bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis and biological activities of potent peptidomimetics selective for somatostatin receptor subtype 2
Authors: Yang Lihu , Berk Scott C. , Rohrer Susan P. , Mosley Ralph T. , Guo Liangqin , Underwood Dennis J. , Arison Byron H. , Birzin Elizabeth T. , Hayes Edward C. , Mitra Sudha W. , Parmar Rupa M. , Cheng Kang , Wu Tsuei-Ju , Butler Bridgette S. , Foor Forrest , Pasternak Alexander , Pan Yanping , Silva Maria , Freidinger Roger M. , Smith Roy G. , Chapman Kevin , Schaeffer James M. , Patchett Arthur A.
Published: July 2002

A series of nonpeptide somatostatin agonists which bind selectively and with high affinity to somatostatin receptor subtype 2 (sst2) have been synthesized. One of these compounds, L-054,522, binds to human sst2 with an apparent dissociation constant of 0.01 nM and at least 3,000-fold selectivity when evaluated against the other somatostatin receptors. L-054,522 is a full agonist based on its inhibition of forskolin-stimulated adenylate cyclase activity in Chinese hamster ovary-K1 cells stably expressing sst2. L-054,522 has a potent inhibitory effect on growth hormone release from rat primary pituitary cells and glucagon release from isolated mouse pancreatic islets. Intravenous infusion of L-054,522 to rats at 50 μg/kg per hr causes a rapid and sustained reduction in growth hormone to basal levels. The high potency and selectivity of L-054,522 for sst2 will make it a useful tool to further characterize the physiological functions of this receptor subtype.
Task-dependent influences of attention on the activation of human primary visual cortex
Authors: Watanabe Takeo , Harner Alexander M. , Miyauchi Satoru , Sasaki Yuka , Nielsen Matthew , Palomo Daniel , Mukai Ikuko
Published: July 2002

There has been a good deal of controversy over whether attention influences area V1—the first cortical area onto which information from the retina is projected. Attention to motion has been found to modulate monkey area MT and the human homolog of MT/MST. Here we show that activation of V1 by attention to motion is task dependent. Our stimulus consisted of a group of translating random dots superimposed over another group of random dots executing expansion motion. Subjects were instructed to pay attention selectively to the translation, expansion, or neither in particular (passive condition). The activity in the human MT/MST homolog measured by functional magnetic resonance imaging (fMRI) was significantly higher in both the translation and the expansion conditions than in the passive condition, while the activity in area V1 was significantly higher only in the translation condition. These results show that attention to motion modulates area V1, and more interestingly that high-level cognitive processing such as attention may directly or indirectly determine the retroactive extent of feedback within the motion pathway in a manner dependent on the type of motion attended.
Evolution of dendritic patterns during alloy solidification: From the initial instability to the steady state
Authors: Losert W. , Shi B. Q. , Cummins H. Z.
Published: July 2002

The evolution of the crystal–melt interface was investigated during directional solidification of a dilute binary alloy, starting at the marginal stability time
t
i
at which the planar interface first becomes unstable. The time delay between
t
i
and the crossover time
t
0
at which the interface modulation becomes observable was determined experimentally. The interface morphology was analyzed as the cellular pattern appeared, and it was followed through the coarsening phase to the final steady-state dendritic pattern. The relevance of the initial instability for steady-state pattern selection was verified experimentally, and some aspects of the coarsening dynamics were measured and compared with theoretical predictions of Warren and Langer [Warren, J. A. & Langer, J. S. (1990)
Phys. Rev. A
42, 3518–3525; Warren, J. A. & Langer, J. S. (1993)
Phys. Rev. E
47, 2702–2712].
Crystal structure of the BTB domain from PLZF
Authors: Ahmad K. Farid , Engel Christian K. , Privé Gilbert G.
Published: July 2002

The BTB domain (also known as the POZ domain) is an evolutionarily conserved protein–protein interaction motif found at the N terminus of 5–10% of C
2
H
2
-type zinc-finger transcription factors, as well as in some actin-associated proteins bearing the kelch motif. Many BTB proteins are transcriptional regulators that mediate gene expression through the control of chromatin conformation. In the human promyelocytic leukemia zinc finger (PLZF) protein, the BTB domain has transcriptional repression activity, directs the protein to a nuclear punctate pattern, and interacts with components of the histone deacetylase complex. The association of the PLZF BTB domain with the histone deacetylase complex provides a mechanism of linking the transcription factor with enzymatic activities that regulate chromatin conformation. The crystal structure of the BTB domain of PLZF was determined at 1.9 Å resolution and reveals a tightly intertwined dimer with an extensive hydrophobic interface. Approximately one-quarter of the monomer surface area is involved in the dimer intermolecular contact. These features are typical of obligate homodimers, and we expect the full-length PLZF protein to exist as a branched transcription factor with two C-terminal DNA-binding regions. A surface-exposed groove lined with conserved amino acids is formed at the dimer interface, suggestive of a peptide-binding site. This groove may represent the site of interaction of the PLZF BTB domain with nuclear corepressors or other nuclear proteins.
A DNA damage and stress inducible G protein-coupled receptor blocks cells in G2/M
Authors: Weng Zhigang , Fluckiger Anne-Catherine , Nisitani Sazuku , Wahl Matthew I. , Le Lu Q. , Hunter Charity A. , Fernal Anthony A. , Le Beau Michelle M. , Witte Owen N.
Published: July 2002

