bluuebunny/crossref_metadata_2025_split huggingface dataset:
Linker mutagenesis in the gene of an outer membrane protein of escherichia coli, LamB
Authors: Bouges‐Bocquet B. , Villarroya H. , Hofnung M. 
Published: December 2004

AbstractIn order to identify sequences involved in the localization of LamB, an outer membrane protein from E coli K12, mutagenesis by linker insertion has been performed on a lamB gene copy carried on a plasmid devised for this purpose. An analysis of the first set of 16 clones constructed by this technique shows that, in these clones, the lamB protein is altered either by frameshift mutations leading to abnormal COOH terminal (usually premature termination) or by in‐phase deletions or small insertions. Except for two in‐phase linker insertions, which only slightly changed the behavior of the protein, the modified proteins are either toxic to cell growth or unstable. In all cases examined so far, the modified proteins were in the outer membrane. We suggest that toxicity is due to incorrect folding, which leads to disruption of the outer membrane. The nature of the genetic alterations leads to the hypothesis that the first 183 amino acids of the LamB mature protein contain, together with the signal sequence, all the instructions needed for proper localization.
Mitochondrial membrane biogenesis: Characterization and use of pet mutants to clone the nuclear gene coding for subunit V of yeast cytochrome c oxidase
Authors: McEwen Joan E. , Cumsky Michael G. , Ko Christine , Power Scott D. , Poyton Robert O. 
Published: December 2004

AbstractA nuclear pet mutant of Saccharomyces cerevisiae that is defective in the structural gene for subunit V of cytochrome c oxidase has been identified and used to clone the subunit V gene (COX5) by complementation. This mutant, E4‐238 [24], and its revertant, JM110, produce variant forms of subunit V. In comparison to the wild‐type polypeptide (Mr = 12,500), the polypeptides from E4‐238 and JM110 have apparent molecular weights of 9,500 and 13,500, respectively. These mutations directly alter the subunit V structural gene rather than a gene required for posttranslational processing or modification of subunit V because they are cis‐acting in diploid cells; that is, both parental forms of subunit V are produced in heteroallelic diploids formed from crosses between the mutant, revertant, and wild type. Several plasmids containing the COX5 gene were isolated by transformation of JM28, a derivative of E4‐238, with DNA from a yeast nuclear DNA library in the vector YEp13. One plasmid, YEp13‐511, with a DNA insert of 4.8 kilobases, was characterized in detail. It restores respiratory competency and cytochrome oxidase activity in JM28, encodes a new form of subunit V that is functionally assembled into mitochondria, and is capable of selecting mRNA for subunit V. The availability of mutants altered in the structural gene for subunit V (COX5) and of the COX5 gene on a plasmid, together with the demonstration that plasmid‐encoded subunit V is able to assemble into a functional holocytochrome c oxidase, enables molecular genetic studies of subunit V assembly into mitochondria and holocytochrome c oxidase.
Rat liver membrane secretory component is larger than free secretory component in bile: Evidence for proteolytic conversion of membrane form to free form
Authors: Kloppel Thomas M. , Brown William R. 
Published: December 2004

AbstractSecretory component is a receptor for polymeric immunoglobulins on epithelial cells and hepatocytes that facilitates transport of polymeric immunoglobulins into external secretions. Little is known about the transcellular migration of secretory component‐polymeric IgA complexes or the membrane forms of secretory component. We therefore examined rat bile and liver membranes to identify and compare the various molecular species of secretory component. Bile or liver membrane proteins were electrophoresed in sodium dodecyl sulfate‐polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Protein profiles on blots were probed with antisecretory, component antiserum, and the immunoreactive bands were visualized by indirect immunoperoxidase staining. Bile collected in the presence of proteolytic inhibitors showed an immunoreactive doublet band (Mr = 82,000 and 78,000) in the molecular weight range of free secretory component. By contrast, free secretory component in bile collected in the absence of proteolytic inhibitors and purified by affinity chromatography migrated as a single protein with an Mr = 70,000. Both components of the free secretory component doublet bound dimeric IgA when blots were probed with human dimeric IgA. Crude liver membranes prepared in the presence of proteolytic inhibitors showed two immunoreactive secretory component‐containing bands, Mr = 107,000 and 99,000, whereas membranes prepared without proteolytic inhibitors showed two smaller immunoreactive bands; one of these proteolytically severed proteins comigrated with the 82,000‐dalton free secretory component in bile. These results indicate that membrane forms of secretory component are present in rat liver. The observations that the membrane secretory component is larger than biliary free secretory component and yields biliary SC‐like forms of secretory component upon proteolysis support the hypothesis that free secretory component in bile is a proteolytic product of larger liver membrane‐associated secretory component.
Effect of extraction time on ability of calmodulin to activate 30S and 14S dynein ATPases
Authors: Blum Jacob J. , Hayes Alvernon 
Published: December 2004

