bluuebunny/crossref_metadata_2025 huggingface dataset:
Hierarchical self-assembly of chiral rod-like molecules as a model for peptide β-sheet tapes, ribbons, fibrils, and fibers
Authors: Aggeli A. , Nyrkova I. A. , Bell M. , Harding R. , Carrick L. , McLeish T. C. B. , Semenov A. N. , Boden N.
Published: July 2002

A generic statistical mechanical model is presented for the self-assembly of chiral rod-like units, such as β-sheet-forming peptides, into helical tapes, which with increasing concentration associate into twisted ribbons (double tapes), fibrils (twisted stacks of ribbons), and fibers (entwined fibrils). The finite fibril width and helicity is shown to stem from a competition between the free energy gain from attraction between ribbons and the penalty because of elastic distortion of the intrinsically twisted ribbons on incorporation into a growing fibril. Fibers are stabilized similarly. The behavior of two rationally designed 11-aa residue peptides, P
11
-I and P
11
-II, is illustrative of the proposed scheme. P
11
-I and P
11
-II are designed to adopt the β-strand conformation and to self-assemble in one dimension to form antiparallel β-sheet tapes, ribbons, fibrils, and fibers in well-defined solution conditions. The energetic parameters governing self-assembly have been estimated from the experimental data using the model. The 8-nm-wide fibrils consist of eight tapes, are extremely robust (scission energy ≈200
k
B
T
), and sufficiently rigid (persistence length
l̃
fibril
≈ 20–70 μm) to form nematic solutions at peptide concentration
c
≈ 0.9 mM (volume fraction ≈0.0009 vol/vol), which convert to self-supporting nematic gels at
c
> 4 mM. More generally, these observations provide a new insight into the generic self-assembling properties of β-sheet-forming peptides and shed new light on the factors governing the structures and stability of pathological amyloid fibrils
in vivo
. The model also provides a prescription of routes to novel macromolecules based on a variety of self-assembling chiral units, and protocols for extraction of the associated energy changes.
Distinct potassium channels on pain-sensing neurons
Authors: Rasband Matthew N. , Park Eunice W. , Vanderah Todd W. , Lai Josephine , Porreca Frank , Trimmer James S.
Published: July 2002

Differential expression of ion channels contributes functional diversity to sensory neuron signaling. We find nerve injury induced by the Chung model of neuropathic pain leads to striking reductions in voltage-gated K
+
(Kv) channel subunit expression in dorsal root ganglia (DRG) neurons, suggesting a potential molecular mechanism for hyperexcitability of injured nerves. Moreover, specific classes of DRG neurons express distinct Kv channel subunit combinations. Importantly, Kv1.4 is the sole Kv1 α subunit expressed in smaller diameter neurons, suggesting that homomeric Kv1.4 channels predominate in Aδ and C fibers arising from these cells. These neurons are presumably nociceptors, because they also express the VR-1 capsaicin receptor, calcitonin gene-related peptide, and/or Na
+
channel SNS/PN3/Nav1.8. In contrast, larger diameter neurons associated with mechanoreception and proprioception express high levels of Kv1.1 and Kv1.2 without Kv1.4 or other Kv1 α subunits, suggesting that heteromers of these subunits predominate on large, myelinated afferent axons that extend from these cells.
Mutation frequency and biological cost of antibiotic resistance in
Helicobacter pylori
Authors: Björkholm Britta , Sjölund Maria , Falk Per G. , Berg Otto G. , Engstrand Lars , Andersson Dan I.
Published: July 2002

Among the several factors that affect the appearance and
spread of acquired antibiotic resistance, the mutation frequency and
the biological cost of resistance are of special importance.
Measurements of the mutation frequency to rifampicin resistance in
Helicobacter pylori
strains isolated from dyspeptic
patients showed that ≈1/4 of the isolates had higher mutation
frequencies than
Enterobacteriaceae
mismatch-repair
defective mutants. This high mutation frequency could explain why
resistance is so frequently acquired during antibiotic treatment of
H. pylori
infections. Inactivation of the
mutS
gene had no substantial effect on the mutation
frequency, suggesting that MutS-dependent mismatch repair is absent in
this bacterium. Furthermore, clarithromycin resistance conferred a
biological cost, as measured by a decreased competitive ability of the
resistant mutants in mice. In clinical isolates this cost could be
reduced, indicating that compensation is a clinically relevant
phenomenon that could act to stabilize resistant bacteria in a
population.
T cell infiltration of the prostate induced by androgen withdrawal in patients with prostate cancer
Authors: Mercader Maria , Bodner Barbara K. , Moser Micheal T. , Kwon Pamela S. , Park Eugene S. Y. , Manecke Ryan G. , Ellis Thomas M. , Wojcik Eva M. , Yang Damu , Flanigan Robert C. , Waters W. Bedford , Kast W. Martin , Kwon Eugene D.
Published: July 2002

