bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Sperm‐Activating Peptide I on Hemicentrotus pulcherrimus Spermatozoa in High Potassium Sea Water
Authors: Harumi Tatsuo , Hoshino Katsuaki , Suzuki Norio
Published: April 2005

The effects of sperm‐activating peptide I (SAP‐I: Gly‐Phe‐Asp‐Leu‐Asn‐Gly‐Gly‐Gly‐Val‐Gly) on Hemicentrotus pulcherrimus spermatozoa in high [K+] sea water were examined. In high [K+] sea water, the respiration rates and motility of H. pulcherrimus spermatozoa were lower than those in normal sea water. SAP‐I did not stimulate the lowered respiration rate or motility, although the peptide bound to the spermatozoa as it does in normal sea water. SAP‐I elevated the sperm cGMP level in 100 mM K+ sea water (from 0.37 to 4.81 pmol/mg wet weight spermatozoa) more than those in normal sea water (from 0.21 to 0.93 pmol/mg wet weight). A phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine (IBMX) and SAP‐I synergistically elevated the cGMP level from 0.35 to 33.08 pmol/mg wet weight in 100 mM K+ sea water. However, in high [K+] sea water, SAP‐I did not increase the cAMP level even in the presence of IBMX. SAP‐I caused rapid, transient elevation of the intracellular pH and Ca2+ concentration of spermatozoa in normal sea water but not in 100mM K+ sea water. SAP‐I did not decrease the apparent molecular weight of sperm guanylate cyclase from 131,000 to 128,000 in high [K+] sea water. These results suggest that the SAP‐I‐induced elevation of the cGMP level in sea urchin spermatozoa occurs before or independently of membrane hyperpolarization induced by the opening of K+ channels.
Changes in Electrophoretic Patterns of Oocyte Proteins during Oocyte Maturation in Oryzias latipes
Authors: Iwamatsu Takashi , Takahashi Susumu Y. , Oh‐ishi Masamichi , Yokochi Takashi , Maeda Hiroko
Published: April 2005

Changes in the two‐dimensional SDS‐electrophoretic patterns of extracts of maturing denuded oocytes of the medaka (Oryzias latipes) were surveyed. In oocytes without follicular constituents several proteins became detectable in the area between the acidic and slightly basic proteins on the two‐dimensional electrophoretograms, while a few of the protein spots disappeared during the process of oocyte maturation. The former proteins were detected also in oocytes that were induced to mature in vivo without breakdown of the germinal vesicle. Several proteins newly observed in extracts of post‐vitellogenic oocytes during maturation after breakdown of the germinal vesicle were also identified by two‐dimensional electrophoresis. Of several proteins that exhibited noticeable changes in maturing oocytes, only one spot incorporated 14C‐labeled amino acid during maturation, suggesting that post‐translational modification of many proteins occurred during oocyte maturation.
On the Development of the Eminentia Mediana of the Hypophysis in Rana temporaria, Studied in Normal, Hypophysectomized, and Thyroidectomized Tadpoles.
Authors: Aronsson Stig , Enemar Anders
Published: April 2005

In the Rana temporaria tadpole, the part of the infundibular floor that eventually becomes the eminentia mediana is very thin throughout premetamorphosis; the supply of aminergic and neurosecretory nerves is poor, and the area is covered by a few fenestrated capillaries. The organ begins to thicken around mid‐prometamorphosis owing to a marked invasion of nerves. At this stage it is also penetrated by capillaries and perivascular space extensions, which together form the neurovascular link structures. These become more pronounced during metamorphic climax, when the eminentia mediana attains the adult appearance. Development of the organ is arrested at the premetamorphic stage after thyroidectomy in the young premetamorphic tadpole. Removal of the adenohypophysial primordium from the embryo of the tailbud stage prevents the vascular—but not the neural—component of the eminentia from developing beyond the premetamorphic stage. By contrast, in the American Rana‐species investigated the neural component also fails to develop after hypophysectomy.
Immuno‐Localization of DEAD Family Proteins in Germ Line Cells of Xenopus Embryos.
Authors: Watanabe Masanao , Itoh Keiji , Abe Kuniya , Akizawa Toshifumi , Ikenishi Kohji , Furusawa Mitsuru
Published: April 2005

In order to investigate whether a vasa‐like protein is present in germ line cells of Xenopus, antibodies were produced which react specifically with synthetic oligopeptides of sequences from near the N‐ or C‐termini or with one including the DEAD box of the Drosophila vasa protein.

Only the antibody against the oligopeptide including the DEAD box reacted strongly with germ plasm (GP) or with cytoplasm of germ line cells of Xenopus embryos by immunofluorescence microscopy. By immunoelectron microscopy, the antibody was demonstrated to react with the GP‐specific structure, germinal granules, in cleaving embryos, and with their derivatives in the germ line cells of embryos at stages extending from gastrula to feeding tadpole. It also reacted with mitochondria not only in the GP and the germ line cells but also in somatic cells, and with myofibrils in muscle cells. By Western blotting, the antibody was shown to react with several bands of Mr 42–69 ± 103 in protein samples from Xenopus embryos. In samples from Drosophila ovaries, it reacted with a Mr 71 ± 103 band which was probably the vasa protein. This indicates the possibility that Xenopus embryos contain several DEAD family proteins. One of these is present on germinal granules, resembling the vasa protein on polar granules of Drosophila.
Mouse Offspring Derived from Fetal Ovaries or Reaggregates Which were Cultured and Transplanted into Adult Females
Authors: Hashimoto Koichiro , Noguchi Motoko , Nakatsuji Norio
Published: April 2005

