bluuebunny/crossref_metadata_2025 huggingface dataset:
Fine root dynamics and trace gas fluxes in two lowland tropical forest soils
Authors: Silver Whendee L. , Thompson Andrew W. , McGroddy Megan E. , Varner Ruth K. , Dias Jadson D. , Silva Hudson , Crill Patrick M. , Keller Michael
Published: February 2005

Fine root dynamics have the potential to contribute significantly to ecosystem‐scale biogeochemical cycling, including the production and emission of greenhouse gases. This is particularly true in tropical forests which are often characterized as having large fine root biomass and rapid rates of root production and decomposition. We examined patterns in fine root dynamics on two soil types in a lowland moist Amazonian forest, and determined the effect of root decay on rates of C and N trace gas fluxes. Root production averaged 229 (±35) and 153 (±27) g m−2 yr−1 for years 1 and 2 of the study, respectively, and did not vary significantly with soil texture. Root decay was sensitive to soil texture with faster rates in the clay soil (k=−0.96 year−1) than in the sandy loam soil (k=−0.61 year−1), leading to greater standing stocks of dead roots in the sandy loam. Rates of nitrous oxide (N2O) emissions were significantly greater in the clay soil (13±1 ng N cm−2 h−1) than in the sandy loam (1.4±0.2 ng N cm−2 h−1). Root mortality and decay following trenching doubled rates of N2O emissions in the clay and tripled them in sandy loam over a 1‐year period. Trenching also increased nitric oxide fluxes, which were greater in the sandy loam than in the clay. We used trenching (clay only) and a mass balance approach to estimate the root contribution to soil respiration. In clay soil root respiration was 264–380 g C m−2 yr−1, accounting for 24% to 35% of the total soil CO2 efflux. Estimates were similar using both approaches. In sandy loam, root respiration rates were slightly higher and more variable (521±206 g C m2 yr−1) and contributed 35% of the total soil respiration. Our results show that soil heterotrophs strongly dominate soil respiration in this forest, regardless of soil texture. Our results also suggest that fine root mortality and decomposition associated with disturbance and land‐use change can contribute significantly to increased rates of nitrogen trace gas emissions.
A spatially interactive simulation of climate change, harvesting, wind, and tree species migration and projected changes to forest composition and biomass in northern Wisconsin, USA
Authors: Scheller Robert M. , Mladenoff David J.
Published: February 2005

In the coming century, forecast climate changes caused by increasing greenhouse gases may produce dramatic shifts in tree species distributions and the rates at which individual tree species sequester carbon or release carbon back to the atmosphere. The species composition and carbon storage capacity of northern Wisconsin (USA) forests are expected to change significantly as a result. Projected temperature changes are relatively large (up to a 5.8°C increase in mean annual temperature) and these forests encompass a broad ecotone that may be particularly sensitive to climate change. Our objective was to estimate the combined effects of climate change, common disturbances, and species migrations on regional forests using spatially interactive simulations. Multiple scenarios were simulated for 200 years to estimate aboveground live biomass and tree species composition. We used a spatially interactive forest landscape model (LANDIS‐II) that includes individual tree species, biomass accumulation and decomposition, windthrow, harvesting, and seed dispersal. We used data from two global circulation models, the Hadley Climate Centre (version 2) and the Canadian Climate Center (version 1) to generate transient growth and decomposition parameters for 23 species. The two climate change scenarios were compared with a control scenario of continuing current climate conditions. The results demonstrate how important spatially interactive processes will affect the aboveground live biomass and species composition of northern Wisconsin forests. Forest composition, including species richness, is strongly affected by harvesting, windthrow, and climate change, although five northern species (Abies balsamea,Betula papyrifera,Picea glauca,Pinus banksiana,P. resinosa) are lost in both climate scenarios regardless of disturbance scenario. Changes in aboveground live biomass over time are nonlinear and vary among ecoregions. Aboveground live biomass will be significantly reduced because of species dispersal and migration limitations. The expected shift towards southern oaks and hickory is delayed because of seed dispersal limitations.
Indole induces the expression of multidrug exporter genes in Escherichia coli
Authors: Hirakawa Hidetada , Inazumi Yoshihiko , Masaki Takeshi , Hirata Takahiro , Yamaguchi Akihito
Published: February 2005

Our comprehensive expression cloning studies previously revealed that 20 intrinsic xenobiotic exporter systems are encoded in the Escherichia coli chromosome, but most of them are not expressed under normal conditions. In this study, we investigated the compounds that induce the expression of these xenobiotic exporter genes, and found that indole induces a variety of xenobiotic exporter genes including acrD, acrE, cusB, emrK, mdtA, mdtE and yceL. Indole treatment of E. coli cells confers rhodamine 6G and SDS resistance through the induction of mdtEF and acrD gene expression respectively. The induction of mdtE by indole is independent of the EvgSA two‐component signal transduction system that regulates the mdtE gene, but mediated by GadX. On the other hand, the induction of acrD and mdtA was mediated by BaeSR and CpxAR, two‐component systems. Interestingly, CpxAR system‐mediated induction required intrinsic baeSR genes, whereas BaeSR‐mediated induction was observed in the cpxAR gene‐deletion mutant. BaeR and CpxR directly bound to different sequences of the acrD and mdtA promoter regions. These observations indicate that BaeR is a primary regulator, and CpxR enhances the effect of BaeR.
Functional interplay between the Bacillus subtilis DnaD and DnaB proteins essential for initiation and re‐initiation of DNA replication
Authors: Bruand Claude , Velten Marion , McGovern Stephen , Marsin Stéphanie , Sérèna Céline , Ehrlich S. Dusko , Polard Patrice
Published: February 2005

