bluuebunny/crossref_metadata_2025 huggingface dataset:
Two distinct proteolytic processes in the generation of a major histocompatibility complex class I-presented peptide
Authors: Craiu Abie , Akopian Tatos , Goldberg Alfred , Rock Kenneth L.
Published: July 2002

Although cellular proteins degraded by proteasomes are the source of most antigenic peptides presented on major histocompatibility complex class I molecules, it is unknown whether the eight- to nine-residue peptides that fit in the binding groove of class I molecules are directly produced by proteasomes alonein vivo. If the eight-residue peptide SIINFEKL from chicken ovalbumin is extended by one or several residues at its C terminus and microinjected into cells or expressed from a minigene, it is processed and presented on major histocompatibility complex class I. However, processing and presentation are inhibited by proteasome inhibitors, such as lactacystin. In contrast, when SIINFEKL is extended by 2 to 25 residues at its N terminus, its presentation is not blocked by proteasome inhibitors. N-terminal processing also can occur when the extended peptide is cotranslationally inserted into the endoplasmic reticulum. Thus, two different proteolytic steps in the generation of an chicken ovalbumin-presented peptide can be distinguished. Cleavage by the proteasome defines the proper C terminus, whereas distinct peptidase(s) in the cytosol or endoplasmic reticulum may generate the appropriate N terminus from extended peptides.
Somatic mutations in the p53 tumor suppressor gene in rheumatoid arthritis synovium
Authors: Firestein Gary S. , Echeverri Fernando , Yeo Michele , Zvaifler Nathan J. , Green Douglas R.
Published: July 2002

The factors that regulate the perpetuation and invasiveness of rheumatoid synovitis have been the subject of considerable inquiry, and the possibility that nonimmunologic defects can contribute to the disease has not been rigorously addressed. Using a mismatch detection system, we report that synovial tissue from the joints of severe chronic rheumatoid arthritis patients contain mutant p53 transcripts, which were not found in skin samples from the same patients or in joints of patients with osteoarthritis. Mutant p53 transcripts also were identified in synoviocytes cultured from rheumatoid joints. The predicted amino acid substitutions in p53 were identical or similar to those commonly observed in a variety of tumors and might influence growth and survival of rheumatoid synoviocytes. Thus, mutations in p53 and subsequent selection of the mutant cells may occur in the joints of patients as a consequence of inflammation and contribute to the pathogenesis of the disease.
Reduced stress defense in heme oxygenase 1-deficient cells
Authors: Poss Kenneth D. , Tonegawa Susumu
Published: July 2002

Stressed mammalian cells up-regulate heme oxygenase 1 (Hmox1; EC1.14.99.3), which catabolizes heme to biliverdin, carbon monoxide, and free iron. To assess the potential role of Hmox1 in cellular antioxidant defense, we analyzed the responses of cells from mice lacking functional Hmox1 to oxidative challenges. CulturedHmox1−/−embryonic fibroblasts demonstrated high oxygen free radical production when exposed to hemin, hydrogen peroxide, paraquat, or cadmium chloride, and they were hypersensitive to cytotoxicity caused by hemin and hydrogen peroxide. Furthermore, young adultHmox1−/−mice were vulnerable to mortality and hepatic necrosis when challenged with endotoxin. Ourin vitroandin vivoresults provide genetic evidence that up-regulation of Hmox1 serves as an adaptive mechanism to protect cells from oxidative damage during stress.
Bioactive and nuclease-resistant
l
-DNA ligand of vasopressin
Authors: Williams Kelly P. , Liu Xiao-Hong , Schumacher Ton N. M. , Lin Herbert Y. , Ausiello Dennis A. , Kim Peter S. , Bartel David P.
Published: July 2002

In vitro
selection experiments have produced nucleic acid ligands (aptamers) that bind tightly and specifically to a great variety of target biomolecules. The utility of aptamers is often limited by their vulnerability to nucleases present in biological materials. One way to circumvent this problem is to select an aptamer that binds the enantiomer of the target, then synthesize the enantiomer of the aptamer as a nuclease-insensitive ligand of the normal target. We have so identified a mirror-image single-stranded DNA that binds the peptide hormone vasopressin and have demonstrated its stability to nucleases and its bioactivity as a vasopressin antagonist in cell culture.
Identification of a small, very acidic constitutive nucleolar protein (NO29) as a member of the nucleoplasmin family
Authors: Zirwes Rudolf F. , Schmidt-Zachmann Marion S. , Franke Werner W.
Published: July 2002

We report the discovery and molecular characterization of a small and very acidic nucleolar protein of an SDS/PAGE mobility corresponding to
M
r
29,000 (NO29). The cDNA-deduced sequence of the
Xenopus laevis
protein defines a polypeptide of a calculated molecular mass of 20,121 and a pI of 3.75, with an extended acidic region near its C terminus, and is related to the major nucleolar protein, NO38, and the histone-binding protein, nucleoplasmin. This member of the nucleoplasmin family of proteins was immunolocalized to nucleoli in
Xenopus
oocytes and diverse somatic cells. Protein NO29 is associated with nuclear particles from
Xenopus
oocytes, partly complexed with protein NO38, and occurs in preribosomes but not in mature ribosomes. The location and the enormously high content of negatively charged amino acids lead to the hypothesis that NO29 might be involved in the nuclear and nucleolar accumulation of ribosomal proteins and the coordinated assembly of pre-ribosomal particles.
Assigning folds to the proteins encoded by the genome of
Mycoplasma genitalium
Authors: Fischer Daniel , Eisenberg David
Published: July 2002

