plane. A two-dimensional InGaAs focal plane array collects light that has been dispersed in wavelength along the horizontal direction, and in time along the vertical direction. The data is demodulated using a high performance computer-based digital signal processor. This parallel approach greatly enhances (by more than 100x) the speed at which spectrally resolved lock-in data can be acquired. The noise performance of a working system optimized for the 1.3 μm wavelength range is analyzed using a laser diode light source. Time-resolved absorption traces are obtained for InAs quantum dots embedded in a GaAs matrix, and for dispersed films of PbSe nanocrystals.
Crystal shapes and crystallization in continuum modeling
Authors: Hütter Markus , Rutledge Gregory C. , Armstrong Robert C.
Published: December 2004

A crystallization model appropriate for application in continuum modeling of complex processes is presented. As an extension to the previously developed Schneider equations [W. Schneider, A. Köppel, and J. Berger, “Non-isothermal crystallization of polymers,” Int. Polym. Proc. 2, 151 (1988)], the model presented here allows one to account for the growth of crystals of various shapes and to distinguish between one-, two-, and three-dimensional growth, e.g., between rod-like, plate-like, and sphere-like growth. It is explained how a priori knowledge of the shape and growth processes is to be built into the model in a compact form and how experimental data can be used in conjunction with the dynamic model to determine its growth parameters. The model is capable of treating transient processing conditions and permits their straightforward implementation. By using thermodynamic methods, the intimate relation between the crystal shape and the driving forces for phase change is highlighted. All these capabilities and the versatility of the method are made possible by the consistent use of four structural variables to describe the crystal shape and number density, irrespective of the growth dimensionality.
Externally controlled pressure and temperature microreactor forin situx-ray diffraction, visual and spectroscopic reaction investigations under supercritical and subcritical conditions
Authors: Diefenbacher Jason , McKelvy Michael , Chizmeshya Andrew V.G. , Wolf George H.
Published: December 2004

A microreactor has been developed for in situ, spectroscopic investigations of materials and reaction processes with full external pressure and temperature control from ambient conditions to 400 °C and 310 bar. The sample chamber is in direct contact with an external manifold, whereby gases, liquids or fluids can be injected and their activities controlled prior to and under investigation conditions. The microreactor employs high strength, single crystal moissanite windows which allow direct probe beam interaction with a sample to investigate in situ reaction processes and other materials properties. The relatively large volume of the cell, along with full optical accessibility and external temperature and pressure control, make this reaction cell well suited for experimental investigations involving any combination of gas, fluid, and solid interactions. The microreactor’s capabilities are demonstrated through an in situ x-ray diffraction study of the conversion of a meta-serpentine sample to magnesite under high pressure and temperature. Serpentine is one of the mineral candidates for the implementation of mineral carbonation, an intriguing carbon sequestration candidate technology.
Marangoni destabilization on a core-annular film flow due to the presence of surfactant
Authors: Wei Hsien-Hung
Published: December 2004

In this paper, the linear stability of a two-fluid, surfactant-laden core-annular flow is asymptotically examined in the thin-annulus limit. The instability of the system is determined by the interplay between interfacial tensions, Marangoni effects and viscosity stratification. A scaling analysis is developed to identify the dominant instability mechanisms in various parameter regimes. With proper scalings, the combined effects of Marangoni forces and viscosity stratification on the leading order stability are examined for both cases in the absence and the presence of the core inertia. For each case, a coupled set of linear evolution equations is derived for the interfacial deflection and the surfactant concentration. In the absence of the core inertia, the system with a more viscous film is always unstable due to the presence of surfactant. When the core fluid is more viscous, the stabilizing effect of the core inertia is compromised by the Marangoni destabilization. The resulting critical Reynolds number, beyond which the instability is completely suppressed by the core inertia, increases with surfactant elasticity.
Resonant interferometric autocorrelator for estimation of laser pulse duration
Authors: McFerran John J. , Winterflood John , Burton Clinton , Luiten Andre N.
Published: December 2004