Initiation and re‐initiation of chromosomal DNA replication in bacteria rely on divergent multiprotein assemblies, which direct the functional delivery of the replicative helicase on single‐stranded DNA (ssDNA) at specific sites. These two processes are triggered either at the single chromosomal origin oriC or at arrested forks by the conserved DnaA and PriA proteins respectively. In Bacillus subtilis, these two pathways further require the three essential proteins DnaB, DnaD and DnaI, restrictively encoded in Gram positive bacteria of low GC content. We have recently shown that DnaI and DnaB act as a pair of loaders of the DnaC replicative helicase. The role of DnaD appeared more enigmatic. It was previously shown to interact with DnaA and to display weak ssDNA binding activity. Here, we report that purified DnaD can interact physically with PriA and with DnaB. We show that the lethality of the temperature‐sensitive dnaD23 mutant can be suppressed by different DnaB point mutants, which were found to be identical to the suppressors of priA null mutants. The DnaD23 protein displays lower ssDNA binding activity than DnaD. Conversely, the DnaB75 protein, the main dnaD23 suppressor, has gained affinity for ssDNA. Finally, we observed that this interplay between DnaD and DnaB is crucial for their concerted interaction with SSB‐coated ssDNA, which is the expected substrate for the loading of the replicative helicase in vivo. Altogether, these results highlight the need for both DnaD and DnaB to interact individually and together with ssDNA during the early stages of initiation and re‐initiation of chromosomal DNA replication. They also point at a main structural role of DnaD in the multiprotein assemblies built during these two essential processes.
Use of the Rhodopseudomonas palustris genome sequence to identify a single amino acid that contributes to the activity of a coenzyme A ligase with chlorinated substrates
Authors: Samanta Sudip K. , Harwood Caroline S.
Published: February 2005

Rhodopseudomonas palustris strain RCB100 degrades 3‐chlorobenzoate (3‐CBA) anaerobically. We purified from this strain a coenzyme A ligase that is active with 3‐CBA and determined its N‐terminal amino acid sequence to be identical to that of a cyclohexanecarboxylate‐CoA ligase encoded by aliA from the R. palustris strain (CGA009) that has been sequenced. Strain CGA009 differs from strain RCB100 in that it does not use 3‐CBA as a sole carbon source. The aliA gene from the 3‐CBA degrading strain differed by a single nucleotide from the aliA gene from strain CGA009, causing the substitution of a serine for a threonine at position 208. Both AliA enzymes, purified as His‐tagged fusion proteins, had comparable activities with cyclohexanecarboxylate. However, AliA from the 3‐CBA degrading strain was 10‐fold more active with 3‐CBA (kcat/Km of 4.3 × 104 M−1 s−1) than the enzyme from the sequenced strain (kcat/Km 0.32 × 104 M−1 s−1). The CGA009 enzyme was not sufficiently active with 3‐CBA to complement an RCB100 aliA mutant for growth on this compound. Here, whole genome sequence information enabled us to identify a single nucleotide among 5.4 million nucleotides that contributes to the substrate preference of a coenzyme A ligase.
Escherichia coli ribosomal RNA transcription: regulatory roles for ppGpp, NTPs, architectural proteins and a polymerase‐binding protein
Authors: Gralla Jay D.
Published: February 2005

Ribosomal RNA transcription can limit the rate of Escherichia coli growth and is subject to complex regulation. Somehow, the cell is able to sense the general nutritional environment and adjust rRNA transcription so that an appropriate number of ribosomes is produced. This review discusses the current state of affairs, including recent information about the involvement of two nucleotide regulators, two architectural protein regulators, one new co‐regulator and stalled ribosomes.
Regulated degradation of chromosome replication proteins DnaA and CtrA in Caulobacter crescentus
Authors: Gorbatyuk Boris , Marczynski Gregory T.
Published: February 2005

DnaA protein binds bacterial replication origins and it initiates chromosome replication. The Caulobacter crescentus DnaA also initiates chromosome replication and the C. crescentus response regulator CtrA represses chromosome replication. CtrA proteolysis by ClpXP helps restrict chromosome replication to the dividing cell type. We report that C. crescentus DnaA protein is also selectively targeted for proteolysis but DnaA proteolysis uses a different mechanism. DnaA protein is unstable during both growth and stationary phases. During growth phase, DnaA proteolysis ensures that primarily newly made DnaA protein is present at the start of each replication period. Upon entry into stationary phase, DnaA protein is completely removed while CtrA protein is retained. Cell cycle arrest by sudden carbon or nitrogen starvation is sufficient to increase DnaA proteolysis, and relieving starvation rapidly stabilizes DnaA protein. This starvation‐induced proteolysis completely removes DnaA protein even while DnaA synthesis continues. Apparently, C. crescentus relies on proteolysis to adjust DnaA in response to such rapid nutritional changes. Depleting the C. crescentus ClpP protease significantly stabilizes DnaA. However, a dominant‐negative clpX allele that blocks CtrA degradation, even when combined with a clpA null allele, did not decrease DnaA degradation. We suggest that either a novel chaperone presents DnaA to ClpP or that ClpX is used with exceptional efficiency so that when ClpX activity is limiting for CtrA degradation it is not limiting for DnaA degradation. This unexpected and finely tuned proteolysis system may be an important adaptation for a developmental bacterium that is often challenged by nutrient‐poor environments.
The acidocalcisome Ca2+‐ATPase (TgA1) of Toxoplasma gondii is required for polyphosphate storage, intracellular calcium homeostasis and virulence
Authors: Luo Shuhong , Ruiz Felix A. , Moreno Silvia N. J.
Published: February 2005

