bluuebunny/crossref_metadata_2025 huggingface dataset:
On the tryptophan residue of smooth muscle myosin that responds to binding of nucleotide
Authors: Onishi Hirofumi , Konishi Kaoru , Fujiwara Keigi , Hayakawa Kazuyoshi , Tanokura Masaru , Martinez Hugo M. , Morales Manuel F.
Published: July 2002

Initially, we asked which (of 10) smooth muscle myosin head
residues responds to MgADP or MgATP binding with enhanced fluorescence
emission (Trp-441 and Trp-512 were leading candidates)? To decide, we
prepared sham-mutated smooth muscle heavy meromyosin (HMM), W441F HMM,
and W512F HMM. On adding MgATP, emission of wild-type and W441F HMMs
increased by 25–27%, but that of W512F HMM by 5%. So, in myosin, 512
is the “sensitive Trp.” Unexpectedly, properties of W512F HMM
[elevated Ca
2+
-ATPase, depressed EDTA
(K
+
)-ATPase, no regulation of its basal or actin-activated
Mg
2+
-ATPase by phosphorylation of its “regulatory”
light chain, limited actin activation, and inability to move actin
filaments in a motility assay] are strikingly like those of smooth
muscle myosin reacted at Cys-717 with thiol reagent. From
crystallography-based [Houdusse, A., Kalabakis, V. N., Himmel,
D., Szent-Györgyi, A. G. & Cohen, C. (1999)
Cell
97, 459–470] simulations, we found that in
wild-type HMM with MgADP added, Trp-512 is in a “hydrophobic
pocket,” but that pocket becomes distorted in W512F HMM. We think
that there is a “path of influence” from 512 to 717 to the active
site. We suggest that the mutational changes at 512 are transmitted
along this path to Cys-717, where they induce changes similar to those
caused by reacting wild-type HMM with thiol reagent.
Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for gag budding from cells
Authors: Kikonyogo Alexandra , Bouamr Fadila , Vana Marcy L. , Xiang Yan , Aiyar Ashok , Carter Carol , Leis Jonathan
Published: July 2002

The late assembly (L) domain of retrovirus Gag, required in the
final steps of budding for efficient exit from the host cell, is
thought to mediate its function through interaction with unknown
cellular factors. Here, we report the identification of the Nedd4-like
family of E3 ubiquitin protein ligases as proteins that specifically
interact with the Rous sarcoma virus (RSV) L domain
in
vitro
and
in vivo
. We screened a chicken embryo
cDNA expression library by using a peptide derived from the RSV p2b
sequence, isolating two unique partial cDNA clones. Neither clone
interacted with a peptide containing mutations known to disrupt
in vivo
RSV L domain function or with human
immunodeficiency virus type 1 (HIV-1) and equine infectious anemia
virus (EIAV) L domain-derived peptides. The WW domain region of one of
the clones, late domain-interacting protein 1 (LDI-1), but not the C2
domain, bound RSV Gag and inhibited RSV Gag budding from human 293
cells in a dominant-negative manner, functionally implicating LDI-1 in
RSV particle budding from cells. RSV Gag can be coimmune precipitated
from cell extracts with an antisera directed at an exogenously
expressed hemagglutinin (HA)-tagged LDI-1 or endogenous
Nedd4 proteins. These findings mechanistically link the cellular
ubiquitination pathway to retrovirus budding.
How patterns of bleached rods and cones become visual perceptual experiences: A proposal
Authors: Galambos Robert , Juhász Gábor
Published: July 2002

In an attempt to increase information about how mammalian visual
systems create a perceptual experience out of a retinal photochemical
bleach pattern, this article brings together recent rat physiological
data acquired with large electrodes, an old cat behavioral experiment,
and two complex human behaviors: reading and the reversible blindness
people experience when the scene being viewed is stabilized on the
retinal surface. The outcome suggests this juxtaposition of disparate
data sets has been logical, reasonable, and informative. The link
between rats and reading is the fact that both rat and human retinas
convert bleach patterns into ganglion cell volleys 3 times a second.
The probable trigger for these episodic retinal volleys is a more or
less abrupt change in the pattern of bleached rods and cones, and we
claim the absence of this trigger when the image is stabilized is
responsible for the blindness. The cat behavioral experiment correlates
performance on visual discrimination tasks with the number of nerve
fibers remaining after lesions of the optic tract. The analysis of the
result, which shows that as few as 2% of the normal number of nerve
fibers supports perfect performance of such tasks, prompts the concept
of a second dynamic visual system, operating in parallel with the
anatomical nervous system pictured in the textbooks. The dynamic visual
system model, which brings into the foreground important old facts that
have been neglected and integrates them with new data, offers a
synthesis that may be useful in interpreting classical visual
behavioral phenomena.
A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA.
Authors: Paillart J C , Skripkin E , Ehresmann B , Ehresmann C , Marquet R
Published: July 2002

RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.
A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase.
Authors: Kochanek S , Clemens P R , Mitani K , Chen H H , Chan S , Caskey C T
Published: July 2002

Adenoviral vector-mediated gene transfer offers significant potential for gene therapy of many human diseases. However, progress has been slowed by several limitations. First, the insert capacity of currently available adenoviral vectors is limited to 8 kb of foreign DNA. Second, the expression of viral proteins in infected cells is believed to trigger a cellular immune response that results in inflammation and in only transient expression of the transferred gene. We report the development of a new adenoviral vector that has all viral coding sequences removed. Thus, large inserts are accommodated and expression of all viral proteins is eliminated. The first application of this vector system carries a dual expression cassette comprising 28.2 kb of nonviral DNA that includes the full-length murine dystrophin cDNA under control of a large muscle-specific promoter and a lacZ reporter construct. Using this vector, we demonstrate independent expression of both genes in primary mdx (dystrophin-deficient) muscle cells.
Amelioration of lupus-like autoimmune disease in NZB/WF1 mice after treatment with a blocking monoclonal antibody specific for complement component C5.
Authors: Wang Y , Hu Q , Madri J A , Rollins S A , Chodera A , Matis L A
Published: July 2002

New Zealand black x New Zealand white (NZB/W) F1 mice spontaneously develop an autoimmune syndrome with notable similarities to human systemic lupus erythematosus. Female NZB/WF1 mice produce high titers of antinuclear antibodies and invariably succumb to severe glomerulonephritis by 12 months of age. Although the development of the immune-complex nephritis is accompanied by abundant local and systemic complement activation, the role of proinflammatory complement components in disease progression has not been established. In this study we have examined the contribution of activated terminal complement proteins to the pathogenesis of the lupus-like autoimmune disease. Female NZB/W F1 mice were treated with a monoclonal antibody (mAb) specific for the C5 component of complement that blocks the cleavage of C5 and thus prevents the generation of the potent proinflammatory factors C5a and C5b-9. Continuous therapy with anti-C5 mAb for 6 months resulted in significant amelioration of the course of glomerulonephritis and in markedly increased survival. These findings demonstrate an important role for the terminal complement cascade in the progression of renal disease in NZB/W F1 mice, and suggest that mAb-mediated C5 inhibition may be a useful approach to the therapy of immune-complex glomerulonephritis in humans.
Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences.
Authors: Barns S M , Delwiche C F , Palmer J D , Pace N R
Published: July 2002

Phylogenetic analysis of ribosomal RNA sequences obtained from uncultivated organisms of a hot spring in Yellowstone National Park reveals several novel groups of Archaea, many of which diverged from the crenarchaeal line of descent prior to previously characterized members of that kingdom. Universal phylogenetic trees constructed with the addition of these sequences indicate monophyly of Archaea, with modest bootstrap support. The data also show a specific relationship between low-temperature marine Archaea and some hot spring Archaea. Two of the environmental sequences are enigmatic: depending upon the data set and analytical method used, these sequences branch deeply within the Crenarchaeota, below the bifurcation between Crenarchaeota and Euryarchaeota, or even as the sister group to Eukaryotes. If additional data confirm either of the latter two placements, then the organisms represented by these ribosomal RNA sequences would merit recognition as a new kingdom, provisionally named "Korarchaeota."
Mexican papita viroid: putative ancestor of crop viroids.
Authors: Martínez-Soriano J P , Galindo-Alonso J , Maroon C J , Yucel I , Smith D R , Diener T O
Published: July 2002

