bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of identical twins by separating two‐cell rat embryos
Authors: Matsumoto K. , Miyake M. , Utsumi K. , Iritani A.
Published: February 2005

Rat identical twins were produced from two‐cell embryos. In the presence of cytochalasin B, rat two‐cell embryos could be separated efficiently into two blastomeres by micromanipulation. Isolated blastomeres, embedded in agar cylinders and cultivated in ligated rat oviducts for 3 days, developed to the morula or blastocyst stage. After removing the agar, pairs of developed one‐half embryos were transferred into Day 1 oviducts or Day 4 uteri of pseudopregnant rats. The percentage of embryos, separated either in the presence or absence of cytochalasin B, that developed into live fetuses was higher in cases of uterine transfer than in cases of oviduct transfer (38% vs. 18%, 31% vs. 15%, respectively). Throughout the present experiment, nine pairs of identical twins were successfully produced. This is the first report of the production of identical rat twins by separating two‐cell embryos.
Age dependence of bovine oocyte activation
Authors: Ware C. B. , Barnes F. L. , Maiki‐Laurila M. , First N. L.
Published: February 2005

The ability of bovine oocytes to undergo parthenogenetic activation using either a Ca++‐Mg++‐H+ ionophore (A23187) or electric shock was investigated, as a prelude to understanding activation potential following nuclear transfer into ooplasm. Oocytes were collected from slaughterhouse ovaries by aspiration of 1–5‐mm follicles. The time of placement into maturation medium was noted, and maturational age (time in culture) measured from that point. After exposure to activating conditions eggs were cultured for a further 12–16 hours, fixed, and stained with aceto‐orcein. Oocytes that progressed to telophase or pronuclear formation were considered activated. Concentrations of A23187 ranging from 100 pM to 100 μM showed that 1–100 μM levels resulted in 94–100% activation at 30 hours maturation. Frequency of activation differed from controls (no ionophore) at 100 nM (49%; P < 0.05). With A23187 maximum response occurred between 26 and 30 hours of maturation (77% and 92%, respectively). A short pulse electric shock, capable of causing oocyte membrane fusion, gave similar results relative to maturational age (82% and 90% activation for 26 and 30 hours, respectively). Therefore, maximum response to the two activating stimuli occurred in oocytes at similar maturational ages. Exposure to activating conditions prior to onset of activating ability (18 hours) followed by another exposure at 26 hours showed that the oocytes were still fully able to activate upon reaching maturational activation competence. Because cytochalasin B is present in the medium used for nuclear transfer, oocytes were incubated with cytochalasin B prior to exposure to an activating stimulus. Frequency of activation was similar to the control treatment (61% and 73%). The effect of mechanical stress of cytoplasm removal and replacement by electrofusion on activation was also not significant. Overall, maturational age of the oocyte was the main determinant of activation ability.
Protein carboxyl methyltransferase activity specific for age‐modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis
Authors: O'Connor Clare M. , Germain Bonnie J. , Guthrie Kathleen M. , Aswad Dana W. , Millette Clarke F.
Published: February 2005

An antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age‐damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell‐deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4–5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5‐fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.
Modification of the zona‐free hamster ova bioassay of boar sperm fertility and correlation with in vivo fertility
Authors: Berger Trish , Parker Kent
Published: February 2005

These studies were designed to evaluate the ability of the zona‐free hamster ova bioassay to detect differences in fertility of boar sperm. In the first study, sperm from two previously infertile boars were compared to sperm from seven previously fertile boars. The percentage of zona‐free hamster ova penetrated by sperm from the previously infertile boars was significantly lower than the percentage of ova penetrated by sperm from previously fertile boars (18% of ova penetrated vs. 83%, P < .001). In the 14 ejaculates from the previously infertile boars that had ejaculate motilities of 50% or greater, the percentage of zona‐free hamster ova penetrated continued to be lower than in ejaculates from the fertile boars. One of the two previously infertile boars consistently had a normal semen analysis. The only two observed manifestations of his reduced fertility were his zero conception rate and the limited ability of his sperm to penetrate zona‐free hamster ova. In the second study, females were inseminated with equal numbers of sperm from two previously fertile males and the paternity of offspring determined at birth. The experiment was replicated with four combinations of six boars. A high correlation was observed between the percentage of offspring sired and the ability to penetrate zona‐free hamster ova (R = .89). Neither morphology nor the ability of the sperm to undergo an acrosome reaction during in vitro incubation was correlated with fertility in the competitive mating situation. These results suggest the zona‐free hamster ova bioassay can improve the in vitro fertility assessment of fresh boar semen.
Pregnant mares' serum gonadotropin source influences fertilization and fresh or thawed embryo development, but the effect is genotype specific
Authors: Schmidt P. M. , Monfort S. L. , Wildt D. E.
Published: February 2005

