bluuebunny/crossref_metadata_2025 huggingface dataset:
A common ancestry for multiple catalytic antibodies generated against a single transition-state analog.
Authors: Miyashita H , Hara T , Tanimura R , Tanaka F , Kikuchi M , Fujii I
Published: June 2006

Immunization with a single haptenic transition-state analog generates a few catalytic antibodies among the dozens of antibodies capable of binding the hapten. The diversity of the immune response has raised some fundamental issues, such as How do catalytic and noncatalytic antibodies differ on a structural basis? To address this issue, the variable region primary sequences of 11 antibodies (including 6 catalytic and 5 noncatalytic antibodies) elicited against a single haptenic transition-state analog were deduced from cDNA sequences. Cluster analyses using phylogenetic trees constructed by the neighbor-joining method have revealed that the amino acid sequences of noncatalytic antibodies bear no relationship to one another, while the catalytic antibodies share significant structural identity. Furthermore, no catalytic antibodies possessing amino acid sequences with high homology to those of noncatalytic antibodies were detected. Five catalytic antibodies examined showed 89-95% and 74-84% sequence homologies in the complete light- and heavy-chain variable regions, respectively. Thus, it seems likely that the catalytic antibodies elicited against a single hapten use the canonical set of variable region genes. Interestingly, one catalytic antibody showed only limited sequence similarity to the other catalytic antibodies and was found to exhibit a distinctly different substrate specificity. From the broad range of their binding constants to the hapten, it is unlikely that highly homologous catalytic antibodies are generated as a result of simple high-affinity choices. These results emphasize the utility of rationally designed transition-state analogs for the induction of antibody molecules with catalytic activity.
Milk composition and lactation of beta-casein-deficient mice.
Authors: Kumar S , Clarke A R , Hooper M L , Horne D S , Law A J , Leaver J , Springbett A , Stevenson E , Simons J P
Published: June 2006

beta-Casein is a major protein component of milk and, in conjunction with the other caseins, it is assembled into micelles. The casein micelles determine many of the physical characteristics of milk, which are important for stability during storage and for milk-processing properties. There is evidence that suggests that beta-casein may also possess other, nonnutritional functions. To address the function of beta-casein, the mouse beta-casein gene was disrupted by gene targeting in embryonic stem cells. Homozygous beta-casein mutant mice are viable and fertile; females can lactate and successfully rear young. beta-Casein was expressed at a reduced level in heterozygotes and was completely absent from the milk of homozygous mutant mice. Despite the deficiency of beta-casein, casein micelles were assembled in heterozygous and homozygous mutants, albeit with reduced diameters. The absence of beta-casein expression was reflected in a reduced total protein concentration in milk, although this was partially compensated for by an increased concentration of other proteins. The growth of pups feeding on the milk of homozygous mutants was reduced relative to those feeding on the milk of wild-type mice. Various genetic manipulations of caseins have been proposed for the qualitative improvement of cow's milk composition. The results presented here demonstrate that beta-casein has no essential function and that the casein micelle is remarkably tolerant of changes in composition.
Evidence for intron capture: an unusual path for the evolution of proteins.
Authors: Golding G B , Tsao N , Pearlman R E
Published: June 2006

Most new genes are thought to evolve from preexisting genes but duplications of entire genes or shuffling of preexisting exons provides only a limited repertoire of new sequences that can be presented to a cell. Only pieces that previously existed can be used in the construction and any further divergence depends on the slow accumulation of mutations. We show here the presence of a small, in-frame intron in a ciliate phosphoglycerate kinase gene and the insertion of an unusually random amino acid sequence at the same position in trypanosome phosphoglycerate kinase. The unusual sequences in trypanosomes were likely to have originally been introns that have been subsequently captured by the protein and have now been incorporated as part of the coding sequence. Via this path a truly unique sequence can be incorporated into an existing protein, leading in time to the evolution of a new, functionally distinct protein.
Excited-state intramolecular proton transfer as a fluorescence probe for protein binding-site static polarity.
Authors: Sytnik A , Kasha M
Published: June 2006

A fluorescence probe is introduced for protein conformation and binding-site monitoring as the proton-transfer (PT) tautomer fluorescence by using 4-hydroxy-5-azaphenanthrene (HAP) as a prototype. A typical grossly-wavelength-shifted PT fluorescence for HAP is observed in the 600-nm spectral region for this UV-absorbing molecule (absorption onset, 400 nm), for which case PT occurs even in protic solvents. It is shown that PT fluorescence of HAP can serve as a protein-binding-site static-polarity calibrator, shifting from a lambda max of 612 nm in cyclohexane to 585 nm in ethanol at 298 K, contrary to the usual dispersion red shift. A small mechanical solvent-cage effect is noted in ethanol at 77 K, but solvent dielectric relaxation is not apparent from the fluorescence spectrum. Thus, HAP serves to distinguish static solvent-cage polarity from dynamical solvent dielectric relaxation and other solvent-cage effects (mechanical restriction of molecular conformation). HAP as a PT-fluorescence probe is applied to human serum albumin (HSA) and beaver apomyoglobin.
Regulation of activity and apical targeting of the Cl-/HCO3- exchanger in rat hepatocytes.
Authors: Benedetti A , Strazzabosco M , Ng O C , Boyer J L
Published: June 2006