Primordial germ cells (PGCs) and gonia could be promising novel targets and vehicles for manipulation of the mammalian germ line. To make such manipulation a practical possibility, PGCs or gonia must be allowed to produce gametes and offspring after they were isolated from embryos and manipulated in culture. As the first step to develop such research strategy, we obtained offspring from mouse oogonia which were isolated from embryonic ovaries and cultured as dispersed cells before transplantation into female mice as reaggregates.
Differential Expression of c‐myc and N‐myc during Oral Organogenesis of the Mouse Embryo
Authors: Yamada Shumpei , Ikeda Masa‐aki , Eto Kazuhiro
Published: April 2005

The expressions of the c‐ and N‐myc proto‐oncogenes during oral development of midgestational mouse embryos were examined by in situ hybridization in order to analyze their roles. In the mandibular rudiment, c‐myc RNA was strongly expressed in the mesenchymal condensation around the ossification center in which high‐level expression of 2 ar (osteopontin) was detected. In tooth germs, c‐myc was strongly expressed in the epithelia at the bud stage, and its expression gradually became restricted to the inner enamel epithelia from the cap to bell stages. In contrast, N‐myc expression was detected in the undifferentiated mesenchymal cells of the dental papilla. Incorporation of BrdU was examined immunohistochemically to study the relationship between the expressions of c‐ and N‐myc and cell proliferation. Unexpectedly, the distribution of BrdU labelled regions was not coincident with the expressions of c‐ and N‐myc. These results suggest that the level of myc expression is not necessarily related to cell proliferation.
Cloning of an Epidermis‐specific Cynops cDNA from Neurula Library
Authors: Takabatake Takashi , Takahashi Tadashi C. , Takeshima Kazuhito
Published: April 2005

A cDNA encoding one of the epidermis‐specific proteins designated as the spot 6 was isolated from the Cynops embryo. Cynops neurula cDNA library was constructed with the plasmid vector containing the promoter sequence for SP6 RNA polymerase. After transcription and translation in vitro the final protein products were screened for the presence of spot 6 by two‐dimensional gel electrophoresis. The total library was digested by 11 restriction endonucleases selected not to destroy the sequence in both the vector and the insert encoding spot 6 protein. The ATP‐dependent DNase digestion eliminated the cDNA population sensitive to these endonucleases. These steps effectively enriched the sequence for spot 6 protein. The resultant sublibrary was repeatedly divided into smaller pools and was screened. The tryptic peptide analysis showed that the isolated clone produced the protein identical to the spot 6 protein originally defined in vivo. Northern analyses showed that the cloned gene was expressed as expected from the developmental behavior of the spot 6 in vivo.
Purification of a Sperm Lectin Extracted from Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus
Authors: Seike Yuuji , Shibata Harumi , Suyemitsu Takashi
Published: April 2005

Hemagglutinating activity for human type A erythrocytes was detected in a sperm extract obtained by treatment with Triton X‐100 of spermatozoa from the sea urchin Hemicentrotus pulcherrimus. Among tested sugars only N‐acetyl‐D‐galactosamine had any inhibitory effect on the hemagglutinating activity of the sperm extract. The lectin was purified by a combination of affinity chromatography and ion‐exchange chromatography. A single band was obtained after SDS‐polyacrylamide gel electrophoresis of the purified lectin, corresponding to an apparent molecular weight of 15,000 daltons. Trypsin‐generated fragments of the surface of eggs significantly inhibited hemagglutination of erythrocytes by the purified lectin. The biological role of the sperm lectin is discussed.
Induction of Heterothallic and Homothallic Zygotes in Dictyostelium discoideum by Ethylene
Authors: Amagai Aiko
Published: April 2005

Dictyostelium mucoroides‐7 (Dm7) and a mutant (MF1) derived from it exhibit homothallic macrocyst formation in the sexual process. As previously shown, the zygote formation during macrocyst formation is induced by a potent plant hormone, ethylene. The present work was undertaken to know if ethylene is also involved in heterothallic and homothallic macrocyst formation in D. discoideum. In heterothallic macrocyst formation between NC4 and V12M2 cells, ethionine, an analogue of methionine, inhibits macrocyst formation through arresting specifically the acquisition process of fusion competence. Such an inhibitory effect of ethionine was almost completely cancelled by co‐application of ACC (1‐aminocyclopropane‐1‐carboxylic acid), the immediate precursor of ethylene. Essentially the same effects of ethionine and ACC were also noticed on homothallic macrocyst formation in D. discoideum AC4. Thus it seems most likely that ethylene is required for the acquisition of fusion competence during macrocyst formation, and that in a variety of strains examined there is a common mechanism regulated by ethylene, beyond the difference of sexual modes.
Differentiation Inducing Phosphatidyl Choline(s) from the Embryos of Rainbow Trout (Salmo gairdneri): Isolation and Structural Elucidation1
Authors: Suzuki Motofumi , Asahi Ken‐ichi , Isono Kiyoshi , Sakurai Akira , Takahashi Nobutaka
Published: April 2005

Phospholipid which is able to induce the differentiation of some undifferentiated tumour cells was isolated from the rainbow trout (Salmo gairdneri) embryos.

As HPLC gave only one peak, the phospholipid seemed to be completely purified. However, the spectra of SIMS and FD‐MS have shown that it was a mixture of two molecular species. By the chemical and enzymic studies their structures were determined as C16:0/C22:6‐and C18:1/C22:6‐phosphatidyl cholines.

The isolated and structurally elucidated phosphatidyl cholines induce haemoglobin synthesis in murine erythroleukemia cells and a rapid decrease in alkaline phosphatase activity in teratocarcinoma cells. Some phosphatidyl cholines which are commercially available have shown no or conspicuously low activities.

