bluuebunny/crossref_metadata_2025 huggingface dataset:
Lysosomal storage disorders
Authors: Vellodi Ashok
Published: February 2005

Although the first description of a lysosomal storage disorder was that of Tay‐Sachs disease in 1881, the lysosome was not discovered until 1955, by Christian De Duve. The first demonstration by Hers in 1963 of a link between an enzyme deficiency and a storage disorder (Pompe's disease) paved the way for a series of seminal discoveries about the intracellular biology of these enzymes and their substrates, culminating in the successful treatment of Gaucher's disease with β‐glucosidase in the early 1990s. It is now recognized that these disorders are not simply a consequence of pure storage, but result from perturbation of complex cell signalling mechanisms. These in turn give rise to secondary structural and biochemical changes, which have important implications for therapy. Significant challenges remain, particularly the treatment of central nervous system disease. It is hoped that recent advances in our understanding of lysosomal biology will enable successful therapies to be developed.
Induction of CCAAT/enhancer binding protein‐δ by cytokinins, but not by retinoic acid, during granulocytic differentiation of human myeloid leukaemia cells
Authors: Ishii Yuki , Kasukabe Takashi , Honma Yoshio
Published: February 2005

Cytokinins, purine derivatives that act as hormones to control many processes in plants, are very effective at inducing the granulocytic differentiation of human myeloid leukaemia cells. Isopentenyladenine (IPA), a potent cytokinin, significantly induced the expression of CCAAT/enhancer‐binding protein (C/EBP)δ, but not C/EBPα protein, whereas all‐trans retinoic acid, a well‐known inducer of granulocytic differentiation, induced C/EBPα but not C/EBPδ protein. Antisense oligonucleotide for C/EBPδ, but not C/EBPα or C/EBPβ, effectively suppressed IPA‐induced differentiation, suggesting that the expression of C/EBPδ protein is necessary for cytokinin‐induced differentiation. Although C/EBPα is known to be crucial for granulocytic differentiation, the function of C/EBPδ has not been well documented in the regulation of haematopoiesis. The role of C/EBPδ in the granulocytic differentiation of myeloid leukaemia cells is discussed.
Differential control of G0 programme in chronic lymphocytic leukaemia: a novel prognostic factor
Authors: Danilov Alexey V. , Klein Andreas K. , Lee Henry J. , Baez Diana Velez , Huber Brigitte T.
Published: February 2005

Chronic lymphocytic leukaemia (CLL) is a unique malignancy where quiescent B cells accumulate in the peripheral blood. Since clinical outcomes in CLL are very heterogeneous, it is of utmost importance to correctly assess the disease prognosis in each individual case. Recently, it has been shown that high ZAP‐70 [Zeta‐chain (T‐cell receptor) associated protein kinase (70 kDa)] expression level strongly correlates with lack of IgVH mutations and poor prognosis in B‐CLL. As CLL malignant cells are arrested in G0, we investigated whether Dipeptidyl Peptidase 2 (DPP2), a serine protease that plays a key role in keeping cells in the quiescent state, is involved in cell‐cycle control in CLL. We have previously shown that specific inhibition of DPP2 results in apoptosis of normal lymphocytes. In this study, cell apoptosis experiments were conducted in 38 patients with B‐CLL. Two distinct subsets of B‐CLL were identified, susceptible and resistant to DPP2‐inhibition‐induced apoptosis. If resistant to apoptosis (42·1%), the CLL cells have higher expression of ZAP‐70 and exhibit a worse prognosis, such as shorter treatment‐free time period. Thus, resistance vs. susceptibility to DPP2‐inhibiton induced apoptosis can be employed as a novel prognostic factor in CLL.
Accuracy of platelet counting haematology analysers in severe thrombocytopenia and potential impact on platelet transfusion
Authors: Segal H. C. , Briggs C. , Kunka S. , Casbard A. , Harrison P. , Machin S. J. , Murphy M. F.
Published: February 2005

Although haematology analysers provide reliable full blood counts, they are known to be inaccurate at enumerating platelets in severe thrombocytopenia. If the thresholds for platelet transfusion, currently set at 10 × 109/l, are to be further reduced, it is vital that the limitations of current analysers are fully understood. The aim of this large multicentre study was to determine the accuracy of haematology analysers in current routine practice for platelet counts below 20 × 109/l. Platelet counts estimated by analysers using optical, impedance and immunological methods were compared with the International Reference Method for platelet counting. The results demonstrated variation in platelet counting between different analysers and even the same type of analyser at different sites. Optical methods for platelet counting on the XE 2100, Advia 120, Cell‐Dyn 4000 and H3* were not superior to impedance methods on the XE 2100, LH750 and Pentra analysers. All analysers except one overestimated the platelet count, which would result in under transfusion of platelets. This study highlights the inaccuracies of haematology analysers in platelet counting in severe thrombocytopenia. It re‐emphasizes the need for external quality control to improve analyser calibration for samples with low platelet counts, and suggests that the optimal thresholds for prophylactic platelet transfusions should be re‐evaluated.
Global change effects on a mechanistic decomposer food web model
Authors: Kuijper Lothar D. J. , Berg Matty P. , Morriën Elly , Kooi Bob W. , Verhoef Herman A.
Published: February 2005

