bluuebunny/crossref_metadata_2025 huggingface dataset:
A transmission electron microscope (TEM) calibration standard sample for all magnification, camera constant, and image/diffraction pattern rotation calibrations
Authors: McCaffrey J. P. , Baribeau J.‐M.
Published: February 2005

A calibration sample for transmission electron microscopy (TEM) has been developed that performs the three major instrument calibrations for a transmission electron microscope: the image magnification calibration for measurements of images, the camera constant calibration for indexing diffraction patterns, and the image/diffraction pattern rotation calibration for relating crystal directions to features in the image. This offers an improvement over commercially available calibration standards, where up to five different samples are required to perform these three calibrations. The new calibration sample consists of an electron‐transparent cross‐sectional TEM sample made from a molecular beam epitaxy (MBE)‐grown, single‐crystal semiconductor wafer. When the calibration structure is viewed in a TEM, it appears as a series of light and dark layers where the layer thicknesses are very accurately known. The calibrated thickness measurements of these light (silicon) and dark (SiGe alloy) layers are based on careful TEM measurements of the {111} lattice spacing of silicon which is visible on the calibration sample itself, and are supported by X‐ray diffraction measurements. Furthermore, the layer thickness variation across the entire silicon wafer has been verified to be less than 1%, allowing all samples prepared from the same wafer to have errors in the given layer thickness values of less than 1%. As the sample is a single crystal of silicon, the calibrations requiring electron diffraction information such as the camera constant calibration and the image/diffraction pattern rotation calibration can also be performed easily and unambiguously. One single calibration sample can therefore be used to provide all three of the major TEM instrument calibrations at all magnifications and all camera lengths. © 1995 Government of Canada.

Exclusive worldwide publication rights in the article have been transferred to Wiley‐Liss, Inc., in perpetuity.
A specimen holder for high‐resolution low‐temperature scanning electron microscopy
Authors: Bastacky Jacob , Lee Charles , Freeman Tony , Weber George , Baeza Armando , Hubbins Ted , Chen Ya
Published: February 2005

A miniature vise built into a 5 mm diameter copper capsule is described that holds small pieces of prefrozen, hydrated specimens at low temperatures within the lens of the Hitachi S900 high‐resolution scanning electron microscope.
Distribution and possible role of perinuclear theca proteins during bovine spermiogenesis
Authors: Oko R. , Maravei D.
Published: February 2005

The perinuclear theca (PT) is a unique cytoskeletal element that encapsulates the mammalian sperm nucleus. It is divided into subacrosomal and postacrosomal regions. The objective of this study was to analyze and stage the intracellular distribution of several promiment bull PT proteins during spermatogenesis. For this purpose, polyclonal antibodies raised and affinitypurified against the 15.5‐, 25‐, 28‐, 32‐, 36‐, and 60‐kDa bull PT polypeptides were used as probes on sections of aldehyde‐fixed testes. Immunoperoxidase staining revealed that the PT polypeptides first appeared early in spermiogenesis, concomitant with early steps of development of the acrosomic system. Immunogold labeling further showed that these polypeptides were peripherally associated with the entire acrosomal membrane, before and during the attachment of the acrosomic vesicle onto the spermatid's nucleus. Once the acrosome had capped the nucleus the labeling resided mainly in the subacrosomal region of the spermatid, between the inner acrosomal membrane and nuclear envelope. Later, during the elongation of the spermatid's nucleus, the labeling with all antibodies except the anti‐15.5‐kDa antibody extended caudally over the assembling postacrosomal sheath. This study suggests that the perinuclear theca proteins play an instrumental role in the attachment, spreading, and binding of the acrosome onto the nucleus of spermatids. © 1995 Wiley‐Liss, Inc.
The acyclic edge chromatic number of a random d‐regular graph is d + 1
Authors: Nešetřil Jaroslav , Wormald Nicholas C.
Published: February 2005

We prove the theorem from the title: the acyclic edge chromatic number of a random d‐regular graph is asymptotically almost surely equal to d + 1. This improves a result of Alon, Sudakov, and Zaks and presents further support for a conjecture that Δ(G) + 2 is the bound for the acyclic edge chromatic number of any graph G. It also represents an analog of a result of Robinson and the second author on edge chromatic number. © 2005 Wiley Periodicals, Inc. J Graph Theory 49: 69–74, 2005
Improved Anti‐Inflammatory and Pharmacokinetic Properties for Superoxide Dismutase by Chemical Glycosidation with Carboxymethylchitin
Authors: Valdivia Aimara , Perez Yunel , Dominguez Amalia , Caballero Julio , Gomez Leissy , Schacht Etienne H. , Villalonga Reynaldo
Published: February 2005

Summary: O‐Carboxymethylchitin (molecular weight = 1.07 × 105, degree of carboxymethylation = 80%, degree of N‐acetylation = 91%) was chemically attached to superoxide dismutase by the formation of amide linkages through a carbodiimide catalyzed reaction. The glycosidated enzyme contained about 1.8 mole of polysaccharide per mole of protein and retained 57% of the initial catalytic activity. The anti‐inflammatory activity of the enzyme was 2.4 times increased after conjugation with the polysaccharide. The modified superoxide dismutase preparation was remarkably more resistant to inactivation with H2O2 and its plasma half‐life time was prolonged from 4.8 min to 69 h.

Superoxide dismutase‐O‐carboxymethylchitin conjugate.