Cell cycle progression is monitored by highly coordinated checkpoint machinery, which is activated to induce cell cycle arrest until defects like DNA damage are corrected. We have isolated an anti-proliferative cell cycle regulator named G2A (for G2accumulation), which is predominantly expressed in immature T and B lymphocyte progenitors and is a member of the seven membrane-spanning G protein-coupled receptor family. G2A overexpression attenuates the transformation potential of BCR-ABL and other oncogenes, and leads to accumulation of cells at G2/M independently of p53 and c-Abl. G2A can be induced in lymphocytes and to a lesser extent in nonlymphocyte cell lines or tissues by multiple stimuli including different classes of DNA-damaging agents and serves as a response to damage and cellular stimulation which functions to slow cell cycle progression.
The optimization principle in phylogenetic analysis tends to give incorrect topologies when the number of nucleotides or amino acids used is small
Authors: Nei Masatoshi , Kumar Sudhir , Takahashi Kei
Published: July 2002

In the maximum parsimony (MP) and minimum evolution (ME) methods of phylogenetic inference, evolutionary trees are constructed by searching for the topology that shows the minimum number of mutational changes required
(M)
and the smallest sum of branch lengths
(S)
, respectively, whereas in the maximum likelihood (ML) method the topology showing the highest maximum likelihood
(A)
of observing a given data set is chosen. However, the theoretical basis of the optimization principle remains unclear. We therefore examined the relationships of
M
,
S
, and
A
for the MP, ME, and ML trees with those for the true tree by using computer simulation. The results show that
M
and
S
are generally greater for the true tree than for the MP and ME trees when the number of nucleotides examined (
n
) is relatively small, whereas
A
is generally lower for the true tree than for the ML tree. This finding indicates that the optimization principle tends to give incorrect topologies when
n
is small. To deal with this disturbing property of the optimization principle, we suggest that more attention should be given to testing the statistical reliability of an estimated tree rather than to finding the optimal tree with excessive efforts. When a reliability test is conducted, simplified MP, ME, and ML algorithms such as the neighbor-joining method generally give conclusions about phylogenetic inference very similar to those obtained by the more extensive tree search algorithms.
Folding and aggregation of designed proteins
Authors: Broglia R. A. , Tiana G. , Pasquali S. , Roman H. E. , Vigezzi E.
Published: July 2002

Protein aggregation is studied by following the simultaneous folding of two designed identical 20-letter amino acid chains within the framework of a lattice model and using Monte Carlo simulations. It is found that protein aggregation is determined by elementary structures (partially folded intermediates) controlled by local contacts among some of the most strongly interacting amino acids and formed at an early stage in the folding process.
PCR-based subtractive hybridization and differences in gene content among strains of
Helicobacter pylori
Authors: Akopyants Natalia S. , Fradkov Arkady , Diatchenko Luda , Hill Jason E. , Siebert Paul D. , Lukyanov Sergey A. , Sverdlov Eugene D. , Berg Douglas E.
Published: July 2002

Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen
Helicobacter pylori
. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated
H. pylori
strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the
H. pylori
population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.
Retinoic acid alters the intracellular trafficking of the mannose-6-phosphate/insulin-like growth factor II receptor and lysosomal enzymes
Authors: Kang Jing X. , Bell Jennifer , Leaf Alexander , Beard Richard L. , Chandraratna Roshantha A. S.
Published: July 2002

Previously, we showed that retinoic acid (RA) binds to the mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) with high affinity, suggesting that M6P/IGF2R may be a receptor for RA. Here, we show that RA, after 2–3 h of incubation with cultured neonatal-rat cardiac fibroblasts, dramatically alters the intracellular distribution of M6P/IGF2R as well as that of cathepsin B (a lysosomal protease bearing M6P). Immunofluorescence techniques indicate that this change in intracellular distribution is characterized by a shift of the proteins from the perinuclear area to cytoplasmic vesicles. The effect of RA was neither blocked by an RA nuclear receptor antagonist (AGN193109) nor mimicked by a selective RA nuclear-receptor agonist (TTNPB). Furthermore, the RA-induced translocation of cathepsin B was not observed in M6P/IGF2R-deficient P388D1 cells but occurred in stably transfected P388D1 cells expressing the receptor, suggesting that the effect of RA might be the result of direct interaction with M6P/IGF2R, rather than the result of binding to the nuclear receptors. These observations not only support the idea that M6P/IGF2R mediates an RA-response pathway but also indicate a role for RA in control of intracellular trafficking of lysosomal enzymes. Therefore, our observations may have important implications for the understanding of the diverse biological effects of retinoids.
Ribosomes can slide over and beyond “hungry” codons, resuming protein chain elongation many nucleotides downstream
Authors: Gallant Jonathan A. , Lindsley Dale
Published: July 2002

In cells subjected to moderate aminoacyl-tRNA limitation, the peptidyl-tRNA–ribosome complex stalled at the “hungry” codon can slide well beyond it on the messenger RNA and resume translation further downstream. This behavior is proved by unequivocal amino acid sequence data, showing a protein that lacks the bypassed sequence encoded between the hungry codon and specific landing sites. The landing sites are codons cognate to the anticodon of the peptidyl-tRNA. The efficiency of this behavior can be as high as 10–20% but declines with the length of the slide. Interposition of “trap” sites (nonproductive landing sites) in the bypassed region reduces the frequency of successful slides, confirming that the ribosome–peptidyl-tRNA complex passes through the untranslated region of the message. This behavior appears to be quite general: it can occur at the two kinds of hungry codons tested, AUA and AAG; the sliding peptidyl-tRNA can be any of three species tested, phenylalanine, tyrosine, or leucine tRNA; the peptidyl component can be either of two very different peptide sequences; and translation can resume at any of the three codons tested.
Inputs to directionally selective simple cells in macaque striate cortex
Authors: De Valois Russell L. , Cottaris Nicolas P.
Published: July 2002