The influence of the source of pregnant mares' serum gonadotropin (PMSG) on the num ber, quality, and in vitro development of mouse embryos before and after freezing was evaluated among three genotypes: N:NIH(S), C57BL/6N, and C3H/HeN‐MTV−. Immature females were given PMSG from one of five commercial sources. Following col lection ( 116 hr later), embryos were evaluated for stage of development, and four‐to eight‐cell embryos were pooled within genotype and assigned to standardized fresh or freeze‐thaw culture trials. Different PMSG sources stimulated the production of different num bers of total embryos (P < 0.05) but not necessarily more embryos suitable for freezing. Differences in embryo production among genotypes indicated that absolute embryo num bers using a single mouse genotype may not accurately reflect the potency of a specific gonadotropin source. The PMSG source also affected the ability of an embryo to survive in culture either immediately after collection or after frozen storage. The effect, however, was genotype specific, with some mouse strains being relatively insensitive to PMSG source, whereas gonadotropin source played a major role in determining in vitro viability in others. Development rates for freshly collected embryos differed, often inconsistently, from those of thawed embryos regardless of the PMSG source used, demonstrating that fresh embryo development cannot be used to estimate expected post‐thaw survival. In vitro development of thawed embryos is influenced not only by genotype, but also the source of the gonadotropin used to promote follicular development and oocyte maturation. These findings may explain, in part, the wide variation in embryo viability and culture rates reported among laboratories and intraspecies animal populations.
Gamete processing in an environmental chamber: Effect on in vitro fertilization outcome
Authors: Gwatkin Ralph B. L. , Quigley Martin M. , Collins Robert L.
Published: February 2005

A study of mouse gamete processing for in vitro fertilization (IVF) under various conditions showed that it is necessary to control the atmosphere if the temperature is raised from 22°C to 37°C. The data suggest that maximum IVF success is attained by processing the gametes at 37°C, under an atmosphere of 5% O2 and 5% CO2, and overlaying the medium with silicone oil.
Influence of cumulus cell processes on oolemma permeability and lethality of isolated mouse oocytes cultured in Ca2+‐free medium
Authors: De Felici Massimo , Dolci Susanna , Siracusa Gregorio
Published: February 2005

Cumulus cell processes remaining in the zona pellucida of mouse occytes mechanically isolated from the ovary have been indirectly visualized by labeling their actin microfilament core with rhodaminyl‐phalloidin. If the isolation of the oocytes is performed in Ca2+‐free medium, the preisence of such processes allows the entry into the cell of low molecular weight molecules (such as 5‐6 carboxyfluorescein) and contributes to the death of the cell in such experimental conditions.

Following dissolution of the zona pellucida (by enzymatic or acidic treatment) the oocyte is no longer permeable to small molecules and becomes resistant to Ca2+‐free medium, probably as a consequence of the collapse of cumulus cell processes. The role of cumulus cell processes and gap junctions in the permeability of mechanically isolated ovarian oocytes is discussed.
Structure of the zona pellucida and cumulus oophorus in three species of native Australian rodents
Authors: McGregor Lesley , Flaherty Sean P. , Breed William G.
Published: February 2005

The sperm head of many Australian hydromyine rodents has three curved hooks projecting from its anterior margin; the structure of the hooks has been characterized, but their function is unknown. In this study, we have investigated whether the hooks might have evolved to assist sperm penetration through more formidable egg vestments, particularly the zona pellucida. Cumulus‐oocyte complexes were obtained from two species that possess a three‐hooked sperm head (Pseudomys australis and P. nanus) and one species that does not (Notomys alexis) and examined by light and electron microscopy. After fixation in the presence of ruthenium red, the zona pellucida was found to consist of a fibrillar meshwork, but there were no interspecific structural differences. A corona radiata was absent, and the cumulus extracellular matrix was composed of filaments and electron‐dense granules in each species. Measurements of the zona thickness in freshly ovulated, unfixed oocytes revealed that it was thinnest (7.8 μm) in P. australis. Which has a three‐hooked sperm head, and thickest (11.4 μm) in N. alexis, the species in which the ventral hooks are absent. Hence, no correlation was found between the thickness of the zona pellucida or the structure of the cumulus‐oocyte complex, and the presence of three hooks on the sperm head. We conclude, therefore, that it is unlikely that the evolution of the three‐hooked sperm head is an adaptation for penetration of increased barriers around the oocyte.
Inhibition of the human sperm acrosome reaction by proteinase inhibitors
Authors: De Jonge C. J. , Mack S. R. , Zaneveld L. J. D.
Published: February 2005