We present a simple design for a Mach–Zehnder interferometric autocorrelator that is aimed at real-time laser pulse diagnostics. The device is based around a symmetric and balanced mechanical oscillator that confers a simple and compact design, while still allowing the measurement of a broad range of pulse durations with high speed and high resolution. The device requires a very low drive level and possesses a very weak mechanical coupling to the environment. The apparatus provides two outputs: one output is proportional to the instantaneous velocity of the interferometer arms, which can be integrated to determine the arm position. This gives the means to calibrate the arm position accurately and thereby account for any nonuniformity of the motion. The second output, from a two-photon photodetector, is used to determine the pulse duration. The present device can measure pulses from the femtosecond through to the picosecond domain.
Commercial aspects of cloning and genetic modification in cattle
Authors: Lewis I. M. , French A. J. , Tecirlioglu R. T. , Vajta G. , McClintock A. E. , Nicholas K. R. , Zuelke K. A. , Holland M. K. , Trounson A. O.
Published: December 2004

A range of potential commercial applications of cloning and genetic modification in cattle has been suggested over the last decade. It includes the rapid multiplication of elite genotypes, production of valuable human proteins, altered production characteristics, increased disease resistance and milk with improved nutritional value and processing capabilities. However, an economic return from the sale of product is far from reality in any of these areas. One impediment to achieving economic sustainability is the extremely low efficiency in producing healthy offspring from transferred cloned embryos. Other significant impediments are societal concerns surrounding such technologies, animal welfare issues and regulatory requirements. This review will focus on current biological limitations and technical capabilities in commercial settings, the changes required to allow the production and sale of products at economically sustainable levels, cryopreservation and the progress towards automation of cloning techniques.
Biosorption of Cadmium by Fucus spiralis
Authors: Sastre de Vicente Manuel Esteban , Herrero Roberto , Lodeiro Pablo , Cordero Bruno
Published: December 2004

Environmental Context. Conventional processes for the removal of heavy metals from wastewaters generally involves chemical precipitation of metals (changing the pH) followed by a period to allow the metal precipitates to settle and be separated. These processes are inefficient when the metals are at a low concentration and still demand handling and disposal of toxic metal sludges. An alternative method for heavy metal removal is adsorption onto a biological material, biosorption. The biological materials, including agricultural byproducts, bacteria, fungi, yeast, and algae, all which take up heavy metals in substantial quantities, are relatively inexpensive, widely available, and from renewable sources. However, biological materials are complex and the active mechanisms often unclear.
Abstract. Cadmium biosorption properties of nonliving, dried brown marine macroalga Fucus spiralis from Galician coast (northwest Spain) have been investigated. The biosorption capacity of the alga strongly depends on solution pH; the uptake is almost negligible at pH ≤ 2 and reaches a plateau at around pH 4.0. Cadmium biosorption kinetics by F. spiralis is relatively fast, with 90% of total adsorption taking place in less than one hour. A pseudo second order mechanism has been proved to be able to predict the kinetic behaviour of the biosorption process. The effect of initial cadmium ion concentration, alga dose, solution pH, and temperature on the biosorption kinetics has been studied. The Langmuir, Freundlich, Langmuir–Freundlich, and Tóth isotherms were used to fit the experimental data and to find out the adsorption parameters. Acid–base properties of the alga have been studied potentiometrically in order to calculate the number of acidic groups and the apparent pK value by using Katchalsky model. The pK obtained is comparable with typical values associated to the ionization of carboxyl groups of alginates, supporting the implication of these groups in the biosorption process.
Atmospheric Oxidation Mechanism of Isoprene
Authors: Fan Jiwen , Zhang Renyi
Published: December 2004

Environmental Context. Many plant species biosynthesize and emit the volatile hydrocarbon isoprene. Once in the atmosphere, isoprene is susceptible to a range of reactions involving potentially hundred of products and intermediate compounds. The products of these reactions in turn may pose a risk to human and plant health and impact the climate through the generation of acids, ozone, and atmospheric aerosols.
Abstract. The atmospheric oxidation mechanism of isoprene initiated by OH, O3, NO3, and Cl, which incorporates the most recent laboratory and theoretical studies, is described. A box model intercomparison between the new mechanism and previous available isoprene oxidation mechanisms has been performed. Ozone and OH concentrations are compared with predictions by the previous mechanisms in high and low NOx scenarios. The O3 and OH sensitivities to the chlorine−isoprene reactions have also been investigated by comparing the box model results with and without the chlorine−isoprene reactions, showing that the ozone production rate and OH concentrations are slightly impacted. The new mechanism facilitates more accurate modelling of isoprene photochemistry in the atmosphere.
Organic Trace Gases in the Atmosphere: An Overview
Authors: Williams Jonathan
Published: December 2004

