bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations in the Escherichia coli UvrB ATPase motif compromise excision repair capacity.
Authors: Seeley T W , Grossman L
Published: May 2006

The Escherichia coli UvrB protein possesses an amino acid sequence motif common to many ATPases. The role of this motif in UvrB has been investigated by site-directed mutagenesis. Three UvrB mutants, with amino acid replacements at lysine-45, failed to confer UV resistance when tested in the UV-sensitive strain N364 (delta uvrB), while five other mutants constructed near this region of UvrB confer wild-type levels of UV resistance. Because even the conservative substitution of arginine for lysine-45 in UvrB results in failure to confer UV resistance, we believe we have identified an amino acid side chain in UvrB essential to nucleotide excision repair in E. coli. The properties of two purified mutant UvrB proteins, lysine-45 to alanine (K45A) and asparagine-51 to alanine (N51A), were analyzed in vitro. While the K45A mutant is fully defective in incision of UV-irradiated DNA, K45A is capable of interaction with UvrA in forming an ATP-dependent nucleoprotein complex. The K45A mutant, however, fails to activate the characteristic increase in ATPase activity observed with the wild-type UvrB in the presence of UvrA and DNA. From these results we conclude that there is a second nucleotide-dependent step in incision following initial complex formation, which is defective in the K45A mutant. This experimental approach may prove of general applicability in the study of function and mechanism of other ATPase motif proteins.
Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster.
Authors: Laski F A , Ganguly S , Sharp P A , RajBhandary U L , Rubin G M
Published: May 2006

Drosophila melanogaster strains with a stably incorporated amber suppressor tRNA gene have been generated. A tRNATyr gene was site specifically mutated to produce an anticodon sequence that recognizes the amber codon and then introduced into Drosophila by using P-element-mediated transformation. Transformants from four integration events were recovered. Two integrations resulted in both male and female sterility, whereas the other two resulted in male sterility but female fertility. Strains derived from the two female-fertile integration events were shown to have a low level of amber-suppressing activity by their ability to suppress an amber mutation in a chloramphenicol acetyltransferase gene.
What is molt-inhibiting hormone? The role of an ecdysteroidogenesis inhibitor in the crustacean molting cycle
Authors: Naya Yoko , Ohnishi Mayumi , Ikeda Midori , Miki Wataru , Nakanishi Koji
Published: May 2006

The
in vivo
molt-inhibitory effects of the ecdysone biosynthesis inhibitors 3-hydroxy-L-kynurenine and xanthurenic acid were investigated. These ecdysone biosynthesis inhibitors, isolated from the eyestalks of blue crabs (
Callinectes sapidus
), were injected into eyestalk-ablated crayfish (
Procambarus clarkii
). The active factor was found to be species-nonspecific within crabs and crayfish. The seasonal profiles of the xanthurenic acid and ecdysone titers exhibited a staggered relationship. Moreover, the activity of a 3-hydroxy-L-kynurenine aminotransferase varied during the molting cycle. The data suggested that 3-hydroxy-L-kynurenine, which is secreted from the X-organ-sinus gland complex of crustaceans, is released into the hemolymph, and after accumulating at the surface of the Y-organ, is converted into the active form, xanthurenic acid. Xanthurenic acid was found to profoundly repress ecdysteroidogenesis
in vitro
.
Expression in Xenopus oocytes of rat liver mRNA coding for a bile salt-dependent cholesteryl ester hydrolase.
Authors: Zolfaghari R , Harrison E H , Ross A C , Fisher E A
Published: May 2006

A catalytically active bile salt-dependent cholesteryl ester hydrolase (CEH) was expressed when Xenopus oocytes were injected with rat liver mRNA. The expressed CEH activity was highly dependent on the presence of trihydroxy bile salts (cholate or one of its conjugates); maximum hydrolytic activity was observed in the presence of 10 mM sodium cholate. The expressed CEH was not activated by dihydroxy bile salts (deoxycholate and its conjugates). In the presence of 10 mM sodium cholate, the CEH activity was maximal near pH 7 but was significant between pH 6 and 8. Monospecific immune IgG raised against rat pancreatic CEH completely inhibited the CEH expressed in Xenopus oocytes. Phenylmethylsulfonyl fluoride, a serine enzyme inhibitor, was inhibitory to the expressed CEH activity, whereas p-chloromercuribenzoate (up to 5 mM), a potent thiol-blocking agent, did not significantly inhibit the expressed activity. These experiments clearly demonstrate that the liver contains an mRNA encoding a bile salt-dependent CEH activity and suggest that the uptake of pancreatic enzyme is not necessarily the source of liver CEH as has been speculated.
Molecular cloning and characterization of a mammalian excision repair gene that partially restores UV resistance to xeroderma pigmentosum complementation group D cells.
Authors: Arrand J E , Bone N M , Johnson R T
Published: May 2006