Recently we isolated another differentiation‐inducing substance(s), which proved to be diglyceride(s) containing the completely same fatty acids as those in the above mentioned phosphatidyl cholines, that is, C16:0/C22:6‐and C18:1/C22:6. These results suggest that the lipids containing C22:6 fatty acid or this fatty acid itself might play an important part in the differentiation and development.
SCN— Blocks Hardening of the Fertilization Envelope of Sea Urchin Eggs and Adhesion of Blastomeres
Authors: Fujiwara Akiko , Yasumasu Ikuo
Published: April 2005

Sea urchin eggs kept in artificial sea water (ASW) containing 0.01–0.3 M NaSCN in place of NaCI from within 2 min after insemination formed thin, enlarged fertilization envelopes, which were broken on mild agitation of egg suspensions more easily than those formed in Ca2+‐free ASW. The blastomeres of almost all embryos derived from eggs treated with 0.2M SCN— for 1 hr dissociated spontaneously, and did not reassociate with other blastomeres appreciably. Thus SCN— probably denaturated some compound(s) participating in blastomere binding and hardening of the fertilization envelope. Abnormal arrangements of blastomeres, probably due to incomplete blastomere dissociation, were observed in embryos derived from eggs treated with 0.1 M SCN— for 1 hr. Treatment of fertilized or unfertilized eggs with 0.05–0.1 M SCN— for a short period caused concentration‐dependent block of morphogenic processes such as formation of the archenteron and pluteus arms in the post‐hatching period. The effects of SCN— on morphogenesis were not inhibited by furosemide or 4,4′‐diisothiocyano 2,2′‐disulfonic stilbene. Presumably, the denaturation of several compounds in the egg surface by SCN— causes abnormal morphogenesis of embryos. The inhibitory effects of SCN— on hardening of the fertilization envelope, blastomere binding and morphogenesis were greater in the absence of Ca2+.
Analysis of the Formation of the Animal‐Vegetal Axis during Xenopus Oogenesis Using Monoclonal Antibodies
Authors: Tabata Makoto J. , Kawahara Akira , Amano Minoru
Published: April 2005

Maternally accumulated materials in Xenopus oocytes, in particular mRNAs and proteins, are considered to participate in the determination of the developmental specification of embryonic cells. In this study, a large number of monoclonal antibodies was raised against bulk oocyte antigens to examine patterns of intracellular distribution of oocyte proteins. Immunohistochemical experiments with mature oocytes showed that there are five different patterns of distribution of oocyte proteins, with enrichment on the animal side (type A1, and A2 ptoteins), vegetal side (type V1 and V2 proteins), and in the peripheral cytoplasm (type P proteins). Clear localization of type A and V antigen proteins occurred at Dumont's stages IV‐VI. However, at the preceding stages, the distributions of these antigen proteins appeared to be homogeneous. By contrast, the pattern of distribution of type P protenis did not change markedly throughout oogenesis. The presence of type A and type V antigen proteins reflected the animal‐vegetal axis in the cytoplasm of the mature oocyte. Furthermore, there were two boundaries of the distributions of proteins at the equatorial region, excluding or including the cytoplasm around the germinal vesicle. Thus, the cytoplasm of mature oocytes was multilayered with respect to the different proteins distributed along the animal‐vegetal axis.
Sperm of the Shrimp Sicyonia ingentis Undergo a Bi‐Phasic Capacitation Accompanied by Morphological Changes
Authors: Wikramanayake Athula H. , Uhlinger Kevin R. , Griffin Fred J. , Clark Wallis H.
Published: April 2005

Sperm removed from seminal receptacles of female Sicyonia ingentis can be induced to undergo a bi‐phasic acrosome reaction (AR), acrosomal exocytosis followed by filament formation, using egg water (EW). Sperm removed from males will not undergo any phase of the AR when incubated with EW, indicating that these sperm undergo a capacitation process after insemination. Freshly molted females (functional virgins) were placed in aquaria with males and monitored for copulation. Mated females were isolated and allowed to carry sperm for specific periods of time. At these time points, sperm were removed and assayed for the ability to undergo the AR using EW. The results indicate that sperm are competent to undergo acrosomal exocytosis after approximately 25 hr, while competency to form acrosomal filaments is not achieved until around 145 hr post‐insemination. Morphological examination of sperm removed from males and sperm removed from females revealed dramatic differences. Microscopic evidence indicates that some of the morphological changes seen during capacitation are necessary for the successful completion of the AR.
Inhibition of Mouse Blastocyst Hatching by Subsite‐Specific Trypsin Inhibitors, Peptidyl Argininals1
Authors: Sawada Hitoshi , Hoshi Motonori , Someno Tetsuya , Suzuki Rika , Yamazaki Kimie
Published: April 2005

To explore the substrate or subsite specificity of a mouse hatching enzyme, effects of leupeptin [acetyl(P4)‐Leu(P3)‐Leu(P2)‐argininal(P1)] and its analogs (peptidyl argininals) on mouse blastocyst hatching were investigated. The compounds containing benzyloxycarbonyl group (Z) in the P4 position inhibited the hatching more strongly than those containing acetyl group or unprotected N‐terminal amino acid. Among five Z‐Leu‐P2‐argininals, a derivative containing a P2 Ser residue was the most potent inhibitor, and the derivatives containing Leu, Thr, Pro, and Gly in the P2 position followed in this order. Then, we synthesized four Z‐P3‐Ser‐argininals and tested their effects on hatching. The result indicated that the compound with Phe residue in the P3 position was the strongest inhibitor, and the Leu‐, Pro‐, and Ala‐containing derivatives were ranked in this order. Thus, among Z‐dipeptidyl‐argininals tested, Z‐Phe‐Ser‐argininal most potently inhibited the mouse embryonic hatching, suggesting the preference of the mouse hatching enzyme for Phe(P3)‐Ser(P2)‐Arg(P1) sequence as a substrate.
Immunological Characterization of cdc2 and wee1 Proteins during the Growth and Differentiation of Dictyostelium discoideum
Authors: Hinze Ehleen , Michaelis Christine , Daya‐Makin Maleki , Pelech Steven , Weeks Gerald
Published: April 2005