Manipulations capable of breaking host tolerance to induce tissue-specific T cell-mediated inflammation are of central importance to tumor immunotherapy and our understanding of autoimmunity. We demonstrate that androgen ablative therapy induces profuse T cell infiltration of benign glands and tumors in human prostates. T cell infiltration is readily apparent after 7–28 days of therapy and is comprised predominantly of a response by CD4+ T cells and comparatively fewer CD8+ T cells. Also, T cells within the treated prostate exhibit restricted TCR Vβ gene usage, consistent with a local oligoclonal response. Recruitment/activation of antigen-presenting cells in treated prostate tissues may contribute to local T cell activation. The induction of T cell infiltration in prostate tissues treated with androgen ablation may have implications for the immunotherapeutic treatment of prostate cancer as well as other hormone-sensitive malignancies, including breast carcinoma.
Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.
Authors: Erickson J D , Schafer M K , Bonner T I , Eiden L E , Weihe E
Published: July 2002

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.
Arachidonate lipoxygenases as essential regulators of cell survival and apoptosis.
Authors: Tang D G , Chen Y Q , Honn K V
Published: July 2002

Arachidonic acid (AA) metabolites derived from both cyclooxygenase (COX) and lipoxygenase (LOX) pathways transduce a variety of signals related to cell growth. Here, we report that the AA LOX pathway also functions as a critical regulator of cell survival and apoptosis. Rat Walker 256 (W256) carcinosarcoma cells express 12-LOX and synthesize 12(S)- and 15(S)-hydroxyeicosatetraenoic acids as their major LOX metabolites. W256 cells transfected with 12-LOX-specific antisense oligonucleotide or antisense oligonucleotides directed to conserved regions of LOXs underwent time- and dose-dependent apoptosis. Likewise, treatment of W256 cells with various LOX but not COX inhibitors induced apoptotic cell death, which could be partially inhibited by exogenous 12(S)- or 15(S)-hydroxyeicosatetraenoic acids. The W256 cell apoptosis induced by antisense oligos and LOX inhibitors was followed by a rapid downregulation of bcl-2 protein, a dramatic decrease in the bcl-2/bax ratio, and could be suppressed by bcl-2 overexpression. In contrast, p53, which is wild type in W256 cells, did not undergo alterations during apoptosis induction. The results suggest that the LOX pathway plays an important physiological role in regulating apoptosis.
Conditional reduction of human immunodeficiency virus type 1 replication by a gain-of-herpes simplex virus 1 thymidine kinase function.
Authors: Smith S M , Markham R B , Jeang K T
Published: July 2002

The development of an effective vaccine for human immunodeficiency virus type 1 (HIV-1) would be a major advance toward controlling the AIDS pandemic. Several disparate strategies for a safe and effective HIV vaccine have been proposed. Recent data suggest that loss-of-function live-attenuated virus could be a safe lentivirus vaccine. Here, we propose a gain-of-function approach that can complement loss-of-function in enhancing the safety profile of a live-attenuated virus. We describe an example in which ganciclovir (GCV) was used to treat effectively nef(-)HIV-1 engineered to express herpes simplex virus (HSV-1) thymidine kinase (TK). This treatment was found to be highly efficient in controlling HIV-1 spread in tissue culture and in a small animal (hu-PBL-SCID) model. We demonstrate that one distinct advantage of GCV-HSV-TK treatment is the elimination of integrated proviruses, a goal not easily achieved with other antiretrovirals.
A human homologue of the Drosophila sex determination factor transformer-2 has conserved splicing regulatory functions.
Authors: Dauwalder B , Amaya-Manzanares F , Mattox W
Published: July 2002

Regulation of gene expression through alternative pre-mRNA splicing appears to occur in all metazoans, but most of our knowledge about splicing regulators derives from studies on genetically identified factors from Drosophila. Among the best studied of these is the transformer-2 (TRA-2) protein which, in combination with the transformer (TRA) protein, directs sex-specific splicing of pre-mRNA from the sex determination gene doublesex (dsx). Here we report the identification of htra-2 alpha, a human homologue of tra-2. Two alternative types of htra-2 alpha cDNA clones were identified that encode different protein isoforms with striking organizational similarity to Drosophila tra-2 proteins. When expressed in flies, one hTRA-2 alpha isoform partially replaces the function of Drosophila TRA-2, affecting both female sexual differentiation and alternative splicing of dsx pre-mRNA. Like Drosophila TRA-2, the ability of hTRA-2 alpha to regulate dsx is female-specific and depends on the presence of the dsx splicing enhancer. These results demonstrate that htra-2 alpha has conserved a striking degree of functional specificity during evolution and leads us to suggest that, although they are likely to serve different roles in development, the tra-2 products of flies and humans have similar molecular functions.
The genetic basis of epidermolysis bullosa simplex with mottled pigmentation.
Authors: Uttam J , Hutton E , Coulombe P A , Anton-Lamprecht I , Yu Q C , Gedde-Dahl T , Fine J D , Fuchs E
Published: July 2002