Global change may affect the structure and functioning of decomposer food webs through qualitative changes in freshly fallen litter. We analyzed the predicted effects of a changing environment on a dynamic model of a donor‐controlled natural decomposer ecosystem near Wekerom, the Netherlands. This system consists of fungi, bacteria, fungivores, bacterivores and omnivores feeding on microbiota and litter as well. The model concentrates on carbon and nitrogen flows through the trophic niches that define this decomposer system, and is designed to predict litter masses and abundances of soil biota. For modeling purposes, the quality of freshly fallen leaf litter is defined in terms of nitrogenous and non‐nitrogenous components, of which refractory and labile forms are present. The environmental impacts of elevated CO2, enhanced UV‐B and eutrophication, each with their own influence on leaf litter quality, are studied. The model predicts steady‐state dynamics exclusively, for all three scenarios. Environmental changes impact most demonstratively on the highest trophic niches, and affect microbiotic abundances and litter decomposition rates to a lesser extent. We conclude that the absence of trophic cascade effects may be attributed to weak trophic links, and that non‐equilibrium dynamics occurring in the system are generally because of encounter rates based on fractional substrate densities in the litter. We set out a number of experimentally testable hypotheses that may improve understanding of ecosystem dynamics.
Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl‐homoserine lactones
Authors: Collins Cynthia H. , Arnold Frances H. , Leadbetter Jared R.
Published: January 2005

LuxR‐type  transcriptional  regulators  play  key  roles in quorum‐sensing systems that employ acyl‐homoserine lactones (acyl‐HSLs) as signal molecules. These proteins mediate quorum control by changing their interactions with RNA polymerase and DNA in response to binding their cognate acyl‐HSL. The evolutionarily related LuxR‐type proteins exhibit considerable diversity in primary sequence and in their response to acyl‐HSLs having acyl groups of differing length and composition. Little is known about which residues determine acyl‐HSL specificity, and less about the evolutionary time scales required to forge new ones. To begin to examine such issues, we have focused on the LuxR protein from Vibrio fischeri, which activates gene transcription in response to binding its cognate quorum signal, 3‐oxohexanoyl‐homoserine lactone (3OC6HSL). Libraries of luxR mutants were screened for variants exhibiting increased gene activation in response to octanoyl‐HSL (C8HSL), with which wild‐type LuxR interacts only weakly. Eight LuxR variants were identified that showed a 100‐fold increase in sensitivity to C8HSL; these variants also displayed increased sensitivities to pentanoyl‐HSL and tetradecanoyl‐HSL, while maintaining a wild‐type or greater response to 3OC6HSL. The most sensitive variants activated gene transcription as strongly with C8HSL as the wild type did with 3OC6HSL. With one exception, the amino acid residues involved were restricted to the N‐terminal, ‘signal‐binding’ domain of LuxR. These residue positions differed from critical positions previously identified via ‘loss‐of‐function’ mutagenesis. We have demonstrated that acyl‐HSL‐dependent quorum‐sensing systems can evolve rapidly to respond to new acyl‐HSLs, suggesting that there may be an evolutionary advantage to maintaining such plasticity.
Mutations in the amino‐terminal domain of λ‐integrase have differential effects on integrative and excisive recombination
Authors: Warren David , Lee Sang Yeol , Landy Arthur
Published: February 2005

Lambda integrase (Int) forms higher‐order protein–DNA complexes necessary for site‐specific recombination. The carboxy‐terminal domain of Int (75–356) is responsible for catalysis at specific core‐type binding sites whereas the amino‐terminal domain (1–70) is responsible for cooperative arm‐type DNA binding. Alanine scanning mutagenesis of residues 64–70, within full‐length integrase, has revealed differential effects on cooperative arm binding interactions that are required for integrative and excisive recombination. Interestingly, while these residues are required for cooperative arm‐type binding on both P′1,2 and P′2,3 substrates, cooperative binding at the arm‐type sites P′2,3 was more severely compromised than binding at arm‐type sites P′1,2 for L64A. Concomitantly, L64A had a much stronger effect on integrative than on excisive recombination. The arm‐binding properties of Int appear to be intrinsic to the amino‐terminal domain because the phenotype of L64A was the same in an amino‐terminal fragment (Int 1–75) as it was in the full‐length protein.
Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus
Authors: Enos‐Berlage Jodi L. , Guvener Zehra T. , Keenan Carrie E. , McCarter Linda L.
Published: February 2005

Vibrio parahaemolyticus isolates display variation in colony morphology, alternating between opaque (OP) and translucent (TR) cell types. Phase variation is the consequence of genetic alterations in the locus encoding the quorum sensing output regulator OpaR. Here, we show that both cell types form stable, but distinguishable biofilms that differ with respect to attachment and detachment profiles to polystyrene, pellicle formation and stability at the air/medium interface, and submerged biofilm architecture and dispersion at a solid/liquid interface. The pellicle, which is a cohesive mat of cells, was exploited to identify mutants having altered or defective biofilm formation. Transposon insertion mutants were obtained with defects in genes affecting multiple cell surface characteristics, including extracellular polysaccharide, mannose‐sensitive haemagglutinin type 4 pili and polar (but not lateral) flagella. Other insertions disrupted genes coding for potential secreted proteins or transporters of secreted proteins, specifically haemolysin co‐regulated protein and an RTX toxin‐like membrane fusion transporter, as well as potential modifiers of cell surface molecules (nagAC operon). The pellicle screen also identified mutants with lesions in regulatory genes encoding H‐NS, a CsgD‐like repressor and an AraC‐like protein. This work initiates the characterization of V. parahaemolyticus biofilm formation in the OP and TR cell types and identifies a diverse repertoire of cell surface elements that participate in determining multicellular architecture.
TUP1 disruption reveals biological differences between MATa and MATα strains of Cryptococcus neoformans
Authors: Lee Hyeseung , Chang Yun C. , Kwon‐Chung K. J.
Published: February 2005