Two different antibody preparations, raised independently against the conserved EGVPSTAIREISLLKE sequence of the protein kinases encoded by the Schizosaccharomyces pombe cdc2 gene and its species homologs, immunoblotted a Dictyostelium protein of 32 kDa (p32). This polypeptide bound to p13suc1‐agarose beads, suggesting that it represents the Dictyostelium cdc2 and / or cdk2 products. The amounts of p32 detectable in cell free extracts and bound to p13suc1‐agarose were unaltered during the growth of cells synchronized for division. Although there was also essentially no change in the level of p32 during differentiation, the protein from the pseudoplasmodial stage of development did not bind to p13suc1‐agarose, implicating a developmentally regulated modification of the kinase. One of the EGVPSTAIREISLLKE antibodies also recognized a protein of 49 kDa (p49) that increased in amount dramatically during aggregation and then remained at elevated levels throughout the remainder of the differentiation process. This protein was present in low amounts throughout the growth of cells synchronized for division and was not absorbed by p13suc1‐agarose.

A 103 kDa protein (p103) was detected by Western blot analysis using antibodies raised against two different peptides corresponding to sequences in the S. pombe protein kinase p105wee1, which is a putative upstream negative regulator of p34cdc2 in fission yeast. The levels of p103 were constant during differentiation and during the growth of cells synchronized for division.
Nuclear Differentiation in Paramecium caudatum: Analysis by the Monoclonal Antibody against a Macronuclear Antigen
Authors: Yanagi Akira
Published: April 2005

To know molecular mechanism controlling the differentiation of somatic nuclei (macronuclei) in Paramecium caudatum, I obtained the monoclonal antibody 36B against a macronuclear antigen. Immunocytochemical observations showed that the antigen 36B was dispersed in macronuclei. But in late stationary phase, large aggregation of the antigen was observed in some stocks, though this was not observed in another stock. In addition, the 36B‐positive portion in macronuclei seemed to contain smaller amount of DNA than the remaining portion. These observations suggest that the antigen 36B exists in nucleoli in the macronucleus. In the late stage of conjugation, the antigen 36B was retained in the degenerating fragments derived from old macronuclei as long as the fragments persisted. In the beginning of the development of new macronuclei, the antigen 36B was not observed in four macronuclear anlagen, but after a while it appeared in all of them. This shows that the macronuclear anlagen newly get the antigen 36B during their development. Since the appearance of the antigen 36B coincides with the stage when macronuclei are known to be transcriptionally active, the antigen 36B may be involved in the expression of genes in macronuclei. This is consistent with the possibility that the antigen 36B may be a component of nucleoli.
Introduction and Expression of Recombinant Genes in Ascidian Embryos
Authors: Hikosaka Akira , Kusakabe Takehiro , Satoh Noriyuki , Makabe Kazuhiro W.
Published: April 2005

In order to examine the expression of exogenous genes introduced into ascidian eggs, two recombinant plasmids pmiwZ and pHrMA4aCAT were microinjected into the cytoplasm of fertilized eggs of Ciona savignyi and Halocynthia roretzi, respectively. The plasmid pmiwZ contains the coding sequence of bacterial β‐galactosidase gene (lac‐Z) fused with animal gene promoters, while pHrMA4aCAT was constructed by fusing about 1.4‐kb long 5′ flanking region of H. roretzi muscle actin gene HrMA4a with bacterial chloramphenicol acetyltransferase gene (CAT). Injection of approximately 160 pl of 10 μg/ml pmiwZ DNA into Ciona eggs did not affect the embryogenesis, although introduction of the same volume of 30 μg/ml pmiwZ DNA resulted in abnormal development of injected eggs. When the expression of lac‐Z was examined by histochemical detection of the enzyme activity, the expression was evident in the early tailbud embryos and later stage embryos, and larvae, irrespective of linear or circular form of the plasmid. The enzyme activity appeared in various cell‐types including epidermis, nervous system, endoderm, mesenchyme, notochord, and muscle. In contrast, when pHrMA4aCAT was introduced into Halocynthia eggs and the appearance of CAT protein was examined later by the anti‐CAT antibody, the CAT expression was restricted to muscle cells. These results indicate that the recombinant genes introduced into ascidian eggs could express during embryogenesis and that the 1.4‐kb long 5′ flanking region of HrMA4a contains regulatory sequences enough for the appropriate spatial and temporal expression of the gene.
Maternal Exogastrula‐Inducing Peptides (EGIPs) and Their Changes during Development in the Sea Urchin Anthocidaris crassispina
Authors: Kinoshita Kounosuke , Fujii Yasushi , Fujita Yoshiaki , Yamasu Kyo , Suyemitsu Takashi , Ishihara Katsutoshi
Published: April 2005

Exogastrula‐inducing activity was examined in eggs and embryos of the sea urchin Anthocidaris crassispina at various stages. During fractionation on a column of DEAE‐cellulose, the exogastrula‐inducing activity was found in the flow‐through fraction at all developmental stages. In particular, the activity present in the flow‐through fraction of unfertilized eggs represents the presence of maternal exogastrula‐inducing peptides (EGIPs). The flow‐through fractions from the column of DEAE‐cellulose were applied to a column of Sephadex G‐100 and the activities in the eluate were assayed. The active low‐molecular‐weight fraction was obtained in all cases with the exception of pluteus larvae, extracts of which contained another active fraction. Immunoblots of protein samples from eggs and embryos probed with antiserum against EGIP‐D indicated that there is a major immunoreactive protein that migrates with an apparent molecular weight of about 6 kDa in all cases with the exception of pluteus larvae, and that there are two major immunoreactive proteins that migrate with apparent molecular weights of 6 kDa and 35 kDa, respectively, in pluteus larvae.
Effect of Microtubular Poisons on Cleavage Furrow Formation and Induction of Furrow‐like Dent in Amphibian Eggs
Authors: Sawai Tsuyoshi
Published: April 2005

The effects of the microtubular poisons colchicine, vinblastine and nocodazole, on cleavage furrow formation and induction of furrow‐like dents in eggs of the newt, Cynops pyrrhogaster, were examined.

