bluuebunny/crossref_metadata_2025 huggingface dataset:
Load Testing of a Deteriorated Concrete Box Girder Bridge
Authors: Yang Jun , DeWolf John T
Published: October 2002

This paper reports on load testing of a continuous three cell concrete box girder bridge. The bridge is non-prismatic, curved in plan, and has skewed supports. The bridge is over thirty years old, and it was designed with a construction joint between two continuous spans and a span with a cantilever. The tests were carried out to evaluate the cause of large cracks extending through most of the height in some of the webs. Evaluation of the test results has shown that the distribution of strains in the bridge is significantly different than assumed in design. This has resulted in a redistribution of the load carrying capacity across the bridge. It is concluded that this is due to the varying cross-sectional dimensions and the reductions in the stiffness that occurs following development of the major cracks. The test results have been used to evaluate the bridge's overall behaviour and condition.
Residual Strength of Concrete Filled RHS Stub Columns after Exposure to High Temperatures
Authors: Han Lin-Hai , Yang Hua , Cheng Shu-Liang
Published: October 2002

Tests are reported on twenty-six concrete filled steel tube of rectangular section after being exposed to high temperatures, to investigate the influence of temperature on section capacity and load-deformation behavior. The main parameter varied is temperature, from 20°C to 900°C. A mechanics model is described in this paper for the behaviour of concrete-filled RHS (Rectangular Hollow Section) columns after exposed to high temperatures, and is a development of the analysis (Han et al, 2001a) used when only normal temperatures apply. The predicted load versus axial strain relationship is in good agreement with stub column test results. Simplified models are derived for the section capacities and the modulus of elasticity of the composite sections. It was found in general, that the higher the exposure temperature, the higher the loss of section capacities and elastic modulus which resulted. The tests have shown the importance of the influence of high temperatures on the performance of concrete filled steel tubes. The work in this paper provides a basis for further theoretical study on the residual strength of concrete filled steel tubular columns.
The combination of the antitumoural pyridyl cyanoguanidine CHS 828 and etoposide in vitro–from cytotoxic synergy to complete inhibition of apoptosis
Authors: Martinsson P , Ekelund S , Nygren P , Larsson R
Published: October 2002

The present study was aimed at elucidating the apoptosis inhibitory properties of the cyanoguanidine CHS 828. CHS 828 exhibits impressive cytotoxic activity in vitro and in vivo. Apoptosis is not its main mode of cytotoxic effect, and we have previously proposed a dual mechanism, where CHS 828 inhibits its own cell death pathways.

Etoposide on the other hand, is a well‐established anticancer agent with documented effect in a number of malignancies, induces apoptosis through extensively studied caspase dependent pathways.

Here we studied the combined effect of the two drugs in the human lymphoma cell line U‐937 GTB. Cytotoxicity was evaluated as total viability measured by the fluorometric microculture cytotoxicity assay (FMCA). Caspase activity was assessed by colorimetric detection of specific cleavage products for caspases 3, 8 and 9, respectively. Morphology was evaluated in May‐Grünwald/Giemsa stained preparations. Interaction analysis based on FMCA results of simple combination exposure revealed impressive synergistic effect on cell kill.

Detailed investigations of the kinetics involved showed that short pre‐exposure (0–12 h) to CHS 828 enhanced caspase activation by etoposide, while longer pre‐exposure (18–48 h) inhibited both caspase activation and apoptotic morphology otherwise induced by etoposide. The present results support the theory that CHS 828 block specific cell death pathways.

The synergistic results are promising for future combination trials in animals, however, different dosing schedules should be considered, in order to investigate whether the above findings translate into the in vivo setting.

British Journal of Pharmacology (2002) 137, 568–573. doi:10.1038/sj.bjp.0704888
Therapeutic effect of the endogenous fatty acid amide, palmitoylethanolamide, in rat acute inflammation: inhibition of nitric oxide and cyclo‐oxygenase systems
Authors: Costa Barbara , Conti Silvia , Giagnoni Gabriella , Colleoni Mariapia
Published: October 2002

The anti‐inflammatory activity of the endogenous fatty acid amide palmitoylethanolamide and its relationship to cyclo‐oxygenase (COX) activity, nitric oxide (NO) and oxygen free radical production were investigated in the rat model of carrageenan‐induced acute paw inflammation and compared with the nonsteroidal anti‐inflammatory drug (NSAID) indomethacin.

Palmitoylethanolamide (1, 3, 5, 10 mg kg−1; p.o.) and indomethacin (5 mg kg−1; p.o.) were administered daily after the onset of inflammation for three days and the paw oedema was measured daily; 24 h after the last dose (fourth day) the rats were killed and the COX activity and the content of nitrite/nitrate (NO2−/NO3−), malondialdehyde (MDA), endothelial and inducible nitric oxide synthase (eNOS and iNOS) were evaluated in the paw tissues.

Palmitoylethanolamide had a curative effect on inflammation, inhibiting the carrageenan‐induced oedema in a dose‐ and time‐dependent manner. This effect was not reversed by the selective CB2 receptor antagonist (N‐[(1S)‐endo‐1,3,3‐trimethylbicyclo[2.2.1]heptan‐2yl]‐5‐(4‐chloro‐3‐methylphenyl)‐1‐(4‐methylbenzyl)pyrazole‐3 carboxamide) (SR144528), 3 mg kg−1 p.o. On the fourth day after carrageenan injection, COX activity and the level of NO2−/NO3−, eNOS and MDA were increased in the inflamed paw, but iNOS was not present. Palmitoylethanolamide (10 mg kg−1) and indomethacin markedly reduced these increases.