Cryptococcus neoformans exists in two mating types MATa and MATα. Although the morphology, growth characteristics and genetic segregation patterns among MATa and MATα strains are indistinguishable in the laboratory, the predominance of MATα strains in nature suggests that MATα strains are better suited for survival in nature. We disrupted the TUP1 gene, a global repressor, to find the possible biological differences in congenic MATα and MATa cells of C. neoformans. Disruption of TUP1 affected neither the yeast nor the hyphal cell morphology but resulted in a similar reduction of mating frequencies in both MATα and MATa cells. Disruption of TUP1, however, functionally manifested itself in several mating type‐dependent phenotypes: (i) MATα cells became more sensitive to 0.8 M KCl while MATa cells showed no change in sensitivity, (ii) a temperature‐dependent growth reduction was exhibited at both 30°C and 25°C in MATa but a similar growth reduction was not observed in MATα cells until the temperature was lowered to 25°C and (iii) the transcriptional level of genes in several different biological pathways was markedly altered in a mating type‐dependent manner. This work is the first case in which non‐mating‐related biological differences are observed between two congenic mating partners in yeast.
Retinal biosynthesis in Eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp. PCC 6803
Authors: Ruch Sandra , Beyer Peter , Ernst Hansgeorg , Al‐Babili Salim
Published: February 2005

Retinal and its derivatives represent essential compounds in many biological systems. In animals, they are synthesized through a symmetrical cleavage of β‐carotene catalysed by a monooxygenase. Here, we demonstrate that the open reading frame sll1541 from the cyanobacterium Synechocystis sp. PCC 6803 encodes the first eubacterial, retinal synthesizing enzyme (Diox1) thus far reported. In contrast to enzymes from animals, Diox1 converts β‐apo‐carotenals instead of β‐carotene into retinal in vitro. The identity of the enzymatic product was proven by HPLC, GC‐MS and in a biological test. Investigations, of the stereospecifity showed that Diox1 cleaved only the all‐trans form of β‐apo‐8′‐carotenal, yielding all‐trans‐retinal. However, Diox1 exhibited wide substrate specificity with respect to chain‐lengths and functional end‐groups. Although with divergent Km and Vmax values, the enzyme converted β‐apo‐carotenals, (3R)‐3‐OH‐β‐apo‐carotenals as well as apo‐lycopenals into retinal, (3R)‐3‐hydroxy‐retinal and acycloretinal respectively. In addition, the alcohols of these substrates were cleaved to yield the corresponding retinal derivatives.
Telomere length control and transcriptional regulation of subtelomeric adhesins in Candida glabrata
Authors: Castaño Irene , Pan Shih‐Jung , Zupancic Margaret , Hennequin Christophe , Dujon Bernard , Cormack Brendan P.
Published: February 2005

The pathogenic yeast Candida glabrata is able to bind in vitro to human epithelial cells. This interaction depends on expression of the adhesin Epa1p. The genome contains a number of EPA1 paralogues which localize to the subtelomeric regions of the C. glabrata. We have identified three hyperadherent mutants of C. glabrata. The first has an insertion adjacent to EPA7, an EPA1‐related adhesin. The others disrupt the SIR3 and RIF1 genes of C. glabrata. We show that SIR3 and RIF1 are required for subtelomeric silencing in C. glabrata and that RIF1 regulates telomere length in C. glabrata. We show that the hyperadherent phenotype of the sir3Δ and rif1Δ deletion strains depends primarily on derepression of two novel members of the EPA gene family –EPA6 and EPA7. The sir3Δ and rif1Δ mutants show increased colonization of the kidney in a murine model of disseminated infection and this hypercolonization depends, at least in part, on derepression of EPA6 and EPA7. The analysis here is the first evidence that multiple EPA genes encode adhesins and demonstrates that transcription of at least two of these adhesins is regulated by subtelomeric silencing.
Inactivation and deficiency of core proteins of photosystems I and II caused by genetical phylloquinone and plastoquinone deficiency but retained lamellar structure in a T‐DNA mutant of Arabidopsis
Authors: Shimada Hiroshi , Ohno Ryoichi , Shibata Masaru , Ikegami Isamu , Onai Kiyoshi , Ohto Masa‐aki , Takamiya Ken‐ichiro
Published: February 2005

Phylloquinone, a substituted 1,4‐naphthoquinone with an 18‐carbon‐saturated phytyl tail, functions as a bound one‐electron carrier cofactor at the A1 site of photosystem I (PSI). A Feldmann tag line mutant, no. 2755 (designated as abc4 hereafter), showed pale‐green young leaves and white old leaves. The mutated nuclear gene encoded 1,4‐dihydroxy‐2‐naphtoic acid phytyltransferase, an enzyme of phylloquinone biosynthesis, and high‐performance liquid chromatography analysis revealed that the abc4 mutant contained no phylloquinone, and only about 3% plastoquinone. Photooxidation of P700 of PSI in the abc4 mutant was not observed, and reduced‐versus‐oxidized difference spectroscopy indicated that the abc4 mutant had no P700. The maximum quantum yield of photosystem II (PSII) in the abc4 mutant was much decreased, and the electron transfer from PSII to PSI in the abc4 mutant did not occur. For the pale‐green leaves of the abc4 mutant plant, the ultrastructure of the chloroplasts was almost the same as that of the wild‐type plant. However, the chloroplasts in the albino leaves of the mutant were smaller and had a lot of grana thylakoids and few stroma thylakoids. The amounts of PSI and PSII core subunits in the abc4 mutant were significantly decreased compared with those in the wild type. These results suggested that a deficiency of phylloquinone in PSI caused the abolishment of PSI and a partial defect of PSII due to a significant decrease of plastoquinone, but did not influence the ultrastructure of the chloroplasts in young leaves.
Cyclization of several linear penta‐ and heptapeptides with different metal ions studied by CD spectroscopy*
Authors: Liu M. , Tang Y.‐C. , Fan K.‐Q , Jiang X. , Lai L.‐H , Ye Y.‐H
Published: February 2005