A hamster DNA repair gene has been isolated by cosmid rescue after two rounds of transfection of an immortalized xeroderma pigmentosum (XP) complementation group D cell line with neomycin-resistance gene (neo)-tagged normal hamster DNA and selection with G418 and ultraviolet irradiation. The functional length of the sequence has been defined as 11.5 kilobase pairs by measurement of the region of overlap between two hamster DNA-containing cosmids, cloned by selection for the integrated neo gene, that are able to confer an increase in resistance to ultraviolet irradiation on two XP-D cell line but not on an XP-A line. Detailed molecular characterization of the hamster repair gene has revealed no obvious similarities to two human excision repair genes (ERCC1 and ERCC2) that correct repair-defective hamster cells but have no effect on XP cells. Hybridization analyses of normal human and XP cell genomic DNAs and mRNAs, using a cosmid-clone probe from which repeated sequences have been removed, show that homologues are present and expressed in all cases.
Reciprocal inhibition of binding between interleukin 3 and granulocyte-macrophage colony-stimulating factor to human eosinophils.
Authors: Lopez A F , Eglinton J M , Gillis D , Park L S , Clark S , Vadas M A
Published: May 2006

125I-labeled recombinant human interleukin 3 (IL-3) bound, at 4 degrees C, to a single class of high-affinity receptors on human eosinophils with an apparent dissociation constant (Kd) of 470 pM, but it did not bind to human neutrophils. 125I-labeled recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) also bound to a single class of high-affinity receptors on eosinophils with an apparent Kd of 44 pM and on neutrophils with an apparent Kd of 70 pM. These binding characteristics were consistent with the biological activities of IL-3 and GM-CSF on eosinophils and with the lack of stimulation of neutrophil function by IL-3. Specificity studies under conditions shown to prevent receptor internalization showed that the binding of 125I-labeled IL-3 to eosinophils was partially inhibited by GM-CSF but not by other cytokines. Reciprocal experiments with 125I-labeled GM-CSF showed that IL-3 but not other cytokines partially inhibited binding to eosinophils. In contrast, the binding of 125I-labeled GM-CSF to neutrophils was not inhibited by IL-3 or other cytokines tested. Quantitative inhibition binding experiments on eosinophils showed that the reciprocal inhibition between IL-3 and GM-CSF was not complete up to a concentration of heterologous ligand of 100 nM. These results show that (i) IL-3 binds to eosinophils but not neutrophils and (ii) IL-3 and GM-CSF specifically interact on the surface of eosinophils, providing a possible mechanism for the overlapping activities of IL-3 and GM-CSF on these cells.
Cell sorting-out is modulated by both the specificity and amount of different cell adhesion molecules (CAMs) expressed on cell surfaces.
Authors: Friedlander D R , Mège R M , Cunningham B A , Edelman G M
Published: May 2006

Cell adhesion molecules (CAMs) are cell surface glycoproteins that may play a variety of roles in morphogenesis and histogenesis, particularly in defining borders of discrete cell populations. To examine the influence of CAM expression on such cell segregation events in vitro, we have transfected cells with cDNAs coding for two calcium-dependent CAMs of different specificity, the liver CAM (L-CAM) and the structurally related molecule N-cadherin. The cDNAs were introduced separately or together into murine sarcoma S180 cells, which normally do not express these molecules, to produce cell lines denoted S180L, S180cadN, and S180L/cadN, respectively. A number of cell lines of each type were produced that differed in their levels of CAM expression. In adhesion assays, S180L and S180cadN cells aggregated specifically via their respective CAMs, and S180L cells did not appear to adhere to S180cadN cells. Cells expressing high levels of each CAM aggregated more rapidly than cells expressing low levels. Segregation between two cell types occurred when they expressed CAMs of different specificity or different levels of the same CAM. S180L and S180cadN cells both sorted out from untransfected cells, and cells expressing high levels of either L-CAM or N-cadherin segregated from cells expressing low levels of the same CAM; in all cases segregation was inhibited by antibodies specific for the transfected CAM. S180L cells sorted out from S180cadN cells, but this segregation was inhibited only when antibodies to both CAMs were applied together. Doubly transfected S180L/cadN cells also sorted out from S180L cells and from S180cadN cells, and the process was inhibited by antibodies to the unshared CAM (N-cadherin or L-CAM, respectively). Cytochalasin D and nocodazole inhibited sorting-out, consistent with the probable role of microfilaments and microtubules in cell movement and in accord with evidence that the action of these CAMs depends on interactions with cortical cytoplasmic components. Using cDNAs for only two CAMs in these studies, we could distinguish at least eight cell lines by their behavior in sorting-out assays. This suggests that qualitative and quantitative differences in the expression in vivo of a relatively small number of CAMs can lead to a large variety of patterns among cell collectives and their borders during tissue formation.
Duplication of large genomic regions during the evolution of vertebrate homeobox genes.
Authors: Schughart K , Kappen C , Ruddle F H
Published: May 2006

The phylogenetic relationships of 21 murine Antp-class (Drosophila mutation Antennapedia-type class) homeobox genes have been analyzed, and several groups of related genes have been identified. The murine Antp-class homeobox genes are localized within four gene clusters. The similar structural organization of the four gene clusters strongly suggests that genes within a group of related Antp-class homeobox genes are derived from duplications of large genomic regions. After the duplication, the gross structures of the homeobox gene clusters have been maintained over a long period of evolutionary time, indicating that the specific organization of genes within a cluster may be of functional importance.
Blocking of pregnancy in mice by immunization with anti-idiotype directed against monoclonal anti-progesterone antibody.
Authors: Wang M W , Heap R B , Taussig M J
Published: May 2006