Our findings show, for the first time, that palmitoylethanolamide has a curative effect in a model of acute inflammation. The inhibition of COX activity and of NO and free radical production at the site of inflammation might account for this activity.

British Journal of Pharmacology (2002) 137, 413–420. doi:10.1038/sj.bjp.0704900
Prediction of structure and function of G protein-coupled receptors
Authors: Vaidehi Nagarajan , Floriano Wely B. , Trabanino Rene , Hall Spencer E. , Freddolino Peter , Choi Eun Jung , Zamanakos Georgios , Goddard William A.
Published: October 2002

G protein-coupled receptors (GPCRs) mediate our sense of vision, smell, taste, and pain. They are also involved in cell recognition and communication processes, and hence have emerged as a prominent superfamily for drug targets. Unfortunately, the atomic-level structure is available for only one GPCR (bovine rhodopsin), making it difficult to use structure-based methods to design drugs and mutation experiments. We have recently developed first principles methods (MembStruk and HierDock) for predicting structure of GPCRs, and for predicting the ligand binding sites and relative binding affinities. Comparing to the one case with structural data, bovine rhodopsin, we find good accuracy in both the structure of the protein and of the bound ligand. We report here the application of MembStruk and HierDock to β1-adrenergic receptor, endothelial differential gene 6, mouse and rat I7 olfactory receptors, and human sweet receptor. We find that the predicted structure of β1-adrenergic receptor leads to a binding site for epinephrine that agrees well with the mutation experiments. Similarly the predicted binding sites and affinities for endothelial differential gene 6, mouse and rat I7 olfactory receptors, and human sweet receptor are consistent with the available experimental data. These predicted structures and binding sites allow the design of mutation experiments to validate and improve the structure and function prediction methods. As these structures are validated they can be used as targets for the design of new receptor-selective antagonists or agonists for GPCRs.
Mechanism of ABC transporters: A molecular dynamics simulation of a well characterized nucleotide-binding subunit
Authors: Jones Peter M. , George Anthony M.
Published: October 2002

ATP-binding cassette (ABC) transporters are membrane-bound molecular pumps that form one of the largest of all protein families. Several of them are central to phenomena of biomedical interest, including cystic fibrosis and resistance to chemotherapeutic drugs. ABC transporters share a common architecture comprising two hydrophilic nucleotide-binding domains (NBDs) and two hydrophobic transmembrane domains (TMDs) that form the substrate pathway across the membrane. The conformational changes in the NBDs induced by ATP hydrolysis and the means by which they are transmitted to the TMDs to effect substrate translocation remain largely unknown. We have performed a molecular dynamics simulation of HisP, the well studied NBD of the bacterial histidine permease, to identify hinges and switches of the NBD conformational transitions and subunit–subunit interfaces. This analysis reveals that the TMDs regulate ATP hydrolysis by controlling conformational transitions of the NBD helical domains, and identifies the conformational changes and the crucial TMD:NBD interface, by which the energy of ATP hydrolysis is transmitted to the TMDs. We also define the conformational transitions of the Q-loop, a key element of the NBD mechanism, and identify pathways by which Q-loop switching is coordinated with TMD and NBD conformational changes. We propose a model for the catalytic cycle of ABC transporters that shows how substrate-binding and transport by the TMDs may be coordinated and coupled with ATP binding and hydrolysis in the NBDs.
Very high growth rate chemical vapor deposition of single-crystal diamond
Authors: Yan Chih-shiue , Vohra Yogesh K. , Mao Ho-kwang , Hemley Russell J.
Published: October 2002

Diamond possesses extraordinary material properties, a result that has given rise to a broad range of scientific and technological applications. This study reports the successful production of high-quality single-crystal diamond with microwave plasma chemical vapor deposition (MPCVD) techniques. The diamond single crystals have smooth, transparent surfaces and other characteristics identical to that of high-pressure, high-temperature synthetic diamond. In addition, the crystals can be produced at growth rates from 50 to 150 μm/h, which is up to 2 orders of magnitude higher than standard processes for making polycrystalline MPCVD diamond. This high-quality single-crystal MPCVD diamond may find numerous applications in electronic devices as high-strength windows and in a new generation of high-pressure instruments requiring large single-crystal anvils.
Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins
Authors: Yabe Daisuke , Brown Michael S. , Goldstein Joseph L.
Published: October 2002

This paper describes insig-2, a second protein of the endoplasmic reticulum that blocks the processing of sterol regulatory element-binding proteins (SREBPs) by binding to SCAP (SREBP cleavage-activating protein) in a sterol-regulated fashion, thus preventing it from escorting SREBPs to the Golgi. By blocking this movement, insig-2, like the previously described insig-1, prevents the proteolytic processing of SREBPs by Golgi enzymes, thereby blocking cholesterol synthesis. The sequences of human insig-1 and -2 are 59% identical. Both proteins are predicted to contain six transmembrane helices. The proteins differ functionally in two respects: (
i
) production of insig-1, but not insig-2, in cultured mammalian cells requires nuclear SREBPs; and (
ii
) at high levels of expression, insig-1, but not insig-2, can block SCAP movement in the absence of exogenous sterols. The combined actions of insig-1 and -2 permit feedback regulation of cholesterol synthesis over a wide range of sterol concentrations.
Telomerase contributes to tumorigenesis by a telomere length-independent mechanism
Authors: Stewart Sheila A. , Hahn William C. , O'Connor Benjamin F. , Banner Elisa N. , Lundberg Ante S. , Modha Poonam , Mizuno Hana , Brooks Mary W. , Fleming Mark , Zimonjic Drazen B. , Popescu Nicholas C. , Weinberg Robert A.
Published: October 2002

