bluuebunny/crossref_metadata_2025 huggingface dataset:
Computer simulations of enzyme catalysis: Finding out what has been optimized by evolution
Authors: Warshel Arieh , Florián Jan
Published: July 2002

The origin of the catalytic power of enzymes is discussed, paying attention to evolutionary constraints. It is pointed out that enzyme catalysis reflects energy contributions that cannot be determined uniquely by current experimental approaches without augmenting the analysis by computer simulation studies. The use of energy considerations and computer simulations allows one to exclude many of the popular proposals for the way enzymes work. It appears that the standard approaches used by organic chemists to catalyze reactions in solutions are not used by enzymes. This point is illustrated by considering the desolvation hypothesis and showing that it cannot account for a large increase in kcatrelative to the corresponding kcagefor the reference reaction in a solvent cage. The problems associated with other frequently invoked mechanisms also are outlined. Furthermore, it is pointed out that mutation studies are inconsistent with ground state destabilization mechanisms. After considering factors that were not optimized by evolution, we review computer simulation studies that reproduced the overall catalytic effect of different enzymes. These studies pointed toward electrostatic effects as the most important catalytic contributions. The nature of this electrostatic stabilization mechanism is far from being obvious because the electrostatic interaction between the reacting system and the surrounding area is similar in enzymes and in solution. However, the difference is that enzymes have a preorganized dipolar environment that does not have to pay the reorganization energy for stabilizing the relevant transition states. Apparently, the catalytic power of enzymes is stored in their folding energy in the form of the preorganized polar environment.
Potent inhibition of respiratory syncytial virus replication using a 2-5A-antisense chimera targeted to signals within the virus genomic RNA
Authors: Player Mark R. , Barnard Dale L. , Torrence Paul F.
Published: July 2002

The 2-5A system is a recognized mechanistic component of the antiviral action of interferon. Interferon-induced 2-5A synthetase generates 2-5A, which, in turn, activates the latent constitutive RNase L that degrades viral RNA. Chemical conjugation of 2-5A to an antisense oligonucleotide can target the 2-5A-dependent RNase L to the antisense-specified RNA and effect its selective destruction. Such a 2-5A-antisense chimera (NIH351) has been developed that targets a consensus sequence within the respiratory syncytial virus (RSV) genomic RNA. NIH351 was 50- to 90-fold more potent against RSV strain A2 than was ribavirin, the presently approved drug for clinical management of RSV infection. It was similarly active against a variety of RSV strains of both A and B subgroups and possessed a cell culture selectivity index comparable to ribavirin. In addition, the anti-RSV activity of NIH351 was shown to be virus-specific and a result of a true antisense effect, because a scrambled nucleotide sequence in the antisense domain of NIH351 caused a significant decrease in antiviral activity. The 2-5A system’s RNase L was implicated in the mechanism of action of NIH351 because a congener with a disabled 2-5A moiety was of greatly reduced anti-RSV effectiveness. These findings represent an innovative approach to the control of RSV replication.
The importance of tRNA backbone-mediated interactions with synthetase for aminoacylation
Authors: McClain William H. , Schneider Jay , Bhattacharya Subhra , Gabriel Kay
Published: July 2002

We have identified six new aminoacylation determinants of
Escherichia coli
tRNA
Gln
in a genetic and biochemical analysis of suppressor tRNA. The new determinants occupy the interior of the acceptor stem, the inside corner of the L shape, and the anticodon loop of the molecule. They supplement the primary determinants located in the anticodon and acceptor end of tRNA
Gln
described previously. Remarkably, the three-dimensional structure of the complex between tRNA
Gln
and glutaminyl-tRNA synthetase shows that the enzyme interacts with the phosphate–sugar backbone but not the base of every new determinant. Moreover, a small protein motif interacts with five of these determinants, and it binds proximal to the sixth. The motif also interacts with the middle base of the anticodon and with the backbones of six other nucleotides. Our results emphasize that synthetase recognition of tRNA is more elaborate than amino acid side chains of the enzyme interacting with nucleotide bases of the tRNA. Recognition also includes synthetase interaction with tRNA backbone functionalities whose distinctive locations in three-dimensional space are exquisitely determined by the tRNA sequence.
RecA binding to a single double-stranded DNA molecule: A possible role of DNA conformational fluctuations
Authors: Leger J. F. , Robert J. , Bourdieu L. , Chatenay D. , Marko J. F.
Published: July 2002

Most genetic regulatory mechanisms involve protein–DNA interactions. In these processes, the classical Watson–Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein–DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA–protein interactions.
Mitochondrial adenine nucleotide translocase is modified oxidatively during aging
Authors: Yan Liang-Jun , Sohal Rajindar S.
Published: July 2002

The purpose of this study was to test the hypothesis that elevation in protein oxidative damage during the aging process is a targeted rather than a stochastic phenomenon. Oxidative damage to proteins in mitochondrial membranes in the flight muscles of the housefly, manifested as carbonyl modifications, was detected immunochemically with anti-dinitrophenyl antibodies. Adenine nucleotide translocase (ANT) was found to be the only protein in the mitochondrial membranes exhibiting a detectable age-associated increase in carbonyls. The age-related elevation in ANT carbonyl content was correlated with a corresponding loss in its functional activity. Senescent flies that had lost the ability to fly exhibited a relatively higher degree of ANT oxidation and a greater loss of functional activity than their cohorts of the same age that were still able to fly. Exposure of flies to 100% oxygen resulted in an increase in the level of ANT carbonyl content and a loss in its activity.
In vitro
treatment of mitochondria with a system that generated hydroxyl free radicals caused an increase in ANT carbonyl level and a decrease in ANT exchange activity. ANT was also the only mitochondrial membrane protein exhibiting adducts of the lipid peroxidation product 4-hydroxynonenal. Results of this study indicate that proteins in mitochondrial membranes are modified selectively during aging.
Aminoguanidine prevents age-related arterial stiffening and cardiac hypertrophy
Authors: Corman Bruno , Duriez Micheline , Poitevin Pierre , Heudes Didier , Bruneval Patrick , Tedgui Alain , Levy Bernard I.
Published: July 2002