Solutions of the poisons were injected beneath the cortex around the small initial furrow, or around the advancing tip of the furrow of eggs during the first cleavage. This resulted in prompt block of the progress of the furrow at the injection site, and subsequent total regression of the furrow or incomplete cleavage.

The ability of the cortex of a cleavage‐arrested blastomere to form a furrow‐like dent was tested by inhibiting furrow formation of one blastomere of two‐cell embryos by injection of the microtubular poisons, and then transplantation of the blastomere under the cortex of the animal half with furrow‐inducing cytoplasm (FIC) taken from normally cleaving eggs. No dent was formed. Moreover, FIC from eggs treated with a poison had no ability to induce a dent on the surface of normally cleaving eggs.

These results show that microtubule structures are directly involved in formation of a cleavage furrow.
Ultrastructural Localization of Calcium in the Presumptive Ectodermal Cells in Gastrulae of the Newt, Cynops pyrrhogaster, as Revealed by Cytochemistry and X‐Ray Microanalysis
Authors: Komazaki Shinji
Published: April 2005

The ultrastructural localization of calcium in the presumptive ectodermal cells of gastrulae of the newt, Cynops pyrrhogaster, was examined by cytochemical methods and X‐ray microanalysis (XMA). The cells were fixed with solutions that contained potassium oxalate, potassium ferricyanide and potassium pyroantimonate to preserve the localization of intracellular calcium in situ and for the analysis of electron density due to calcium. Electron‐dense deposits associated with the localization of calcium were observed under the electron microscope. Specificially, pigment granules, round vacuoles, endoplasmic reticulum and mitochondria as well as the extracellular matrix were observed to contain calcium. In addition, XMA clearly demonstrated the localization of calcium in all of these electron‐dense organelles and yolk granules.
Fertilization Reaction without Changes in Intracellular Ca2+ in Medaka Eggs—An Experiment with Acetone‐Treated Eggs
Authors: Iwamatsu Takashi , Kikuyama Munehiro , Hiramoto Yukio
Published: April 2005

To investigate whether or not causal relationship exists between the increase in intracellular Ca2+ and other cortical reactions at fertilization in the medaka, Oryzias latipes, intracellular Ca2+ was determined from luminescence of aequorin previously microinjected into cortical cytoplasm in acetone‐treated eggs, when they were inseminated or activated by microinjection of Ca2+. Neither an increase in cytoplasmic calcium nor exocytosis of cortical alveoli occurred in eggs treated with acetone, though other events of fertilization i.e. completion of meiosis, fusion of pronuclei, and accumulation of cortical cytoplasm with intact cortical alveoli in the animal pole region were observed in normal time sequence in these eggs. When denuded eggs were treated with acetone, contraction of the egg and slow resumption of meiosis (extrusion of polar body) were observed without insemination. When denuded eggs were inseminated immediately after acetone‐treatment, the number of spermatozoa that penetrated into the egg was greater in the animal hemisphere than in the vegetal hemisphere. These results may indicate that acetone inactivates the egg plasma membrane or its adjacent cortical cytoplasm so that it cannot participate in a propagative increase in intracellular Ca2+ and exocytosis, while it also induces cytoplasmic activation leading to egg contraction, resumption of meiosis and formation of pronuclei. The present results suggest that sperm penetration, resumption of meiosis and ooplasmic segregation are regulated separately from the release of intracellular Ca2+ and exocytosis.
Cis‐Acting Elements for Proper Ontogenic Expression of Arylsulfatase Gene of Sea Urchin Embryo
Authors: Yamada Kazumi , Eguchi Sachiko , Yamamoto Takashi , Akasaka Koji , Shimada Hiraku
Published: April 2005

To define the cis‐acting regulatory elements required for proper ontogenic expression of the Arylsulfatase gene in the sea urchin (Hemicentrotus pulcherrimus) embryo, we have constructed the fusion genes composed of bacterial CAT gene and various regions from the 5′ flanking sequence of the Ars gene, injected them into unfertilized sea urchin eggs and monitored their expression during development. A wild type construct containing the 5′ flanking region of the Ars gene spanning from —2 to —3160 was expressed under proper temporal regulation following injection into unfertilized eggs. By deletion analysis the transcriptional enhancers were localized between —1280 and —2680 of the Ars gene. In addition, sequences acting as a negative regulator as well as a minimal promoter for expression of the Ars gene were detected by internal deletion analysis.
Primary actinomycosis of the anterior abdominal wall: Case report and review of the literature
Authors: FILIPOVIĆ BRANKA , MILINIĆ NIKOLA , NIKOLIĆ GORAN , RAN?ELOVIĆ TOMISLAV
Published: April 2005

Abstract  Actinomycosis of the anterior abdominal wall is a rare infection caused by different Actinomyces species. We report a case of primary actinomycosis localized on the anterior abdominal wall, diagnosed by computed tomography (CT) scan, postoperatively confirmed by histopathological examination and treated by surgical resection combined with postoperative antibiotic therapy. The patient has been free of recurrence after 1 year. A review of the available literature is also presented.
Interleukin‐17 and lipopolysaccharides synergistically induce cyclooxygenase‐2 expression in human intestinal myofibroblasts
Authors: ZHANG ZHUOBIN , ANDOH AKIRA , INATOMI OSAMU , BAMBA SHIGEKI , TAKAYANAGI ATSUSHI , SHIMIZU NOBUYOSHI , FUJIYAMA YOSHIHIDE
Published: April 2005

Background:  Colonic subepithelial myofibroblasts (SEMF) play a role in the modulation of mucosal inflammatory responses via the secretion of various inflammatory mediators. In the present study the effects of interleukin (IL)‐17 and lipopolysaccharides (LPS) on cyclooxygenase (COX) expression in colonic SEMF were investigated.