Passive transfer of monoclonal anti-progesterone antibodies shortly after mating blocks the onset of pregnancy in different species (mouse, rat, and ferret). Here we report that BALB/c mice can be actively immunized against progesterone, and hence against pregnancy, by means of rabbit anti-idiotypic antibodies specific for a mouse monoclonal anti-progesterone antibody, DB3. Some of the anti-idiotypic antibodies reacted with the steroid-combining site on the DB3 molecule. In response to repeated anti-idiotypic immunization, mice produced serum anti-progesterone antibodies (up to 100 micrograms/ml) that resembled DB3 in idiotypy, affinity, and specificity for progesterone and other steroid ligands. Thus an anti-idiotype can mimic the antigenicity of a steroid hormone with a high degree of accuracy. Compared with immunization with a progesterone-bovine serum albumin conjugate, the anti-progesterone response to anti-idiotype was considerably lower and clonally restricted. When mated after completion of the immunization course, the fertility rate of anti-idiotype-immunized mice was reduced to 30% from a control level of 91%. The anti-fertility effect was correlated with the circulating anti-progesterone concentration in individual animals and persisted for 4 or 5 estrous cycles. Active immunization with progesterone-bovine serum albumin was a highly effective means of rendering mice infertile; it reduced the fertility rate to zero over 16 or 17 estrous cycles. Our results suggest that anti-idiotypes may form the basis of contraceptive vaccines.
CD4 antigen-based antireceptor peptides inhibit infectivity of human immunodeficiency virus in vitro at multiple stages of the viral life cycle.
Authors: Nara P L , Hwang K M , Rausch D M , Lifson J D , Eiden L E
Published: May 2006

Benzylated derivatives of peptides corresponding to residues 81 through 92 of the CD4 molecule [CD4-(81-92)] inhibit human immunodeficiency virus 1 (HIV-1)-induced cell fusion and infection in vitro. If such peptides are to be considered as candidates in the therapy of HIV infection, it is crucial to know if the anti-HIV efficacy of CD4-based peptides is limited to blockade of infection and virus-induced cell fusion or if other stages of the viral life cycle are affected by these compounds. Accordingly, an in vitro quantitative microassay for acute HIV infection was divided into two kinetic phases corresponding to the two general stages of the viral life cycle: (i) viral infection and (ii) transmission of virus and viral protein products through cell contact or release of free virions. CEM-SS cell cultures were treated with peptide during either the infection or the transmission phase of the assay. When peptides were present during the infection phase, inhibition of syncytium formation correlated with decreased expression of viral core protein p24 and lack of infectious cell centers when cells exposed to virus were washed and replated onto fresh uninfected indicator cells. These data are consistent with complete inhibition of viral infection when peptide is present only during initial exposure to virus. Unexpectedly, parallel inhibition of syncytium formation, decreased p24 levels, and inhibition of infectious cell center formation were also seen even when peptides were added as late as 48 hr after inoculation, during the transmission period of the assay. Since viral binding and penetration are completed well before 48 hr in this assay system, CD4-(81-92) peptide derivatives appear to exert a virostatic effect on cultures already infected with HIV-1, decreasing p24 production, cytopathicity, and cell-mediated infectivity.
Disease tropism of c-erbB: effects of carboxyl-terminal tyrosine and internal mutations on tissue-specific transformation.
Authors: Pelley R J , Maihle N J , Boerkoel C , Shu H K , Carter T H , Moscovici C , Kung H J
Published: May 2006

Avian leukosis virus induces erythroleukemia in chickens by proviral insertional mutation of the protooncogene c-erbB. The product of the insertionally activated c-erbB locus lacks the extracellular ligand-binding domain and is strictly leukemogenic. It has previously been demonstrated that the disease spectrum associated with aberrant c-erbB expression can be expanded by structural perturbation of the cytoplasmic domain of this protein. In this report, we use mutagenesis and retroviral vectors to identify specific mutations in the carboxyl-terminal domain of the insertionally activated c-erbB product that are sufficient to activate the sarcomagenic potential of this protein. Interestingly, a point mutation in the kinase domain appears to be sufficient for sarcomagenic activation. However, removal of the terminal tyrosine residue of the c-erbB product, implicated in modulating kinase activity, does not lead to a fully transforming phenotype. These studies suggest that there are multiple ways to activate the fibroblast-transforming potential of the insertionally activated c-erbB product. The conformation of this protein may play a more significant role in oncogenic activation than the phosphorylation status of the putative carboxyl-terminal autophosphorylation site.
Rapidly and slowly replicating human immunodeficiency virus type 1 isolates can be distinguished according to target-cell tropism in T-cell and monocyte cell lines.
Authors: Schwartz S , Felber B K , Fenyö E M , Pavlakis G N
Published: May 2006

Human immunodeficiency virus type 1 (HIV-1) isolates from various patients were divided into two major groups, rapid/high and slow/low, according to their replication properties in vitro. Rapid/high isolates grow well in cell lines and induce the formation of syncytia in peripheral blood mononuclear cells. In contrast, slow/low isolates do not replicate in cell lines and rarely induce syncytia in peripheral blood mononuclear cells. To understand the differences in replicative capacity of these isolates, a panel of indicator cell lines was used. These cell lines were generated for sensitive detection of HIV-1 isolates and show characteristics of T-lymphoid or monocytoid cells. As a result of infection, chloramphenicol acetyltransferase expression is activated. Rapid/high viruses activate chloramphenicol acetyltransferase expression in T-cell and monocytoid indicator cell lines, whereas slow/low isolates activate chloramphenicol acetyltransferase expression only in monocytoid cell lines. The block in infection of T-lymphoid cells by the slow/low isolates appears to occur early in the infection cycle, prior to the production of the virally encoded tat protein. HIV-1 isolates can thus be distinguished according to target-cell tropism. Monocyte-derived cells seem to be a more general target for the various HIV-1 isolates.
An RNA polymerase II transcription factor has an associated DNA-dependent ATPase (dATPase) activity strongly stimulated by the TATA region of promoters.
Authors: Conaway R C , Conaway J W
Published: May 2006