To test the hypothesis that rat hepatocyte canalicular Cl-/HCO3- exchange activity might be regulated by HCO3- or protein kinase-induced changes in the apical targeting of vesicles, isolated rat hepatocytes were cultured in the presence or absence of HCO3-/CO2.Cl-/HCO3- exchange activity increased in cells cultured in the presence of HCO3-/CO2 or when stimulated by dibutyryl cAMP. Both of these effects were blocked by either colchicine or the protein kinase C agonist phorbol 12,13-dibutyrate. Fluorescence and confocal microscopy, respectively, revealed increased pericanalicular-apical membrane localization of two canalicular markers, peanut agglutinin and a 110-kDa canalicular ecto-ATPase, when hepatocyte couplets were preincubated in HCO3-/CO2-containing medium, an effect that was again blocked by colchicine. Dibutyryl cAMP also stimulated canalicular localization of the 110-kDa protein. These findings suggest that hepatocyte Cl-/HCO3- exchange activity is regulated by HCO3-/CO2 and by protein kinase A and protein kinase C agonists through microtubule-dependent targeting of vesicles containing this exchanger to the canalicular domain.
Isolation of cDNAs encoding guanine nucleotide-binding protein beta-subunit homologues from maize (ZGB1) and Arabidopsis (AGB1).
Authors: Weiss C A , Garnaat C W , Mukai K , Hu Y , Ma H
Published: June 2006

We have isolated cDNAs from maize (ZGB1) and Arabidopsis (AGB1) encoding proteins homologous to beta subunits of guanine nucleotide-binding protein (G protein). The predicted ZGB1 and AGB1 gene products are 76% identical to each other and 41% or more identical to animal G protein beta subunits. Both predicted proteins contain seven repeats of the so-called "WD-40" motif, where WD is Trp-Asp. RNA blot analysis indicates that ZGB1 mRNA is present in the root, leaf, and tassel and that AGB1 mRNA is expressed in the root, leaf, and flower. DNA blot hybridizations indicate that maize and Arabidopsis genomes contain no other genes that are highly similar to ZGB1 and AGB1, respectively, suggesting that the newly isolated G protein beta-subunit homologues are likely to have unique functions. Furthermore, these G protein beta-subunit homologues are conserved among other plant species and may play important role(s) in plant signaling.
Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage.
Authors: Choo Y , Klug A
Published: June 2006

We have used two selection techniques to study sequence-specific DNA recognition by the zinc finger, a small, modular DNA-binding minidomain. We have chosen zinc fingers because they bind as independent modules and so can be linked together in a peptide designed to bind a predetermined DNA site. In this paper, we describe how a library of zinc fingers displayed on the surface of bacteriophage enables selection of fingers capable of binding to given DNA triplets. The amino acid sequences of selected fingers which bind the same triplet are compared to examine how sequence-specific DNA recognition occurs. Our results can be rationalized in terms of coded interactions between zinc fingers and DNA, involving base contacts from a few alpha-helical positions. In the paper following this one, we describe a complementary technique which confirms the identity of amino acids capable of DNA sequence discrimination from these positions.
Infectious amyloid precursor gene sequences in primates used for experimental transmission of human spongiform encephalopathy.
Authors: Cervenáková L , Brown P , Goldfarb L G , Nagle J , Pettrone K , Rubenstein R , Dubnick M , Gibbs C J , Gajdusek D C
Published: June 2006

Based on the analysis of genomic DNA from single healthy animals of each of five primate species, nucleotide and predicted amino acid sequences of the infectious amyloid precursor gene of higher apes (Gorilla and Pan) and Old World (Macaca) and New World (Ateles, Saimiri) monkeys showed 95-99% homology to the human sequences, corresponding to their phylogenetic distance from humans. Two of 18 amino acids that differed from humans resulted from nucleotide changes at sites of mutations in humans with familial forms of spongiform encephalopathy (a deleted codon within the codon 51-91 region of 24 bp repeats and a substitution at codon 198). In each of the five animals, codon 129 specified methionine, the more common of the two polymorphic genotypes in humans. Because genotypic homology did not correlate with experimental transmission rates of human spongiform encephalopathy, primary structural similarity of the infectious amyloid precursor protein in humans and experimental primates may not be an important factor in disease transmissibility.
Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras, and Rap.
Authors: Spaargaren M , Bischoff J R
Published: June 2006

To identify proteins that bind to the Ras-related protein R-ras we performed a yeast two-hybrid cDNA library screen. Several clones were obtained encoding the C-terminal region of the guanine nucleotide dissociation stimulator for Ral (RalGDS). The R-ras-binding domain of RalGDS (RalGDS-RBD) is distinct from the conserved catalytic exchange factor regions. Using the two-hybrid system, we show that RalGDS-RBD interacts with H-ras, K-ras, and Rap, and with active but not with inactive point mutants of these Ras-like GTPases. Moreover, using purified proteins, we demonstrate the direct GTP-dependent interaction of the Ras-like GTPases with RalGDS-RBD and full-length RalGDS in vitro. Furthermore, we show that RalGDS-RBD and the Ras-binding domain of Raf-1 compete for binding to the Ras-like GTPases. These data indicate that RalGDS is a putative effector molecule for R-ras, H-ras, K-ras, and Rap.
Molecular cloning of bullfrog saxiphilin: a unique relative of the transferrin family that binds saxitoxin.
Authors: Morabito M A , Moczydlowski E
Published: June 2006