AbstractCilia from the protozoan Tetrahymena pyriformis were demembranated and then extracted for 5 min with a buffer containing 0.5 M NaCl. The briefly extracted axonemal pellet was then reextracted for about 20 hr. The soluble material obtained from each extraction was resolved into 14S and 30S dynein ATPases by sedimentation on sucrose density gradients and tested for sensitivity to added calmodulin. The 14S dynein obtained by a 5‐min extraction was generally insensitive to added calmodulin, whereas that obtained by 20‐hr extraction of the 5‐min extracted axonemes was activated by calmodulin, the activation being much larger in the “light” 14S fractions than in the “heavy” fractions. The 30S dynein ATPase obtained by a 5‐min extraction was generally activated over 1.6‐fold by added calmodulin, whereas that obtained by the subsequent long extraction was usually activated only 1.3‐fold. After further purification of the 5‐min extracted 30S dynein and of the 5‐min to 20‐hr‐extracted 14S dynein on DEAE‐Sephacel, these dyneins retained much of their calmodulin activatability. The ATPase activity of both 14S and 30S dyneins was inhibited more strongly by erythro‐9‐[3‐(2‐hydroxynonyl)] adenine and by vanadate in the presence of added calmodulin than in its absence. These data suggest that the only ATPase activity present in the fractions studied is that of the dyneins and demonstrate that both the 14S and 30S dynein ATPases may be obtained in forms mat are activated by added calmodulin as well as in forms that are insensitive to added calmodulin.
Electrophoretic analysis of the cytoplasmic and nuclear protein changes after induction of differentiation in WEHI‐3B myelomonocytic leukemia cells
Authors: Cooper Paul C. , Burgess Antony W. 
Published: December 2004

AbstractIn response to a differentiation factor (G‐CSF) the myelomonocytic leukemia cell line (WEHI‐3B(D+) differentiates to form mature macrophages and neutrophils. The effect of G‐CSF on WEHI‐3B(D+) differentiation was augmented by low concentrations (5 ng/ml) of actinomycin D. Quantitative binding of an antineutrophil serum was used to segregate the differentiated cells from the leukemic blast cells. Molecular markers of later myeloid differentiation were detected in myelocytes and macrophages purified from differentiating WEHI‐3B(D+ ) cells. To study the initial molecular processes associated with the initiation of WEHI‐3B(D+) cells to differentiation, the protein changes were analyzed using gel electrophoresis. Quantitative analysis of the fluorographs from the two‐dimensional (2D) electrophorograms of the 35S‐labeled proteins revealed major changes in the biosynthetic rates for 16 proteins within 5 hr: The biosynthesis of six proteins was increased and another ten proteins were synthesized at a reduced rate. Two of the proteins (17K and 36K daltons) were located in the nucleus. Pulse‐chase experiments indicated that protein turnover for these proteins was rapid but the degradation of four proteins was suppressed. At least six of the proteins (16K to 120K daltons) were acidic and were associated with the cytoplasm. Electrophoretic analysis of the 35S‐labeled proteins indicated that a 35K protein induced by G‐CSF was found in high abundance only in purified cells of intermediate differentiation (eg, myelocytes). Other proteins (eg, a very high molecular weight protein, and a 16K dalton protein) were obviously late markers of differentiated neutrophils or macrophages.
Rat liver 6‐phosphofructo 2‐kinase/ fructose 2,6‐bisphosphatase: A review of relationships between the two activities of the enzyme
Authors: El‐Maghrabi M. Raafat , Pilkis Simon J. 
Published: December 2004