A large proportion of intracellular Ca2+ in Toxoplasma gondii tachyzoites is stored within acidocalcisomes. These organelles are characterized by their acidic nature and high calcium and polyphosphate (polyP) content. The activity of a Ca2+/H+‐ATPase named TgA1 may be important for the accumulation of Ca2+ in these organelles. This enzyme belongs to a group of plasma membrane Ca2+‐ATPase (PMCA) that lack a calmodulin‐binding domain and have vacuolar localization. To investigate the role of this enzyme, we have generated T. gondii mutants deficient in TgA1 through gene disruption. Proliferation of these mutants decreased dramatically because of deficient cell invasion. In addition, these cells had reduced virulence in a mouse model. Biochemical analysis revealed that the cell polyP content was drastically reduced, and the basal calcium levels were increased and unstable. Microneme secretion under the conditions of stimulation by ionophores was altered. Complementation of null mutants with TgA1 restored most functions. In summary, these results establish a link between TgA1, calcium homeostasis, polyP storage and virulence.
Building a bacterial orisome: emergence of new regulatory features for replication origin unwinding
Authors: Leonard Alan C. , Grimwade Julia E.
Published: February 2005

Triggering new rounds of chromosomal DNA replication during the bacterial cell cycle is exquisitely regulated, ensuring both proper timing and one round per cycle stringency. A critical first step is stable unwinding of oriC, the chromosomal replication origin, by multiprotein orisome complexes comprising the AAA+ initiator DnaA and modulator proteins that bend DNA. Recently identified oriC–DnaA interactions in Escherichia coli raise important questions regarding the molecular mechanisms that regulate origin unwinding in bacteria. We describe staged binding of E. coli origin recognition proteins and suggest an unwinding switch based on interactions between DnaA‐ATP and specialized oriC sites that must be filled during orisome assembly. By focusing multiple regulatory pathways on only a few key oriC DNA–protein interactions, this model includes an efficient way to control unwinding followed by orisome inactivation during the cell cycle. Future studies will determine whether this regulatory scheme is correct and whether it is generally applicable to other bacterial types.
pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum
Authors: Hayward Rhys , Saliba Kevin J. , Kirk Kiaran
Published: February 2005

Efforts to control malaria worldwide have been hindered by the development and expansion of parasite populations resistant to many first‐line antimalarial compounds. Two of the best‐characterized determinants of drug resistance in the human malaria parasite Plasmodium falciparum are pfmdr1 and pfcrt, although the mechanisms by which resistance is mediated by these genes is still not clear. In order to determine whether mutations in pfmdr1 associated with chloroquine resistance affect the capacity of the parasite to persist when drug pressure is removed, we conducted competition experiments between P. falciparum strains in which the endogenous pfmdr1 locus was modified by allelic exchange. In the absence of selective pressure, the component of chloroquine resistance attributable to mutations at codons 1034, 1042 and 1246 in the pfmdr1 gene also gave rise to a substantial fitness cost in the intraerythrocytic asexual stage of the parasite. The loss of fitness incurred by these mutations was calculated to be 25% with respect to an otherwise genetically identical strain in which wild‐type polymorphisms had been substituted at these three codons. At least part of the fitness loss may be attributed to a diminished merozoite viability. These in vitro results support recent in vivo observations that in several countries where chloroquine use has been suspended because of widespread resistance, sensitive strains are re‐emerging.
The Yak1p kinase controls expression of adhesins and biofilm formation in Candida glabrata in a Sir4p‐dependent pathway
Authors: Iraqui Ismail , Garcia‐Sanchez Susana , Aubert Sylvie , Dromer Françoise , Ghigo Jean‐Marc , D’Enfert Christophe , Janbon Guilhem
Published: February 2005

Biofilm is the predominant type of microbial development in natural environments, and potentially represents a major form of resistance or source of recurrence during host infection. Although a large number of studies have focussed on the genetics of bacterial biofilm formation, very little is known about the genes involved in this type of growth in fungi. A genetic screen for Candida glabrata Biofilm mutants was performed using a 96‐well plate model of biofilm formation. Study of the isolated mutant strains allowed the identification of four genes involved in biofilm formation (RIF1, SIR4, EPA6 and YAK1). Epa6p is a newly identified adhesin required for biofilm formation in this pathogenic yeast. EPA6 and its close paralogue EPA7 are located in subtelomeric regions and their transcription is regulated by Sir4p and Rif1p, two proteins involved in subtelomeric silencing. Biofilm growth conditions induce the transcription of EPA6 and EPA7: this is dependent on the presence of an intact subtelomeric silencing machinery and is independent of the Mpk1p signalling pathway. Finally, the kinase Yak1p is required for expression of both adhesin genes and acts through a subtelomeric silencing machinery‐dependent pathway.
Nuclear fusion occurs during mating in Candida albicans and is dependent on the KAR3 gene
Authors: Bennett Richard J. , Miller Mathew G. , Chua Penelope R. , Maxon Mary E. , Johnson Alexander D.
Published: February 2005