Plasma and tissue of certain vertebrates contain a protein called saxiphilin that specifically binds the neurotoxin saxitoxin with nanomolar affinity. We describe the isolation of a cDNA clone of saxiphilin from liver of the North American bullfrog (Rana catesbeiana). The cDNA sequence encodes a protein that is evolutionarily related to members of the transferrin family of Fe(3+)-binding proteins. Pairwise sequence alignment of saxiphilin with various transferrins reveals amino acid identity as high as 51% and predicts 14 disulfide bonds that are highly conserved. The larger size of saxiphilin (91 kDa) versus serum transferrin (approximately 78 kDa) is primarily due to a unique insertion of 144 residues. This insertion contains a 49-residue domain classified as a type 1 repetitive element of thyroglobulin, which is shared by a variety of membrane, secreted, and extracellular matrix proteins. Saxiphilin also differs from transferrins in 9 of 10 highly conserved amino acids in the two homologous Fe3+/HCO3-binding sites of transferrin. Identification of saxiphilin implies that transferrin-like proteins comprise a diverse superfamily with functions other than iron binding.
Origin of the foraminifera.
Authors: Wray C G , Langer M R , DeSalle R , Lee J J , Lipps J H
Published: June 2006

We report nuclear small-subunit rDNA sequences for two species of the benthic foraminifer Ammonia. Because of their abundance in present and past seas, the foraminifera are a very important group of organisms used in a wide range of geologic studies; however, the ancestry of this group was, until now, unknown. Difficulties in purifying foraminiferal DNA and avoiding PCR contamination led us to develop DNA.DNA in situ hybridization techniques to verify our sequence data. Phylogenetic analysis of verified foraminiferal DNA sequence indicates these taxa are a divergent, "alveolate" lineage, within the major eukaryotic radiation. Our findings cast doubt upon the assumption that all foraminifera are derived from an amoebalike ancestor. Cytological features and the DNA sequences presented herein suggest that the foraminifera were derived from a heterokaryotic flagellated marine protist, probably sometime in the later Proterozoic.
Recombinant vesicular stomatitis viruses from DNA.
Authors: Lawson N D , Stillman E A , Whitt M A , Rose J K
Published: June 2006

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.
Nascent polypeptide-associated complex protein prevents mistargeting of nascent chains to the endoplasmic reticulum.
Authors: Lauring B , Sakai H , Kreibich G , Wiedmann M
Published: June 2006

We show that, after removal of the nascent polypeptide-associated complex (NAC) from ribosome-associated nascent chains, ribosomes synthesizing proteins lacking signal peptides are efficiently targeted to the endoplasmic reticulum (ER) membrane. After this mistargeting, translocation across the ER membrane occurs, albeit less efficiently than for a nascent secretory polypeptide, perhaps because the signal peptide is needed to catalyze the opening of the translocation pore. The mistargeting was prevented by the addition of purified NAC and was shown not to be mediated by the signal recognition particle and its receptor. Instead, it appears to be a consequence of the intrinsic affinity of ribosomes for membrane binding sites, since it can be blocked by competing ribosomes that lack associated nascent polypeptides. We propose that, when bound to a signalless ribosome-associated nascent polypeptide, NAC sterically blocks the site in the ribosome for membrane binding.
Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells.
Authors: Frey M R , Matera A G
Published: June 2006

Coiled bodies (CBs) are nuclear organelles whose structures appear to be highly conserved in evolution. In rapidly cycling cells, they are typically located in the nucleoplasm but are often found in contact with the nucleolus. The CBs in human cells contain a unique protein, called p80-coilin. Studies on amphibian oocyte nuclei have revealed a protein within the "sphere" organelle that shares significant structural similarity to p80-coilin. Spheres and CBs are also highly enriched in small nuclear ribonucleoproteins and other RNA-processing components. We present evidence that, like spheres, CBs contain U7 small nuclear RNA (snRNA) and associate with specific chromosomal loci. Using biotinylated 2'-O-methyl oligonucleotides complementary to the 5' end of U7 snRNA and fluorescence in situ hybridization, we show that U7 is distributed throughout the nucleoplasm, excluding nucleoli, and is concentrated in CBs. Interestingly, we found that CBs often associate with subsets of the histone, U1, and U2 snRNA gene loci in interphase HeLa-ATCC and HEp-2 monolayer cells. However, in a strain of suspension-grown HeLa cells, called HeLa-JS1000, we found a much lower rate of association between CBs and snRNA genes. Possible roles for CBs in the metabolism of these various histone and snRNAs are discussed.
Adenoviral E1B-55kDa protein inhibits yeast mRNA export and perturbs nuclear structure.
Authors: Liang S , Hitomi M , Tartakoff A M
Published: June 2006