AbstractBoth the synthesis and the degradation of Fru‐2,6‐P 2 are catalyzed by a single enzyme protein; ie, the enzyme is bifunctional. This protein, which we have designated 6‐phosphofructo 2‐kinasc/fructose 2,6‐bisphosphatase is an important enzyme in the regulation of hepatic carbohydrate metabolism since its activity determines the steady‐slate concentration of fructose 2,6‐P2, an activator of 6‐phosphofructo 1‐kinase and an inhibitor of fructose 1,6‐bisphosphatase. Regulation of the bifunctional enzyme in intact cells is a complex function of both covalent modification via phosphorylation/dephosphorylation and the influence of substrates and low molecular weight effectors.Recent evidence suggests that both reactions may proceed by two‐step transfer mechanisms with different phosphoenzyme intermediates. The enzyme catalyzes exchange reactions between ADP and ATP and between fructose 6‐P and fructose 2,6‐P2. A labeled phosphoenzyme is formed rapidly during incubation with [2‐32P]Fru‐2,6‐P2. The labeled residue has been identified as 3‐phosphohistidinc. However, it was not possible to demonstrate significant labeling of the enzyme directly from [γ‐32P]ATP. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E‐P is labile) and a fructose 2,6‐bisphosphatase site which is readily phosphorylated by fructose 2,6‐P2. Additional evidence in support of two active sites include: (1) limited proteolysis with thermolysin results in loss of 6‐phosphofructo 2‐kinase activity and activation of fructose 2,6‐bisphosphatase, (2) mixed function oxidation results in inactivation of the 6‐phosphofructo 2‐kinase but no affect on the fructose 2,6‐bisphosphatase, (3) N‐ethylmaleimide treatment also inactivates the kinase but docs not affect the bisphosphatase, and (4) p‐chlorom‐ercuribenzoate immediately inactivates the fructose 2,6‐bisphosphatase but not the 6‐phosphofructo 2‐kinasc. Our findings indicate that the bifunctional enzyme is a rather complicated enzyme; a dimer, probably with two catalytic sites reacting with sugar phosphate, and with an unknown number of regulatory sites for most of its substrates and products. Three enzymes from Escherichia coli, isocitric dehydrogenase kinase/phosphatase, glutamine‐synthetase adenylyltransferase, and the uridylyltransferase for the regulatory protein PU in the glutamine synthetase cascade system also catalyze opposing reactions probably at two discrete sites. All four enzymes are important in the regulation of metabolism and may represent a distinct class of regulatory enzymes.
An improved purification method for cytoplasmic dynein
Authors: Pratt M. M. , Hisanaga S. , Begg D. A. 
Published: December 2004

AbstractAn improved method has been devised for the purification of cytoplasmic dynein from sea urchin eggs (Strongylocentrotus droebachiensis and S purpuratus). This protocol introduces three changes over a previously published procedure (Hisanaga and Sakai: J Biochem 93:87, 1983)—the substitution of diethylaminoethyl (DEAE)‐cellulose for hydroxylapatite chromatography, the elimination of sucrose density gradient centrifugation, and the use of phosphoceliulose chromatography. These changes reduce the time and increase the efficiency of the purification procedure. The purified egg cytoplasmic dynein has enzymatic properties in common with axonemal dynein, including ionic specificity (Ca++ATPase/ Mg++ATPase = 0.8) and inhibition by sodium vanadate and erythro‐9‐2,3‐hydroxynonyl adenine (EHNA). As assayed by silver staining of polyacrylamide gels, the cytoplasmic dynein is composed of two high molecular weight polypeptides ( > 300 kilodaltons) that comigrate with flagellar dynein heavy chains, and lesser amounts of three lower molecular weight bands. None of these polypeptides appears to contain bound carbohydrate. The purification procedure can be modified slightly to allow the preparation of cytoplasmic dynein in only 2 days from as little as 3–5 ml of packed eggs, a 20‐fold reduction over the previous method. This more rapid and efficient method will facilitate the investigation of cytoplasmic dynein in other systems where starting material is limited, including tissue culture cells and nerve axoplasm.
Evidence for transsynaptic regulation of neuronal cell surface heparan sulfate proteoglycan in developing rat superior cervical ganglion
Authors: Greif Karen F. , Trenchard Holly I. 
Published: December 2004

AbstractThe effect of neonatal deafferentation on the expression of a neuronal cell surface heparan sulfate proteoglycan (HeS‐PG) was investigated in the developing rat superior cervical ganglion. Two monoclonal antibodies, one directed against the core protein of HeS‐PG, and one to a determinant associated with a heparan sulfate side‐chain, were used to monitor postnatal increases of HeS‐PG by ra‐dioiminunoassay. Following neonatal deafferentation by section of the cervical sympathetic trunk, total protein per ganglion was slightly reduced at survival times of 7, 14, and 30 days. Expression of the core protein determinant on HeS‐PG was not altered in deafferented ganglia. In contrast, levels of side‐chain determinant were significantly reduced at 14 and 30 days. These results suggest that processing of HeS‐PG side‐chains by principal ganglionic neurons is partially regulated by transsynaptic influences during development. Transsynaptic regulation of neuronal development may be a more general process than was believed previously, with effects not limited to molecules associated with synaptic development.
Detection of mRNAs containing regulatory peptide coding sequences using synthetic oligodeoxynucleotides
Authors: Gozes Illana , Bodner Mordechai , Shani Yael , Fridkin Mati 
Published: December 2004

