bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine‐treated fibroblasts as donor cells
Authors: Lai Liangxue , Park Kwang‐Wook , Cheong Hee‐Tae , Kühholzer Birgit , Samuel Melissa , Bonk Aaron , Im Gi‐Sun , Rieke August , Day Billy N. , Murphy Clifton N. , Carter David B. , Prather Randall S. 
Published: August 2002

AbstractFetal‐derived fibroblast cells were transduced with replication defective vectors containing the enhanced green fluorescent protein (EGFP). The transgenic cells were treated with colchicine, which theoretically would synchronize the cells into G2/M stage, and then used as donor nuclei for nuclear transfer. The donor cells were transferred into the perivitalline space of enucleated in vitro matured porcine oocytes, and fused and activated with electrical pulses. A total of 8.3% and 28.6% of reconstructed oocytes showed nuclear envelope breakdown and premature chromosome condensation 0.5 and 2 hr after activation, respectively. Percentage of pronuclear formation was 62.5, 12 hr after activation. Most (91.4%) of the 1‐cell embryos with pronuclei did not extrude a polar body. Most (77.2%) embryos on day 5 were diploid. Within 2 hr after fusion, strong fluorescence was detectable in most reconstructed oocytes (92.3%). The fluorescence in all NT embryos became weak 15 hr after fusion and disappeared when culture to 48 hr. But from day 3, cleaved embryos at the 2‐ to 4‐cell stage started to express EGFP again. On day 7, 85.8% of cleaved embryos expressed EGFP. A total of 9.4% of reconstructed embryos developed to blastocyst stage and 71.5% of the blastoctysts expressed EGFP. After 200 reconstructed 1‐cell stage embryos were transferred into four surrogate gilts, three recipients were found to be pregnant. One of them maintained to term and delivered a healthy transgenic piglet expressing EGFP. Our data suggest that the combination of transduction of somatic cells by a replication defective vector with the nuclear transfer of colchicine‐treated donors is an alternative to produce transgenic pigs. Furthermore, the tissues expressing EGFP from descendents of this pig may be very useful in future studies using pigs that require genetically marked cells. Mol. Reprod. Dev. 62: 300–306, 2002. © 2002 Wiley‐Liss, Inc.
Cloning and sequencing of cDNA encoding for a novel human testis‐specific contraceptive vaccinogen: Role in immunocontraception
Authors: Naz Rajesh K. , Zhu Xiaolong , Kadam Arjun L. 
Published: August 2002

AbstractSperm‐specific antigens are attractive candidates for the development of a contraceptive vaccine. Using the subtractive cDNA hybridization technology, the present study was conducted to obtain a human sperm‐specific antigen. The 32P‐labeled single stranded cDNA of human testis, subtracted with poly(A)+ RNA of human peripheral white blood cells, was used to screen the human testis cDNA‐ZAP II library. The putative positive clones were further screened for binding with the solubilized human oocyte zona pellucida preparation (HZP). After screening 107 colonies, one positive clone, designated contraceptive vaccinogen (CV), was obtained. It had an insert of ∼1.3 kb, that was cloned and sequenced. The sense strand was identified by using the in vitro transcription and translation procedures, and the full‐length sequence was obtained by using the 5′ rapid amplification of 5′ ‐cDNA ends (5′‐RACE) procedure. The full‐length CV cDNA has an ORF of 312 amino acids (aa) with the first ATG Met start codon at nucleotide (nt) 35 and the stop codon TAA, at nt 959. The translated protein has a calculated molecular mass of 35.3 kD and four potential N‐linked glycosylation and several phosphorylation sites. Hydropathy plot generated from the deduced aa sequence showed it to be a membrane‐anchored peptide. Extensive computer search in the database did not find any homology of existing sequences with CV both for nt and aa. Northern blot analysis indicated the human testis‐specific expression of CV antigen. The coding region of CV cDNA was subcloned into pET22b(+) vector and expressed. The expressed recombinant (r)CV protein had a molecular size of ∼44 kD, and it specifically reacted with the ZP3 component of HZP. Rabbit rCV antibodies recognized the rCV, and a cognate antigen of ∼64 kD in the human sperm extract. The antibodies showed binding with the live and methanol‐fixed human sperm, and significantly (P < 0.001) inhibited human sperm penetration of zona‐free hamster oocytes, as well as human sperm binding to human oocyte zona pellucida. These findings indicate that the testis/sperm‐ specific CV antigen has a role in human sperm function and may find clinical applications in the contra ceptive vaccine development and in the specific diagnosis and treatment of male infertility. Mol. Reprod. Dev. 60: 116–127, 2001. © 2001 Wiley‐Liss, Inc.
Expression of retinol‐binding protein messenger RNA and retinoic acid receptors in preattachment bovine embryos
Authors: Mohan M. , Malayer J.R. , Geisert R.D. , Morgan G.L. 
Published: August 2002