It is clear that the initial analysis of visual motion takes place in the striate cortex, where directionally selective cells are found that respond to local motion in one direction but not in the opposite direction. Widely accepted motion models postulate as inputs to directional units two or more cells whose spatio-temporal receptive fields (RFs) are approximately 90° out of phase (quadrature) in space and in time. Simple cells in macaque striate cortex differ in their spatial phases, but evidence is lacking for the varying time delays required for two inputs to be in temporal quadrature. We examined the space-time RF structure of cells in macaque striate cortex and found two subpopulations of (nondirectional) simple cells, some that show strongly biphasic temporal responses, and others that are weakly biphasic if at all. The temporal impulse responses of these two classes of cells are very close to 90° apart, with the strongly biphasic cells having a shorter latency than the weakly biphasic cells. A principal component analysis of the spatio-temporal RFs of directionally selective simple cells shows that their RFs could be produced by a linear combination of two components; these two components correspond closely in their respective latencies and biphasic characters to those of strongly biphasic and weakly biphasic nondirectional simple cells, respectively. This finding suggests that the motion system might acquire the requisite temporal quadrature by combining inputs from these two classes of nondirectional cells (or from their respective lateral geniculate inputs, which appear to be from magno and parvo lateral geniculate cells, respectively).
Double-stranded RNA induces mRNA degradation inTrypanosomabrucei
Authors: Ngô Huân , Tschudi Christian , Gull Keith , Ullu Elisabetta
Published: July 2002

Double-stranded RNA (dsRNA) recently has been shown to give rise to genetic interference inCaenorhabditis elegansand also is likely to be the basis for phenotypic cosuppression in plants in certain instances. While constructing a plasmid vector for transfection of trypanosome cells, we serendipitously discovered thatin vivoexpression of dsRNA of the α-tubulin mRNA 5′ untranslated region (5′ UTR) led to multinucleated cells with striking morphological alterations and a specific block of cytokinesis. Transfection of synthetic α-tubulin 5′ UTR dsRNA, but not of either strand individually, caused the same phenotype. On dsRNA transfection, tubulin mRNA, but not the corresponding pre-mRNA, was rapidly and specifically degraded, leading to a deficit of α-tubulin synthesis. The transfected cells were no longer capable of carrying out cytokinesis and eventually died. Analysis of cytoskeletal structures from these trypanosomes revealed defects in the microtubules of the flagellar axoneme and of the flagellar attachment zone, a complex cortical structure that we propose is essential for establishing the path of the cleavage furrow at cytokinesis. Last, dsRNA-mediated mRNA degradation is not restricted to α-tubulin mRNA but can be applied to other cellular mRNAs, thus establishing a powerful tool to genetically manipulate these important protozoan parasites.
Structure of a biological oxygen sensor: A new mechanism for heme-driven signal transduction
Authors: Gong Weimin , Hao Bing , Mansy Sheref S. , Gonzalez Gonzalo , Gilles-Gonzalez Marie A. , Chan Michael K.
Published: July 2002

The FixL proteins are biological oxygen sensors that restrict the expression of specific genes to hypoxic conditions. FixL’s oxygen-detecting domain is a heme binding region that controls the activity of an attached histidine kinase. The FixL switch is regulated by binding of oxygen and other strong-field ligands. In the absence of bound ligand, the heme domain permits kinase activity. In the presence of bound ligand, this domain turns off kinase activity. Comparison of the structures of two forms of the
Bradyrhizobium japonicum
FixL heme domain, one in the “on” state without bound ligand and one in the “off” state with bound cyanide, reveals a mechanism of regulation by a heme that is distinct from the classical hemoglobin models. The close structural resemblance of the FixL heme domain to the photoactive yellow protein confirms the existence of a PAS structural motif but reveals the presence of an alternative regulatory gateway.
X-ray-induced mutations in mouse embryonic stem cells
Authors: Thomas James W. , LaMantia Christian , Magnuson Terry
Published: July 2002

Deletion complexes consisting of multiple chromosomal deletions induced at single loci can provide a means for functional analysis of regions spanning several centimorgans in model genetic systems. A strategy to identify and map deletions at any cloned locus in the mouse is described here. First, a highly polymorphic, germ-line competent F
1(129/Sv-+

