bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a gene trap insertion into a novel gene, cordon‐bleu, expressed in axial structures of the gastrulating mouse embryo
Authors: Gasca Stephan , Hill David P. , Klingensmith John , Rossant Janet
Published: May 2005

We have used a gene trap (GT) vector and embryonic stem (ES) cell chimeras to screen for insertions of the lacZ reporter gene into transcription units that are spatially and temporally regulated during early mouse embryogenesis. GT vectors which can act as both a reporter and a mutagen have been previously used to isolate new genes that are essential for mouse development. In this paper we describe a GT insertion which displays a very restricted pattern of expression in the gastrulating embryo. β‐Galactosidase activity was first detected at 7.5 days post‐coitum (E7.5) in the node region of the embryo and extended to the midline structures at E8.0. At E9.5 expression was restricted to the floor plate, the notochord, the roof of the gut, and the liver anlage. Expression appeared in the somites at E10.0 and later became more widespread. We used rapid amplification of cDNA ends‐polymerase chain reaction (RACE‐PCR) to clone a partial 360 base pair (bp) cDNA representing an endogenous sequence and containing an open reading frame (ORF) fused in frame to the lacZ reporter gene. The sequence showed no homology to any known protein or protein domain. An overlapping 1,200 bp fragment from a wild‐type cDNA library was cloned and it detected the same pattern of expression as the reporter gene in E7.5, E8.5, and E9.5 wild‐type embryos. It hybridized to a 5.4 kb lacZ fusion transcript and to an endogenous transcript of 6.5 kb. The gene was mapped to chromosome 11 and was named cordon‐bleu (cobl). No phenotype was detected in mice homozygous for the insertion. However, the insertion may not cause a complete disruption of the gene function. The pattern of expression of cobl is very similar to that of hepatic nuclear factor 3β (HNF3β) and sonic hedgehog (Shh), both of which are involved in axial patterning. Therefore, the product of the cobl gene may also prove to be an important component of the genetic pathway regulating vertebrate axis formation. © 1995 Wiley‐Liss, Inc.
Epithelial proliferation and differentiation in the mammary gland do not correlate with cFABP gene expression during early pregnancy
Authors: Binas B. , Gusterson B. , Wallace R. , Clark A. J.
Published: May 2005

Cardiac fatty acid binding protein (cFABP) is abundantly expressed in the nondividing, functionally differentiated mammary ephithelium. It is very closely related, if not identical to, a previously described protein termed mammary derived growth inhibitor (MDGI). In vitro studies suggest that low concentrations of diffusible cFABP/MDGI may play a hormone‐like role in limiting proliferative activity and promoting functional differentiation of this tissue, but no in vivo data to support this idea have been published. To test this hypothesis, we compared the levels of cFABP mRNA with both the epithelial DNA labelling index and levels of β‐casein mRNA in wild‐type mice. We also investigated the effect of a precocious experimental increase of cFABP levels in the mammary gland of transgenic mice on the labelling index and β‐casein mRNA levels. This was accomplished by expressing a bovine cFABP cDNA under the control of the ovine β‐lactoglobulin (BLG) gene promoter. We found that although both the DNA labelling index, β‐casein mRNA levels, and cFABP mRNA levels in wild‐type mice are developmentally regulated, they do not correlate with each other during early pregnancy in individual mice. Moreover, a three‐ to fourfold increase of total cFABP mRNA in two transgenic lines did not affect the DNA labelling index or the levels of β‐casein mRNA, an established marker of differentiation of the mammary epithelium, at this developmental stage. These data suggest that epithelial DNA synthesis, β‐casein gene expression, and expression of the cFABP gene are regulated independently in the proliferatively active mammary gland and that the rapidly dividing mammary epithelial cells are not susceptible to the action of cFABP during early pregnancy. © 1995 Wiley‐Liss, Inc.
Epigenetic programming of differential gene expression in development and evolution
Authors: Monk Marilyn
Published: May 2005

This review covers data on changing patterns of DNA methylation and the regulation of gene expression in mouse embryonic development. Global demethylation occurs from the eight‐cell stage to the blastocyst stage in pre‐implantation embryos, and global de novo methylation begins at implantation. We have used X‐chromosome inactivation in female embryos as a model system to study specific CpG sites in the X‐linked Pgk‐1 and Gópd housekeeping genes and in the imprinted regulatory Xist gene to elucidate the role of methylation in the initiation and maintenance of differential gene activity. Methyl‐ation of the X‐linked housekeeping genes occurs very close in time to their inactivation, thus raising the question as to whether methylation could be causal to inactivation, as well as being involved in its maintenance. A methylation difference between sperm and eggs in the promoter region of the Xist gene, located at the X‐chromosome inactivation centre, is correlated with imprinted preferential inactivation of the paternal X chromosome in extra‐embryonic tissues. Based on our data, a picture of the inheritance of methylation imprints and speculation on the significance of the Xist imprint in development is presented. On a more general level, an hypothesis of evolution by “adaptive epige‐netic/genetic inheritance” is considered. This proposes modification of germ line DNA in response to a change in environment and mutation at the site of modification (e.g., of methylated cytosine to thymine). Epigenetic inheritance could function to shift patterns of gene expression to buffer the evolving system against changes in environment. If the altered patterns of gene activity and inactivity persist, the modifications may become “fixed” as mutations; alternatively, previously silenced gene networks might be recruited into function, thus appearing as if they are “acquired characteristics.” An extension of this hypothesis is “foreign gene acquisition and sorting” (selection or silencing of gene function according to use). “Kidnapping” and sorting of foreign genes in this way could explain the observation that increased complexity in evolution is associated with more “junk” DNA. Adaptive epigenetic/genetic inheritance challenges the “central dogma” that information is unidirectional from the DNA to protein and the idea that Darwinian random mutation and selection are the sole mechanisms of evolution. © 1995 Wiley‐Liss, Inc.
Genetic conflict and evolution of mammalian X‐chromosome inactivation
Authors: Moore Tom , Hurst Laurence D. , Reik Wolf
Published: May 2005