AbstractIn cattle, retinoic acid (RA) has been indirectly associated with developmental potential of the embryo. RA is transported by retinol‐binding protein (RBP) and actions of RA are mediated by several subtypes of nuclear retinoic acid receptors (RAR). Bovine embryos, produced in vitro from oocytes harvested from ovaries collected at a local abattoir, were frozen in liquid nitrogen at the oocyte, 2‐, 4‐, 8‐, 16 to 20‐cell, morula, blastocyst, and hatched blastocyst stages. Employing reverse transcription polymerase chain reaction (RT‐PCR) we investigated mRNA expression for RBP, RARα, RARβ, RARγ, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Total RNA was extracted from 25 pooled embryos at each stage and RT‐PCR analysis was repeated thrice. GAPDH transcript was detected in all stages. Transcripts for RBP, RARα, and RARγ were also detected in all stages from the oocyte through to the hatched blastocyst. Expression of RARβ was not detected at any stage. Whole‐mount immunohistochemistry was performed with intact and hatched blastocysts using polyclonal antibodies against RARα and RARγ2 to investigate if these embryonic mRNAs were translated to the mature protein. Strong immunostaining was observed for both RARα and RARγ2 in the inner cell mass and trophectoderm of intact and hatched blastocysts. Expression of mRNA for RBP, RARα, RARγ, and of the RARα and RARγ2 receptor proteins in the bovine embryo suggests that RA is likely to directly regulate gene expression during preimplantation development in that species. Mol. Reprod. Dev. 60: 289–296, 2001. © 2001 Wiley‐Liss, Inc.
Solubilized zona pellucida proteins and progesterone induce calcium influx and the acrosome reaction in capacitated dog spermatozoa
Authors: Brewis I.A. , Morton I.E. , Moore H.D.M. , England G.C.W. 
Published: August 2002

AbstractSpermatozoa from the sperm‐rich fractions of the semen of 6 beagle dogs were capacitated and the effect of both zona pellucida (ZP) proteins and progesterone on calcium flux and the acrosome reaction measured. Sperm calcium flux was determined using the dual wavelength calcium probe indo‐1/AM (6 μM) in a flow cytometric assay (one ejaculate from each dog examined; n = 6). No calcium flux was observed in the negative control treatments (RPMI medium or DMSO). Both heat‐solubilized bitch ZP proteins and progesterone caused a similar response characterized by a gradual but marked influx of calcium ions which was sustained over 2 min. Acrosomal status was assessed by indirect immunofluorescence using a specific monoclonal antibody following 1 hr incubation for each treatment (four ejaculates from each dog examined; n = 24). The level of acrosomal exocytosis was very high for samples treated with ZP proteins (70.3 ± 2.1%) and progesterone (84.6 ± 1.5%) and was significantly different from the respective controls (P < 0.001). Interestingly the patterns of calcium flux in response to both ZP proteins and progesterone were in contrast to the situation in other species studied to date raising the possibility that the mechanism for triggering the acrosome reaction may be different in dog spermatozoa. In addition the high degree of progesterone‐induced acrosomal exocytosis compared to other species raises the probability that the majority of dog spermatozoa are already undergoing the acrosome reaction before they reach the egg ZP. Mol. Reprod. Dev. 60: 491–497, 2001. © 2001 Wiley‐Liss, Inc.
Acrocentric centromere organization within the chromocenter of the human sperm nucleus
Authors: Gurevitch Merav , Amiel Aliza , Ben‐Zion Moshe , Fejgin Moshe , Bartoov Benjamin 
Published: August 2002