The mechanisms of export of RNA from the nucleus are poorly understood; however, several viral proteins modulate nucleocytoplasmic transport of mRNA. Among these are the adenoviral proteins E1B-55kDa and E4-34kDa. Late in infection, these proteins inhibit export of host transcripts and promote export of viral mRNA. To investigate the mechanism by which these proteins act, we have expressed them in Saccharomyces cerevisiae. Overexpression of either or both proteins has no obvious effect on cell growth. By contrast, overexpression of E1B-55kDa bearing a nuclear localization signal (NLS) dramatically inhibits cell growth. In this situation, the NLS-E1B-55kDa protein is localized to the nuclear periphery, fibrous material is seen in the nucleoplasm, and poly(A)+ RNA accumulates in the nucleus. Simultaneous overexpression of E4-34kDa bearing or lacking an NLS does not modify these effects. We discuss the mechanisms of selective mRNA transport.
Olfactory transduction is intrinsically noisy.
Authors: Lowe G , Gold G H
Published: June 2006

The sources of noise that limit olfactory signal detection were investigated in dissociated rat olfactory receptor cells. Near-threshold odorant-evoked currents exhibited large random fluctuation. However, similar fluctuations were observed in the absence of applied odorants when currents were induced by elevating the intracellular cyclic AMP concentration. This suggests that the fluctuations reflect noise intrinsic to the transduction mechanism, rather than the quantal nature of an odorant stimulus. For many odorants, this intrinsic noise may preclude the reliable detection of single odorant molecules.
Membrane disposition of the M5-M6 hairpin of Na+,K(+)-ATPase alpha subunit is ligand dependent.
Authors: Lutsenko S , Anderko R , Kaplan J H
Published: June 2006

Extensive proteolytic digestion of Na+,K(+)-ATPase (EC 3.6.1.37) by trypsin produces a preparation where most of the extramembrane portions of the alpha subunit have been digested away and the beta subunit remains essentially intact. The fragment Gln-737-Arg-829 of the Na+,K(+)-ATPase alpha subunit, which includes the putative transmembrane hairpin M5-M6, is readily, selectively, and irreversibly released from the posttryptic membrane preparation after incubation at 37 degrees C for several minutes. Once released from the membrane, the fragment aggregates but remains water soluble. Occlusion of K+ or Rb+ specifically prevents release of the Gln-737-Arg-829 fragment into the supernatant. Labeling of the posttryptic membrane preparation with cysteine-directed reagents revealed that Cys-802 (which is thought to be located within the M6 segment) is protected against the modification by Rb+ while this fragment is in the membrane but can be readily modified upon release. Cation occlusion apparently alters the folding and/or disposition of the M5-M6 fragment in the membrane in a way that does not occur when the fragment migrates to the aqueous phase. The ligand-dependent disposition of the M5-M6 hairpin in the membrane along with recent labeling studies suggest a key role for this segment in cation pumping by Na+,K(+)-ATPase.
Follicular lymphomas can be induced to present alloantigen efficiently: a conceptual model to improve their tumor immunogenicity.
Authors: Schultze J L , Cardoso A A , Freeman G J , Seamon M J , Daley J , Pinkus G S , Gribben J G , Nadler L M
Published: June 2006

In the tumor-bearing host, T cells invariably fail to induce a clinically significant antitumor immune response. Although model systems support the existence of tumor peptide antigens, the molecular interactions critical for antigen presentation by the tumor cell remain unresolved. Here, we demonstrate that human follicular lymphoma cells are highly inefficient at presenting alloantigen despite their strong expression of major histocompatibility complex and low-to-intermediate expression of some adhesion and B7 costimulatory molecules. Activation of follicular lymphoma cells via CD40 induces or up-regulates both adhesion and B7 costimulatory molecules essential to repair this defect. More importantly, once primed, alloreactive T cells efficiently recognize unstimulated follicular lymphoma cells. Thus, correction of defective tumor immunity requires not only expression of major histocompatibility complex but also sufficient expression of multiple adhesion and costimulatory molecules.
Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide.
Authors: George A J , Jamar F , Tai M S , Heelan B T , Adams G P , McCartney J E , Houston L L , Weiner L M , Oppermann H , Peters A M
Published: June 2006

We describe a method to facilitate radioimaging with technetium-99m (99mTc) by genetic incorporation of a 99mTc chelation site in recombinant single-chain Fv (sFv) antibody proteins. This method relies on fusion of the sFv C terminus with a Gly4Cys peptide that specifically coordinates 99mTc. By using analogues of the 26-10 anti-digoxin sFv as our primary model, we find that addition of the chelate peptide, to form 26-10-1 sFv', does not alter the antigen-binding affinity of sFv. We have demonstrated nearly quantitative chelation of 0.5-50 mCi of 99mTc per mg of 26-10-1 sFv' (1 Ci = 37 GBq). These 99mTc-labeled sFv' complexes are highly stable to challenge with saline buffers, plasma, or diethylenetriaminepentaacetic acid. We find that the 99mTc-labeled 741F8-1 sFv', specific for the c-erbB-2 tumor-associated antigen, is effective in imaging human ovarian carcinoma in a scid mouse tumor xenograft model. This fusion chelate methodology should be applicable to diagnostic imaging with 99mTc and radioimmunotherapy with 186Re or 188Re, and its use could extend beyond the sFv' to other engineered antibodies, recombinant proteins, and synthetic peptides.
The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction.
Authors: Li R , Bianchet M A , Talalay P , Amzel L M
Published: June 2006

