bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective incorporation of (15S)-hydroxyeicosatetraenoic acid in phosphatidylinositol of human neutrophils: agonist-induced deacylation and transformation of stored hydroxyeicosanoids.
Authors: Brezinski M E , Serhan C N
Published: May 2006

The uptake and mobilization of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE), a major product of arachidonic acid metabolism, was examined with human neutrophils (polymorphonuclear leukocytes; PMNs). Upon exposure to labeled 15-HETE, PMNs rapidly (15 sec to 20 min) incorporated approximately 20% of the label into phosphatidylinositol, while less than 4% was associated with other phospholipid classes and neutral lipids. This pattern was distinct from that of either labeled arachidonate or labeled(5S)-hydroxy-8,11,14-cis-6-trans-eicosatetraenoic acid (5-HETE), which within 20 min were predominantly associated with triglycerides and phosphatidylcholine. After reversed-phase HPLC, greater than 98% of the label in phosphatidylinositol, isolated from PMNs, was released with phospholipase A2. Upon exposure to either chemotactic peptide (FMLP), phorbol 12-myristate 13-acetate, or an ionophore (A23187), 15-HETE-labeled PMNs released 15-HETE from phosphatidylinositol and displayed an impaired ability to generate leukotriene B4 (LTB4), 20-OH-LTB4, and 20-COOH-LTB4. Deacylated [3H]15-HETE was converted to (5S,15S)-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid (5,15-DHETE), lipoxin A4, and lipoxin B4, each carrying 3H label. PMNs labeled with 5-HETE also released and transformed this HETE when stimulated. However, the profile of labeled products differed between PMNs with either esterified 15-HETE or 5-HETE. When activated, 5-HETE-labeled PMNs generated both 5,20-DHETE and 5,15-DHETE but not labeled lipoxins. Threshold aggregation induced by FMLP with 15-HETE-labeled PMNs was inhibited (approximately 2 orders of magnitude), while the threshold response was relatively unimpaired with either A23187 or phorbol 12-myristate 13-acetate-induced aggregation. Results indicate that 15-HETE is rapidly esterified into phosphatidylinositol of PMNs, which can be mobilized and transformed upon exposure of the cells to a second signal. Moreover, they suggest that eicosanoid intermediates other than arachidonic acid can be stored by cells, released via signal transduction, and oxygenated to generate alternative profiles of eicosanoids.
Complete sequence of the Drosophila nonmuscle myosin heavy-chain transcript: conserved sequences in the myosin tail and differential splicing in the 5' untranslated sequence.
Authors: Ketchum A S , Stewart C T , Stewart M , Kiehart D P
Published: May 2006

We have sequenced a cDNA that encodes the nonmuscle myosin heavy chain from Drosophila melanogaster. An alternatively spliced exon at the 5' end generates two distinct heavy-chain transcripts: the longer transcripts inserts an additional start codon upstream of the primary translation start site and encodes a myosin heavy chain with a 45-residue extension at its amino terminus. The remainder of the coding sequence reveals extensive homology with other conventional myosins, especially metazoan nonmuscle and smooth muscle myosin isoforms. Comparisons among available myosin heavy-chain sequences establish that characteristic differences in sequence throughout the length of both the globular myosin head and extended rod-like tail readily distinguish nonmuscle and smooth muscle myosins from striated muscle isoforms and predict a basis for their functional diversity.
Peptides on phage: a vast library of peptides for identifying ligands.
Authors: Cwirla S E , Peters E A , Barrett R W , Dower W J
Published: May 2006