Abstract:  A cyclic pentapeptide c(Tyr‐Leu‐Ala‐Gly‐Pro) (I), which was isolated and identified from Pseudostellaria heterophylla medicinal herbs, and two cyclic heptapeptides, c(Gly‐Tyr‐Gly‐Gly‐Pro‐Phe‐Pro) (II) and c(Gly‐Ile‐Pro‐Tyr‐Ile‐Ala‐Ala) (III), which were isolated and identified from Stellaria yunnanensis Franch (M), were synthesized by using 3‐(diethoxyphosphoryloxy)‐1,2,3‐benzotriazin‐4(3 H)‐one (DEPBT) as a coupling reagent in solution, and mediated by different metal ions, from their linear peptide precursors H‐Tyr‐Leu‐Ala‐Gly‐Pro‐OH (I‐1) and H‐Ala‐Gly‐Pro‐Tyr‐Leu‐OH (I‐2), H‐Gly‐Tyr‐Gly‐Gly‐Pro‐Phe‐Pro‐OH (II‐1) and H‐Gly‐Ile‐Pro‐Tyr‐Ile‐Ala‐Ala‐OH (III‐1), respectively. The results show that alkali metal ions can improve the cyclization yields and/or the cyclization rates of linear peptide precursors, such as Na+ ion is favorable for the cyclization of linear pentapeptides and Cs+ ion is favorable for the cyclization of linear heptapeptides, while some bivalent and trivalent metal ions, such as Mg2+, Ca2+, Zn2+, Fe2+, Ni2+ and Cr3+ reduced/inhibited both the cyclization yields and the cyclization rates of the linear peptide precursors. The circular dichroism spectra of I‐1, II‐1 and III‐1 with different metal ions were studied to elucidate the changes in their secondary structures. It is shown that Cs+ can induce and stabilize the type I β‐turn conformation in the linear heptapeptide II‐1 and the type II β‐turn conformation in the linear heptapeptide III‐1.
Solvent effects on coupling yields during rapid solid‐phase synthesis of CGRP(8‐37) employing in situ neutralization*
Authors: Taylor C.K. , Abel P.W. , Hulce M. , Smith D.D.
Published: February 2005

Abstract:  The success of solid‐phase peptide synthesis is often dependent upon solvation of the resin and the growing resin‐bound peptide chain. We investigated the relationship between solvent properties and solvation of the resin and peptide‐resin in order to obtain satisfactory coupling yields for the rapid solid‐phase peptide synthesis, using butyloxycarbonyl‐(Boc)‐amino acid derivatives, of human‐α‐calcitonin gene‐related peptide(8‐37) (CGRP(8‐37)). Solvation of (p‐methylbenzhydrylamine)copoly(styrene–1% divinylbenzene (DVB) (resin) and resin covalently bound to the fully protected amino acid sequence of CGRP(8‐37) (peptide–resin) was correlated to solvent Hildebrand solubility (δ) and hydrogen‐bonding (δh) parameters. Contour solvation plots of δh vs. δ revealed maximum solvation regions of resin and peptide–resin. Maximum resin solvation occurred with N‐methylpyrrolidinone (NMP), NMP : dimethylsulfoxide (DMSO) (8 : 2) and DMSO. Inefficient solvation of the peptide–resin occurred with these solvents and resulted in poor syntheses with average coupling yields of 78.1, 88.9 and 91.8%, respectively. Superior peptide–resin solvation was obtained using dimethylacetamide (DMA) and dimethylformamide (DMF), resulting in significantly higher average coupling yields of 98.0 and 99.5%, respectively. Thus, the region of maximum peptide–resin solvation shifts to solvents with higher δh values. DMF provided the most effective peptide–resin solvation and was the only solvent from which CGRP(8‐37) was obtained as a single major product in the crude cleaved material.
An asymmetric conformation of 1,3,5‐benzene tricarbonyl [Aib4OMe]3*
Authors: Karle I.L. , Ranganathan D.
Published: February 2005

Abstract:  Molecules of 1,3,5‐benzene tricarbonyl [Aib4OMe]3 do not possess any internal symmetry, neither exact nor approximate, in the crystalline state. The Aib4OMe moieties each form a 310‐helix with an appropriate pair of hydrogen bonds but the sense of rotation is right‐handed for two of the helices and left‐handed for the third one. The helices are not evenly positioned around the benzene ring, and their helix axes are inclined toward one side of the plane of the benzene ring, giving the molecule the shape of a shallow bowl with an irregular periphery. The molecules are largely surrounded by water and dimethyl sulfoxide (DMSO) solvent molecules that form hydrogen bonds with the CO and NH moieties that protrude from the surfaces of the peptide molecule. The space group is Cc with a = 23.618(4) Å, b = 19.708(6) Å, c = 17.939(7) Å and β = 100.09(3)°.
Azobenzene as photoresponsive conformational switch in cyclic peptides*
Authors: Renner C. , Kusebauch U. , Löweneck M. , Milbradt A.G. , Moroder L.
Published: February 2005

Abstract:  Over the last decades azobenzene has been the most widely used optical trigger for the synthesis of photoresponsive systems ranging from poly‐α‐amino acids to innovative materials with light‐controlled mechanical and optical properties. More recently, its use in form of appropriate derivatives allowed to generate cyclic peptide structures of constraint conformational space and thus to exploit its reversible photoisomerization to induce well defined transitions between different conformational states. These can be characterized in detail in both photostationary states making such systems ideal substrates for ultrafast spectroscopic analysis of conformational transitions. Moreover, the changes in biophysical properties that occur as a consequence of the different conformational states can be exploited for a photo‐control of a large variety of molecular recognition processes.
ULTRASTRUCTURAL STUDY ON THE SCLEROBLAST OF ORYZIAS LATIPES DURING ETHISTERONE‐INDUCED ANAL‐FIN PROCESS FORMATION
Authors: UWA HIROSHI
Published: January 2005