The existence of parentally imprinted gene expression in the somatic tissues of mammals and plants can be explained by a theory of intragenomic genetic conflict, which is a logical extension of classical parent‐offspring conflict theory. This theory unites conceptually the phenomena of autosomal imprinting and X‐chromosome inactivation. We argue that recent experimental studies of X‐chromosome inactivation and andro‐genetic development address previously published predictions of the conflict theory, and we discuss possible explanations for the occurrence of random X‐inactivation in the somatic tissues of eutherians. © 1995 Wiley‐Liss, Inc.
Parent‐of‐origin specific effects on the methylation of a transgene in the zebrafish, Danio rerio
Authors: Martin C. Cristofre , McGowan Ross
Published: May 2005

We have examined the inheritance of a transgene locus in the zebrafish, Daniorerio and demonstrated that its methylation is af fected by the sex of the parent contributing the allele. This parent‐of‐origin effect on the zebrafish transgene appears to be identical to imprinting as seen in mammals except that in zebrafish, passage of the locus through a female tended to decreased its methylation, whereas passage through a male increased it. Methylation of the transgene in gametic tissues differed from somatic tissue with the locus being hypomethylated in sperm and hypermethylated in the unfertilized egg. The potential identification of imprinting in the zebrafish has important ramifications with respect to the evolution of the process as well as for understanding the role of imprinting in mammals. © 1995 Wiley‐Liss, Inc.
Chromatin structure and imprinting: Developmental control of DNase‐I sensitivity in the mouse insulin‐like growth factor 2 gene
Authors: Feil Robert , Handel Mary Ann , Allen Nicholas D. , Reik Wolf
Published: May 2005

The insulin‐like growth factor 2 (Igf2) gene on distal mouse chromosome 7 is expressed predominantly from the paternal allele. In previous studies we identified two regions of paternal allele‐specific methylation; one at ˜ 3 kb upstream of promoter 1, and a second in the 3′, coding portion of the gene. The 3′ region is methylated in an expressing tissue (fetal liver), whereas in a non‐expressing tissue (fetal brain), it is not methylated. By contrast, in the 5′ region, the paternal allele is highly methylated in all tissues. Here, we have studied another characteristic of chromatin, namely, sensitivity to DNase‐1 and have focused our developmental analysis on the two differentially methylated regions of Igf2. In the upstream region, four clustered DNase‐I hypersensitive sites (HSS) were detected in embryonic stem (ES) cells and in midgestation embryos, but not in neonatal liver or brain. In promoter 1 (P1), at β 0.3 kb upstream of exon 1, we detected a tissue‐specific HSS that was present in neonatal liver, in which P1 is active, but was absent in ES cells, the embryo, and in neonatal brain. No DNase‐I HSS were detected in the 3′ differentially methylated region of Igf2. In all these regions, we did not detect differences in DNase‐I sensitivity between the parental chromosomes. These results establish major developmental and tissue‐specific control of chromatin in the Igf2 locus. The presence of the HSS upstream of Igf2 precedes transcriptional activation of the Igf2 gene and may be indicative of a promoter for another transcript that is transcribed in the opposite direction. The HSS in P1 is largely liver‐specific; this promoter therefore is differently regulated than the more general fetal promoters P2 and P3. Whereas methylation can be allele‐specific, presumably reflecting the gene imprint, the nuclease sensitivity, as detected by our assay, is not. These results, taken together with previous observations, reveal developmental and tissue‐specific complexity in the expression of the parental imprint at the level of chromatin and transcription. We propose that epigenetic features of tissue‐specific control and of the control of allelic expression are intricately linked. © 1995 Wiley‐Liss, Inc.
Differential amplification of pheromone genes of the ciliate Euplotes raikovi
Authors: La Terza Antonietta , Miceli Cristina , Luporini Pierangelo
Published: May 2005

In hypotrich ciliates, the entire silent chromosomal genome of the germinal nucleus (micronucleus) undergoes extensive DNA rearrangements that, during the development of the somatic nucleus (macronucleus) at the beginning a new cell life cycle, eventually result in the production of linear DNA molecules. These molecules represent functional genes, each one consisting of a central coding region flanked by two shorter regions, which apparently lack canonical elements for regulation of replication and transcription. These are amplified to thousands of copies in the “adult” macronucleus of the vegetative cell. We defined the extent of this amplification for allelic codominant genes which, in the macronucleus of Euplotes raikovi, encode polypeptide cell recognition factors (pheromones). This amplification was shown to be allele‐specific. The copy numbers of genes coding for pheromones Er‐1, Er‐2, and Er‐10 were determined to be 2.5 ‐ 2.9 × 104, 0.9 ‐ 1.2 × 104, 1.6 ‐ 1.85 × 104 respectively, and these numbers did not appreciably vary during the vegetative cell proliferation. This differential amplification of pheromone genes was (i) independent of whether two genes coexisted in the same heterozy‐gous cell or were separated in the corresponding homozygotes, and (ii) directly correlated with quantitative variations in mRNA synthesis and pheromone secretion. On the basis of these results, it is suggested that a mechanism of gene‐specific amplification may be used by hypotrich ciliates to modulate gene expression. © 1995 Wiley‐Liss, Inc.
Two isoforms of xenopus retinoic acid receptor γ2 (B) exhibit differential expression and sensitivity to retinoic acid during embryogenesis
Authors: Crawford Michael J. , Liversage Richard A. , Varmuza Susannah L.
Published: May 2005