A crucial step in exploiting the information inherent in genome sequences is to assign to each protein sequence its three-dimensional fold and biological function. Here we describe fold assignment for the proteins encoded by the small genome of
Mycoplasma genitalium
. The assignment was carried out by our computer server (
http://www.doe-mbi.ucla.edu/people/frsvr/frsvr.html
), which assigns folds to amino acid sequences by comparing sequence-derived predictions with known structures. Of the total of 468 protein ORFs, 103 (22%) can be assigned a known protein fold with high confidence, as cross-validated with tests on known structures. Of these sequences, 75 (16%) show enough sequence similarity to proteins of known structure that they can also be detected by traditional sequence–sequence comparison methods. That is, the difference of 28 sequences (6%) are assignable by the sequence–structure method of the server but not by current sequence–sequence methods. Of the remaining 78% of sequences in the genome, 18% belong to membrane proteins and the remaining 60% cannot be assigned either because these sequences correspond to no presently known fold or because of insensitivity of the method. At the current rate of determination of new folds by x-ray and NMR methods, extrapolation suggests that folds will be assigned to most soluble proteins in the next decade.
Selection for spiral waves in the social amoebae
Dictyostelium
Authors: Pálsson Eiríkur , Lee Kyoung J. , Goldstein Raymond E. , Franke Jakob , Kessin Richard H. , Cox Edward C.
Published: July 2002

Starving
Dictyostelium
amoebae emit pulses of the chemoattractant cAMP that are relayed from cell to cell as circular and spiral waves. We have recently modeled spiral wave formation in
Dictyostelium
. Our model suggests that a secreted protein inhibitor of an extracellular cAMP phosphodiesterase selects for spirals. Herein we test the essential features of this prediction by comparing wave propagation in wild type and inhibitor mutants. We find that mutants rarely form spirals. The territory size of mutant strains is approximately 50 times smaller than wild type, and the mature fruiting bodies are smaller but otherwise normal. These results identify a mechanism for selecting one wave symmetry over another in an excitable system and suggest that the phosphodiesterase inhibitor may be under selection because it helps regulate territory size.
Insulin-like growth factors-I and -II differentially regulate endogenous acetylcholine release from the rat hippocampal formation
Authors: Kar S. , Seto D. , Doré S. , Hanisch U.-K. , Quirion R.
Published: July 2002

Insulin-like growth factors-I and -II (IGF-I and -II) are structurally related mitogenic polypeptides with potent growth promoting effects. These peptides and their corresponding IGF-I and -II receptors are selectively localized in the brain. To date, most of the effects of IGFs are believed to be mediated by IGF-I receptors whereas the significance of IGF-II receptor in mediating biological responses remains unclear. In the present study, we characterized the distribution of IGF-I and IGF-II receptor sites and investigated the effects of both factors on endogenous acetylcholine (ACh) release in adult rat hippocampus. [
125
I]IGF-I receptor binding sites are recognized by IGF-I> IGF-II> insulin, whereas [
125
I]IGF-II binding was competed potently by IGF-II> IGF-I but not by insulin. At the cellular level, IGF-I receptor sites were primarily noted in the molecular layer of the dentate gyrus and the CA2-CA3 subfields of the Ammon’s horn whereas IGF-II sites were localized predominantly in the pyramidal cell layer of the CA1-CA3 subfields and in the granular cell layer of the dentate gyrus. IGF-I (10
−14
–10
−8
M) and des(1–3) IGF-I (10
−10
–10
−8
M) were found to inhibit whereas IGF-II (10
−14
–10
−8
M) potentiated K
+
-evoked ACh release from hippocampal slices. Tetrodotoxin altered the effects of IGF-I but not those of IGF-II suggesting that IGF-I acts indirectly via the release of other modulators whereas IGF-II acts directly on or in close proximity to the cholinergic terminals. The inhibitory effects of IGF-I were also observed in the frontal cortex but not in the striatum. In contrast, the stimulatory effects of IGF-II were evident both in the frontal cortex and striatum. Taken together, these results reveal the differential localization of IGF-I and IGF-II receptor sites in the hippocampal formation and the opposite role for these growth factors in the acute regulation of ACh release likely via two distinct mechanisms. Additionally, these data provide the first evidence for a direct role for IGF-II and its receptors in the regulation of transmitter release in the central nervous system.
Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells
Authors: O’Gorman Stephen , Dagenais Nicole A. , Qian Min , Marchuk Yelena
Published: July 2002

The production of subtle or conditional mutations in mice through the combined use of site-specific and homologous recombination has become an increasingly widespread experimental paradigm in mammalian genetics. Embryonic stem cells containing recombinase transgenes that were expressed in the male germ line, but not in other tissues or in the embryonic stem cells themselves, would substantially simplify the production of such alleles. Here we show that transgenes comprised of the mouse protamine 1 promoter and the Cre recombinase coding sequence mediate the efficient recombination of a Cre target transgene in the male germ line, but not in other tissues. Embryonic stem cell lines generated from one of these transgenic strains were transfected with targeting vectors that included loxP-flanked selectable markers, and homologously recombined alleles containing the marker and functional loxP sites were isolated. These results establish the potential of the system for substantially reducing the time, effort, and resources required to produce homologously recombined alleles in mice that have been secondarily rearranged by a site-specific recombinase.
Cloning and sequence analysis of a
Bothrops jararaca
cDNA encoding a precursor of seven bradykinin-potentiating peptides and a C-type natriuretic peptide
Authors: Murayama Nobuhiro , Hayashi Mirian A. F. , Ohi Hiroaki , Ferreira Luiza A. F. , Hermann Vivian V. , Saito Hiromi , Fujita Yoshiaki , Higuchi Shigesada , Fernandes Beatriz L. , Yamane Tetsuo , de Camargo Antonio C. M.
Published: July 2002