Environmental Context. The major carbon-containing atmospheric gases (carbon dioxide, carbon monoxide, and methane) are found in the atmosphere at the parts-per-million levels, where they affect physical phenomena such as the greenhouse effect. There are however many more carbon-containing gases at much lower levels with many and varied roles; in the main these gases are more chemically active and affect principally chemical phenomena such as the ozone budget. Abstract. An overview of atmospheric organic trace gases is presented. This work is suited to those new to the field and to those seeking to place related activities in a broader context.
105 EXPRESSION OF PLURIPOTENCY-DETERMINING FACTORS Oct-4 AND NANOG IN PRE-IMPLANTATION GOAT EMBRYOS
Authors: He S. , Pant D. , Bischoff S. , Gavin W. , Melican D. , Keefer C.
Published: December 2004

The objective of this study was to determine the expression patterns of the pluripotency-determining factors, Oct-4 and Nanog, in pre-implantation goat embryos. The POU octamer-binding domain transcription factor Oct-4 and the homeobox transcription factor Nanog have been shown to play key roles in the maintenance of pluripotency in the inner cell mass (ICM) of pre-implantation mouse embryos and in embryonic stem cells. As Oct-4 protein has been observed in human, monkey, bovine, and porcine pre-implantation embryos, its role in embryonic development and differentiation may be conserved across these species. The patterns of mRNA expression for Oct-4 and Nanog have not been reported for ruminant embryos. In this study, total RNA was extracted from 10 in vivo-derived goat embryos at each stage (8-cell, morula, and blastocyst) using an Absolutely RNA Nanoprep Kit (Stratagene, La Jolla, CA, USA). The first-strand cDNAs were synthesized using Superscript III (Invitrogen, Carlsbad, CA, USA) and cDNAs were amplified with PfuUltra hotstart PCR master mix (Stratagene). Oct-4 primers were designed based on bovine Oct-4 open-reading sequence, while Nanog primers were designed based on the human Nanog open-reading sequence. Expression screening by PCR was performed. Oct-4 mRNA expression was detected at the 8-cell, morula and blastocyst stages. Sequencing of the 1.1-kb PCR product with Oct-4 primers revealed 87% homology to human cDNA sequence and 96% homology to the bovine sequence. Protein localization of Oct-4 as observed by immunocytochemistry was diffuse at the morula stage, but moved to a more nuclear location at the blastocyst stage. Oct-4 protein and mRNA expression were detected in both the ICM and trophectoderm of expanded blastocysts. This pattern of protein expression is similar to that reported by others in the pig and cow. As caprine, bovine, and porcine embryos all show extensive proliferation and elongation of the trophectoderm, continued expression of Oct-4 protein in the trophectoderm may be necessary to prevent premature differentiation of the trophectoderm. Nanog mRNA was detected at the morula and blastocyst stages. Nanog mRNA was detected in the ICM but not the trophectoderm of expanded goat blastocysts, a pattern that follows the expression observed in mice. Sequencing of the 698 bp PCR product obtained by RT-PCR from goat blastocysts confirmed that the mRNA detected was Nanog. Sequence alignment (ClustalW) showed that the cDNA sequence identities were 96% between goat and human and 70% between goat and mouse. The amino acid identities were 93% between goat and human and 52% between goat and mouse. To our knowledge this is the first report of detection of Nanog in domestic animals. These results are supportive of the premise that core components involved in the control of pluripotency are analogous across vertebrate species.
112 THE PRESENCE OF LAMIN A/C ANTIGENS IN PORCINE EMBRYOS
Authors: Lee K. , Fodor W.L. , Machaty Z.
Published: December 2004