Aging is associated with cardiac hypertrophy and arterial stiffening possibly associated with accumulation of advanced glycation end products (AGEs). We evaluated the effect of aminoguanidine, an inhibitor of AGE production, on end-stage alterations of renal and cardiovascular systems. Normotensive WAG/Rij rats were treated from 24 to 30 mo with aminoguanidine and compared with a control group. Aminoguanidine did not modify body and kidney weights but prevented the age-related cardiac hypertrophy (heart weight: 1276 ± 28 mg and 1896 ± 87 mg in 24- and 30-mo-old control animals and 1267 ± 60 mg in 30-mo-old treated rats,
P
< 0.01). The increase in mesangial surface in aging rats was reduced by 30% by aminoguanidine. Collagen content of the arterial wall increased between 24 and 30 mo whereas elastin content, media thickness, and smooth muscle cell number remained unchanged. Aminoguanidine did not affect these parameters; however, the age-related increase in aortic impedance (12.4 ± 1.4 and 18.2 ± 1.9 10
3
⋅dyne⋅sec⋅cm
−5
in control 24- and 30-mo-old rats,
P
< 0.01) and the decrease in carotid distensibility (0.79 ± 0.11 and 0.34 ± 0. 07 mm Hg
−1
in control 24- and 30-mo-old rats,
P
< 0.01) were prevented by aminoguanidine. The prevention of arterial stiffening and cardiac hypertrophy in the absence of changes in collagen and elastin content suggests that the effect of aminoguanidine is related to a decrease in the AGE-induced cross-linking of the extracellular matrix.
Accelerated reaction of nitric oxide with O
2
within the hydrophobic interior of biological membranes
Authors: Liu Xiaoping , Miller Mark J. S. , Joshi Mahesh S. , Thomas Douglas D. , Lancaster Jack R.
Published: July 2002

We demonstrate herein dramatic acceleration of aqueous nitric oxide (NO) reaction with O
2
within the hydrophobic region of either phospholipid or biological membranes or detergent micelles and demonstrate that the presence of a distinct hydrophobic phase is required. Per unit volume, at low amounts of hydrophobic phase, the reaction of NO with O
2
within the membranes is approximately 300 times more rapid than in the surrounding aqueous medium. In tissue, even though the membrane represents only 3% of the total volume, we calculate that 90% of NO reaction with O
2
will occur there. We conclude that biological membranes and other tissue hydrophobic compartments are important sites for disappearance of NO and for formation of NO-derived reactive species and that attenuation of these potentially damaging reactions is an important protective action of lipid-soluble antioxidants such as vitamin E.
Etk/Bmx, a tyrosine kinase with a pleckstrin-homology domain, is an effector of phosphatidylinositol 3′-kinase and is involved in interleukin 6-induced neuroendocrine differentiation of prostate cancer cells
Authors: Qiu Yun , Robinson Dan , Pretlow Tom G. , Kung Hsing-Jien
Published: July 2002

Etk/Bmx is the newest member of Btk tyrosine kinase family that contains a pleckstrin homology domain, an src homology 3 domain, an src homology 2 domain, and a catalytic domain. Unlike other members of the Btk family kinases, which are mostly hemopoietic cell-specific, Etk/Bmx is preferentially expressed in epithelial and endothelial cells. We first identified this kinase in prostate cancer [Robinson, D., He, F., Pretlow, T. & Kung, H. J. (1996)Proc. Natl. Acad. Sci. USA93, 5958–5962). Here we report that Etk is engaged in phosphatidylinositol 3-kinase (PI3-kinase) pathway and plays a pivotal role in interleukin 6 (IL-6) signaling in a prostate cancer cell line, LNCaP. Our evidence that PI3-kinase is involved in Etk activation includes: (i) Wortmannin, a specific inhibitor of PI3-kinase, abolished the activation of Etk by IL-6; (ii) a constitutively active p110 subunit of PI3-kinase was able to activate Etk in the absence of IL-6; and (iii) a dominant negative p85 subunit of PI3-kinase mutant blocked the activation of Etk by IL-6. Interestingly, IL-6 treatment of LNCaP induced a remarkable neuroendocrine-like differentiation phenotype, with neurite extension and enhanced expression of neuronal markers. This phenotype could be abrogated by the overexpression of a dominant-negative Etk, indicating Etk is required for this differentiation process.
Femtosecond dynamics of DNA-mediated electron transfer
Authors: Wan Chaozhi , Fiebig Torsten , Kelley Shana O. , Treadway Christopher R. , Barton Jacqueline K. , Zewail Ahmed H.
Published: July 2002