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Superoxide dismutase‐O‐carboxymethylchitin conjugate.
Alkaline and Enzymatic Degradation of L‐Lactide Copolymers, 1
Authors: Tsuji Hideto , Tezuka Yasufumi
Published: February 2005

Summary: Films of poly(L‐lactide) [i.e., poly(L‐lactic acid) (PLLA)] and L‐lactide copolymers with glycolide [P(LLA‐GA)(81/19)], ε‐caprolactone [P(LLA‐CL)(82/18)], D‐lactide [P(LLA‐DLA)(95/5), (77/23), and (50/50)] were prepared and a comparative study on the effects of comonomer type and content on alkaline and proteinase K‐catalyzed hydrolyses of the films was carried out. The hydrolyzed films were investigated using gravimetry (weight loss and water absorption), differential scanning calorimetry (DSC), polarimetry, and gel permeation chromatography (GPC). To exclude the effects of molecular weight and crystallinity on the hydrolysis, the films were prepared from polymers having similar molecular weights and made amorphous by melt‐quenching. It was found that incorporation of hydrophilic glycolide units in L‐lactide chains raises the alkaline and enzymatic hydrolyzabilities, whereas incorporation of hydrophobic ε‐caprolactone units in L‐lactide chains reduces the alkaline and enzymatic hydrolyzabilities. On the other hand, incorporation of D‐lactide units with the same hydrophilicity of L‐lactide units increases the alkaline hydrolyzability but decreases the enzymatic hydrolyzability. The alkaline hydrolyzability of the films of L‐lactide copolymers with different kinds of comonomers and P(LLA‐DLA) with different D‐lactide unit contents can be closely related to their hydrophilicity. On the other hand, the enzymatic hydrolyzability of L‐lactide copolymer films with different kinds of comonomers is mainly determined by hydrophilicity, while that of P(LLA‐DLA) films is determined by the averaged L‐lactyl and D‐lactyl unit sequence lengths. The catalytic effect of proteinase K relative to that of alkali on the hydrolysis of P(LLA‐DLA)(77/23) and P(LLA‐GA)(81/19) films normalized by that of PLLA was lower than unity, whereas the normalized relative catalytic effect of proteinase K on the hydrolysis of P(LLA‐CL)(82/18) film was higher than unity, meaning that despite low absolute alkaline and enzymatic hydrolyzability of the P(LLA‐CL)(82/18) film, the catalytic effect of proteinase K may be maintained for this copolymer film, probably because of its blocky structure.

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Cover Picture: Macromol. Biosci. 2/2005
Authors: Kameda Tsunenori , Miyazawa Mitsuhiro , Ono Hiroshi , Yoshida Mitsuru
Published: February 2005

Cover: High‐resolution 13C solid‐state NMR revealed two hydrogen bond systems in α‐chitin. The carbonyl oxygen was either intermolecularly hydrogen‐bonded to NH (blue; carbonyl 13C NMR peak at 173.5 ppm) or hydrogen‐bonded both intermolecularly to NH and intramolecularly to C(6)‐OH (red; 175.8 ppm peak). The former accounts for about 60% of the carbonyl groups and the latter for about 40%.

Further details can be found in the Communication by T. Kameda,* M. Miyazawa, H. Ono, and M. Yoshida on page 103.
Supramolecular Assembly of Gold Nanoparticles Mediated by Polypseudorotaxane with Thiolated β‐Cyclodextrin
Authors: Liu Yu , Zhao Yan‐Li , Chen Yong , Wang Min
Published: February 2005

Summary: A water‐soluble gold nanoparticle aggregate 2 was prepared by chloroauric acid and a polypseudorotaxane 1 of mono‐6‐thio‐β‐cyclodextrin with poly(propylene glycol) bis(2‐aminopropyl ether) ($\overline M _{\rm w}$ ≈ 2 000) in the presence of sodium borohydride in N,N‐dimethylformamide (DMF) solution. The investigative results indicated that the gold nanoparticle aggregate 2 might act as an efficient DNA‐cleavage reagent.

A typical TEM image of gold nanoparticle aggregate 2.

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A typical TEM image of gold nanoparticle aggregate 2.
Selective NMR Pulse Sequences for the Study of Solid Hydrogen‐Containing Fluoropolymers
Authors: Ando Shinji , Harris Robin K. , Hazendonk Paul , Wormald Philip
Published: February 2005

Summary: Fluorine‐19 NMR spectra of solids have some special features, which are discussed in this article. In particular, they generally contain two abundant spin baths (protons and fluorine nuclei). This situation throws up some special operational requirements, as does the study of heterogeneous samples. The relaxation characteristics of heterogeneous systems, which are briefly described herein, frequently permit the use of specific pulse sequences to obtain subspectra for individual components. Various possible selective sequences for use in fluorinated heterogeneous organic solids are listed and their actions rationalized on the basis of molecular mobility. Semicrystalline hydrogen‐containing fluoropolymers form especially suitable systems for such operations, and in order to understand their domain structures it is essential to obtain subspectra of the amorphous and crystalline domains. Examples are given of the use of selective pulse sequences for studying fluoropolymers, especially for poly(vinylidene fluoride) (PVDF) and the copolymer P(VDF75/TrFE25) (TrFE = trifluoroethylene).

DIVAM/CP spectra of the vinylidene fluoride/trifluorethylene copolymer as a function of the minipulse angle used. Top: Unfiltered spectrum. Middle: the amorphous domains. Bottom: the crystalline domains.