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant skin diseases characterized by blistering, due to mechanical stress-induced degeneration of basal epidermal cells. It is now well-established that the three major subtypes of EBS are genetic disorders of the basal epidermal keratins, keratin 5 (K5) and keratin 14 (K14). Here we show that a rare subtype, referred to as EBS with mottled pigmentation (MP), is also a disorder of these keratins. Affected members of two seemingly unrelated families with EBS-MP had a C to T point mutation in the second base position of codon 24 of one of two K5 alleles, leading to a Pro: Leu mutation. This mutation was not present in unaffected members nor in 100 alleles from normal individuals. Linkage analyses mapped the defect to this type II keratin gene (peak logarithm of odds score at phi = 0 of 3.9), which is located on chromosome 12q11-q13. This provides strong evidence that this mutation is responsible for the EBS-MP phenotype. Only conserved between K5 and K6, and not among any of the other type II keratins, Pro-24 is in the nonhelical head domain of K5, and only mildly perturbs the length of 10-nm keratin filaments assembled in vitro. However, this part of the K5 head domain is likely to protrude on the filament surface, perhaps leading to additional aberrations in intermediate filament architecture and/or in melanosome distribution that are seen ultrastructurally in patients with the mutation.
Crystal structure of phage P22 tailspike protein complexed with Salmonella sp. O-antigen receptors.
Authors: Steinbacher S , Baxa U , Miller S , Weintraub A , Seckler R , Huber R
Published: July 2002

The O-antigenic repeating units of lipopolysaccharides from Salmonella serogroups A, B, and D1 serve as receptors for the phage P22 tailspike protein, which also has receptor destroying endoglycosidase (endorhamnosidase) activity, integrating the functions of both hemagglutinin and neuraminidase in influenza virus. Crystal structures of the tailspike protein in complex with oligosaccharides, comprising two O-antigenic repeating units from Salmonella typhimurium, Salmonella enteritidis, and Salmonella typhi 253Ty were determined at 1.8 A resolution. The active-site topology with Asp-392, Asp-395, and Glu-359 as catalytic residues was identified. Kinetics of binding and cleavage suggest a role of the receptor destroying endorhamnosidase activity primarily for detachment of newly assembled phages.
Functions of interleukin 1 receptor antagonist in gene knockout and overproducing mice.
Authors: Hirsch E , Irikura V M , Paul S M , Hirsh D
Published: July 2002

Interleukin 1 receptor antagonist (IL-1ra) is a cytokine whose only known action is competitive inhibition of the binding of interleukin 1 (IL-1) to its receptor. To investigate the physiological roles of endogenously produced IL-1ra, we generated mice that either lack IL-1ra or overproduce it under control of the endogenous promoter. Mice lacking IL-1ra have decreased body mass compared with wild-type controls. They are more susceptible than controls to lethal endotoxemia but are less susceptible to infection with Listeria monocytogenes. Conversely, IL-1ra overproducers are protected from the lethal effects of endotoxin but are more susceptible to listeriosis. Serum levels of IL-1 following an endotoxin challenge are decreased in IL-1ra nulls and increased in IL-1ra overproducers in comparison to controls. These data demonstrate critical roles for endogenously produced IL-1ra in growth, responses to infection and inflammation, and regulation of cytokine expression.
Symmetry arguments in chemistry
Authors: Dunitz Jack D.
Published: July 2002

The use (and misuse) of symmetry arguments in constructing
molecular models and in the interpretation of experimental observations
bearing on molecular structure (spectroscopy, diffraction, etc.) is
discussed. Examples include the development of point groups and space
groups for describing the external and internal symmetry of crystals,
the derivation of molecular symmetry by counting isomers (the benzene
structure), molecular chirality, the connection between macroscopic and
molecular chirality, pseudorotation, the symmetry group of nonrigid
molecules, and the use of orbital symmetry arguments in discussing
aspects of chemical reactivity.
Conversion of inactive glycosylation inhibiting factor to bioactive derivatives by modification of a SH group
Authors: Nakano Tatsumi , Watarai Hiroshi , Liu Yun-Cai , Oyama Yukihito , Mikayama Toshifumi , Ishizaka Kimishige
Published: July 2002

Escherichia coli-derived recombinant human glycosylation inhibiting factor (rhGIF) contains three cysteine residues (Cys-57, -60, and -81). All SH groups in the cysteine residues are free, and the GIF molecule had no biologic activity. Carboxymethylation of the SH group of Cys-60 in the molecule resulted in the generation of bioactivity, although the activity of the carboxymethylated GIF was 10- to 20-fold less than that of suppressor T cell (Ts)-derived GIF. However, treatment of the inactive rhGIF with ethylmercurithiosalicylate or 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in the generation of derivatives whose bioactivity was comparable to that of the Ts-derived bioactive GIF. The activity of these derivatives was lost by treatment with DTT. Isolation and chemical analysis of the DTNB-treated GIF derivative revealed that binding the 5-thio-2-nitrobenzoic acid group with Cys-60 was responsible for the generation of the highly bioactive derivative. Inactive cytosolic GIF from mammalian cells could also be converted to bioactive derivative by treatment with the SH reagent, while Ts-derived bioactive GIF was inactivated by DTT. These results, together with an x-ray crystal structure of GIF molecules, strongly suggest that the generation of bioactivity of GIF in Ts cells is due to posttranslational modifications that result in conformational changes in the molecule.
Likelihood-mapping: A simple method to visualize phylogenetic content of a sequence alignment
Authors: Strimmer Korbinian , von Haeseler Arndt
Published: July 2002