Once immortalized, human cells are susceptible to transformation by introduction of an oncogene such as
ras
. Several lines of evidence now suggest that the maintenance of telomere length is a major determinant of replicative lifespan in human cells and thus of the immortalized state. The majority of human tumor cells acquire immortality through expression of the catalytic subunit of telomerase (hTERT), whereas others activate an alternative mechanism of telomere maintenance (ALT) that does not depend on the actions of telomerase. We have examined whether ALT could substitute for telomerase in the processes of transformation
in vitro
and tumorigenesis
in vivo
. Expression of oncogenic H-Ras in the immortal ALT cell line GM847 did not result in their transformation. However, subsequent ectopic expression of hTERT in these cells imparted a tumorigenic phenotype. Indeed, this outcome was also observed after introduction of a mutant hTERT that retained catalytic activity but was incapable of maintaining telomere length. These studies indicate that hTERT confers an additional function that is required for tumorigenesis but does not depend on its ability to maintain telomeres.
Molecular indetermination in the transition to error catastrophe: Systematic elimination of lymphocytic choriomeningitis virus through mutagenesis does not correlate linearly with large increases in mutant spectrum complexity
Authors: Grande-Pérez A. , Sierra S. , Castro M. G. , Domingo E. , Lowenstein P. R.
Published: October 2002

Studies with several RNA viruses have shown that enhanced mutagenesis resulted in decreases of infectivity or virus extinction, as predicted from virus entry into error catastrophe. Here we report that lymphocytic choriomeningitis virus, the prototype arenavirus, is extremely susceptible to extinction mutagenesis by the base analog 5-fluorouracil. Virus elimination was preceded by increases in complexity of the mutant spectra of treated populations. However, careful molecular comparison of the mutant spectra of several genomic segments suggests that the largest increases in mutation frequency do not predict virus extinction. Highly mutated viral genomes have escaped detection presumably because lymphocytic choriomeningitis virus replicates at or near the error threshold, and genomes in the transition toward error catastrophe may have an extremely short half-life and escape detection with state-of-the-art cloning and sequencing technologies.
A molecular profile of a hematopoietic stem cell niche
Authors: Hackney Jason A. , Charbord Pierre , Brunk Brian P. , Stoeckert Christian J. , Lemischka Ihor R. , Moore Kateri A.
Published: October 2002

The hematopoietic microenvironment provides a complex molecular milieu that regulates the self-renewal and differentiation activities of stem cells. We have characterized a stem cell supportive stromal cell line, AFT024, that was derived from murine fetal liver. Highly purified
in vivo
transplantable mouse stem cells are maintained in AFT024 cultures at input levels, whereas other primitive progenitors are expanded. In addition, human stem cells are very effectively supported by AFT024. We suggest that the AFT024 cell line represents a component of an
in vivo
stem cell niche. To determine the molecular signals elaborated in this niche, we undertook a functional genomics approach that combines extensive sequence mining of a subtracted cDNA library, high-density array hybridization and in-depth bioinformatic analyses. The data have been assembled into a biological process oriented database, and represent a molecular profile of a candidate stem cell niche.
Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains
Authors: Ho C. Kiong , Shuman Stewart
Published: October 2002

RNA ligases participate in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. Bacteriophage T4 RNA ligase (gp63) is the best-studied member of this class of enzymes, which includes yeast tRNA ligase and trypanosome RNA-editing ligases. Here, we identified another RNA ligase from the bacterial domain—a second RNA ligase (Rnl2) encoded by phage T4. Purified Rnl2 (gp24.1) catalyzes intramolecular and intermolecular RNA strand joining through ligase-adenylate and RNA-adenylate intermediates. Mutational analysis identifies amino acids required for the ligase-adenylation or phosphodiester synthesis steps of the ligation reaction. The catalytic residues of Rnl2 are located within nucleotidyl transferase motifs I, IV, and V that are conserved in DNA ligases and RNA capping enzymes. Rnl2 has scant amino acid similarity to T4 gp63. Rather, Rnl2 exemplifies a distinct ligase family, defined by variant motifs, that includes the trypanosome-editing ligases and a group of putative RNA ligases encoded by eukaryotic viruses (baculoviruses and an entomopoxvirus) and many species of archaea. These findings have implications for the evolution of covalent nucleotidyl transferases and virus-host dynamics based on RNA restriction and repair.
The natural center of chromaticity space is not always achromatic: A new look at color induction
Authors: Ekroll Vebjørn , Faul Franz , Niederée Reinhard , Richter Eike
Published: October 2002

Although current theories of color vision differ in many respects, they all assume the existence of a uniquely defined neutral point in chromaticity space. It generally is assumed that this point satisfies several criteria simultaneously. One of these criteria is that it is perceived as achromatic. A further criterion shared by most theories is the structural assumption that lines in chromaticity space of constant hue converge on the neutral point. The basic assumption that these two criteria coincide is clearly true for isolated spots of light presented in darkness, and it usually is taken for granted that this coincidence generalizes to more complex visual stimuli. Here, we show that this is not the case. Our experiments with infields in chromatic surrounds revealed that the point in chromaticity space that appears gray is clearly different from the point on which lines of constant hue converge. A plausible interpretation of this apparently paradoxical finding in terms of color scission is proposed.
The heterotrimeric G protein α subunit acts upstream of the small GTPase Rac in disease resistance of rice
Authors: Suharsono Utut , Fujisawa Yukiko , Kawasaki Tsutomu , Iwasaki Yukimoto , Satoh Hikaru , Shimamoto Ko
Published: October 2002