Methods:  The expression of COX‐1 and ‐2 proteins and mRNAs were determined by western and northern blotting, respectively. Nuclear factor (NF)‐κB DNA binding activities were evaluated by electrophoretic gel mobility shift assays (EMSA).

Results:  The expression of COX‐2 protein and mRNA was rapidly induced by the addition of IL‐17 and LPS, whereas COX‐1 expression was not affected by these factors. The effects of IL‐17 and LPS were detected in a dose‐ and time‐dependent manner. Furthermore, IL‐17 and LPS synergistically induced COX‐2 mRNA and protein expression. The EMSA demonstrated that the addition of IL‐17 and LPS induced NF‐κB activation within 1.5 h after stimulation, and a blockade of NF‐κB activation by a recombinant adenovirus containing a stable form of IκBa markedly reduced the IL‐17‐ and LPS‐induced COX‐2 mRNA expression. In these cells, the expression of Toll‐like receptor (TLR)‐4, which is a cellular receptor for LPS, was detected.

Conclusion:  Interleukin‐17 and LPS play an important role in the induction of COX‐2 in SEMF. These findings suggest that COX‐2 expression and prostaglandin synthesis might be regulated by both T‐cell‐derived factor (IL‐17) and bacterial products (LPS) in the inflamed mucosa.
Electrogastrography: Basic knowledge, recording, processing and its clinical applications
Authors: CHANG FULL‐YOUNG
Published: April 2005

Abstract The slow wave (SW) of the gastrointestinal (GI) tract mainly functions to trigger the onset of spike to elicit smooth muscle contraction, which provides the essential power of motility. Smooth muscle myogenic control activity or SW is believed to originate in the interstitial cells of Cajal (ICC). The electrical coupling promotes interaction between muscle cells, and ICC additionally contribute to SW rhythmicity. Stomach SW originates in the proximal body showing the continuous rhythmic change in the membrane potential and propagates normally to the distal antrum with a regular rhythm of approximately 3 c.p.m. A technique using electrodes positioned on the abdominal skin to pick up stomach rhythmic SW refers to electrogastrography (EGG). The stomach SW amplitude is very weak, while many visceral organs also produce rhythmic electricities, for example heartbeat, respiration, other organs of the GI tract and even body movements. Thus noise other than SW should be filtered out during the recording, while motion artifacts are visually examined and deleted. Finally, the best signal among all recordings is selected to compute EGG parameters based on spectral analysis. The latter is done not only to tranform frequency domain to time domain but also to provide information of time variability in frequency. Obtained EGG parameters include dominant frequency/power, % normal rhythm, % bradygastria, % tachygastria, instability coefficient and power ratio. Clinical experience in EGG has been markedly accumulated since its rapid evolution. In contrast, lack of standardized methodology in terms of electrode positions, recording periods, test meals, analytic software and normal reference values makes the significance of EGG recording controversial. Unlike imaging or manometrical studies, stomach motility disorders are not diagnosed based only on abnormal EGG parameters. Limitations of EGG recording, processing, computation, acceptable normal parameters, technique and reading should be known to conduct subjective assessments when EGG is used to resolve stomach dysfunction. Understanding basic SW physiology, recording methodology and indications may open EGG as a new domain to approach the stomach motor dysfunction.
Calcitonin gene‐related peptide released by capsaicin suppresses myoelectrical activity of gastric smooth muscle
Authors: MIZUGUCHI SUMITO , OHNO TAKASHI , HATTORI YOUICHIRO , KAMATA KAZUHISA , ARAI KATSUHARU , SAEKI TAKEO , SAIGENJI KATSUNORI , HAYASHI IZUMI , KURIBAYASHI YOSHIKAZU , MAJIMA MASATAKA
Published: April 2005

Background:  It is widely accepted that the inhibition of gastric motor activity as well as the maintenance of gastric mucosal blood flow and mucous secretion are important for the homeostasis of the gastric mucosa. The present study was performed to ascertain whether or not capsaicin, which can protect the stomach from noxious stimuli, affects gastric motor activity.

Methods:  Male Sprague–Dawley rats were anesthetized with urethane, and the stomach was cannulated by two catheters from esophageal and duodenal sides. A biopolar electrode was fixed to the serosal surface of the antrum and myoelectrical activity was recorded during the instillation of a small volume of solutions.

Results:  The myoelectrical activity of rat gastric smooth muscle was increased at intragastric pressures of >2 cmH2O. Replacement of intragastric physiological saline with 1.6 mmol/L capsaicin solution significantly suppressed this myoelectrical activity by 50%. Intragastric capsaicin administration caused a significant release of substance P (SP) and calcitonin gene‐related peptide (CGRP). The maximum released levels of CGRP in the gastric perfusates were 100‐fold those of SP. The myoelectrical activity observed at an intragastric pressure of 2 cmH2O was avoided by continuous infusion of CGRP (0.1–3.0 nmol/kg per min) into the gastric artery in a dose‐dependent manner, but not by that of SP (1.0 nmol/kg per min). Continuous CGRP‐(8‐37) infusion into the gastric artery completely blocked the reduction by intragastric capsaicin of myoelectrical activity.