The potato spindle tuber disease was first observed early in the 20th century in the northeastern United States and shown, in 1971, to be incited by a viroid, potato spindle tuber viroid (PSTVd). No wild-plant PSTVd reservoirs have been identified; thus, the initial source of PSTVd infecting potatoes has remained a mystery. Several variants of a novel viroid, designated Mexican papita viroid (MPVd), have now been isolated from Solanum cardiophyllum Lindl. (papita güera, cimantli) plants growing wild in the Mexican state of Aguascalientes. MPVd's nucleotide sequence is most closely related to those of the tomato planta macho viroid (TPMVd) and PSTVd. From TPMVd, MPVd may be distinguished on the basis of biological properties, such as replication and symptom formation in certain differential hosts. Phylogenetic and ecological data indicate that MPVd and certain viroids now affecting crop plants, such as TPMVd, PSTVd, and possibly others, have a common ancestor. We hypothesize that commercial potatoes grown in the United States have become viroid-infected by chance transfer of MPVd or a similar viroid from endemically infected wild solanaceous plants imported from Mexico as germplasm, conceivably from plants known to have been introduced from Mexico to the United States late in the 19th century in efforts to identify genetic resistance to the potato late blight fungus, Phytophthora infestans.
Highly plastic chromosomal organization in Salmonella typhi.
Authors: Liu S L , Sanderson K E
Published: July 2002

Gene order in the chromosomes of Escherichia coli K-12 and Salmonella typhimurium LT2, and in many other species of Salmonella, is strongly conserved, even though the genera diverged about 160 million years ago. However, partial digestion of chromosomal DNA of Salmonella typhi, the causal organism of typhoid fever, with the endonuclease I-CeuI followed by separation of the DNA fragments by pulsed-field gel electrophoresis showed that the chromosomes of independent wild-type isolates of S. typhi are rearranged due to homologous recombination between the seven rrn genes that code for ribosomal RNA. The order of genes within the I-CeuI fragments is largely conserved, but the order of the fragments on the chromosome is rearranged. Twenty-one different orders of the I-CeuI fragments were detected among the 127 wild-type strains we examined. Duplications and deletions were not found, but transpositions and inversions were common. Transpositions of I-CeuI fragments into sites that do not change their distance from the origin of replication (oriC) are frequently detected among the wild-type strains, but transpositions that move the fragments much further from oriC were rare. This supports the gene dosage hypothesis that genes at different distances from oriC have different gene dosages and, hence, different gene expression, and that during evolution genes become adapted to their specific location; thus, cells with changes in gene location due to transpositions may be less fit. Therefore, gene dosage may be one of the forces that conserves gene order, although its effects seem less strong in S. typhi than in other enteric bacteria. However, both the gene dosage and the genomic balance hypotheses, the latter of which states that the origin (oriC) and terminus (TER) of replication must be separated by 180 degrees C, need further investigation.
Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein.
Authors: Mebatsion T , Conzelmann K K
Published: July 2002

A recombinant rabies virus (RV) mutant deficient for the surface spike glycoprotein (G) gene was used to study the incorporation of envelope proteins from HIV-1 expressed from transfected plasmids. A hybrid HIV-1 protein in which the cytoplasmic domain was replaced with that of RV G was incorporated into the virus envelope and rescued the infectivity of the RV mutant. The RV(HIV-1) pseudotype viruses could infect only CD4+ cells, and their infectivity was neutralized specifically by anti-HIV-1 sera. In contrast to the chimeric protein, wild-type HIV-1 envelope protein or mutants with truncated cytoplasmic domains failed to produce pseudotyped particles. This indicates the presence of a specific signal in the RV G cytoplasmic domain, allowing correct incorporation of a spike protein into the envelope of rhabdovirus particles. The possibility of directing the cell tropism of RV by replacement of the RV G with proteins of defined receptor specificity should prove useful for future development of targetable gene delivery vectors.
Development of HIV vectors for anti-HIV gene therapy.
Authors: Poeschla E , Corbeau P , Wong-Staal F
Published: July 2002

Current gene therapy protocols for HIV infection use transfection or murine retrovirus mediated transfer of antiviral genes into CD4+ T cells or CD34+ progenitor cells ex vivo, followed by infusion of the gene altered cells into autologous or syngeneic/allogeneic recipients. While these studies are essential for safety and feasibility testing, several limitations remain: long-term reconstitution of the immune system is not effected for lack of access to the macrophage reservoir or the pluripotent stem cell population, which is usually quiescent, and ex vivo manipulation of the target cells will be too expensive and impractical for global application. In these regards, the lentivirus-specific biologic properties of the HIVs, which underlie their pathogenetic mechanisms, are also advantageous as vectors for gene therapy. The ability of HIV to specifically target CD4+ cells, as well as non-cycling cells, makes it a promising candidate for in vivo gene transfer vector on one hand, and for transduction of non-cycling stem cells on the other. Here we report the use of replication-defective vectors and stable vector packaging cell lines derived from both HIV-1 and HIV-2. Both HIV envelopes and vesicular stomatitis virus glycoprotein G were effective in mediating high-titer gene transfer, and an HIV-2 vector could be cross-packaged by HIV-1. Both HIV-1 and HIV-2 vectors were able to transduce primary human macrophages, a property not shared by murine retroviruses. Vesicular stomatitis virus glycoprotein G-pseudotyped HIV vectors have the potential to mediate gene transfer into non-cycling hematopoietic stem cells. If so, HIV or other lentivirus-based vectors will have applications beyond HIV infection.
On the molecular mechanism of chloroquine's antimalarial action.
Authors: Sullivan D J , Gluzman I Y , Russell D G , Goldberg D E
Published: July 2002