We have constructed a vast library of peptides for finding compounds that bind to antibodies and other receptors. Millions of different hexapeptides were expressed at the N terminus of the adsorption protein (pIII) of fd phage. The vector fAFF1, derived from the tetracycline resistance-transducing vector fd-tet, allows cloning of oligonucleotides in a variety of locations in the 5' region of gene III. A library of 3 x 10(8) recombinants was generated by cloning randomly synthesized oligonucleotides. The library was screened for high-avidity binding to a monoclonal antibody (3-E7) that is specific for the N terminus of beta-endorphin (Tyr-Gly-Gly-Phe). Fifty-one clones selected by three rounds of the affinity purification technique called panning were sequenced and found to differ from previously known ligands for this antibody. The striking finding is that all 51 contained tyrosine as the N-terminal residue and that 48 contained glycine as the second residue. The binding affinities of six chemically synthesized hexapeptides from this set range from 0.35 microM (Tyr-Gly-Phe-Trp-Gly-Met) to 8.3 microM (Tyr-Ala-Gly-Phe-Ala-Gln), compared with 7.1 nM for a known high-affinity ligand (Tyr-Gly-Gly-Phe-Leu). These results show that ligands can be identified with no prior information concerning antibody specificity. Peptide libraries are also likely to be useful in finding ligands that bind to other classes of receptors and in discovering pharmacologic agents.
The 60- to 90-kDa parietal cell autoantigen associated with autoimmune gastritis is a beta subunit of the gastric H+/K(+)-ATPase (proton pump).
Authors: Toh B H , Gleeson P A , Simpson R J , Moritz R L , Callaghan J M , Goldkorn I , Jones C M , Martinelli T M , Mu F T , Humphris D C
Published: May 2006

Autoantibodies in the sera of patients with pernicious anemia recognize, in addition to the alpha subunit of the gastric H+/(+)-ATPase, an abundant gastric microsomal glycoprotein of apparent Mr 60,000-90,000. Herein we have colocalized the glycoprotein and the alpha subunit of the gastric H+/K(+)-ATPase to the tubulovesicular membranes of the parietal cell by immunogold electron microscopy. Moreover, the glycoprotein and the alpha subunit were coimmunoprecipitated, and copurified by immunoaffinity chromatography, with an anti-glycoprotein monoclonal antibody. The pig glycoprotein was purified by chromatography on tomato lectin-Sepharose, and five tryptic peptides from the purified glycoprotein were partially sequenced. The complete amino acid sequence, deduced from the nucleotide sequence of overlapping cDNA clones, showed 33% similarity to the sequence of the beta subunit of the pig kidney Na+/K(+)-ATPase. We therefore propose that the 60- to 90-kDa glycoprotein autoantigen is the beta subunit of the gastric H+/K(+)-ATPase and that the alpha and beta subunits of the proton pump are major targets for autoimmunization in autoimmune gastritis.
In vitro assembly of relaxosomes at the transfer origin of plasmid RP4.
Authors: Pansegrau W , Balzer D , Kruft V , Lurz R , Lanka E
Published: May 2006

During initiation of conjugative transfer of DNA containing the transfer origin (oriT) of the promiscuous plasmid RP4, the proteins TraI, TraJ, and TraH interact and assemble a specialized nucleoprotein complex (the relaxosome) at oriT. The structure can be visualized on electron micrographs. Site- and strand-specific nicking at the transfer origin in vitro is dependent on the proteins TraI and TraJ and on Mg2+ ions. Substrate specificity is directed exclusively towards the cognate transfer origin: the RP4-specified TraJ protein cannot recognize the closely related oriT of plasmid R751. After nicking, TraI protein remains attached to the 5'-terminal 2'-deoxycytidyl residue at the nic site [Pansegrau, W., Ziegelin, G. & Lanka, E. (1990) J. Biol. Chem. 265, 10637-10644]. Nicking and relaxosome formation require supercoiled DNA. Thus, a complicated structure involving multiple plasmid-specified proteins and a defined region of DNA must be formed at the transfer origin to prepare the plasmid for generating the single strand to be transferred.
Similarities in transthyretin gene expression and differences in transcription factors: liver and yolk sac compared to choroid plexus.
Authors: Costa R H , Van Dyke T A , Yan C , Kuo F , Darnell J E
Published: May 2006

The serum thyroxine-binding protein, transthyretin (TTR), is made by hepatocytes and by choroid plexus epithelium in adults and by yolk sac cells in embryogenesis. Four hepatocyte nuclear factors (HNF-1, -3, and -4 and C/EBP) that are present in liver but not in most other adult tissues bind DNA sites in the TTR gene that are sufficient to direct transgenic expression. Three of these proteins were also found in yolk sac cells, which also express the transgene. A limited transgenic construct is not active in the choroid plexus and a TTR-producing choroid plexus tumor lacks three of the liver-enriched DNA-binding proteins. We conclude that cell-specific expression of TTR is regulated at least in part by the differential cellular distribution of positive-acting transcription factors.
GTP-binding proteins in rat liver nuclear envelopes.
Authors: Rubins J B , Benditt J O , Dickey B F , Riedel N
Published: May 2006

Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membrane vesicles required for reassembly of the nucleus after mitosis.
Growth of Plasmodium falciparum in human erythrocytes containing abnormal membrane proteins.
Authors: Schulman S , Roth E F , Cheng B , Rybicki A C , Sussman I I , Wong M , Wang W , Ranney H M , Nagel R L , Schwartz R S
Published: May 2006

To evaluate the role of erythrocyte (RBC) membrane proteins in the invasion and maturation of Plasmodium falciparum, we have studied, in culture, abnormal RBCs containing quantitative or qualitative membrane protein defects. These defects included hereditary spherocytosis (HS) due to decreases in the content of spectrin [HS(Sp+)], hereditary elliptocytosis (HE) due to protein 4.1 deficiency [HE(4.1(0))], HE due to a spectrin alpha I domain structural variant that results in increased content of spectrin dimers [HE(Sp alpha I/65)], and band 3 structural variants. Parasite invasion, measured by the initial uptake of [3H]hypoxanthine 18 hr after inoculation with merozoites, was normal in all of the pathologic RBCs. In contrast, RBCs from six HS(Sp+) subjects showed marked growth inhibition that became apparent after the first or second growth cycle. Preincubation of HS(Sp+) RBCs in culture for 3 days did not alter these results. Normal parasite growth was observed in RBCs from one HS subject with normal membrane spectrin content. The extent of decreased parasite growth in HS(Sp+) RBCs closely correlated with the extent of RBC spectrin deficiency (r = 0.90). Homogeneous subpopulations of dense HS RBCs exhibited decreased parasite growth to the same extent as did HS whole blood. RBCs from four HE subjects showed marked parasite growth inhibition, the extent of which correlated with the content of spectrin dimers (r = 0.94). RBCs from two unrelated subjects with structural variants of band 3 sustained normal parasite growth. Decreased growth in the pathologic RBCs was not the result of decreased ATP or glutathione levels or of increased RBC hemolysis. We conclude that abnormal parasite growth in these RBCs is not the consequence of metabolic or secondary defects. Instead, we suggest that a functionally and structurally normal host membrane is indispensable for parasite growth and development.
The structure of DNA in a nucleosome.
Authors: Hayes J J , Tullius T D , Wolffe A P
Published: May 2006

We describe the application of the hydroxyl radical footprinting technique to examine the histone-DNA interactions of a nucleosome that includes part of the 5S ribosomal RNA gene of Xenopus borealis. We establish that two distinct regions of DNA with different helical periodicities exist within the nucleosome and demonstrate a change in the helical periodicity of this DNA upon nucleosome formation. In particular, we find that on average the helical periodicity of DNA in this nucleosome is 10.18 +/- 0.05 base pairs per turn. The same DNA, when bound to a calcium phosphate surface, has a periodicity of 10.49 +/- 0.05 base pairs per turn, similar to that of random sequence DNA. Modulations in minor groove width within the naked DNA detected by the hydroxyl radical are maintained and exaggerated in nucleosomal DNA. These features correlate with regions in the DNA previously suggested to be important for nucleosome positioning.
A 140-base-pair repetitive sequence element in the mouse rRNA gene spacer enhances transcription by RNA polymerase I in a cell-free system.
Authors: Kuhn A , Deppert U , Grummt I
Published: May 2006