It is now well established that mating can occur between diploid a and α cells of Candida albicans. There is, however, controversy over when, and with what efficiency, nuclear fusion follows cell fusion to create stable tetraploid a/α cells. In this study, we have analysed the mating process between C. albicans strains using both cytological and genetic approaches. Using strains derived from SC5314, we used a number of techniques, including time‐lapse microscopy, to demonstrate that efficient nuclear fusion occurs in the zygote before formation of the first daughter cell. Consistent with these observations, zygotes micromanipulated from mating mixes gave rise to mononuclear tetraploid cells, even when no selection for successful mating was applied to them. Mating between different clinical isolates of C. albicans revealed that while all isolates could undergo nuclear fusion, the efficiency of nuclear fusion varied in different crosses. We also show that nuclear fusion in C. albicans requires the Kar3 microtubule motor protein. Deletion of the CaKAR3 gene from both mating partners had little or no effect on zygote formation but reduced the formation of stable tetraploids more than 600‐fold, as determined by quantitative mating assays. These findings demonstrate that nuclear fusion is an active process that can occur in C. albicans at high frequency to produce stable, mononucleate mating products.
A polymorphic motif in the small subunit of ADP‐glucose pyrophosphorylase modulates interactions between the small and large subunits
Authors: Cross Joanna M. , Clancy Maureen , Shaw Janine R. , Boehlein Susan K. , Greene Thomas W. , Schmidt Robert R. , Okita Thomas W. , Curtis Hannah L.
Published: February 2005

The heterotetrameric, allosterically regulated enzyme, adenosine‐5′‐diphosphoglucose pyrophosphorylase (AGPase) catalyzes the rate‐limiting step in starch synthesis. Despite vast differences in allosteric properties and a long evolutionary separation, heterotetramers of potato small subunit and maize large subunit have activity comparable to either parent in an Escherichia coli expression system. In contrast, co‐expression of maize small subunit with the potato large subunit produces little activity as judged by in vivo activity stain. To pinpoint the region responsible for differential activity, we expressed chimeric maize/potato small subunits in E. coli. This identified a 55‐amino acid motif of the potato small subunit that is critical for glycogen production when expressed with the potato large subunit. Potato and maize small subunit sequences differ at five amino acids in this motif. Replacement experiments revealed that at least four amino acids of maize origin were required to reduce staining. An AGPase composed of a chimeric potato small subunit containing the 55‐amino acid maize motif with the potato large subunit exhibited substantially less affinity for the substrates, glucose‐1‐phosphate and ATP and an increased Ka for the activator, 3‐phosphoglyceric acid. Placement of the potato motif into the maize small subunit restored glycogen synthesis with the potato large subunit. Hence, a small polymorphic motif within the small subunit influences both catalytic and allosteric properties by modulating subunit interactions.
Overexpression of a petunia zinc‐finger gene alters cytokinin metabolism and plant forms
Authors: Nakagawa Hitoshi , Jiang Chang‐Jie , Sakakibara Hitoshi , Kojima Mikiko , Honda Ichiro , Ajisaka Hidetoshi , Nishijima Takaaki , Koshioka Masaji , Homma Tamaki , Mander Lewis N. , Takatsuji Hiroshi
Published: February 2005

Shoot branching and plant height are among the key factors that define the overall architecture of plants. We found that overexpression of a cDNA for a zinc‐finger protein of petunia, designated Lateral shoot‐Inducing Factor (LIF), in transgenic petunia plants resulted in a dramatic increase in lateral shoots and reduced plant height. LIF overexpression also caused a decrease in the number of cells in the stem, leaf, and flower, accompanied by enlargement of cells. trans‐Zeatin was decreased while N6‐(Δ2‐isopentenyl)adenine was increased in the leaves of LIF‐overexpressed petunia. Most of the riboside, ribotide, and glucoside forms were also increased. Expression analysis using a LIF::GUS fusion gene and RT‐PCR suggested that LIF is specifically expressed around the bases of axillary buds and weakly in basal part of flowers in wild‐type petunia. GFP‐LIF‐GUS fusion proteins were translocated into the nucleus when transiently expressed in onion epidermal cells. LIF overexpression resulted in enhanced branching also in tobacco and Arabidopsis, indicating the conservation of the response to LIF overexpression among dicotyledonous plants. On the basis of these results we discuss about possible functions of LIF.
Genetic variation in epigenetic inheritance of ribosomal RNA gene methylation in Arabidopsis
Authors: Riddle Nicole C. , Richards Eric J.
Published: February 2005