Chloroquine is thought to exert its antimalarial effect by preventing the polymerization of toxic heme released during proteolysis of hemoglobin in the Plasmodium digestive vacuole. The mechanism of this blockade has not been established. We incubated cultured parasites with subinhibitory doses of [3H]chloroquine and [3H] quinidine. These [3H]quinoline compounds became associated with hemozoin as assessed by electron microscope autoradiography and subcellular fractionation. In vitro, binding of [3H]quinoline inhibitors to the hemozoin chain depended on the addition of heme substrate. These data counter previous conclusions regarding the lack of quinoline association with hemozoin, explain the exaggerated accumulation of quinolines in the plasmodium digestive vacuole, and suggest that a quinoline heme complex incorporates into the growing polymer to terminate chain extension, blocking further sequestration of toxic heme.
The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.
Authors: Linder M , Teeri T T
Published: July 2002

Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes. The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate. In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose. The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H. Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved. Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures. The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution. The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.
A fast marching level set method for monotonically advancing fronts.
Authors: Sethian J A
Published: July 2002

A fast marching level set method is presented for monotonically advancing fronts, which leads to an extremely fast scheme for solving the Eikonal equation. Level set methods are numerical techniques for computing the position of propagating fronts. They rely on an initial value partial differential equation for a propagating level set function and use techniques borrowed from hyperbolic conservation laws. Topological changes, corner and cusp development, and accurate determination of geometric properties such as curvature and normal direction are naturally obtained in this setting. This paper describes a particular case of such methods for interfaces whose speed depends only on local position. The technique works by coupling work on entropy conditions for interface motion, the theory of viscosity solutions for Hamilton-Jacobi equations, and fast adaptive narrow band level set methods. The technique is applicable to a variety of problems, including shape-from-shading problems, lithographic development calculations in microchip manufacturing, and arrival time problems in control theory.
A role for the Msx-1 homeodomain in transcriptional regulation: residues in the N-terminal arm mediate TATA binding protein interaction and transcriptional repression.
Authors: Zhang H , Catron K M , Abate-Shen C
Published: July 2002

In a previous study we showed that the murine homeodomain protein Msx-1 is a potent transcriptional repressor and that this activity is independent of its DNA binding function. The implication of these findings is that repression by Msx-1 is mediated through its association with certain protein factors rather than through its interaction with DNA recognition sites, which prompted investigation of the relevant protein factors. Here we show that Msx-1 interacts directly with the TATA binding protein (TBP) but not with several other general transcription factors. This interaction is mediated by the Msx-1 homeodomain, specifically through residues in the N-terminal arm. These same N-terminal arm residues are required for repression by Msx-1, suggesting a functional relationship between TBP association and transcriptional repression. This is further supported by the observation that addition of excess TBP blocks the repressor action of Msx-1 in in vitro transcription assays. Finally, DNA binding activity is separable from both TBP interaction and repression, which further shows that these other activities of the Msx-1 homeodomain are distinct. Therefore, these findings define a role for the Msx-1 homeodomain, particularly the N-terminal arm residues in protein-protein interaction and transcriptional repression, and implicate a more complex role overall for homeodomains in transcriptional regulation.
Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium.
Authors: Shea J E , Hensel M , Gleeson C , Holden D W
Published: July 2002

Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7. This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12. Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type III secretion system. To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2. SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90% identity with those of the E. coli ydhE and pykF genes. Thus SPI2 was probably acquired horizontally by insertion into a region corresponding to that between the ydhE and pykF genes of E. coli. Virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice.
Ecdysone-inducible gene expression in mammalian cells and transgenic mice.
Authors: No D , Yao T P , Evans R M
Published: July 2002