The analogue of the second messenger cAMP, dibutyryl cAMP (dbcAMP), was shown to induce the human sperm acrosome reaction to the same extent as calcium ionophore A23187, providing preliminary evidence for the involvement of the adenylate cydase system in the acrosome reaction (AR) of human spermatozoa. Using the human synchronous acrosome reaction system, proteinase inhibitors were tested for their effect on the dbcAMP‐induced human sperm acrosome reaction. The proteinase inhibitor 4′‐acctamidophenyl4‐guanidinoben‐zoate (AGB), an inhibitor of proacrosin activation and of acrosin, when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, significantly (P < 0.01) inhibited the acrosome reaction at final concentrations of 1 × 10−4 M to 1 × 10−6 M in comparison to dbcAMP treatment alone. At concentrations less than 1 × 10−6 M, no significant inhibitory effect was seen. Similarly, para‐aminobenzamidine (pAB), also an inhibitor of proacrosin activation and of acrosin, significantly (P < 0.01) inhibited the dbcAMP‐induced acrosome reaction at final concentrations of 1 × 10‐4 M to I × 10‐6 M when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, in comparison to stimulation by dbcAMP alone. However, at concentrations less than 1 × 10−6 M, no significant (P > 0.05) inhibitory effect was seen. These results indicate that a serine proteinase, most likely acrosin, has a role in the human sperm acrosome reaction and suggest that the enzyme functions after the involvement of the adenylate cyclase system.
Development in vitro of investment‐free marsupial embryos during cleavage and early blastocyst formation
Authors: Selwood Lynne
Published: February 2005

Twenty embryos at the cleavage‐arrested four‐cell stage of the brown antechinus, Antechinus stuartii, were used to develop a method to obtain investment‐free embryos by incubation at 35°C in 2.5% pancreatin, 0.05% trypsin, and 0.05% pronase. Control intact embryos and the investment‐free embryos were incubated in Dulbccco's Modified Eagle's Medium with high glucose and 10% foetal calf serum in 5% CO2 in air at 35°C for 48 h. Release of embryos from the investments (shell, muooid, and zona pcllucida) took 4–4½ h in pancreatin, 6 h in trypsin, and 55 min to 1¼ h in pronase. Embryonic survival in vitro was best following pronase digestion. Pronase concentrations of 0.05%, 0.1 %, 0.25%, 0.5%, and 1.0% were used to free one‐to 16‐cell stage embryos of brown antechinus and the stripe‐faced dunnart, Sminthopsis macroura. from their investments. Postincubation survival and development of embryos in vitro was best using between 0.05% and 0.25% pronase. Blastomeres in both intact and investment‐free embryos shared similar characteristics during cleavage and blastocyst formation. The combined effect of these characteristics was that in intact embryos, no morula was formed, and the blastocyst developed, when cell numbers were high enough (about 32 cells), as a unilaminar structure flattened against the zona pellucida and without an inner cell mass. In investment‐free embryos, the blastomeres dispersed over the base of the culture vessel. The investments were necessary to confine the blastomeres, to provide a surface for blastomere flattening, and to allow normal cellular associations to develop.
Chemical N‐desulfation of heparin negates its ability to capacitate bovine spermatozoa
Authors: Milter David J. , Ax Roy L.
Published: February 2005

Heparin specifically and saturably binds to bovine spermatozoa and stimulates capacitation as assessed by the ability of spermatozoa to undergo a zona pellucida‐induced acrosome reaction (AR) in vitro. However, the structural features of heparin important for capacitation are poorly understood The purpose of this study was to determine the importance of the sulfatc content of heparin for its potency to bind to bull spermatozoa and promote agglutination and capacitation. The pyridine salt of heparin was Nndesulfated, which reduced its mean sulfate content from 19.7% to 11.6%. The N‐desulfated heparin was then resulfated by incubation with trimcthylamine sulfur trioxide for 6,12, or 24 hr, raising sulfate to original concentrations. Heparin but not N‐desulfated beparin competed with [3H]‐heparin to bind to spermatozoa. Heparin at 11.6 μg/ml reduced [3H]‐heparin binding by half when competing with a saturating concentration of the radidabeled compound (12 μg/ml). N‐desulfated heparin did not displace [3H]‐heparin. Heparin, resulfated 6 hr or 12 hr, was equal to native heparin in binding potency. Heparin at 50,100, or 250 μg/ml caused more than 40% of the cells to head‐to‐head agglutinate in aggregates of 8 or more. N‐desulfated heparin did not cause agglutination. After spermatozoa were incubated with 0, 5,10, 50, 100, or 250 μg/ml of heparin for 4 hr, 100 μg/ml of lysophosphatidylcholine (LPC)‐induccd AR within 20 min in 21.3, 37.7, 27.8, 45.3, 54.2, or 42.5% of the cells, respectively. Sperm exposed to the same concentrations of N‐dcsulfated heparin exhibited AR of 17.7,27.3,24.3,22.5,27.7, or 33.8%, respectively, following exposure to LPG Resulfated heparin did not agglutinate or capacitate spermatozoa. In conclusion, N‐desulfation of heparin abolished heparin's ability to bind to, agglutinate, and capacitate bovine spermatozoa. Resulfation of N‐desulfated heparin restored binding activity but not agglutination or capacitation activity.
Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos
Authors: Malter Henry E. , Cohen Jacques
Published: February 2005