Cytodifferentiation of the scleroblast during ethisterone‐induced anal‐fin process formation in adult females of Oryzias latipes was observed electron‐microscopically. Scleroblasts seem to arise from immature mesenchymal cells located not only along the postero‐external margin of the joint plates, but also in the space between paired joint plates. These cells contained a small nucleus with condensed chromatin and a very small amount of cytoplasm with a marked paucity of formed organelles. These cells proliferated and/or migrated to the process‐forming site of the joint plates and aggregated to the mass. Young scleroblasts were spindle‐shaped in profile and contained an enlarged nucleus with dispersed chromatin. Rough‐surfaced endoplasmic reticulum was developed in the cytoplasm. Scleroblasts became oval in shape and commenced to secrete bone matrix. Well‐developed scleroblasts were characterized by an extensive development of rough‐surfaced endoplasmic reticulum. This seems to correlate with the active protein synthesis required by the scleroblast for laying down bone matrix. As the young horny process developed, scleroblasts covered its apex, and became flattened. Some scleroblasts which became enclosed in the developing matrix had much the same characteristics as when they were lying on its surface.
INTERACTIONS OF 1‐METHYLADENINE AND THE SURFACE OF STARFISH OOCYTE
Authors: SHORTRIDGE KATHRYN , LOSEK BARBARA , LEE HAROLD H.
Published: January 2005

Three metabolic inhibitors, mycostatin, concanavalin A (Con A) and cytochalasin B (CB) were used to study the interactions between l‐methyladenine (1‐MA) and the starfish oocyte surface leading to germinal vesicle breakdown (GVB). Mycostatin and Con A had no obvious effects on GVB. CB did not inhibit, but did delay GVB. This delaying effect was interpreted as having multiple 1‐MA reactive “sites” on the surface. The results also suggested that not all of them were needed to react with 1‐MA to bring about GVB.
SOME OBSERVATIONS OF ABNORMAL EMBRYOS INDUCED BY SHORT‐PERIOD TREATMENT WITH CHLORAMPHENICOL DURING EARLY DEVELOPMENT OF SEA URCHIN
Authors: FUJIWARA AKIKO , YASUMASU IKUO
Published: January 2005

Sea urchin embryos, which were treated with 5 × 10−3 M chloramphenicol for 1 to 4 hr at certain stages before hatching, developed to several types of abnormal embryos. No significant effect on the shape of the embryo was observed when the concentrations of chloramphenicol used in the short‐period treatment were lower than 2 × 10−3 M. Embryos up to the 2‐cell stage, treated with 5 × 10−3 M chloramphenicol for a short period, became small blastulae filled with mesenchyme‐like cells (type A). A similar effect of puromycin (2 μg/ml) was also observed at this stage. When the chloramphenicol treatment (for 1 to 4 hr) was applied at 8 ∽ 32‐cell stages, vegetalized larvae were produced (type B). Embryos treated with chloramphenicol at 7 hr after insemination at 20°C, developed to another type of abnormal larva different from the previous types (type C). A concentration of puromycin (2 μg/ml) which inhibited protein synthesis to the same degree as 5 × 10−3 M chloramphenicol, induced only type A. Between these chloramphenicol‐sensitive stages, there were chloramphenicol‐insensitive stages for forming abnormal embryos.
THE MITOTIC APPARATUS ISOLATED IN GLYCEROL‐CONTAINING MEDIUM
Authors: SAKAI HIKOICHI , KURIYAMA RYOKO
Published: January 2005

The mitotic apparatus of the sea urchin egg was isolated at 30°C in an isolation medium containing glycerol which is known to stabilize microtubules. After isolation in the 1 m glycerol‐isolation medium, the mitotic apparatus was stabilized on addition of glycerol to a final concentration of 3 to 4 m. Without the addition, the chromosomes were disjoined from the spindle and the interzonal region between separating chromosomes was fragile resulting in separation of half spindles. Lowering the temperature of the isolation medium to 20°C or below, the isolation procedure allowed to isolate spindles. The isolated spindle behaved in a manner similar to the mitotic apparatus on the effect of glycerol concentration.

The glycerol‐mitotic apparatus contained tubulin which was extractable with the isolation medium containing Ca ions or an organic mercurial. Tubulin was also extracted upon lowering the temperature to 0°C in the presence of GTP. Addition of KCl to a final concentration of 0.6 m immediately dispersed the mitotic apparatus. The extract revealed a colchicine binding of 0.001 mole per 105,000 ×g of protein. The colchicine binding complex was found to have a molecular weight of 105,000. The DEAE Sephadex column chromatography of the KCl extract allowed to elute tubulin fraction which bound 0.1 mole colchicine per 105,000 ×g of protein. The mitotic apparatus tubulin was shown to contain α and β subunits with mobilities quite identical with those of brain tubulin subunits. The molecular weights of the α and β subunits were 55,000 ± 1,000 and 51,000 ± 1,000, respectively.
EFFECTS OF CULTURE MEDIA ON THE “FOREIGN” DIFFERENTIATION OF LENS AND PIGMENT CELLS FROM NEURAL RETINA IN VITRO
Authors: ARAKI MASASUKE , OKADA T. S.
Published: January 2005

Neural retinal cells of 3.5‐day‐old quail embryos were cultured as a monolayer to examine their potentials for differentiation in vitro. The “foreign” differentiation into lentoid and pigment cells was much affected by the choice of medium (Eagle's MEM and Ham's F–12); in Eagle's MEM, neural retinal cells differentiated extensively into lentoid bodies and pigment cells, as previously reported in cultures of chick neural retinal cells, while in Ham's F–12, though the cells proliferated as well as in Eagle's MEM, the “foreign” differentiation is inhibited. When primary cultures were transferred to secondary cultures, the occurrence of “foreign” differentiation did not depend on the medium used for the primary culturing, but wholly on the medium used for secondary cultures. This difference in differentiation in two different media was quantitatively substantiated by measuring the amounts of α‐, δ‐crystallins and melanins of cultured cells.
HISTOCHEMICAL AND ELECTRON MICROSCOPIC OBSERVATIONS ON THE STEROID HORMONE‐SECRETING CELLS IN THE TESTIS OF THE JAPANESE RED‐BELLIED NEWT, CYNOPS PYRRHOGASTER PYRRHOGASTER
Authors: IMAI KATSUTOSHI , TANAKA SHIGEYASU
Published: January 2005