We introduce a graphical method, likelihood-mapping, to visualize the phylogenetic content of a set of aligned sequences. The method is based on an analysis of the maximum likelihoods for the three fully resolved tree topologies that can be computed for four sequences. The three likelihoods are represented as one point inside an equilateral triangle. The triangle is partitioned in different regions. One region represents star-like evolution, three regions represent a well-resolved phylogeny, and three regions reflect the situation where it is difficult to distinguish between two of the three trees. The location of the likelihoods in the triangle defines the mode of sequence evolution. If
n
sequences are analyzed, then the likelihoods for each subset of four sequences are mapped onto the triangle. The resulting distribution of points shows whether the data are suitable for a phylogenetic reconstruction or not.
Molecular delineation of the smallest commonly deleted region of chromosome 5 in malignant myeloid diseases to 1–1.5 Mb and preparation of a PAC-based physical map
Authors: Zhao Nanding , Stoffel Archontoula , Wang Pauline W. , Eisenbart James D. , Espinosa Rafael , Larson Richard A. , Le Beau Michelle M.
Published: July 2002

Loss of a whole chromosome 5 or a deletion of the long arm, del(5q), is a recurring abnormality in malignant myeloid diseases. In previous studies, we delineated a commonly deleted segment of ≈4 Mb within band 5q31 that was flanked by
IL9
on the proximal side and D5S166 on the distal side. We have generated a physical map of P1 (PAC), bacterial (BAC), and yeast artificial chromosome (YAC) clones of this interval. The contig consists of 108 clones (78 PACs, 2 BACs, and 28 YACs) to which 125 markers (5 genes, 11 expressed sequence tags, 12 polymorphisms, and 97 sequence-tagged sites) have been mapped. Using PAC clones for fluorescence
in situ
hybridization analysis of leukemia cells with a del(5q), we have narrowed the commonly deleted segment to 1–1.5 Mb between D5S479 and D5S500. To search for allele loss, we used 7 microsatellite markers within and flanking the commonly deleted segment to examine leukemia cells from 28 patients with loss of 5q, and 14 patients without cytogenetically detectable loss of 5q. In the first group of patients, we detected hemizygous deletions, consistent with the cytogenetically visible loss; no homozygous deletions were detected. No allele loss was detected in patients without abnormalities of chromosome 5, suggesting that allele loss on 5q is the result of visible chromosomal abnormalities. The development of a stable PAC contig and the identification of the smallest commonly deleted segment will facilitate the molecular cloning of a myeloid leukemia suppressor gene on 5q.
Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats
Authors: Bernabeu Ramon , Bevilaqua Lia , Ardenghi Patricia , Bromberg Elke , Schmitz Paulo , Bianchin Marino , Izquierdo Ivan , Medina Jorge H.
Published: July 2002

cAMP/cAMP-dependent protein kinase (PKA) signaling pathway has been recently proposed to participate in both the late phase of long term potentiation in the hippocampus and in the late, protein synthesis-dependent phase of memory formation. Here we report that a late memory consolidation phase of an inhibitory avoidance learning is regulated by an hippocampal cAMP signaling pathway that is activated, at least in part, by D
1
/D
5
receptors. Bilateral infusion of SKF 38393 (7.5 μg/side), a D
1
/D
5
receptor agonist, into the CA
1
region of the dorsal hippocampus, enhanced retention of a step-down inhibitory avoidance when given 3 or 6 h, but not immediately (0 h) or 9 h, after training. In contrast, full retrograde amnesia was obtained when SCH 23390 (0.5 μg/side), a D
1
/D
5
receptor antagonist, was infused into the hippocampus 3 or 6 h after training. Intrahippocampal infusion of 8Br-cAMP (1.25 μg/side), or forskolin (0.5 μg/side), an activator of adenylyl cyclase, enhanced memory when given 3 or 6 h after training. KT5720 (0.5 μg/side), a specific inhibitor of PKA, hindered memory consolidation when given immediately or 3 or 6 h posttraining. Rats submitted to the avoidance task showed learning-specific increases in hippocampal
3
H-SCH 23390 binding and in the endogenous levels of cAMP 3 and 6 h after training. In addition, PKA activity and P-CREB (phosphorylated form of cAMP responsive element binding protein) immunoreactivity increased in the hippocampus immediately and 3 and 6 h after training. Together, these findings suggest that the late phase of memory consolidation of an inhibitory avoidance is modulated cAMP/PKA signaling pathways in the hippocampus.
Genes, peoples, and languages
Authors: Cavalli-Sforza L. Luca
Published: July 2002