Tyr

+

p

× CAST/Ei)
mouse embryonic stem cell line was established. Then, x-ray and UV-induced mutagenesis was performed to determine the feasibility of generating deletion complexes throughout the mouse genome. Reported here are the selection protocols, induced mutation frequencies, cytogenetic and extensive molecular analysis of mutations at the X-chromosome-linked hypoxanthine phosphoribosyltransferase (
Hprt
) locus and at the neural cell adhesion molecule (
Ncam
) locus located on chromosome 9. Mutation analysis with PCR-based polymorphic microsatellite markers revealed deletions of <3 cM at the
Hprt
locus, whereas results consistent with deletions covering >28 cM were observed at the
Ncam
locus. Fluorescence
in situ
hybridization with a chromosome 9 paint revealed that some of the
Ncam
deletions were accompanied by complex chromosome rearrangements. In addition, deletion mapping in combination with loss of heterozygosity of microsatellite markers revealed a putative haploinsufficient region distal to
Ncam
. These data indicate that it is feasible to generate x-ray-induced deletion complexes in mouse embryonic stem cells.
Prostate stem cell antigen: A cell surface marker overexpressed in prostate cancer
Authors: Reiter Robert E. , Gu Zhennen , Watabe Tetsuro , Thomas George , Szigeti Kinga , Davis Elizabeth , Wahl Matthew , Nisitani Sazuku , Yamashiro Joyce , Le Beau Michelle M. , Loda Massimo , Witte Owen N.
Published: July 2002

The identification of cell surface antigens is critical to the development of new diagnostic and therapeutic modalities for the management of prostate cancer. Prostate stem cell antigen (PSCA) is a prostate-specific gene with 30% homology to stem cell antigen 2, a member of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA encodes a 123-aa protein with an amino-terminal signal sequence, a carboxyl-terminal GPI-anchoring sequence, and multiple N-glycosylation sites. PSCA mRNA expression is prostate-specific in normal male tissues and is highly up-regulated in both androgen-dependent and -independent prostate cancer xenografts.
In situ
mRNA analysis localizes PSCA expression in normal prostate to the basal cell epithelium, the putative stem cell compartment of the prostate. There is moderate to strong PSCA expression in 111 of 126 (88%) prostate cancer specimens examined by
in situ
analysis, including high-grade prostatic intraepithelial neoplasia and androgen-dependent and androgen-independent tumors. Flow cytometric analysis demonstrates that PSCA is expressed predominantly on the cell surface and is anchored by a GPI linkage. Fluorescent
in situ
hybridization analysis localizes the PSCA gene to chromosome 8q24.2, a region of allelic gain in more than 80% of prostate cancers. A mouse homologue with 70% amino acid identity and similar genomic organization to human PSCA has also been identified. These results support PSCA as a target for prostate cancer diagnosis and therapy.
Mapping a disease locus by allelic association
Authors: Collins A. , Morton N. E.
Published: July 2002

Allelic association provides a means to map disease genes that, in a dense map of polymorphic markers, has considerably higher resolution than linkage methods. We describe here a composite likelihood estimate of location for a disease gene against a high-resolution marker map by using allele frequencies at linked loci. Data may be family-based, as in the transmission disequilibrium test, or from a case-control study. χ
2
tests, logarithm of odds, standard errors, and information weights are provided. The method is illustrated by analysis of published cystic fibrosis haplotypes, in which ΔF508 is more accurately localized than by other association studies. This differs from current approaches by adopting a more general Malecot model for isolation by distance, where distance here is between marker and disease locus, allowance for errors in the map and model, and freedom from assumptions about demography, systematic pressures, and the ratio of physical to genetic distance. When these assumptions are introduced the number of generations since the original mutation may be estimated, but this is not required to determine location and its standard error, so that evidence from allelic association may be efficiently combined with linkage evidence to identify a region for positional cloning of a disease gene.
A pineal regulatory element (PIRE) mediates transactivation by the pineal/retina-specific transcription factor CRX
Authors: Li Xiaodong , Chen Shiming , Wang Qingliang , Zack Donald J. , Snyder Solomon H. , Borjigin Jimo
Published: July 2002

The circadian hormone melatonin is synthesized predominantly in the pineal gland by the actions of two pineal-specific enzymes: serotonin
N
-acetyltransferase (NAT) and hydroxyindole-
O
-methyltransferase (HIOMT). Pineal night-specific ATPase (PINA), another pineal- and night-specific protein we recently identified, is produced as a truncated form of the Wilson disease gene (
Atp7b
) product. To identify the regulatory elements required for pineal-specific gene expression, we isolated sequences upstream of the rat PINA gene and discovered a cis-acting element that is recognized by a novel pineal/retina-specific nuclear factor. This pineal regulatory element (PIRE) has a consensus of TAATC/T and is present in six copies in the 5′ regulatory region of the PINA gene, at least three copies in the rat NAT promoter, and at least one copy in each of the putative HIOMT promoters A and B. A recently identified retina-specific protein, cone rod homeobox (CRX), binds to PIRE
in vitro
and transactivates PIRE-reporter constructs. These data suggest that
Crx
may play a crucial role in regulating pineal gene expression through interactions with PIRE.
C-C chemokines, pivotal in protection against HIV type 1 infection
Authors: Zagury Daniel , Lachgar Abderrahim , Chams Vida , Fall Lat S. , Bernard Jacky , Zagury Jean-François , Bizzini Bernard , Gringeri Alessandro , Santagostino Elena , Rappaport Jay , Feldman Michael , O’Brien Stephen J. , Burny Arsene , Gallo Robert C.
Published: July 2002