Hatching in vitro was studied following zona drilling of 507 two‐cell mouse embryos using three methods: 1) acidic Tyrode's (AT), 2) partial zona dissection (PZD) using a sharp micronecdle, and 3) zona chiseling (CH), using a large beveled needle. PZD and CH were performed while the embryos were kept in a sucrose/PBS solution. Hatching was compared to 191 umnicromanipulated controls. The incidences of cavitation and completion of hatching did not differ between groups, however more micromanipulated embryos (20–25%) hatched partially than controls (9%). The zona pellucida thinned in 59/59 (100%) control blastocysts during expansion, but in only 3/205 (2%) micromanipulated blastocysts. The hatching gap was wide in all control embryos, but smaller in 96/129 (75%) micromanipulated embryos. Partially hatched blastocysts with a ‘figure‐8’ shape were found in 59/129 (46%) micromanipulated embryos and in none of the 39 hatching controls. Hatching usually occurred a day earlier in micromanipulated embryos as 214/218 (98%) had started extruding on day 5 as compared to 20/59 (27%) control blastocysts. Fifty percent of 1‐day‐old human oocytes were fertilized following PZD and reinsemination and 15/31 (48%) were monospermic. Thirteen monospermic embryos cleaved, six compacted and four cavitatcd—of these, three extruded through the PZD incision upon expansion. The zonae did not thin and one blastocyst twinned spontaneously as it was caught between the thick ridges of the PZD hole. Results indicate that the hatching process is abnormal following zona drilling; more embryos start hatching, extrusion occurs earlier, and many become trapped which may lead to artificial twinning or the formation of trophoblastic vesicles.
Development of human male pronucleus: Ultrastructure and timing
Authors: Tesarik Jan , Kopecny Vaclav
Published: February 2005

The process of human male pronuclear formation was studied using an experimental model based on in vitro inseminated human zona‐free eggs prepared from oocytes that failed to fertilize in a clinical in vitro fertilization program. The main ultrastructural changes in penetrated sperm nuclei transforming into pronuclei were used to define four stages of pronuclear development. The first two stages, representing partial (Stage 1) and total (Stage 2) sperm chromatin decondensation, appeared as early as 1 hr after mixing of gametes. This rapid initial phase was followed by a more lengthy array of events leading to transformation of decondensed sperm nuclei into fully developed male pronuclei (Stages 3 and 4). Stage 3 was characterized by reformation of the nuclear envelope, reorganization of chromatin, and the assembly of nuclcolar precursors. It was not completed until 12 hr after in vitro insemination when fully developed male pronuclei (Stage 4) were first observed. In some eggs pronuclei did not reach Stage 4 at all. The results of this study provide a morphological background for further research into molecular aspects of human male pronuclear development and its regulation.
Production of bovine tetrapolid embryos by electrofusion and their developmental capability in vitro
Authors: Iwasaki S. , Kono T. , Fukastu H. , Nakahara T.
Published: February 2005

Optimal conditions of electrofusion for blastomeres of two‐cell bovine embryos to produce tetraploid embryos were investigated. The high fusion rate (73–95%), viability, and develop mental capacity were obtained under a field strength of 1.0 kV/cm with direct current pulses of 10 or 25 μsec duration applied twice. Cytological study showed that 78.6% (11/14 embryos) of embryos exposed to electrofusion had tetraploid chromosome sets and the others were diploid or hexaploid. The tetraploid embryos had the capability to develop up to morulae stage in vitro.
Immunogold distribution of actin during sperimiogenesis in the normal rabbit and after experimental cryptorchidism
Authors: Fouquet Jean‐Pierre , Kann Marie‐Louise , Courtens Jean‐Luc , Ploöen Leif
Published: February 2005

Immunogold procedures for actin detection were used in combination with experimental cryptorchidism in the rabbit as a modei to shed more light on the function of subacrosomal actin during spermiogenesis. In the normal testis, actin was localized in the perinuclcar substance (PNS) from round spermatid onward but it was not detected in late spermatids. Actin labeling in each type of spermatid was essentially unmodified after 24 hr of cryptorchidism. However, among relevant immediate and delayed effects, discontinuous acrosomes overlying a continuous PNS with normal actin labeling were noted. Nuclear invaginations were seen in combination with subacrosomal dilatations: at this site actin labeling was found only in the PNS closely apposed to the nuclear envelope. In subacrosomal areas lacking PNS, actin labeling also was lacking. These results suggest that the subacrosomal actin (F‐actin) is a component of the PNS that is tightly bound to the nuclear envelope rather than the overlying inner acrosomal membrane. Therefore, a function for the subacrosomal actin either in anchoring the acrosome to the nucleus or in capping the inner acrosomal membrane appears unlikely. The data rather suggest a capping function for the nuclear membrane during spermiogenesis.
Structural features of isolated generative cells and their protoplasts from pollen of some liliaceous plants
Authors: Tanaka Ichiro , Nakamura Sumio , Miki‐Hirosige Hisako
Published: February 2005