Conclusion:  These results suggest that the suppression of the myoelectrical activity of gastric smooth muscle by capsaicin is attributable to the endogenous CGRP released.
Usefulness of combined measurement of serum bile acids and ferritin as additional prognostic markers to predict failure to reach sustained response to antiviral treatment in chronic hepatitis C
Authors: JORQUERA FRANCISCO , MONTE MARIA J , GUERRA JOSE , SANCHEZ‐CAMPOS SONIA , MERAYO JOSE A , OLCÓZ JOSE L , GONZÁLEZ‐GALLEGO JAVIER , MARIN JOSE J G
Published: April 2005

Aim: To investigate the relationship between serum levels of ferritin and bile acids (BA) and the response to antiviral treatment in chronic hepatitis C (HCV).

Methods: A retrospective study was carried out on 35 control volunteers and 50 patients receiving interferon alpha‐2b alone or plus ribavirin for 48 weeks. These were classified as sustained responders (SR) for >6 months after therapy (n = 17), non‐responders (NR) (n = 27) and relapsers (RL) (n = 6). Before treatment, serum ferritin levels were determined by immunoturbidometry, 3α‐hydroxyl‐BA levels (S‐3α‐OH‐BA) were assayed enzymatically and total (desulfated, deglucuronidated and deamidated) BA concentrations (STBA) by gas chromatography‐mass spectrometry.

Results: STBA were lower in controls than in patients (SR < NR + RL). The highest levels of cholic acid and chenodeoxycholic acid families were found in NR + RL. Levels of cholic acid family were similar in controls and SR, whereas those of chenodeoxycholic acid family were higher in SR than in controls. A significant correlation between STBA (but not S‐3α‐OH‐BA) and ferritin was found. Apparent value to predict the absence of a sustained response was calculated by combining elevated ferritin (>300 µg/mL) and STBA or individual BA species at different cut‐off values. The best degree of certainty (100% specificity) was obtained using STBA >15 µM.

Conclusion: These results recommend that larger prospective trials should be performed in chronic HCV patients to evaluate the usefulness of combined measuring of STBA and ferritin as additional prognostic markers to predict the existence of a very low probability of a sustained response to the current standard treatment, i.e. pegylated interferon in combination with ribavirin.
Agenesis of the gallbladder: Difficulties in management
Authors: BANI‐HANI KAMAL E
Published: April 2005

Abstract  Gallbladder agenesis is a rare congenital biliary anomaly that may be associated with other biliary and extrabiliary congenital anomalies. Awareness of this entity by clinicians and radiologists is essential because many of these patients present with biliary symptoms and have unnecessary operations. In the present article, the relative epidemiological, etiological (embryology and development), pathophysiological, diagnostic tools and pitfalls and management aspects of this rare anatomic anomaly are briefly discussed through review of the literature. Particular reference to the difficulty in preoperative diagnosis is highlighted. The importance of the possibility of preoperative diagnosis to avoid unnecessary surgery is stressed.
Carcinoid of the ampulla of Vater
Authors: HARTEL MARK , WENTE MORITZ N , SIDO BERND , FRIESS HELMUT , BÜCHLER MARKUS W
Published: April 2005

Abstract  Endocrine neoplasms only rarely occur at the ampulla of Vater, comprising mostly carcinoids and malignant carcinoids, as well as few cases of poorly differentiated endocrine carcinomas (small cell carcinomas). Only 105 cases are reported in the literature, most as single case reports. For many years, the neoplasms of the disseminated neuroendocrine cell system of the gastrointestinal tract have been subsumed as ‘carcinoids’. Instead, in the latest World Health Organization (WHO) classification published in 2000, it is recommended to distinguish between (i) well‐differentiated endocrine tumors (carcinoids); (ii) well‐differentiated endocrine carcinomas (malignant carcinoids); and (iii) poorly differentiated endocrine carcinomas (small cell carcinomas). Patients with carcinoid tumors of the ampulla of Vater are very often free of clinical and laboratory findings that belong to the carcinoid syndrome. Approximately 26% of all patients with carcinoid tumor reported in the literature had neurofibromatosis. Besides endoscopic retrograde cholangiopancreatography, endosonography, computed tomography or magnetic resonance imaging may complete the staging approach of this tumor. The Kausch–Whipple procedure or pylorus‐preserving pancreaticoduodenectomy is considered the treatment of choice for ampullary, well‐differentiated carcinoids >2.0 cm and for ampullary neuroendocrine carcinomas. However, it should be considered that long‐term survival of patients with ampullary carcinoids is also reported after local tumor excision (5‐year survival rate of 90%). The dilemma is that the differentiation of neuroendocrine tumors cannot be assessed intraoperatively in most cases. Therefore, considering that the 5‐year survival rate in patients with neuroendocrine carcinomas of the ampulla of Vater is very low without radical resection, neuroendocrine tumors of the ampulla of Vater without definite histological differentiation should undergo extended surgery.
Clinical features of HBsAg‐negative but anti‐HBc‐positive hepatocellular carcinoma in a hepatitis B virus endemic area
Authors: SHIM JAEJUN , KIM BYUNG‐HO , KIM NAM HOON , DONG SEOK HO , KIM HYO JONG , CHANG YOUNG WOON , LEE JOUNG IL , CHANG RIN
Published: April 2005

Backgound:  The presence of antibody to the hepatitis B core antigen (anti‐HBc) IgG in serum usually means a past infection of the hepatitis B virus (HBV). The clinical characteristics of patients with hepatocellular carcinoma (HCC), who have only a marker for past HBV infection, were investigated.