Diverse biophysical and biochemical studies have sought to understand electron transfer (ET) in DNA in part because of its importance to DNA damage and its repair. However, the dynamics and mechanisms of the elementary processes of ET in this medium are not fully understood and have been heavily debated. Two fundamental issues are the distance over which charge is transported and the time-scale on which the transport through the π-stack of the DNA base pairs may occur. With femtosecond resolution, we report direct observation in DNA of ultrafast ET, initiated by excitation of tethered ethidium (E), the intercalated electron acceptor (A); the electron donor (D) is 7-deazaguanine (Z), a modified base, placed at different, fixed distances from A. The ultrafast ET between these reactants in DNA has been observed with time constants of 5 ps and 75 ps and was found to be essentially independent of the D–A separation (10–17 Å). However, the ET efficiency does depend on the D–A distance. The 5-ps decay corresponds to direct ET observed from 7-deazaguanine but not guanine to E. From measurements of orientation anisotropies, we conclude that the slower 75-ps process requires the reorientation of E before ET, similar to E/nucleotide complexes in water. These results reveal the nature of ultrafast ET and its mechanism: in DNA, ET cannot be described as in proteins simply by a phenomenological parameter, β. Instead, the involvement of the base pairs controls the time scale and the degree of coherent transport.
Receptors for oxidized low-density lipoprotein on elicited mouse peritoneal macrophages can recognize both the modified lipid moieties and the modified protein moieties: Implications with respect to macrophage recognition of apoptotic cells
Authors: Bird David A. , Gillotte Kristin L. , Hörkkö Sohvi , Friedman Peter , Dennis Edward A. , Witztum Joseph L. , Steinberg Daniel
Published: July 2002

It has been shown previously that the binding of oxidized low-density lipoprotein (OxLDL) to resident mouse peritoneal macrophages can be inhibited (up to 70%) by the apoprotein B (apoB) isolated from OxLDL, suggesting that macrophage recognition of OxLDL is primarily dependent on its modified protein moiety. However, recent experiments have demonstrated that the lipids isolated from OxLDL and reconstituted into a microemulsion can also strongly inhibit uptake of OxLDL (up to 80%). The present studies show that lipid microemulsions prepared from OxLDL bind to thioglycollate-elicited macrophages at 4°C in a saturable fashion and inhibit the binding of intact OxLDL and also of the apoB from OxLDL. Reciprocally, the binding of the OxLDL-lipid microemulsions was strongly inhibited by intact OxLDL. A conjugate of synthetic 1-palmitoyl 2(5-oxovaleroyl) phosphatidylcholine (an oxidation product of 1-palmitoyl 2-arachidonoyl phosphatidylcholine) with serum albumin, shown previously to inhibit macrophage binding of intact OxLDL, also inhibited the binding of both the apoprotein and the lipid microemulsions prepared from OxLDL. Finally, a monoclonal antibody against oxidized phospholipids, one that inhibits binding of intact OxLDL to macrophages, also inhibited the binding of both the resolubilized apoB and the lipid microemulsions prepared from OxLDL. These studies support the conclusions that: (i) at least some of the macrophage receptors for oxidized LDL can recognizeboththe lipid and the protein moieties; and (ii) oxidized phospholipids, in the lipid phase of the lipoprotein and/or covalently linked to the apoB of OxLDL, likely play a role in that recognition.
A dimeric mutant of human pancreatic ribonuclease with selective cytotoxicity toward malignant cells
Authors: Piccoli Renata , Di Gaetano Sonia , De Lorenzo Claudia , Grauso Michela , Monaco Carmen , Spalletti-Cernia Daniela , Laccetti Paolo , Cinátl Jaroslav , Matoušek Josef , D’Alessio Giuseppe
Published: July 2002

Monomeric human pancreatic RNase, devoid of any biological activity other than its RNA degrading ability, was engineered into a dimeric protein with a cytotoxic action on mouse and human tumor cells, but lacking any appreciable toxicity on mouse and human normal cells. This dimeric variant of human pancreas RNase selectively sensitizes to apoptotic death cells derived from a human thyroid tumor. Because of its selectivity for tumor cells, and because of its human origin, this protein represents a potentially very attractive, novel tool for anticancer therapy.
Bacillus subtilis
aconitase is an RNA-binding protein
Authors: Alén Claudia , Sonenshein Abraham L.
Published: July 2002

The aconitase protein of
Bacillus subtilis
was able to bind specifically to sequences resembling the iron response elements (IREs) found in eukaryotic mRNAs. The sequences bound include the rabbit ferritin IRE and IRE-like sequences in the
B. subtilis
operons that encode the major cytochrome oxidase and an iron uptake system. IRE binding activity was affected by the availability of iron both
in vivo
and
in vitro
. In eukaryotic cells, aconitase-like proteins regulate translation and stability of iron metabolism mRNAs in response to iron availability. A mutant strain of
B. subtilis
that produces an enzymatically inactive aconitase that was still able to bind RNA sporulated 40× more efficiently than did an aconitase null mutant, suggesting that a nonenzymatic activity of aconitase is important for sporulation. The results support the idea that bacterial aconitases, like their eukaryotic homologs, are bifunctional proteins, showing aconitase activity in the presence of iron and RNA binding activity when cells are iron-deprived.
The MAPKKK Ste11 regulates vegetative growth through a kinase cascade of shared signaling components
Authors: Lee Bee Na , Elion Elaine A.
Published: July 2002