Exposure to HIV type 1 (HIV-1) does not usually lead to infection. Although this could be because of insufficient virus titer, there is now abundant evidence that some individuals resist infection even when directly exposed to a high titer of HIV. This protection recently has been correlated with homozygous mutations of an HIV-1 coreceptor, namely CCR5, the receptor for the β-chemokines. Moreover, earlier results already had shown that the same chemokines markedly suppress the nonsyncitial inducing variants of HIV-1, the chief virus type transmitted from person to person. CCR5 mutation, as a unique mechanism of protection, is, however, suspect because HIV-1 variants can use other chemokine receptors as their coreceptor. Moreover, recent results have established that infection can indeed sometimes occur with such mutations. Here, we report on transient natural resistance over time of most of 128 hemophiliacs who were inoculated repeatedly with HIV-1-contaminated Factor VIII concentrate from plasma during 1980–1985 before the development of the HIV blood test. Furthermore, and remarkably, 14 subjects remain uninfected to this date, and in these subjects we found homozygous CCR5 mutations in none but in most of them overproduction of β chemokines.
In vitro
experiments confirmed the potent anti-HIV suppressive effect of these chemokines.
Genetic analysis using genomic representations
Authors: Lucito Robert , Nakimura Mariko , West Joseph A. , Han Ying , Chin Koei , Jensen Kendall , McCombie Richard , Gray Joe W. , Wigler Michael
Published: July 2002

Analysis of the genetic changes in human tumors is often problematical because of the presence of normal stroma and the limited availability of pure tumor DNA. However, large amounts of highly reproducible “representations” of tumor and normal genomes can be made by PCR from nanogram amounts of restriction endonuclease cleaved DNA that has been ligated to oligonucleotide adaptors. We show here that representations are useful for many types of genetic analyses, including measuring relative gene copy number, loss of heterozygosity, and comparative genomic hybridization. Representations may be prepared even from sorted nuclei from fixed and archived tumor biopsies.
The developing renal, reproductive, and respiratory systems of the African elephant suggest an aquatic ancestry
Authors: Gaeth A. P. , Short R. V. , Renfree M. B.
Published: July 2002

The early embryology of the elephant has never been studied before. We have obtained a rare series of African elephant (Loxodonta africana) embryos and fetuses ranging in weight from 0.04 to 18.5 g, estimated gestational ages 58–166 days (duration of gestation is ≈660 days). Nephrostomes, a feature of aquatic vertebrates, were found in the mesonephric kidneys at all stages of development whereas they have never been recorded in the mesonephric kidneys of other viviparous mammals. The trunk was well developed even in the earliest fetus. The testes were intra-abdominal, and there was no evidence of a gubernaculum, pampiniform plexus, processus vaginalis, or a scrotum, confirming that the elephant, like the dugong, is one of the few primary testicond mammals. The palaeontological evidence suggests that the elephant’s ancestors were aquatic, and recent immunological and molecular evidence shows an extremely close affinity between present-day elephants and the aquatic Sirenia (dugong and manatees). The evidence from our embryological study of the elephant also suggests that it evolved from an aquatic mammal.
Shedding light on the dark and weakly fluorescent states of green fluorescent proteins
Authors: Weber Wolfgang , Helms Volkhard , McCammon J. Andrew , Langhoff Peter W.
Published: July 2002

Recent experiments on various similar green fluorescent protein (GFP) mutants at the single-molecule level and in solution provide evidence of previously unknown short- and long-lived “dark” states and of related excited-state decay channels. Here, we present quantum chemical calculations on cis-trans photoisomerization paths of neutral, anionic, and zwitterionic GFP chromophores in their ground and first singlet excited states that explain the observed behaviors from a common perspective. The results suggest that favorable radiationless decay channels can exist for the different protonation states along these isomerizations, which apparently proceed via conical intersections. These channels are suggested to rationalize the observed dramatic reduction of fluorescence in solution. The observed single-molecule fast blinking is attributed to conversions between the fluorescent anionic and the dark zwitterionic forms whereas slow switching is attributed to conversions between the anionic and the neutral forms. The predicted nonadiabatic crossings are seen to rationalize the origins of a variety of experimental observations on a common basis and may have broad implications for photobiophysical mechanisms in GFP.
Glutamine synthetase inactivation by protein–protein interaction
Authors: García-Domínguez Mario , Reyes José C. , Florencio Francisco J.
Published: July 2002

Glutamine synthetase (GS; EC
6.3.1.2
) is the pivotal enzyme of nitrogen metabolism in prokaryotes. Control of bacterial GS activity by reversible adenylylation has provided one of the classical paradigms of signal transduction by cyclic cascades. By contrast, in the present work we show that cyanobacterial GS is controlled by a different mechanism that involves the interaction of two inhibitory polypeptides with the enzyme. Both inactivating factors (IFs), named IF7 and IF17, are required
in vivo
for complete GS inactivation. Inactive GS-IF7 and GS-IF17 complexes were reconstituted
in vitro
by using
Escherichia coli
-expressed purified proteins. Our data suggest that control of GS activity is exerted by regulating the levels of IF7 and IF17.
A visceral pain pathway in the dorsal column of the spinal cord
Authors: Willis William D. , Al-Chaer Elie D. , Quast Michael J. , Westlund Karin N.
Published: July 2002