During metamorphosis of Drosophila melanogaster, a cascade of morphological changes is triggered by the steroid hormone 20-OH ecdysone via the ecdysone receptor, a member of the nuclear receptor superfamily. In this report, we have transferred insect hormone responsiveness to mammalian cells by the stable expression of a modified ecdysone receptor that regulates an optimized ecdysone responsive promoter. Inductions reaching 4 orders of magnitude have been achieved upon treatment with hormone. Transgenic mice expressing the modified ecdysone receptor can activate an integrated ecdysone responsive promoter upon administration of hormone. A comparison of tetracycline-based and ecdysone-based inducible systems reveals the ecdysone regulatory system exhibits lower basal activity and higher inducibility. Since ecdysone administration has no apparent effect on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene in transgenic mice and for gene therapy.
Membrane lipid perturbation modifies the set point of the temperature of heat shock response in yeast.
Authors: Carratù L , Franceschelli S , Pardini C L , Kobayashi G S , Horvath I , Vigh L , Maresca B
Published: July 2002

Addition of a saturated fatty acid (SFA) induced a strong increase in heat shock (HS) mRNA transcription when cells were heat-shocked at 37 degrees C, whereas treatment with an unsaturated fatty acid (UFA) reduced or eliminated the level of HS gene transcription at 37 degrees C. Transcription of the delta 9-desaturase gene (Ole1) of Histoplasma capsulatum, whose gene product is responsible for the synthesis of UFA, is up-regulated in a temperature-sensitive strain. We show that when the L8-14C mutant of Saccharomyces cerevisiae, which has a disrupted Ole1 gene, is complemented with its own Ole1 coding region under control of its own promoter or Ole1 promoters of H. capsulatum, the level of HS gene transcription depends on the activity of the promoters. Fluorescence anisotropy of mitochondrial membranes of completed strains corresponded to the different activity of the Ole1 promoter used. We propose that the SFA/UFA ratio and perturbation of membrane lipoprotein complexes are involved in the perception of rapid temperature changes and under HS conditions disturbance of the preexisting membrane physical state causes transduction of a signal that induces transcription of HS genes.
rRNA-like sequences occur in diverse primary transcripts: Implications for the control of gene expression
Authors: Mauro Vincent P. , Edelman Gerald M.
Published: July 2002

Many eukaryotic mRNAs contain sequences that resemble segments of 28S and 18S rRNAs, and these rRNA-like sequences are present in both the sense and antisense orientations. Some are similar to highly conserved regions of the rRNAs, whereas others have sequence similarities to expansion segments. In particular, four 18S rRNA-like sequences are found in several hundred different genes, and the location of these four sequences within the various genes is not random. One of these rRNA-like sequences is preferentially located within protein coding regions immediately upstream of the termination codon of a number of genes. Northern blot analysis of poly(A)
+
RNA from different vertebrates (chicken, cattle, rat, mouse, and human) revealed that a large number of discrete RNA molecules hybridize at high stringency to cloned probes prepared from the 28S or 18S rRNA sequences that were found to match those in mRNAs. Inhibition of polymerase II activity, which prevents the synthesis of most mRNAs, abolished most of the hybridization to the rRNA probes. We consider the hypotheses that rRNA-like sequences may have spread throughout eukaryotic genomes and that their presence in primary transcripts may differentially affect gene expression.
Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons
Authors: Du Yansheng , Bales Kelly R. , Dodel Richard C. , Hamilton-Byrd Elizabeth , Horn Jeffrey W. , Czilli Dan L. , Simmons Linda K. , Ni Binhui , Paul Steven M.
Published: July 2002

Neurotoxicity induced by overstimulation of
N
-methyl-
d
-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca
2+
; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration- and time-dependent activation of the interleukin 1β-converting enzyme (ICE)/CED-3-related protease, CPP32/Yama/apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamate-induced apoptosis of CGNs is mediated by a posttranslational activation of the ICE/CED-3-related cysteine protease caspase 3.
Fluorescence correlation spectroscopy: Diagnostics for sparse molecules
Authors: Maiti Sudipta , Haupts Ulrich , Webb Watt W.
Published: July 2002