In haploid
Saccharomyces cerevisiae
, the mating and invasive growth (IG) pathways use the same mitogen-activated protein kinase kinase kinase kinase (MAPKKKK, Ste20), MAPKKK (Ste11), MAPKK (Ste7), and transcription factor (Ste12) to promote either G
1
arrest and fusion or foraging in response to distinct stimuli. This exquisite specificity is the result of pathway-specific receptors, G proteins, scaffold protein, and MAPKs. It is currently not thought that the shared signaling components function under the basal conditions of vegetative growth. We tested this hypothesis by searching for mutations that cause lethality when the
STE11
gene is deleted. Strikingly, we found that Ste11, together with Ste20, Ste7, Ste12, and the IG MAPK Kss1, functions in a third pathway that promotes vegetative growth and is essential in an
och1
mutant that does not synthesize mannoproteins. We term this pathway the
STE
vegetative growth (SVG) pathway. The SVG pathway functions, in part, to promote cell wall integrity in parallel with the protein kinase C pathway. During vegetative growth, the SVG pathway is inhibited by the mating MAPK Fus3. By contrast, the SVG pathway is constitutively activated in an
och1
mutant, suggesting that it senses intracellular changes arising from the loss of mannoproteins. We predict that general proliferative functions may also exist for other MAPK cascades thought only to perform specialized functions.
Neural restrictive silencer factor recruits mSin3 and histone deacetylase complex to repress neuron-specific target genes
Authors: Naruse Yoshihisa , Aoki Tsutomu , Kojima Takuya , Mori Nozomu
Published: July 2002

Accumulative evidence suggests that more than 20 neuron-specific genes are regulated by a transcriptional cis-regulatory element known as the neural restrictive silencer (NRS). A trans-acting repressor that binds the NRS, NRSF [also designated RE1-silencing transcription factor (REST)] has been cloned, but the mechanism by which it represses transcription is unknown. Here we show evidence that NRSF represses transcription of its target genes by recruiting mSin3 and histone deacetylase. Transfection experiments using a series of NRSF deletion constructs revealed the presence of two repression domains, RD-1 and RD-2, within the N- and C-terminal regions, respectively. A yeast two-hybrid screen using the RD-1 region as a bait identified a short form of mSin3B.
In vitro
pull-down assays and
in vivo
immunoprecipitation-Western analyses revealed a specific interaction between NRSF-RD1 and mSin3 PAH1-PAH2 domains. Furthermore, NRSF and mSin3 formed a complex with histone deacetylase 1, suggesting that NRSF-mediated repression involves histone deacetylation. When the deacetylation of histones was inhibited by tricostatin A in non-neuronal cells, mRNAs encoding several neuronal-specific genes such as SCG10, NMDAR1, and choline acetyltransferase became detectable. These results indicate that NRSF recruits mSin3 and histone deacetylase 1 to silence neural-specific genes and suggest further that repression of histone deacetylation is crucial for transcriptional activation of neural-specific genes during neuronal terminal differentiation.
Serial microanalysis of renal transcriptomes
Authors: Virlon Bérangère , Cheval Lydie , Buhler Jean-Marie , Billon Emmanuelle , Doucet Alain , Elalouf Jean-Marc
Published: July 2002

Large-scale gene expression studies can now be routinely performed on macroamounts of cells, but it is unclear to which extent current methods are valuable for analyzing complex tissues. In the present study, we used the method of serial analysis of gene expression (SAGE) for quantitative mRNA profiling in the mouse kidney. We first performed SAGE at the whole-kidney level by sequencing 12,000 mRNA tags. Most abundant tags corresponded to transcripts widely distributed or enriched in the predominant kidney epithelial cells (proximal tubular cells), whereas transcripts specific for minor cell types were barely evidenced. To better explore such cells, we set up a SAGE adaptation for downsized extracts, enabling a 1,000-fold reduction of the amount of starting material. The potential of this approach was evaluated by studying gene expression in microdissected kidney tubules (50,000 cells). Specific gene expression profiles were obtained, and known markers (e.g., uromodulin in the thick ascending limb of Henle's loop and aquaporin-2 in the collecting duct) were found appropriately enriched. In addition, several enriched tags had no databank match, suggesting that they correspond to unknown or poorly characterized transcripts with specific tissue distribution. It is concluded that SAGE adaptation for downsized extracts makes possible large-scale quantitative gene expression measurements in small biological samples and will help to study the tissue expression and function of genes not evidenced with other high-throughput methods.
Localization of non-Mhccollagen-induced arthritis susceptibility loci in DBA/1j mice
Authors: McIndoe Richard A. , Bohlman Beverly , Chi Emil , Schuster Eugene , Lindhardt Molly , Hood Leroy
Published: July 2002

One approach to understanding common human diseases is to determine the genetic defects responsible for similar diseases in animal models and place those defective genes in their corresponding biochemical pathways. Our laboratory is working with an animal model for human rheumatoid arthritis called collagen-induced arthritis (CIA). We are particularly interested in determining the location of disease-predisposing loci. To that end, we performed experiments to localize susceptibility loci for CIA in an F2 cross between the highly susceptible mouse strain DBA/1j and the highly resistant mouse strain SWR/j. Specifically, a quantitative trait locus analysis was performed to localize regions of the mouse genome responsible for susceptibility/severity to CIA. One susceptibility locus,Cia1in the major histocompatibility locus, had been identified previously. Two additional loci were detected in our analysis that contribute to CIA severity (Cia2,Cia3) on chromosomes 2 and 6. A third locus was detected that contributes to the age of onset of the disease. This locus (Cia4) was located on chromosome 2 and was linked to the same region asCia2. Determining the identity of these loci may provide insights into the etiology of human rheumatoid arthritis.
Femtosecond dynamics of dative bonding: Concepts of reversible and dissociative electron transfer reactions
Authors: Zhong Dongping , Zewail Ahmed H.
Published: July 2002