AbstractTo understand the regulation of the production of peptide hormones, it is vital to elucidate their biosynthetic pathways. We chose to study a major regulatory peptide, vasoactive intestinal peptide (VIP), a peptide possessing both neurotransmitter and neurohormone actions. To identify the specific peptide mRNA we are using, as hybridization probes, radiolabeled synthetic oligodcoxynucleotides with sequence complementary to the predicted peptide mRNA sequence. Employing this approach, we identified and partially purified a ∼ 1600‐base long mRNA containing VIP related sequences which can be translated in vitro into VIP‐immunoreactive polypeptides. Such mRNA was detected in normal VIP producing tissue (rat brain), as well as in a tumor producing VIP (human buccal tumor). This mRNA differs in size from a known VIP‐mRNA identified in human neuro‐blastoma cells, suggesting the possibility of different VIP‐mRNAs in different cell types.
Microcompartmentation of cAMP in wild‐type and memory‐mutant dunce strains of Drosophila melanogaster
Authors: Friedrich Peter , Solti Magda , Gyurkovics Henrik 
Published: December 2004

AbstractThe “enzyme‐probe” method [Solti M, Friedrich P: Eur J Biochem 95:551, 1979] has been applied to characterize the cyclic AMP pool in wild‐type Canton‐S and memory‐mutant dunceM11 strains of Drosophila melanogaster. The kinetics of cyclic AMP breakdown in whole fly homogenates by endogenous cyclic nucleo‐tide phosphodiesterase(s) indicate that the cyclic AMP pool is divided into free and bound fractions. The bound fraction in Canton‐S and dunceM11 is 0.5 and 1.5 pmole/mg fly, respectively. Considering the total cyclic AMP content of the two strains, 1.6 and 10 pmole/mg fly, respectively, we conclude that the bulk of excess cyclic AMP in the mutant is free nucleotide.
Characteristics of the core protein of the aggregating proteoglycan from the Swarm rat chondrosarcoma
Authors: Stevens Jeff W. , Oike Yasuteru , Handley Christopher , Hascall Vincent C. , Hampton Anne , Caterson Bruce 
Published: December 2004

AbstractA ternary complex of hyaluronic acid‐binding region and link protein bound to hyaluronic acid was isolated from limit clostripain digests of proteoglycan aggregates isolated from the Swarm rat chondrosarcoma. Under these conditions, the hyaluronic acid‐binding region has a molecular weight of ≅ 65,000 (HA‐BR65). N‐terminal amino acids in the complex were selectively l4C‐carbamylated. The resulting derivatized HA‐BR65 was isolated, and tryptic peptide maps were prepared and developed on two‐dimensional TLC sheets. A single, labeled peptide was obtained which gave a Mr by ≅ 8,000 by SDS‐PAGE. Chymotrypsin digestion of the ternary complex reduced the molecular weight of HA‐BR65 to a polypeptide of ≅ 55,000 (HA‐BR55) which still retains the same N‐terminal tryptic peptide. Partial digestion of proteoglycan aggregates with clostripain generated a series of larger intermediates with the hyaluronic acid‐binding region. Direct SDS‐PAGE analysis revealed one major intermediate with Mr ≅ 109,000 (HA‐BR109) as well as HA‐BR65. After chondroitinase digestion, two additional prominent intermediates were observed on a SDS‐PAGE gel at Mr ≅ 120,000 (HA‐BR120) and ≅ 140,000 (HA‐BR140). All the intermediates were recognized by a monoclonal antibody specific for the hyaluronic acid‐binding region, and all of them contained the same N‐terminal tryptic peptide. The results indicate that the N terminus of the core protein is at the hyaluronic acid‐binding end of the proteoglycan and that the chondroitin sulfate chains are first present on the core protein in a region between 109,000 and 120,000 molecular weight away from the N terminus.
Three classes of signalling molecules on B‐cell membranes
Authors: Corley Ronald B. , LoCascio N. J. , Ovnic Mariana , Arnold Larry W. , Pillai P. S. , Scott David W. , Haughton Geoffrey 
Published: December 2004