The robust glow of molecular fluorescence renders even sparse
molecules detectable and susceptible to analysis for concentration,
mobility, chemistry, and photophysics. Correlation spectroscopy, a
statistical-physics-based tool, gleans quantitative information from
the spontaneously fluctuating fluorescence signals obtained from small
molecular ensembles. This analytical power is available for studying
molecules present at minuscule concentrations in liquid solutions (less
than one nanomolar), or even on the surfaces of living cells at less
than one macromolecule per square micrometer. Indeed, routines are
becoming common to detect, locate, and examine individual molecules
under favorable conditions.
Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis
Authors: Lee Ning , MacDonald Heather , Reinhard Christoph , Halenbeck Robert , Roulston Anne , Shi Tim , Williams Lewis T.
Published: July 2002

Apoptosis is a highly regulated form of cell death, characterized by distinctive features such as cellular shrinkage and nuclear condensation. We demonstrate here that proteolytic activation of hPAK65, a p21-activated kinase, induces morphological changes and elicits apoptosis. hPAK65 is cleaved both
in vitro
and
in vivo
by caspases at a single site between the N-terminal regulatory p21-binding domain and the C-terminal kinase domain. The C-terminal cleavage product becomes activated, with a kinetic profile that parallels caspase activation during apoptosis. This C-terminal hPAK65 fragment also activates the c-Jun N-terminal kinase pathway
in vivo
. Microinjection or transfection of this truncated hPAK65 causes striking alterations in cellular and nuclear morphology, which subsequently promotes apoptosis in both CHO and Hela cells. Conversely, apoptosis is delayed in cells expressing a dominant-negative form of hPAK65. These findings provide a direct evidence that the activated form of hPAK65 generated by caspase cleavage is a proapoptotic effector that mediates morphological and biochemical changes seen in apoptosis.
Arabidopsis thaliana CBF1
encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit
Authors: Stockinger Eric J. , Gilmour Sarah J. , Thomashow Michael F.
Published: July 2002

Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeat/dehydration responsive element (DRE), that stimulates transcription in response to low temperature and water deficit. Here we report the isolation of an
Arabidopsis thaliana
cDNA that encodes a C-repeat/DRE binding factor, CBF1 (
C
-repeat/DRE
B
inding
F
actor
1
). Analysis of the deduced CBF1 amino acid sequence indicates that the protein has a molecular mass of 24 kDa, a potential nuclear localization sequence, and a possible acidic activation domain. CBF1 also has an AP2 domain, which is a DNA-binding motif of about 60 aa present in the
Arabidopsis
proteins APETALA2, AINTEGUMENTA, and TINY; the tobacco ethylene response element binding proteins; and numerous other plant proteins of unknown function. The transcript levels for
CBF1
, which appears to be a single or low copy number gene, did not change appreciably in plants exposed to low temperature or in detached leaves subjected to water deficit. Binding of CBF1 to the C-repeat/DRE was demonstrated in gel shift assays using recombinant CBF1 protein expressed in
Escherichia coli
. Moreover, expression of CBF1 in yeast was found to activate transcription of reporter genes containing the C-repeat/DRE as an upstream activator sequence but not mutant versions of the DNA element. We conclude that CBF1 can function as a transcriptional activator that binds to the C-repeat/DRE DNA regulatory element and, thus, is likely to have a role in cold- and dehydration-regulated gene expression in
Arabidopsis
.
A novel arachidonate-preferring acyl-CoA synthetase is present in steroidogenic cells of the rat adrenal, ovary, and testis
Authors: Kang Man-Jong , Fujino Takahiro , Sasano Hironobu , Minekura Hiroyuki , Yabuki Noritaka , Nagura Hiroshi , Iijima Hiroaki , Yamamoto Tokuo T.
Published: July 2002

We report herein the cDNA cloning of a novel rat acyl-CoA synthetase (ACS) that preferentially uses arachidonate and eicosapentaenoate. This newly identified ACS (designated ACS4) contains 670 amino acids and is 68% identical to rat ACS3, a previously characterized ACS that is highly expressed in brain. ACS4 was overproduced in
Escherichia coli
and the resulting enzyme was purified to homogeneity. The purified enzyme utilizes arachidonate and eicosapentaenoate most preferentially among C
8
–C
22
saturated fatty acids and C
14
–C
22
unsaturated fatty acids. Kinetic analyses revealed that the enzyme has a high affinity for arachidonate and eicosapentaenoate and low affinity for palmitate. ACS4 transcripts are detectable in a wide range of tissues, with the highest level in adrenal gland. Immunoreactivity to ACS4 was detected in the zona fasciculata and reticularis of adrenal gland, in the corpus luteum and stromal luteinized cells in ovary, and in the Leydig cells of testis.
Guanylyl cyclase expression in specific sensory neurons: A new family of chemosensory receptors
Authors: Yu Sidney , Avery Leon , Baude Eric , Garbers David L.
Published: July 2002