With fs time, speed, and angular resolution of the elementary steps in electron transfer reactions, we report direct observation of reversible and dissociative processes for dative bonding involving covalent and ionic characters. For bimolecular reactions of various donors and acceptors we find strong correlation between the structure and the dynamics. The dynamics from the transition state to final products involve two elementary processes, with different reaction times, speed, and angular distributions. For example, for the R
2
S⋅I
2
(R = C
2
H
5
) system, it is shown that after charge separation, the reversible electron transfer occurs in less than 150 fs (fastest trajectory) and is followed by the rupture of the I—I bond with the release of the first I-atom in 510 fs. However, the second process of the remaining and trapped I-atom takes 1.15 ps with its speed (500 m/s) being much smaller than the first one (1,030 m/s). The S—I—I average angle is 130°. These findings, on this and the other systems reported here, elucidate the mechanism and address some concepts of nonconcertedness, caging, and restricted energy redistribution.
Regulation of p53 expression by thymidylate synthase
Authors: Ju Jingfang , Pedersen-Lane Joan , Maley Frank , Chu Edward
Published: July 2002

Previous studies showed that thymidylate synthase (TS), as an RNA binding protein, regulates its own synthesis by impairing the translation of TS mRNA. In this report, we present evidence that p53 expression is affected in a similar manner by TS. For these studies, we used a TS-depleted human colon cancer HCT-C cell that had been transfected with either the human TS cDNA or the
Escherichia coli
TS gene. The level of p53 protein in transfected cells overexpressing human TS was significantly reduced when compared with its corresponding parent HCT-C cells. This suppression of p53 expression was the direct result of decreased translational efficiency of p53 mRNA. Similar results were obtained upon transfection of HCT-C cells with pcDNA 3.1 (+) containing the
E. coli
TS gene. These findings provide evidence that TS, from diverse species, specifically regulates p53 expression at the translational level. In addition, TS-overexpressing cells with suppressed levels of p53 are significantly impaired in their ability to arrest in G
1
phase in response to exposure to a DNA-damaging agent such as γ-irradiation. These studies provide support for the
in vivo
biological relevance of the interaction between TS and p53 mRNA and identify a molecular pathway for controlling p53 expression.
A second calcineurin binding site on the NFAT regulatory domain
Authors: Park Sheldon , Uesugi Motonari , Verdine Gregory L.
Published: July 2002

NFATc (a member of the family of nuclear factors of activated T cells) is a transcriptional factor responsible for the Ca
2+
-inducible activation of cytokine genes during the immune response. In resting T cells, NFATc is retained in the cytoplasm by a mechanism that depends on multiple phosphorylations in an N-terminal regulatory domain. Physical interaction with and dephosphorylation by Ca
2+
-activated calcineurin (Cn) allows the protein to enter the nucleus, where it binds to specific sites in cytokine gene promoters. Previous studies had identified a peptide segment in NFATc that binds Cn stably. Here we report the identification of a second Cn-binding element in NFATc, which synergizes with the previously identified element. Although these sequences are conserved in all isoforms of NFAT, we find that the two sites contribute differentially to the overall affinity for Cn in an isoform-dependent manner. The regulatory domain of NFAT also was found to be entirely devoid of structure, both in the phosphorylated and unphosphorylated state. This finding suggests that the NFAT regulatory domain does not undergo phosphorylation-induced conformational switching, but instead requires partner proteins to control accessibility of the NFAT nuclear localization and nuclear export signals.
Regulated expression of P210 Bcr-Abl during embryonic stem cell differentiation stimulates multipotential progenitor expansion and myeloid cell fate
Authors: Era Takumi , Witte Owen N.
Published: July 2002

P210 Bcr-Abl is an activated tyrosine kinase oncogene encoded by the Philadelphia chromosome associated with human chronic myelogenous leukemia (CML). The disease represents a clonal disorder arising in the pluripotent hematopoietic stem cell. During the chronic phase, patients present with a dramatic expansion of myeloid cells and a mild anemia. Retroviral gene transfer and transgenic expression in rodents have demonstrated the ability of Bcr-Abl to induce various types of leukemia. However, study of human CML or rodent models has not determined the direct and immediate effects of Bcr-Abl on hematopoietic cells from those requiring secondary genetic or epigenetic changes selected during the pathogenic process. We utilized tetracycline-regulated expression of Bcr-Abl from a promoter engineered for robust expression in primitive stem cells through multilineage blood cell development in combination with the
in vitro
differentiation of embryonal stem cells into hematopoietic elements. Our results demonstrate that Bcr-Abl expression alone is sufficient to increase the number of multipotent and myeloid lineage committed progenitors in a dose-dependent manner while suppressing the development of committed erythroid progenitors. These effects are reversible upon extinguishing Bcr-Abl expression. These findings are consistent with Bcr-Abl being the sole genetic change needed for the establishment of the chronic phase of CML and provide a powerful system for the analysis of any genetic change that alters cell growth and lineage choices of the hematopoietic stem cell.
Macroporous polymer foams by hydrocarbon templating
Authors: Shastri Venkatram Prasad , Martin Ivan , Langer Robert
Published: July 2002