We used rice
dwarf1
(
d1
) mutants lacking a single-copy Gα gene and addressed Gα's role in disease resistance.
d1
mutants exhibited a highly reduced hypersensitive response to infection by an avirulent race of rice blast. Activation of
PR
gene expression in the leaves of the mutants infected with rice blast was delayed for 24 h relative to the wild type. H
2
O
2
production and
PR
gene expression induced by sphingolipid elicitors (SE) were strongly suppressed in
d1
cell cultures. Expression of the constitutively active OsRac1, a small GTPase Rac of rice, in
d1
mutants restored SE-dependent defense signaling and resistance to rice blast. Gα mRNA was induced by an avirulent race of rice blast and SE application on the leaf. These results indicated the role of Gα in
R
gene-mediated disease resistance of rice. We have proposed a model for the defense signaling of rice in which the heterotrimeric G protein functions upstream of the small GTPase OsRac1 in the early steps of signaling.
Topical colchicine selection of keratinocytes transduced with the multidrug resistance gene (
MDR1
) can sustain and enhance transgene expression
in vivo
Authors: Pfützner W. , Terunuma A. , Tock C. L. , Snead E. K. , Kolodka T. M. , Gottesman M. M. , Taichman L. , Vogel J. C.
Published: October 2002

For skin gene therapy, achieving prolonged high-level gene expression in a significant percentage of keratinocytes (KC) is difficult because we cannot selectively target KC stem cells. We now demonstrate that topical colchicine treatment can be used to select,
in vivo
, KC progenitor cells transduced with the multidrug resistance gene (
MDR1
). When human skin equivalents containing
MDR1
-transduced KC were grafted onto immunocompromised mice, topical colchicine treatments significantly increased (7-fold) the percentage of KC expressing MDR1, compared to vehicle-treated controls, for up to 24 wk. Topical colchicine treatment also significantly enhanced the amount of MDR1 protein expressed in individual KC. Furthermore, quantitative real-time PCR analysis of
MDR1
transgene copy number demonstrates that topical colchicine treatment selects and enriches for KC progenitor cells in the skin that contain and express MDR1. For clinical skin gene therapy applications, this
in vivo
selection approach promises to enhance both the duration and expression level of a desired therapeutic gene in KC, by linking its expression to the
MDR1
selectable marker gene.
Olfactory search at high Reynolds number
Authors: Balkovsky Eugene , Shraiman Boris I.
Published: October 2002

Locating the source of odor in a turbulent environment—a common behavior for living organisms—is nontrivial because of the random nature of mixing. Here we analyze the statistical physics aspects of the problem and propose an efficient strategy for olfactory search that can work in turbulent plumes. The algorithm combines the maximum likelihood inference of the source position with an active search. Our approach provides the theoretical basis for the design of olfactory robots and the quantitative tools for the analysis of the observed olfactory search behavior of living creatures (e.g., odor-modulated optomotor anemotaxis of moths).
Structures of bromoalkanes' photodissociation in solution by means of ultrafast extended x-ray absorption fine-structure spectroscopy
Authors: Oulianov D. A. , Tomov I. V. , Dvornikov A. S. , Rentzepis P. M.
Published: October 2002

The structures of initial and final products of bromoalkanes' photodisociation reaction in cyclohexane solution have been measured with a bond length accuracy of 0.02 Å by means of ultrafast time-resolved extended x-ray absorption fine structure spectroscopy. The photoredaction mechanism is also discussed.
Acceleration of genomic evolution caused by enhanced mutation rate in endocellular symbionts
Authors: Itoh Takeshi , Martin William , Nei Masatoshi
Published: October 2002

Endosymbionts, which are widely observed in nature, have undergone reductive genome evolution because of their long-term intracellular lifestyle. Here we compared the complete genome sequences of two different endosymbionts,Buchneraand a protist mitochondrion, with their close relatives to study the evolutionary rates of functional genes in endosymbionts. The results indicate that the rate of amino acid substitution is two times higher in symbionts than in their relatives. This rate increase was observed uniformly among different functional classes of genes, although strong purifying selection may have counterbalanced the rate increase in a few cases. Our data suggest that, contrary to current views, neither the Muller's ratchet effect nor the slightly deleterious mutation theory sufficiently accounts for the elevated evolutionary rate. Rather, the elevated evolutionary rate appears to be mainly due to enhanced mutation rate, although the possibility of relaxation of purifying selection cannot be ruled out.
Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis
Authors: Walker Kevin , Fujisaki Shingo , Long Robert , Croteau Rodney
Published: October 2002