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DIVAM/CP spectra of the vinylidene fluoride/trifluorethylene copolymer as a function of the minipulse angle used. Top: Unfiltered spectrum. Middle: the amorphous domains. Bottom: the crystalline domains.
Nerve stimulation boosts botulinum toxin action in spasticity
Authors: Frasson Emma , Priori Alberto , Ruzzante Barbara , Didonè Giuseppe , Bertolasi Laura
Published: February 2005

Spasticity leads to functional and structural changes in nerves and muscles, which alter skeletal muscle function. To evaluate whether short‐term electrical nerve stimulation (NS) improves the effect of botulinum toxin in spastic skeletal muscle, we studied changes in the amplitude of the compound muscle action potential (CMAP) recorded from the extensor digitorum brevis (EDB) muscle in response to peroneal nerve stimulation at the ankle after injection of botulinum toxin type A (BTXA) alone or combined with short‐term NS. In paraparetic patients, both EDB muscles were injected with BTXA; and NS was applied to one EDB muscle alone. All patients received a 30‐minute session of electrical NS once a day for 5 consecutive days after BTXA injection. We used two different stimulation frequencies (low‐frequency, 4 Hz; and high‐frequency, 25 Hz). EDB‐CMAP amplitudes were evaluated before BTXA injection (day 0) and changes in CMAP amplitude, expressed as a percentage (CMAP%), were measured at various time points over a 30‐day period after BTXA injection. We compared changes in the CMAP% amplitude on the stimulated and contralateral nonstimulated sides. We also studied the electromyographic activity recorded from EDB muscles over a 30‐day period. CMAP% amplitudes measured at all time points after BTXA injections were significantly reduced in both EDB muscles. On days 4, 10, and 15, the CMAP% amplitude reduction was significantly greater for the low‐frequency stimulated EDB than for the contralateral nonstimulated EDB. No significant differences in CMAP% were observed for the high‐frequency stimulated and nonstimulated EDB. After BTXA injection, spontaneous activity appeared in both EDB muscles; but it appeared earlier and involved larger areas in the stimulated than in the nonstimulated EDB. In conclusion, short‐term NS accelerates the effectiveness of intramuscular BTXA injections on the neuromuscular blockade in patients with spastic paraparesis and could induce a rapid and persistent improvement in spasticity. Its action probably arises mainly from low‐frequency NS. © 2005 Movement Disorder Society
Driving in Parkinson's disease: Mobility, accidents, and sudden onset of sleep at the wheel
Authors: Meindorfner Charlotte , Körner Yvonne , Möller Jens Carsten , Stiasny‐Kolster Karin , Oertel Wolfgang Hermann , Krüger Hans‐Peter
Published: February 2005

Only few studies have addressed driving ability in Parkinson's disease (PD) to date. However, studies investigating accident proneness of PD patients are urgently needed in the light of motor disability in PD and—particularly—the report of “sleep attacks” at the wheel. We sent a questionnaire about sudden onset of sleep (SOS) and driving behavior to 12,000 PD patients. Subsequently, of 6,620 complete data sets, 361 patients were interviewed by phone. A total of 82% of those 6,620 patients held a driving license, and 60% of them still participated in traffic. Of the patients holding a driving license, 15% had been involved in and 11% had caused at least one accident during the past 5 years. The risk of causing accidents was significantly increased for patients who felt moderately impaired by PD, had an increased Epworth Sleepiness Scale (ESS) score, and had experienced SOS while driving. Sleep attacks at the wheel usually occurred in easy driving situations and resulted in typical fatigue‐related accidents. Those having retired from driving had a more advanced (subjective) disease severity, higher age, more frequently female gender, an increased ESS score, and a longer disease duration. The study revealed SOS and daytime sleepiness as critical factors for traffic safety in addition to motor disabilities of PD patients. The results suggest that real sleep attacks without any prior sleepiness are rare. However, our data underline the importance of mobility for patients and the need for further studies addressing the ability to drive in PD. © 2005 Movement Disorder Society
Alanine and leucine transport in unfertilized pig oocytes and early blastocysts
Authors: Prather R. S. , Peters M. S. , Van Winkle L. J.
Published: February 2005

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. In mouse oocytes and cleavage‐stage conceptus Na+‐dependent L‐alanine and L‐leucine transport are nearly undetectable. Sodium‐dependent transport via system BO,+ in the mouse conceptus increases greatly between the 8‐cell and blastocyst stages. By contrast, data presented here for the pig show that L‐alanine and L‐leucine transport is mainly Na+‐dependent in the oocyte; this Na+‐dependent component of transport becomes undetectable by the blastocyst stage. The Na+‐dependent component of transport in oocytes is inhibited by BCH (2‐aminoendo‐bicyclo[2.2.1] hexane‐2‐carboxylic acid) and L‐lysine and thus could be a form of system BO,+. In both oocytes and blastocysts Na+‐independent L‐leucine transport is inhibited by BCH, which is consistent with the presence of system L. The dramatic decrease in Na+‐dependent amino acid transport activity could occur in pig conceptuses in association with the onset of RNA synthesis during the 4‐cell stage. Regardless of the precise time during development at which it occurs, however, this dramatic, developmentally regulated decrease in Na+‐dependent alanine and leucine transport activity contrasts sharply with the large increase in Na+‐dependent system BO,+ activity that occurs during preimplantation development of murine conceptuses. Elucidation of the molecular mechanisms by which these changes occur should contribute to an understanding of regulation of gene expression during early development. © 1993 Wiley‐Liss, Inc.
Roles of pH and cyclic adenosine monophosphate in the acquisition of potential for sperm motility during migration from the sea to the river in chum salmon
Authors: Morisawa Sachiko , Ishida Katsumi , Okuno Makoto , Morisawa Masaaki
Published: February 2005