Porous polymeric media (polymer foams) are utilized in a wide range of applications, such as thermal and mechanical insulators, solid supports for catalysis, and medical devices. A process for the production of polymer foams has been developed. This process, which is applicable to a wide range of polymers, uses a hydrocarbon particulate phase as a template for the precipitation of the polymer phase and subsequent pore formation. The use of a hydrocarbon template allows for enhanced control over pore structure, porosity, and other structural and bulk characteristics of the polymer foam. Polymer foams with densities as low as 120 mg/cc, porosity as high as 87%, and high surface areas (20 m
2
/g) have been produced. Foams of poly(
l
-lactic acid), a biodegradable polymer, produced by this process have been used to engineer a variety of different structures, including tissues with complex geometries such as in the likeness of a human nose.
Collaborative Tasks, Collaborative Children: An analysis of reciprocity during peer interaction at Key Stage 1
Authors: Ogden Lynn
Published: July 2002

The introduction of Literacy and Numeracy Hours in primary schools requires children to work with their peers in pairs or groups. Peer collaboration requires reciprocal interaction as children share ideas and gain a joint conception of what they are trying to achieve. This article examines the reciprocal nature of contributions made by 36 pairs of infant school peers as they worked together on shared tasks in the classroom. Qualitative and quantitative analyses were used to examine the frequency and length of sequences of responsive turns. Findings indicate that whilst pairs of Reception and Year 1 children demonstrate a similar level of reciprocal interaction, Year 2 children were significantly more responsive and demonstrated a greater capacity for extended reciprocal interaction. The findings are discussed in relation to children's developing ability to collaborate and the implications this might have for group work in the classroom.
The Estimation of Rates of Unemployment for Small Spatial Units
Authors: Jones Calvin
Published: July 2002

This paper details a methodology for estimating the rate of unemployment at a spatial level below that currently available. It suggests that a reasonable estimate of the unemployment rate, based on residents, can be made, using available data, for small areas. Using Cardiff as an example, the paper shows how, once published data has been manipulated to achieve an age and gender disaggregated population base for electoral wards, estimates of activity rates can be applied to achieve a total resident economically active population. This estimate is then used, in conjunction with the claimant count, to produce a resident-based unemployment rate applicable to small areas within the county. The results are evaluated against alternative approaches.
Technology Transfer from Publicly Funded Research for Improved Natural Resource Management: Analysis and Australian Examples
Authors: Tisdell Clem
Published: July 2002

Considerable public funding is provided for research and development intended to improve the management and use of shared natural resources, such as water. In Australia, the Land and Water Research and Development Corporation (LWRRDC) and Environment Australia are significant providers of such funds. These providers tend to judge the value of R&D projects supported by them on the basis of whether or not significant technology transfer and adoption takes place. Researchers involved in these projects are expected to be the prime movers of such transfer. However, it seems that research funders have been guided by over-simplified models of processes of technology transfer and by false analogies with the transfer of industrial technology. There has been a failure to recognise that much of the new technology developed to improve the management of shared resources, such as water, affects the supply of social or collective commodities, a factor which materially alters the technology transfer process. Here, processes of transferring publicly funded intellectual knowledge are discussed and modelled, dynamic patterns of adoption of new technology are considered along with factors influencing adoption rates and barriers to adoption, particularly when the supply of social or collective commodities such as water, are involved. Some points from the analysis are illustrated by observations from a sample of LWRRDC-supported research projects.
Cytokine secretion by decidual lymphocytes in transient hypertension of pregnancy and pre‐eclampsia
Authors: Wilczyński Jacek R. , Tchórzewski Henryk , Głowacka Ewa , Banasik Małgorzata , Lewkowicz Przemyslaw , Szpakowski Marian , Zeman Krzysztof , Wilczyński Jan
Published: July 2002

Background: Transient hypertension (TH) and pre‐eclampsia (PE) are believed to have different patho‐physiology. However, 15‐25% of pregnant women initially diagnosed as having TH develop PE. To clarify the immuno‐pathogenetical connections between the two syndromes, we studied the pattern of T helper cell (Th)1/Th2 cytokine balance disturbances existing inside maternal decidua in normal pregnancy (NP) and pregnancies complicated with TH and PE.

Methods: Third‐trimester decidual tissue was obtained by curettage of uterine cavity during elective caesarean sections in NP
(n = 11), TH (n = 17) and PE (n = 21) patients. Cell suspensions were prepared by an electromechanical dispersal method and centrifugated using a standard gradient sedimentation technique. Isolated lymphocytes were placed in medium (RPMI 1640, 10% fetal calf serum, L‐glutamine, penicillin, streptomycin) and cultured for 72 h with or without mitogen phytohaemaglutinine (PHA). The enzyme‐linked immunosorbent assay method was used for estimation of interleukin (IL)‐2, IL‐4, IL‐6, IL‐10, IL‐12 and interferon‐γ (IFNγ) in culture supernatant.

Statistical analysis: The Kruskal‐Wallis and the Mann‐Whitney U tests were used (p < 0.05).

Results: Both spontaneous and PHA‐stimulated secretion of Th2‐type cytokines IL‐6 and IL‐10 was decreased in PE patients compared with TH and NP patients. The concentration of Th1‐type cytokine IFNγ was increased in patients suffering both from TH and PE.