The structural pharmacophore of Taxol, responsible for binding the N terminus of the β-subunit of tubulin to arrest cell proliferation, comprises, in part, the 13-
O
-(
N
-benzoyl-3-phenylisoserinoyl) side chain. To identify the side chain transferase of Taxol biosynthesis, a set of transacylases obtained from an enriched cDNA library (constructed from mRNA isolated from
Taxus cuspidata
cells induced with methyl jasmonate for Taxol production) was screened. A cDNA clone (designated TAX7) encoding a taxoid C-13
O
-phenylpropanoyltransferase was isolated which yielded a recombinant enzyme that catalyzes the selective 13-
O
-acylation of baccatin III with β-phenylalanoyl CoA as the acyl donor to form
N
-debenzoyl-2′-deoxytaxol. This enzymatic product was converted to 2′-deoxytaxol by chemical
N
-benzoylation, and the identity of this derivative was confirmed by spectrometric analyses. The full-length cDNA has an ORF of 1,335 bases and encodes a 445-aa protein with a calculated molecular weight of 50,546. Evaluation of kinetic parameters revealed
K
m
values of 2.4 ± 0.5 μM and 4.9 ± 0.3 μM for baccatin III and β-phenylalanoyl-CoA, respectively. The pH optimum for the recombinant
O
-(3-amino-3-phenylpropanoyl)transferase is at 6.8. Identification of this clone completes acquisition of the five aroyl/acyltransferases involved in the biosynthesis of Taxol. Application of these transacylase genes in suitable host cells can improve the production yields of Taxol and could enable the preparation of second-generation Taxol analogs possessing greater bioactivity and improved water solubility.
The Neandertal type site revisited: Interdisciplinary investigations of skeletal remains from the Neander Valley, Germany
Authors: Schmitz Ralf W. , Serre David , Bonani Georges , Feine Susanne , Hillgruber Felix , Krainitzki Heike , Pääbo Svante , Smith Fred H.
Published: October 2002

The 1856 discovery of the Neandertal type specimen (Neandertal 1) in western Germany marked the beginning of human paleontology and initiated the longest-standing debate in the discipline: the role of Neandertals in human evolutionary history. We report excavations of cave sediments that were removed from the Feldhofer caves in 1856. These deposits have yielded over 60 human skeletal fragments, along with a large series of Paleolithic artifacts and faunal material. Our analysis of this material represents the first interdisciplinary analysis of Neandertal remains incorporating genetic, direct dating, and morphological dimensions simultaneously. Three of these skeletal fragments fit directly on Neandertal 1, whereas several others have distinctively Neandertal features. At least three individuals are represented in the skeletal sample. Radiocarbon dates for Neandertal 1, from which a mtDNA sequence was determined in 1997, and a second individual indicate an age of ≈40,000 yr for both. mtDNA analysis on the same second individual yields a sequence that clusters with other published Neandertal sequences.
Physical and functional interactions of histone deacetylase 3 with TFII-I family proteins and PIASxβ
Authors: Tussié-Luna María Isabel , Bayarsaihan Dashzeveg , Seto Edward , Ruddle Frank H. , Roy Ananda L.
Published: October 2002

TFII-I family proteins are characterized structurally by the presence of multiple reiterated I-repeats, each containing a putative helix–loop–helix domain. Functionally, they behave as multifunctional transcription factors that are activated by a variety of extracellular signals. In studying their subcellular localization, we noticed that these transcription factors frequently reside in subnuclear domains/dots. Because nuclear dots are believed often to harbor components of histone deacetylase enzymes (HDACs), we investigated whether TFII-I family proteins colocalize and interact with HDACs. Here, we show that TFII-I and its related member hMusTRD1/BEN physically and functionally interact with HDAC3. The TFII-I family proteins and HDAC3 also show nearly identical expression patterns in early mouse development. Consistent with our earlier observation that TFII-I family proteins also interact with PIASxβ, a member of the E3 ligase family involved in the small ubiquitin-like modifier (SUMO) pathway, we show further that PIASxβ physically and functionally interacts with HDAC3 and relieves the transcriptional repression exerted by HDAC3 upon TFII-I-mediated gene activation. These results suggest a complex interplay between two posttranslational pathways—histone modification and SUMOylation—brokered in part by TFII-I family proteins.
Insect feeding mobilizes a unique plant defense protease that disrupts the peritrophic matrix of caterpillars
Authors: Pechan Tibor , Cohen Allen , Williams W. Paul , Luthe Dawn S.
Published: October 2002

Plants frequently respond to herbivorous insect attack by synthesizing defense proteins that deter insect feeding and prevent additional herbivory. Maize (
Zea mays
L.) lines, resistant to feeding by a number of lepidopteran species, rapidly mobilize a unique 33-kDa cysteine protease in response to caterpillar feeding. The accumulation of the 33-kDa cysteine protease in the maize mid-whorl was correlated with a significant reduction in caterpillar growth that resulted from impaired nutrient utilization. Black Mexican Sweetcorn callus transformed with
mir1
, the gene encoding the 33-kDa cysteine protease, expressed the protease and growth of caterpillars reared on the transgenic callus was reduced 60–80%. Scanning electron microscopy was used to examine the effect of plant material expressing the 33-kDa cysteine protease on the structure of the caterpillar peritrophic matrix. Because the peritrophic matrix surrounds the food bolus, assists in digestive processes, and protects the caterpillar midgut from physical and chemical damage, disruption of peritrophic matrix may reduce caterpillar growth. The results indicated that the peritrophic matrix was severely damaged when caterpillars fed on resistant maize plants or transgenic Black Mexican Sweetcorn. The accumulation of the 33-kDa cysteine protease in response to caterpillar feeding, and its ability to damage the insect peritrophic matrix, represents an unusual host–plant resistance mechanism that may have applications in agricultural biotechnology.
Functional correction of adult
mdx
mouse muscle using gutted adenoviral vectors expressing full-length dystrophin
Authors: DelloRusso Christiana , Scott Jeannine M. , Hartigan-O'Connor Dennis , Salvatori Giovanni , Barjot Catherine , Robinson Ann S. , Crawford Robert W. , Brooks Susan V. , Chamberlain Jeffrey S.
Published: October 2002