A transcription factor required for synthesis of accurately initiated run-off transcripts by RNA polymerase II has been purified and shown to have an associated DNA-dependent ATPase (dATPase) activity that is strongly stimulated by the TATA region of promoters. This transcription factor, designated delta, was purified more than 3000-fold from extracts of crude rat liver nuclei and has a native molecular mass of approximately 230 kDa. DNA-dependent ATPase (dATPase) and transcription activities copurify when delta is analyzed by hydrophobic interaction and ion-exchange HPLC, arguing that transcription factor delta possesses an ATPase (dATPase) activity. ATPase (dATPase) is specific for adenine nucleotides; ATP and dATP, but not CTP, UTP, or GTP, are hydrolyzed. ATPase (dATPase) is stimulated by both double-stranded and single-stranded DNAs, including pUC18, ssM13, and poly(dT); however, DNA fragments containing the TATA region of either the adenovirus 2 major late or mouse interleukin 3 promoters stimulate ATPase as much as 10-fold more effectively than DNA fragments containing nonpromoter sequences. These data suggest the intriguing possibility that delta plays a critical role in the ATP (dATP)-dependent activation of run-off transcription through a direct interaction with the TATA region of promoters.
Modulation of the stability of a gene-regulatory protein dimer by DNA and cAMP.
Authors: Brown A M , Crothers D M
Published: May 2006

We describe an experimental approach to the measurement of protein subunit exchange in which biotinylated subunits mediate attachment of 35S-labeled subunits to a streptavidin column as a result of the exchange process. Application of the method to Escherichia coli catabolite activator protein (CAP) revealed that in the absence of cAMP, the dimerization equilibrium constant is 3 x 10(10) M-1, with a dimer lifetime of 300 min. Exchange of CAP subunits is accelerated at least 1000-fold by the presence of nonspecific DNA, under low ionic strength conditions. Catalysis of exchange also occurs at physiological ionic conditions. In contrast, physiological concentrations of cAMP stabilize CAP with respect to subunit exchange in either the presence or the absence of DNA. We discuss the functional implications of monomerization of gene-regulatory proteins resulting from kinetic and thermodynamic lability of their dimers.
Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor.
Authors: Weiser W Y , Temple P A , Witek-Giannotti J S , Remold H G , Clark S C , David J R
Published: May 2006

A cDNA encoding a human macrophage migration inhibitory factor (MIF) was isolated, through functional expression cloning in COS-1 cells, from a cDNA library prepared from a lectin-stimulated T-cell hybridoma, T-CEMB. The 115-amino acid polypeptide encoded by the MIF cDNA (p7-1) was effectively released from the transfected COS-1 cells and yielded readily detectable MIF activity in the culture supernatant despite the apparent lack of a classical protein secretory sequence. Insertional mutational analysis and elution of MIF activity from polyacrylamide gel slices demonstrated that the Mr 12,000 protein with MIF activity released by the COS-1 cells is encoded by p7-1. The p7-1 cDNA hybridized with a 700-base mRNA expressed by Con-A-stimulated lymphocytes but not unstimulated lymphocytes. The availability of the MIF cDNA clone and recombinant MIF will facilitate the analysis of the role of this lymphokine in cell-mediated immunity, immunoregulation, and inflammation.
Role of thrombin and thromboxane A2 in reocclusion following coronary thrombolysis with tissue-type plasminogen activator.
Authors: Fitzgerald D J , Fitzgerald G A
Published: May 2006

Reocclusion of the coronary artery occurs after thrombolytic therapy of acute myocardial infarction despite routine use of the anticoagulant heparin. However, heparin is inhibited by platelet activation, which is greatly enhanced in this setting. Consequently, it is unclear whether thrombin induces acute reocclusion. To address this possibility, we examined the effect of argatroban [MCI9038, (2R,4R)-4-methyl-1-[N alpha-(3-methyl-1,2,3,4-tetrahydro-8- quinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid], a specific thrombin inhibitor, on the response to tissue-type plasminogen activator in a closed-chest canine model of coronary thrombosis. MCI9038 prolonged the thrombin time and shortened the time to reperfusion (28 +/- 2 min vs. 59 +/- 7 min in controls; mean +/- SEM, n = 5, P less than 0.01). At the highest dose, 2.5 mg/kg per hr, complete reocclusion was prevented in four of the five experimental animals, whereas reocclusion occurred in all five controls. However, reperfusion was complicated by cycles of decrease flow, which were abolished by the thromboxane A2 antagonist, GR32191. GR32191 at 1 mg/kg combined with MCI9038 at 0.5 mg/kg per hr prevented reocclusion, whereas, at these doses, either drug alone was without effect. In addition, thromboxane A2 biosynthesis, determined as excretion of its metabolite 2,3-dinor-thromboxane B2, was increased after reperfusion at all doses of MCI9038. These data demonstrate that thrombin impairs thrombolysis induced by tissue-type plasminogen activator in vivo and induces acute reocclusion. Furthermore, the response to thrombin inhibition may be impaired by continued formation of thromboxane A2.
Transformation of siphon responses during conditioning of Aplysia suggests a model of primitive stimulus-response association.
Authors: Walters E T
Published: May 2006