AbstractIt has recently been reported that in human sperm cells, the centromeres are clustered in a chromocenter in the interior region of the nucleus. The aim of the present study was to determine the intra‐chromocenter organization of the five centromeres of the acrocentric chromosomes responsible for the biosynthesis of rRNA. The acrocentric centromeres were labeled by fluorescence in situ hybridization (FISH) after mild decondensation of the sperm nuclei to preserve the tail structure. The tail was used as a topographical marker for the orientation of the nucleus. The following results were obtained: (a) the association among the five centromeres was higher than expected from random distribution; (b) all the centromeres observed were randomly located within the chromocenter, occupying about 87% of the total area of the internal nucleus; (c) a major subpopulation of centromeres was located in a preferred area occupying 8.3% of the total nuclear area, with a peak 0.6 μm on the lateral axis and 1.0 μm on the apical side of the longitudinal axis; and (d) The dispersion of the centromeres was not influenced by the degree of the nuclear decondensation. We conclude that in human sperm nuclei, the acrocentric centromeres are organized within a nonlocalized structural element in the chromocenter. The chromocenter can range from an expanded size of 87% of the whole nucleus to a preferred size of 8.3% independent of the degree of nuclear decondensation. These findings have important implications for nuclear function (rRNA) that is not directly related to sperm cell function or early embryo development. Mol. Reprod. Dev. 60: 507–516, 2001. © 2001 Wiley‐Liss, Inc.
Apoptosis in the preimplantation mouse embryo: Effect of strain difference and in vitro culture
Authors: Kamjoo Marziel , Brison Daniel R. , Kimber Susan J. 
Published: August 2002

AbstractCell death by apoptosis occurs predominantly in the inner cell mass (ICM) of the blastocyst, the cell population which carries the germ line and gives rise to the foetus. The frequency of apoptosis in blastocysts varies widely within outbred species such as human and cow. We have addressed the basis of this variation by examining the relative influence of strain difference and in vitro culture conditions on apoptosis, using embryos from two different strains of mice (MF1 and C57BL6/CBA) in two different culture media (M16 and kSOM). In both strains and all crosses apoptosis was first detected by nuclear fragmentation or TUNEL [Terminal deoxynucleotidyl transferase mediated d‐UTP nick end‐labelling] labelling at the early blastocyst stage. This was true for embryos which had developed in vivo, and in vitro in both M16 and kSOM. The apoptotic index in blastocysts was found to be significantly different between both media and strain (P < 0.0001). Blastocysts from MF1 × MF1 at equivalent stages had an apoptotic index of 32.4% in M16 and 20.3% in kSOM. Blastocysts from C57BL6/CBA × C57BL6/CBA had an apoptotic index of 19.3% in M16 and 14.4% in kSOM. When embryos of similar cell number were compared, a significantly greater apoptotic index was found for cultured MF1 × MF1 embryos with a cell number between 40 and 59 compared to similar directly flushed C57BL6/CBA embryos (P = 0.001), and MF1 embryos (P < 0.0005). MF1 × MF1 embryos and C57BL6/CBA × MF1 embryos of 60–79 cells had a greater apoptotic index in M16 than kSOM (P < 0.0005) but the difference between media was not significant for C57BL6/CBA × C57BL6/CBA. When strain was compared MF1 × MF1 embryos of 60–79 cells had a significantly greater apoptotic index than C57BL6/CBA × MF1 in both media (P < 0.0005 M16; P = 0.002 kSOM) and than C57BL6/CBA × C57BL6/CBA in M16 (P = 0.019). Our data suggest that genetic make‐up and the chemical composition of simple medium are equally important in determining the level of apoptosis. Mol. Reprod. Dev. 61: 67–77, 2002. © 2002 Wiley‐Liss, Inc.
Ultrastructural studies on developing follicles of the spotted ray Torpedo marmorata
Authors: Prisco Marina , Loredana Ricchiari , Piero Andreuccetti 
Published: August 2002

AbstractLight and ultrastructural investigations on sub‐adult and adult sexually mature females, demonstrates that in Torpedo marmorata folliculogenesis starts in the early embryo and that the two ovaries in the adult contain developing follicles of various sizes and morphology. Initially, the follicle is constituted by a small oocyte, surrounded by a single layer of squamous follicle cells. The organization is completed by a basal lamina and, more externally, by a theca, that at this stage is composed by a network of collagen fibers. As the oocyte growth goes on, during previtellogenesis and vitellogenesis, the organization of the basal lamina and of the oocyte nucleus does not change significantly. The basal lamina, infact, remains acellular and constituted by fibrils intermingled in an amorphous matrix; the nucleus always shows an extended network of chromatin due to the lampbrush chromosomes, and one or two large nucleoli. By contrast, the granulosa (or follicular epithelium), the ooplasm, and the theca cells significantly change. The granulosa shows the most relevant modifications becoming multi‐layered and polymorphic for the progressive appearance of intermediate and pyriform‐like cells, located respectively next to the vitelline envelope, or spanning the whole granulosa. The appearance of intermediate cells follows that of intercellular bridges between small follicle cells and the oocyte so that one can postulate that, as in other vertebrates, small cells differentiate into intermediate, and then pyriform‐like cells, once they have fused their plasma membrane with that of the oocyte. Regarding the ooplasm, one can observe as in previtellogenic follicles, it is characterized by the presence of intermediate vacuoles containing glycogen, while in vitellogenic follicles by an increasing number of yolk globules. The theca also undergoes significant changes: initially, it is constituted by a network of collagen fibers, but later, an outermost theca esterna containing cuboidal cells and an interna, with flattened cells, can be recognized. The role of the different constituents of the ovarian follicle in the oocyte growth is discussed. Mol. Reprod. Dev. 61: 78–86, 2002. © 2002 Wiley‐Liss, Inc.
Transition to adulthood: Supporting young adults to access social, employment, and civic pursuits
Authors: Hughes Carolyn 
Published: August 2002