We investigated the fidelity of epigenetic inheritance in crosses between three accessions of the flowering plant Arabidopsis thaliana (Canary Islands, Cape Verde Islands, and Columbia). Specifically, we examined the cytosine methylation content of the ribosomal RNA genes at the two nucleolus organizer regions (NOR2 and NOR4) in F1 and F2 hybrid individuals derived from reciprocal crosses between the high NOR methylation strain, Columbia, and the two other accessions, both of which have less NOR methylation. In crosses between the Columbia and Cape Verde Islands strains, the cytosine methylation content segregated as an additive Mendelian trait: the high NOR methylation state was tightly associated with the inheritance of the two Columbia‐derived NOR loci. First‐generation hybrid individuals between the Canary Islands and Columbia strains also showed a cytosine methylation content at the NORs intermediate between the parental values, consistent with the epigenetic inheritance of parental methylation patterns. Interestingly, mapping data from F2 individuals derived from a Canary Islands × Columbia cross revealed that NOR2 accounted for nearly all of the NOR methylation variation segregating in the population. NOR4 retains a significant effect on total NOR methylation content only through a complex epistatic interaction with NOR2. Our results indicate that the inheritance of differential cytosine methylation states at NOR loci can be modified by their genetic context, opening up the possibility of genetic dissection of epigenetic inheritance.
A CELD‐fusion method for rapid determination of the DNA‐binding sequence specificity of novel plant DNA‐binding proteins
Authors: Xue Gang‐Ping
Published: February 2005

The current focus of many functional genomic studies is on the elucidation of gene regulatory networks. The functional analyses of transcription factors and their DNA‐binding sites, in conjunction with genome‐wide expression profiling, are crucial in understanding of gene regulatory networks. This paper describes an efficient and easy method for characterizing the DNA‐binding sequence specificity of novel plant transcription factors. This new method is based on the fusion of a DNA‐binding protein (DBP) to 6×His‐tagged cellulase D (CELD), which serves both as a means for affinity purification of DBP‐DNA complex in the selection of binding sites from a pool of biotinylated random‐sequence oligonucleotides and as a reporter for measurement of DNA‐binding activity. Thus, it eliminates the use of radioactivity and gel electrophoresis techniques currently used for purification of DBP‐DNA complexes and assays of DNA‐binding activity. The effectiveness of this method was demonstrated by the success of simultaneous selection of the binding sites of nine plant DBPs from four superfamilies (AP2, bHLH, NAC and MYB). The high‐throughput capacity of CELD‐based DNA‐binding assays allows the quantitative analysis of the binding sequence specificity from a large number of DBP‐selected oligonucleotides. The binding sequence specificity of three novel transcription factors (rice OsbHLH66, wheat TaNAC69 and TaMYB80), determined with this method, is presented. This new method provides the capacity of high‐throughput analysis on the DNA‐binding sequence specificity of a large number of putative transcription factors, predicted on the basis of conserved DNA‐binding domains.
K+ currents through SV‐type vacuolar channels are sensitive to elevated luminal sodium levels
Authors: Ivashikina Natalya , Hedrich Rainer
Published: February 2005

Non‐selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch‐clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150‐fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient‐driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.
Aromatic interactions in tryptophan‐containing peptides: crystal structures of model tryptophan peptides and phenylalanine analogs*
Authors: Sengupta A. , Mahalakshmi R. , Shamala N. , Balaram P.
Published: February 2005

Abstract:  The crystal structures of the peptides, Boc‐Leu‐Trp‐Val‐OMe (1), Ac‐Leu‐Trp‐Val‐OMe (2a and 2b), Boc‐Leu‐Phe‐Val‐OMe (3), Ac‐Leu‐Phe‐Val‐OMe (4), and Boc‐Ala‐Aib‐Leu‐Trp‐Val‐OMe (5) have been determined by X‐ray diffraction in order to explore the nature of interactions between aromatic rings, specifically the indole side chain of Trp residues. Peptide 1 adopts a type I β‐turn conformation stabilized by an intramolecular 4→1 hydrogen bond. Molecules of 1 pack into helical columns stabilized by two intermolecular hydrogen bonds, Leu(1)NH…O(2)Trp(2) and IndoleNH…O(1)Leu(1). The superhelical columns further pack into the tetragonal space group P43 by means of a continuous network of indole–indole interactions. Peptide 2 crystallizes in two polymorphic forms, P21 (2a) and P212121 (2b). In both forms, the peptide backbone is extended, with antiparallel β‐sheet association being observed in crystals. Extended strand conformations and antiparallel β‐sheet formation are also observed in the Phe‐containing analogs, Boc‐Leu‐Phe‐Val‐OMe (3) and Ac‐Leu‐Phe‐Val‐OMe (4). Peptide 5 forms a short stretch of 310‐helix. Analysis of aromatic–aromatic and aromatic–amide interactions in the structures of peptides, 1, 2a, 2b are reported along with the examples of 14 Trp‐containing peptides from the Cambridge Crystallographic Database. The results suggest that there is no dramatic preference for a preferred orientation of two proximal indole rings. In Trp‐containing peptides specific orientations of the indole ring, with respect to the preceding and succeeding peptide units, appear to be preferred in β‐turns and extended structures.
Identification of Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐amino acid‐OH as new impurities in Fmoc‐protected amino acid derivatives*
Authors: Hlebowicz E. , Andersen A.J. , Andersson L. , Moss B.A.
Published: February 2005