The genetic history of a group of populations is usually analyzed
by reconstructing a tree of their origins. Reliability of the
reconstruction depends on the validity of the hypothesis that genetic
differentiation of the populations is mostly due to population fissions
followed by independent evolution. If necessary, adjustment for major
population admixtures can be made. Dating the fissions requires
comparisons with paleoanthropological and paleontological dates, which
are few and uncertain. A method of absolute genetic dating recently
introduced uses mutation rates as molecular clocks; it was applied to
human evolution using microsatellites, which have a sufficiently high
mutation rate. Results are comparable with those of other methods and
agree with a recent expansion of modern humans from Africa. An
alternative method of analysis, useful when there is adequate
geographic coverage of regions, is the geographic study of frequencies
of alleles or haplotypes. As in the case of trees, it is necessary to
summarize data from many loci for conclusions to be acceptable. Results
must be independent from the loci used. Multivariate analyses like
principal components or multidimensional scaling reveal a number of
hidden patterns and evaluate their relative importance. Most patterns
found in the analysis of human living populations are likely to be
consequences of demographic expansions, determined by technological
developments affecting food availability, transportation, or military
power. During such expansions, both genes and languages are spread to
potentially vast areas. In principle, this tends to create a
correlation between the respective evolutionary trees. The correlation
is usually positive and often remarkably high. It can be decreased or
hidden by phenomena of language replacement and also of gene
replacement, usually partial, due to gene flow.
Importance of epistasis as the genetic basis of heterosis in an elite rice hybrid
Authors: Yu S. B. , Li J. X. , Xu C. G. , Tan Y. F. , Gao Y. J. , Li X. H. , Zhang Qifa , Maroof M. A. Saghai
Published: July 2002

The genetic basis of heterosis was investigated in an elite rice hybrid by using a molecular linkage map with 150 segregating loci covering the entire rice genome. Data for yield and three traits that were components of yield were collected over 2 years from replicated field trials of 250 F
2:3
families. Genotypic variations explained from about 50% to more than 80% of the total variation. Interactions between genotypes and years were small compared with the main effects. A total of 32 quantitative trait loci (QTLs) were detected for the four traits; 12 were observed in both years and the remaining 20 were detected in only one year. Overdominance was observed for most of the QTLs for yield and also for a few QTLs for the component traits. Correlations between marker heterozygosity and trait expression were low, indicating that the overall heterozygosity made little contribution to heterosis. Digenic interactions, including additive by additive, additive by dominance, and dominance by dominance, were frequent and widespread in this population. The interactions involved large numbers of marker loci, most of which individually were not detectable on single-locus basis; many interactions among loci were detected in both years. The results provide strong evidence that epistasis plays a major role as the genetic basis of heterosis.
Characterization of the antiproliferative signal mediated by the somatostatin receptor subtype sst5
Authors: Cordelier Pierre , Estève Jean-Pierre , Bousquet Corinne , Delesque Nathalie , O’Carroll Anne-Marie , Schally Andrew V. , Vaysse Nicole , Susini Christiane , Buscail Louis
Published: July 2002

We investigated cell proliferation modulated by cholecystokinin (CCK) and somatostatin analogue RC-160 in CHO cells bearing endogenous CCK
A
receptors and stably transfected by human subtype sst5 somatostatin receptor. CCK stimulated cell proliferation of CHO cells. This effect was suppressed by inhibitor of the soluble guanylate cyclase, LY 83583, the inhibitor of the cGMP dependent kinases, KT 5823, and the inhibitor of mitogen-activated protein (MAP) kinase kinase, PD 98059. CCK treatment induced an increase of intracellular cGMP concentrations, but concomitant addition of LY 83583 virtually suppressed this increase. CCK also activated both phosphorylation and activity of p42-MAP kinase; these effects were inhibited by KT 5823. All the effects of CCK depended on a pertussis toxin-dependent G protein. Somatostatin analogue RC-160 inhibited CCK-induced stimulation of cell proliferation but it did not potentiate the suppressive effect of the inhibitors LY 83583 and KT 5823. RC-160 inhibited both CCK-induced intracellular cGMP formation as well as activation of p42-MAP kinase phosphorylation and activity. This inhibitory effect was observed at doses of RC-160 similar to those necessary to occupy the sst5 recombinant receptor and to inhibit CCK-induced cell proliferation. We conclude that, in CHO cells, the proliferation and the MAP kinase signaling cascade depend on a cGMP-dependent pathway. These effects are positively regulated by CCK and negatively influenced by RC-160, interacting through CCK
A
and sst5 receptors, respectively. These studies provide a characterization of the antiproliferative signal mediated by sst5 receptor.
Increased interleukin 12 production in progressive multiple sclerosis: Induction by activated CD4+T cells via CD40 ligand
Authors: Balashov Konstantin E. , Smith Derek R. , Khoury Samia J. , Hafler David A. , Weiner Howard L.
Published: July 2002