Nuclear lamins are components of the nuclear lamina, and their primary role is to support the nuclear envelope and provide anchorage sites for the chromatin. While type B lamins are expressed in all cells, type A lamins (including lamins A and C) are developmentally regulated and expressed in differentiated cells only. There are conflicting results about the presence of lamin A/C in early mammalian embryos. Lamin A/C was found to localize in the nuclear envelope of bovine, pig, and mouse embryos, while recently it has been reported that early mouse and bovine embryos lacked lamin A/C antigens. It has also been suggested that the existence of lamin A/C in the pronuclei of mouse and bovine nuclear transfer embryos indicated faulty reprogramming. The aim of this study was to investigate the presence of lamin A/C in porcine embryos of different origins (in vivo, parthenogenetic, and nuclear transfer). Embryos of various developmental stages were collected from inseminated gilts. For the production of parthenogenetic embryos, mature oocytes were electroporated and cultured for up to seven days. Fibroblast cells served as differentiated controls; progenitor cells from the olfactory bulb of a porcine fetus were used as undifferentiated controls. Lamin A/C was visualized by immunocytochemistry. Olfactory bulb progenitor cells lacked lamin A/C (0 out of 50 cells showed staining) while all fibroblast nuclei (n = 50) reacted positively with the antibody. GV-stage oocytes, being terminally differentiated cells, also possessed lamin A/C antigens (30/30). Lamin A/C was not detectable in any of the mature oocytes examined (n = 30), but it was found in early cleavage-stage embryos [both in vivo (20/20) and parthenogenetic (30/30)] with the signal becoming weaker in blastocysts (15/15). After nuclear transfer, the lamin A/C signal from fibroblast nuclei disappeared (20/20), consistent with nuclear envelope breakdown. Later it became detectable again; all nuclear transfer embryos reconstructed with either fibroblast or progenitor cells displayed lamin A/C staining in their pronuclei and at all stages examined (n = 65). This suggests that recipient oocytes remodel the donor nuclei and reassemble the nuclear envelopes of both differentiated and undifferentiated cells with type A lamins. Activated oocytes (n = 30) and early embryos (n = 40) were then incubated in the presence of actinomycin D (an inhibitor of RNA polymerase II) or cycloheximide (a protein synthesis inhibitor) for 14 h. Lamin A/C assembly was not perturbed by either treatment, indicating that the assembly did not result from de novo gene transcription but rather from solubilized lamins already in the cytoplasm. The results imply that lamin A/C is present in early pig embryos and that its presence after nuclear transfer is not an indicator of erroneous reprogramming, unlike that reported in cattle and mouse.
128 SERUM TRANSAMINASE ACTIVITIES IN ENDOMETRITIC, REPEAT BREEDER AND ESTRUS CROSSBRED COWS
Authors: Tareen K.D. , Kakar M.A. , Qureshi Z.I. , Lodhi L.A. , Samad H.A.
Published: December 2004