We show that the repetitive 140-base-pair (bp) elements present in the spacer of mouse rRNA genes function as enhancers for RNA polymerase I. Attachment of these elements to the rDNA promoter stimulates rRNA synthesis both in vivo and in vitro. The cis-activating effect of the spacer repeats is orientation-independent and increases with increasing numbers of the 140-bp elements. Competition experiments demonstrate that the spacer repeats bind one or more of the transcription factors interaction with the rDNA promoter. Both the 140-bp elements and the core promoter act cooperatively and thus are functionally linked. The 60/81-bp enhancer repeats from Xenopus laevis rDNA compete for a murine transcription factor(s) and stimulate transcription often fused to the mouse rDNA promoter. The results indicate that despite the marked species specificity of rDNA transcription initiation, common factors may interact with both the rDNA promoter and the enhancer.
Identification and preliminary characterization of protein-cysteine farnesyltransferase.
Authors: Manne V , Roberts D , Tobin A , O'Rourke E , De Virgilio M , Meyers C , Ahmed N , Kurz B , Resh M , Kung H F
Published: May 2006

Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus (Cys-186 in mammalian Ras p21 proteins) in order to exert their biological activity. Previous studies indicate that an intermediate in the mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group. Inhibition of mevalonate synthesis reverts the abnormal phenotypes induced by the mutant RAS2Val-19 gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus oocytes induced by an oncogenic Ras p21 protein of human origin. These results have raised the possibility of using inhibitors of the mevalonate pathway to block the transforming properties of ras oncogenes. Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, we describe an enzymatic activity(ies) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro at the correct (Cys-186) residue. This farnesylating activity is heat-labile, requires Mg2+ or Mn2+ ions, is linear with time and with enzyme concentration, and is present in all mammalian cell lines and tissues tested. Gel filtration analysis of a partially purified preparation of protein farnesyltransferase revealed two peaks of activity at 250-350 kDa and 80-130 kDa. Availability of an in vitro protein farnesyltransferase assay should be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway.
Direct detection of linker DNA bending in defined-length oligomers of chromatin.
Authors: Yao J , Lowary P T , Widom J
Published: May 2006

Linker DNA, which connects between nucleosomes in chromatin, is short and, therefore, may be essentially straight and inflexible. We have carried out hydrodynamic and electron microscopic studies of dinucleosomes--fragments of chromatin containing just two nucleosomes--to test the ability of linker DNA to bend. We find that ionic conditions that stabilize the folding of long chromatin cause linker DNA in dinucleosomes to bend, bringing the two nucleosomes into contact. The results uphold a key prediction of the solenoid model of chromosome folding and suggest a mechanism by which proteins that are separated along the DNA can interact by direct contact.
2'-O-alkyl oligoribonucleotides as antisense probes.
Authors: Iribarren A M , Sproat B S , Neuner P , Sulston I , Ryder U , Lamond A I
Published: May 2006

2'-O-Methyl oligoribonucleotides have recently been introduced as antisense probes for studying RNA processing and for affinity purification of RNA-protein complexes. To identify RNA analogues with improved properties for antisense analysis, 2'-O-alkyl oligoribonucleotides were synthesized in which the alkyl moiety was either the three-carbon linear allyl group or the five-carbon branched 3,3-dimethylallyl group. Both these analogues were found to be completely resistant to degradation by either DNA- or RNA-specific nucleases. Use of biotinylated derivatives of the probes to affinity-select ribonucleoprotein particles from crude HeLa cell nuclear extracts showed that the presence of the bulky 3,3-dimethylallyl group significantly reduces affinity selection, whereas the allyl derivative binds rapidly and stably to targeted sequences and affinity-selects efficiently. The allyl derivatives also showed an increase in the level of specific binding to targeted sequences compared with 2'-O-methyl probes of identical sequence. These properties indicate that the 2'-O-allyl oligoribonucleotides are particularly well suited for use as antisense probes.
Wound-inducible nuclear protein binds DNA fragments that regulate a proteinase inhibitor II gene from potato.
Authors: Palm C J , Costa M A , An G , Ryan C A
Published: May 2006