The testis of the Japanese red‐bellied newt, Cynops pyrrhogaster pyrrhogaster, caught in the middle of May was investigated in order to demonstrate the presence of steroid hormone (SH)‐secreting cells, by means of histochemical and electron microscopic techniques. The newt testis is divided into two main parts of spermatogenetic stages. The anterior part is immature and consists of many seminal cysts containing spermatogonia and spermatocytes. The posterior part is mature and made up of many cysts containing mature spermatozoa and glandular tissue.

Histochemical studies revealed Δ5‐3β‐hydroxysteroid dehydrogenase (Δ5‐3β‐HSD) activity in the pericystic cells of the mature part, weak in those before spermiation, then gradually becoming more intense in the pericystic cells just after spermiation. Activity was most pronounced in the outer layer of cells of the glandular tissue which originate from the pericystic cells. In contrast to this, the Δ5‐3β‐HSD reaction was entirely negative in the pericystic cells of the immature part, the Sertoli cells, and the inner cells of the glandular tissue originating from the Sertoli cells.

Electron microscopic observation of the pericystic cells and glandular tissue gave further information on the histochemical features. The pericystic cells in the immature part are fibroblast‐like cells. The cell organelles of these cells are poorly developed except for rough‐surfaced endoplasmic reticulum and rod‐shaped mitochondria. In the mature part, the fibroblast‐like cells are transformed into cells characteristic of SH‐secreting cells. In the cytoplasm, the amount of smooth‐surfaced endoplasmic reticulum increases and typical globular mitochondria with tubular cristae take the place of the rod‐shaped mitochondria. In addition, a number of lipid droplets and lipofuscin granules appear. Most of the fine structures of the outer layer of the cells of the glandular tissue were found to be similar in appearance to those of the SH‐secreting cells in mammals and other vertebrates. These results strongly suggest that, as in the other urodeles, the SH‐secreting cells in the testis of the Japanese red‐bellied newt are the pericystic cells in the mature part and in the outer layer of cells of the glandular tissue.
Mesodermalizing Factors in Fetal Calf Serum
Authors: SUZUKI MICHIHIRO
Published: January 2005

The inducing activities of fetal calf serum (FCS) and fetal calf serum heated for a short time (HFCS) were tested with early gastrula presumptive ectoderm of the newt, Cynops pyrrhogaster as a reactor. Inducing activity was measured by sitting‐drop culture of pieces of ectoderm before and after treatment with Ca, Mg‐free solution (CMF) in culture medium contained FCS or HFCS (10–50%) as inductor. Results showed that FCS did not induce mesodermalization of ectoderm, before or after treatment with CMF, and that HFCS induced mesodermalization only of ectoderm treated with CMF. The treated ectoderm cells differentiated into mesoderm cells such as muscle and notochord. The induction increased with increase in the duration of CMF treatment and with the concentration of HFCS in the medium. This indicates that FCS changes into mesodermalizing material when heated for a short time and that mesodermal induction by HFCS depends on some effect of CMF on presumptive ectoderm. Since CMF was found to be a neuralizing factor, activation of presumptive ectoderm with a neuralizing factor is probably a prerequisite for mesodermal induction with HFCS.
Transmission of Homoiogenetic Induction in Presumptive Ectoderm of Newt Embryo
Authors: KURIHARA KAZUSHIGE , SASAKI NAOI
Published: January 2005

Using newt, Cynops pyrrhogaster, embryos, the production and/or transmission of a homoiogenetic effect of the induction within a presumptive ectoderm was investigated. The primary inductor was the swimbladder of a crucian carp, Caracius auratus. In the first experiment, a piece of presumptive ectoderm was isolated from an early gastrula and one‐third of its inner surface was placed in contact with the swimbladder for 30 min. After removal of the inductor, the ectodermal piece was allowed to stand by itself for several hours (pre‐cultivation), and then it was divided into three parts of equal size, i.e. the end that had been placed in contact with the swimbladder (P1), the next middle (P2) and the end part (P3).

The tissues produced in each part were examined after 10 days' cultivation in Holtfreter's solution. The induction was evoked not only in P1, but also in P2 and P3 which had been free from the inductor. The incidences of tissue differentiation in P2 and P3 increased with the lapse of the pre‐cultivation time, and the rise in P3 came after P2. These results suggested that the mesodermal tissues in the parts not placed in contact with the swimbladder (P2 and P3) were evoked by the homoiogenetic stimulus which came from P1 and P2, respectively.

In the next experiment, P2 of the ectodermal piece was substituted by an aged ectoderm which had lost its primary competence. In this system, the mesodermal induction was not evoked in P3. This suggested that the production of the homoiogenetic activity of the ectoderm might be associated with its competenece.
Localization of Acrosomal Proteinase after Vesculation of the Acrosome
Authors: SAKAI YASUHIRO , YASUDA KENJIRO
Published: January 2005

The freshly ovulated egg is covered by the zona pellucida and follicular‐cell layer. The former is reported to dissolved by acrosomal proteinase and the latter may be dispersed by hyaluronidase. If these two enzymes have different targets, two possibilities can be taken into consideration concerning the timing of release of the two enzymes from the acrosome.