Conclusion: On the base of decidual cytokine secretion, both PE and TH are syndromes of local Th1/Th2 cytokine balance disturbances as compared with NP, and TH seems to be an intermediate step to PE.
Leukocyte migration in experimental inflammatory bowel disease
Authors: Van Rees E. P. , Palmen M. J. H. J. , Van De Goot F. R. W. , Macher B. A. , Dieleman L. A.
Published: July 2002

Emigration of leukocytes from the circulation into tissue by transendothelial migration, is mediated subsequently by adhesion molecules such as selectins, chemokines and integrins. This multistep paradigm, with multiple molecular choices at each step, provides a diversity in signals. The influx of neutrophils, monocytes and lymphocytes into inflamed tissue is important in the pathogenesis of chronic inflammatory bowel disease. The importance of each of these groups of adhesion molecules in chronic inflammatory bowel disease, either in human disease or in animal models, will be discussed below. Furthermore, the possibilities of blocking these different steps in the process of leukocyte extravasation in an attempt to prevent further tissue damage, will be taken into account.
Interleukin‐4 (IL‐4) enhances and soluble interleukin‐4 receptor (sIL‐4R) inhibits histamine release from peripheral blood basophils and mast cells in vitro and in vivo
Authors: Niggemann B. , Zuberbier T. , Herz U. , Enssle K. , Wahn U. , Renz H.
Published: July 2002

The aim of the study was to analyse the effect of interleukin‐4 (IL‐4) on allergen and anti‐IgE mediated histamine release from basophils and human skin mast cells and to assess whether soluble recombinant interleukin‐4 receptor (sIL4R) can inhibit these effects. Anti‐IgE stimulated histamine release from peripheral blood basophils and mast cells of atopic donors was enhanced after preincubation with IL‐4, whereas after preincubation with sIL‐4R it was inhibited. These effects were even more pronounced when samples were stimulated with a clinically relevant allergen. In IL‐4 preincubated skin mast cells, there was a similar enhancement of anti‐IgE stimulated histamine release, which could again be inhibited by sIL‐4R. The effects of IL‐4 and sIL4R were dose‐ and time‐dependent. Mice sensitized to ovalbumin and treated with soluble recombinant murine sIL‐4R showed significantly reduced immediate‐type cutaneous hypersensitivity responses compared with untreated mice.
These in vivo effects were IgE independent, since there were no significant differences in total and allergen specific IgE/IgG1 antibody titres between treated and untreated mice. This indicates that IL4 exerts priming effects on histamine release by effector cells of the allergic response and that these effects are potently antagonized by soluble IL‐4R both
in vitro and in vivo.
Mast cell amines and inosineinduced vasoconstriction in the rat hind limb
Authors: Northover A. M. , Northover B. J.
Published: July 2002

Under certain circumstances injected inosine causes a net vasoconstrictive effect on the arterioles, which has been attributed to 5‐hydroxytryptamine (5HT) released in response to adenosine type 3 (A3) receptor stimulation of mast cells residing in the adventitia. We have sought further evidence for this hypothesis using blood vessels of the rat hind limb perfused in vitro at constant rate with a gelatin‐containing physiological salt solution. Injection of inosine (2.7 mg) caused a rise in perfusion pressure, which was only slightly increased by inclusion of N‐nitro‐L‐arginine methyl ester (100 μM) in the perfusate. Inclusion in the perfusate of cyproheptadine (1 μM), compound 48 80 (1 μg ml), 8‐phenyltheophylline (1 μM) or 8‐cyclopentyl‐1,3 dipropylxanthine (0.1 μM) greatly reduced the pressor response to inosine. The pressor effect of injected 5HT (400 μg) was abolished by pre‐treatment with cyproheptadine, but not by pre‐treatment with compound 48 80. These results suggest that the net pressor response to injected inosine was mainly the result of an A1 receptor‐mediated release of 5HT, most probably from mast cells. No evidence was found for an involvement of A3 receptor stimulation.
Lung eicosanoids in perinatal rats with congenital diaphragmatic hernia
Authors: Ijsselstijn H. , Zijlstra F. J. , Van Dijk J. P. M. , De Jongste J. C. , Tibboel D.
Published: July 2002

Abnormal levels of pulmonary eicosanoids have been reported in infants with persistent pulmonary hypertension (PPH) and congenital diaphragmatic hernia (CDH). We hypothesized that a dysbalance of vasoconstrictive and vasodilatory eicosanoids is involved in PPH in CDH patients. The levels of several eicosanoids in lung homogenates and in bronchoalveolar lavage fluid of controls and rats with CDH were measured after caesarean section or spontaneous birth. In controls the concentration of the stable metabolite of prostacyclin (6‐keto‐PGF1α), thromboxane
A2 (TxB2), prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) decreased after spontaneous birth. CDH pups showed respiratory insufficiency directly after birth. Their lungs had higher levels of 6‐ keto‐PGF1α, reflecting the pulmonary vasodilator prostacyclin
(PGI2), than those of controls. We conclude that in CDH abnormal lung eicosanoid levels are present perinatally. The elevated levels of 6‐keto‐PGF1α in CDH may reflect a compensation mechanism for increased vascular resistance.
Rooperol, an inhibitor of cytokine synthesis, decreases the respiratory burst in human and rat leukocytes and macrophages
Authors: Guzdek A. , Turyna B. , Allison A. C. , Sladek K. , Koj A.
Published: July 2002