We report the isolation of two retinoic acid receptor isoforms (RARγ), which differ only in the 5′ untranslated and putative N‐terminus A regions. The two isoforms appear to serve as early markers for the presumptive neural axis; however, their expression patterns differ. RARγ2, 1 is first expressed at gastrulation at the dorsal lip and subsequently along the presumptive neural axis. RARγ2.2 represents the full‐length sequence of a receptor cDNA already partially characterized and present as a maternal transcript [Ellinger‐Ziegelbauer and Dreyer (1991); Genes Dev 5:94‐104, (1993): Mech Dev 41:31‐46; Pfeffer and DeRobertis, (1994) Mech Dev: 45:147‐153]. Unlike RARγ2.2, the 2.1 variant is not expressed either in pre‐somitic mesoderm or notochord. RARγ2.1 is strongly expressed in branchial arches and to a lesser extent in the neural floor plate. The two isoforms also exhibit differential sensitivity to retinoic acid. Constitutive expression of RARγ2.2 following neurulation appears to be depressed by treatment with retinoic acid, but domains of highest expression, namely, the head and tail, remain relatively unaffected, as do patterns of expression prior to late neurulation. By contrast, RARγ2.1 is not transcribed in retinoid‐inhibited structures. Using microinjection techniques, we show that changes of RARγ2.1 expression in presumptive head structures occur as an early and local consequence of retinoic acid administration. Since RARγ2.1 expression is inhibited by retinoic acid, we tested to see if other treatments that perturb axis formation had any effect. Surprisingly, UV irradiation did not suppress expression of the RARγ2.1 transcript, suggesting that its inhibition by retinoic acid is not due solely to inhibition of anterior neural development. These experiments demonstrate a new subdivision of isoforms that undergo differential expression during development and that exhibit differential sensitivity to retinoic acid and to UV. This sensitivity and the presence of this isoform variant in regions that are known to exhibit polarizing activity strengthen the hypothesis that these receptors play a primary role during morphogenesis.
Comparative biochemical and stress analysis of genetically selected drosophila strains with different longevities
Authors: Force Allan G. , Staples Tiffany , Soliman Sherif , Arking Robert
Published: May 2005

We have performed a comparative analysis of the effects of age of reproduction on the biochemical (protein, lipid, and glycogen content) and stress resistance (ability to survive starvation, desiccation, and exogenous paraquat) parameters on 10 sister lines of five different Drosophila strains. Four pairs of these sister lines were selected under different regimens for either early or delayed reproduction; the fifth pair was maintained in a nonselected state and served as the baseline strain to which all others were compared. It is generally accepted that the early regimens give rise to short‐lived phenotypes, whereas the delayed regimens give rise to long‐lived phenotypes. Our results suggest that a mechanism involving lipid and starvation resistance is not operative in our long‐lived strains. In addition, a mechanism involving glycogen content and desiccation resistance is only weakly supported. Finally, there is strong support for a mechanism that gives rise to enhanced paraquat resistance and therefore may involve regulatory changes in the pattern of ADS gene expression. In addition, the 15‐day early age at reproduction regimen (M type) shows qualitatively similar responses to that of the late age at reproduction regimen (L type). These results suggest that correlations between biochemical traits and longevity must be interpreted with caution. We discuss possible reasons for these results, including the possibility of multiple mechanisms, each leading to a different extended longevity phenotype.
Paraquat resistance and starvation conditions in the selection for longevity extremes in drosophila melanogaster populations previously selected for long and short developmental period
Authors: Cunha Gilson Luis Da , Mânica Da Cruz Ivana Beatrice , Fiorino Patrícia , De Oliveira Alice Kalisz
Published: May 2005

This paper analyzes the interaction between resistance to free radicals, development under starvation conditions, sex, and its consequences to the lifespan of Drosophila mela‐nogaster populations selected for developmental time and longevity. Our data suggest that the interaction between these physiological and environmental parameters is modulated largely by the pre‐imaginal developmental time, since the response to selection for longevity extremes depends strongly on the previous selection for developmental time extremes.
Calixarene‐Based Picolinamide Extractants for Selective An/Ln Separation from Radioactive Waste
Authors: Casnati Alessandro , Della Ca' Nicola , Fontanella Marco , Sansone Francesco , Ugozzoli Franco , Ungaro Rocco , Liger Karine , Dozol Jean‐Francois
Published: May 2005

Eleven novel ligands having picolinamide or thiopicolinamide binding groups at the upper or lower rim of calix[4]‐, calix[6]‐ and calix[8]arenes have been synthesised. The conformational properties of some of these ligands were studied in solution by means of NMR spectroscopy and in the solid state by X‐ray diffraction. Their ability to extract AmIII or EuIII from water to NPHE (o‐nitrophenyl hexyl ether) was tested at different concentrations of LiNO3, HNO3 and of a lipophilic dicarbollide anion (BrCosan). The data show a high cooperation between binding sites of the calixarene ligands in comparison to data obtained with the model ligand N‐butyl picolinamide (19). Some of these ligands show very high distribution coefficients for Am3+ (DAm > 300) and good Am/Eu selectivity at pH ≥ 3, which open up interesting perspectives for their application in the treatment of low acidity radioactive waste. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
From Amino Acids to Enantiopure Bicyclic Isoxazolidinylpyridin‐4(1H)‐ones through Intramolecular Nitrone Cycloadditions
Authors: Romeo Roberto , Iannazzo Daniela , Piperno Anna , Chiacchio Maria A. , Corsaro Antonino , Rescifina Antonio
Published: May 2005