A 1.8-kb cDNA clone was isolated from a
Bothrops jararaca
venom gland cDNA library that encodes a 256-aa precursor for bradykinin-potentiating peptides (angiotensin-converting enzyme inhibitors) and a C-type natriuretic peptide (CNP). The seven bradykinin-potentiating peptides are aligned tandemly after the hydrophobic signal peptide sequence, followed by a putative intervening sequence and a CNP at the C terminus. Northern blot analysis indicated the predominant expression of a 1.8-kb mRNA in the venom glands as well as in the spleen and the brain. Two lower intensity mRNA bands of 3.5 kb and 5.7 kb also hybridized to the cDNA clone. Radioimmunoassay for the CNP was performed using the antiserum against rat CNP. The presence of CNP immunoreactivity was detected in the low molecular weight fraction of the
Bothrops jararaca
venom.
Systematic isolation of peptide signal molecules regulating development in hydra: LWamide and PW families
Authors: Takahashi Toshio , Muneoka Yojiro , Lohmann Jan , de Haro Maria S. Lopez , Solleder Gaby , Bosch Thomas C. G. , David Charles N. , Bode Hans R. , Koizumi Osamu , Shimizu Hiroshi , Hatta Masayuki , Fujisawa Toshitaka , Sugiyama Tsutomu
Published: July 2002

To isolate new peptide signal molecules involved in regulating developmental processes in hydra, a novel screening project was developed. Peptides extracted from the tissue of
Hydra magnipapillata
were systematically purified to homogeneity using HPLC. A fraction of each purified peptide was examined by differential display–PCR for its ability to affect gene expression in hydra. Another fraction was used to determine the tentative structure using an amino acid sequence analyzer and/or a mass spectrometer. Based on the results, peptides of potential interest were selected for chemical synthesis, followed by confirmation of the identity of the synthetic with the native peptides using HPLC. Using this approach, 286 peptides have been isolated, tentative amino acid sequences have been determined for 95 of them, and 19 synthetic peptides identical to native ones were produced. The 19 synthetic peptides were active in a variety of biological tests. For example, Hym-54 stimulated muscle contraction in adult polyps of hydra and sea anemone,
Anthopleura fuscoviridis
, and induced metamorphosis of planula, the larval stage, into polyps in a marine hydrozoan species,
Hydractinia serrata
. Another peptide, Hym-33H, inhibited nerve cell differentiation in hydra and induced tissue contraction in planula of
Hydractinia serrata
. The evidence obtained so far suggests that hydra contains a large number (>350) of peptide signal molecules involved in regulating developmental or other processes in cnidaria. These peptides can be isolated and their functions examined systematically with the new approach developed in this study.
Allergen-induced bronchial hyperreactivity and eosinophilic inflammation occur in the absence of IgE in a mouse model of asthma
Authors: Mehlhop Paul D. , van de Rijn Matthijs , Goldberg Alison B. , Brewer Joanne P. , Kurup Viswanath P. , Martin Thomas R. , Oettgen Hans C.
Published: July 2002

In patients with asthma, elevations of IgE correlate both with allergic inflammation of the airways and with bronchial hyperreactivity (BHR). Several investigations, using mouse models of this disease, have indicated a central role for IgE in the pathogenesis of the eosinophilic inflammation as well as in the obstructive airway physiology of BHR. Some diagnostic studies and therapeutic strategies for asthma are based on the putative role of IgE in asthma pathogenesis. Here, we use mice with a null mutation of the Cɛ locus to show that bronchial inflammation and BHR in response to allergen inhalation both can occur in the absence of IgE. We demonstrate that the eosinophilic bronchial inflammation elicited in an established mouse model of hypersensitivity toAspergillus fumigatus(Af) is accompanied by the asthmatic physiology of BHR. Wild-type and IgE-deficient mice were sensitized intranasally withAfextract. Both groups of animals developed bronchoalveolar lavage eosinophilia and pulmonary parenchymal eosinophilia. This was accompanied by increased serum levels of total andAf-specific IgE in the wild-type animals only. ThisAf-sensitization protocol resulted in significant BHR in both wild-type mice and IgE-deficient mice. Interestingly, unsensitized IgE-deficient mice had increased bronchial responsiveness compared with unsensitized wild-type controls. We conclude that BHR and airways inflammation can be fully expressed via IgE-independent mechanisms. These may involve the activation of mast cells by factors other than IgE as well as a mucosal lymphocyte-mediated immune response to allergen.
Opiate receptor knockout mice define μ receptor roles in endogenous nociceptive responses and morphine-induced analgesia
Authors: Sora Ichiro , Takahashi Nobuyuki , Funada Masahiko , Ujike Hiroshi , Revay Randal S. , Donovan David M. , Miner Lucinda L. , Uhl George R.
Published: July 2002