Abstract&ldquo;Transition support&rdquo; is a concept new to the field of secondary transition. This paper describes the development of a model of transition support as derived from the empirical literature &lpar;Hughes and Carter. &lsqb;2000&rsqb; The Transition Handbook. Baltimore, MD: Paul H. Brookes&rpar;. The components of the Transition Support Model are both empirically based and field&hyphen;tested among practitioners. The over 500 transition support strategies that compose the model have been used by practitioners in the field to improve educational and postschool outcomes for high school students. These strategies are grouped according to two main goals &lpar;i.e., &ldquo;developing support in the environment&rdquo; and &ldquo;increasing students&apos; competence&rdquo;&rpar; and corresponding components. Literature supporting the Transition Support Model is described as well as the use of the model in the transition planning process. Suggestions are made for application of transition support in high schools and the need for future research. MRDD Research Reviews 7:84&ndash;90, 2001. &copy; 2001 Wiley&hyphen;Liss, Inc.
Glutamate signaling and the fetal alcohol syndrome
Authors: Olney J.W. , Wozniak D.F. , Jevtovic‐Todorovic V. , Ikonomidou Chrysanthy 
Published: August 2002

AbstractIt has been known for three decades that ethanol, the most widely abused drug in the world, has deleterious effects on the developing human brain, but progress has been slow in developing animal models that are optimal for studying this problem, and the underlying mechanisms have remained elusive. Recently, we have shown that during the synaptogenesis period, also known as the brain growth spurt period, ethanol has the potential to trigger widespread neuronal suicide (apoptosis), deleting many millions of neurons from the in vivo mammalian brain. It appears that ethanol triggers apoptotic neurodegeneration by a dual mechanism (blockade of NMDA glutamate receptors and excessive activation of GABAA receptors), in that ethanol has both NMDA antagonist and GABAmimetic properties; we have shown that other drugs which have either of these properties trigger apoptotic neurodegeneration in the developing brain. The brain growth spurt period in humans spans the last trimester of pregnancy and the first several years after birth. Thus, our findings provide a likely explanation for the reduced brain mass and neurobehavioral disturbances associated with the human fetal alcohol syndrome. Furthermore, since NMDA antagonist and GABAmimetic drugs are sometimes abused by pregnant women and also are used as anticonvulsants, sedatives, or anesthetics in pediatric medicine, our findings suggest the possibility that exposure of the developing brain to these various drugs either pre or postnatally could contribute to mental disability syndromes that have heretofore been attributed to unknown causes. In addition, the observation that ethanol and related drugs trigger massive neuronal apoptosis in the developing brain provides an unprecedented opportunity to study both neuropathological aspects and molecular mechanisms of apoptotic neurodegeneration in the in vivo mammalian brain. MRDD Research Reviews 2001;7:267–275. © 2001 Wiley‐Liss, Inc.
Bulk magnetic susceptibility effects on the assessment of intra‐ and extramyocellular lipids in vivo
Authors: Szczepaniak Lidia S. , Dobbins Robert L. , Stein Daniel T. , McGarry J. Denis 
Published: August 2002