Luminol‐enhanced chemiluminescence was measured in fresh whole human blood, or human neutrophils isolated from heparinized blood, human alveolar macrophages and rat alveolar macrophages stimulated with bacterial endotoxin (LPS). Tetraacetate esters of rooperol, a dicatechol showing anticytokine activity, added to cells simultaneously with LPS inhibited the respiratory burst. The effective concentrations of rooperol were in the range of 1‐10 μM depending on cell type and corresponded well with inhibition of nitric oxide production by rat alveolar macrophages. Thus rooperol may reduce some effects of excessive phagocytic activity and inflammatory reaction but by quenching free radicals production may also diminish the resistance to bacterial infections.
Systemic inflammatory response syndrome (SIRS) induced by carbon tetrachloride in rats
Authors: Gergely J. , Sipka S. , Csípő J. , Udvardy M. , Szegedi Gy. , Kulcsár A.
Published: July 2002

We have observed the symptoms of systemic inflammatory response syndrome (SIRS) in male rats intoxicated by carbon tetrachloride (CCl4). Severe hypothermia, tachypnoea and increase in the heart beat min were diagnosed. These symptoms developed in the first hour of intoxication. The hepatic dysfunction was characterized by elevated bilirubin levels. In the sera we have measured increases in the activity of secretable (group II) phospholipase A2 sPLA2 (2,8x) and 6‐ketoprostaglandin F1α (KPGF) (1,44x). Supposedly the free radicals derived from CCl4—mainly trichloromethyl—could induce the prompt reaction of SIRS and the release of sPLA2 as well as the formation of KPGF. Our findings show that in the early phase of CCl4 intoxication the symptoms of SIRS can be related to elevation of sPLA2 and the products of cyclooxygenase II.
Vaccinia virus‐regulated acute phase cytokine production in human fibroblasts, U937 cells and endothelium
Authors: Rokita H. , Kupiec T. , Guzik K. , Koj A.
Published: July 2002

The production of acute phase cytokines, interleukin 6 (IL‐6), tumour necrosis factor (TNF α ) and interleukin 1 (IL‐1β ), was studied in primary cultures of human skin fibroblasts, human monocytic cell line U937 and primary cultures of human umbilical vein endothelial cells (HUVEC) after in vitro infection with vaccinia virus. Significant increase in IL‐6 mRNA followed by enhanced protein secretion into the culture media was found in fibroblasts, U937 cells, and HUVEC. TNFα increased production in vaccinia virus infected U937 cells resembled closely the pattern of IL‐6 production observed in the infected cells. Transient increase in NF‐κB binding activity was found in the infected U937 (at 90 min) and endothelial (at 30 min) cells. Vaccinia virus induced cytokine production appeared to be transcriptional.
Evaluation of the Reproductive Toxicity and Embryotoxicity of Insulin Glargine (LANTUS) in Rats and Rabbits
Authors: Hofmann Thomas , Horstmann Georg , Stammberger Ingo
Published: July 2002

Insulin glargine (LANTUS) is a new insulin analog that has a prolonged duration of action with no pronounced peak of activity, rendering it an ideal basal insulin for the treatment of diabetes. The aim of these studies was to assess the reproductive and embryotoxicity of insulin glargine. Reproductive toxicity was assessed in 25 male and 25 female Wistar rats per group treated with a daily subcutaneous injection of control; 1 IU/kg, 3 IU/kg, and 10 IU/kg insulin glargine; or 3 IU/kg NPH insulin in the premating and mating periods, and throughout pregnancy and lactation in the females. Embryotoxicity was assessed in 20 female rats per group injected with daily subcutaneous doses of control; 2 IU/kg, 6.3 IU/kg, and 20 IU/kg insulin glargine; or 6.3 IU/kg NPH insulin from the 7th to 18th day of pregnancy. Embryotoxicity was also assessed in 20 female rabbits per group treated with 0 IU/kg, 0.5 IU/kg, 1 IU/kg, and 2 IU/kg insulin glargine, or 1 IU/kg NPH insulin from the 6th to 18th day of pregnancy. The data demonstrated that, with the exception of toxicologic effects induced by hypoglycemia in response to high doses of insulin glargine and NPH insulin (including the premature dropout of female rats in the reproductive toxicity study, and increased incidence of abortions, early intrauterine deaths, and single anomalies in the rabbit embryotoxicity study), insulin glargine had no effects on reproduction, embryofetal development, and postnatal development in rats. Maternal and embryofetal toxicity in rabbits treated with middle and high doses of insulin glargine was related to the hypoglycemic effect of insulin.
Acute Toxicity of Tbt and Irgarol in Artemia salina
Authors: Panagoula Bakoulia , Panayiota Marcouli , Iliopoulou-Georgudaki Joan
Published: July 2002

A 24-hour LC50 bioassay method was carried out to study acute toxicity of Tributyltin (TBT) and IRGAROL (C11H19N5S) in Artemia salina. Five graded levels of both biocides were tested. According to the range test, the doses of TBT were 11.6 ng·l–1, 21.0 ng·l–1, 37.3 ng·l–1, 65.2 ng·l–1, and 116.5 ng·l–1, and for IRGAROL were 1.0 mg·l–1, 1.8 mg·l–1, 3.2 mg·l–1, 5.6 mg·l–1, and 10 mg·l–1. The determined LC50 values were 41.41 ng·l–1 and 1.62 mg·l–1 respectively. These results indicate that in this system TBT is acutely more toxic than IRGAROL; however, both are proven environmentally toxic substances.