Morphine produces analgesia at opiate receptors expressed in nociceptive circuits. μ, δ, and κ opiate receptor subtypes are expressed in circuits that can modulate nociception and receive inputs from endogenous opioid neuropeptide ligands. The roles played by each receptor subtype in nociceptive processing in drug-free and morphine-treated states have not been clear, however. We produced homologous, recombinant μ, opiate receptor, heterozygous and homozygous knockout animals that displayed ≈54% and 0% of wild-type levels of μ receptor expression, respectively. These mice expressed κ receptors and δ receptors at near wild-type levels. Untreated knockout mice displayed shorter latencies on tail flick and hot plate tests for spinal and supraspinal nociceptive responses than wild-type mice. These findings support a significant role for endogenous opioid–peptide interactions with μ opiate receptors in normal nociceptive processing. Morphine failed to significantly reduce nociceptive responses in hot plate or tail flick tests of homozygous μ receptor knockout mice, and heterozygote mice displayed right and downward shifts in morphine analgesia dose–effect relationships. These results implicate endogenous opioid–peptide actions at μ opiate receptors in several tests of nociceptive responsiveness and support μ receptor mediation of morphine-induced analgesia in tests of spinal and supraspinal analgesia.
The two α subunits of
Escherichia coli
RNA polymerase are asymmetrically arranged and contact different halves of the DNA upstream element
Authors: Murakami Katsuhiko , Kimura Makoto , Owens Jeffrey T. , Meares Claude F. , Ishihama Akira
Published: July 2002

RNA polymerase core enzyme of
Escherichia coli
is composed of two α subunits and one each of the β and β′ subunits. The C-terminal domain of the RNA polymerase α subunit plays a key role in molecular communications with class I transcription factors and upstream (UP) elements of promoter DNA, using the same protein surface. To identify possible differences in the functional roles of the two α subunits, we have developed a reconstitution method for hybrid RNA polymerases containing two distinct α subunit derivatives in a defined orientation (“oriented α-heterodimer”). The binding sites of two α C-terminal domains on the UP element DNA were determined by hydroxyl radical-based DNA cleavage mediated by (
p
-bromoacetamidobenzyl)-EDTA·Fe, which was bound at Cys-269 on the UP recognition surface of one or both α subunits. The results clearly indicated that the two α subunits bind in tandem to two helix turns of the
rrnB
P1 UP element, and that the β′-associated α subunit is bound to the promoter–distal region.
A heparin-binding synthetic peptide of heparin/heparan sulfateinteracting protein modulates blood coagulation activities
Authors: Liu Shouchun , Zhou Fanyu , Höök Magnus , Carson Daniel D.
Published: July 2002

We have previously identified and characterized a heparin-binding cell surface protein (heparin/heparan sulfate-interacting protein, or HIP) present on epithelial and endothelial cells. A synthetic peptide mimicking a heparin-binding domain of HIP is now shown to bind a small subset of heparin molecules with high affinity and, therefore, presumably recognizes a specific structural motif in the heparin molecule. Further analyses revealed that the heparin molecules exhibiting a high affinity for the HIP peptide also show an extremely high affinity for antithrombin III (AT-III), a cofactor required for heparin’s anticoagulant activity. The HIP peptide was shown to compete with AT-III for binding to heparin and to neutralize the anticoagulant activity of heparin in blood plasma assays. Furthermore, the heparin subfraction that binds to the HIP peptide with high affinity exhibits an extremely high anticoagulant activity. We conclude that although the HIP peptide shows no sequence similarity with AT-III, the two proteins recognize the same or similar structural motifs in heparin.
Discovery and analysis of inflammatory disease-related genes using cDNA microarrays
Authors: Heller Renu A. , Schena Mark , Chai Andrew , Shalon Dari , Bedilion Tod , Gilmore James , Woolley David E. , Davis Ronald W.
Published: July 2002

cDNA microarray technology is used to profile complex diseases and discover novel disease-related genes. In inflammatory disease such as rheumatoid arthritis, expression patterns of diverse cell types contribute to the pathology. We have monitored gene expression in this disease state with a microarray of selected human genes of probable significance in inflammation as well as with genes expressed in peripheral human blood cells. Messenger RNA from cultured macrophages, chondrocyte cell lines, primary chondrocytes, and synoviocytes provided expression profiles for the selected cytokines, chemokines, DNA binding proteins, and matrix-degrading metalloproteinases. Comparisons between tissue samples of rheumatoid arthritis and inflammatory bowel disease verified the involvement of many genes and revealed novel participation of the cytokine interleukin 3, chemokine Groα and the metalloproteinase matrix metallo-elastase in both diseases. From the peripheral blood library, tissue inhibitor of metalloproteinase 1, ferritin light chain, and manganese superoxide dismutase genes were identified as expressed differentially in rheumatoid arthritis compared with inflammatory bowel disease. These results successfully demonstrate the use of the cDNA microarray system as a general approach for dissecting human diseases.
A putative angiogenin receptor in angiogenin-responsive human endothelial cells
Authors: Hu Guo-fu , Riordan James F. , Vallee Bert L.
Published: July 2002

Angiogenin stimulates both [
3
H]thymidine incorporation and proliferation of human endothelial cells in sparse cultures. Under these conditions, a 170-kDa cell surface protein can be detected that binds angiogenin specifically. Angiogenin-stimulated cell growth is concentration-dependent and is completely inhibited by an anti-angiogenin monoclonal antibody, but not by a nonimmune control antibody. It is not affected by the nonangiogenic homolog, RNase A, nor by other angiogenic proteins, such as basic fibroblast growth factor and its antibody. Results suggest that under specific conditions, endothelial cells express an angiogenin receptor that may mediate angiogenin-stimulated DNA synthesis and proliferation and play an important role in angiogenin-induced angiogenesis.
Downstream of Crk adaptor signaling pathway: Activation of Jun kinase by v-Crk through the guanine nucleotide exchange protein C3G
Authors: Tanaka Shinya , Ouchi Toru , Hanafusa Hidesaburo
Published: July 2002