A semi-intact preparation was used to study the effects of classical conditioning on the type of siphon response elicited by a conditioned stimulus to the mantle of Aplysia. Five pairings of the conditioned stimulus with an unconditioned stimulus to nerves from the tail transformed the constricting alpha response of the siphon into a conditioned flaring response resembling the unconditioned response to stimulation of the tail nerves. Although some pseudoconditioning occurred, an associative component was indicated by the significantly greater incidence of flaring responses after paired training than after unpaired presentations of the conditioned and unconditioned stimulus or the unconditioned stimulus alone. Previously described cellular plasticity in the underlying neural circuits suggests a testable model based on cell-wide rather than synapse-specific mechanisms, which can account for specific conditioned responses. In this model, effective stimulus-response associations are produced by a concatenation of stimulus-specific facilitation of sensory neurons (a mechanism for alpha conditioning) and response-specific facilitation of motor neurons (a mechanism for pseudoconditioning).
Epidermal growth factor deficiency associated with diabetes mellitus.
Authors: Kasayama S , Ohba Y , Oka T
Published: May 2006

The production of epidermal growth factor (EGF) in the submandibular gland and its circulating level were studied in diabetic mice. In genetically diabetic (C57BL/KsJ db/db) mice, EGF concentrations in the submandibular gland and plasma were reduced to 13% and 30% of the control levels, respectively. In streptozotocin-treated diabetic mice, they were reduced to 18% and 20% of controls, respectively, 5 weeks after the drug injection. Furthermore, levels of submandibular prepro-EGF mRNA in these diabetic mice were decreased almost in parallel with the glandular EGF concentrations, while there was no change in the levels of submandibular beta-actin mRNA and kidney prepro-EGF mRNA. In addition, histological examination of the submandibular glands indicated that the size of the granular convoluted tubules, which produce EGF, was substantially reduced in the diabetic mice. Insulin administration to streptozotocin-treated mice almost completely reversed the decrease in EGF content in the submandibular gland, substantially elevated the level of the glandular prepro-EGF mRNA and plasma EGF concentration, and increased the size of the granular convoluted tubules in the gland. These results indicate that EGF deficiency occurs in diabetes mellitus and that insulin may be important in maintaining the normal level of EGF in the submandibular gland and plasma.
Hydroxylation of aspartic acid in domains homologous to the epidermal growth factor precursor is catalyzed by a 2-oxoglutarate-dependent dioxygenase.
Authors: Stenflo J , Holme E , Lindstedt S , Chandramouli N , Huang L H , Tam J P , Merrifield R B
Published: May 2006

3-Hydroxyaspartic acid and 3-hydroxyasparagine are two rare amino acids that are present in domains homologous to the epidermal growth factor precursor in vitamin K-dependent plasma proteins as well as in proteins that do not require vitamin K for normal biosynthesis. They are formed by posttranslational hydroxylation of aspartic acid and asparagine, respectively. The first epidermal growth factor-like domain in factor IX (residues 45-87) was synthesized with aspartic acid in position 64, replacing 3-hydroxyaspartic acid. It was used as substrate in a hydroxylase assay with rat liver microsomes as the source of enzyme and reaction conditions that satisfy the requirements of 2-oxoglutarate-dependent dioxygenases. The synthetic peptide stimulated the 2-oxoglutarate decarboxylation in contrast to synthetic, modified epidermal growth factor (Met-21 and His-22 deleted and Glu-24 replaced by Asp) and synthetic peptides corresponding to residues 60-71 in human factor IX. This indicates that the hydroxylase is a 2-oxoglutarate-dependent dioxygenase with a selective substrate requirement.
A highly potent insulin: des-(B26-B30)-[AspB10,TyrB25-NH2]insulin(human).
Authors: Schwartz G P , Burke G T , Katsoyannis P G
Published: May 2006

An insulin analogue that embodies two distinct structural modifications, each of which independently increases insulin activity, has been synthesized and evaluated for biological activity. The analogue, des-(B26-B30)-[AspB10,TyrB25-NH2]insulin is the most potent insulin analogue yet described; it displays an 11- to 13-fold higher activity than natural insulin. The findings are discussed with regard to the receptor-binding domains of insulin.
Dynamic aspects of intermediate filament networks in BHK-21 cells.
Authors: Vikstrom K L , Borisy G G , Goldman R D
Published: May 2006

A procedure was developed for the conjugation of vimentin with biotin. Biotinylated vimentin was then microinjected into BHK-21 cells and the fate of the labeled protein was determined at various times postinjection by indirect immunofluorescence. Microinjected vimentin could be traced through a specific sequence of morphological changes ultimately resulting in the formation of a filamentous network. The injected protein was first detected in spots dispersed throughout the cytoplasm. Subsequently, these spots appeared to cluster near the nucleus where they merged into a diffuse "cap." This cap coincided with a concentration of endogenous intermediate filaments and eventually gave rise to a filamentous network that was coincident with the endogenous intermediate filament network as determined by double-label immunofluorescence. The results indicate that the incorporation of exogenous vimentin into a filamentous network is initiated in a perinuclear region and progresses in a polarized fashion toward the cell surface.
Evidence for the involvement of opioid neuropeptides in the adherence and migration of immunocompetent invertebrate hemocytes.
Authors: Stefano G B , Leung M K , Zhao X H , Scharrer B
Published: May 2006