A guanylyl cyclase (GC-D) was recently shown to be expressed in a subclass of neurons within the neuroepithelim of the rat, but given that only a single cyclase was discovered, whether it represents an odorant/pheromone receptor as has been suggested for the large family of seven-transmembrane receptors remains unclear. Through cloning and expression of cDNA we now demonstrate that at least 29 genomic or cDNA sequences found in
Caenorhabditis elegans
represent guanylyl cyclases. Many of the membrane forms retain cysteine residues conserved within the extracellular, ligand-binding domain of known cyclase receptors. Of eight orphan cyclase receptor::GFP (green fluroescence protein) fusion constructs for which signals were obtained, all were expressed in specific sensory neurons. Furthermore, a cyclase/GFP fusion protein (GCY-10/GFP) was principally expressed in the sensory cilium, suggesting these cyclases function as primary chemosensory receptors. For the first time, we also found that chemosensory neurons (ASE), known to be bilaterally symmetric, demonstrate absolute right or left sidedness with respect to the expression of three different cyclases. Thus, the guanylyl cyclases represent an unexpectedly large and new family of sensory neuron receptors that may complement the 7-transmembrane family of odorant/pheromone receptors.
Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance
Authors: Arriza Jeffrey L. , Eliasof Scott , Kavanaugh Michael P. , Amara Susan G.
Published: July 2002

Although a glutamate-gated chloride conductance with the properties of a sodium-dependent glutamate transporter has been described in vertebrate retinal photoreceptors and bipolar cells, the molecular species underlying this conductance has not yet been identified. We now report the cloning and functional characterization of a human excitatory amino acid transporter, EAAT5, expressed primarily in retina. Although EAAT5 shares the structural homologies of the EAAT gene family, one novel feature of the EAAT5 sequence is a carboxy-terminal motif identified previously in
N
-methyl-
d
-aspartate receptors and potassium channels and shown to confer interactions with a family of synaptic proteins that promote ion channel clustering. Functional properties of EAAT5 were examined in the
Xenopus
oocyte expression system by measuring radiolabeled glutamate flux and two-electrode voltage clamp recording. EAAT5-mediated
l
-glutamate uptake is sodium- and voltage-dependent and chloride-independent. Transporter currents elicited by glutamate are also sodium- and voltage-dependent, but ion substitution experiments suggest that this current is largely carried by chloride ions. These properties of EAAT5 are similar to the glutamate-elicited chloride conductances previously described in retinal neurons, suggesting that the EAAT5-associated chloride conductance may participate in visual processing.
β-dystrobrevin, a member of the dystrophin-related protein family
Authors: Blake Derek J. , Nawrotzki Ralph , Loh Nellie Y. , Górecki Dariusz C. , Davies Kay E.
Published: July 2002

The importance of dystrophin and its associated proteins in normal muscle function is now well established. Many of these proteins are expressed in nonmuscle tissues, particularly the brain. Here we describe the characterization of β-dystrobrevin, a dystrophin-related protein that is abundantly expressed in brain and other tissues, but is not found in muscle. β-dystrobrevin is encoded by a 2.5-kb alternatively spliced transcript that is found throughout the brain. In common with dystrophin, β-dystrobrevin is found in neurons of the cortex and hippocampal formation but is not found in the brain microvasculature. In the brain, β-dystrobrevin coimmunoprecipitates with the dystrophin isoforms Dp71 and Dp140. These data provide evidence that the composition of the dystrophin-associated protein complex in the brain differs from that in muscle. This finding may be relevant to the cognitive dysfunction affecting many patients with Duchenne muscular dystrophy.
Biodiversity and ecosystem functioning: A mechanistic model
Authors: Loreau Michel
Published: July 2002

