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Showing papers by "Laboratory of Molecular Biology published in 1991"


Journal ArticleDOI
TL;DR: The results suggest that a single large phage display library can be used to isolate human antibodies against any antigen, by-passing both hybridoma technology and immunization.

2,678 citations


Journal ArticleDOI
15 Aug 1991-Nature
TL;DR: Using a random combinatorial library of the rearranged heavy and kappa light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), diverse libraries of antibody fragments are displayed on the surface of fd phage and elicited many more pairings with strong binding activities.
Abstract: To by-pass hybridoma technology and animal immunization, we are trying to build antibodies in bacteria by mimicking features of immune selection. Recently we used fd phage to display antibody fragments fused to a minor coat protein, allowing enrichment of phage with antigen. Using a random combinatorial library of the rearranged heavy (VH) and kappa (V kappa) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), we have now displayed diverse libraries of antibody fragments on the surface of fd phage. After a single pass over a hapten affinity column, fd phage with a range of phOx binding activities were detected, at least one with high affinity (dissociation constant, Kd = 10(-8) M). A second pass enriched for the strong binders at the expense of the weak. The binders were encoded by V genes similar to those found in anti-phOx hybridomas but in promiscuous combinations (where the same V gene is found with several different partners). By combining a promiscuous VH or V kappa gene with diverse repertoires of partners to create hierarchical libraries, we elicited many more pairings with strong binding activities. Phage display offers new ways of making antibodies from V-gene libraries, altering V-domain pairings and selecting for antibodies with good affinities.

2,141 citations


Journal ArticleDOI
12 Jul 1991-Cell
TL;DR: Evidence is provided that E-cadherin acts as an invasion suppressor molecule in epithelial tumor cell lines of dog kidney or mouse mammary gland origin.

1,706 citations


Journal ArticleDOI
08 Feb 1991-Cell
TL;DR: Point mutagenesis of the nuclear targeting sequence of nucleoplasmin has identified two interdependent basic domains separated by 10 intervening "spacer" amino acids that tolerate point mutations and some insertions.

1,477 citations


Journal ArticleDOI
24 Jan 1991-Nature
TL;DR: This work has shown that monoclonal antibodies can be genetically engineered and endowed with new properties and could be extended to production of 'in vitro' repertoires of variable domain genes, and obviate the immunization of animals.
Abstract: Monoclonal antibodies can now be genetically engineered and endowed with new properties. In the future, gene technology could enable antigen-binding fragments to be made by exploiting repertoires of variable domain genes derived from immunized animals and expressed in bacteria. How readily can this approach be extended to production of 'in vitro' repertoires of variable domain genes, and obviate the immunization of animals?

1,170 citations


Journal ArticleDOI
31 Oct 1991-Nature
TL;DR: The structure of the δ-endotoxin from Bacillus thuringiensis subsp.
Abstract: The structure of the delta-endotoxin from Bacillus thuringiensis subsp. tenebrionis that is specifically toxic to Coleoptera insects (beetle toxin) has been determined at 2.5 A resolution. It comprises three domains which are, from the N- to C-termini, a seven-helix bundle, a three-sheet domain, and a beta sandwich. The core of the molecule encompassing all the domain interfaces is built from conserved sequence segments of the active delta-endotoxins. Therefore the structure represents the general fold of this family of insecticidal proteins. The bundle of long, hydrophobic and amphipathic helices is equipped for pore formation in the insect membrane, and regions of the three-sheet domain are probably responsible for receptor binding.

740 citations


Journal ArticleDOI
TL;DR: The prospects of TNF as an antitumor drug can be improved on the one hand by agents such as LI+, which synergizes, and on the other hand by inhibitors of the systemic toxicity which do not interfere with the antitumors efficacy.