Crk, which belongs to the adaptor family of proteins composed of Src homology 2 (SH2) and SH3 domains, has a putative role in signaling. However, the downstream events of Crk signaling remain unclear. In this study, we found that Jun kinase (JNK) is moderately activated by v-Crk in both NIH 3T3 cells and chicken embryo fibroblasts. Transient expression of v-Crk, c-Crk-I, or c-Crk-II activated JNK1 in human embryo kidney cells, 293T. Coexpression of a guanine nucleotide exchange protein C3G, which specifically binds to Crk’s SH3 domain, further enhanced the JNK activity as well as growth rate and anchorage-independent growth of v-Crk NIH 3T3 cells. Furthermore, overexpression of a dominant-negative form of C3G lacking the guanine nucleotide exchange domain abolished both the JNK activity and the colony forming potential of v-Crk NIH 3T3 cells. The requirement for JNK activation in v-Crk induced transformation was demonstrated by the suppression of colony forming activity of v-Crk NIH 3T3 cells when a dominant-negative form of JNK kinase, Sek1/MKK4 is expressed in these cells. These data strongly suggest the existence of a novel signaling cascade involving an adaptor protein v-Crk, which transmits signals through C3G toward JNK activation.
A novel seizure-induced synaptotagmin gene identified by differential display
Authors: Babity Joseph M. , Armstrong John N. , Plumier J.-Christophe L. , Currie R. William , Robertson Harold A.
Published: July 2002

Systemic administration of kainic acid, a cyclic analogue of glutamate, produces many of the clinical features of human temporal lobe epilepsy and status epilepticus in rats, including the induction of motor convulsions and the degeneration of neurons in the hippocampus and piriform cortex. Differential display PCR was used to identify mRNAs that are differentially expressed between degenerating and nondegenerating tissues in the brain after kainic acid-induced seizure activity. A novel cDNA fragment expressed in the degenerating hippocampus and piriform cortex, but not in the nondegenerating parietal cortex, was identified, cloned, and sequenced. This novel cDNA fragment identified a new member of the synaptotagmin gene family that is rapidly and transiently induced in response to seizure activity. Differential expression of this synaptotagmin gene,
syt X
, was confirmed by Northern blot analysis and
in situ
hybridization. This novel, inducible synaptotagmin gene may provide a direct link between seizure-induced neuronal gene expression and subsequent modulation of synaptic structure and function.
Crystal structure of varicella-zoster virus protease
Authors: Qiu Xiayang , Janson Cheryl A. , Culp Jeffrey S. , Richardson Susan B. , Debouck Christine , Smith Ward W. , Abdel-Meguid Sherin S.
Published: July 2002

Varicella-zoster virus (VZV), an α-herpes virus, is the causative agent of chickenpox, shingles, and postherpetic neuralgia. The three-dimensional crystal structure of the serine protease from VZV has been determined at 3.0-Å resolution. The VZV protease is essential for the life cycle of the virus and is a potential target for therapeutic intervention. The structure reveals an overall fold that is similar to that recently reported for the serine protease from cytomegalovirus (CMV), a herpes virus of the β subfamily. The VZV protease structure provides further evidence to support the finding that herpes virus proteases have a fold and active site distinct from other serine proteases. The VZV protease catalytic triad consists of a serine and two histidines. The distal histidine is proposed to properly orient the proximal histidine. The identification of an α-helical segment in the VZV protease that was mostly disordered in the CMV protease provides a better definition of the postulated active site cavity and reveals an elastase-like S′ region. Structural differences between the VZV and CMV proteases also suggest potential differences in their oligomerization states.
Reciprocal interactions between occlusion and motion computations
Authors: Anderson Barton L. , Sinha Pawan
Published: July 2002

The “aperture problem” refers to the inherent ambiguity of the motion generated by an untextured contour moving within an aperture. The limited spatial extent of the receptive fields of neurons in cortical areas like V1 and MT render them susceptible to this problem. Most psychophysical experiments have probed how the visual system overcomes the aperture problem by presenting moving contours behind one or more simulated apertures. The assumption has been that the computational ambiguities that arise in resolving these displays are equivalent to the computational problems created by receptive fields that sample a small region of visual space. Evidence is presented here that challenges this view. We demonstrate that a fundamental computational difference in the interpretation of contour terminators arises in these two variants of the aperture problem. When the aperture is a receptive field, and a moving contour extends beyond its boundaries, the contour “terminators” delimit the boundaries of the receptive field, not the ends of the contour. In contrast, when a moving contour is viewed through a simulated aperture, the contour terminators are generated by the occluding edges of the aperture. In a series of experiments, we show that reciprocal interactions arise between computations of occlusion and those of motion direction and integration. Our results demonstrate that the visual system solves the aperture problem by decomposing moving contours into moving segments, and unpaired terminators that arise from the accretion and deletion of contours behind occluding edges, generating both coherent motion and illusory occluding surfaces.
Refolding chromatography with immobilized mini-chaperones
Authors: Altamirano Myriam M. , Golbik Ralph , Zahn Ralph , Buckle Ashley M. , Fersht Alan R.
Published: July 2002