AbstractThe question of whether surface immunoglobulin and Ia molecules have a signalling function in helper T cell‐dependent activation of B cells has been evaluated. Two sources of B cells have been used, one a purified population of haptenbinding B cells, the other a B‐cell lymphoma, CH12, with known antigen specificity. Evidence is presented that both immunoglobulin and Ia molecules are receptors actively involved in the initial activation of resting B cells. Nevertheless, the requirements for ligand binding to either receptor can be bypassed under appropriate conditions, and the implications of this result for the function of these molecules is discussed. With respect to B‐cell Ia, the authors present data that demonstrate two distinct functions of this molecule, one as a restricting element for T‐cell activation, the second as a signalling receptor for B‐cell excitation. On the CH12 surface, the I‐A molecule fulfills the former function, but T‐cell interactions with I‐A fail to result in B‐cell stimulation, suggesting that B‐cell la may limit helper T cell‐B cell interactions. We suggest that the binding of antigen surface immunoglobulin and binding of helper T‐cell receptors to the appropriate Ia molecule(s) results in the activation of genes that encode for a third class of membrane B‐cell receptors, those that bind B‐cell stimulating factors.
Regulation of gonadotropin release, GnRH receptors, and gonadotrope responsiveness: A role for GnRH receptor microaggregation
Authors: Conn P. Michael , Rogers Deloris C. , Seay Sallie G. , Jinnah Hyder , Bates Michael , Luscher Diana 
Published: December 2004

AbstractGonadotropin‐releasing hormone (GnRH) stimulates luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) release from pituitary gonadotrope cells. Additional receptor‐mediated actions of the releasing hormone include homologous regulation of both the GnRH receptor and of cell responsiveness. While it is apparent that the release mechanism is Ca2+ mediated, it remains unclear how this receptor‐mediated action is integrated with regulation of the receptor and with cell responsiveness. It is the purpose of this review to describe the requirements for gonadotropin release as well as for receptor and response regulation in order to prepare an integrated model for these actions of the releasing hormone.
Comparisons of evolutionarily distinct fibronectins: Evidence for the origin of plasma and fibroblast cellular fibronectins from a single gene
Authors: Akiyama Steven K. , Yamada Kenneth M. 
Published: December 2004

AbstractPlasma and fibroblast cellular fibronectins from three different species were compared for structural similarities and differences. Partial tryptic digestion of either human or chicken plasma and cellular fibronectins yields homologous protease‐resistant domains within a species but few homologies between species regardless of the source. Within a species, human or chicken plasma and fibroblast cellular fibronectins are immunologically indistinguishable as determined by the ELISA technique. There is limited immunological cross‐reactivity between species. Two‐dimensional tryptic peptide maps of fibroblast cellular and plasma fibronectins from the same species are also very similar: 85–95% of the spots on such maps comigrate. When peptide maps from different species are compared no more than 10% of the spots comigrate.Three models for the genetic origin of cellular and plasma fibronectins in vertebrates are considered. A model in which both fibroblast cellular and plasma fibronectins arise from the same gene is the simplest that is consistent with the data.
Domain structure of neurofilament subunits as revealed by monoclonal antibodies
Authors: Angeletti Ruth Hogue , Trojanowski John Q. , Garden Martin , Schlaepfer William W. , Lee Virginia M.‐Y. 
Published: December 2004

AbstractMonoclonal antibodies have been prepared against purified neurofilament (NF) subunits (NF68, NF150, and NF200). From 25 fusions, several hundred strongly positive antibodies have been obtained. Among them are antibodies against the specific subunits as well as antibodies recognizing common antigenic determinants. These have all been characterized according to the following properties: ELISA (enzyme‐linked immunosorbant assay) testing against each subunit, immunoblots against enriched neurofilament preparation, immunoblots of cyanogen bromide or chymotrypsin‐treated neurofilaments, immunofluorescence with PC12 cells, and immunohistochemistry of cerebellum. Whereas the antibodies against the NF68 and NF150 appear to react with single cyanogen bromide fragments, the antibodies against the NF200 react with multiple cyanogen bromide fragments. These data are consistent with the hypothesis that the NF200 is partially computed of several repeated structural determinants. Furthermore, all of the antibodies that react with the NF200 recognize the solubilized “sidearm” domain from limited chymotryptic digestions. The locations of the common and variable domains of the three subunits are discussed in light of these results.