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.
Glial cell line-derived neurotrophic factor promotes the survival and morphologic differentiation of Purkinje cells.
Authors: Mount H T , Dean D O , Alberch J , Dreyfus C F , Black I B
Published: June 2006

Glial cell line-derived neurotrophic factor (GDNF) promotes survival of midbrain dopaminergic neurons and motoneurons. Expression of GDNF mRNA in cerebellum raises the possibility that cells within this structure might also respond to GDNF. To examine potential trophic activities of GDNF, dissociated cultures of gestational day 18 rat cerebellum were grown for < or = 21 days in the presence of factor. GDNF increased Purkinje cell number without affecting the overall number of neurons or glial cells. A maximal response (50% above control) was elicited with GDNF at 1 pg/ml. Effects of GDNF on Purkinje cell differentiation were examined by scoring the morphologic maturation of cells in treated and control cultures. GDNF increased the proportion of Purkinje cells that displayed relatively mature morphologies, characterized by dendritic thickening and the development of spines and filopodial extensions. Morphologic maturation of the overall neuronal population was unaffected. In sum, our data indicate that GDNF is a potent survival and differentiation factor for Purkinje cells, the efferent neurons of cerebellar cortex. Together with its other actions, these findings raise the possibility that GDNF might be a critical trophic factor at multiple loci in neuronal circuits that control motor function.
Antibody-targeted superantigens are potent inducers of tumor-infiltrating T lymphocytes in vivo.
Authors: Dohlsten M , Hansson J , Ohlsson L , Litton M , Kalland T
Published: June 2006

Recruitment of antigen-specific tumor-infiltrating lymphocytes (TILs) is a major goal for immunotherapy of malignant tumours. We now describe that T-cell-activating superantigens targeted to a tumor by monoclonal antibodies induced large numbers of pseudospecific TILs and eradication of micrometastases. As a model for tumor micrometastases, syngeneic B16 melanoma cells transfected with the human colon carcinoma antigen C215 were injected intravenously into C57BL/6 mice and therapy with an anti-C215 Fab fragment-staphylococcal enterotoxin A (C215Fab-SEA) fusion protein reacting with the C215 antigen was initiated when visible lung metastases were established. More than 90% reduction of the number of lung metastases was observed when mice carrying 5-day-old established lung metastases were treated with C215Fab-SEA. The antitumor effect of C215Fab-SEA was shown to be T-cell-dependent since no therapeutic effect was seen in T-cell-deficient nude mice. Depletion of T-cell subsets by injection of monoclonal antibody demonstrated that CD8+ cells were the most prominent effector cells although some contribution from CD4+ cells was also noted. C215Fab-SEA treatment induced massive tumor infiltration of CD4+ and CD8+ T cells, while only scattered T cells were observed in untreated tumors. SEA treatment alone induced a slight general inflammatory response in the lung parenchyme, but no specific accumulation of T cells was seen in the tumor. TILs induced by C215Fab-SEA were mainly CD8+ but a substantial number of CD4+ cells were also present. Immunohistochemical analysis showed strong production of the tumoricidal cytokines tumor necrosis factor alpha and interferon gamma in the tumor. Thus, the C215Fab-SEA fusion protein targets effector T lymphocytes to established tumors in vivo and provokes a strong local antitumor immune response.
Sample size determination in combinatorial chemistry.
Authors: Zhao P L , Zambias R , Bolognese J A , Boulton D , Chapman K
Published: June 2006

Combinatorial chemistry is gaining wide appeal as a technique for generating molecular diversity. Among the many combinatorial protocols, the split/recombine method is quite popular and particularly efficient at generating large libraries of compounds. In this process, polymer beads are equally divided into a series of pools and each pool is treated with a unique fragment; then the beads are recombined, mixed to uniformity, and redivided equally into a new series of pools for the subsequent couplings. The deviation from the ideal equimolar distribution of the final products is assessed by a special overall relative error, which is shown to be related to the Pearson statistic. Although the split/recombine sampling scheme is quite different from those used in analysis of categorical data, the Pearson statistic is shown to still follow a chi2 distribution. This result allows us to derive the required number of beads such that, with 99% confidence, the overall relative error is controlled to be less than a pregiven tolerable limit L1. In this paper, we also discuss another criterion, which determines the required number of beads so that, with 99% confidence, all individual relative errors are controlled to be less than a pregiven tolerable limit L2 (0 < L2 < 1).
Down-regulation of NF-kappa B protein levels in activated human lymphocytes by 1,25-dihydroxyvitamin D3.
Authors: Yu X P , Bellido T , Manolagas S C
Published: June 2006