Mini-chaperones (e.g., a peptide consisting of residues 191-345 of GroEL) that are immobilized on agarose have very efficient chaperoning activity with several proteins that are otherwise recalcitrant to renaturation by conventional methods. We have used immobilized mini-chaperones both in column chromatography and batchwise to renature an insoluble protein from an inclusion body, to refold apparently irreversibly denatured proteins, and to recondition enzymes that have lost activity on storage. Refolding chromatography offers an efficient and simple means to renature proteins in high yield and with biological activity.
Charge separation in the reaction center of photosystem II studied as a function of temperature
Authors: Groot Marie-Louise , van Mourik Frank , Eijckelhoff Camiel , van Stokkum Ivo H. M. , Dekker Jan P. , van Grondelle Rienk
Published: July 2002

In photosystem II of green plants the key photosynthetic reaction consists of the transfer of an electron from the primary donor called P680 to a nearby pheophytin molecule. We analyzed the temperature dependence of this reaction by subpicosecond transient absorption spectroscopy over the temperature range 20–240 K using isolated photosystem II reaction centers from spinach. After excitation in the red edge of the Q
y
absorption band, the decay of the excited state can conveniently be described by two kinetic components that both accelerate with temperature. This temperature behavior differs remarkably from that observed in purple bacterial reaction centers. We attribute the first component, which accelerates from 2.6 ps at 20 K to 0.4 ps at 240 K, to charge separation after direct excitation of P680, and explain its temperature dependence by an intermediate that lies in energy above the singlet-excited P680 and that possibly has charge-transfer character. The second component accelerates from 120 ps at 20 K to 18 ps at 240 K and is attributed to charge separation after direct excitation of the “trap” state near-degenerate with P680 and subsequent slow energy transfer from this trap state to P680. We suggest that the slow energy transfer from the trap state to P680 plays an important role in the kinetics of radical pair formation at room temperature.
Highly specific protein sequence motifs for genome analysis
Authors: Nevill-Manning Craig G. , Wu Thomas D. , Brutlag Douglas L.
Published: July 2002

We present a method for discovering conserved sequence motifs from
families of aligned protein sequences. The method has been implemented
as a computer program called
emotif
(
http://motif.stanford.edu/emotif
). Given an aligned set of
protein sequences,
emotif
generates a set of motifs with a
wide range of specificities and sensitivities.
emotif
also
can generate motifs that describe possible subfamilies of a protein
superfamily. A disjunction of such motifs often can represent the
entire superfamily with high specificity and sensitivity. We have used
emotif
to generate sets of motifs from all 7,000 protein
alignments in the
blocks
and
prints
databases.
The resulting database, called
identify
(
http://motif.stanford.edu/identify
), contains more than 50,000
motifs. For each alignment, the database contains several motifs having
a probability of matching a false positive that range from
10
−10
to 10
−5
. Highly specific motifs are
well suited for searching entire proteomes, while generating very few
false predictions.
identify
assigns biological functions to
25–30% of all proteins encoded by the
Saccharomyces
cerevisiae
genome and by several bacterial genomes. In
particular,
identify
assigned functions to 172 of proteins
of unknown function in the yeast genome.
A human fibrosarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1
Authors: Volpert Olga V. , Lawler Jack , Bouck Noël P.
Published: July 2002

Concomitant tumor resistance refers to the ability of some large primary tumors to hold smaller tumors in check, preventing their progressive growth. Here, we demonstrate this phenomenon with a human tumor growing in a nude mouse and show that it is caused by secretion by the tumor of the inhibitor of angiogenesis, thrombospondin-1. When growing subcutaneously, the human fibrosarcoma line HT1080 induced concomitant tumor resistance, preventing the growth of experimental B16/F10 melanoma metastases in the lung. Resistance was due to the production by the tumor cells themselves of high levels of thrombospondin-1, which was present at inhibitory levels in the plasma of tumor-bearing animals who become unable to mount an angiogenic response in their corneas. Animals carrying tumors formed by antisense-derived subclones of HT1080 that secreted low or no thrombospondin had weak or no ability to control the growth of lung metastases. Although purified human platelet thrombospondin-1 had no effect on the growth of melanoma cells
in vitro
, when injected into mice it was able to halt the growth of their experimental metastases, providing clear evidence of the efficacy of thrombospondin-1 as an anti-tumor agent.
Disease sequence from mutant
rhodopsin
allele to rod and cone photoreceptor degeneration in man
Authors: Cideciyan Artur V. , Hood Donald C. , Huang Yijun , Banin Eyal , Li Zong-Yi , Stone Edwin M. , Milam Ann H. , Jacobson Samuel G.
Published: July 2002