Evidence for the participation of opioid neuropeptides in immunoregulatory activities, especially cellular adherence and migration, has been obtained in representatives of two phyla of invertebrates, the mollusc Mytilus edulis and the insect Leucophaea maderae. The injection of a synthetic analog of [Met]enkephalin [( D-Ala2,Met5]enkephalinamide, DAMA; 10(-6) M) had a stimulatory, naloxone-reversible effect on the directed migration of immunocompetent hemocytes. Incubation of hemolymph in the presence of exogenous or endogenous opioid material significantly enhanced the adherence of hemocytes on albumin-coated slides as demonstrated by use of indirect Zeiss-Zonax reflectance computer analysis. Conversely, hemocyte adherence was markedly reduced by the addition of naloxone (10(-8) M) to the incubation medium, either alone or in combination with DAMA. The antagonistic effects of naloxone on the stimulatory activities of opioids indicate that, like those previously reported in mammals, they are receptor-mediated. The presence of an endogenous [Met]enkephalin-like material was demonstrated in cell-free hemolymph as well as sequestered hemocytes by use of high-pressure liquid chromatography and radioimmunoassay. These results demonstrate that the capacity of immunocytes to release and respond to opioid neuropeptide messengers is not restricted to mammalian organisms but was developed early in the course of evolution.
De novo expression of intercellular-adhesion molecule 1 in melanoma correlates with increased risk of metastasis.
Authors: Johnson J P , Stade B G , Holzmann B , Schwäble W , Riethmüller G
Published: May 2006

The 89-kDa cell surface glycoprotein, P3.58, is detectable on advanced human melanomas in situ but not on benign melanocytes or early melanomas. cDNA cloning of P3.58 from melanoma cells was accomplished by screening a lambda zap expression vector library with monoclonal antibodies produced against the denatured antigen. Nucleotide sequencing of the clones revealed that P3.58 is identical to the intercellular-adhesion molecule 1. No qualitative differences in P3.58 mRNA species could be seen between melanoma cells and hematopoietic cells and no differences in gene organization were observed between peripheral blood leukocytes and melanoma cells. Inspection of the deduced amino acid sequence of P3.58 indicated the presence of the consensus sequence characteristic for complement-binding proteins. The acquisition of this cell-adhesion molecule during the process of tumor progression is speculated to contribute to the development of metastasis in melanoma.
Alternative mRNA splicing generates multiple forms of peptidyl-glycine alpha-amidating monooxygenase in rat atrium.
Authors: Stoffers D A , Green C B , Eipper B A
Published: May 2006

Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the conversion of a variety of glycine-extended peptides into biologically active alpha-amidated product peptides in a reaction dependent on copper, ascorbate, and molecular oxygen. We have isolated and sequenced cDNAs representing the two major classes of PAM mRNA in the adult rat heart atrium. The two types of cDNA, rPAM-1 and rPAM-2, are identical except for the deletion of a 315-base-pair segment within the protein coding region in rPAM-2, suggesting that rPAM-1 and rPAM-2 arise by alternative splicing. Northern analysis using a cDNA probe derived from within the 315-base-pair region deleted in rPAM-2 visualized the larger of the PAM mRNAs in adult rat atrium and not the smaller, indicating that the presence or absence of this 315-nucleotide segment is a major feature distinguishing the two size forms of PAM mRNA. The 105 amino acid segment that distinguishes the two forms of atrial PAM contains a consensus N-glycosylation site and a paired basic amino acid site of potential importance in endoproteolytic processing. Comparison of the nucleotide sequences of rat, frog, and bovine PAM cDNAs reveals an extremely well conserved segment in the 3' untranslated region. The high degree of conservation in amino acid sequence throughout the catalytic, intragranular, and cytoplasmic domains of rat atrium, bovine pituitary, and frog skin PAM suggests that both the catalytic and noncatalytic domains of the protein subserve important functions.
Protein-electroblotting and -microsequencing strategies in generating protein data bases from two-dimensional gels.
Authors: Bauw G , Van Damme J , Puype M , Vandekerckhove J , Gesser B , Ratz G P , Lauridsen J B , Celis J E
Published: May 2006

Coomassie blue-stained, heat-dried, and computer-imaged two-dimensional gels used to develop comprehensive human protein data bases served as the protein source to generate partial amino acid sequences. The protein spots were collected from multiple gels, rehydrated, concentrated by stacking into a new gel, electroblotted onto inert membranes, and in situ-digested with trypsin. Peptides eluting from the membranes were separated by HPLC and sequenced. Using this procedure, it was possible to generate partial sequences from 13 human proteins recorded in the amnion cell protein data base. Eight of these sequences matched those of proteins stored in data bases, demonstrating that a systematic analysis of proteins by computerized two-dimensional gel electrophoresis can be directly linked to protein microsequencing methods. The latter technique offers a unique opportunity to link information contained in protein data bases derived from the analysis of two-dimensional gels with forthcoming DNA sequence data on the human genome.
Avian proto-myc genes promoted by defective or nondefective retroviruses are single-hit transforming genes in primary cells.
Authors: Zhou R P , Duesberg P H
Published: May 2006