Abstract:  During the manufacture of a proprietary peptide drug substance a new impurity appeared unexpectedly. Investigation of its chemical structure established the impurity as a β‐Ala insertion mutant of the mother peptide. The source of the β‐Ala was identified as contamination of the Fmoc‐Ala‐OH raw material with Fmoc‐β‐Ala‐Ala‐OH. Further studies also demonstrated the presence of β‐Ala in other Fmoc‐amino acids, particularly in Fmoc‐Arg(Pbf)‐OH. In this case, it was due to the presence of both Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐Arg(Pbf)‐OH. It is concluded that β‐Ala contamination of Fmoc‐amino acid derivatives is a general and hitherto unrecognized problem to suppliers of Fmoc‐amino acid derivatives. The β‐Ala is often present as Fmoc‐β‐Ala‐OH and/or as a dipeptide, Fmoc‐β‐Ala‐amino acid‐OH. In collaboration with the suppliers, new specifications were introduced, recognizing the presence of β‐Ala‐related impurities in the raw materials and limiting them to acceptable levels. The implementation of these measures has essentially eliminated β‐Ala contamination as a problem in the manufacture of the drug substance.
Proposed intracellular regulatory functions of glutathione transferases by recognition and binding to S‐glutathiolated proteins
Authors: Listowsky I.
Published: February 2005

Abstract:  A general reaction scheme is considered in which structurally diverse compounds can enhance transcription of glutathione S‐transferase (GST) genes. Many of those compounds have the capacity to promote S‐glutathiolation reactions with cysteine residues of proteins. The binding sites of GSTs, which are highly specific for binding of the tripeptide glutathione (GSH), can accommodate many structurally different substituents linked to GSH. Accordingly, it is suggested that GSH transferases can function by stoichiometric binding to S‐glutathiolated proteins that are generated by oxidative stress or by reactive compounds. Binding to a GST could influence properties and regulate cellular functions of those proteins.
Development Zones, Foreign Investment, and Global City Formation in Shanghai*
Authors: WEI YEHUA DENNIS , LEUNG CHI KIN
Published: February 2005

ABSTRACT  The rapid economic ascent of China and the increasing integration of the world economy in the past two decades have made metropolises in China such as Shanghai and Beijing emerging global cities. Foreign investment is a central force underlying the emergence and transformation of the Chinese metropolises into global cities. This is especially true in Shanghai, which has experienced massive infusion of foreign investment. Varied forms of foreign investment or development zones have been created to promote foreign investment inflows, yet remain under‐studied. This paper analyzes structure, performance, and underlying factors of development zones in Shanghai, and discusses the implications for global city‐formation; it unfolds the variations among development zones, and illustrates the significant role of the state and local conditions. As the literature on global cities dwells primarily on the experiences of advanced economies, this paper further contributes to a better understanding of the dynamics of emerging global cities in the developing world.
The New Retail Economy of Shanghai
Authors: WANG SHUGUANG , ZHANG YONGCHANG
Published: February 2005

ABSTRACT  As Shanghai strives to build itself into an international center of finance, trade, and commerce, a new retail economy has evolved accordingly. In the past two decades, its retail sector has been transformed from a simple and inefficient distribution system to a much more complex and highly competitive market‐oriented economy. The new retail economy in many ways resembles the contemporary capitalist retail economy in the Western cities, but it also exhibits significant differences with Chinese characteristics. While the affluent consumer market is the necessary condition for sustained retail growth, it is the retail deregulation that has been the fundamental driving force for the structural changes in Shanghai's retail sector. Its liberal policies attracted major international retailers to either choose Shanghai as the gateway city to enter the China market, or locate their China headquarters offices in Shanghai to command their operations throughout the country. Indeed, the retail transformation in post‐reform Shanghai is a clear testimony of the Economic Transition Model. The main data sources for this empirical study are the 1999 Census of Commercial Activity in Shanghai and the Shanghai Statistical Yearbook. They are supplemented by data collected from reputable Web sites and through field work in Shanghai.
Reduced neuronal expression of synaptic transmission modulator HNK‐1/neural cell adhesion molecule as a potential consequence of amyloid β‐mediated oxidative stress: a proteomic approach
Authors: Thomas Stefani N. , Soreghan Brian A. , Nistor Mihaela , Sarsoza Floyd , Head Elizabeth , Yang Austin J.
Published: February 2005

Oxidative stress imparted by reactive oxygen species (ROS) is implicated in the pathogenesis of Alzheimer's disease (AD). Given that amyloid β (Aβ) itself generates ROS that can directly damage proteins, elucidating the functional consequences of protein oxidation can enhance our understanding of the process of Aβ‐mediated neurodegeneration. In this study, we employed a biocytin hydrazide/streptavidin affinity purification methodology followed by two‐dimensional liquid chromatography tandem mass spectrometry coupled with SEQUEST bioinformatics technology, to identify the targets of Aβ‐induced oxidative stress in cultured primary cortical mouse neurons. The Golgi‐resident enzyme glucuronyltransferase (GlcAT‐P) was a carbonylated target that we investigated further owing to its involvement in the biosynthesis of HNK‐1, a carbohydrate epitope expressed on cell adhesion molecules and implicated in modulating the effectiveness of synaptic transmission in the brain. We found that increasing amounts of Aβ, added exogenously to the culture media of primary cortical neurons, significantly decreased HNK‐1 expression. Moreover, in vivo, HNK‐1 immunoreactivity was decreased in brain tissue of a transgenic mouse model of AD. We conclude that a potential consequence of Aβ‐mediated oxidation of GlcAT‐P is impairment of its enzymatic function, thereby disrupting HNK‐1 biosynthesis and possibly adversely affecting synaptic plasticity. Considering that AD is partly characterized by progressive memory impairment and disordered cognitive function, the data from our in vitro studies can be reconciled with results from in vivo studies that have demonstrated that HNK‐1 modulates synaptic plasticity and is critically involved in memory consolidation.
Regulation of cholinergic gene expression by nerve growth factor depends on the phosphatidylinositol‐3′‐kinase pathway
Authors: Madziar Beata , Lopez‐Coviella Ignacio , Zemelko Victoria , Berse Brygida
Published: February 2005