This study was carried out to determine the serum transaminase activities in endometritic, repeat breeder, and anestrus crossbred (Sahiwal Friesian) cows. A total of 60 blood samples were studied for enzyme activity in serum, with a Spectronic 20 using the commercial diagnostic kit adjusted at 546 nm wavelengths. The performance of liver and the intensity of liver damage were studied by iodine flocculation test. The highest mean (142.2 ± 2.10 μ/L) value of (GOT) activity was observed in the anestrus group, while the lowest mean (58.36 ± 2.1 μ/L) value of GOT was seen in the control group. Analysis of variance revealed a significant (P < 0.01) difference for GOT activity in anestrus compared to control, repeat breeder, and endometritic groups. The highest mean (18.45 ± 3.05 μ/L) value of GPT activities was recorded in repeat breeders while the lowest mean (11.81 ± 1.99 μ/L) value was noted in the control. The occurrence of moderate liver damage was 41.67%, whereas the prevalences of mild and normal damage were 41.67 and 16.66%, respectively. The highest activity (134.80 ± 1.27 μ/L) of GOT was recorded in cows with moderate liver damage, while the lowest activity (50.54 ± 2.72 μ/L) of GOT was observed in cows with normal livers. Mild liver-damaged animals had lower mean (62.13 ± 3.14 μ/L) GOT values compare to moderate liver-damaged animals. Analysis of variance revealed that the moderate liver-damaged group differed significantly in GOT activity from the normal and mild liver-damaged groups. The highest mean values of GOP (91.42 ± 2.8 μ/L) and GPT (20.83 ± 3.40 μ/L) activities were seen in cows with weak body condition, while lowest activities of GOP (82.56 ± 6.42 μ/L) and GPT (13.25 ± 1.55 μ/L) activities were recorded in animals with satisfactory body condition. When the milk yield was considered, the highest mean value of GOT (91.73 ± 3.59 μ/L) and GPT (16.09 ± 3.23 μ/L) activities were recorded in high yielder and the lowest mean values of GOP (72.58 ± 4.79 μ/L) and GPT (13.05 ± 1.99 μ/L) activities were observed in low yielder cows. Based on the findings of this study, it was concluded that although the occurrence of moderate fatty liver was 41.67%, this could be associated with the infertility condition of crossbred dairy cows. It is quite possible that cows showing mild liver damage were in recovery stage after severe or moderate liver damage, as the postpartum period in cross bred cows under study varied from 1.5 to 8 months.
133 INSULIN, TRANSFERRIN AND SELENIUM WITH OR WITHOUT BSA IN A SERUM-FREE CULTURE SYSTEM FOR BOVINE EMBRYO, AND ITS SUITABILITY FOR EMBRYOS CULTURED IN SMALL GROUPS
Authors: Daniaux C. , Verhaeghe B. , Donnay I.
Published: December 2004

Serum in embryo culture medium may be a potential cause of abnormal accumulation of lipid droplets, which is correlated to a higher sensitivity to cryopreservation. Moreover, serum may introduce pathogens. With the aim of developing a serum-free culture medium, we first (Experiment 1) investigated the effect of adding ITS (5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium) as a serum substitute in SOF medium on embryos cultured in large groups (20 embryos per culture drop of 20 μL) and we then (Experiment 2) analyzed the effect of adding BSA. In this second experiment, our serum-free culture media were also tested on embryos cultured in small numbers (5 embryos per drop of 20 μL) in order to mimic ovum pickup (OPU) conditions. Embryos were obtained from slaughterhouse oocytes, matured in vitro for 24 h in a serum-free enriched 199 medium (Donnay et al. 2004 Reprod. Fertil. Dev. 16, 274) containing ITS, and fertilized for 18 h. In experiment 1, embryos were cultured in SOF (Holm et al. 1999 Theriogenology 52, 683–700) supplemented with 0.1 mg/mL polyvinylpyrrolidane (PVP) without (SOF) or with ITS (SOF-ITS), or with 5% FCS (SOF-FCS). Cavitation occurred earlier in presence of serum (Table). Adding ITS to SOF increased blastocyst rates at Day 7 and Day 8 post-insemination (p.i.) and also the hatching rate. In experiment 2, embryos were cultured in SOF-FCS, SOF-ITS, or SOF-ITS supplemented with 4 mg/mL fatty acid free BSA (SOF-ITS-BSA). Within each condition, no differences were observed for blastocyst and hatching rates between embryos cultured in large or in small groups. Adding BSA to SOF-ITS increased blastocyst rate at Day 6 p.i. and also the hatching rate. At Days 7 and 8 p.i., blastocyst rates were higher in SOF-FCS than in SOF-ITS and tended to be higher than in SOF-ITS-BSA, especially for embryos cultured in small groups. Cell numbers of the resulting embryos were unaffected. These results indicate that: (1) ITS as supplement to SOF medium promotes embryo development in vitro. (2) BSA as protein supplement to SOF-ITS medium accelerates blastulation and improves hatching rate. (3) SOF-ITS and SOF-ITS-BSA are two serum-free culture media that can sustain development of embryos, also when cultured in small number, even though SOF-FCS tended to afford better rates of development. Further studies will include evaluation of other quality parameters including resistance to cryopreservation.