Lymphomas of certain strains of chickens infected by retroviruses frequently contain recombinant transforming genes in which the promoter of the cellular proto-myc gene is replaced by that of a defective rather than an intact retrovirus. Here we ask whether the resulting hybrid genes are sufficient for tumorigenic transformation like viral myc genes. Further, we ask whether retroviruses must be defective in order to mutate proto-myc to a transforming gene or whether the defectiveness plays a transformation-independent function in tumorigenesis. For this purpose the defective provirus of proviral-proto-myc recombinants from lymphomas were repaired, or intact proviruses were recombined with proto-myc genes in vitro, and then compared to recombinant proto-myc genes with defective proviruses for transforming function in quail embryo fibroblasts. It was found that a single copy of a provirus-proto-myc recombinant gene with an intact provirus is sufficient to transform a quail embryo cell in vitro. Moreover, our analyses showed that multiple internal retroviral deletions [corrected] eliminate or inhibit provirus expression. The effect of these deletions [corrected] was detectable only because the inactive proviruses were linked to the selectable, transforming proto-myc gene marker. It is consistent with our results that proviral defectiveness of recombinant proto-myc genes is necessary in vivo for the clonal growth of a transformed cell into a tumor to escape antiviral immunity. The large discrepancy between the probabilities of provirus insertion and tumorigenesis is suggested to reflect the low probabilities of spontaneous deletion of the provirus and of rare, strain-specific defects of tumor-resistance genes of the host.
Molecular complementation of a genetic marker in Dictyostelium using a genomic DNA library.
Authors: Dynes J L , Firtel R A
Published: May 2006

We constructed a partial Sau3A Dictyostelium genomic DNA library in a shuttle vector that replicates extrachromosomally in Dictyostelium cells. This library was used to complement Dictyostelium strain HPS400, which lacks thymidylate synthase activity and requires exogenous thymidine for growth. We have used a modified high-frequency transformation protocol that allows the introduction of the library into a sufficient number of Dictyostelium cells to select complementing plasmids. Using this approach, we have isolated a gene (Thy1) that complements the thymidine growth requirement of HPS400. The gene encodes a 1.2-kilobase RNA and the derived amino acid sequence shows no homology to thymidylate synthase, a protein highly conserved throughout evolution, or any other protein sequence in the data base examined. Thy1 provides an important selectable marker for transforming Dictyostelium cells. In addition, this work suggests that it will be possible to isolate genes that are essential for developmental processes in Dictyostelium by complementation.
Immunoglobulin double-isotype expression by trans-mRNA in a human immunoglobulin transgenic mouse.
Authors: Shimizu A , Nussenzweig M C , Mizuta T R , Leder P , Honjo T
Published: May 2006

We have studied immunoglobulin double-isotype expression in a transgenic mouse (TG.SA) in which expression of the endogenous immunoglobulin heavy chain locus is almost completely excluded by a nonallelic rearranged human mu transgene. By flow-cytometric analyses, we have shown that a small, but significant, portion (about 4%) of transgenic spleen cells expresses human mu (transgene) and mouse gamma (endogenous) chains when cultured in vitro with bacterial lipopolysaccharide and interleukin 4. By using amplification of cDNA by the polymerase chain reaction, followed by cloning and sequencing of the amplified cDNA fragment, we have demonstrated expression of trans-mRNA consisting of the transgenic variable and endogenous constant (gamma 1) region sequences. Such trans-mRNA could be produced by either switch recombination or trans-splicing between the transgene and endogenous sterile gamma 1-gene transcripts. These results indicate that trans-splicing might be a possible mechanism for the immunoglobulin double-isotype expression in normal B lymphocytes that have not rearranged the second expressed constant region gene.
One-way analysis of variance with unequal variances.
Authors: Rice W R , Gaines S D
Published: May 2006

We have designed a statistical test that eliminates the assumption of equal group variances from one-way analysis of variance. This test is preferable to the standard technique of trial-and-error transformation and can be shown to be an extension of the Behrens-Fisher T test to the case of three or more means. We suggest that this procedure be used in most applications where the one-way analysis of variance has traditionally been applied to biological data.
Vectorial expansion of the involucrin gene and the relatedness of the hominoids.
Authors: Djian P , Green H
Published: May 2006

In higher primates, the coding region of the gene for involucrin, an epidermal protein, is mostly composed of a recently generated (modern) segment of repeats of a sequence of 10 codons. While the rest of the coding region has evolved only by nucleotide substitutions, the modern segment has evolved by successive addition of repeats. This process has not taken place randomly; instead, the expansion of the modern segment has been progressive from 3' to 5' end, thus adding vectorially regions that have been defined as early, middle, and late. The relatedness of the human, chimpanzee, and gorilla may be analyzed with greatest sensitivity by comparing their middle regions. The chimpanzee involucrin gene is more closely related to that of the gorilla than to that of the human.
Inhibition of the replication of hepatitis B virus by the carbocyclic analogue of 2'-deoxyguanosine.
Authors: Price P M , Banerjee R , Acs G
Published: May 2006