724 citations


Journal Article
TL;DR: The affinity profiles of several of these antagonists at five cloned human muscarinic receptors stably expressed in Chinese hamster ovary cells are determined.
Abstract: A variety of muscarinic antagonists are currently used as tools to pharmacologically subclassify muscarinic receptors into M1, M2 and M3 subtypes. In the present study, we have determined the affinity profiles of several of these antagonists at five cloned human muscarinic receptors (m1-m5) stably expressed in Chinese hamster ovary cells (CHO-K1). At all five receptors, the (R)-enantiomers of trihexyphenidyl and hexbutinol displayed considerably higher affinities (up to 525-fold) than their corresponding (S)-isomers. The stereoselectivity ratios [inhibition constant(S)/inhibition constant(R)] for both pairs of enantiomers were lowest at m2 receptors, suggesting that less stringent configurational demands are made by this receptor subtype. The "M1-selective" antagonist (R)-trihexyphenidyl displayed high affinities for m1 and m4 receptors. The "M2-selective" antagonists himbacine, (+-)-5,11-dihydro-11- ([(2-[(dipropylamino)methyl]-1- piperidinyl)ethyl)amino]carbonyl)-6H-pyrido(2,3-b)(1,4)benzodiazepine-6- one (AF-DX 384), 11-[4-[4-(diethylamino)butyl]-1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b) (1,4)benzodiazepine-6-one (AQ-RA 741) and (+)-(11-[2-[(diethylamino) methyl]-1-piperidinyl)acetyl)-5,11-di-hydro-6H-pyrido(2,3-b)(1,4) benzodiazepine-6-one [AF-DX 250; the (+)-enantiomer of AF-DX 116] exhibited high affinities for m2 and m4, intermediate affinities for m1 and m3 and low affinities for m5 receptors. This selectivity profile was most prominent for AQ-RA 741, which displayed 195- and 129-fold higher affinities for m2 and m4 receptors than for m5 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

627 citations


Journal ArticleDOI
20 Sep 1991-Cell
TL;DR: The finding that IL5 and GM-CSF share a receptor subunit provides a molecular basis for the observation that these cytokines can partially interfere with each other's binding and have highly overlapping biological activities on eosinophils.

567 citations


Journal ArticleDOI
22 Feb 1991-Science
TL;DR: Vertebrate retinal photoreceptors recover from photoexcitation-induced hydrolysis of guanosine 3', 5'-monophosphate (cyclic GMP) by resynthesizing cyclicGMP, which reopens cation channels that have been closed by light.
Abstract: Vertebrate retinal photoreceptors recover from photoexcitation-induced hydrolysis of guanosine 3', 5'-monophosphate (cyclic GMP) by resynthesizing cyclic GMP, which reopens cation channels that have been closed by light. Activation of guanylate cyclase by light-induced depletion of cytosolic calcium is a key event in this recovery process. This cyclase has now been shown to be regulated by a 23-kilodalton calcium binding protein. The protein is present in both rod and cone photoreceptors and was named recoverin because it promotes recovery of the dark state. The amino acid sequence of recoverin exhibits three potential calcium binding sites (EF hands). That recoverin binds calcium was confirmed with calcium-45 and by observing calcium-induced changes in its tryptophan fluorescence. Recoverin activated guanylate cyclase when free calcium was lowered from 450 to 40 nM, an effect that was blocked by an antibody to recoverin. Thus, guanylate cyclase in retinal rods is stimulated during recovery by the calcium-free form of recoverin. A comparison of recoverin with other calcium binding proteins reveals that it may represent, along with the protein visinin, a family of proteins that are regulated by submicromolar calcium concentrations.

549 citations


Journal ArticleDOI
TL;DR: Insight is provided into the neuroanatomical basis of the differential effects of drugs that act on D1 or D2 receptors by mapping the cellular expression of the corresponding mRNAs in rat brain by in situ hybridization histochemistry.
Abstract: Physiological and pharmacological criteria have divided dopamine receptors into D1 and D2 subtypes, and genes encoding these subtypes have recently been cloned Based on the sequences of the cloned receptors, we prepared oligodeoxynucleotide probes to map the cellular expression of the corresponding mRNAs in rat brain by in situ hybridization histochemistry These mRNAs showed largely overlapping yet distinct patterns of expression The highest levels of expression for both mRNAs were observed in the caudate-putamen, nucleus accumbens, and olfactory tubercle Within the caudate-putamen, 47 +/- 6% and 46 +/- 5% of the medium-sized neurons (10-15 microns) expressed the D1 and D2 mRNAs, respectively, and only the D2 mRNA was observed in the larger neurons (greater than 20 microns) The D1 and D2 mRNAs were expressed in most cortical regions, with the highest levels in the prefrontal and entorhinal cortices Within neocortex, D1 mRNA was observed primarily in layer 6 and D2 mRNA in layers 4-5 Within the amygdala, D1 mRNA was observed in the intercalated nuclei, and D2 mRNA in the central nucleus Within the hypothalamus, D1 mRNA was observed in the suprachiasmatic nucleus and D2 mRNA in many of the dopaminergic cell groups Within the septum, globus pallidus, superior and inferior colliculi, mammillary bodies, and substantia nigra only D2 mRNA was detected These data provide insight into the neuroanatomical basis of the differential effects of drugs that act on D1 or D2 receptors