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system postulated to be a cell-mediated autoimmune disease in which interferon γ (IFN-γ) plays an important role. There is increased IFN-γ secretion in MS, and IFN-γ administration induces exacerbations of disease. We found that interleukin 12 (IL-12) was responsible for raised IFN-γ secretion in MS as anti-IL-12 antibodies reversed raised anti-CD3-induced IFN-γ in MS patients to normal levels. Furthermore, we found a marked increase in T cell receptor-mediated IL-12 secretion in progressive MS patients vs. controls (24.8 ± 7.7 pg/ml vs. 1.5 ± 1.0 pg/ml,P= 0.003) and vs. relapsing–remitting patients (3.7 ± 1.4 pg/ml,P< 0.05). Investigation of the cellular basis for raised IL-12 demonstrated that T cells from MS patients induced IL-12 secretion from non-T cells, and that T cells from MS patients could even drive non-T cells from normal subjects to produce increased IL-12. Anti-CD40 ligand antibody completely blocked IL-12 secretion induced by activated T cells, and we found increased CD40 ligand expression by activated CD4+T cells in MS patients vs. controls. The CD40 ligand-dependent Th1-type immune activation was observed in the progressive but not in the relapsing–remitting form of MS, suggesting a link to disease pathogenesis and progression and providing a basis for immune intervention in the disease.
N-terminal sequences from
Autographa californica
nuclear polyhedrosis virus envelope proteins ODV-E66 and ODV-E25 are sufficient to direct reporter proteins to the nuclear envelope, intranuclear microvesicles and the envelope of occlusion derived virus
Authors: Hong Tao , Summers Max D. , Braunagel Sharon C.
Published: July 2002

Baculovirus occlusion-derived virus (ODV) derives its envelope from an intranuclear membrane source. N-terminal amino acid sequences of the
Autographa californica
nuclear polyhedrosis virus (
Ac
MNPV) envelope proteins, ODV-E66 and ODV-E25 (23 and 24 amino acids, respectively) are highly hydrophobic. Recombinant viruses that express the two N-terminal amino acid sequences fused to green fluorescent protein (23GFP or 24GFP) provided visual markers to follow protein transport and localization within the nucleus during infection. Autoflourescence was first detected along the cytoplasmic periphery of the nucleus and subsequently localized as foci to discrete locations within the nucleus. Immunoelectron microscopy confirmed that these foci predominantly contained intranuclear microvesicles and the reporter fusion proteins were also detected in cytoplasmic membranes near the nucleus, and the outer and inner nuclear membrane. Therefore, these defined hydrophobic domains are sufficient to direct native and fusion proteins to induced membrane microvesicles within a baculovirus-infected cell nucleus and the viral envelope. In addition, these data suggest that movement of these proteins into the nuclear envelope may initiate through cytoplasmic membranes, such as endoplasmic reticulum, and that transport into the nucleus may be mediated through the outer and inner nuclear membrane.
Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity
Authors: Sibson Nicola R. , Dhankhar Ajay , Mason Graeme F. , Rothman Douglas L. , Behar Kevin L. , Shulman Robert G.
Published: July 2002

To determine the relationship between cerebral Glc metabolism and glutamatergic neuronal function, we used
13
C NMR spectroscopy to measure, simultaneously, the rates of the tricarboxylic acid cycle and Gln synthesis in the rat cortex
in vivo
. From these measurements, we calculated the rates of oxidative Glc metabolism and glutamate–neurotransmitter cycling between neurons and astrocytes (a quantitative measure of glutamatergic neuronal activity). By measuring the rates of the tricarboxylic acid cycle and Gln synthesis over a range of synaptic activity, we have determined the stoichiometry between oxidative Glc metabolism and glutamate–neurotransmitter cycling in the cortex to be close to 1:1. This finding indicates that the majority of cortical energy production supports functional (synaptic) glutamatergic neuronal activity. Another implication of this result is that brain activation studies, which map cortical oxidative Glc metabolism, provide a quantitative measure of synaptic glutamate release.
Controlling signaling with a specifically designed G
i
-coupled receptor
Authors: Coward Peter , Wada H. Gary , Falk Matthew S. , Chan Sam D. H. , Meng Fan , Akil Huda , Conklin Bruce R.
Published: July 2002

We are developing a system to control G protein signaling
in vivo
to regulate a broad range of physiologic responses. Our system utilizes G protein-coupled peptide receptors engineered to respond exclusively to synthetic small molecule ligands and not to their natural ligand(s). These engineered receptors are designated RASSLs (receptor activated solely by a synthetic ligand). We have made two prototype RASSLs that are based on the human κ opioid receptor. Small molecule drugs that activate the κ receptor are nonaddictive and safe to administer
in vivo
. Binding and signaling assays reveal 200–2000-fold reductions in the ability of our RASSLs to bind or be activated by dynorphin, an endogenous peptide ligand of the κ opioid receptor. In a high-throughput signaling assay, these prototype RASSLs expressed in Chinese hamster ovary K1 cells showed little or no response to a panel of 21 opioid peptides but still signaled normally in response to small molecule drugs such as spiradoline. Activation of a RASSL by spiradoline also caused proliferation of rat-1a tissue culture cells. These data provide evidence that G protein-coupled receptors can be made into RASSLs. The potential
in vivo
applications for RASSLs include the positive enrichment of transfected cells and the development of new animal models of disease.
Persistent inhibition of cell respiration by nitric oxide: Crucial role ofS-nitrosylation of mitochondrial complex I and protective action of glutathione
Authors: Clementi Emilio , Brown Guy Charles , Feelisch Martin , Moncada Salvador
Published: July 2002

Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of nitric oxide (NO) by cells. Using J774 cells, we have studied the effect of long-term exposure to NO on different enzymes of the respiratory chain. Our results show that, although NO inhibits complex IV in a way that is always reversible, prolonged exposure to NO results in a gradual and persistent inhibition of complex I that is concomitant with a reduction in the intracellular concentration of reduced glutathione. This inhibition appears to result fromS-nitrosylation of critical thiols in the enzyme complex because it can be immediately reversed by exposing the cells to high intensity light or by replenishment of intracellular reduced glutathione. Furthermore, decreasing the concentration of reduced glutathione accelerates the process of persistent inhibition. Our results suggest that, although NO may regulate cell respiration physiologically by its action on complex IV, long-term exposure to NO leads to persistent inhibition of complex I and potentially to cell pathology.
The 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis
Authors: Liu Xuesong , Li Peng , Widlak Piotr , Zou Hua , Luo Xu , Garrard William T. , Wang Xiaodong
Published: July 2002

We report here the reconstitution of a pathway that leads to the apoptotic changes in nuclei by using recombinant DNA fragmentation factor (DFF), a heterodimeric protein of 40 and 45 kDa. Coexpression of DFF40 and DFF45 is required to generate recombinant DFF, which becomes activated when DFF45 is cleaved by caspase-3. The cleaved fragments of DFF45 dissociate from the DFF40, the active component of DFF. Purified DFF40 exhibited an intrinsic DNase activity that was markedly stimulated by chromatin-associated proteins histone H1 and high mobility group proteins. DFF40 also triggered chromatin condensation when incubated with nuclei. These data suggest that DFF40 is sufficient to trigger both DNA fragmentation and chromatin condensation during apoptosis.
A bacterial cytokine
Authors: Mukamolova Galina V. , Kaprelyants Arseny S. , Young Danielle I. , Young Michael , Kell Douglas B.
Published: July 2002

Viable cells of
Micrococcus luteus
secrete a factor, which promotes the resuscitation and growth of dormant, nongrowing cells of the same organism. The resuscitation-promoting factor (Rpf) is a protein, which has been purified to homogeneity. In picomolar concentrations, it increases the viable cell count of dormant
M. luteus
cultures at least 100-fold and can also stimulate the growth of viable cells. Rpf also stimulates the growth of several other high G+C Gram-positive organisms, including
Mycobacterium avium, Mycobacterium bovis
(BCG),
Mycobacterium kansasii
,
Mycobacterium smegmatis,
and
Mycobacterium tuberculosis
. Similar genes are widely distributed among high G+C Gram-positive bacteria; genome sequencing has uncovered examples in
Mycobacterium leprae
and
Mb. tuberculosis
and others have been detected by hybridization in
Mb. smegmatis
,
Corynebacterium glutamicum,
and
Streptomyces
spp. The mycobacterial gene products may provide different targets for the detection and control of these important pathogens. This report is thus a description of a proteinaceous autocrine or paracrine bacterial growth factor or cytokine.
Gene discovery in the wood-forming tissues of poplar: Analysis of 5,692 expressed sequence tags
Authors: Sterky Fredrik , Regan Sharon , Karlsson Jan , Hertzberg Magnus , Rohde Antje , Holmberg Anders , Amini Bahram , Bhalerao Rupali , Larsson Magnus , Villarroel Raimundo , Van Montagu Marc , Sandberg Göran , Olsson Olof , Teeri Tuula T. , Boerjan Wout , Gustafsson Petter , Uhlén Mathias , Sundberg Björn , Lundeberg Joakim
Published: July 2002

A rapidly growing area of genome research is the generation of expressed sequence tags (ESTs) in which large numbers of randomly selected cDNA clones are partially sequenced. The collection of ESTs reflects the level and complexity of gene expression in the sampled tissue. To date, the majority of plant ESTs are from nonwoody plants such as
Arabidopsis
,
Brassica
, maize, and rice. Here, we present a large-scale production of ESTs from the wood-forming tissues of two poplars,
Populus tremula
L. ×
tremuloides
Michx. and
Populus trichocarpa
‘Trichobel.’ The 5,692 ESTs analyzed represented a total of 3,719 unique transcripts for the two cDNA libraries. Putative functions could be assigned to 2,245 of these transcripts that corresponded to 820 protein functions. Of specific interest to forest biotechnology are the 4% of ESTs involved in various processes of cell wall formation, such as lignin and cellulose synthesis, 5% similar to developmental regulators and members of known signal transduction pathways, and 2% involved in hormone biosynthesis. An additional 12% of the ESTs showed no significant similarity to any other DNA or protein sequences in existing databases. The absence of these sequences from public databases may indicate a specific role for these proteins in wood formation. The cDNA libraries and the accompanying database are valuable resources for forest research directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.
Tumor cells expressing a retroviral envelope escape immune rejectionin vivo
Authors: Mangeney Marianne , Heidmann Thierry
Published: July 2002