In the natural process of the migration of chum salmon from the sea to the river, spermatozoa moved from the testis to the sperm duct, and the pH value of seminal plasma, concentration of cyclic adenosine monophosphate (AMP) in the sperm cells, and potential for sperm motility increased. Cyclic AMP levels and the potential for motility gradually increased when testis spermatozoa with no capacity for movement were incubated in the artificial seminal plasma of which the pH was much the same as, or higher than, the pH of natural seminal plasma from the sperm duct. Such correlation in motility, pH, and cyclic AMP suggests that the increases in seminal pH and intracellular cyclic AMP level during passage of spermatozoa from the testis to the sperm duct cause the acquisition of potential for motility. Motility of testicular spermatozoa demembranated with Triton X‐100 was very low in fish caught in the sea, while motility of spermatozoa from the posterior portion of the sperm duct was much higher in fish caught in the river. Furthermore, nondemembranated, intact spermatozoa showed a lag in the timing of the acquisition of potential for motility vs. demembranated spermatozoa: The demembranated sperm exhibited the potential earlier than the nondemembranated sperm. These data suggest that increase in activity of the motile apparatus, the axoneme, is a prerequisite, in part, for the acquisition of sperm motility, whereas the development of some function of the plasma membrane also contributes to this phenomenon. © 1993 Wiley‐Liss, Inc.
Production of cumulus expansion enabling factor by mouse oocytes grown in vitro: Preliminary characterization of the factor
Authors: Eppig John J. , Peters Antoine H. F. M. , Telfer Evelyn E. , Wigglesworth Karen
Published: February 2005

The objective of this study was to determine whether fully grown oocytes, obtained after isolation from preantral follicles and growth in vitro, secrete paracrine factors affecting granulosa cell development and function. If so, the relative ease in producing oocytes in this way could facilitate the identification and characterization of the factors. As a test of this idea, the ability of in vitro grown oocytes to produce a paracrine factor that is known to enable the isolated cumulus oophorus to undergo expansion in response to follicle stimulating hormone (FSH) was determined. Initial experiments compared culture systems, which differed in the orientation of the oocyte‐granulosa cell complexes from preantral follicles to an extracellular matrix, for their ability to support oocyte growth and the acquisition of competence to resume meiosis. The systems for culture on the surface of the matrix produced larger oocytes and the highest percentage of oocytes having competence to resume meiosis. Oocytes grown using this system secreted active cumulus expansion enabling factor, albeit at levels about half that of oocytes grown in vivo. A preliminary characterization of the cumulus expansion enabling factor secreted by the oocytes grown in vitro showed that activity was lost upon treatment with either heat (65°C for 15 min) or proteinase K. Activity did not pass through a membrane having a nominal molecular weight limit (NMWL) of 100 kd but did pass through a membrane having a NMWL of 300 kd. It is concluded that cumulus expansion enabling factor is secreted by oocytes grown in vitro. This factor is probably a protein or depends upon a protein for its activity. The ease in obtaining relatively large numbers of GVB‐competent oocytes using techniques for growth in vitro combined with the demonstration that these produce cumulus expansion enabling factor indicates that these protocols can be used to produce oocytes for the collection and characterization of oocyte secretory products some of which are paracrine regulators of granulosa cells. © 1993 Wiley‐Liss, Inc.
Expression of proto‐oncogenes in mouse eggs and preimplantation embryos
Authors: Pal Subrata K. , Crowell Richard , Kiessling Ann A. , Cooper Geoffrey M.
Published: February 2005

The expression of several protooncogenes has been investigated in mouse eggs and preimplantation embryos using reverse transcription coupled to amplification of cDNAs by the polymerase chain reaction (RT‐PCR). The genes chosen for analysis included both cytoplasmic (c‐raf‐1, rasH, rasK, and rasN) and nuclear (c‐fos and c‐myc) proto‐oncogenes encoding proteins involved in the transduction of signals from protein‐tyrosine kinase growth factor receptors. Transcripts of the cytoplasmic proto‐oncogenes were detected both as maternal and embryonic mRNAs at levels (ca. 1,000 copies per egg or embryo) approximately comparable to their levels of transcription in somatic cells. Transcripts of c‐fos and c‐myc were also detected in both eggs and embryos, although at more variable levels: Maternal transcripts were present at very low levels (ca. 1–10 copies per egg) in growing oocytes and ovulated eggs; embryonic transcription of c‐myc increased, reaching mRNA levels of approximately 100–1,000 copies per embryo in four‐cell embryos, mórula, and blastocysts; in contrast the transcription of c‐fos remained at low, barely detectable levels throughout preimplantation development. Although the significance of the low levels of c‐fos mRNA is unclear, these results indicate that preimplantation embryos possess the basic intracellular signaling apparatus required to respond to polypeptide growth factors. © 1993 Wiley‐Liss, Inc.
Inositol tri‐phosphate in human and ascidian spermatozoa
Authors: Tosti Elisabetta , Palumbo Anna , Dale Brian
Published: February 2005

Using a specific protein binding assay we have shown that a spermatozoon of the ascidian Ciona intestinalis contains 1.58 ± 0.74 × 10−19 moles of inositol 1,4,5‐tri‐phosphate (InsP3), while a human spermatozoon contains 6.4 ± 0.14 × 10−19 moles. Induction of the acrosome reaction (AR) in both species, by exposure to the calcium ionophore A23187, does not significantly alter levels of InsP3, suggesting that phosphatidylinositol (PI) turnover is not necessary for the calcium ionophore induced AR. Furthermore, PI turnover in ascidian spermatozoa appears to be insensitive to lithium and phorbol ester. The high intracellular concentration of InsP3 in spermatozoa, corresponding to 50–200 μM, suggests it may play a role in egg activation. © 1993 Wiley‐Liss, Inc.
Zona‐free sperm penetration assay and inducers of the acrosome reaction: A model for sperm microinjection under the zona pellucida
Authors: Tarín Juan J. , Trounson Alan O.
Published: February 2005