Large quantities of intact generative cells and their protoplasts were isolated from pollen protoplasts of four liliaceous plants, and their structural features were investigated. The generative cells, liberated from the vegetative cell cytoplasm of the pollen protoplasts, were initially spindle‐shaped with two long, oppositely oriented extensions, and were surrounded by two cell membranes, one on each side of a wall of uniform thickness. The generative nuclei, stained with 4′,6‐diamidino‐2‐phenylindole (DAPI), showed ellipsoidal and highly condensed chromatin, whereas the generative cell cytoplasm, whose quantity was widely different from species to species, showed no fluorescence, suggesting the absence of plastid and mitochondria! DNA, although many mitochondria were present. The isolated generative cells, which were spindle‐shaped at first, became spherical in shape in vitro. Immunocytochemistry and transmission electron microscopy revealed that this change was associated with the depolymerization of an axial array of microtubules present in generative cells in situ. These results are discussed in relation to the function of the generative cell within the bicellular pollen of angiosperms.
Devlopment of chimaeric two‐cell mouse embryos produced by allogenic exchange of single nucleus from two‐ and eight‐cell embryos
Authors: Kono Tomohireo , Tsunoda Yukio , Watanabe Tadao , Nakahara Tatsuo
Published: February 2005

Synchronous or asynchronous chimaeras were produced by transplanting a single nucleus of two‐ and eight‐cell embryos from CD‐lxCD‐1 or BALB/CxBALB/C albino strains into one enucleated blastomere of a late Fl (C57/BLxCBA) × Fl two‐cell embryo. The cytoplasmic volume of the blastomere was reduced in some instances by 50%. These chimaeric embryos were cultured in vitro and transferred to pseudopregnant recipients. The distribution of each component to the pups and to the day‐10 embryos after transfer to recipients was determined by examining their coat color and by glucose phosphate isomerase analysis, respectively. The contribution of progeny of the nuclear‐transplanted cell with nonreduced cytoplast to the pups was 83% when synchronous; this proportion decreased to 43% when asynchronous because the progeny tended to migrate to the trophoblast and/or to the primitive endoderm. When the recipient cytoplast was reduced by 50%, the contribution of the nuclear‐transplanted cell progeny to the pups was 79% when synchronous and 80% when asynchronous. This shows that allogenic exchange of a single nucleus at the two‐cell stage by nuclear transfer is an effective procedure for producing highly asynchronous mouse chimaeras and suggests that larger and advanced blastomeres tend to be excluded from the inner cell mass of the embryo, but smaller, advanced blastomeres do not.
Development of a noninvasive ultramicrofluorometric method for measuring net uptake of glutamine by single preimplantation mouse embryos
Authors: Gardner D. K. , Clarke R. N. , Lechene C. P. , Biggers J. D.
Published: February 2005

A noninvasive ultramicrofluorometric method for measuring net uptake of glutamine by single preimplantation mouse embryos is described. A linear relationship was found between fluorescence intensity of NADH produced and glutamine concentration (R2 = 0.985). Single embryos were placed in 20 nl drops of medium containing 0.5 mM glutamine, and the medium was sampled after 2 hr incubation at 37°C. Changes in net glutamine uptake were determined in one‐cell, two‐cell, and eight‐cell embryos and blastocysts incubated in medium containing no energy substrates (glucose, pyruvate, and lactate). The median glutamine uptake increased significantly from 0.480 and 0.270 pmoles/embryo/2 hr at the one‐cell and two‐cell stages, respectively, to 1.610 pmoles/embryo/2 hr at the blastocyst stage. Mean glutamine uptake was compared in the presence or absence of energy substrates at several developmental stages. A highly significant reduction of glutamine uptake in the presence of substrates was observed at the one‐cell and two‐cell stages of development. At the eight‐cell stage, glutamine uptake was only marginally reduced in the presence of substrates, and no effect was found at the blastocyst stage. These data may partially explain the beneficial effect of glutamine on the culture of early mouse embryos through the two‐cell block of development.
Changes in the rat sperm head during epididymal transit
Authors: Fornés Miguel W. , de Rosas Juan C.
Published: February 2005

Morphological changes in sperm are one aspect of a maturation process during epididymal transit in marnrnals. The literature mentions only, for different strains of rats, a remodeling and decrease in size of the acrosome. In the present work, the sperm were obtained from caput, corpus, and cauda epididymis of the albino rat. Samples were processed for scanning electron microscopy, with routine techniques, and for light microscopy and video microscopy. It appeared, with these techniques, that the acrosomal curvature and the whole head surface area of the rat sperm decrease during the epididymal transit. To measure these changes, a geometrical method was designed, and surface measurements were made using a computer program. It was found that the caput sperm head has the greatest surface area and a sharper acrosome bend than the cauda sperm. In an attempt to explain the above‐mentioned changes, the suggestion is offered that some compactation of the nucleus and acrosomal material could be related to the decrease of the surface area.
Correlated errors in biased surrogates: study designs and methods for measurement error correction
Authors: Spiegelman D. , Zhao B. , Kim J.
Published: February 2005