AbstractLocalized proton spectroscopy provides a novel method for noninvasive measurement of lipid content in skeletal muscle. It has been suggested that the chemical shift difference between lipid signals from distinct compartments in skeletal muscle might be caused by bulk magnetic susceptibility (BMS) differences from lipids stored in intra‐ (IMCL) and extramyocellular (EMCL) compartments. Direct evidence is provided to confirm the theoretical prediction that compartment symmetry is responsible for discrimination between resonances of IMCL and EMCL. Phantoms imitating lipids in skeletal muscle were constructed using soybean oil to represent EMCL, and Intralipid™, an intravenous fat emulsion of fine droplets, to represent IMCL. It was found that the chemical shift of Intralipid™ is independent of the BMS effects, while the resonance of soybean oil shifts in a predictable manner determined by the geometry of the compartment. Magn Reson Med 47:607–610, 2002. © 2002 Wiley‐Liss, Inc.
White matter mapping using diffusion tensor MRI
Authors: Tench C.R. , Morgan P.S. , Wilson M. , Blumhardt L.D. 
Published: August 2002

AbstractDiffusion tensor MRI is used to define trajectories that reflect the long‐range order of in vivo white matter (WM) fiber tracts. Fiber tracking is particularly prone to cumulative error from noise and partial volume along the length of the trajectory paths, but the overall shape of each path is anatomically meaningful. By considering only the long‐range similarity of path shapes, a method of constructing 3D maps of specific WM structures has been developed. A trajectory is first computed from an operator‐selected seed voxel, located within the anatomical structure of interest (SOI). Voxels from the same structure are then automatically identified based on the similarity of trajectory path shapes, assessed using Pearson's correlation coefficient. The corpus callosum and pyramidal tracts in 14 patients with multiple sclerosis, and in 10 healthy controls were mapped by this method, and the apparent diffusion coefficient (ADC) was measured. The ADC was significantly higher in patients than in controls, and higher in the corpus callosum than in the pyramidal tracts for both groups. Using this method the different functional structures in the WM may be identified and mapped. Within these maps, MRI parameters can be measured for subsequent comparison with relevant clinical data. Magn Reson Med 47:967–972, 2002. © 2002 Wiley‐Liss, Inc.
In vivo 1H NMR spectroscopy of the human brain at 7 T
Authors: Tkáč Ivan , Andersen Peter , Adriany Gregor , Merkle Hellmut , Uǧurbil Kâmil , Gruetter Rolf 
Published: August 2002

AbstractIn vivo 1H NMR spectra from the human brain were measured at 7 T. Ultrashort echo‐time STEAM was used to minimize J‐modulation and signal attenuation caused by the shorter T2 of metabolites. Precise adjustment of higher‐order shims, which was achieved with FASTMAP, was crucial to benefit from this high magnetic field. Sensitivity improvements were evident from single‐shot spectra and from the direct detection of glucose at 5.23 ppm in 8‐ml volumes. The linewidth of the creatine methyl resonance was at best 9 Hz. In spite of the increased linewidth of singlet resonances at 7 T, the ability to resolve overlapping multiplets of J‐coupled spin systems, such as glutamine and glutamate, was substantially increased. Characteristic spectral patterns of metabolites, e.g., myo‐inositol and taurine, were discernible in the in vivo spectra, which facilitated an unambiguous signal assignment. Magn Reson Med 46:451–456, 2001. © 2001 Wiley‐Liss, Inc.
T2 relaxation reveals spatial collagen architecture in articular cartilage: A comparative quantitative MRI and polarized light microscopic study
Authors: Nieminen Miika T. , Rieppo Jarno , Töyräs Juha , Hakumäki Juhana M. , Silvennoinen Johanna , Hyttinen Mika M. , Helminen Heikki J. , Jurvelin Jukka S. 
Published: August 2002

AbstractIt has been suggested that orientational changes in the collagen network of articular cartilage account for the depthwise T2 anisotropy of MRI through the magic angle effect. To investigate the relationship between laminar T2 appearance and collagen organization (anisotropy), bovine osteochondral plugs (N = 9) were T2 mapped at 9.4T with cartilage surface normal to the static magnetic field. Collagen fibril arrangement of the same samples was studied with polarized light microscopy, a quantitative technique for probing collagen organization by analyzing its ability to rotate plane polarized light, i.e., birefringence (BF). Depthwise variation of safranin O‐stained proteoglycans was monitored with digital densitometry. The spatially varying cartilage T2 followed the architectural arrangement of the collagen fibril network: a linear positive correlation between T2 and the reciprocal of BF was established in each sample, with r = 0.91 ±
0.02 (mean ± SEM, N = 9). The current results reveal the close connection between the laminar T2 structure and the collagen architecture in histologic zones. Magn Reson Med 46:487–493, 2001. © 2001 Wiley‐Liss, Inc.