Duchenne muscular dystrophy is a lethal X-linked recessive disorder caused by mutations in the dystrophin gene. Delivery of functionally effective levels of dystrophin to immunocompetent, adult
mdx
(dystrophin-deficient) mice has been challenging because of the size of the gene, immune responses against viral vectors, and inefficient infection of mature muscle. Here we show that high titer stocks of three different gutted adenoviral vectors carrying full-length, muscle-specific, dystrophin expression cassettes are able to efficiently transduce muscles of 1-yr-old
mdx
mice. Single i.m. injections of viral vector restored dystrophin production to 25–30% of mouse limb muscle 1 mo after injection. Furthermore, functional tests of virally transduced muscles revealed almost 40% correction of their high susceptibility to contraction-induced injury. Our results show that functional abnormalities of dystrophic muscle can be corrected by delivery of full-length dystrophin to adult, immunocompetent
mdx
mice, raising the prospects for gene therapy of muscular dystrophies.
FlgM gains structure in living cells
Authors: Dedmon Matthew M. , Patel Chetan N. , Young Gregory B. , Pielak Gary J.
Published: October 2002

Intrinsically disordered proteins such as FlgM play important roles in biology, but little is known about their structure in cells. We use NMR to show that FlgM gains structure inside living
Escherichia coli
cell
s
and under physiologically relevant conditions
in vitro
, i.e., in solutions containing high concentrations (≥400 g/liter) of glucose, BSA, or ovalbumin. Structure formation represents solute-induced changes in the equilibrium between the structured and disordered forms of FlgM. The results provide insight into how the environment of intrinsically disordered proteins could dictate their structure and, in turn, emphasize the relevance of studying proteins in living cells and
in vitro
under physiologically realistic conditions.
Calpain as an effector of the Gq signaling pathway for inhibition of Wnt/β-catenin-regulated cell proliferation
Authors: Li Guangnan , Iyengar Ravi
Published: October 2002

Signaling pathways interact to integrate and regulate information flow in evoking complex cellular responses. We have studied the mechanisms and consequences of interactions between the Gq and Wnt/β-catenin pathways. In human colon carcinoma SW480 cells, activation of the Gq pathway inhibits β-catenin signaling as determined by transcriptional reporter and cell proliferation assays. Ca
2+
release from internal stores results in nuclear export and calpain-mediated degradation of β-catenin in the cytoplasm. Gαq does not inhibit the effects of constitutively activated ΔN-XTCF3-VP16 chimera in SW480 cells. Similarly, in HEK293 cells the Gq pathway suppresses β-catenin–T cell factor/lymphocyte enhancer factor-1 transcriptional activity induced by Wnt/Frizzled interaction or glycogen synthase kinase-3β-resistant β-catenin, but not ΔN-XTCF3-VP16. We conclude that Gq signaling promotes nuclear export and calpain-mediated degradation of β-catenin, which therefore contributes to the inhibition of Wnt/β-catenin pathway.
Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu
Authors: Xu Wanping , Marcu Monica , Yuan Xitong , Mimnaugh Edward , Patterson Cam , Neckers Len
Published: October 2002

Overexpression of the transmembrane receptor tyrosine kinase ErbB2 is common in multiple malignancies, including breast and ovarian cancer. ErbB2 is resistant to degradation mediated by c-Cbl, the E3 ubiquitin ligase responsible for ligand-induced ubiquitination of ErbB1 (epidermal growth factor receptor). Because of its resistance to degradation, ErbB2 is the preferred dimerization partner for other members of the ErbB family, and its overexpression
in vivo
is associated with poor prognosis. We now show that the chaperone-binding ubiquitin ligase CHIP efficiently ubiquitinates and down-regulates ErbB2. CHIP expression shortens the half-life of both nascent and mature ErbB2 protein.
In vitro
ubiquitination assay shows that CHIP serves as a ubiquitin ligase for ErbB2, and both exogenously expressed and endogenous CHIP coprecipitate with the kinase. Furthermore, CHIP association with ErbB2 requires a chaperone intermediate and is increased by the chaperone-binding drug geldanamycin, a potent stimulator of ErbB2 ubiquitination and degradation. These data describe a previously unrecognized pathway, amenable to pharmacologic manipulation, that mediates ErbB2 stability.
Perinatal switch from synchronous to asynchronous activity of motoneurons: Link with synapse elimination
Authors: Buffelli Mario , Busetto Giuseppe , Cangiano Lorenzo , Cangiano Alberto
Published: October 2002

Synaptic competition is a basic feature of developing neural connections. To shed light on its dependence on the activity pattern of competing inputs, we investigated
in vivo
rat motoneuronal firing during late embryonic and early neonatal life, when synapse elimination occurs in muscle. Electromyographic recordings with floating microelectrodes from tibialis anterior and soleus muscles revealed that action potentials of motoneurons belonging to the same pool have high temporal correlation. The very tight linkage, a few tens of milliseconds, corresponds to the narrow time windows of published paradigms of activity-dependent synaptic plasticity. A striking change occurs, however, soon after birth when motoneuronal firing switches to the adult uncorrelated type. The switch precedes the onset of synapse elimination, whose time course was determined with confocal microscopy. Interestingly, the soleus muscle, whose motoneurons switch to desynchronized activity later than those of the tibialis anterior muscle, also exhibits delayed synapse elimination. Our findings support a developmental model in which synchronous activity first favors polyneuronal innervation, whereas an asynchronous one subsequently promotes synapse elimination.
Development of
17
O NMR approach for fast imaging of cerebral metabolic rate of oxygen in rat brain at high field
Authors: Zhu Xiao-Hong , Zhang Yi , Tian Run-Xia , Lei Hao , Zhang Nanyin , Zhang Xiaoliang , Merkle Hellmut , Ugurbil Kamil , Chen Wei
Published: October 2002