This work was supported by the Ministery of Agriculture of the Region wallonne de Belgique.
145 COMPARISON OF DEVELOPMENT AND QUALITY OF PORCINE EMBRYOS CULTURED IN DIFFERENT OXYGEN CONCENTRATIONS
Authors: Ock S.-A. , Kim J.-G. , Shi L.-Y. , Jin H.-F. , Mohana Kumar B. , Choe S.-Y. , Rho G.-J.
Published: December 2004

In spite of valuable uses of pig IVP embryos, including IVF and cloned embryos for production of valuable offspring that provide recombinant protein and xenotransplantation, several problems limit the success. In cattle embryo culture, lowering the oxygen concentration resulted in increased development, quality, and cell number, and reduced apoptosis incidence. The high O2 concentration in in vitro culture caused overproduction of reactive oxygen specias (ROS), causing damage to the cell membrane or DNA. The present study, therefore, evaluated the developmental ability of porcine embryos by culturing in either low or high oxygen concentration on the rates of cleavage, development, cell number, and apoptosis. These values were then compared to parthenote embryos, which were activated with electric stimulation by 2 DC pulses of 2.0 kV/cm, 30 μs in 0.3 M mannitol containing 100 μM CaCl2 and 100 μM MgCl2 and then transferred into NCSU23 medium supplemented with 7.5 μg/mL cytochalasin B for 6 h. Cumulus-oocyte complexes from slaughterhouse ovaries were matured in TCM 199 medium supplemented with 10 ng/mL EGF, 0.5 μg/mL FSH, 0.5 μg/mL LH, 0.57 mM cysteine, and 0.91 mM Na pyruvate for 24 h and further cultured in the same medium without FSH and LH for 20 h at 38.5°C, 5% CO2 in air. IVF was carried out in mTBM for 5 h with 1 × 105 sperm/mL by following the previously reported protocols (2004 Methods Mol. Biol. 253, 227–234) with minor modifications. Zygotes were allocated two O2 concentrations; zygotes were cultured in NCSU23 medium supplemented with 0.17 mM Na pyruvate, 2.73 mM Na lactate, and 0.4% BSA for 54 h and subsequently cultured in the same medium supplemented with 5.55 mM glucose for 90 h at 38.5°C, 5% CO2, 5% O2 and 90% N2 (Treatment 1) or 5% CO2, 20% O2 in air (Treatment 2). Statistical analysis was performed with one-way ANOVA by SPSS 10.0 (P < 0.05). Most oocytes (>82%) cleaved and the rates did not differ between groups. However, the rates of blastocyst development from oocytes used as parthenote controls (5% O2, 40.0 ± 13.0 (70/162); 20% O2, 34.5 ± 11.2 (56/161)) were significantly (P < 0.05) higher than those in IVF embryos (5% O2, 27.9 ± 8.0 (70/247); 20% O2, 27.1 ± 7.2 (68/249)). Similarly cell number and apoptosis index by TUNEL staining of blastocysts in parthenote (5% O2, 42 and 15%; 20% O2, 39 and 20%) were significantly (P < 0.05) higher than those in IVF (5% O2, 32 and 13%; 20% O2, 27 and 14%, all respectively). In conclusion, no differences between low and high oxygen concentrations in culture of porcine IVF and parthenote embryos were observed on the rates of cleavages and development into blastocyst cell number and apoptosis incidence. Further research should be carried out to develop a reliable IVP system.
This work was supported by grant No. R05-2004-000-10702-0 from Ministry of Science & Technology, Republic of Korea.
147 EFFECT OF CULTURE SYSTEM ON THE DEVELOPMENT OF IN VITRO-FERTILIZED OR DNA-INJECTED BOVINE EMBRYOS
Authors: Park Y.S. , Min G.S.
Published: December 2004