Journal ArticleDOI
TL;DR: This is the first crystal structure of a member of the serpin family in an uncleaved form and the peptide that is homologous to the reactive centre of inhibitory serpins adopts an alpha-helical conformation.

Journal ArticleDOI
TL;DR: A simple in vitro system that carries out the integration reaction and the use of this system to probe the mechanism of integration provides a simple screen for testing candidate inhibitors of HIV IN protein; some such inhibitors might have useful antiviral activity.
Abstract: Growth of human immunodeficiency virus (HIV) after infection requires the integration of a DNA copy of the viral RNA genome into a chromosome of the host. Here we present a simple in vitro system that carries out the integration reaction and the use of this system to probe the mechanism of integration. The only HIV protein necessary is the integration (IN) protein, which has been overexpressed in insect cells and then partially purified. DNA substrates are supplied as oligonucleotides that match the termini of the linear DNA product of reverse transcription. In the presence of HIV IN protein, oligonucleotide substrates are cleaved to generate the recessed 3' ends that are the precursor for integration, and the cleaved molecules are efficiently inserted into a DNA target. Analysis of reaction products reveals that HIV IN protein joins 3' ends of the viral DNA to 5' ends of cuts made by IN protein in the DNA target. We have also used this assay to characterize the sequences at the ends of the viral DNA involved in integration. The assay provides a simple screen for testing candidate inhibitors of HIV IN protein; some such inhibitors might have useful antiviral activity.

Journal ArticleDOI
TL;DR: The results show that the transport of structurally unrelated molecules can be mediated by members of different families of membrane transporters.
Abstract: Bacillus subtilis cells selected for their resistance to rhodamine 6G demonstrated a multidrug-resistance (MDR) phenotype resembling that of mammalian MDR cells Like MDR in mammalian cells, MDR in bacteria was mediated by the efflux of the drugs from the cells The bacterial multidrug efflux system transported similar drugs and was sensitive to similar inhibitors as the mammalian multidrug transporter, P-glycoprotein The gene coding for the bacterial multidrug transporter, like the P-glycoprotein gene in mammalian MDR cells, was amplified in the resistant bacteria On the other hand, the bacterial multidrug transporter showed no sequence similarity to P-glycoprotein but exhibited an obvious homology to tetracycline efflux pumps and carbohydrate-ion symporters These results show that the transport of structurally unrelated molecules can be mediated by members of different families of membrane transporters

Journal ArticleDOI
TL;DR: In this paper, the rhombotin gene family was defined as a class of T-cell oncogenes with duplicated cysteine-rich LIM domains, which showed homology to zinc-binding proteins and to iron-sulfur centers of ferredoxins.
Abstract: A chromosomal translocation in a T-cell leukemia involving the short arm of human chromosome 11 at band 11p15 disrupts the rhombotin gene. This gene encodes a protein with duplicated cysteine-rich regions called LIM domains, which show homology to zinc-binding proteins and to iron-sulfur centers of ferredoxins. Two homologues of the rhombotin gene have now been isolated. One of these, designated Rhom-2, is located on human chromosome 11 at band 11p13, where a cluster of T-cell leukemia-specific translocations occur; all translocation breakpoints at 11p13 are upstream of the Rhom-2 gene. Human and mouse Rhom-2 are highly conserved and, like rhombotin, encode two tandem cysteine-rich LIM domains. Rhom-2 mRNA is expressed in early mouse development in central nervous system, lung, kidney, liver, and spleen but only very low levels occur in thymus. The other gene, designated Rhom-3, is not on chromosome 11 but also retains homology to the LIM domain of rhombotin. Since the Rhom-2 gene is such a common site of chromosomal damage in T-cell tumors, the consistency of translocations near the rhombotin gene was further examined. A second translocation adjacent to rhombotin was found and at the same position as in the previous example. Therefore, chromosome bands 11p15 (rhombotin) and 11p13 (Rhom-2) are consistent sites of chromosome translocation in T-cell leukemia, with the 11p15 target more rarely involved. The results define the rhombotin gene family as a class of T-cell oncogenes with duplicated cysteine-rich LIM domains.

Journal ArticleDOI
22 Nov 1991-Science
TL;DR: Recombinant toxins target cell surface receptors and antigens on tumor cells by mechanisms different from conventional chemotherapy, so that cross resistance to conventional chemotherapeutic agents should not be a problem.
Abstract: Recombinant toxins target cell surface receptors and antigens on tumor cells They kill by mechanisms different from conventional chemotherapy, so that cross resistance to conventional chemotherapeutic agents should not be a problem Furthermore, they are not mutagens and should not induce secondary malignancies or accelerate progression of benign malignancies They can be mass-produced cheaply in bacteria as homogeneous proteins Either growth factor-toxin fusions or antibody-toxin fusions can be chosen, depending on the cellular target

Journal ArticleDOI
01 Nov 1991-Cell

Journal ArticleDOI
TL;DR: A sequence assembly and editing program for managing large and small projects is being used to sequence complete cosmids and has substantially reduced the time taken to process the data.
Abstract: We describe a sequence assembly and editing program for managing large and small projects. It is being used to sequence complete cosmids and has substantially reduced the time taken to process the data. In addition to handling conventionally derived sequences it can use data obtained from Applied Biosystems,Inc. 373A and Pharmacia A.L.F. fluorescent sequencing machines. Readings are assembled automatically. All editing is performed using a mouse operated contig editor that displays aligned sequences and their traces together on the screen. The editor, which can be used on single contigs or for joining contigs, permits rapid movement along the aligned sequences. Insertions, deletions and replacements can be made in individual aligned readings and global changes can be made by editing the consensus. All changes are recorded. A click on a mouse button will display the traces covering the current cursor position, hence allowing quick resolution of problems. Another function automatically moves the cursor to the next unresolved character. The editor also provides facilities for annotating the sequences. Typical annotations include flagging the positions of primers used for walking, or for marking sites, such as compressions, that have caused problems during sequencing. Graphical displays aid the assessment of progress.

Journal ArticleDOI
TL;DR: Immunohistochemistry shows complementary distributions of tau and MAP2, with tau being concentrated in axons and high molecular mass MAP2 being confined to dendrites in nerve cells.

Journal ArticleDOI
TL;DR: It is reported herein thatPHFs and SFs form hybrid filaments displaying both morphologies, that PHFs andSFs share surface epitopes, and that computed maps reveal a similar C-shaped morphological unit in PHFsand SFs, though differing in relative arrangement in the two types of filament.
Abstract: The presence of abundant neurofibrillary tangles in certain areas of the brain constitutes one of the defining pathological characteristics of Alzheimer disease. The predominant component of the tangle is an abnormal fibrous assembly known as the paired helical filament (PHF). The PHF is formed by a twisted double-helical ribbon of subunits that gives rise to an image alternating in width between 8 nm and 20 nm with a cross-over spacing of 80 nm. Also found in tangles is the straight filament (SF), a different kind of abnormal filament, about 15 nm wide, that does not exhibit the marked modulation in width shown by the PHF. It is reported herein that PHFs and SFs form hybrid filaments displaying both morphologies, that PHFs and SFs share surface epitopes, and that computed maps reveal a similar C-shaped morphological unit in PHFs and SFs, though differing in relative arrangement in the two types of filament. The observations imply that the SF is a structural variant of the PHF and establish a common unit of assembly for these two pathological filaments.

Journal ArticleDOI
29 Aug 1991-Nature
TL;DR: It is shown that just two chains, the α and β associated chains, are sufficient to reconstitute an IgM surface receptor in fibroblasts and pro-pose that B-cell antigen receptors consist of a common α/β heterodimer associated with each immunoglobulin class.
Abstract: Several proteins associate with surface IgM to form the antigen receptor. We show that just two, the α and β associated chains, are sufficient to reconstitute an IgM surface receptor in fibroblasts. Contrary to expectation, α common a chain associ-ates with all five immunoglobulin classes. We pro-pose that B-cell antigen receptors consist of a common α/β heterodimer associated with each immunoglobulin class. But the classes differ both in the glycosylation of their associated α chain and in their dependence on α/β for surface transport.

Journal ArticleDOI
08 Aug 1991-Nature
TL;DR: There was a shift in the antibody repertoire after the primary response towards an immunoglobulin family with an extremely high on-rate constant, consistent with B-lymphocyte proliferation being subject to a kinetic selection, with a premium on binding target antigens rapidly, in parallel with a thermodynamic selection based on binding tightly.
Abstract: IS the affinity maturation of antibodies under thermodynamic or kinetic control, or both? We compared the physical constants of hapten binding by antibodies from 2-phenyl-5-oxazolone-specific hybridomas from primary, secondary and tertiary responses. In addition to an increase in equilibrium constant, there was a shift in the antibody repertoire after the primary response towards an immunoglobulin family with an extremely high on-rate constant. This shift occurred in spite of the average or below-average affinity of this group of antibodies. This is consistent with B-lymphocyte proliferation being subject to a kinetic selection, with a premium on binding target antigens rapidly, in parallel with a thermodynamic selection based on binding tightly

Journal ArticleDOI
TL;DR: The 229 kilobase pair DNA genome of human cytomegalovirus (HCMV) strain AD169 is the largest contiguous sequence determined to date, and as such provides a realistic benchmark for assessing the practical rate of DNA sequencing as opposed to theoretical calculations which are usually much greater.
Abstract: In the first part of this article we review what has been learnt from the analysis of the sequence of HCMV. A summary of this information is presented in the form of an updated map of the viral genome. HCMV is representative of a major lineage of herpesviruses distinct from previously sequenced members of this viral family and demonstrates striking differences in genetic content and organization. The virus encodes approximately 200 genes, including nine gene families, a large number of glycoprotein genes, and homologues of the human HLA class I and G protein-coupled receptor genes. The HCMV sequence thus provides a sound basis for future molecular studies of this highly complex eukaryotic virus. The second part discusses the practical rate of DNA sequencing as deduced from this and other studies. The 229 kilobase pair DNA genome of human cytomegalovirus (HCMV) strain AD169 is the largest contiguous sequence determined to date, and as such provides a realistic benchmark for assessing the practical rate of DNA sequencing as opposed to theoretical calculations which are usually much greater. The sequence was determined manually and we assess the impact of new developments in DNA sequencing.

Journal ArticleDOI
TL;DR: Investigation of chimeras between the two proteins revealed that the transmembrane segment of ST specifies Golgi retention, which appears to involve recognition of an extended region of the protein within and on both sides of the bilayer.
Abstract: The glycosyltransferase alpha-2,6-sialyltransferase (ST) is a Type II membrane protein localized to the Golgi apparatus. The first 44 amino acids of this protein were able to specify Golgi retention of a fused marker protein, lysozyme. This section of ST contains a transmembrane segment which serves as a non-cleaved signal anchor. When lysozyme was fused to an equivalent region of a cell surface protein it now appeared on the cell surface. Analysis of chimeras between the two proteins revealed that the transmembrane segment of ST specifies Golgi retention. Furthermore, altering this segment in full-length ST results in the protein accumulating on the cell surface. However, the retaining effect of the transmembrane domain of ST is augmented by the presence of adjacent lumenal and cytoplasmic sequences from ST. If these sequences are spaced apart by a transmembrane domain of the same length as that of ST they too can specify Golgi retention. Thus retention in the Golgi of ST appears to involve recognition of an extended region of the protein within and on both sides of the bilayer.


Journal ArticleDOI
TL;DR: Peter Vandenabeele and Walter Fiers propose possible intracerebral sources of cytokines and acute phase proteins in microglia, astrocytes, neurons and cells of the choroid plexus of Alzheimer's disease.

Journal ArticleDOI
TL;DR: This work optimized the design of the PCR primers for human V genes and used them to amplify cDNA from human peripheral blood lymphocytes and identified a region conserved within V gene families, but differing between families, and used this to design family‐specific oligonucleotide probes.
Abstract: In recent work, the polymerase chain reaction (PCR) has been used to amplify rearranged mouse and human immunoglobulin heavy and kappa light chain variable (V) genes. Here we have optimized the design of the PCR primers for human V genes and used them to amplify cDNA from human peripheral blood lymphocytes. Cloning and sequencing revealed a diverse repertoire of V genes, and the presence of members of each human V gene family. After alignment of the sequences, we identified a region conserved within V gene families, but differing between families, and used this to design family-specific oligonucleotide probes.

Journal ArticleDOI
TL;DR: This finding gives further evidence for the hypothesis that the magnesium ion is bound to the pro-R oxygen in the transition state of the hammerhead cleavage reaction.
Abstract: The chemical synthesis is described of oligoribonucleotides containing a single phosphorothioate linkage of defined Rp and Sp configuration. The oligoribonucleotides were used as substrates in the study of the mechanism of cleavage of an RNA hammerhead domain having the phosphorothioate group at the cleavage site. Whereas the Rp isomer was cleaved only very slowly in the presence of magnesium ion, the rate of cleavage of the Sp isomer was only slightly reduced from that of the unmodified phosphodiester. This finding gives further evidence for the hypothesis that the magnesium ion is bound to the pro-R oxygen in the transition state of the hammerhead cleavage reaction. Also, inversion of configuration at phosphorus is confirmed for a two-stranded hammerhead.

Journal ArticleDOI
TL;DR: Both recombinant immunotoxins were shown to be cytotoxic specifically to carcinoma cell lines that express the B3 antigen on their surface; B3(Fv)-PE38KDEL was significantly more active and caused complete regression of human epidermoid carcinomas growing subcutaneously in immunodeficient mice.
Abstract: The genes encoding the heavy- and light-chain Fv regions of the monoclonal murine antibody B3, which recognizes a carbohydrate antigen on the surface of many human carcinomas, were cloned by PCR techniques and used to generate single-chain immunotoxins containing Pseudomonas exotoxin (PE) The light and heavy chains were connected by a flexible linker to form a single-chain antigen-binding protein, B3(Fv), which was in turn fused to truncated forms of PE lacking the cell-binding domain The single-chain Fv and two different B3(Fv) immunotoxins, B3(Fv)-PE40 and B3(Fv)-PE38KDEL, were expressed in Escherichia coli and the single-chain immunotoxins were purified to near homogeneity Both recombinant immunotoxins were shown to be cytotoxic specifically to carcinoma cell lines that express the B3 antigen on their surface; B3(Fv)-PE38KDEL was significantly more active Furthermore, intravenous administration of B3(Fv)-PE38KDEL caused complete regression of human epidermoid carcinomas growing subcutaneously in immunodeficient mice

Journal ArticleDOI
TL;DR: The application of the hydroxyl radical footprinting technique is described to examine the contribution of the core histone tails and of histones H3 and H4 to the structure of DNA in the nucleosome.
Abstract: We describe the application of the hydroxyl radical footprinting technique to examine the contribution of the core histone tails and of histones H3 and H4 to the structure of DNA in the nucleosome. We first establish that, as was previously determined for a nucleosome containing a unique sequence of DNA, mixed-sequence nucleosomes contain two distinct regions of DNA structure. The central three turns of DNA in the nucleosome have a helical periodicity of approximately 10.7 base pairs per turn, while flanking regions have a periodicity of approximately 10.0 base pairs per turn. Removal of the histone tails does not change the hydroxyl radical cleavage pattern in either mixed- or unique-sequence nucleosome samples. A tetramer of histones H3 and H4, (H3/H4)2, organizes the central 120 base pairs of DNA identically to that found in the nucleosome. Moreover, "tailless" octamers and the (H3/H4)2 tetramer recognize the same nucleosome positioning signals as the intact octamer.