Nerve growth factor (NGF) exerts anti‐apoptotic, trophic and differentiating actions on sympathetic neurons and cholinergic cells of the basal forebrain and activates the expression of genes regulating the synthesis and storage of the neurotransmitter acetylcholine (ACh). We have been studying the intracellular signaling pathways involved in this process. Although, in the rat pheochromocytoma cell line PC12, NGF strongly activates the mitogen‐activated protein kinase (MAPK) pathway, prolonged inhibition of MAPK kinase (MEK) activity by PD98059 or U0126 did not affect the ability of NGF to up‐regulate choline acetyltransferase (ChAT) or to increase intracellular ACh levels. In contrast, the treatment with the phosphatidylinositol 3′‐kinase (PI3K) inhibitor LY294002, but not with its inactive analogue LY303511, completely abolished the NGF‐induced production of ACh. Inhibition of PI3K also eliminated the NGF effect on the intracellular ACh level in primary cultures of septal neurons from E18 mouse embryos. Blocking the PI3K pathway prevented the activation of cholinergic gene expression, as demonstrated in RT/PCR assays and in transient transfections of PC12 cells with cholinergic locus promoter‐luciferase reporter constructs. These results indicate that the PI3K pathway, but not the MEK/MAPK pathway, is the mediator of NGF‐induced cholinergic differentiation.
Proteasome inhibition protects HT22 neuronal cells from oxidative glutamate toxicity
Authors: Van Leyen Klaus , Siddiq Ambreena , Ratan Rajiv R. , Lo Eng H.
Published: February 2005

Oxidative stress caused by glutathione depletion after prolonged exposure to extracellular glutamate leads to a form of neuronal cell death that exhibits morphologically mixed features of both apoptosis and necrosis. However, specific downstream executioners involved in this form of cell death have yet to be identified. We report here that glutamate exposure does not activate caspase‐3 in the HT22 neuronal cell line. Furthermore, no cytoprotection was achieved with either the pan‐caspase inhibitor Z‐VAD‐fmk or the caspase‐3‐specific inhibitor DEVD‐CHO. In contrast, inhibition of the proteasome by lactacystin protected both HT22 cells and rat primary neuronal cells against cell lysis. In parallel, oxidatively altered and ubiquitinated proteins accumulated in the mitochondrial fraction of cells after proteasome inhibition. These findings suggest that caspases can be decoupled from oxidative stress under some conditions, and implicate the ubiquitin/proteasome pathway in neuronal cell death caused by oxidative glutamate toxicity.
A role for caveolin‐1 in post‐injury reactive neuronal plasticity
Authors: Gaudreault Sophie B. , Blain Jean‐François , Gratton Jean‐Philippe , Poirier Judes
Published: February 2005

Remodeling and plasticity in the adult brain require cholesterol redistribution and synthesis for the formation of new membrane components. Caveolin‐1 is a cholesterol‐binding membrane protein involved in cellular cholesterol transport and homeostasis. Evidence presented here demonstrates an up‐regulation of caveolin‐1 in the hippocampus, which was temporally correlated with an increase in synaptophysin during the reinnervation phase in a mouse model of hippocampal deafferentation. Using an in vitro model of neuronal reactive plasticity, we examined the effect of virally mediated overexpression of caveolin‐1 on injured differentiated PC12 cells undergoing terminal remodeling. Three days post lesion, caveolin‐1‐overexpressing cells revealed increases in synaptophysin and GAP‐43, two markers of neurite sprouting and synaptogenesis. Morphologically, caveolin‐1‐overexpressing cells showed a decrease in primary neurite outgrowth and branching as well as an increase in neurite density. Caveolin‐1‐overexpressing cells also revealed the presence of terminal swelling and beading along processes, consistent with a possible alteration of microtubules stability. Moreover, a focal enrichment of caveolin‐1 immunofluorescence was observed at the bases of axonal and dendritic terminals of mouse primary hippocampal neurons. Altogether, these results indicate that caveolin‐1 plays an active role in the regulation of injury‐induced synaptic and terminal remodeling in the adult CNS.
Cytochrome c release from rat brain mitochondria is proportional to the mitochondrial functional deficit: implications for apoptosis and neurodegenerative disease
Authors: Clayton Rebecca , Clark John B. , Sharpe Martyn
Published: February 2005