A measurement error model proposed previously allows for correlations between subject‐specific biases and between random within‐subject errors in the surrogates obtained from two modes of measurement. However, most of these model parameters are not identifiable from the standard validation study design, including, importantly, the attenuation factor needed to correct for bias in relative risk estimates due to measurement error. We propose validation study designs that permit estimation and inference for the attenuation factor and other parameters of interest when these correlations are present. We use an estimating equations framework to develop semi‐parametric estimators for these parameters, exploiting instrumental variables techniques. The methods are illustrated through application to data from the Nurses' Health Study and Health Professionals' Follow‐up Study, and comparisons are made to more restrictive models. Copyright © 2005 John Wiley & Sons, Ltd.
Methods for Fabricating Microarrays of Motile Bacteria
Authors: Rozhok Sergey , Shen Clifton K.‐F. , Littler Pey‐Lih H. , Fan Zhifang , Liu Chang , Mirkin Chad A. , Holz Richard C.
Published: February 2005

Motile bacterial cell microarrays were fabricated by attaching Escherichia coli K‐12 cells onto predesigned 16‐mercaptohexadecanoic acid patterned microarrays, which were covalently functionalized with E. coli antibodies or poly‐L‐lysine. By utilizing 11‐mercaptoundecyl‐penta(ethylene glycol) or 11‐mercapto‐1‐undecanol as passivating molecules, nonspecific binding of E. coli was significantly reduced. Microcontact printing and dip‐pen nanolithography were used to prepare microarrays for bacterial adhesion, which was studied by optical fluorescence and atomic force microscopy. These data indicate that single motile E. coli can be attached to predesigned line or dot features and binding can occur via the cell body or the flagella of bacteria. Adherent bacteria are viable (remain alive and motile after adhesion to patterned surface features) for more than four hours. Individual motile bacterial cells can be placed onto predesigned surface features that are at least 1.3 μm in diameter or larger. The importance of controlling the adhesion of single bacterial cell to a surface is discussed with regard to biomotor design.
Accuracy of magnetic resonance imaging for measuring fetal sheep lungs and other organs
Authors: Jani J. , Breysem L. , Maes F. , Boulvain M. , Roubliova X. , Lewi L. , Vaast P. , Biard J.‐M. , Cannie M. , Deprest J.
Published: February 2005

Objective

Lung volume measurement by fetal magnetic resonance imaging (fetMRI) has been used to predict survival of fetuses with isolated congenital diaphragmatic hernia (CDH). So far, the accuracy and precision of fetMRI for volumetry of either the normal or hypoplastic developing lung has not been formally studied.

Methods

A total of nine sheep carrying 14 fetuses underwent fetMRI under general anesthesia at a mean of 118 days' gestational age (term = 145 days). A total of 61 organs were measured in nine normal fetal sheep and five that underwent surgical creation of diaphragmatic hernia (DH), so as to induce pulmonary hypoplasia. Lungs were measured on T2‐WI (weighted images) in three different planes, while liver and kidneys were measured in the axial (T1‐WI) and sagittal (T2‐WI) planes, respectively. Necropsy was done within 24 h after fetMRI to determine the volume postmortem by the water displacement method. Values were linearly correlated and a Bland and Altman analysis was done for volume measurement comparison, calculating means ± SD, bias (mean of the difference of volume measurements), precision (SD of the difference) and absolute and proportionate limits of agreement for both methods. The accuracy of fetMRI volume measurement was determined for different organ groups by calculating the median relative error and precision index, both being measures of error in proportion to the magnitude of the volume measured, as a clinically relevant proxy of potential errors.

Results

The fetMRI volume measurements were on average larger than postmortem volumes, except for the kidneys. Kidney volume determination had a relative error of 29%, while measurements of larger organs had larger relative errors (42% for liver). Normal lungs were less accurately measured in the coronal or sagittal than in the axial plane (relative error 53%, 73% and 38%, respectively; P < 0.05 for sagittal vs. axial). Axially‐measured lung volumes were more accurate for lungs of normal sheep compared to DH lungs (relative error 38% vs. 73%, respectively; P < 0.05).

Conclusion

FetMRI measured systematically higher volumes for organs such as fetal liver or lung. This may be related to fluid loss or lack of perfusion at the time of necropsy. Measurement of lung volume by fetMRI was most accurate in the axial plane. Measurements of lung and liver volumes by fetMRI in normal sheep were both in agreement with volumes measured at necropsy. Loss of accuracy for DH‐lungs in comparison with the accuracy when measuring other similarly small organs, such as kidneys, suggests that fetMRI measurements can be less accurate for hypoplastic lungs related to CDH. With improving hardware, it might become easier to render the fetal lung and determine its volume reliably. Copyright © 2005 ISUOG. Published by John Wiley & Sons, Ltd.
Lipid composition and fluidity in the jejunal brush-border membrane of spontaneously hypertensive rats. Effects on activities of membrane-bound proteins
Authors: Vázquez Carmen M. , Zanetti Rosana , Ruiz-Gutierrez Valentina
Published: February 2005