Homochiral bicyclic isoxazolidinylpyridin‐4(1H)‐ones have been synthesised by an intramolecular nitrone cycloaddition process, starting from homochiral β‐amino acids. Stereoselection at C2 or C3 of the acyclic substrate appears to give the best results in the control of the stereochemistry of the new formed chiral centres. The synthetic approach has been further directed towards functionalised piperidones. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
Palladium‐Mediated 11C‐Carbonylative Cross‐Coupling of Alkyl/Aryl Iodides with Organostannanes: An Efficient Synthesis of Unsymmetrical Alkyl/Aryl [11C‐carbonyl]Ketones
Authors: Karimi Farhad , Barletta Julien , Långström Bengt
Published: May 2005

[11C]Carbon monoxide in low concentration, palladium complexes, alkyl/aryl iodides, and organostannanes are utilized in the synthesis of twenty alkyl [carbonyl‐11C]ketones. The activated palladium(0) species [Pd{P(o‐Tol)3}2] was generated in situ from tris(dibenzylideneacetone)palladium(0) [Pd2(dba)3] and a large excess of tri‐o‐tolylphosphane [P(o‐Tol)3]. The Stille coupling reactions were performed in a micro‐autoclave system. Radiochemical yields of 11C‐labelled alkyl/aryl ketones were in the range of 37–98 % with specific radioactivity up to 300 GBq μmol–1. Using this method, 4'‐aminoacetophen[13C‐arbonyl)one 6 was synthesised in order to confirm the position of labelling (δ = 196.7 ppm, CDCl3). The presented approach is an efficient way for synthesising 11C‐labelled alkyl/aryl ketones with acceptable radiochemical yield and is generally applicable in 13C‐labelling syntheses. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
Efficient Synthesis of the Azabicyclo[3.3.1]nonane Ring System in the Alkaloid Methyllycaconitine Using Bis(alkoxymethyl)alkylamines as Aminoalkylating Agents in a Double Mannich Reaction
Authors: Brimble Margaret A. , Brocke Constanze
Published: May 2005

The double Mannich reaction of cyclic β‐keto esters with bis(alkoxymethyl)alkylamines provides an efficient and versatile method for the construction of azabicyclo[3.3.1]nonanes and azabicyclo[3.2.1]octanes. The optimum conditions for efficient reaction involve use of the activator trichloromethylsilane in acetonitrile as solvent at ambient temperature. The utility of this synthetic method is further demonstrated by the facile synthesis of several AE ring analogues 39, 42 of the alkaloid methyllycaconitine by appendage of the key N‐(methylsuccininimido)anthranilate pharmacophore to the N‐(3‐phenylpropyl)‐substituted double Mannich adducts 18, 27. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
Highly Diasteroselective and Remarkably π‐Facially Selective Lewis Acid‐Catalysed Diels–Alder Cycloaddition Reactions: Access to Novel 1,3,4‐Trisubstituted 2‐Azetidinones
Authors: Bhargava Gaurav , Mahajan Mohinder P. , Saito Takao , Otani Takashi , Kurashima Mai , Sakai Ken
Published: May 2005

Diastereoselective and π‐facially selective Lewis acid‐catalysed intermolecular Diels–Alder reactions of 3‐butadienylazetidin‐2‐ones with a variety of symmetrical dienophiles (maleic anhydride, N‐phenylmaleimide, N‐p‐tolylmaleimide, benzoquinone and naphthoquinone) resulting in the synthesis of diastereomerically pure 1,3,4‐trisubstituted 2‐azetidinones are reported. The effects of different Lewis acids on the yields of the selectively formed diastereoisomers under different reaction conditions are also examined. Preferential chelation between the Lewis acid complexes of the different dienophiles and the carbonyl oxygens in the dienylazetidin‐2‐ones has been invoked to explain the observed π‐facial selectivity. The Lewis acid complexes of the dienophiles have been shown to approach preferentially from the si‐faces of the dienes, resulting in the formation of π‐facially selective “endo” adducts. The structures of these “endo” adducts have even been supported by X‐ray diffraction studies. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
XNA (xylo Nucleic Acid): A Summary and New Derivatives
Authors: Ravindra Babu B. , Raunak  , Poopeiko Nicolai E. , Juhl Martin , Bond Andrew D. , Parmar Virinder S. , Wengel Jesper
Published: May 2005