Since hyaluronidase is considered to be released immediately after the acrosomal reaction at the follicular‐cell layer, this work was undertaken to examine the timing of release of acrosomal proteinase after vesiculation of the acrosome, by demonstrating the site of the proteinase histochemically. The results indicated that the acrosomal proteinase disappeared immediately after vesiculation of the acrosome. This result suggests that spermatozoa that show vesiculation of the acrosome in the follicular‐cell layer probably lose acrosomal proteinase in this layer. Thus the spermatozoon probably does not attack the follicular‐cell layer and zona pellucida, one by one by release sequential of hyaluronidase and acrosomal proteinase.
Different Growth Responses of Hamster Dermal Fibroblasts and Chondrocytes to Platelet–derived Growth Facter in Cell Culture
Authors: KATOH Y. , TAKAYAMA S.
Published: January 2005

The growth responses of dermal fibroblasts and chondrocytes obtained from 1‐week‐old hamsters to a growth factor from platelets [platelet‐derived growth factor (PDGF)] were compared. Autoradiography showed that most of the whole nuclei of dermal fibroblasts were labeled with 3H‐thymidine in 5% whole blood serum (WBS), which contains platelet releasate, but only a few percent were labeled in 5% plasma‐derived serum (PDS), which is free of platelet releasate. Addition of platelet releasate to PDS restored the growth stimulatory activity of the serum for dermal fibroblasts. In contrast, most of the nuclei of chondrocytes were labeled with 3H‐thymidine both in 5% WBS and 5% PDS. Dermal fibroblasts grew in 5% WBS, but not in 5% PDS, whereas chondrocytes grew in both 5% WBS and 5% PDS. Partially purified PDGF added to the medium with 0.5% fetal calf serum stimulated DNA synthesis in dermal fibroblasts, but not in chondrocytes.
Developmental Changes of Ecdysteroids in the Eggs of the Silkworm, Bombyx mori
Authors: MIZUNO TAKASHI , WATANABE KUNIO , OHNISHI EIJI
Published: January 2005

Ecdysteroids were studied in relation to embryonic development and diapause of the silkworm, Bombyx mori. The majority of the ecdysteroids was found to be in the conjugated form, and minor part, in the free form. In the developing eggs, 2‐deoxyecdysone, 2‐deoxy‐20‐hydroxy‐ecdysone and 3‐epi‐ecdysone were found to have the free ecdysteroid form as well as the conjugated ecdysteroid form. Ecdysone and 20‐hydroxyecdysone almost exclusively had the conjugated form. The concentration of ecdysteroids in the embryo was higher than that in yolkplasm in the early embryonic stages. During the embryonic diapause, the concentration of free ecdysteroids decreased to a low level while the conjugated form maintained the original level.
Studies on Unequal Cleavage in Sea Urchins II. Surface Differentiation and the Direction of Nuclear Migration
Authors: DAN KATSUMA , ENDO SACHIKO , UEMURA ISAO
Published: January 2005

Cortical features of the meso‐ and macromeres differ from those of the micromeres in sea urchins. At the end of the 8‐cell stage, the four animal cells have a continuous row of vesicles lining the free surface of the cell by transmission electron microscopy (TEM) and the nuclei and the resulting mitotic apparatuses (MA) remain at the cell centers and eventually divide equally into eight mesomeres. In the four vegetal cells, narrow gaps can be seen in the vesicular rows near the vegetal pole. The resting nuclei migrate to these gaps and on forming the spindles, they point directly to the gaps. The result is formation of vesicle‐free micromeres and vesicle‐covered macromeres by unequal divisions.
Cellular Basis of Neuralization of Induced Neurectoderm in Amphibian Embryogenesis: Changes of Cell Shape, Cell Size, and Cytodifferentiation of the Neurectoderm after Neural Induction
Authors: SUZUKI AKIO S. , MIKI KAYOKO
Published: January 2005

Cellular alterations of the neurectoderm after primary embryonic induction were examined by measuring several indices of shape, volume, and cytodifferentiation of the neurectodermal cells of Cynops embryos during gastrulation and early neurulation.

Results showed that cellular alterations occurs just after the 18 hr embryo stage (stage 13b). The thickness of the neurectoderm layer decreases like that of the epidermal ectoderm during early and middle gastrulation. After the 18 hr embryo stage, however, the neurectoderm thickens, mainly due to formation of columnar cells. Measurement of cell volume indicates that the neurectoderm of the early and middle gastrulae consists of a cell population of heterogeneous size. The heterogeneity diminishes sharply after the 18 hr embryo stage and the neural plate of the 36 hr embryo (stage 18) consists of cells of homogeneous size.

Stages before the 12 hr embryo (stage 12b) and after the 18 hr embryo (stage 13b) also showed differed in cell adhesion to the culture flask and in cytodifferentiating potency. Single cells dissociated from the neurectoderm of 18 hr embryos could adhere to the substratum and differentiate into both nerve‐like cells and pigment cells. Both capacities increase during further development.