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2)D3], a steroid hormone with immunomodulating properties, on nuclear factor kappa B (NF-kappa B) proteins was examined in in vitro activated normal human lymphocytes by Western blot analysis. Over a 72-hr period of activation, the expression of the 50-kDa NF-kappa B, p50, and its precursor, p105, was increased progressively. When cells were activated in the presence of 1,25(OH)2D3, the levels of the mature protein as well as its precursor were decreased. The effect of the hormone on the levels of p50 was demonstrable in the cytosolic and nuclear compartments; it required between 4 and 8 hr and was specific, as 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were ineffective. Besides p50, 1,25(OH)2D3 decreased the levels of another NF-kappa B protein, namely c-rel. In addition, 1,25(OH)2D3 decreased the abundance of a specific DNA-protein complex formed upon incubation of nuclear extracts from activated lymphocytes with a labeled NF-kappa B DNA binding motif. Further, 1,25(OH)2D3 inhibited the transcriptional activity of NF-kappa B in Jurkat cells transiently transfected with a construct containing four tandem repeats of the NF-kappa B binding sequence of the immunoglobulin kappa light chain gene linked to the chloramphenicol acetyltransferase reporter gene. These observations demonstrate directly that there is de novo synthesis of NF-kappa B during human lymphocyte activation and suggest that this process is hormonally regulated.
A sauvagine/corticotropin-releasing factor receptor expressed in heart and skeletal muscle.
Authors: Kishimoto T , Pearse R V , Lin C R , Rosenfeld M G
Published: June 2006

Corticotropin-releasing factor (CRF) mediates many critical aspects of the physiological response to stress. These effects are elicited by binding to specific high-affinity receptors, which are coupled to guanine nucleotide stimulatory factor (Gs)-response pathways. Recently, a gene encoding a receptor for CRF, expressed in pituitary and the central nervous system (PC-CRF receptor), was isolated and characterized. Here we report the identification and characterization of a second, distinct CRF receptor that is expressed primarily in heart and skeletal muscle and exhibits a specific ligand preference and antagonist sensitivity compared with the PC-CRF receptor. We refer to this second receptor as the heart/muscle (HM)-CRF receptor.
A macrophage receptor for oxidized low density lipoprotein distinct from the receptor for acetyl low density lipoprotein: partial purification and role in recognition of oxidatively damaged cells.
Authors: Ottnad E , Parthasarathy S , Sambrano G R , Ramprasad M P , Quehenberger O , Kondratenko N , Green S , Steinberg D
Published: June 2006

The binding and uptake of oxidatively modified low density lipoprotein (OxLDL) by mouse peritoneal macrophages occurs, in part, via the well characterized acetyl LDL receptor. However, several lines of evidence indicate that as much as 30-70% of the uptake can occur via a distinct receptor that recognizes OxLDL with a higher affinity than it recognizes acetyl LDL. We describe the partial purification and characterization of a 94- to 97-kDa plasma membrane protein from mouse peritoneal macrophages that specifically binds OxLDL. This receptor is shown to be distinct from the acetyl LDL receptor as well as from two other macrophage proteins that also bind OxLDL--the Fc gamma RII receptor and CD36. We suggest that this OxLDL-binding membrane protein participates in uptake of OxLDL by murine macrophages and also represents a receptor responsible for macrophage binding and phagocytosis of oxidatively damaged cells.
Membrane permeabilization by Listeria monocytogenes phosphatidylinositol-specific phospholipase C is independent of phospholipid hydrolysis and cooperative with listeriolysin O.
Authors: Goldfine H , Knob C , Alford D , Bentz J
Published: June 2006

We have examined potential cooperative interactions of Listeria monocytogenes phosphatidylinositol-specific phospholipase C (PI-PLC) and listeriolysin O (LLO), a pore-forming hemolysin, in a liposome lysis assay. Large unilamellar vesicles, approximately 0.1 micron in diameter, encapsulating the fluorescent probe calcein, were treated with PI-PLC or LLO at pH 6.0, and each was capable of causing dye release. With phosphatidylcholine/phosphatidylinositol/cholesterol liposomes at 0.1 microM lipid, minimal release of dye was observed on addition of 80 pM LLO or 7 nM PI-PLC. Addition of the two proteins together produced rapid dye release. Unexpectedly, essentially identical results were obtained with phosphatidylcholine/cholesterol liposomes. Thus, the effect of PI-PLC did not depend on lipid hydrolysis. Both proteins also released inulin (M(r) 5200) from liposomes. Membrane permeabilization was not accompanied by membrane fusion. Very little dye release from phosphatidylcholine/phosphatidylinositol/cholesterol liposomes was seen with PI-PLC from Bacillus thuringiensis, and addition of this enzyme to LLO produced no additional dye release; however PI-PLC from L. monocytogenes cooperated with perfringolysin O from Clostridium perfringens. PI-PLC from L. monocytogenes and LLO bind to phosphatidylcholine/cholesterol liposomes, and the rate of binding of each protein was not influenced by the presence of the other. These data support a postulated accessory role for PI-PLC with LLO in lysing the primary phagosome of a macrophage.
Cystic fibrosis epithelial cells have a receptor for pathogenic bacteria on their apical surface.
Authors: Imundo L , Barasch J , Prince A , Al-Awqati Q
Published: June 2006