Mutations in the gene encoding rhodopsin, the visual pigment in rod photoreceptors, lead to retinal degeneration in species from
Drosophila
to man. The pathogenic sequence from rod cell-specific mutation to degeneration of rods and cones remains unclear. To understand the disease process in man, we studied heterozygotes with 18 different rhodopsin gene mutations by using noninvasive tests of rod and cone function and retinal histopathology. Two classes of disease expression were found, and there was allele-specificity. Class A mutants lead to severely abnormal rod function across the retina early in life; topography of residual cone function parallels cone cell density. Class B mutants are compatible with normal rods in adult life in some retinal regions or throughout the retina, and there is a slow stereotypical disease sequence. Disease manifests as a loss of rod photoreceptor outer segments, not singly but in microscopic patches that coalesce into larger irregular areas of degeneration. Cone outer segment function remains normal until >75% of rod outer segments are lost. The topography of cone loss coincides with that of rod loss. Most class B mutants show an inferior-nasal to superior-temporal retinal gradient of disease vulnerability associated with visual cycle abnormalities. Class A mutant alleles behave as if cytotoxic; class B mutants can be relatively innocuous and epigenetic factors may play a major role in the retinal degeneration.
Direct visualization of antigen-specific T cells: HTLV-1 Tax11–19- specific CD8+T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients
Authors: Greten Tim F. , Slansky Jill E. , Kubota Ryuji , Soldan Samantha S. , Jaffee Elizabeth M. , Leist Thomas P. , Pardoll Drew M. , Jacobson Steven , Schneck Jonathan P.
Published: July 2002

Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11–19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2–Ig complex to directly visualize HTLV-1 Tax11–19-specific T cells from peripheral blood and cerebrospinal fluid withoutin vitrostimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8+lymphocytes specific for the HTLV-1 Tax11–19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11–19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11–19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11–19-specific CD8+T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-α and γ-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11–19-specific CD8+T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.
sud1
+
targets cyclin-dependent kinase-phosphorylated Cdc18 and Rum1 proteins for degradation and stops unwanted diploidization in fission yeast
Authors: Jallepalli Prasad V. , Tien Deborah , Kelly Thomas J.
Published: July 2002

In the fission yeast
Schizosaccharomyces pombe
, S phase is limited to a single round per cell cycle through cyclin-dependent kinase phosphorylation of critical replication factors, including the Cdc18 replication initiator protein. Because defects in Cdc18 phosphorylation lead to a hyperstable and hyperactive form of Cdc18 that promotes high levels of overreplication
in vivo
, we wished to identify the components of the Cdc18 proteolysis pathway in fission yeast. In this paper we describe one such component, encoded by the
sud1
+
gene.
sud1
+
shares homology with the budding yeast
CDC4
gene and is required to prevent spontaneous re-replication in fission yeast. Cells lacking
sud1
+
accumulate high levels of Cdc18 and the CDK inhibitor Rum1, because they cannot degrade these two key cell cycle regulators. Through genetic analysis we show that hyperaccumulation of Rum1 contributes to re-replication in
Δsud1
cells, but is not the cause of the defect in Cdc18 proteolysis. Rather, Sud1 itself is associated with the ubiquitin pathway in fission yeast and binds to Cdc18
in vivo
. Most importantly, Sud1-Cdc18 binding requires prior phosphorylation of the Cdc18 polypeptide at CDK consensus sites. These results provide a biochemical mechanism for the phosphorylation-dependent degradation of Cdc18 and other cell cycle regulators, including Rum1. Evolutionary conservation of the Sud1/CDC4 pathway suggests that phosphorylation-coupled proteolysis may be a general feature of nearly all eukaryotic cell cycles.
ADP ribosylation factor regulates spectrin binding to the Golgi complex
Authors: Godi Anna , Santone Ivana , Pertile Paolo , Devarajan Prasad , Stabach Paul R. , Morrow Jon S. , Di Tullio Giuseppe , Polishchuk Roman , Petrucci Tamara C. , Luini Alberto , De Matteis Maria Antonietta
Published: July 2002

Homologues of two major components of the well-characterized erythrocyte plasma-membrane-skeleton, spectrin (a not-yet-cloned isoform, βIΣ* spectrin) and ankyrin (Ank
G119
and an ≈195-kDa ankyrin), associate with the Golgi complex. ADP ribosylation factor (ARF) is a small G protein that controls the architecture and dynamics of the Golgi by mechanisms that remain incompletely understood. We find that activated ARF stimulates the
in vitro
association of βIΣ* spectrin with a Golgi fraction, that the Golgi-associated βIΣ* spectrin contains epitopes characteristic of the βIΣ2 spectrin pleckstrin homology (PH) domain known to bind phosphatidylinositol 4,5-bisphosphate (PtdInsP
2
), and that ARF recruits βIΣ* spectrin by inducing increased PtdInsP
2
levels in the Golgi. The stimulation of spectrin binding by ARF is independent of its ability to stimulate phospholipase D or to recruit coat proteins (COP)-I and can be blocked by agents that sequester PtdInsP
2
. We postulate that a PH domain within βIΣ* Golgi spectrin binds PtdInsP
2
and acts as a regulated docking site for spectrin on the Golgi. Agents that block the binding of spectrin to the Golgi, either by blocking the PH domain interaction or a constitutive Golgi binding site within spectrin’s membrane association domain I, inhibit the transport of vesicular stomatitis virus G protein from endoplasmic reticulum to the medial compartment of the Golgi complex. Collectively, these results suggest that the Golgi-spectrin skeleton plays a central role in regulating the structure and function of this organelle.
Activation of preexisting T cell clones by targeted interleukin 2 therapy
Authors: thor Straten Per , Guldberg Per , Seremet Tina , Reisfeld Ralph A. , Zeuthen Jesper , Becker Jürgen C.
Published: July 2002