Fully modified homopyrimidine 2'‐deoxy‐xylo nucleic acid (dXNA) form triple helixes with complementary DNA/RNA with thermal stabilities comparable to those of the corresponding DNA:DNA and DNA:RNA duplexes. However, a single or few insertions of dXNA monomers in a DNA strand significantly lower duplex stabilities. The dXNA monomers are known to adopt predominantly an N‐type furanose conformation in solution. With a desire to increase the binding affinity, seven sugar‐modified XNA monomers (H, F, N, M, K, P and Q) have been synthesised and their effect on hybridization towards DNA and RNA complements studied. The introduction of 2'‐fluoro and 2'‐hydroxy substituents was expected to induce conformational restriction towards C3'‐endo‐type furanose conformation of monomer F derived from 1‐(2'‐deoxy‐2'‐fluoro‐β‐D‐xylofuranosyl)thymine and monomer H derived from 1‐(β‐D‐xylofuranosyl)thymine. The presence of functionalites facing the minor groove as in 1‐(2'‐amino‐2'‐deoxy‐2'‐N,4'‐C‐methylene‐β‐D‐xylofuranosyl)thymine (monomer N), 1‐[4‐C‐(N‐methylpiperazinyl)methyl‐β‐D‐xylofuranosyl]thymine (monomer P), 1‐(4‐C‐piperazinylmethyl‐β‐D‐xylofuranosyl)thymine (monomer Q), 1‐(4‐C‐hydroxymethyl‐β‐D‐xylofuranosyl)thymine (monomer M) and 9‐(4‐C‐hydroxymethyl‐β‐D‐xylofuranosyl)adenine (monomer K) was studied, with monomer N being locked in an N‐type furanose conformation. Besides, an efficient synthesis of known xylo‐LNA phosphoramidite 19, required for the incorporation of 1‐(2'‐O,4'‐C‐methylene‐β‐D‐xylofuranosyl)thymine (monomer L) is described. For comparison, hydridization data of various XNAs reported in the literature are included in the discussion section. The thermal denaturation studies show that XNAs containing conformationally locked monomers (N and L) display improved binding affinity, and that partially modified DNA/XNA chimera, or fully modified XNA display preferential hybridization towards RNA complements. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
Domino Heck–Diels–Alder Reactions of Differently Substituted Cyclopropylallenes
Authors: Knoke Mario , de Meijere Armin
Published: May 2005

Several substituted cyclopropylallenes were coupled under Heck conditions with aryl iodides and aryl bromides, respectively, and the 1,3,5‐hexatrienes resulting with ring‐opening of the cyclopropyl group, were captured in situ by a number of reactive dienophiles to yield oligosubstituted cyclohexene derivatives (8–86 %, 33 examples) with diastereomeric ratios ranging from 1.4:1 to 4.9:1. The influences of the number, the nature and the pattern of substituents have been elaborated. The [4+2] cycloaddition most probably proceeds in two steps through a mesomerically well stabilized 1,6‐zwitterionic intermediate. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
Studies on the Synthesis of Macrocyclic Allenes by Ring Closing Metathesis and Doering–Moore–Skattebøl Reaction
Authors: Janßen Christian E. , Krause Norbert
Published: May 2005

Several efficient synthetic approaches to allenic cyclophanes of the general structure 2/3, which are of interest as chiral ligands or host molecules, are described. The combination of the ruthenium‐catalyzed ring‐closing metathesis and the Doering–Moore–Skattebøl (DMS) reaction using the Seyferth reagent PhHgCBr3 provided a straightforward access to the allenic cyclophane 8. The macrocycles 13 and 17 with the allenic bridge between the aromatic units were also obtained efficiently by copper‐promoted SN2'‐substitution of propargylic acetates and ring‐closing metathesis. Alternatively, macrocyclic allenes can be synthesized by ring‐closing metathesis of α,ω‐bisallenes, as was demonstrated by the formation of the products 20, 21, and 23. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
Cover Picture: A New Glucose: Towards Conformationally Locked Hexoses through Annulation (Eur. J. Org. Chem. 11/2005)
Authors: Mehta Goverdhan , Ramesh Senaiar S.
Published: May 2005

The cover picture shows the concept of carbocyclic annulation (trans‐ring fusion) to lock hexoses, such as β‐glucose, in their unnatural axial‐rich conformation, while retaining their natural configuration. The bipolarofacial nature of these novel cyclohexane ring annulated sugar entities is reflected in their channel‐like supramolecular assembly in the solid state, consisting of a hydrophilic interior and a hydrophobic exterior. Details are discussed in the article by G. Mehta and S. S. Ramesh on p. 2225 ff.
Effect of variation of normal force on seismic performance of resilient sliding isolation systems in highway bridges
Authors: Iemura Hirokazu , Taghikhany Touraj , Takahashi Yoshikazu , Jain Sarvesh K.
Published: May 2005

In this study, a series of shaking table tests are carried out on scaled models of two seismically isolated highway bridges to investigate the effect of rocking motion and vertical acceleration on seismic performance of resilient sliding isolators. In addition, performance of RSI is compared with system having solely natural rubber bearings. Test results show that variation of normal force on sliders due to rocking effect and vertical acceleration makes no significant difference in response of RSI systems. In addition, analytical response of prototype isolated bridge and the model used in experiments is obtained analytically by using non‐linear model for isolation systems. It is observed that for seismically isolated bridges, dynamic response of full‐scale complex structures can be predicted with acceptable accuracy by experiments using a simple model of the structure. Copyright © 2005 John Wiley & Sons, Ltd.
Nerve growth factor does not activate Na+/H+ exchange in PC12 pheochromocytoma cells
Authors: Chandler Charles E. , Cragoe Edward J. , Glaser Luis
Published: May 2005