Recent experiments have provided some evidence that loss of biodiversity may impair the functioning and sustainability of ecosystems. However, we still lack adequate theories and models to provide robust generalizations, predictions, and interpretations for such results. Here I present a mechanistic model of a spatially structured ecosystem in which plants compete for a limiting soil nutrient. This model shows that plant species richness does not necessarily enhance ecosystem processes, but it identifies two types of factors that could generate such an effect: (
i
) complementarity among species in the space they occupy below ground and (
ii
) positive correlation between mean resource-use intensity and diversity. In both cases, the model predicts that plant biomass, primary productivity, and nutrient retention all increase with diversity, similar to results reported in recent field experiments. These two factors, however, have different implications for the understanding of the relationship between biodiversity and ecosystem functioning. The model also shows that the effect of species richness on productivity or other ecosystem processes is masked by the effects of physical environmental parameters on these processes. Therefore, comparisons among sites cannot reveal it, unless abiotic conditions are very tightly controlled. Identifying and separating out the mechanisms behind ecosystem responses to biodiversity should become the focus of future experiments.
Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites
in vivo
Authors: Rompel Annette , Cinco Roehl M. , Latimer Matthew J. , McDermott Ann E. , Guiles R. D. , Quintanilha Alexandre , Krauss Ronald M. , Sauer Kenneth , Yachandra Vittal K. , Klein Melvin P.
Published: July 2002

The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inflection point energy for cysteine and cystine are observed. For methionine sulfoxide the inflection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfur-containing components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.
Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes
Authors: Szabò Ildikò , Lepple-Wienhues Albrecht , Kaba Kristen N. , Zoratti Mario , Gulbins Erich , Lang Florian
Published: July 2002

CD95/Fas/APO-1 mediated apoptosis is an important mechanism in the regulation of the immune response. Here, we show that CD95 receptor triggering activates an outwardly rectifying chloride channel (ORCC) in Jurkat T lymphocytes. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces activation of ORCC in cell-attached patch clamp experiments. Activation is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor herbimycin A or by genetic deficiency of p56lck.In vitroincubation of excised patches with purified p56lck results in activation of ORCC, which is partially reversed upon addition of anti-phosphotyrosine antibody. Inhibition of ORCC by four different drugs correlates with a 30–65% inhibition of apoptosis. Intracellular acidification observed upon CD95 triggering is abolished by inhibition of either ORCC or p56lck. The results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes.
α-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies
Authors: Spillantini Maria Grazia , Crowther R. Anthony , Jakes Ross , Hasegawa Masato , Goedert Michel
Published: July 2002

Lewy bodies and Lewy neurites are the defining neuropathological characteristics of Parkinson’s disease and dementia with Lewy bodies. They are made of abnormal filamentous assemblies of unknown composition. We show here that Lewy bodies and Lewy neurites from Parkinson’s disease and dementia with Lewy bodies are stained strongly by antibodies directed against amino-terminal and carboxyl-terminal sequences of α-synuclein, showing the presence of full-length or close to full-length α-synuclein. The number of α-synuclein-stained structures exceeded that immunoreactive for ubiquitin, which is currently the most sensitive marker of Lewy bodies and Lewy neurites. Staining for α-synuclein thus will replace staining for ubiquitin as the preferred method for detecting Lewy bodies and Lewy neurites. We have isolated Lewy body filaments by a method used for the extraction of paired helical filaments from Alzheimer’s disease brain. By immunoelectron microscopy, extracted filaments were labeled strongly by anti-α-synuclein antibodies. The morphologies of the 5- to 10-nm filaments and their staining characteristics suggest that extended α-synuclein molecules run parallel to the filament axis and that the filaments are polar structures. These findings indicate that α-synuclein forms the major filamentous component of Lewy bodies and Lewy neurites.
The making of the architecture of the plant cell wall: How cells exploit geometry
Authors: Emons Anne Mie C. , Mulder Bela M.
Published: July 2002

Cell wall deposition is a key process in the formation, growth, and differentiation of plant cells. The most important structural components of the wall are long cellulose microfibrils, which are synthesized by synthases embedded in the plasma membrane. A fundamental question is how the microfibrils become oriented during deposition at the plasma membrane. The current textbook explanation for the orientation mechanism is a guidance system mediated by cortical microtubules. However, too many contraindications are known in secondary cell walls for this to be a universal mechanism, particularly in the case of helicoidal arrangements, which occur in many situations. An additional construction mechanism involves liquid crystalline self-assembly [A. C. Neville (1993)
Biology of Fibrous Composites: Development Beyond the Cell Membrane
(Cambridge Univ. Press, Cambridge, U.K.)], but the required amount of bulk material that is able to equilibrate thermally is not normally present at any stage of the wall deposition process. Therefore, we have asked whether the complex ordered texture of helicoidal cell walls can be formed in the absence of direct cellular guidance mechanisms. We propose that they can be formed by a mechanism that is based on geometrical considerations. It explains the genesis of the complicated helicoidal texture and shows that the cell has intrinsic, versatile tools for creating a variety of textures. A compelling feature of the model is that local rules generate global order, a typical phenomenon of life.