The induction of an immunological antitumor response capable of eradicating metastatic tumors is the ultimate goal of immunotherapy. We have recently shown that this can be achieved by interleukin 2 (IL-2) therapy directed to the tumor microenvironment by a recombinant antibody–IL-2 fusion protein. It is not known, however, whether this curative treatment is associated with a predominance of T cells carrying specific T cell receptor variable β regions (TCRBV) or the presence of clonally expanded T cells. To address this question, we have used a quantitative reverse transcriptase-coupled PCR method to analyze the TCRBV region repertoire in tumor-infiltrating lymphocytes of treated and untreated animals. As controls the TCRBV region repertoire was analyzed in blood and skin from disease-free animals. The results indicate an overexpression of TCRBV5 in the tumors of all treated mice and an additional overexpression of individual regions in each tumor. Direct sequencing of these TCRBV regions did not reveal any evidence of clonal expansions. However, since clonal expansions could exist as subpopulations in highly expressed regions, not detectable by direct sequencing, a denaturing gradient gel electrophoresis assay was used for clonal analysis of TCRBV PCR products. Denaturing gradient gel electrophoresis analysis of selected TCRBV regions revealed the presence of clonotypic T cells in tumors from both treated and untreated animals. These data indicate that targeted IL-2 therapy in this model does not induce clonal T cell responsesde novo, rather it acts as an activator for an already existing population of clonotypic T cells.
Transcriptional activating activity of Smad4: Roles of SMAD hetero-oligomerization and enhancement by an associating transactivator
Authors: Shioda Toshi , Lechleider Robert J. , Dunwoodie Sally L. , Li Huchun , Yahata Tetsuro , de Caestecker Mark P. , Fenner Martin H. , Roberts Anita B. , Isselbacher Kurt J.
Published: July 2002

Smad4 plays a pivotal role in signal transduction of the transforming growth factor β superfamily cytokines by mediating transcriptional activation of target genes. Hetero-oligomerization of Smad4 with the pathway-restricted SMAD proteins is essential for Smad4-mediated transcription. We provide evidence that SMAD hetero-oligomerization is directly required for the Smad4 C-terminal domain [Smad4(C)] to show its transcriptional transactivating activity; this requirement obtains even when Smad4(C) is recruited to promoters by heterologous DNA-binding domains and in the absence of the inhibitory Smad4 N-terminal domain. Defined mutations of GAL4 DNA-binding domain fusion of Smad4(C) that disrupt SMAD hetero-oligomerization suppressed transcriptional activation. Importantly, we found that an orphan transcriptional activator MSG1, a nuclear protein that has strong transactivating activity but apparently lacks DNA-binding activity, functionally interacted with Smad4 and enhanced transcription mediated by GAL4 DNA-binding domain-Smad4(C) and full-length Smad4. Transcriptional enhancement by MSG1 depended on transforming growth factor β signaling and was suppressed by Smad4(C) mutations disrupting SMAD hetero-oligomerization or by the presence of Smad4 N-terminal domain. Furthermore, Smad4(C) did not show any detectable transactivating activity in yeast when fused to heterologous DNA-binding domains. These results demonstrate additional roles of SMAD hetero-oligomerization in Smad4-mediated transcriptional activation. They also suggest that the transcriptional-activating activity observed in the presence of Smad4 in mammalian cells may be derived, at least in part, from endogenously expressed separate transcriptional activators, such as MSG1.
New susceptibility locus for rheumatoid arthritis suggested by a genome-wide linkage study
Authors: Cornélis François , Fauré Sabine , Martinez Maria , Prud’homme Jean-François , Fritz Pierre , Dib Colette , Alves Helena , Barrera Pilar , de Vries Niek , Balsa Alejandro , Pascual-Salcedo Dora , Maenaut Kristin , Westhovens René , Migliorini Paola , Tran Tuyet-Hoa , Delaye Arnaud , Prince Nathalie , Lefevre Caroline , Thomas Gaëlle , Poirier Murielle , Soubigou Stéphane , Alibert Olivier , Lasbleiz Sandra , Fouix Sylvaine , Bouchier Christiane , Lioté Frédéric , Loste Marie-Noëlle , Lepage Virginia , Charron Dominique , Gyapay Gabor , Lopes-Vaz Antonio , Kuntz Daniel , Bardin Thomas , Weissenbach Jean
Published: July 2002

Rheumatoid arthritis (RA), the most common autoimmune disease, is associated in families with other autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM). Its genetic component has been suggested by familial aggregation (λs = 5), twin studies, and segregation analysis.
HLA
, which is the only susceptibility locus known, has been estimated to account for one-third of this component. The aim of this paper was to identify new RA loci. A genome scan was performed with 114 European Caucasian RA sib pairs from 97 nuclear families. Linkage was significant only for
HLA
(
P
< 2.5⋅10
−5
) and nominal for 19 markers in 14 other regions (
P
< 0.05). Four of the loci implicated in IDDM potentially overlap with these regions: the putative
IDDM6, IDDM9, IDDM13
, and
DXS998
loci. The first two of these candidate regions, defined in the RA genome scan by the markers
D18S68-D18S61-D18S469
(18q22–23) and
D3S1267
(3q13), respectively, were studied in 194 additional RA sib pairs from 164 nuclear families. Support for linkage to chromosome 3 only was extended significantly (
P
= 0.002). The analysis of all 261 families provided a linkage evidence of
P
= 0.001 and suggested an interaction between this putative RA locus and
HLA
. This locus could account for 16% of the genetic component of RA. Candidate genes include those coding for CD80 and CD86, molecules involved in antigen-specific T cell recognition. In conclusion, this first genome scan in RA Caucasian families revealed 14 candidate regions, one of which was supported further by the study of a second set of families.