In order to minimize the percentage of false‐negative results in the zona‐free sperm penetration assay (SPA), a wide range of substances and/or physical agents capable of inducing the acrosome reaction (AR) have been incorporated in the incubation medium. These agents can also be used for treatment of severe male infertility using the technique of sperm microinjection under the zona pellucida (SMUZ). In the present review, the percentages of acrosome‐reacted spermatozoa induced by several physiological, biochemical or physical agents published in the literature are compared in order to find the most efficient method(s) of inducing the AR In human sperm as a previous requirement for optimizing the technique of SMUZ. A working estimate of the level of efficiency of a given AR inducer is calculated by adding up its range score in each of three different arrangements from the highest to the lowest value of percentages of AR and differences in percentages of AR and penetration indexes between treated and control groups in SPA. The agents able to induce the AR by nonphysiological (electropermeabilization, lysophosphatidyl choline, and freezing‐thawing) have better positions in this hierarchical system than those ones which require the active participation of sperm membrane receptors or second messenger systems (progesterone, zona pellucida, and stimulators of protein kinase A). Electropermeabilization appears to be the most efficient AR inducer. However, more possibilities need to be explored to enhance the relatively low percentages of acrosome‐reacted spermatozoa shown by infertile men. © 1993 Wiley‐Liss, Inc.
Gene transfer in bovine blastocysts using replication‐defective retroviral vectors packaged with gibbon ape leukemia virus envelopes
Authors: Kim Teoan , Leibfried‐Rutledge Mary L. , First Neal L.
Published: February 2005

With this work we demonstrate that murine leukemia virus (MLV)‐based replication‐defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins. In a test of internal promoter activity in an MLV retroviral vector, the rat β‐actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E. coli β‐galactosidase marker gene in bovine target cells. By co‐culture of bovine blastocysts and virus‐producing cells, or by culture of embryos in the medium harvested from virus‐producing cells, we transferred the E. coli β‐galactosidase gene into trophoblasts and also into inner cell mass (ICM) cells of a bovine embryo through the infection of the MLV‐based replication‐defective retroviruses encapsidated with GaLV envelope proteins. The infection was confirmed by the expression of the E. coli β‐galactosidase gene under a β‐actin internal promoter. In addition, co‐culture of ICM cells with virus‐producing cells resulted in differentiation of ICM cells into embryoid bodies expressing the marker genes. © 1993 Wiley‐Liss, Inc.
Quantitative analysis of protein synthesis in mouse embryos. II: Differentiation of endoderm, mesoderm, and ectoderm
Authors: Latham Keith E. , Beddington Rosa S. P. , Solter Davor , Garrels James I.
Published: February 2005

The changes in protein synthesis that occur during differentiation of the primitive germ layers were examined by high‐resolution, two‐dimensional gel electrophoresis of proteins synthesized in 6.5 and 7.5 days postcoitum (d.p.c.) mouse embryos. For 6.5 d.p.c. embryos, protein synthesis patterns were compared between whole extraembryonic and embryonic regions and between embryonic visceral endoderm and embryonic ectoderm. For 7.5 d.p.c. embryos, comparisons were made between extraembryonic and embryonic regions and between isolated embryonic endoderm, mesoderm, and ectoderm. Each of the isolated 7.5 d.p.c. germ layers was divided into anterior and posterior fragments in order to evaluate possible regional differences in gene expression along the anterior–posterior axis. Comparisons of protein synthesis patterns revealed the greatest difference between isolated endoderm and ectoderm, indicating that by as early as 6.5 d.p.c. patterns of gene expression differ significantly between these tissues. The greatest similarities were found between ectoderm and whole embryonic regions and between endoderm and whole extraembryonic regions, which most likely reflects the overall cellular compositions of the embryonic and extraembryonic regions. Based on their patterns of synthesis, four groups of proteins were identified that were preferentially synthesized in either endoderm or ectoderm. These provide useful markers for studying differentiation in these tissues. One other protein, migrating at the position expected for vimentin, was synthesized at an elevated rate in isolated mesoderm. We also observed differences in rates of synthesis of α‐tubulin and tropomyosin‐5 indicative of potential differences in cytoskeletal composition among the germ layers beyond those previously described. The difference in overall protein synthesis patterns between anterior and posterior regions was greatest in the embryonic endoderm, indicating that differentiation along the anterior–posterior axis may be initiated sooner or may proceed more rapidly in the endoderm than in the other germ layers. These data provide the first quantitative evaluation of the degree to which differentiation of the three primitive germ layers affects protein synthesis patterns and reveal potentially useful markers of endoderm and ectoderm differentiation. © 1993 Wiley‐Liss, Inc.
Rat Sertoli cell extracellular matrix regulates glycosaminoglycan synthesis by peritubular myoid cells in vitro
Authors: Raychoudhury Samir S. , Blackshaw Alan W. , Irving Michael G.
Published: February 2005