The lipid composition and fluidity of jejunal brush-border membrane vesicles (BBMV) have been studied in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. The activities of both Na+-dependent D-glucose cotransport and Na+-H+ antiport have also been determined. A significant increase in the level of free cholesterol was observed in jejunal BBMV from SHR compared to WKY rats. Since phospholipid values did not change in either group of animals, a significant enhancement in the free cholesterol/phospholipid ratio was observed in SHR. A decrease in the levels of phosphatidylethanolamine together with an increase in the values of phosphatidylserine was observed in hypertensive rats. Although the content of phosphatidylcholine (PC) and sphingomyelin (SM) was not singificantly altered in SHR, the ratio PC/SM significantly increased in these animals when compared to WKY rats. The major fatty acids present in bursh-border membranes prepared from SHR and WKY rats were palmitic (16:0), stearic (18:0), oleic (18:1, n-9) and linoleic (18:2, n-6), and the fatty acid composition was not modified by the hypertension. A decreased fluorescence polarization, i.e., increased membrane fluidity, was observed in SHR, which was not correlated to the increased ratio of cholesterol/phospholipid found in the brush-border membrane isolated from these animals. These structural changes found in SHR were associated to an enhancement in both Na+-dependent D-glucose transport and Na+-H+ antiport activity in the jejunal BBMV of SHR.
UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts
Authors: Lirvall Margareta , Ljungqvist-Höddelius Pia , Wasteson Åke , Magnùsson Karl-Eric
Published: February 2005

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2±0.2 × 10−10 cm2/s, and 1.8±0.2 × 10−10 cm2/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3±0.3 × 10−10 cm2/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1±0.8 × 10−10 cm2/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.
Effect of insulin on the incorporation of 3H-inositol into the inositol phospholipids (PI, PIP, PIP2) and Glycosyl-phoshatidylinositols (GPIs) of Tetrahymena pyriformis
Authors: Kovács P. , Csaba G.
Published: February 2005

The unicellular tetrahymena contains inositol phospholipids (PI, PIP, PIP2) and GPIs. Treatment with 10−5M insulin decreases the total 3H-inositol incorporation and incorporation into PI. 24 h after 10−6M insulin treatment there is an elevation of these parameters. Second treatment with 10−6M insulin doubles and 10−5M decreases these levels. This means that the effect on phosphoinositide turnover by insulin in Tetrahymena is rather concentration dependent. Inositol incorporation into GPIs is also influenced by insulin.
Different secretory actions of pancreastatin in bovine and human parathyroid cells
Authors: Ridefelt P. , Hellman P. , Stridsberg M. , Åkerström G. , Rastad J.
Published: February 2005

Chromogranin A is an acidic protein that is costored and cosecreted with parathyroid hormone (PTH) from parathyroid cells. Pancreastatin (PST), is derived from chromogranin A, and inhibits secretion from several endocrine/neuroendocrine tissues. Effects of different pancreastatin peptides were investigated on dispersed cells from bovine and human parathyroid glands. Bovine PST(1–47) and bovine PST(32–47) inhibited PTH release from bovine cells in a dose-dependent manner. The former peptide was more potent and suppressed the secretion at 1–100 nM. This inhibition was evident in 0.5 and 1.25 mM, but not in 3.0 mM external Ca2+. Both peptides failed to alter the concentration of cytoplasmic Ca2+([Ca2+]i) of bovine cells. Human PST(1–52) and PST(34–52) did not affect PTH release or [Ca2+]i of parathyroid cells from patients with hyperparathyroidism, nor [Ca2+]i of normal human parathyroid cells. Furthermore, bovine PST(1–47) and bovine PST(32–47) failed to alter the secretion of abnormal human parathyroid cells. The study indicates that PST exerts secretory inhibition on bovine but not human parathyroid cells, and that this action does not involve alterations of [Ca2+]i.
Peptide T from human immunodeficiency virus does not interact with VIP receptor-effector system in immunocompetent cells of rat and mouse
Authors: Pozo D. , Segura J. J. , Guerrero J. M. , Calvo J. R.
Published: February 2005

Human immunodeficiency virus (HIV) infection is initiated by attachment of the virus to specific target cells. An octapeptide sequence contained within the envelope of HIV, peptide T, mediates the viral binding. Since there is an appreciable structural similarity between peptide T and an eight amino acid sequence of VIP, it is interesting to investigate the interaction of peptide T with the VIP receptor-effector system of immunocompetent cells from both rat and mouse. In this paper, we show the lack of interaction between peptide T and VIP receptor-effector system in peripheral blood lymphocytes, spleen lymphocytes and macrophages of rat and in macrophages of mouse. These results do not support the hypothesis that HIV through peptide T may interact with the VIP receptor-effector system present in immunocompetent cells.
Parallelization of divide-and-conquer in the Bird-Meertens formalism
Authors: Gorlatch Sergei , Lengauer Christian
Published: February 2005