Chronic colonization and infection of the lung with Pseudomonas aeruginosa is the major cause of morbidity and mortality in cystic fibrosis (CF) patients. We found that polarized CF bronchial and pancreatic epithelia bound P. aeruginosa in a reversible and dose-dependent manner. There was significantly greater binding to CF bronchial and pancreatic cells than to their matched pairs rescued with the wild-type CF transmembrane conductance regulator. Bound P. aeruginosa were easily displaced by unlabeled P. aeruginosa but not by Escherichia coli, an organism that does not cause significant pulmonary disease in CF. In contrast, Staphylococcus aureus, a frequent pathogen in CF, could effectively displace bound P. aeruginosa from its receptor. We found undersialylation of apical proteins and a higher concentration of asialoganglioside 1 (aGM1) in apical membranes of CF compared with rescued epithelia. Incubation of P. aeruginosa with aGM1 reduced its binding, as did treatment of the epithelia with the tetrasaccharide moiety of this ganglioside (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc). Finally, an antibody to aGM1 effectively displaced P. aeruginosa from its binding site and blocked binding of S. aureus to CF cells but not to rescued cells. These results show that the tetrasaccharide of aGM1 is a receptor for P. aeruginosa and S. aureus and that its increased abundance in the apical membrane of CF epithelia makes it a likely contributor to the pathogenesis of bacterial infections in the CF lung.
Non-clathrin-coat protein alpha is a conserved subunit of coatomer and in Saccharomyces cerevisiae is essential for growth.
Authors: Gerich B , Orci L , Tschochner H , Lottspeich F , Ravazzola M , Amherdt M , Wieland F , Harter C
Published: June 2006

To complete the molecular characterization of coatomer, the preformed cytosolic complex that is involved in the formation of biosynthetic transport vesicles, we have cloned and characterized the gene for non-clathrin-coat protein alpha (alpha-COP) from Saccharomyces cerevisiae. The derived protein, molecular weight of 135,500, contains four WD-40 repeated motifs (Trp/Asp-containing motifs of approximately 40 amino acids). Disruption of the yeast alpha-COP gene is lethal. Comparison of the DNA-derived primary structure with peptides from bovine alpha-COP shows a striking homology. alpha-COP is localized to coated transport vesicles and coated buds of Golgi membranes derived from CHO cells.
Differential modulation of Th1- and Th2-related cytokine mRNA expression by a synthetic peptide homologous to a conserved domain within retroviral envelope protein.
Authors: Haraguchi S , Good R A , James-Yarish M , Cianciolo G J , Day N K
Published: June 2006

The influence of a synthetic retroviral peptide, CKS-17, on T helper type 1 (Th1)- or Th2-related cytokines was investigated in human blood mononuclear cells. Cells were stimulated with staphylococcal enterotoxin A, anti-CD3 plus anti-CD28 monoclonal antibodies, or lipopolysaccharide to induce cytokine mRNA. mRNA was detected by a reverse transcription-polymerase chain reaction or Northern blot analysis. CKS-17 down-regulated stimulant-induced mRNA accumulation for interferon gamma (IFN-gamma), interleukin (IL)-2, and p40 heavy and p35 light chains of IL-12, a cytokine that mediates development of Th1 response. CKS-17 up-regulated stimulant-induced mRNA accumulation of IL-10 and did not suppress Th2-related cytokine (IL-4, IL-5, IL-6, or IL-13) mRNA expression. A reverse sequence of CKS-17 peptide, used as a control, showed no such action. Anti-human IL-10 monoclonal antibody blocked ability of CKS-17 to inhibit mRNA accumulation for IFN-gamma but not the CKS-17 suppressive activity of IL-12 p40 heavy chain mRNA. Thus, CKS-17-mediated suppression of IFN-gamma mRNA expression is dependent upon augmentation of IL-10 production by CKS-17. This conserved component of several retroviral envelope proteins, CKS-17, may act as an immunomodulatory epitope responsible for cytokine dysregulation that leads to suppression of cellular immunity.
What Can Be Learned from Introducing Settlement Typology into Urban Poverty Analysis: The Case of the Tomsk Region, Russia
Authors: Alexandrova Anastassia , Hamilton Ellen L. , Kuznetsova Polina
Published: June 2006

This study uses household survey data from the Tomsk oblast (region) to analyse how and why household welfare differs among the distinct urban settlement types. Urban settlements patterns in the Soviet Union were unusual because they were determined by government priorities not market forces. As a result, transition brought widespread change in urban fortunes to different types of urban settlement. In Tomsk oblast, smaller cities dependent on timber and other non-oil-related economic activities were hardest hit. Poorly located and increasingly economically disadvantaged, these cities have become geographical poverty traps for their residents. Households living in the oblast capital with its diverse economy and good geographical linkages to other places have fared better, while households living in smaller cities dependent on oil production have done best of all.
An archaeometric application of SANS to pottery finds
Authors: Botti A. , Sodo A. , Ricci M.A.
Published: June 2006