Apoptosis may be initiated in neurons via mitochondrial release of the respiratory protein, cytochrome c. The mechanism of cytochrome c release has been studied extensively, but little is known about its dynamics. It has been claimed that release is all‐or‐none, however, this is not consistent with accumulating evidence of cytosolic mechanisms for ‘buffering’ cytochrome c. This study has attempted to model an underlying disease pathology, rather than inducing apoptosis directly. The model adopted was diminished activity of the mitochondrial respiratory chain complex I, a recognized feature of Parkinson's disease. Titration of rat brain mitochondrial respiratory function, with the specific complex I inhibitor rotenone, caused proportional release of cytochrome c from isolated synaptic and non‐synaptic mitochondria. The mechanism of release was mediated, at least in part, by the mitochondrial outer membrane component Bak and voltage‐dependent anion channel rather than non‐specific membrane rupture. Furthermore, preliminary data were obtained demonstrating that in primary cortical neurons, titration with rotenone induced cytochrome c release that was subthreshold for the induction of apoptosis. Implications for the therapy of neurodegenerative diseases are discussed.
A gene‐targeted mouse model for chorea‐acanthocytosis
Authors: Tomemori Yuko , Ichiba Mio , Kusumoto Akira , Mizuno Emiko , Sato Daisuke , Muroya Shinji , Nakamura Masayuki , Kawaguchi Hiroaki , Yoshida Hiroki , Ueno Shu‐ichi , Nakao Kazuki , Nakamura Kenji , Aiba Atsu , Katsuki Motoya , Sano Akira
Published: February 2005

Chorea‐acanthocytosis (CHAC) is a hereditary neurodegenerative disorder with autosomal recessive transmission, in which selective degeneration of striatum has been reported in brain pathology. Clinically, CHAC shows Huntington's disease‐like neuropsychiatric symptoms and red blood cell acanthocytosis. Recently, we identified the gene, CHAC, encoding a novel protein, chorein, in which a deletion mutation was found in Japanese families with CHAC. In the present study, we have identified the mouse CHAC cDNA sequence and the exon–intron structures of the gene and produced a CHAC model mouse introducing no. 60–61 exon deletion corresponding to a human disease mutation by a gene‐targeting technique. The mice began to show acanthocytosis and motor disturbance in old age. In behavioral observations, locomotor activity was significantly decreased and the contact time at social interaction test was decreased significantly in the model mice. In the brain pathology, many apoptotic cells were observed in the striatum of the mutant mice. In neurochemical determinations, the dopamine metabolite, homovanillic acid, concentration decreased significantly in the portion including the midbrain of the mutant mice. These findings are consistent with the human results reported elsewhere and indicate that the CHAC model mice showed a mild phenotype with late adult onset. The CHAC model mouse therefore provides a good model system to study the human disease.
Identification of autotaxin as a neurite retraction‐inducing factor of PC12 cells in cerebrospinal fluid and its possible sources
Authors: Sato Koichi , Malchinkhuu Enkhzol , Muraki Takeshi , Ishikawa Koichi , Hayashi Kensuke , Tosaka Masahiko , Mochiduki Akikazu , Inoue Kinji , Tomura Hideaki , Mogi Chihiro , Nochi Hiromi , Tamoto Koichi , Okajima Fumikazu
Published: February 2005

Cerebrospinal fluid (CSF) induced neurite retraction of differentiated PC12 cells; the action was observed in 15 min (a rapid response) and the activity further increased until 6 h (a long‐acting response) during exposure of CSF to the cells. The CSF action was sensitive to monoglyceride lipase and diminished by homologous desensitization with lysophosphatidic acid (LPA) and by pretreatment with an LPA receptor antagonist Ki16425. Although fresh CSF contains LPA to some extent, the LPA content in the medium was increased during culture of PC12 cells with CSF. The rapid response was mimicked by exogenous LPA, and a long‐acting response was duplicated by a recombinant autotaxin, lysophospholipase D (lyso‐PLD). Although the lyso‐PLD substrate lysophosphatidylcholine (LPC) was not detected in CSF, lyso‐PLD activity and an ∼120‐kDa autotaxin protein were detected in CSF. On the other hand, LPC but not lyso‐PLD activity was detected in the conditioned medium of a PC12 cell culture without CSF. Among neural cells examined, leptomeningeal cells expressed the highest lyso‐PLD activity and autotaxin protein. These results suggest that leptomeningeal cells may work as one of the sources for autotaxin, which may play a critical role in LPA production and thereby regulate axonal and neurite morphological change.
Mutational studies using a cation‐conducting GABAA receptor reveal the selectivity determinants of the Cys‐loop family of ligand‐gated ion channels
Authors: Jensen Marianne L. , Pedersen Lone N. , Timmermann Daniel B. , Schousboe Arne , Ahring Philip K.
Published: February 2005

The present study evaluates how four key amino acid residue positions (− 4′ to − 1′) within the M1–M2 linker of the GABAA receptor β subunit influences ion selectivity of a cation‐conducting GABA receptor. Cation selectivity was found to be highly dependent on the side‐chains of the amino acid residues present. The critical factor for cation selectivity was the presence of a negatively charged Glu or Asp residue in the − 1′ position. Receptors containing the neutral amino acids Gln or Asn or a positively charged Arg residue were anion selective. In the presence of a −1′ Glu residue, the amino acids in adjacent positions were also found to be important determinants of cation selectivity. Moreover, the length of the M1–M2 linker as well as the presence of a Pro residue within this segment also affected ion selectivity, suggesting that the local environment and three‐dimensional position of the −1′ Glu are essential determinants of cation permeation. Conversely, no specific amino acid residues were found to be essential for anion selectivity, suggesting that the basic architecture of the selectivity segment of this class of receptor channels is optimally suited for anion conduction.