A limited midline myelotomy at T10 can relieve pelvic cancer pain
in patients. This observation is explainable in light of strong
evidence in support of the existence of a visceral pain pathway that
ascends in the dorsal column (DC) of the spinal cord. In rats and
monkeys, responses of neurons in the ventral posterolateral thalamic
nucleus to noxious colorectal distention are dramatically reduced after
a lesion of the DC at T10, but not by interruption of the spinothalamic
tract. Blockade of transmission of visceral nociceptive signals through
the rat sacral cord by microdialysis administration of morphine or
6-cyano-7-nitroquinoxaline-2,3-dione shows that postsynaptic DC neurons
in the sacral cord transmit visceral nociceptive signals to the gracile
nucleus. Retrograde tracing studies in rats demonstrate a concentration
of postsynaptic DC neurons in the central gray matter of the L6-S1
spinal segments, and anterograde tracing studies show that labeled
axons ascend from this region to the gracile nucleus. A similar
projection from the midthoracic spinal cord ends in the gracile and
cuneate nuclei. Behavioral experiments demonstrate that DC lesions
reduce the nocifensive responses produced by noxious stimulation of the
pancreas and duodenum, as well as the electrophysiological responses of
ventral posterolateral neurons to these stimuli. Repeated regional
blood volume measurements were made in the thalamus and other brain
structures in anesthetized monkeys in response to colorectal distention
by functional MRI. Sham surgery did not reduce the regional blood
volume changes, whereas the changes were eliminated by a DC lesion at
T10.
Chemokines and activated macrophages in HIV gp120-induced neuronal apoptosis
Authors: Kaul Marcus , Lipton Stuart A.
Published: July 2002

HIV-1 glycoprotein gp120 induces injury and apoptosis in rodent and human neurons
in vitro
and
in vivo
and is therefore thought to contribute to HIV-associated dementia. In addition to CD4, different gp120 isolates bind to the α- or β-chemokine receptors CXCR4 and CCR5, respectively. These and other chemokine receptors are on brain macrophages/microglia, astrocytes, and neurons. Thus, apoptosis could occur via direct interaction of gp120 with neurons, indirectly via stimulation of glia to release neurotoxic factors, or via both pathways. Here we show in rat cerebrocortical cultures that recapitulate the type and proportion of cells normally found in brain, i.e., neurons, astrocytes, and macrophages/microglia, that the β-chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and macrophage inflammatory protein (MIP-1β) protect neurons from gp120
SF2
-induced apoptosis. The gp120
SF2
isolate prefers binding to CXCR4 receptors, similar to the physiological α-chemokine ligands, stromal cell-derived factor (SDF)-1α/β. SDF-1α/β failed to prevent gp120
SF2
neurotoxicity, and in fact also induced neuronal apoptosis. We could completely abrogate gp120
SF2
-induced neuronal apoptosis with the tripeptide TKP, which inhibits activation of macrophages/microglia. In contrast, TKP or depletion of macrophages/microglia did not prevent SDF-1 neurotoxicity. Inhibition of p38 mitogen-activated protein kinase ameliorated both gp120
SF2
- and SDF-1-induced neuronal apoptosis. Taken together, these results suggest that gp120
SF2
and SDF-1 differ in the cell type on which they stimulate CXCR4 to induce neuronal apoptosis, but both ligands use the p38 mitogen-activated protein kinase pathway for death signaling. Moreover, gp120
SF2
-induced neuronal apoptosis depends predominantly on an indirect pathway via activation of chemokine receptors on macrophages/microglia, whereas SDF-1 may act directly on neurons or astrocytes.
Subpopulations of chloroplast ribosomes change during photoregulated development of
Zea mays
leaves: Ribosomal proteins L2, L21, and L29
Authors: Zhao You-Yang , Xu Tao , Zucchi Paola , Bogorad Lawrence
Published: July 2002

Seedlings grown in darkness, i.e., etiolated seedlings, lack chlorophyll and most other components of the photosynthetic apparatus. On illumination, the plastids become photosynthetically competent through the production of chlorophylls and proteins encoded by certain chloroplast and nuclear genes. There are two types of photosynthetic cells in leaves of the C4 plant maize: bundle sheath cells (BSC) and adjacent mesophyll cells (MC). Some proteins of the maize photosynthetic machinery are solely or preferentially localized in MC and others in BSC. A particular gene may be photoregulated up in one cell type and down in the other. Transcripts of the nuclear gene
rpl29,
encoding the chloroplast ribosomal protein L29, increase in abundance about 17-fold during light-induced maturation of plastids. There is about 1.5 times more L29 protein in ribosomes of greening leaves than in ribosomes of unilluminated leaves; the L29 contents of MC and BSC are about the same. However, L21 is present about equally in plastid ribosomes of unilluminated and illuminated seedlings. In contrast to both L29 and L21, the fraction of the ribosome population containing L2 is about the same in MC and BSC of etiolated leaves but, on illumination, the proportion of the ribosome population with L2 increases in BSC but not in MC. The existence of different subpopulations of plastid ribosomes—e.g., those with and without L21 and/or L29 during development—evokes interesting, but as yet unanswered, questions about the roles of different types of ribosomes in differentiation.
Direct evidence that the rifamycin polyketide synthase assembles polyketide chains processively
Authors: Yu Tin-Wein , Shen Yuemao , Doi-Katayama Yukiko , Tang Li , Park Cheonseok , Moore Bradley S. , Richard Hutchinson C. , Floss Heinz G.
Published: July 2002