We have previously reported metabolic cooperation between Sertoli and peritubular myoid cells in terms of synthesis of one of the main testicular extracellular matrix (ECM) constituents, glycosaminoglycans (GAG). This study concerns Sertoli cell ECM‐peritubular myoid cell interactions in terms of GAG synthesis. We have examined the responses of hormones and other regulatory agents such as a combination of follicle‐stimulating hormone (FSH), insulin, retinol, and testosterone (FIRT) on peritubular myoid cells, and tested if Sertoli cell ECM or serum factor substitute for the stimulation by FIRT. Testicular peritubular myoid cells cultured on Sertoli cell ECM showed significant increases in the levels of cell‐ and ECM‐associated GAG over that when cultured on uncoated plastic. This indicates a specific cell‐substratum interaction between Sertoli cell ECM and peritubular myoid cells in the testis in terms of GAG synthesis. Moreover, in terms of cell‐associated GAG synthesis, peritubular myoid cells cultured on Sertoli cell ECM or on plastic in the presence of serum substituted for the stimulatory response of FIRT on peritubular myoid cells cultured on uncoated plastic. The data are discussed in relation to the possible role of cell‐substratum interaction in maintaining peritubular myoid cell functions. © 1993 Wiley‐Liss, Inc.
Cytoskeletal organization of rat oocytes during metaphase II arrest and following abortive activation: A study by confocal laser scanning microscopy
Authors: Zernicka‐Goetz Magdalena , Kubiak Jacek Z. , Antony Claude , Maro Bernard
Published: February 2005

In metaphase II arrested rat oocytes (M il), microtubles were found in the taper‐shaped meiotic spindle and in the cytoplasm as asters and free microtubules. Whereas spindle microtubules were acetylated, those located in the cytoplasm were not. Cytoplasmic microtubules were also labile as assessed by mild cooling. In contast to mouse oocytes, rat microtubule organizing centers (MTOCs) did not react with MPM‐2 antibody by immunofluorescence despite the fact that this antibody reacts with several proteins as shown by immunoblot. However, cytoplasmic MTOCs in M II‐arrested rat oocytes could be detected by their nucleating capacity in the presence of taxol, a drug that induced the formation of numerous cytoplasmic asters. In addition, taxol caused a change in the spindle shape and the formation of astral microtubules at the spindle poles. Meiotic spindles (as well as chromosomes devoid of microtubules after nocodazoletreatment) were overlaid by an actin‐rich domain. Spontaneous abortive activation led to the extrusion of the second polar body followed by another metaphase arrest— metaphase III; however, normal spindles did not form and dispersed chromosomes surrounded by microtubles were observed. Electron microscopic studies confirmed these observations and revealed that the kinetochores are located deep within the chromosomes in contrast to mouse kinetochores, and this might be responsible for the absence of a metaphase III spindle in the rat oocyte. Induced activation caused transition to interphase with the formation of a characteristic microtubule network. This study shows that there are several significant differences in the cytoskeletal organization of rat and mouse oocytes. © 1993 Wiley‐Liss, Inc.
Binding characteristics and immunolocalization of porcine seminal protein, PSP‐I
Authors: Kwok Simon C. M. , Soares Michael J. , McMurtry John P. , Yurewicz Edward C.
Published: February 2005

PSP‐l, a 13 kDa protein purified from boar seminal plasma, was found to have about 50% amino acid sequence homology with a family of zona pellucida‐binding proteins known as spermadhesins. These proteins are produced by the accessory gland(s) of the male reproductive tract and coat the spermatozoa during ejaculation. In this study, we have investigated the possible biological functions of PSP‐I using a solid‐phase protein binding assay and its site of synthesis using both Western blot and immunocytochemical analyses. PSP‐I was found to bind a number of proteins including endo‐β‐galactosidase digested ZP3, soybean trypsin inhibitor, lgA, lgG and α‐casein, indicating that it may have multiple functions. The protein or carbohydrate structures were not critical in the binding, since polyvinyl sulfate could effectively inhibit the binding of PSP‐l to these proteins. Western blot analysis using specific antiserum to PSP‐l showed that the protein was present in the seminal vesicle but not in the testes, epididymis or prostate. The protein was revealed by immunocytochemical analysis in the epithelium of seminal vesicles but not in the testes or the epididymis. It is concluded that PSP‐I is synthesized by the epithelium of the seminal vesicles, secreted into the semen during ejaculation, and may be involved in various reproductive functions, such as preventing premature acrosome reaction and immunosuppression. © 1993 Wiley‐Liss, Inc.
Changes in ovarian follicles and in vitro sex hormone release in the lizard Podarcis sicula sicula
Authors: Ciarcia Gaetano , Paolucci Marina , Di Fiore Maria Maddalena
Published: February 2005

An in vitro superfusion method was used to test sex hormone release from different kinds of ovarian follicle (growing follicles, postovulatory follicles, and atretic follicles) in the lizard Podarcis sicula sicula. Sex hormone output changes with the stage of follicle evolution and sexual cycle. Previtellogenetic follicles prevail in early‐spring quiescent ovaries and secrete mainly progesterone, which is probably utilized at that phase to delay ovarian resumption. In the active ovary, progesterone output from previtellogenetic follicles decreases, whereas vitellogenetic follicles produce a significant amount of 17β‐estradiol, which is necessary for sustaining vitellogenin synthesis by the liver and oviduct growth. As follicles become ripe, progesterone production is resumed, and it increases in young postovulatory follicles. This is in line with the functions assigned to the hormone at that phase of the sexual cycle, i.e., the induction of oocyte maturation and the regulation of egg retention in the oviduct. Postovulatory follicles can also synthetize 17β‐estradiol. After oviposition, this hormone, which is secreted by the old postovulatory follicles, can reinitiate vitellogenin synthesis, allowing the development of a new oocyte set. Our data confirm that active, although ephemeral, corpora lutea are also formed in oviparous species. A limited contribution to ovarian sex steroid production derives also from atretic follicles, at least at the early stages of the breeding cycle. © 1993 Wiley‐Liss, Inc.
Lysophosphatidylcholine disrupts the acrosome of tammar wallaby (macropus eugenii) spermatozoa
Authors: Sistina Yulia , Lin Minjie , Rodger John C.
Published: February 2005