We have reexamined the possible role of the Na+/H+ antiport in the cellular response by PC12 pheochromaytoma cells to nerve growth factor (NGF). In contrast to previous reports, we observe no activation of Na+/H+ exchange in these cells, using a very sensitive assay based on the measurement of cytoplasmic pH with dimethylfluorescein dextran (Rothenberg et al., J. Biol Chem., 258: 4883–4809, 1983). Our measurements indicate that the PC12 pheochromacytoma cells, under all conditions tested, show a high rate of Na+/H+ exchange. The discrepancy between these observations and previous experiments could be due to differences in cells in different laboratories, but also to changes in cell adhesion induced by NGF. We describe conditions where intracellular pH and rates of Na+ uptake can be measured reliably in PC12 cells with adequate controls for cell adhesion. We conclude that activation of Na+/H+ exchange is neither sufficient nor required for the differentiation of PC12 cells induced by NGF.
Identification of noncollagenous components of calf lens capsule: Evaluation of their adhesion‐promoting activity
Authors: Cammarata Patrick R. , Spiro Robert G.
Published: May 2005

Extraction of calf anterior and posterior lens capsules with 5 M guanidine HCI resulted in the solubilization of protein (12% of total) with a noncollagenous amino acid composition leaving behind the collagen matrix. Polyacrylamide gel electrophoresis of the solubilzed materal revealed a number of components, all of which were susceptible to trypsin but resistant to collagenase digestion. Fractionation of the extracted proteins by Sepharose CL‐6B filtration as well as by affinity chromatography was undertaken, and laminin, fibronectin, entactin, and β‐crystallin were identified by electrophoresis and solid‐phase radiommunoassay in both anterior and posterior capsules. An entactin (Mr = 150,000), which constituted the most prominent component on electrophoresis, was purified after Sepharose CL‐6B filtration by a two‐step lectin affinity chromatography procedure, which was based on the failure of this protein to bind to Bandeiraea simplicifolia I but its positive reactivity with wheat germ lectin. Neither the mixture of proteins extracted from lens capsules by guanidine nor fractions prepared there from were table to enhance lens epithelial cell attachment to type I or type IV collagen‐coated surfaces or to guanidine‐prepared lens capsules; adhesion‐stimulating activity could not be demonstrated even when cycloheximide‐treated cells were employed. Furthermore, the cells were observed to attach as effectively to guanidine extracted as to native capsules. These observations indicate that noncollagenous proteins are not essenital for the in vitro attachment of epithelial cells to lens capsule; it appears that the collagen component itself provides an optimal surface for cell‐basement membrane interaction.
Transformation of mouse bone marrow cells by transfection with a human oncogene related to c‐myc is associated with the endogenous production of macrophage colony stimulating factor 1
Authors: Sklar Marshall D. , Tereba Allan , Chen Ben D.‐M. , Walker William S.
Published: May 2005

We recently derived a series of transformed cell lines by transfecting mouse bone marrow cells highly enriched for macrophage progenitors with a newly described human gene, R‐myc, which has homology to the c‐myc oncogene. In this report, we show that these lines share some features characteristic of cells of the mononuclear phagocyte lineage. Specifically, all cell lines had macrophage‐or monocytelike morphology, contained nonspecific esterase, were phagocytic for latex beads, secreted lysozyme, bore the Mac‐1 antigen, and contained a minority of cells with Fc receptors. However, only a single monocytelike clone had appreciable numbers of cells which bore complement receptor 1, and none were phagocytic for antibody or complementcoated particles, or constitutively secreted Interleukin‐1. All these cell lines secreted a growth factor capable of supporting the in vitro proliferation of bone marrow macrophages. Radiommunoassay and receptor binding studies indicate that this factor is colony stimulating factor 1.
The role of gangliosides in the interaction of a growth inhibitor with mouse lm cells
Authors: Bascom Charles C. , Sharifi Behrooz G. , Melkerson Lyla J. , Rintoul David A. , Johnson Terry C.
Published: May 2005

We have isolated and characterized glycopeptides, derived from mouse and bovine cerebral cortex cells, that inhibit protein synthesis and cell growth of normal but not transformed cells. The inhibitor binds to target cell surfaces, and gangliosides have previously been shown to influence cell sensitivity to the glycopeptides. Preincubation with 3.0 μg/ml ganglioside GM1 at 0°C for 3 hr sensitized the mouse L‐cell line to the inhibitor, as determined by protein synthesis assays. Preincubation of LM cells with ganglioside GM1 alone did not affect protein synthesis rates. In addition, the gangliosides GD1a and GM3 also sensitized the LM cells to the protein synthesis inhibitory effect of the glycopeptide inhibitor. Binding experiments were performed with 3T3 (sensitive) and LM (insensitive) cells to determine if sensitivity to the glycopeptide inhibitor was reflected in binding of the inhibitor to these cells. Binding of 125I‐labeled inhibitor to 3T3 cells was maximal after 60 min at 0°C and saturable at approximately 1 × 104 molecules/cell. Furthermore, binding of the inhibitor was dose‐dependent, with half‐maximal binding at 1.5‐2.0 nM and saturation at 8.0‐10.0 nM. Scatchard plot analysis indicated that the Kd was about 1 × 10−9 M and that there are 1 × 104 receptors/cell. Binding of the inhibitor to LM cells was maximal after 30 min at 0°C and saturation occurred at 5 × 103 molecules/cell. We then examined the possibility that gangliosides are the cellular receptor or co‐receptor for the glycopeptide inhibitor. Binding of the inhibitor to ganglioside GM1 was first examined after the ganglioside had been preadsorbed to polystyrene tubes. These experiments indicated that the ganglioside did not bind the inhibitor. Ganglioside‐containing liposomes from phosphatidylcholine or LM cell membrane components were also prepared; these artificial membranes did not bind appreciable amounts of the iodinated inhibitor. Competition experiments showed that the gangliosides GM1 and GD1a did not neutralize the protein synthesis inhibitory activity of the glycopeptides, indicating that gangliosides do not directly interact with the glycopeptide inhibitor. In addition, binding of the inhibitor to LM cells preincubated with ganglioside GM1 was studied. Although the binding of the inhibitor to LM cells was one‐half that observed for 3T3 cells, incorporation of exogenous gangliosides into LM cells did not result in increased binding of the inhibitor. Therefore, we conclude that the ganglioside‐induced sensitization of LM cells cannot be due to gangliosides serving as the cell‐surface receptor or co‐receptor for the glycopeptide inhibitor.
Growth factor regulation of membrane transport in human fibroblasts and its relationship to stimulation of DNA synthesis
Authors: Tupper Joseph T. , Smith Janet W.
Published: May 2005