An SPMD parallel implementation schema for divide-and-conquer specifications is proposed and derived by formal refinement (transformation) of the specification schema. The specification is in the form of a mutually recursive functional definition. In a first phase, a parallel functional program schema is constructed which consists of a communication tree and a functional program that is shared by all nodes of the tree. The fact that this phase proceeds by semantics-preserving transformations in the Bird-Meertens formalism of higher-order functions guarantees the correctness of the resulting functional implementation. A second phase yields an imperative distributed message-passing implementation of this schema. The derivation process is illustrated with an example: a two-dimensional numerical integration algorithm.
Proof rules for recursive procedures
Authors: Hesselink Wim H.
Published: February 2005

Four proof rules for recursive procedures in a Pascal-like language are presented. The main rule deals with total correctness and is based on results of Gries and Martin. The rule is easier to apply than Martin's. It is introduced as an extension of a specification format for Pascal-procedures, with its associated correctness and invocation rules. It uses well-founded recursion and is proved under the postulate that a procedure is semantically equal to its body.

This rule for total correctness is compared with Hoare's rule for partial correctness of recursive procedures, in which no well-founded relation is needed. Both rules serve to prove correctness, i.e. sufficiency of certain preconditions. There are also two rules for proving necessity of preconditions. These rules can be used to give formal proofs of nontermination and refinement. They seem to be completely new.
Petri net based verification of distributed algorithms: An example
Authors: Kindler Ekkart , Reisig Wolfgang , Völzer Hagen , Walter Rolf
Published: February 2005

A technique to model and to verify distributed algorithms is suggested. This technique (based on Petri nets) reduces the modelling and analysis effort to a reasonable level. The paper outlines the technique using the example of a typical network algorithm, the
echo algorithm
.
A mechanically verified incremental garbage collector
Authors: Russinoff David M.
Published: February 2005

As an application of a system designed for concurrent program verification, we describe a formalisation and mechanical proof of the correctness of Ben-Ari's incremental garbage collection algorithm. The proof system is based on the Manna-Pnueli model of concurrency and is implemented as an extension of the Boyer-Moore prover. The correctness of the garbage collector is represented by two theorems, stating a) that nothing except garbage is ever collected (safety), and b) that all garbage is eventually collected (liveness). We compare our mechanised treatment with several published proofs of the same results.
Inductive families
Authors: Dybjer Peter
Published: February 2005

A general formulation of inductive and recursive definitions in Martin-Löf's type theory is presented. It extends Backhouse's ‘Do-It-Yourself Type Theory’ to include inductive definitions of families of sets and definitions of functions by recursion on the way elements of such sets are generated. The formulation is in natural deduction and is intended to be a natural generalisation to type theory of Martin-Löf's theory of iterated inductive definitions in predicate logic.

Formal criteria are given for correct formation and introduction rules of a new set former capturing definition by strictly positive, iterated, generalised induction. Moreover, there is an inversion principle for deriving elimination and equality rules from the formation and introduction rules. Finally, there is an alternative schematic presentation of definition by recursion.

The resulting theory is a flexible and powerful language for programming and constructive mathematics. We hint at the wealth of possible applications by showing several basic examples: predicate logic, generalised induction, and a formalisation of the untyped lambda calculus.
Coquand's calculus of constructions: A mathematical foundation for a proof development system
Authors: Seldin Jonathan P.
Published: February 2005

The calculus of constructions of Coquand, which is a version of higher order typedλ-calculus based on the dependent function type, is considered from the perspective of its use as the mathematical foundation for a proof development system. The paper considers formulations of the calculus, the underlying consistency of the formalism (i.e., the strong normalisation theorem), and the proof theory of adding assumptions for notions from logic and set theory. Proofs are not given, but references to them are.
Denotational semantics of an object-oriented programming language with explicit wrappers
Authors: Hense Andreas V.
Published: February 2005

Object-oriented languages have traditionally been described by method-lookup-semantics. Their denotational semantics have appeared and matured only recently. Cook's wrapper semantics without state shows the essence of inheritance much clearer than method-lookup-semantics.

In this article, we show how wrapper semantics can describe an object-oriented languagewith statewhile keeping its original clear structure. We then extend our object-oriented language by so calledexplicit wrappers. Wrappers that are used for the description of the semantics of an “ordinary” object-oriented language emerge from the semantics level and are included into the language itself. This unusual step is being justified by a greater reusability of code. With explicit wrappers and single inheritance, one variety of multiple inheritance can be expressed.
Asynchronous communication model based on linear logic
Authors: Kobayashi Naoki , Yonezawa Akinori
Published: February 2005

We propose a new framework called ACL for concurrent computation based on linear logic. ACL is a kind oflinear logic programmingframework, where its operational semantics is described in terms ofproof constructionin linear logic. We also give a model-theoretic semantics based onphase semantics, a model of linear logic. Our framework well captures concurrent computation based on asynchronous communication. It will, therefore, provide us with a new insight into other models of asynchronous concurrent computation from alogicalpoint of view. We also expect ACL to become a formal framework for analysis, synthesis and transformation of concurrent programs by the use of techniques for traditional logic programming. ACL's attractive features for concurrent programming paradigms are also discussed.