The acrosomal status of wallaby spermatozoa was evaluated by light and electron microscopy after incubation in 1–100 μM lysophosphatidylcholine (LPC) for up to 120 min. Treatment with 1 and 10 μM LPC for 120 min did not lead to acrosomal loss, or detectable alteration to the acrosome, as detected by Bryan's staining and light microscopy. Incubation with 25 μM LPC had little effect on acrosomal loss, however statistically significant changes (P < 0.05) in the acrosomal matrix (altered) were detected after 10‐min incubation by light microscopy. Around 50% of acrosomes were altered after 20‐min incubation in 50 μM LPC (P < 0.001), and 40% of spermatozoa had lost their acrosome after 60‐min incubation (P < 0.001). Treatment with 75 and 100 μM LPC led to rapid acrosomal loss from around 50% of spermatozoa within 10 min (P < 0.001), and by 60 min acrosomal loss was 70–80%. LPC, like the diacylglycerol DiC8 (1,2‐di‐octanoyl‐sn‐glycerol), is thus an effective agent to induce loss of the relatively stable wallaby sperm acrosome, and it also induces changes within the acrosomal matrix.

Ultrastructure of the LPC‐treated spermatozoa revealed that the plasma membrane and the acrosomal membranes were disrupted in a manner similar to that seen after detergent treatment (Triton X‐100). There was no evidence of point fusion between the plasma membrane overlying the acrosome and the outer acrosomal membrane. The plasma membrane was the first structure to disappear from the spermatozoa. The acrosomal membranes and matrix showed increasing disruption with time and LPC concentration. Wallaby spermatozoa incubated with LPC at concentrations that induced significant acrosomal loss also underwent a rapid decline in motility that suggested that acrosomal loss may be due to cell damage, rather than a physiological AR. This study concluded that LPC‐induced acrosomal loss from tammar wallaby spermatozoa is due to its action as a natural detergent and not as a phosphoinositide pathway intermediate. The study further demonstrates the unusual stability of the marsupial acrosomal membranes. © 1993 Wiley‐Liss, Inc.
Biochemical characterization and epididymal localization of the maturation‐dependent ram sperm surface antigen ESA152
Authors: Weaver Frances E. , Dino Judith E. , Germain Bonnie J. , Wolf David E. , Fairbanks Grant
Published: February 2005

We examine here the biochemical properties and epididymal localization of a maturation dependent ram sperm surface antigen. A monoclonal antibody, ESA152, identifies an antigen that is present on the surface of ejaculated sperm, but is absent from testicular sperm. Crosslinking of the ESA152 antigen with bivalent antibodies induces the acrosome reaction, redistributing the antigen into the anterior region of the sperm head where it associates with the fusion product of the plasma membrane and the outer acrosomal membrane. The ESA152 antigen appears as a polypeptide of 18 kDa on immunoblots of SDS‐polyacrylamide gels. The ESA152 epitope includes the sialic acid termini of N‐linked oligosaccharides, as shown by its sensitivity to neuraminidase and endoglycosidase F. The ESA152 antigen is a highly hydrophobic integral membrane protein that resists aqueous extraction, partitions into the detergent phase of Triton‐X‐114, and solubilizes in chloroform‐methanol mixtures. The anchoring of ESA152 is unaffected by phosphtidylinositol specific phospholipase C. The antigen is absent from extracts of caput and corpus epididymidis but appears abruptly in the first segment of the cauda. Immunofluorescence reveals that the ESA152 epitope first appears in clusters of cells in the luminal epithelium of the proximal cauda, prior to or concurrent with its appearance on sperm. © 1993 Wiley‐Liss, Inc.
Molecular dynamics of insulin/IGF‐I receptor transmembrane signaling
Authors: Pessin Jeffrey E. , Frattali Anne L.
Published: February 2005

To examine the molecular basis of ligand‐stimulated intramolecular β‐subunit autophosphorylation, hybrid receptors composed of wild‐type and mutantinsulin and insulin‐like growth factor‐1 (lGF‐l) half‐receptor precursors were characterized. Previous studies have demonstrated that assembly of the IGF‐I wild‐type half‐receptor (αβWT) with a kinase‐defective half‐receptor (αβA/K) produced a substrate kinase‐inactive holoreceptor in vitro [Treadway et al. (1991): Proc Natl Acad Sci USA 88:214–218]. To extend these studies, the vaccinia virus/ bacteriophage T7 expression system was used to generate various hybrid receptor complexes in cultured cells. As was observed for hybrid receptors assembled in vitro, the wild‐type/mutant hybrid receptors formed in situ were also incapable of phosphorylating several peptide substrates. However, ligand‐stimulated β‐subunit autophosphorylation was still observed. To determine the molecular basis for this discrepancy, hybrid receptors were assembled from a truncated β‐subunit insulin half‐receptor (αβδ43). and a kinase‐defective half‐receptor (αβA/K). Under these conditions, insulin‐stimulated autophosphorylation primarily oc‐curred on the full‐length kinase‐inactive β‐subunit (αβA/K) without significant labeling of the kinase‐active truncated β‐subunit (αβδ43). A similar IGF‐I hybrid receptor species was characterized, and the same pattern of autophosphorylation was observed in response to IGF‐I. These data demonstrate that both insulin and IGF‐I stimulate an intramolecular trans‐autophosphorylation reaction between two adjacent β‐subunits within the holoreceptors. Furthermore, our data suggest that trans‐phosphorylation between two functional β‐subunits is required for substrate kinase activation of the insulin and IGF‐I holoreceptors. These results suggest a molecular basis for the dominant‐negative phenotype observed in heterozygous, insulin‐resistant patients possessing one kinase‐defective insulin receptor allele. © 1993 Wiley‐Liss, Inc.
IGF binding proteins and their functions
Authors: Clemmons David R.
Published: February 2005