Small angle neutron scattering has been employed to extract microscopic parameters of ceramic samples prepared under controlled conditions, in the attempt of establishing a protocol for deriving information on the firing process of pottery finds. This protocol has been applied to a series of samples from the early middle ages coming from the Italian region of Miseno. The variation of the microscopical features observed on these samples could mirror a technological evolution in the period spanning from VI to XII century A.D.
Integrated non-invasive technologies for the diagnosis and conservation of the cultural heritage
Authors: Bacci M. , Casini A. , Picollo M. , Radicati B. , Stefani L.
Published: June 2006

The necessity of non-invasive measurements is stressed for studies of works of art. The integration of different techniques can supply a large quantity of information without any sampling. As a case study, Leonardo da Vinci’s Madonna of the Yarn Winder has been considered. The preliminary results of an investigation with fibre optic reflectance spectroscopy and hyper-spectral image spectroscopy are reported and discussed.
Texture and structure studies on marbles from Villa Adriana via neutron diffraction technique
Authors: Filabozzi A. , Andreani C. , de Pascale M.P. , Gorini G. , Pietropaolo A. , Perelli Cippo E. , Senesi R. , Tardocchi M. , Kockelmann W.
Published: June 2006

The primary objective of this work is the characterisation of ancient roman marble fragments through neutron diffraction, a non-destructive experimental method. The neutron diffractometer ROTAX, operating at the pulsed neutron source ISIS, in the UK has been used to determine composition down to a 0.5 wt% level and to obtain information on preferred orientations of grains in the marble tiles.
Neutrons in cultural heritage research
Authors: Kockelmann W. , Chapon L.C. , Engels R. , Schelten J. , Neelmeijer C. , Walcha H.-M. , Artioli G. , Shalev S. , Perelli-Cippo E. , Tardocchi M. , Gorini G. , Radaelli P.G.
Published: June 2006

Neutron radiation is a highly penetrating probe, routinely used for non-destructive testing of engineering components and for analysis of the structure and dynamics of novel advanced materials. The same analytical techniques can be employed for characterising objects of art, museum pieces and archaeological findings. In particular, neutron radiography, neutron diffraction and neutron activation analysis provide valuable and complementary information to address questions of provenance, authenticity, fabrication techniques and conservation. This paper surveys some basic concepts and recent applications of neutrons in cultural heritage research at the ISIS neutron spallation source.
Mesoscopic structure of marble determined by combined USANS and SANS
Authors: Triolo R. , Lo Celso F. , Gorgoni C. , Pallante P. , Schwahn D. , Baron M.
Published: June 2006

Marbles, carbonatic (i.e. calcitic–dolomitic) rocks deriving from the metamorphic evolution of previous carbonates, are often rather similar to each other in many respects (i.e. mineralogical, physical–structural and chemical), and thus difficult to identify. In search for a diagnostic method to provenance marbles, we have selected a limited number of Italian white marbles representing a wide array of metamorphic (i.e. thermal) levels. The mesoscale structural arrangement of these samples was derived by means of combined Ultra Small Angle and Small Angle Neutron Scattering experiments. The parameters of the model used to fit the data have been correlated with the metamorphic history of the samples. These interesting preliminary results, to be confirmed with new analyses, throw new light on the strict relationship between the formation history of a marble and its intermediate mesoscopic structure, which is of potential interest for other applications, such as the source identification and authentication of ancient marble artifacts.
Villa Adriana RiVa project—preliminary results of a study dedicated to an unveiling and restoring of new areas of the complex
Authors: Cinque G.E.
Published: June 2006

The RiVa project, where RiVa stands for Rediscovering Hadrian’s villa, is aimed at defining a multidisciplinary methodology to approach the problem of representing, reconstructing and restoring archaeological complexes. This methodology should serve as a common base to finalize efforts and contributions of different expertise towards the same goal. Within the RiVa project experts from the areas of engineering, physics, chemistry, architecture and archaeology find a common base to share findings and define strategies for future studies. RiVa serves as a pilot project to take maximum benefits from different expertise in tracing the origin of ancient materials, their scope and function within a structural assembly, their archaeological value, their position within a coherent framework progressively reconstructed with the aid of surveying and artificial data imaging.
The Holocaust in Italian Collective Memory: Il giorno della memoria, 27 January 2001
Authors: Gordon Robert S. C.
Published: June 2006

The article examines the first official, national Holocaust memorial day in Italy, the so-called Giorno della memoria (Day of memory), marked on the 56th anniversary of the liberation of Auschwitz on 27 January 2001. It looks at the ways in which the day acted as a filter for issues of national collective memory and identity, in particular state and public negotiation of the legacy of Fascism, as well as addressing broader issues of Holocaust remembrance. The article looks first at the origins of the Giorno della memoria in political and in legislative terms. Then it sketches in the events of the day itself, at national and local levels, in political, educational and cultural terms. This is followed by an analysis of media coverage and contemporary commentary, showing how various lines of interpretation addressed general issues related to the Holocaust and specifically Italian historical, ideological and contemporary questions.