The assembly of the polyketide backbone of rifamycin B on the type I rifamycin polyketide synthase (PKS), encoded by the
rifA–rifE
genes, is terminated by the product of the
rifF
gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam. Inactivation of
rifF
gives a rifamycin B nonproducing mutant that still accumulates a series of linear polyketides ranging from the tetra- to a decaketide, also detected in the wild type, demonstrating that the PKS operates in a processive manner. Disruptions of the
rifD
module 8 and
rifE
module 9 and module 10 genes also result in accumulation of such linear polyketides as a consequence of premature termination of polyketide assembly. Whereas the tetraketide carries an unmodified aromatic chromophore, the penta- through decaketides have undergone oxidative cyclization to the naphthoquinone, suggesting that this modification occurs during, not after, PKS assembly. The structure of one of the accumulated compounds together with
18
O experiments suggests that this oxidative cyclization produces an 8-hydroxy-7,8-dihydronaphthoquinone structure that, after the stage of proansamycin X, is dehydrogenated to an 8-hydroxynaphthoquinone.
MSE55, a Cdc42 effector protein, induces long cellular extensions in fibroblasts
Authors: Burbelo Peter D. , Snow Dianne M. , Bahou Wadie , Spiegel Sarah
Published: July 2002

Cdc42 is a member of the Rho GTPase family that regulates multiple cellular activities, including actin polymerization, kinase-signaling activation, and cell polarization. MSE55 is a nonkinase CRIB (Cdc42/Rac interactive-binding) domain-containing molecule of unknown function. Using glutathioneS-transferase-capture experiments, we show that MSE55 binds to Cdc42 in a GTP-dependent manner. MSE55 binding to Cdc42 required an intact CRIB domain, because a MSE55 CRIB domain mutant no longer interacted with Cdc42. To study the function of MSE55 we transfected either wild-type MSE55 or a MSE55 CRIB mutant into mammalian cells. In Cos-7 cells, wild-type MSE55 localized at membrane ruffles and increased membrane actin polymerization, whereas expression of the MSE55 CRIB mutant showed fewer membrane ruffles. In contrast to these results, MSE55 induced the formation of long, actin-based protrusions in NIH 3T3 cells as detected by immunofluorescence and live-cell video microscopy. MSE55-induced protrusion formation was blocked by expression of dominant-negative N17Cdc42, but not by expression of dominant-negative N17Rac. These findings indicate that MSE55 is a Cdc42 effector protein that mediates actin cytoskeleton reorganization at the plasma membrane.
Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region
Authors: Kudo Nobuaki , Matsumori Nobuaki , Taoka Hiroshi , Fujiwara Daisuke , Schreiner Erwin P. , Wolff Barbara , Yoshida Minoru , Horinouchi Sueharu
Published: July 2002

The cellular target of leptomycin B (LMB), a nuclear export inhibitor, has been identified as CRM1 (exportin 1), an evolutionarily conserved receptor for the nuclear export signal of proteins. However, the mechanism by which LMB inhibits CRM1 still remains unclear. CRM1 in a
Schizosaccharomyces pombe
mutant showing extremely high resistance to LMB had a single amino acid replacement at Cys-529 with Ser. The mutant gene, named
crm1-K1,
conferred LMB resistance on wild-type
S. pombe
, and Crm1-K1 no longer bound biotinylated LMB.
1
H NMR analysis showed that LMB bound
N
-acetyl-
l
-cysteine methyl ester through a Michael-type addition, consistent with the idea that LMB binds covalently via its α,β-unsaturated δ-lactone to the sulfhydryl group of Cys-529. When HeLa cells were cultured with biotinylated LMB, the only cellular protein bound covalently was CRM1. Inhibition by
N
-ethylmaleimide (NEM), an alkylating agent, of CRM1-mediated nuclear export probably was caused by covalent binding of the electrophilic structure in NEM to the sulfhydryl group of Cys-529, because the
crm1-K1
mutant showed the normal rate for the export of Rev nuclear export signal-bearing proteins in the presence of not only LMB but also NEM. These results show that the single cysteine residue determines LMB sensitivity and is selectively alkylated by LMB, leading to CRM1 inactivation.
Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication
Authors: Aparicio Oscar M. , Stout Anita M. , Bell Stephen P.
Published: July 2002

Chromosomes are replicated in characteristic, temporal patterns during S phase. We have compared the timing of association of replication proteins at early- and late-replicating origins of replication. Minichromosome maintenance proteins assemble simultaneously at early- and late-replicating origins. In contrast, Cdc45p association with late origins is delayed relative to early origins. DNA polymerase α association is similarly delayed at late origins and requires Cdc45p function. Activation of the S phase checkpoint inhibits association of Cdc45p with late-firing origins. These studies suggest that Cdc45p is poised to serve as a key regulatory target for both the temporal and checkpoint-mediated regulation of replication origins.
An inverse relationship between T cell receptor affinity and antigen dose during CD4+T cell responsesin vivoandin vitro
Authors: Rees William , Bender Jeremy , Teague T. Kent , Kedl Ross M. , Crawford Frances , Marrack Philippa , Kappler John
Published: July 2002

Multimeric peptide/class II MHC staining reagents were synthesized and shown to bind with appropriate specificity to T cell hybridomas. A small, expanded population of T cells detected with one of these reagents in peptide-immunized C57BL/10 mice persisted for several months. This population expanded further on secondary immunization. Equating the extent of binding of this reagent to T cell receptor affinity, we saw little correlation of immunizing peptide dose to T cell receptor affinity at the peak of the primary response. However, there was an inverse relation between peptide dose and the apparent receptor affinity of the T cells that were present several months after a primary response or after a secondary stimulation eitherin vivoorin vitro.
Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents
Authors: Hickman Mark J. , Samson Leona D.
Published: July 2002