The insulin‐like growth factor (IGF) binding proteins (IGFBPs) have several functions, including transporting the IGFs in the circulation, mediating IGF transport out of the vascular compartment, localizing the IGFs to specific cell types, and modulating both IGF binding to receptors and growth‐promoting actions. The functions of IGFBPs appear to be altered by posttranslational modifications. IGFBP‐3, ‐4, ‐5, and ‐6 have been shown to be glycosylated. Likewise all the IGFBPs have a complex disulfide bond structure that is required for maintenance of normal IGF binding. IGFBP‐2, ‐3, ‐4, and ‐5 are proteolytically cleaved, and specific proteases have been characterized for IGFBP‐3, ‐4, and ‐5. Interestingly, attachment of IGF‐I or II to IGFBP‐4 results in enhancement of proteolysis, whereas attachment of either growth factor to IGFBP‐5 results in inhibition of proteolytic cleavage. Cleavage of IGFBP‐3 results in the appearance of a 31 kDa fragment that is 50‐fold reduced in its affinity for the IGF‐I or IGF‐II. In spite of the reduction in its affinity, this fragment is capable of potentiating the effect of IGF‐I on cell growth responses; therefore, proteolysis may be a specific mechanism that alters IGFBP modulation of IGF actions. Other processes that result in a reduction in IGF binding protein affinity are associated with potentiation of cellular responses to IGF‐I and ‐II. Specifically, the binding of IGFBP‐3 to cell surfaces is associated with its ability to enhance IGF action and with a ten‐ to 12‐fold reduction in its affinity for IGF‐I and IGF‐II. Likewise, binding of IGFBP‐5 to extracellular matrix (ECM) results in an eightfold reduction in its affinity and a 60% increase in cell growth in response to IGF‐I. Another post‐translational modification that modifies IGFBP activity is phosphorylation. IGFBP‐1, ‐2, ‐3, and ‐5 have been shown to be phosphorylated. Phosphorylation of IGFBP‐1 results in a sixfold enhancement in its affinity for IGF‐I and ‐II. Following this enhancement of IGFBP‐1 affinity, this binding protein loses its capacity to potentiate IGF‐I growth‐promoting activity. Future studies using site‐directed mutagenesis to modify these proteins should enable us to determine the effect of these posttranslational modifications on the ability of IGFBPs to modulate IGF biologic activity. © 1993 Wiley‐Liss, Inc.
Fertilization and early embryonic development in the blue fox (Alopex lagopus)
Authors: Farstad W. , Hyttel P. , Grøndahl C. , Mondain‐Monval M. , Smith A. J.
Published: February 2005

A total of 15 blue fox vixens aged 1–6 years were mated, 12 once on the first day of estrus and three a second time 48 hr after the first mating, and were killed 4 hr to 8 days following mating. Ova were collected from the oviducts, evaluated by stereomicroscopy, and studied by transmission (TEM; N = 49, 12 vixens) or scanning (SEM, N = 11, three vixens) electron microscopy. At 0–3 days after ovulation, the ova had not cleaved and were at different stages of meiotic maturation. In about one‐half of these ova, representing all stages of meiotic maturation, a decondensing sperm head without nuclear envelope or a small pronucleus with partial nuclear envelope was observed. No clear relationship was found between maternal meiotic stage and the stage of paternal pronucleus formation. Sperm tails were never identified in the ooplasm. Cortical granules were released after sperm penetration at early stages of meiotic maturation. Thus the block against polyspermic penetration was activated during maturation of the oocyte. The first two‐cell stage appeared 4 days after ovulation (3 days after mating), the first four‐cell stage the following day (day 5), and the first eight‐cell stage 6 days after ovulation (5 days after mating). In a single vixen mated late (7 days postovulation) two‐ to four‐cell stages appeared the following day (day 8). This indicates that the time required for the first cleavage division decreases with increasing interval from ovulation to mating. The development of a functional nucleolus with fibrillar centers and fibrillar and granular components at the eight‐cell stage indicates activation of embryonic RNA synthesis in fox embryos at the six‐ to eight‐cell stage, suggesting that the embryonic genome is activated at this stage. © 1993 Wiley‐Liss, Inc.
Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos
Authors: Graves Kathy H. , Moreadith Randall W.
Published: February 2005

We have derived putative embryonic stem (ES) cell lines from preimplantation rabbit embryos and report here their initial characterization. Two principal cell types emerged following serial passage of explanted embryos, and each has subsequently given rise to immortalized cell lines. One cell type has morphology identical to primary outgrowths of trophectoderm, is strictly feeder‐cell dependent, and spontaneously forms trophectodermal vesicles at high cell density. The second type appears to represent pluripotent ES cells derived from the inner cell mass as evidenced by (1) ability to grow in an undifferentiated state on feeder layers, (2) maintenance of a predominantly normal karyotype through serial passage (over 1 year), and (3) ability to form embryoid bodies, which form terminally differentiated cell types representative of ectoderm, mesoderm, and endoderm. These ES cells may ultimately be suitable for introduction of germline mutations (via homologous recombination). The rabbit's size, reproductive capability, and well‐characterized physiology make it suitable for a wide range of investigations, particularly for development of large animal models of human disease. © 1993 Wiley‐Liss, Inc.