DNA microinjection has become the most widely applied method for gene transfer in mammals. However, the production of the transgenic bovine is relatively inefficient with the pronuclear microinjection technology. This experiment was designed to compare the two different in vitro production systems the serum-containing system (IVM, IVF, and IVC; TCM199, TALP, and CR1aa) and the Serum-free system (IVM, IVF, and IVC; IVMD101, IVF100 and, IVMD101), on the development of in vitro-fertilized embryos (Experiment 1) and DNA-microinjected embryos (Experiment 2). Korean Native Cow (KNC) ovaries were obtained from slaughterhouse and cumulus oocyte complexes (COCs) were aspirated from 2- to 8-mm follicles. In the serum-containing system, groups of 15 COCs were matured in TCM199 supplemented with 10% fetal calf serum (FBS), 1 μg/mL FSH, 10 μg/mL LH, and 1 μg/mL estradiol-17β for 18 h. In vitro matured oocytes were fertilized using frozen-thawed percoll separated spermatozoa (Day 0) in fer-TALP medium for 20 h. Presumptive zygotes were cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10% FBS (after Day 3). In the serum-free system, groups of 15 COCs were matured in IVMD101 medium, fertilized in IVF100 medium and cultured in IVMD101 medium (Hochi et al. 2003 Theriogenology 59, 675–685). The DNA used for microinjection was a green fluorescent protein. The zygotes were centrifuged in TALP medium at 15,000g for 7 min, and then were microinjected into the pronucleus. All cultures were maintained in an incubator at 39°C, 5%CO2 in air with maximum humidity. Data from three replicates were analyzed by chi-square test. In Experiment 1, there were no differences in the cleavage rates between treatments (71.8% v. 65.3%, respectively). The development rate to the 8-cell stage was significantly higher in the serum-free system than in the serum-containing system (P < 0.05; 47.2% v. 24.7%, respectively). However, the development rates to the blastocyst stage were not different (20.4% v. 16.0%, respectively). In Experiment 2, the development rates to the cleavage, 8-cell, and blastocyst stages were significantly higher in the serum-free system than in the serum-containing system (P < 0.05; 47.2, 25.0, and 5.6% vs. 16.5, 3.5, and 0%, respectively). The results of this study suggest that the serum-free system was not effective on the development of in vitro-fertilized embryos, but it was effective on the development of DNA-microinjected embryos.
This study is supported by Technology Development Program for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea.
169 CULTURE OF MURINE EMBRYONIC STEM CELLS ON NWPF DISCS
Authors: Cetinkaya G. , Arat S. , Odaman Mercan H. , Onur M.A. , Tumer A.
Published: December 2004

Murine embryonic stem cells derived from the inner cell mass of mouse blastocysts can be maintained in culture for extended periods by using feeder layers and leukemia inhibitory factor (LIF). Maintenance of undifferentiated status occurs via LIF-mediated signalling pathways. In this study we cultured embryonic stem (ES) cells in Knockout-DMEM with serum replacement on a three-dimensional matrix, non-woven polyester fabric (NWPF), which is formed from non-arrayed polyethylene teraphthalate fibers. The surface of the fibers was modified by immobilizing LIF. While stimulating the matrix-bound form of LIF in vitro, we also tried to induce LIF-mediated signalling pathways continually. Our goal was to constitute a synthetic microenvironment that would support the undifferentiated growth of murine ES cells. Experimental groups were examined according to colony morphology, alkaline phosphatase activity, SSEA-1 antibody immunoreactivity, and SEM analyses. It was shown that three dimensional macroporous fibrous matrix, NWPF could support growth of undifferentiated ES cells. However, the ratio of undifferentiated colonies was higher on feeder layers than an polymeric surfaces (93% on mouse embryonic fibroblasts; 63,7% on hydrolized polymeric surface, P < 0,05). Results showed that LIF-immobilized surfaces supported undifferentiated growth of ES cells better than hydrolyzed surfaces. Colonies cultured on LIF-immobilized surfaces, had higher alkaline phosphatase activity and undifferentiated phenotype ratio than those on hydrolyzed surfaces. When the soluble or the matrix-bound form of LIF was used, the number of undifferentiated colonies increased in the polymeric groups (77.8% soluble LIF; 81.6% matrix bound LIF P < 0,05). On NWPF discs, ES cells formed big cell aggregates which had high alkaline phosphatase activity but low SSEA-1 immunoreactivity . When they were passaged to feeder layers, SSEA-1 activity increased. We managed to obtain undifferentiated colonies on NWPF discs by using LIF but the skeletal structure of polymeric matrix would be more convenient for differentiation studies.
This study was performed in TUBITAK-RIGEB and supported by a part of grant from Hacettepe University (0102601001).