Methods:  A total of 565 HCC patients were classified according to their markers for HBV and the hepatitis C virus (HCV). The clinical features and the survival rate of hepatitis B surface antigen (HBsAg)(–)/anti‐HBc(+) patients were compared to those of HBsAg(+) patients.

Results:  Four hundred and three patients were positive for HBsAg (B group, 71.3%), 64 were positive for anti‐HCV (11.3%), and 90 were negative for both HBsAg and anti‐HCV (N group, 15.9%). In the N group, 71 were positive for anti‐HBc (PB group, 12.6% of total patients). The clinical characteristics of the PB group were different from those of the B group: age at diagnosis (60.6 ± 9.6 vs 53.3 ± 10.6 years, P < 0.001), habitual drinking (59.2% vs 23.6%, P < 0.001), family history of liver disease (9.9% vs 38.9%, P < 0.005), detection with periodic screening (28.2% vs 50.4%, P < 0.001), and elevated α‐fetoprotein (53.5% vs 76.2%, P < 0.001). In both the PB group and the B group, liver cirrhosis was accompanied by a similar high prevalence (74.6% vs 89.1%). However, there was no significant difference in the cumulative survival rate.

Conclusions:  The prevalence of HBsAg(–)/anti‐HBc(+) HCC is not rare or more common than that of anti‐HCV(+) HCC in Korea, a high HBV endemic area. Although some differences in clinical characteristics may imply a different pathogenesis, chronic HBV infection or habitual drinking may be major contributing factors in the development of HCC in these patients.
Clinicopathologic features of patients with primary malignant hepatic tumors seropositive for α‐fetoprotein‐L3 alone in comparison with other patients seropositive for α‐fetoprotein‐L3
Authors: OKUDA HIROAKI , SAITO AKIKO , SHIRATORI KEIKO , YAMAMOTO MASAKAZU , TAKASAKI KEN , NAKANO MASAYUKI
Published: April 2005

Background:  Patients with hepatocellular carcinoma (HCC) seropositive for lectin‐reactive α‐fetoprotein (AFP‐L3) have a poor prognosis. However, there have been no studies of the clinicopathologic features of patients seronegative for both AFP and des‐γ‐carboxy prothrombin (DCP), and seropositive for AFP‐L3 alone in comparison with other patients seropositive for AFP‐L3.

Methods:  Patients with primary malignant hepatic tumors seropositive for AFP‐L3 who underwent hepatectomy (n = 84) were divided into four groups, and their clinicopathologic features were compared: (i) group A, seronegative for AFP <100 ng/mL and DCP <40 mAU/mL; (ii) group B, seropositive for AFP ≥100 ng/mL and seronegative for DCP; (iii) group C, seronegative for AFP and seropositive for DCP ≥40 mAU/mL; and (iv) group D, seropositive for AFP and DCP.

Results:  Among the 14 group A patients seropositive for AFP‐L3 alone with low AFP concentrations, three had intrahepatic cholangiocarcinoma (ICC), one had a cholangiolocellular carcinoma, one had combined HCC and ICC, and one had undifferentiated hepatic sarcoma. Group A had a higher incidence of non‐HCC tumors (P < 0.001) and tumors derived from cholangiocytes (P < 0.001) than the other three groups. They also had a high frequency of poorly differentiated tumors and sarcomatous changes, and showed a poor prognosis.

Conclusions:  Patients with primary malignant hepatic tumors seropositive for AFP‐L3 alone with low AFP concentrations have unique clinicopathologic features. Thus, we should be aware of these patients and should measure AFP‐L3 levels, at least once, even in those seronegative for both AFP and DCP.
Pegylated versus standard interferon‐α in antiviral regimens for post‐transplant recurrent hepatitis C: Comparison of tolerability and efficacy
Authors: TONIUTTO PIERLUIGI , FABRIS CARLO , FUMO ELISABETTA , APOLLONIO LUCA , CALDATO MAYA , AVELLINI CLAUDIO , MINISINI ROSALBA , PIRISI MARIO
Published: April 2005

Background: In the treatment of hepatitis C virus (HCV) infection, regimens including pegylated interferon‐α are superior to those including standard interferon; the present retrospective study was performed to verify whether the same is applicable to biopsy‐proven recurrent hepatitis C (genotype 1b) after liver transplantation (OLT).

Methods: Twenty‐four patients (16 male) were studied. Twelve had received interferon‐α2b (IFN), 9 MU weekly and 12 received pegylated interferon‐α2b (PEG‐IFN), 0.5 µg/kg weekly. All had received oral ribavirin 600–800 mg/day. Treatment duration was intended for 12 months. A repeat liver biopsy, with evaluation of the Ishak grading and staging scores, was obtained at 1 year.

Results: Only 12/24 patients (50%) completed a full year of therapy; 17 (71%) experienced side‐effects requiring a 50% dosage reduction or discontinuation of the IFN, PEG‐IFN and/or ribavirin. This was observed in 6/12 patients (50%) treated with IFN in comparison to 11/12 patients (92%) treated with PEG‐IFN (P < 0.05). The difference was mainly accounted for by anemia and leukopenia that were reported in 4/12 IFN patients (33%) versus 9/12 PEG‐IFN patients (75%; P < 0.05), respectively. End‐of‐treatment viral response (ETVR) and histological response were always associated and occurred in 4/24 patients (17%), two in each treatment arm. Patients with ETVR were younger, had always completed 1 year of therapy, had had recurrent hepatitis later after transplantation and presented a higher baseline grading score.

Conclusions: In the OLT setting, the potential benefits of antiviral treatments including PEG‐IFN may be limited by the poor tolerability of the adopted drugs.