These results are discussed in relation to the neuralizing determination of neurectoderm after primary embryonic induction.
Reactivity of a Monoclonal Antibody Raised against Human Leukemic Cells to Embryonic and Adult Tissues of the Mouse and Teratocarcinomas
Authors: SATO MASAHIRO , OGATA SHUN‐ICHIROH , UEDA RYUZO , NAMIKAWA REIKO , TAKAHASHI TOSHITADA , NAKAMURA TAKAO , SATO EIICHI , MURAMATSU TAKASHI
Published: January 2005

C‐9‐1, a monoclonal IgM antibody raised against human null cell acute lymphocytic leukemia cells reacted with restricted regions of embryonic and adult tissues of the mouse. The antigen positive sites in the embryos included embryonic ectoderm, visceral endoderm, trophoblastic cells invading the maternal decidua of 5∼7‐day embryos, primordial germ cells of 10∼12‐day embryos, epithelium of nasal chamber, the bronchus, Mullerian duct, epididymis and bladder of 12∼17‐day embryos. In the adult mice, C‐9‐1 antigen was detected in renal tubules, a part of stomach, bladder, endometrium and epididymal sperm. Embryonal carcinoma cells, but not endodermal cells of teratocarcinoma expressed the antigen. Thus, C‐9‐1 antigen showed distribution similar to SSEA‐1. However, C‐9‐1 antigen was not detected in preimplantation embryos, nor in oviduct, both of which are positive for SSEA‐1.
Ultrastructural Changes of the Two Types of Differentiated Cells during the Migration and Early Culmination Stages of Dictyostelium discoideum
Authors: TASAKA MASAO , MAEDA YASUO
Published: January 2005

Changes of fine structure during prolonged migration of Dictyostelium discoideum slugs were studied by electronmicroscopy. Prespore specific vacuoles of cells located near the substratum gradually degenerated and the prespore antigen contained in them was lost. During the process, mitochondria in the prespore cells were transformed dramatically: as the mitochondrion elongates, its central part becomes thinner and the cristae become localized at its two ends. Then it bends and its two ends fuse to segregate part of the cytoplasm. The cristae then accumulate in the original ends. Similar mitochondrial transformation was observed in prespore cells of cell masses induced to culminate after a long period of migration.
In Vitro Growth of Rat Blastocyst under the Effect of Azathioprine
Authors: FEIN AMOS , TODER VLADIMIR , NEBEL LASLO
Published: January 2005

Rat blastocysts were isolated from the uterus on the 5th day after fertilization and set in culture. The effect of azathioprine (Imuran) on the blastocyst's development and on the early trophoblastic differentiation in vitro was investigated. Azathioprine, added to the medium of the blastocyst culture at various concentrations, dose dependently arrested development and had definite cytotoxic effect. In order to study the mechanism of action, a minimal dose of 5 μg/ml, which allowed the survival of about 60% of the blastocysts, was added to the medium after 48 hr of culturing. Under the effect of the substance the area of the spreading blastocyst cells was significantly restricted.

It was found, by autoradiographic methods, that the azathioprine affects the development by restraining DNA synthesis in the throphoblastic cells. Concomitantly RNA synthesis was inhibited and protein synthesis was reduced. The observations indicate, that the impairment of the in vitro differentiation of the blastocysts can be a result of the intracellular inhibitory action of the substance.
Genesis of Epidermal Action Potentials in Cytokinesis Arrested Xenopus Embryos
Authors: TAKAMUNE KAZUFUMI , ITO SHIZUO
Published: January 2005

When Xenopus embryos were treated continuously with cytochalasin B (3–10 μg/ml) from the 8 cell stage, cleavage arrested embryos in various degrees were observed. In 3–5 μg/ml cytochalasin B, cytokinesis was inhibited at the midblastula stage and pigment granules remained at the cell cortex of the animal pole. These cells showed epidermal like action potentials when the control embryos (St. 26/28) generated epidermal action potentials. In 5–7 μg/ml cytochalasin B, furrows, following their formation at early cleavage stages, regressed and no further cleavage from the 16 cell stage to morula stage took place. The pigment granules were dispersed throughout the interior of the cytoplasm. These cells showed no epidermal action potentials. Thus, it is considered that cytokinesis per sé, following the midblastula stage, is not a prerequisite for the genesis of epidermal action potentials, and that chronological times corresponding to the tailbud larva stage and a stable structure of the cellular cortex are required to bring about these potentials.
Pathways of Retinotectal Projection in Developing Xenopus Tadpoles Revealed by Selectively Labeling of Retinal Axons with Horseradish Peroxidase (HRP)
Authors: FUJISAWA HAJIME
Published: January 2005

Anatomical mapping was made of the retinal central pathways from the chiasm to the targets within the tectum in the developing Xenopus tadpoles, after labeling a specific regional population of retinal axons with horseradish peroxidase (HRP). In the tadpoles at stage 50, pathway sorting of retinal axons within the optic tract was clear for the dorsoventral axis of the retina, but not for the nasotemporal axis. Most nasal retinal axons and some dorsal and ventral retinal axons invaded the tectum directly at the diencephalotectal junction, and arrived at their correct sites of innervation after running through ectopic parts of the tectum. These findings indicate that the pathway orientation before targets is not a prerequisite factor for establishment of the orderly map of the retinotectal projection. Rather, a direct interaction between ingrowing retinal axons and tectal cells seems to be a predominant factor for specification of retinal central connections.
Translation of RNA from Eggs During Post‐Diapause Development of Bombyx mori
Authors: SAITO AKIRA , SUGIMOTO YASUSHI , KOGA KATSUMI , SAKAGUCHI BUNGO
Published: January 2005

Eggs of Bombyx mori are aroused from diapause by long‐term chilling and develop when transferred to 25°C. During the first 20 hr of post‐diapause development, the polysome content and the presumed rate of protein synthesis increase about 3‐fold, while the ribosome content and the total RNA content increase only 1.1‐fold. In this study, total RNAs were extracted from chilled eggs (termed 0 hr of development), and post‐diapause eggs at 10 and 20 hr of development. The RNAs were purified further by high pressure liquid chromatography to remove RNA‐like oligonucleotides. On translation in a protein‐synthesizing system derived from wheat germ with a subsaturating amount of RNA, no difference was found in the relative amounts of translatable mRNA activity at 10 and 20 hr of development from that at 0 hr. Moreover, the translation products of the different RNA preparations in a rabbit reticulocyte lysate system appeared very similar when separated by gel electrophoresis and located by fluorography. These facts suggest that protein synthesis in early post‐diapause development is controlled at a translational level.