Serum stimulation of serum‐deprived or density‐inhibited normal cells enhances the level of various nutrient and ionic transport systems. Certain of these systems have been implicated in the regulation of cell proliferation. However, the use of serum stimulation to activate quiescent cells leads to enhancement of numerous transport systems with little understanding of which component or components of serum are related to activation of which transport systems. In this study we attempt to identify the specific effect of three known growth promoting factors (insulin, dexamethasone and epidermal growth factor [EGF]) on the activation of four membrane transport systems (A‐amino acids, L‐amino acids, glucose and K+) in normal and SV40‐transformed WI38 human fibroblasts. We have also evaluted the effect of these growth factors on the stimulation of DNA synthesis in growth factor deprived cells. Thus, we can correlate the effect on a given transport system with the relative mitogenic stimulation produced by the growth factor. We conclude (a) that a growth factor can effect a transport system differently in a normal versus transformed cell, (b) that a specific growth factor can effect multiple transport systems and, (c) with the exception of K+ transport, enhanced transport induced by a given growth factor does not necessarily correlate with the mitogenic potency of the growth factor. This latter point is of particular significance since the activation of K+ transport reflects, based on other studies, activation of the Na+ ‐H+ exchanger which has been implicated in cell‐cycle activation.
Insulin stimulation of glucose transport and metabolism in a human Wilms' tumor‐derived myoblast‐like cell Line: Modulation of hormone effects by glucose deprivation
Authors: Lemmon Sandra K. , Buse Maria G. , Sens Donald A.
Published: May 2005

The effects of insulin and glucose on parameters of metabolism were investigated in myoblast‐like (MBL) cells, a human myoblast‐like cell line derived from a Wilms' tumor. Insulin responses were studied after 4 hr pre‐incubation in serum free media, with or without 5 mM glucose. Insulin was added during the last 2 hr. Glucose starvation markedly increased basal glucose transport (measured as 2‐deoxyglucose uptake) as well as the net uptake of [14C]glucose and [14C]glucose incorporation into glycogen. Insulin stimulated net glucose uptake and incorporation into glycogen in a dose‐dependent manner in glucose‐fed and starved cells. These insulin responses were markedly enhanced in glucose‐starved cells. Insulin accelerated 2‐deoxyglucose transport in glucose‐fed cells but did not further stimulate basal glucose transport in glucose‐deprived cells. Insulin increased the incorporation of [3H]leucine into protein in glucose‐fed or ‐starved MBL cells equally. The dose of insulin required for half‐maximal insulin responses was similar for all parameters studied. Cycloheximide did not prevent the increased basal glucose incorporation in glucose‐starved cells, but markedly inhibited the insulin response, while in glucose‐fed cells, cycloheximide stimulated basal glucose incorporation. We conclude that MBL cells resemble fibroblasts in their insulin‐independent stimulation of glucose transport in response to glucose‐deprivation; when provided with glucose, they respond to insulin like fibroblasts. However, after brief glucose‐starvation, the stimulated glucose transport system is no longer insulin‐responsive in MBL cells, while pathways leading to the synthesis of macromolecules demonstrate preserved or enhanced stimulation by insulin, suggesting that these cells may serve as models to study the regulation of receptor‐response coupling by the metabolic milieu.
Density‐dependent arrest of DNA replication is accompanied by decreased levels of c‐myc mRNA in myogenic but not in differentiation‐defective myoblasts
Authors: Sejersen Thomas , Sümegi Janos , Ringertz Nils R.
Published: May 2005

Myoblasts from primary rat cultures and established mouse (Cl10) and rat (L6, Ama 420) cell lines were examined for c‐oncogene expression during exponential growth and under conditions which allowed myogenic differentiation. The abundance of c‐Ki‐ras transcripts in mRNA from confluent, quiescent cultures was reduced to 15–40% of that in mRNA from exponentially growing cells. This reduction was found both in primary myoblast cultures, myoblast lines that formed mytubes (L6 and Cl10) and in a differentiation defective subline (Ama 420). The level of c‐myc transcripts was lowered when myogenic rat L6 myoblasts reached a high cell density, stopped DNA synthesis and formed myotubes. At the same cell density, growth arrested myoblasts of differentiation defective Ama 420 cells maintained a high level of c‐myc expression. This shows that DNA replication and c‐myc expression are independently regulated. All myoblast lines also showed expression of c‐abl during exponential growth phase. Reduced expression was seen in differentiated L6 and Cl10 cultures. No expression was detected when mRNA from multiplying and differentiating myoblasts cultures were probed for c‐myb, c‐erbA, c‐erbB, c‐mos, c‐fes, and c‐src. The observations are consistent with a role for c‐Ki‐ras in myoblast proliferation and suggest that a reduction in c‐myc expression may be a necessary prerequisite for terminal myogenic differentiation.
25‐Hydroxycholesterol‐induced elevations in 45Ca uptake: Permeability changes in P815 cells
Authors: Boissonneault Gilbert A. , Heiniger Hans‐Jörg
Published: May 2005