All cells are unavoidably exposed to chemicals that can alkylate DNA to form genotoxic damage. Among the various DNA lesions formed,
O
6
-alkylguanine lesions can be highly cytotoxic, and we recently demonstrated that
O
6
-methylguanine (
O
6
MeG) and
O
6
-chloroethylguanine (
O
6
CEG) specifically initiate apoptosis in hamster cells. Here we show, in both hamster and human cells, that the MutSα branch of the DNA mismatch repair pathway (but not the MutSβ branch) is absolutely required for signaling the initiation of apoptosis in response to
O
6
MeGs and is partially required for signaling apoptosis in response to
O
6
CEGs. Further,
O
6
MeG lesions signal the stabilization of the p53 tumor suppressor, and such signaling is also MutSα-dependent. Despite this, MutSα-dependent apoptosis can be executed in a p53-independent manner. DNA mismatch repair status did not influence the response of cells to other inducers of p53 and apoptosis. Thus, it appears that mismatch repair status, rather than p53 status, is a strong indicator of the susceptibility of cells to alkylation-induced apoptosis. This experimental system will allow dissection of the signal transduction events that couple a specific type of DNA base lesion with the final outcome of apoptotic cell death.
Orexin A activates locus coeruleus cell firing and increases arousal in the rat
Authors: Hagan Jim J. , Leslie Ron A. , Patel Sara , Evans Martyn L. , Wattam Trevor A. , Holmes Steve , Benham Christopher D. , Taylor Stephen G. , Routledge Carol , Hemmati Panida , Munton Richard P. , Ashmeade Tracey E. , Shah Ajit S. , Hatcher Jonathan P. , Hatcher Paula D. , Jones Declan N. C. , Smith Martin I. , Piper David C. , Hunter A. Jackie , Porter Rod A. , Upton Neil
Published: July 2002

The localization of orexin neuropeptides in the lateral hypothalamus has focused interest on their role in ingestion. The orexigenic neurones in the lateral hypothalamus, however, project widely in the brain, and thus the physiological role of orexins is likely to be complex. Here we describe an investigation of the action of orexin A in modulating the arousal state of rats by using a combination of tissue localization and electrophysiological and behavioral techniques. We show that the brain region receiving the densest innervation from orexinergic nerves is the locus coeruleus, a key modulator of attentional state, where application of orexin A increases cell firing of intrinsic noradrenergic neurones. Orexin A increases arousal and locomotor activity and modulates neuroendocrine function. The data suggest that orexin A plays an important role in orchestrating the sleep-wake cycle.
GTP-dependent activation of urease apoprotein in complex with the UreD, UreF, and UreG accessory proteins
Authors: Soriano Aileen , Hausinger Robert P.
Published: July 2002

Syntheses of metal-containing enzymes often require the participation of accessory proteins. The roles played by many of these accessory proteins are poorly characterized.
Klebsiella aerogenes
urease, a nickel-containing enzyme, provides an ideal system to study metallocenter assembly. Here, we describe a method for isolating a complex containing urease apoprotein and the UreD, UreF, and UreG accessory proteins. We demonstrate that urease apoprotein in this complex is activated to near wild-type enzyme levels when incubated with nickel ions and high (≈100 mM) concentrations of bicarbonate. Significantly, we also observed nickel-dependent activation at physiologically relevant (≈100 μM) bicarbonate levels, but only in the presence of GTP. Based on studies involving a nonhydrolyzable analog of GTP, we conclude that nucleotide hydrolysis, not just binding, is required for this process. The critical nucleotide-binding site was localized to UreG on the basis of experiments using a variant complex. These studies highlight the relevance of the UreD-UreF-UreG-urease apoprotein complex to nickel metallocenter assembly and explain the previously identified
in vivo
energy requirement for urease activation.
Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi
Authors: Nohturfft Axel , DeBose-Boyd Russell A. , Scheek Sigrid , Goldstein Joseph L. , Brown Michael S.
Published: July 2002

The proteolytic cleavage of sterol regulatory element-binding proteins (SREBPs) is regulated by SREBP cleavage-activating protein (SCAP), which forms complexes with SREBPs in membranes of the endoplasmic reticulum (ER). In sterol-depleted cells, SCAP facilitates cleavage of SREBPs by Site-1 protease, thereby initiating release of active NH
2
-terminal fragments from the ER membrane so that they can enter the nucleus and activate gene expression. In sterol-overloaded cells, the activity of SCAP is blocked, SREBPs remain bound to membranes, and transcription of sterol-regulated genes declines. Here, we provide evidence that sterols act by inhibiting the cycling of SCAP between the ER and Golgi. We use glycosidases, glycosidase inhibitors, and a glycosylation-defective mutant cell line to demonstrate that the N-linked carbohydrates of SCAP are modified by Golgi enzymes in sterol-depleted cells. After modification, SCAP returns to the ER, as indicated by experiments that show that the Golgi-modified forms of SCAP cofractionate with ER membranes on density gradients. In sterol-overloaded cells, the Golgi modifications of SCAP do not occur, apparently because SCAP fails to leave the ER. Golgi modifications of SCAP are restored when sterol-overloaded cells are treated with brefeldin A, which causes Golgi enzymes to translocate to the ER. These studies suggest that sterols regulate the cleavage of SREBPs by modulating the ability of SCAP to transport SREBPs to a post-ER compartment that houses active Site-1 protease.