A comprehensive technique was developed for using three-dimensional
17
O magnetic resonance spectroscopic imaging at 9.4T for rapidly imaging the cerebral metabolic rate of oxygen consumption (CMRO
2
) in the rat brain during a two-min inhalation of
17
O
2
. The CMRO
2
value (2.19 ± 0.14 μmol/g/min,
n
= 7) was determined in the rat anesthetized with α-chloralose by independent and concurrent
17
O NMR measurements of cerebral H
2
17
O content, arterial input function, and cerebral perfusion. CMRO
2
values obtained were consistent with the literature results for similar conditions. Our results reveal that, because of its superior sensitivity at ultra-high fields, the
17
O magnetic resonance spectroscopic imaging approach is capable of detecting small dynamic changes of metabolic H
2
17
O during a short inhalation of
17
O
2
gas, and ultimately, for imaging CMRO
2
in the small rat brain. This study provides a crucial step toward the goal of developing a robust and noninvasive
17
O NMR approach for imaging CMRO
2
in animal and human brains that can be used for studying the central role of oxidative metabolism in brain function under normal and diseased conditions, as well as for understanding the mechanisms underlying functional MRI.
The hypocholesterolemic agent LY295427 up-regulates INSIG-1, identifying the INSIG-1 protein as a mediator of cholesterol homeostasis through SREBP
Authors: Janowski Bethany A.
Published: October 2002

Oxysterols regulate cholesterol homeostasis through liver X receptor (LXR; cholesterol-lowering)- and sterol regulatory element-binding protein (SREBP; cholesterol-raising)-mediated signaling pathways. Previously we reported that the hypocholesterolemic agent LY295427 (4α-allylcholestan-3α-ol) reverses oxysterol-mediated suppression of SREBP processing. We now report that LY295427 increases expression of insulin-induced gene-1 (INSIG-1) and restores SREBP processing in cells treated with oxysterols. In cells overexpressing the INSIG-1 gene, by contrast, SREBP processing is suppressed and oxysterol regulation is disrupted. SREBP processing is not restored by addition of LY295427, but is restored by increasing the levels of SREBP cleavage-activating protein (SCAP). These findings suggest that the INSIG-1 protein alters sterol balance by modulating SREBP processing jointly with SCAP. To test whether the action of oxysterols on SREBP processing is mediated through endogenous INSIG-1 protein, we used RNAi to lower the expression of the INSIG-1 gene, and found that reduced INSIG-1 protein levels caused the loss of SREBP regulation by oxysterols. We conclude that: (
i
) INSIG-1 gene expression is suppressed by oxysterols; (
ii
) LY295427 treatment counters the suppressive effects of oxysterols on SREBP processing, resulting in the expression of the INSIG-1 gene; and (
iii
) INSIG-1 gene expression affects SREBP processing. Taken together, these data suggest that INSIG-1 plays a critical role in regulating cholesterol concentrations in the cell.
Mitochondrial respiratory control is lost during growth factor deprivation
Authors: Gottlieb Eyal , Armour Sean M. , Thompson Craig B.
Published: October 2002

The ability of cells to maintain a bioenergetically favorable ATP/ADP ratio confers a tight balance between cellular events that consume ATP and the rate of ATP production. However, after growth factor withdrawal, the cellular ATP/ADP ratio declines. To investigate these changes, mitochondria from growth factor-deprived cells isolated before the onset of apoptosis were characterized
in vitro
. Mitochondria from growth factor-deprived cells have lost their ability to undergo matrix condensation in response to ADP, which is accompanied by a failure to perform ADP-coupled respiration. At the time of analysis, mitochondria from growth factor-deprived cells were not depleted of cytochrome
c
and cytochrome
c
-dependent respiration was unaffected, demonstrating that the inhibition of the respiratory rate is not due to loss of cytochrome
c
. Agents that disrupt the mitochondrial outer membrane, such as digitonin, or maintain outer membrane exchange of adenine nucleotide, such as Bcl-x
L
, restored ADP-dependent control of mitochondrial respiration. Together, these data suggest that the regulation of mitochondrial outer membrane permeability contributes to respiratory control.
Quantitative imaging of cis-regulatory reporters in living embryos
Authors: Dmochowski Ivan J. , Dmochowski Jane E. , Oliveri Paola , Davidson Eric H. , Fraser Scott E.
Published: October 2002