A model system for thein vivocontrol of tumor cell proliferation by the immune system has been used to assay for the possible immunosuppressive activity of retroviral proteins. Expression vectors for the entire or the transmembrane subunit of the Moloney murine leukemia virus envelope protein were constructed, as well as control vectors for irrelevant transmembrane proteins—or no protein. They were introduced either into MCA205 murine tumor cells, which do not proliferate upon s.c. injection into an allogeneic host, or into CL8.1 murine tumor cells, which overexpress class I antigens and are rejected in a syngeneic host. In both cases, expression of the complete envelope protein or of the transmembrane subunit resulted in tumor growthin vivo, with no effect of control vectors. Tumor cell growth results from inhibition of the host immune response, as the envelope-dependent effect was no more observed for MCA205 cells in syngeneic mice or for CL8.1 cells in x-irradiated mice. This inhibition is local because it is not observed at the level of control tumor cells injected contralaterally. These results suggest a noncanonical function of retroviral envelopes in the “penetrance” of viral infections, as well as a possible involvement of the envelope proteins of endogenous retroviruses in tumoral processes.
The barrier-to-autointegration protein is a host factor for HIV type 1 integration
Authors: Chen Hongmin , Engelman Alan
Published: July 2002

In vivo
, retroviral integration is mediated by a large nucleoprotein complex, termed the preintegration complex (PIC). PICs isolated from infected cells display
in vitro
integration activity. Here, we analyze the roles of different host cell factors in the structure and function of HIV type 1 (HIV-1) PICs. PICs purified by size exclusion after treatment with high salt lost their integration activity, and adding back an extract from uninfected cells restored this activity. In parallel, the native protein–DNA intasome structure detected at the ends of HIV-1 by Mu-mediated PCR footprinting was abolished by high salt and restored by the crude cell extract. Various purified proteins previously implicated in retroviral PIC function then were analyzed for their effects on the structure and function of salt-treated HIV-1 PICs. Whereas relatively low amounts (5–20 nM) of human barrier-to-autointegration factor (BAF) protein restored integration activity, substantially more (5–10 μM) human host factor HMG I(Y) was required. Similarly high levels (3–8 μM) of bovine RNase A, a DNA-binding protein used as a nonspecific control, also restored activity. Mu-mediated PCR footprinting revealed that of these three purified proteins, only BAF restored the native structure of the HIV-1 protein–DNA intasome. We suggest that BAF is a natural host cofactor for HIV-1 integration.
Calibrated functional MRI: Mapping the dynamics of oxidative metabolism
Authors: Davis Timothy L. , Kwong Kenneth K. , Weisskoff Robert M. , Rosen Bruce R.
Published: July 2002

MRI was extended to the measurement of changes in oxidative metabolism in the normal human during functionally induced changes in cellular activity. A noninvasive MRI method that is model-independent calibrates the blood oxygen level dependent (BOLD) signal of functional MRI (fMRI) against perfusion-sensitive MRI, using carbon dioxide breathing as a physiological reference standard. This calibration procedure provides a regional measurement of the expected sensitivity of the fMRI BOLD signal to changes in the cellular activity of the brain. Maps of the BOLD signal calibration factor showed regional heterogeneity, indicating that the magnitude of functionally induced changes in the BOLD signal will be dependent on both the local change in blood flow and the local baseline physiology of the cerebral cortex. BOLD signal magnitude is shown to be reduced by 32% from its expected level by the action of oxygen metabolism. The calibrated fMRI technique was applied to stimulation of the human visual cortex with an alternating radial checkerboard pattern. With this stimulus oxygen consumption increased 16% whereas blood flow increased 45%. Although this result is consistent with previous findings of a significant difference between the increase in blood flow and oxygen consumption, it does indicate clearly that oxidative metabolism is a significant component of the metabolic response of the brain to functionally induced changes in cellular activity.
Structure comparison of human glioma pathogenesis-related protein GliPR and the plant pathogenesis-related protein P14a indicates a functional link between the human immune system and a plant defense system
Authors: Szyperski T. , Fernández C. , Mumenthaler C. , Wüthrich K.
Published: July 2002

The human glioma pathogenesis-related protein (GliPR) is highly expressed in the brain tumor glioblastoma multiforme and exhibits 35% amino acid sequence identity with the tomato pathogenesis-related (PR) protein P14a, which has an important role for the plant defense system. A molecular model of GliPR was computed with the distance geometry programdianaon the basis of a P14a–GliPR sequence alignment and a set of 1,200 experimental NMR conformational constraints collected with P14a. The GliPR structure is represented by a group of 20 conformers with small residualdianatarget function values, lowamber-energies after restrained energy-minimization with the programopal, and an average rms deviation relative to the mean of 1.6 Å for the backbone heavy atoms. Comparison of the GliPR model with the P14a structure lead to the identification of a common partially solvent-exposed spatial cluster of four amino acid residues, His-69, Glu-88, Glu-110, and His-127 in the GliPR numeration. This cluster is conserved in all known plant PR proteins of class 1, indicating a common putative active site for GliPR and PR-1 proteins and thus a functional link between the human immune system and a plant defense system.
Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2
Authors: Aragay A. M. , Mellado M. , Frade J. M. R. , Martin A. M. , Jimenez-Sainz M. C. , Martinez-A C. , Mayor F.
Published: July 2002

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called β-adrenergic kinase 1 or βARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein β-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant βARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines.