bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-idiotypic antibodies as probes of protein active sites: application to cholera toxin subunit B.
Authors: Ludwig D S , Finkelstein R A , Karu A E , Dallas W S , Ashby E R , Schoolnik G K
Published: May 2006

Since Jerne proposed a "network" theory of immune regulation, the properties of anti-idiotypic antibodies (anti-IdAb) have been investigated widely. Anti-IdAb raised against antibodies to a variety of ligands have been shown to bind the ligands' receptors. Thus, the combining site of an anti-IdAb may contain information regarding the three-dimensional structure of an antigen. However, this remarkable property of "internal imagery" has not been exploited for structural investigation at the molecular level. In the present report, a monoclonal "auto"-anti-IdAb was raised against ganglioside GM1 (a cell-surface glycolipid that binds cholera toxin) and was shown to crossreact with the B subunit of cholera toxin. This antibody was presumed to recognize amino acid residues located within the GM1 binding domain. To identify these residues, the antibody was screened against homologous toxins purified from enterotoxigenic strains of Escherichia coli and chimeric peptides produced by recombinant methods. Amino acid variation at position 4 from the N terminus of these proteins was found to disrupt antibody binding. Since the toxins and chimera are all closely related in structure and function, the residue at position 4 (an asparagine in cholera toxin B subunit) appears to be in the epitope of the antibody and, by implication, in the GM1 binding site. Of particular significance, this structural detail could not be deduced with GM1 alone. It would seem that ligand and anti-ligand anti-IdAb encode similar stereochemical information but do so with different "chemical alphabets," giving rise to distinct binding specificities.
Expression of functionally active human antithrombin III.
Authors: Stephens A W , Siddiqui A , Hirs C H
Published: May 2006

Human antithrombin III cDNA was cloned into an expression vector suitable for transient expression in COS cells. Upon transfection COS cells secreted a single immunoreactive 58-kDa protein. Quantitation of secretion levels by ELISA indicated that at 44 hr posttransfection cells were secreting 48 +/- 5 ng of antithrombin III per 10(6) cells per 24 hr. Heparin-agarose chromatography resulted in the elution of the COS-derived protein as a broad band between 0.3 and 1.0 M NaCl. 35S-labeled medium from transfected cells reacted with human thrombin (1.5 ng/ml) in the absence of heparin. In 40 min, greater than 80% of the immunoreactive material was found as a higher molecular weight species, consistent with stoichiometric covalent complex formation. In a two-stage chromogenic thrombin inactivation assay, under pseudo-first-order conditions, at 16 nM antithrombin III the t1/2 was 74 min and 50 min for plasma and COS cell-derived antithrombin III, respectively, in the absence of heparin. In the presence of 17.4 nM high-affinity heparin, the t1/2 was 5.2 min and 2.2 min, respectively.
Ribosomal RNA-encoding DNA introgression across a narrow hybrid zone between two subspecies of grasshopper
Authors: Arnold M. L. , Shaw D. D. , Contreras N.
Published: May 2006

A ribosomal RNA-encoding DNA (rDNA) cloned sequence, consisting of a 0.8-kilobase fragment from the 26S/nontranscribed spacer region, was used to identify diagnostic restriction enzyme fragments that distinguish the Moreton and Torresian subspecies of the grasshopper
Caledia captiva
. These restriction fragments were then used to study patterns of rDNA variation across a narrow geographical hybrid zone between the two subspecies. The pattern of rDNA variation that emerged after the analysis of over 250 individuals clearly demonstrates the asymmetrical introgression of the Moreton ribosomal RNA genes into the Torresian subspecies. This asymmetric movement of genetic material occurs even though there exists extreme postmating F
2
and backcross inviability between the two subspecies. From our data, as well as those of previous chromosomal and allozymic studies, we are able to support the occurrence of nonrandom processes such as biased gene conversion and/or natural selection. Because the rDNA loci in the Moreton and Torresian individuals are located in different regions on chromosomes 10 and 11, it should be possible to determine the relative contributions of conversion, natural selection, and these sorts of processes to the pattern of introgression of the Moreton rDNA into the Torresian subspecies.
Mutational analysis of potato spindle tuber viroid reveals complex relationships between structure and infectivity
Authors: Hammond Rosemarie W. , Owens Robert A.
Published: May 2006

Viroids are single-stranded, covalently closed circular RNA pathogens that can be isolated from certain higher plants afflicted with specific diseases. Their small size (246-375 nucleotides;
M
r
0.8-1.3 × 10
5
) and ability to replicate autonomously make viroids a unique model system in which to study the relationships between the structure of an RNA and its biological function. The demonstrated infectivity of certain cloned viroid cDNAs allows the use of site-specific mutagenesis techniques to probe structure-function relationships suggested by comparative sequence analysis. Several site-specific mutations that disrupt base pairing in either the native structure or secondary hairpin I destroyed the ability of potato spindle tuber viroid cDNA to initiate infection. Alterations in the terminal loops of the native structure also abolished cDNA infectivity. One pseudorevertant, a mutant cDNA containing compensating changes that restore base pairing in the native structure, was marginally infectious; a second pseudorevertant in which base pairing was restored within the stem of secondary hairpin I was not infectious. The behavior of these mutants dramatically demonstrates the effect of remarkably small structural changes on viroid infectivity and emphasizes the importance of the conserved rod-like native structure for viroid function.
Poliovirus polypeptide precursors: expression in vitro and processing by exogenous 3C and 2A proteinases.
Authors: Nicklin M J , Kräusslich H G , Toyoda H , Dunn J J , Wimmer E
Published: May 2006

Plasmids have been constructed to generate substrates for the study of proteinases 2A and 3C of poliovirus. They contain the P1 (capsomer precursor) region of the poliovirus genome or P1 and part of P2 (a nonstructural precursor), which can be transcribed and translated in vitro. A transcript containing the entire 5' nontranslated region and the P1 region of the viral RNA gave poor translation in a reticulocyte translation system. Truncation of the 5' nontranslated region to its 3'-most segment gave acceptably good yields of radiolabeled P1. P1 was specifically processed to yield capsomer proteins by enzymes supplied in a postmitochondrial supernatant from poliovirus-infected cells. Thus, proteinase 3C can be supplied exogenously (in trans) and effect processing. This system may be used to provide P1 for the assay of proteinase 3C. Precursors that lacked either the 1A or 1D regions were poor substrates for proteinase 3C--observations that demonstrated a stringent structural requirement in processing by 3C. The translation product of a transcript encoding P1 and part of P2 was rapidly cleaved at the P1-P2 site in the absence of infected-cell extract. A transcript that contained a mutated 2A region gave a stable P1-P2 precursor that could be processed specifically by exogenous proteinase from infected-cell fractions. Processing of P1 appeared to require cleavage of the P1-P2 bond. These results support our previous data that 2A is the second polioviral proteinase and also provides a means of assaying proteinase 2A in vitro.
Import and processing of human ornithine transcarbamoylase precursor by mitochondria from Saccharomyces cerevisiae.
Authors: Cheng M Y , Pollock R A , Hendrick J P , Horwich A L
Published: May 2006

Expression of the subunit precursor of the human mitochondrial matrix enzyme ornithine transcarbamoylase (OTCase; EC 2.1.3.3) was programmed in Saccharomyces cerevisiae from a 2-micron plasmid by using an inducible galactose operon promoter. In the presence of the inducing sugar (galactose), two polypeptides were specifically precipitable with anti-OTCase antiserum: the human OTCase precursor (40 kDa); and the mature OTCase subunit (36 kDa). When yeast cells containing these species were lysed and fractionated, the OTCase precursor was found to be associated with mitochondrial membranes, while the mature subunit was found partly with mitochondrial membranes and partly in the soluble mitochondrial matrix-containing fraction. When OTCase enzymatic activity was assayed in fractions similarly derived from an S. cerevisiae strain devoid of yeast OTCase activity (an arg3 mutant) but expressing human OTCase, activity was detected specifically in the mitochondrial matrix fraction. A mutant human OTCase precursor containing an artificial mutation in the NH2-terminal leader peptide (arginine-23 to glycine) was similarly examined. As was previously observed with mammalian mitochondria, this precursor failed both to reach the matrix compartment and to be proteolytically processed; it also failed to exhibit OTCase enzymatic activity. Presence of OTCase enzymatic activity in an arg3 strain expressing wild-type precursor was utilized to obtain selective growth in a medium devoid of arginine but supplemented with the OTCase substrate ornithine. We conclude that, during evolution, the pathway of mitochondrial import utilized by the human OTCase precursor is conserved between yeast and humans, and that, by using selective growth conditions, it may be possible to examine genetically this pathway in S. cerevisiae.
Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide gamma-chain-specific monoclonal antibody.
Authors: Ioannides C G , Itoh K , Fox F E , Pahwa R , Good R A , Platsoucas C D
Published: May 2006

We developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the gamma chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, we identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from 125I-labeled denatured lysates of T3+ WT31- lymphocytes expanded in culture from a SCID patient. These polypeptide chains were not disulfide linked and were not present on human peripheral blood lymphocytes from normal donors cultured for 5 days with phytohemagglutinin or for 2 weeks with rIL-2 and polyclonal activators or on cells of the Jurkat lymphoblastoid human T-cell line. Chemical crosslinking of 125I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These results were confirmed by sequential immunoprecipitation with anti-Leu-4 mAb followed by 9D7 anti-P13K mAb. The 9D7 anti-P13K mAb immunoprecipitated two polypeptide chains of 43 and 64 kDa from denatured lysates of lymphocytes from a patient with severe common variable immunodeficiency (CVI) that were expanded in culture with rIL-2 and Con A. Thus, this second TCR may be composed of two polypeptide chains (gamma gamma'), both of which appear to be the product of the gamma-chain gene. These experiments were done with polyclonal cell populations. Cloned T3+ WT31- cell populations are required to determine whether this TCR contains two gamma polypeptide chains. In contrast, only one polypeptide chain of 56 kDa was immunoprecipitated by the 9D7 anti-P13K mAb from peripheral blood lymphocytes from a patient with mild CVI expanded in culture with rIL-2 and polyclonal activators. Using the same 9D7 anti-P13K mAb and immunoblotting analysis, we identified a 35 kDa gamma-chain polypeptide under reducing conditions expressed on purified L3T4- Lyt2- BALB/c mouse thymocytes. This gamma-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions.
Expression of the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor by Escherichia coli transformants.
Authors: Gershoni J M
Published: May 2006

Restriction fragments of DNA derived from a cDNA clone of the alpha subunit of the acetylcholine receptor were subcloned in Escherichia coli by using the trpE fusion vector, pATH2. Transformants expressing the amino acid sequences 166-315 or 166-200 are shown to produce a chimeric protein that bound alpha-bungarotoxin. Moreover, it is shown that sufficient amounts of toxin-binding proteins can be generated by individual colonies of bacteria. This provides a new approach for gene selection via functional expression--i.e., ligand overlays of colony blots.
Regulated expression of multiple chicken erythroid membrane skeletal protein 4.1 variants is governed by differential RNA processing and translational control.
Authors: Ngai J , Stack J H , Moon R T , Lazarides E
Published: May 2006

Protein 4.1 is an extrinsic membrane protein that facilitates the interaction of spectrin and actin in the erythroid membrane skeleton and exists as several structurally related polypeptides in chickens. The ratio of protein 4.1 variants is developmentally regulated during terminal differentiation of chicken erythroid and lenticular cells. To examine the mechanisms by which multiple chicken protein 4.1 variants are differentially expressed, we have isolated cDNA clones specific for chicken erythroid protein 4.1. We show that a single protein 4.1 gene gives rise to multiple 6.6-kilobase mRNAs by differential RNA processing. Furthermore, the ratios of protein 4.1 mRNAs change during chicken embryonic erythropoiesis. We observe a quantitative difference in variant ratios when protein 4.1 is synthesized in vivo or in a rabbit reticulocyte lysate in vitro. Our results show that the expression of multiple protein 4.1 polypeptides is regulated at the levels of translation and RNA processing.
Characterization of avian and viral p60src proteins expressed in yeast.
Authors: Kornbluth S , Jove R , Hanafusa H
Published: May 2006

Avian and viral p60src proteins were expressed from a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. Both the viral and cellular src proteins produced in yeast cells were myristoylated at their amino termini, as is the case for src proteins expressed in chicken embryo fibroblasts. The viral src protein produced in yeast autophosphorylated at tyrosine-416 in vivo and had approximately the same level of in vitro kinase activity as p60v-src expressed in Rous sarcoma virus-transformed cells. Unlike p60c-src expressed in chicken cells, which is phosphorylated on tyrosine in vivo almost exclusively at tyrosine-527, p60c-src expressed in yeast was phosphorylated 2.5-3 times more at tyrosine-416 than at tyrosine-527. The specific activity of the p60c-src produced in yeast was 2.5-5.0 times higher than that of p60c-src overexpressed from a retroviral vector in chicken cells, implicating the altered state of in vivo phosphorylation in modulation of the in vitro kinase activity. The expression of p60v-src substantially slowed down the growth of the yeast cells, suggesting that phosphorylation of yeast proteins essential for cell growth may have interfered with their proper functioning.
Visualization of Ca2+-induced phospholipid domains.
Authors: Haverstick D M , Glaser M
Published: May 2006

Large vesicles (5-15 microns) were formed by hydrating a dried lipid film containing phospholipids labeled with a fluorophore in one fatty acid chain. By using a fluorescence microscope attached to a low-light-intensity charge-coupled-device camera and digital-image processor, the vesicles were easily viewed and initially showed uniform fluorescence intensity across the surface. The fluorescence pattern of vesicles made with a fluorophore attached to phosphatidylcholine or phosphatidylethanolamine was unaffected by the presence of divalent cations such as Ca2+, Mg2+, Mn2+, Zn2+, or Cd2+. The fluorescence pattern of vesicles containing a fluorophore attached to the acidic phospholipids phosphatidylserine or phosphatidic acid showed distinct differences when treated with Ca2+ or Cd2+, although they were unaffected by Mg2+, Mn2+, or Zn2+. Treatment with 2.0 mM Ca2+ or Cd2+ resulted in the movement of the fluorophore to a single large patch on the surface of the vesicle. When vesicles were formed in the presence of 33 mol % cholesterol, patching was seen at a slightly lower Ca2+ concentration (1.0 mM). The possibility of interactions between Ca2+ and acidic phospholipids in plasma membranes was investigated by labeling erythrocytes and erythrocyte ghosts with fluorescent phosphatidic acid. When Ca2+ was added, multiple (five or six) small patches were seen per individual cell. The same pattern was observed when vesicles formed from whole lipid extracts of erythrocytes were labeled with fluorescent phosphatidic acid and then treated with Ca2+. This shows that the size and distribution of the Ca2+-induced domains depend on phospholipid composition.
In vitro homing of hemopoietic stem cells is mediated by a recognition system with galactosyl and mannosyl specificities.
Authors: Aizawa S , Tavassoli M
Published: May 2006

We synthesized a number of neoglycoprotein probes by covalently linking three biologically relevant sugars (mannose, galactose, and fucose) to a protein molecule so as to retain the pyranose (ring) form of sugars necessary for their interaction with lectins. In the presence of galactosyl and mannosyl but not fucosyl probes, the production of CFU-S [colony-forming unit(s) in spleen] and total cells was halted in murine long-term marrow cultures. Cobblestone areas disappeared in these cultures, indicating the inhibition of binding of hemopoietic cells to the stroma. Electron microscopy revealed no alterations of the stroma, and the probes did not have direct cytotoxic or inhibitory effects on the growth of CFU-S or CFU-C [colony-forming unit(s) in culture]. Stroma grown for 5 weeks in the presence of the probes could subsequently support the growth of hemopoietic progenitor cells when the probes were removed from the medium. Conversely, the proliferative capacity of CFU-S in the supernate, grown in the presence of the probes, was retained upon grafting to control stroma. Galactosyl and mannosyl but not fucosyl probes differentially agglutinated CFU-S in whole-marrow-cell suspensions, suggesting the presence of membrane lectins with specificity for these sugars on the surface of CFU-S. We conclude that the binding of CFU-S to marrow stroma (homing) is mediated by a recognition system with galactosyl and mannosyl specificities.
Tumor cell heterogeneity: divided-colony assay for measuring drug response.
Authors: Kuczek T , Axelrod D E
Published: May 2006

In vitro tests for predicting the response of tumors to chemotherapeutic agents might be improved if they were modified to take into account tumor-cell heterogeneity. We have studied the heterogeneity of cellular growth rate and drug response in mouse fibroblast NIH 3T3 cells and in NIH 3T3 cells transformed with the human HRAS gene (homologue of the Harvey sarcoma virus oncogene v-Ha-ras) from the EJ human bladder carcinoma cell line. Growth-rate heterogeneity was detected as a broad distribution of numbers of cells per colony. In spite of this heterogeneity, secondary colonies have numbers of cells per colony that resemble that of the primary colony from which they were derived. The variance between unrelated secondary colonies is increased by HRASEJ. Colony-size measurements are reliable because primary colonies divided in half formed two groups of secondary colonies (on two separate plates) that had indistinguishable mean colony sizes. Based on these observations, a divided-colony procedure was devised to detect the drug response of heterogeneous cell populations. Primary colonies are divided into two groups of cells, one of which is treated with a drug and the other is left untreated as a control. The size distribution of treated secondary colonies is then compared to that of the untreated control and to that of the primary colony from which it was derived. The divided-colony procedure is proposed as a modification of the human-tumor-cloning system to increase the sensitivity and reliability of in vitro procedures used to determine the drug response of heterogeneous tumor-cell populations.
Reduced tumorigenicity of a spontaneous mouse lung carcinoma following H-2 gene transfection.
Authors: Bahler D W , Frelinger J G , Harwell L W , Lord E M
Published: May 2006

Cultured cells of the murine lung carcinoma called line 1 express very low levels of H-2 class I antigens and are resistant to lysis mediated by alloreactive T cells. In order to investigate how the expression of class I antigens affects the in vivo growth of this spontaneous tumor, H-2Dp genes were transferred into line 1 cells. Cloned transfectants that displayed H-2Dp surface antigens were identified using flow cytometry. The transfected H-2Dp antigens appeared normal by two-dimensional gel electrophoresis and could also function as excellent targets for T-cell-mediated lysis in vitro. Marked differences in tumorigenicity (defined as tumor growth in immunologically competent hosts) were observed between the Dp transfected cells and untransfected or control transfected line 1 cells in syngeneic mice only if the animals had previously received injections of irradiated Dp transfectants. Expression of Dp antigens did not appreciably affect the growth of line 1 tumors in immunologically naive syngeneic mice or necessarily cause rejection in allogeneic mice. Our in vivo results show that increased expression of class I antigens can reduce the growth of tumors like line 1 that lack all class I antigens. Our results also suggest that increasing class I antigens alone on some spontaneous tumors deficient in expression will not by itself be sufficient for tumor rejection.
Therapeutic vaccination against adjuvant arthritis using autoimmune T cells treated with hydrostatic pressure.
Authors: Lider O , Karin N , Shinitzky M , Cohen I R
Published: May 2006

An ideal treatment for autoimmune diseases would be a nontoxic means of specifically neutralizing the autoreactive lymphocytes responsible for the disease. This goal has been realized in experimental autoimmunity models by immunizing rats or mice against their own autoimmune cells such that the animals generate an immune response specifically repressive to the disease-producing lymphocytes. This maneuver, termed lymphocyte vaccination, was demonstrated to be effective using some, but not all, autoimmune helper T-lymphocyte lines. We now report that T lymphocytes, otherwise incapable of triggering an immune response, can be transformed into effective immunogens by treating the cells in vitro with hydrostatic pressure. Clone A2b, as effector clone that recognized cartilage proteoglycan and caused adjuvant arthritis in Lewis rats, is such a cell. Untreated A2b could not trigger an immune response, but inoculating rats with pressure-treated A2b induced early remission of established adjuvant arthritis as well as resistance to subsequent disease. Specific resistance to arthritis was associated with anti-idiotypic T-cell reactivity to clone A2b and could be transferred from vaccinated rats to naive recipients using donor lymphoid cells. Aggregation of T-lymphocyte membrane components appeared to be important for an immune response because the effects of hydrostatic pressure could be reproduced by treatment of A2b with chemical cross-linkers or with agents disrupting the cytoskeleton. Populations of lymph node cells from antigen-primed rats, when treated with hydrostatic pressure, could also induce suppression of disease. Thus, effective vaccines can be developed without having to isolate the autoimmune T lymphocytes as lines or clones. These results demonstrate that effector T lymphocytes suitably treated may serve as agents for specifically controlling the immune system.
Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters.
Authors: Morin J E , Lubeck M D , Barton J E , Conley A J , Davis A R , Hung P P
Published: May 2006

Recombinant adenoviruses carrying the hepatitis B virus surface antigen coding sequence in the adenovirus E3 region were constructed using DNA from either adenovirus type 5 or an adenovirus type 5 E3-region deletion mutant. Both of these recombinant adenoviruses replicated as efficiently as wild-type adenovirus in all human cells tested, including the human diploid cell strain WI-38. This indicates that insertion of the hepatitis B virus surface antigen gene into the E3 region does not significantly affect viral replication. Human cells infected with these recombinant adenoviruses secreted immunoreactive hepatitis B virus surface antigen. Since a practical small animal model for human adenoviruses was lacking, a hamster model was developed to evaluate the immunogenic potential of these recombinant adenoviruses. Upon intranasal inoculation, both wild-type adenovirus and the adenovirus E3-region deletion mutant replicated in the lungs of these animals and induced an antibody response against adenovirus. Hamsters similarly immunized with the live recombinant adenoviruses produced antibody against both adenovirus and hepatitis B virus surface antigen.
Atomic resolution images of solid-liquid interfaces
Authors: Giambattista Brian , McNairy W. W. , Slough C. G. , Johnson A. , Bell L. D. , Coleman R. V. , Schneir J. , Sonnenfeld R. , Drake B. , Hansma P. K.
Published: May 2006

A scanning tunneling microscope (STM) can provide atomic-resolution images of solids covered with a variety of liquids, including cryogenic fluids, both polar and nonpolar solvents, conductive aqueous solutions, oils, and even greases. This short overview includes images of solids covered with liquid nitrogen, liquid helium, paraffin oil, silicone oil, microscope immersion oil, silicone vacuum grease, fluorocarbon grease, glycerol, and salt water. These images show atoms, charge-density waves, grains in an evaporated metal film, and even corrosion processes as they occur in real time. The future includes not only basic research in surface science but also applied research in lithography, lubrication, catalysis, corrosion, electrochemistry, and perhaps even biology.
On the biologically active structures of cholecystokinin, little gastrin, and enkephalin in the gastrointestinal system.
Authors: Pincus M R , Carty R P , Chen J , Lubowsky J , Avitable M , Shah D , Scheraga H A , Murphy R B
Published: May 2006

The biologically active conformations of a series of four peptides [four cholecystokinin (CCK)-related peptides and enkephalin] in their interactions with gastrointestinal receptors have been deduced using conformational computational analysis. The two peptides that interact exclusively with peripheral-type CCK receptors are the heptapeptide COOH-terminal fragment from CCK (CCK-7) and the analogous sequence from cerulein (CER-7) in which threonine replaces the methionine proximal to the NH2 terminus. The two peptides that interact exclusively with the gastrin receptor in the stomach are the active COOH-terminal fragment of little gastrin and the COOH-terminal tetrapeptide sequence common to all of these peptides, CCK-4. We find that preferred conformations for the peripherally active peptides CCK-7 and CER-7 are principally beta-bends, whereas little gastrin and CCK-4 are fundamentally helical. In the class of lowest energy structures for both CCK-7 and CER-7, the aromatic rings of the tyrosine and phenylalanine lie close to one another whereas the tryptophan indole ring points in the opposite direction. This structure is superimposable on the structures of a set of rigid indolyl benzodiazepine derivatives that interact with complete specificity and high affinity with peripheral CCK receptors further suggesting that the computed beta-bends are the biologically active conformation. The biologically active conformation for CCK-4 and the little gastrin hexapeptide has also been deduced. By excluding conformations common to CCK-7 and CCK-4, which do not bond to each other's receptors, and then by selecting conformations in common to CCK-4 and the gastrin-related hexapeptide, which do bind to each other's receptors, we deduce that the biologically active conformation at the gastrin receptor is partly helical and one in which the indole of tryptophan and the aromatic ring of phenylalanine are close to one another while the methionine and aspartic acid side chains point in the opposite direction. These major differences in preferred structures between the common CCK-7/CER-7 peptides and the common CCK-4/little gastrin peptides explain the mutually exclusive activities of these two sets of peptides. We have observed that [Met]enkephalin strongly antagonizes the action of the naturally occurring peripherally active CCK-8 (CCK-7 with an NH2-terminal aspartic acid residue added). The computed lowest energy structures for this opiate peptide closely resemble key features of the computed CCK-7/CER-7 structure, further supporting the proposed structure.
Direct demonstration of the isomerization component of the monoterpene cyclase reaction using a cyclopropylcarbinyl pyrophosphate substrate analog.
Authors: Wheeler C J , Croteau R B
Published: May 2006

The tightly coupled nature of the reaction sequence catalyzed by monoterpene cyclases has precluded direct observation of the topologically required isomerization step leading from geranyl pyrophosphate to the presumptive, enzyme-bound, tertiary allylic intermediate linalyl pyrophosphate, which ultimately cyclizes to the various monoterpene skeletons. By using a partially purified monoterpene cyclase preparation and 2,3-cyclopropylgeranyl pyrophosphate, a substrate analog designed to uncouple the reaction sequence, the production of the corresponding tertiary homoallylic pyrophosphate isomer was demonstrated. This provides direct evidence for the usually cryptic isomerase component of the overall catalytic cycle. A number of other related products generated by reaction of cyclase with the analog were also identified, the structures and proportions of which were consistent with the intermediacy in catalysis of a cyclopropylcarbinyl cation X pyrophosphate anion pair. Kinetic parameters for the analog were compared with those of the natural substrate geranyl pyrophosphate. The results presented confirm mechanistic similarities in the enzymatic ionization and subsequent transformation of allylic pyrophosphate and cyclopropylcarbinyl pyrophosphate intermediates of isoprenoid metabolism.
Aberrant transmembrane signal transduction in Dictyostelium cells expressing a mutated ras gene.
Authors: Van Haastert P J , Kesbeke F , Reymond C D , Firtel R A , Luderus E , Van Driel R
Published: May 2006

Dictyostelium discoideum cells contain a single ras gene (Dd-ras) that is highly homologous to mammalian ras genes. Cell transformation with a vector carrying a ras gene with a (glycine----threonine) missense mutation at position 12 causes an altered morphogenesis. Extracellular cAMP signals regulate morphogenesis and induce chemotaxis and the activation and subsequent desensitization of adenylate and guanylate cyclase. cAMP signal transduction was investigated in Dd-ras-transformed cells. Transformants that overexpress the mutated Dd-ras-Thr12 gene show normal activation and desensitization of adenylate cyclase and normal activation of guanylate cyclase. However, cAMP induces a stronger desensitization of guanylate cyclase stimulation in the Dd-ras-Thr12 transformant than in transformants overexpressing the Dd-ras-Gly12 wild-type gene or in untransformed cells. This effect was correlated with a reduced chemotactic sensitivity of the transformant expressing the mutated Dd-ras-Thr12 gene.
High activity of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase and cytolytic perforin in cloned cell lines is not demonstrable in in-vivo-induced cytotoxic effector cells.
Authors: Dennert G , Anderson C G , Prochazka G
Published: May 2006

Recent observations have suggested striking similarities between complement-mediated and cell-mediated lysis. Both pathways share the terminal insertion of channels into target membranes, and unique esterases have been postulated to participate in the activation of cytolytic effector molecules. Since killer-specific esterases and channel-forming proteins can be demonstrated in in vitro cell lines, it is important to ascertain that the described esterase and channel-forming proteins are also present in killer cells from in vivo sources. Results presented here show that killer-cell-specific N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase is induced in vitro concomitant with the sensitization of cytotoxic effector cells. In contrast, in-vivo-primed cytotoxic T cells or natural killer (NK) cells fail to express high levels of this enzyme. Assay of different cytotoxic effector cells reveals the presence of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase in clones with T killer and NK activity, but enzyme levels do not correlate with cytolytic activity nor does inhibition of esterase activity interfere with granule-mediated cell lysis. A similar result is seen with granule-mediated cytolytic activity. Cloned NK and T killer cell lines possess granules that are able to lyse erythrocyte targets. However, T killer cells sensitized in mixed lymphocyte culture or in vivo have no detectable cytotoxic granules. Cytotoxic granules are also not detected in NK cells isolated from animals.
Primary structure of human corticosteroid binding globulin, deduced from hepatic and pulmonary cDNAs, exhibits homology with serine protease inhibitors.
Authors: Hammond G L , Smith C L , Goping I S , Underhill D A , Harley M J , Reventos J , Musto N A , Gunsalus G L , Bardin C W
Published: May 2006

We have isolated and sequenced cDNAs for corticosteroid binding globulin (CBG) prepared from human liver and lung mRNAs. Our results indicate that CBG mRNA is relatively abundant in the liver but is also present in the lung, testis, and kidney. The liver CBG cDNA contains an open reading frame for a 405-amino acid (Mr 45,149) polypeptide. This includes a predominantly hydrophobic, leader sequence of 22 residues that precedes the known NH2-terminal sequence of human CBG. We, therefore, predict that the mature protein is composed of 383 amino acids and is a polypeptide of Mr 42,646. A second, in-frame, 72-base-pair cistron of unknown significance exists between the TAA termination codon for CBG and a possible polyadenylylation signal (AATAAA) located 16 nucleotides before the polyadenylylation site. The deduced amino acid sequence of mature CBG contains two cysteine residues and consensus sequences for the attachment of six possible N-linked oligosaccharide chains. The sequences of the human lung and liver CBG cDNAs differ by only one nucleotide within the proposed leader sequence, and we attribute this to a point mutation. No sequence homology was found between CBG and other steroid binding proteins, but there is a remarkable similarity between the amino acid sequences of CBG and of alpha 1-antitrypsin, and this extends to other members of the serpin (serine protease inhibitor) superfamily.
Nucleotide sequence of the gene coding for human factor VII, a vitamin K-dependent protein participating in blood coagulation.
Authors: O'Hara P J , Grant F J , Haldeman B A , Gray C L , Insley M Y , Hagen F S , Murray M J
Published: May 2006

Activated factor VII (factor VIIa) is a vitamin K-dependent plasma serine protease that participates in a cascade of reactions leading to the coagulation of blood. Two overlapping genomic clones containing sequences encoding human factor VII were isolated and characterized. The complete sequence of the gene was determined and found to span about 12.8 kilobases. The mRNA for factor VII as demonstrated by cDNA cloning is polyadenylylated at multiple sites but contains only one AAUAAA poly(A) signal sequence. The mRNA can undergo alternative splicing, forming one transcript containing eight segments as exons and another with an additional exon that encodes a larger prepro leader sequence. The latter transcript has no known counterpart in the other vitamin K-dependent proteins. The positions of the introns with respect to the amino acid sequence encoded by the eight essential exons of factor VII are the same as those present in factor IX, factor X, protein C, and the first three exons of prothrombin. These exons code for domains generally conserved among members of this gene family. The comparable introns in these genes, however, are dissimilar with respect to size and sequence, with the exception of intron C in factor VII and protein C. The gene for factor VII also contains five regions made up of tandem repeats of oligonucleotide monomer elements. More than a quarter of the intron sequences and more than a third of the 3' untranslated portion of the mRNA transcript consist of these minisatellite tandem repeats.
Millisecond Fourier-transform infrared difference spectra of bacteriorhodopsin's M412 photoproduct.
Authors: Braiman M S , Ahl P L , Rothschild K J
Published: May 2006

We have obtained room-temperature transient infrared difference spectra of the M412 photoproduct of bacteriorhodopsin (bR) by using a "rapid-sweep" Fourier-transform infrared (FT-IR) technique that permits the collection of an entire spectrum (extending from 1000 to 2000 cm-1 with 8-cm-1 resolution) in 5 ms. These spectra exhibit less than 10(-4) absorbance unit of noise, even utilizing wet samples containing approximately 10 pmol of bR in the measuring beam. The bR----M transient difference spectrum is similar to FT-IR difference spectra previously obtained under conditions where M decay was blocked (low temperature or low humidity). In particular, the transient spectrum exhibits a set of vibrational difference bands that were previously attributed to protonation changes of several tyrosine residues on the basis of isotopic derivative spectra of M at low temperature. Our rapid-sweep FT-IR spectra demonstrate that these tyrosine/tyrosinate bands are also present under more physiological conditions. Despite the overall similarity to the low-temperature and low-humidity spectra, the room-temperature bR----M transient difference spectrum shows significant additional features in the amide I and amide II regions. These features' presence suggests that a small alteration of the protein backbone accompanies M formation under physiological conditions and that this conformational change is inhibited in the absence of liquid water.
Solution structure of murine epidermal growth factor: determination of the polypeptide backbone chain-fold by nuclear magnetic resonance and distance geometry.
Authors: Montelione G T , Wüthrich K , Nice E C , Burgess A W , Scheraga H A
Published: May 2006

The polypeptide backbone fold in the solution structure of murine epidermal growth factor has been determined by nuclear magnetic resonance spectroscopy and distance geometry calculations. The results are based on nearly complete sequence-specific resonance assignments and on 333 distance and dihedral-angle constraints; these were determined from nuclear Overhauser effect measurements, identification of hydrogen-bonded amide protons, the known locations of disulfide bonds, and backbone vicinal spin-spin coupling constants. The polypeptide chain of the protein is arranged into two distinct domains. The structures of these domains were determined independently in separate calculations and then combined to obtain an overall view of the protein. The backbone fold thus determined includes the regular backbone structure elements that were previously identified using different techniques for the analysis of the nuclear magnetic resonance data. The distance geometry calculations also provided additional details about the conformations of bends and loops and about the twists of the beta-sheets.
Microtubule polarity reversal accompanies regrowth of amputated neurites.
Authors: Baas P W , White L A , Heidemann S R
Published: May 2006

Intact chicken sensory neurites have the same microtubule polarity reported for nongrowing axons in intact organisms. The assembly-favored or + ends of the microtubules are found at the distal terminal (growth cone) of the neuron. After amputation of chicken sensory neurites, the fragment removed from the cell body collapsed to a bead of axoplasm from which neurites rapidly regrew. In nine such regrown neurites analyzed for microtubule polarity, the + ends of microtubules faced the newly formed growth cones, i.e.,--ends of microtubules were now at the original distal terminal of the neuron. These results indicate that microtubules reorganized concomitant with neurite regrowth to form a uniformly polar microtubule array but with reversed polarity. This suggests that mechanisms within the neurite, independent of the cell body, are sufficient for organization of microtubule assembly during axonal elongation. Our data also indicate that microtubule + ends were correlated with growth cone formation in the following three experimental classes of neurites: normal, regrown, and amputated but extended. We speculate that + ends of microtubules are a requirement for growth cone formation and advance.
Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence.
Authors: Millan J L , Driscoll C E , LeVan K M , Goldberg E
Published: May 2006

The sequence and structure of human testis-specific L-lactate dehydrogenase [LDHC4, LDHX; (L)-lactate: NAD+ oxidoreductase, EC 1.1.1.27] has been derived from analysis of a complementary DNA (cDNA) clone comprising the complete protein coding region of the enzyme. From the deduced amino acid sequence, human LDHC4 is as different from rodent LDHC4 (73% homology) as it is from human LDHA4 (76% homology) and porcine LDHB4 (68% homology). Subunit homologies are consistent with the conclusion that the LDHC gene arose by at least two independent duplication events. Furthermore, the lower degree of homology between mouse and human LDHC4 and the appearance of this isozyme late in evolution suggests a higher rate of mutation in the mammalian LDHC genes than in the LDHA and -B genes. Comparison of exposed amino acid residues of discrete antigenic determinants of mouse and human LDHC4 reveals significant differences. Knowledge of the human LDHC4 sequence will help design human-specific peptides useful in the development of a contraceptive vaccine.
B-cell variant of mouse T200 (Ly-5): evidence for alternative mRNA splicing.
Authors: Thomas M L , Reynolds P J , Chain A , Ben-Neriah Y , Trowbridge I S
Published: May 2006

The complete cDNA sequence of mouse T200 glycoprotein from the pre-B-cell line 70Z/3 has been determined. The deduced protein sequence differs from that previously reported for a T-cell form of the molecule [Saga, Y., Tung, J.-S., Shen, F.-W. & Boyse, E. A. (1986) Proc. Natl. Acad. Sci. USA 83, 6940-6944] by the insertion of 139 amino acid residues in the amino-terminal region of the molecule. RNA transfer blotting using a cDNA probe encoding this sequence established that the predominant T200 mRNA species from B cells and cytotoxic T-cell clones but not T-helper cell clones or thymocytes contain all or part of the insert. Overlapping genomic clones of murine T200 were isolated and analysis of the intron-exon structure at the 5' end of the gene provides evidence that variants of T200 glycoprotein are generated by alternative mRNA splicing.
3'-orf and sor genes of human immunodeficiency virus: in vitro transcription-translation and immunoreactive domains.
Authors: Arya S K
Published: May 2006

An in vitro transcription and translation procedure was designed to translate multiple open reading frames from cloned DNAs. For human immunodeficiency virus (HIV) cloned DNA carrying three open reading frames (sor, tat, and 3'-orf), the approach yielded three authentic polypeptides. Clearly, the internal initiation codons can be used for reinitiation of translation of the downstream open reading frames. However, the downstream open reading frames were translated with relatively lower translational efficiencies. In general, the translational efficiency of RNAs depended significantly on their structures. The in vitro approach was utilized further to map the immunoreactive domains of the 3'-orf and sor gene products of HIV. Deletion clones were constructed with deletions within the open reading frames. Translation products of these clones reacted differentially with anti-3'-orf and anti-sor rabbit immune sera and human sera from individuals with acquired immunodeficiency syndrome and related disorders. Apparently, recombinant 3'-orf and sor polypeptides used to immunize rabbits express many more immunogenic epitopes and/or different set of epitopes than is the case for the native proteins in humans infected with HIV. Immunoreactivity and immunogenicity of these gene products were significantly dependent on their structure and/or conformation.
Dynorphin A selectively reduces a large transient (N-type) calcium current of mouse dorsal root ganglion neurons in cell culture.
Authors: Gross R A , Macdonald R L
Published: May 2006

Opioid receptors are differentially coupled to ion channels. Mu- and delta-opioid receptors are coupled to calcium- and/or voltage-dependent potassium channels and kappa-opioid receptors are coupled to voltage-dependent calcium channels. Using the single-electrode voltage-clamp technique, we investigated the effect of the kappa-opioid receptor agonist dynorphin A on somatic calcium currents of mouse dorsal root ganglion (DRG) neurons in culture. Three different calcium currents were recorded: a small transient current activated positive to -60 mV; a large, inactivating current activated positive to -50 mV; and a moderate, slowly inactivating current activated positive to -40 mV. The first was less sensitive to cadmium block than the others. These calcium currents were similar to those described in other cells, which have been designated T, N, and L calcium currents, respectively. The opioid peptide dynorphin A reduced calcium current by selectively reducing the large inactivating (N) calcium current. Naloxone, an opioid receptor antagonist, reversed this action of dynorphin A. N calcium current is the predominant calcium current in DRG neurons. If N calcium channels are present in primary afferent terminals, and if they are coupled to kappa-opioid receptors as in the soma, these results suggest a mechanism by which dynorphin A inhibits calcium influx and neurotransmitter release.