Certain oxysterols are capable of suppressing the activity of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase. We have previously demonstrated that treatment of P815 cells with 1 μg 25‐hydroxycholesterol/ml culture results in a rapid influx of 45Ca, and supplemental cholesterol prevents this from occurring. In this paper, we report on investigations into the means whereby this influx of calcium takes place. Through the use of respiratory inhibitors which prevent mitochondrial retention of calcium it was determined that the large increase in slow phase (intracellular) calcium uptake caused by 25‐hydroxycholesterol treatment was related to mitochrondrial uptake. The effects of various inhibitors of calcium uptake into cells, including verapamil, diltiazem, quinidine, ruthenium red, Co++, Mn++, were tested. Of these only Co++ and ruthenium red had any effect on 45Ca uptake. 25‐Hydroxycholesterol has been shown to be capable of membrane insertion and this could result in plasma membrane permeability changes. To test this hypothesis P815 cells were treated with 1 μg 25‐hydroxycholesterol/ml or 5 μg mevinolin/ml culture. Mevinolin, being a water soluble competitive inhibitor of HMG‐CoA reductase, should be unable to disrupt membrane architecture in a manner analogous to 25‐hydroxycholesterol. While both inhibitors rapidly supressed the synthesis of digitonin‐precipitable sterols, only 25‐hydroxycholesterol was able to increase 45Ca influx. The implications of these findings are discussed.
Characterization of protein kinase C activity in interferon gamma treated murine peritoneal macrophages
Authors: Becton David L. , Adams Dolph O. , Hamilton Thomas A.
Published: May 2005

Macrophage activation for tumoricidal and microbicidal functions can be achieved in part by treatment with recombinant interferon gamma (IFNγ) in vitro. We have previously demonstrated that IFNγ treatment of murine peritoneal macrophages results in a two‐ to five‐fold increase in the activity of Ca++, phospholipid dependent protein kinase C (Hamilton et al., J. Biol. Chem., 260: 1378, 1985). We now report that this effect was not dependent upon continuing protein synthesis since treatment with cycloheximide under conditions where normal protein synthesis was inhibited by greater than 95% had no effect upon the development of increased enzyme activity. Examination of Ca++ and phospholipid requirements revealed no differences between enzyme isolated from control or IFNγ‐treated cells could not be distinguished in terms of the diacyglycerol (DG) or phorbol diester (PMA) concentration required for stimulation of activity. Kinetic analysis of the ATP (as substrate)concentration dependence revealed that both control and treated enzyme preparations (either basal or stimulated) had comparable Km values. Maximum velocity (Vmax) was increased both by IFNγ treatment and also by stimulation with DG or PMA. The major difference which could be discerned between protein kinase C derived from control versus IFNγ‐treated macro‐phages was the magnitude of the response to DG or PMA; IFNγ treatment increased the stimulation index (i.e., ratio of basal to stimulated activity) by a factor of two to four fold. These results suggest that IFNγ treatment leads to reversible modulation of existing protein kinase C resulting in increased catalytic efficiency when exposed to an appropriate stimulant.
Effect of age and dietary restriction on protein synthesis by isolated kidney cells
Authors: Ricketts Wilbert G. , Birchenall‐Sparks Maria C. , Hardwick James P. , Richardson Arlan
Published: May 2005

The influence of age and food restriction on kidney protein synthesis was studied in Fischer F344 rats. The rate of total protein synthesis by suspensions of kiney cells declined 60% between 4 and 31 months of age. The rate of protein synthesis by kidney cells isolated from 19‐month old rats fed a restricted diet (60% of diet consumed by rats fed ad libitum) was 45% higher than the rate of protein synthesis by kidney cells isolated from 19‐month old rats fed ad libitum. The excretion of protein in the urine was measured to assess the effect of the age related decline in protein synthesis on kidney function. A dramatic increase in proteinuria was observed with increasing age, and rats fed the restricted diet excreted significantly less protein in the urine than rats fed ad libitum.
Characterization of permeation pathways in the plasma membrane of human erythrocytes infected with early stages of Plasmodium falciparum: Association with parasite development
Authors: Kutner S. , Breuer W. V. , Ginsbururg H. , Aley S. B. , Cabantchik Z. I.
Published: May 2005

Human intraerythrocytic malarial parasites (Plasmodium falciparum) induce permeability changes in the membrane of their host cells. The differential permeability of infected erythrocytes at various stages of parastie growth, in combination with density gradient centrifugation, was used to fractionate parasitized cells according to their developmental stage. By this method it was possible to obtain cell fractions consisting essentially of erythrocytes infected with the youngest parasite stage (i.e., rings). These preparations were used for the measurement of transport of various solutes. It is shown that permeabilization of host erythrocyte membrane appers as early as 6 h after parasite invasion of the erythrocyte and increases gradually with parasite maturation. Since the selectivity for several different solutes and the enthalpy of activation of transport remain unaltered with maturation‐related increase of permeability, it is concluded that the number of transport agencies in the host cell membrane increases with parasite maturation. Evidence is presented to indicate the need for parasite protein synthesis as an essential factor for the generation of the new permeability pathways.