We report that treatment of 2.2.15, a human hepatoblastoma-derived cell line in which hepatitis B virus is actively replicating, with the carbocyclic analogue of 2'-deoxyguanosine [Shealy, Y. F., O'Dell, C. A., Shannon, W. M. & Arnett, G. (1984) J. Med. Chem. 27, 1416-1421] resulted in the nearly complete cessation of viral replication, as monitored by the absence of both intracellular episomal and secreted viral DNAs and by the absence of viral DNA polymerase activity. The drug was nontoxic in concentrations up to 200 times the minimum effective inhibitory concentration.
Direct visualization and cellular localization of D1 and D2 dopamine receptors in rat forebrain by use of fluorescent ligands.
Authors: Ariano M A , Monsma F J , Barton A C , Kang H C , Haugland R P , Sibley D R
Published: May 2006

The regional and cellular localization of the two subtypes of dopamine receptors, D1 and D2, have been ascertained in rat forebrain by use of fluorescent dopaminergic antagonist ligands. (R,S)-5-(4'-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3- methyl-[1H]-3-benzazepin-7-ol, the 4'-amino derivative of the high-affinity D1-specific antagonist SCH 23390, and the D2 selective antagonist N-(p-aminophenethyl)spiperone were chemically derivatized using the fluorescent compound tetramethylrhodamine. The modification of these antagonist ligands has allowed the specific, cellular resolution of the D1 and D2 receptor binding sites in intact, highly organized regions of forebrain slices in a very rapid experimental time frame. The regional localization of receptors labeled by the fluorescent probes is in agreement with previous receptor autoradiography studies. Moreover, the specific cellular binding patterns for both receptors can now be compared and contrasted to one another in the same tissue by using these fluorescent ligands. D1 receptor sites are most evident within the striatum and exhibit regions of intense "patch" fluorescence corresponding to receptor reactivity in cells and their processes. The distribution of D1 receptor binding is highly analogous to the pattern of dopamine terminal histofluorescence in the caudate nucleus. D2 receptor sites are less prevalent overall and may be localized to a subpopulation of the D1 fluorescent neurons in the caudate nucleus and nucleus accumbens regions.
Overreplication of the origin region in the dnaB37 mutant of Bacillus subtilis: postinitiation control of chromosomal replication.
Authors: Henckes G , Harper F , Levine A , Vannier F , Séror S J
Published: May 2006

When the Bacillus subtilis dnaB37 mutant, defective in initiation, is returned to permissive temperature after accumulation of initiation proteins at 45 degrees C, we have shown, by extensive DNA.DNA hybridization analysis, that the origin region is replicated in excess (approximately 2-fold). However, this replication is limited to a region of about 120-175 kilobases on either side of the origin. This has been confirmed by autoradiographic analysis of the overreplicated region. During the second round of synchronized replication at 30 degrees C, replication in fact appears to resume from the stalled forks on either side of the origin. We propose that in B. subtilis, in addition to a first level of control at the origin, a second level of control exists downstream of the origin in order to limit overreplication of the chromosome. These two controls might normally be tightly coupled. We suggest that the second level of control is exerted through the reversible inhibition of replisome movement at specific regions on either side of the origin.
DNA binding site of the growth factor-inducible protein Zif268.
Authors: Christy B , Nathans D
Published: May 2006

Zif268, a zinc finger protein whose mRNA is rapidly activated in cells exposed to growth factors or other signaling agents, is thought to play a role in regulating the genetic program induced by extracellular ligands. We report that Zif268 has one of the characteristics of a transcriptional regulator, namely, sequence-specific binding to DNA. Zif268 synthesized in Escherichia coli bound to two sites upstream of the zif268 gene and to sites in the promoter regions of other genes. The nucleotide sequences responsible for binding were defined by DNase I footprinting, by methylation interference experiments, and by use of synthetic oligonucleotides. From these results we derived the following consensus sequence for a Zif268 high-affinity binding site: GCGTGGGGCG.
In vivo rearrangement of mitochondrial DNA in Saccharomyces cerevisiae.
Authors: Clark-Walker G D
Published: May 2006

A revertant (SPR1) from a high-frequency petite strain of Saccharomyces cerevisiae has been shown by mapping and sequence analysis to have a rearranged mitochondrial genome. In vivo rearrangement has occurred through a subgenomic-recombination pathway involving the initial formation of subgenomic molecules in nascent petite mutants, recombination between these molecules to form an intermediate with direct repeats, and subsequent excision of the resident or symposed duplication to yield a molecule with three novel junctions and a changed gene order. Sequencing of the novel junctions shows that intramolecular recombination in each case occurs by means of G + C-rich short direct repeats of 40-51 base pairs. Mapping and sequence analysis also reveal that the SPR1 mitochondrial genome lacks three sectors of the wild-type molecule of 4.4, 1.7, and 0.5 kilobases. Each of these sectors occurs in nontemplate, base-biased DNA, that is over 90% A + T. Absence of these sectors together with a rearranged gene order does not appear to affect the phenotype of SPR1, as colony morphology and growth rate on a number of different substrates are not detectably different from the wild type. Lack of phenotypic change suggests that mitochondrial gene expression has not been noticeably disrupted in SPR1 despite deletion of the consensus nonomer promoter upstream from the glutamic acid tRNA gene. Dispensability of DNA sectors and the presence of recombinogenic short, direct repeats are mandatory features of the subgenomic-recombination pathway for creating rearrangements in baker's yeast mtDNA. It is proposed that, in other organisms, organelle genomes containing these elements may undergo rearrangement by the same steps.