A confocal laser scanning microscopy method has been developed for the quantitation of green fluorescent protein (GFP) as a reporter of gene activity in living three-dimensional structures such as sea urchin and starfish embryos. This method is between 2 and 50 times more accurate than conventional confocal microscopy procedures depending on the localization of GFP within an embryo. By using coinjected Texas red dextran as an internal fluorescent standard, the observed GFP intensity is corrected for variations in laser excitation and fluorescence collection efficiency. To relate the recorded image intensity to the number of GFP molecules, the embryos were lysed gently, and a fluorometric analysis of their contents was performed. Confocal laser scanning microscopy data collection from a single sea urchin blastula required less than 2 min, thereby allowing gene expression in dozens of embryos to be monitored in parallel with high spatial and temporal resolution.
Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers
Authors: Picart C. , Mutterer J. , Richert L. , Luo Y. , Prestwich G. D. , Schaaf P. , Voegel J.-C. , Lavalle P.
Published: October 2002

The structure of poly(
l
-lysine) (PLL)/hyaluronan (HA) polyelectrolyte multilayers formed by electrostatic self-assembly is studied by using confocal laser scanning microscopy, quartz crystal microbalance, and optical waveguide lightmode spectroscopy. These films exhibit an exponential growth regime where the thickness increases exponentially with the number of deposited layers, leading to micrometer thick films. Previously such a growth regime was suggested to result from an “in” and “out” diffusion of the PLL chains through the film during buildup, but direct evidence was lacking. The use of dye-conjugated polyelectrolytes now allows a direct three-dimensional visualization of the film construction by introducing fluorescent polyelectrolytes at different steps during the film buildup. We find that, as postulated, PLL diffuses throughout the film down into the substrate after each new PLL injection and out of the film after each PLL rinsing and further after each HA injection. As PLL reaches the outer layer of the film it interacts with the incoming HA, forming the new HA/PLL layer. The thickness of this new layer is thus proportional to the amount of PLL that diffuses out of the film during the buildup step, which explains the exponential growth regime. HA layers are also visualized but no diffusion is observed, leading to a stratified film structure. We believe that such a diffusion-based buildup mechanism explains most of the exponential-like growth processes of polyelectrolyte multilayers reported in the literature.
Autologous transplantation of genetically modified iris pigment epithelial cells: A promising concept for the treatment of age-related macular degeneration and other disorders of the eye
Authors: Semkova Irina , Kreppel Florian , Welsandt Gerhard , Luther Thomas , Kozlowski Jolanta , Janicki Hanna , Kochanek Stefan , Schraermeyer Ulrich
Published: October 2002

Age-related macular degeneration (ARMD) is the leading cause for visual impairment and blindness in the elder population. Laser photocoagulation, photodynamic therapy and excision of neovascular membranes have met with limited success. Submacular transplantation of autologous iris pigment epithelial (IPE) cells has been proposed to replace the damaged retinal pigment epithelium following surgical removal of the membranes. We tested our hypothesis that the subretinal transplantation of genetically modified autologous IPE cells expressing biological therapeutics might be a promising strategy for the treatment of ARMD and other retinal disorders. Pigment epithelium-derived factor (PEDF) has strong antiangiogenic and neuroprotective activities in the eye. Subretinal transplantation of PEDF expressing IPE cells inhibited pathological choroidal neovascularization in rat models of laser-induced rupture of Bruch's membrane and of oxygen induced ischemic retinopathy. PEDF expressing IPE transplants also increased the survival and preserved rhodopsin expression of photoreceptor cells in the RCS rat, a model of retinal degeneration. These findings suggest a promising concept for the treatment of ARMD and other retinal disorders.
A signal transduction protein cues proteolytic events critical to
Caulobacter
cell cycle progression
Authors: Hung Dean Y. , Shapiro Lucy
Published: October 2002

Temporally controlled proteolysis of the essential response regulator, CtrA, is critical for cell cycle progression in
Caulobacter crescentus
. CtrA binds to and silences the origin of replication in swarmer cells. The initiation of replication depends on the proteolysis of CtrA. We present evidence that DivK, an essential single-domain response regulator, contributes to the control of the G
1
–S transition by signaling the temporally controlled proteolysis of CtrA. In a
divK-cs
mutant at the restrictive temperature, the initiation of DNA replication is blocked because of the retention of CtrA. A shift of cells from restrictive to permissive temperature results in rapid degradation of CtrA, initiation of DNA replication, and the resumption of cell cycle progression, including the ordered expression of genes involved in chromosome replication and polar organelle biogenesis. CtrA binds to and regulates the promoters of two genes critical to its temporally controlled proteolysis,
divK
and
clpP
, providing a transcriptional feedback loop for the control of cell cycle progression.
Kir6.2 is required for adaptation to stress
Authors: Zingman Leonid V. , Hodgson Denice M. , Bast Peter H. , Kane Garvan C. , Perez-Terzic Carmen , Gumina Richard J. , Pucar Darko , Bienengraeber Martin , Dzeja Petras P. , Miki Takashi , Seino Susumu , Alekseev Alexey E. , Terzic Andre
Published: October 2002

Reaction to stress requires feedback adaptation of cellular functions to secure a response without distress, but the molecular order of this process is only partially understood. Here, we report a previously unrecognized regulatory element in the general adaptation syndrome. Kir6.2, the ion-conducting subunit of the metabolically responsive ATP-sensitive potassium (K
ATP
) channel, was mandatory for optimal adaptation capacity under stress. Genetic deletion of Kir6.2 disrupted K
ATP
channel-dependent adjustment of membrane excitability and calcium handling, compromising the enhancement of cardiac performance driven by sympathetic stimulation, a key mediator of the adaptation response. In the absence of Kir6.2, vigorous sympathetic challenge caused arrhythmia and sudden death, preventable by calcium-channel blockade. Thus, this vital function identifies a physiological role for K
ATP
channels in the heart.