Deletion analysis from the 3' to the 5' end of the promoter region of the wound-inducible potato proteinase inhibitor IIK gene has identified a 421-base sequence at -136 to -557 that is necessary for expression. Utilizing DNA band-shift assays, a 10-base sequence within the 421-base region was found to bind a nuclear protein from wounded tomato leaves. This 10-base sequence is adjacent to an 8-base consensus sequence at -147 to -155 that is present in the promoter region of several elicitor-inducible genes from various other plants. The evidence suggests that a complex set of cis- and trans-acting elements within the -136 to -165 region of the potato IIK gene may be involved with the signaling mechanisms that regulate the inducibility of this gene in response to pest and pathogen attacks.
Gangliosides normalize distorted single-cell intracellular free Ca2+ dynamics after toxic doses of glutamate in cerebellar granule cells.
Authors: de Erausquin G A , Manev H , Guidotti A , Costa E , Brooker G
Published: May 2006

Glutamate-induced delayed neurotoxicity after abusive and paroxismal activation of its receptors has been proposed to depend upon a sustained increase in intracellular free Ca2+ [( Ca2+]i). To elucidate the temporal and causal relationship between glutamate-induced changes in [Ca2+]i and neuronal death, we simultaneously studied the dynamics of [Ca2+]i changes in single neurons with the acetoxymethyl ester of fura-2 and the cell viability by imaging the nuclear penetration of propidium iodide. The main difference between toxic (50 microM) and nontoxic (5 microM) doses of glutamate is the lack of regulation in [Ca2+]i 20 min after glutamate is removed. This protracted rise in [Ca2+]i in a single cell is correlated with (r = 0.87, P less than 0.01, Spearman's test), and consequently predictive of, the time of appearance of neuronal death, as measured by propidium iodide fluorescence. In addition, the glutamate receptor antagonists dibenzocyclohepteneimine (MK-801) and 3,3-(2-carboxypiperazine-4-yl)propyl 1-phosphate reduce the acute increase of [Ca2+]i induced by glutamate but fail to revert the protracted increase of [Ca2+]i, elicited by toxic doses of glutamate. In contrast, the ganglioside GM1 and the semisynthetic lysoGM1 with N-acetylsphingosine (LIGA-4) and lysoGM1 with N-dichloroacetylsphingosine (LIGA-20) failed to change the immediate rise of [Ca2+]i elicited by glutamate but prevented the protracted increase in [Ca2+]i after toxic doses of glutamate. Voltage-dependent Ca2+ channel blockers (nifedipine, etc.) did not change the initial or protracted responses to glutamate.
Size polymorphism of chicken major histocompatibility complex-encoded B-G molecules is due to length variation in the cytoplasmic heptad repeat region.
Authors: Kaufman J , Salomonsen J , Skjødt K , Thorpe D
Published: May 2006

B-G antigens are cell-surface molecules encoded by a highly polymorphic multigene family located in the chicken major histocompatibility complex (MHC). Rabbit antisera to B-G molecules immunoprecipitate 3-6 bands from iodinated erythrocytes by sodium dodecyl sulfate (SDS) gels under reducing conditions. These are all B-G molecules because they all map to the B-G region of the chicken MHC in congenic and recombinant chickens, most are directly recognized by the antisera, most form disulfide-linked dimers, and none bear N-linked carbohydrate. Both apparent homodimers and heterodimers are found, which bear intrachain disulfide bonds. All 3-6 bands have different mobilities in SDS gels between different haplotypes, ranging from 30 to 55 kDa. This size polymorphism is not affected by glycosidase treatment or addition of protease inhibitors. Partial proteolysis of cell surface-iodinated B-G molecules generates extremely similar patterns of spots, both within and between haplotypes. These surface-iodinated peptides bear either interchain or intrachain disulfide bonds. Additional peptides are generated by proteolysis of B-G molecules iodinated after isolation. Thus, it appears that the extracellular regions of these molecules are very similar and that the length polymorphism is due to variations in the cytoplasmic regions. Inspection of the cDNA-derived protein sequence in this region shows many heptad repeats, which may allow variation in length by step deletion and alternative splicing. The repeats indicate an alpha-helical coiled-coil structure, which could form an interaction between subunits of the dimer or with the cytoskeleton or both.
Superhelical torsion in cellular DNA responds directly to environmental and genetic factors.
Authors: McClellan J A , Boublíková P , Palecek E , Lilley D M
Published: May 2006

Superhelical tension of DNA in living bacteria is believed to be partially constrained by interaction with proteins. Yet DNA topology is a significant factor in a number of genetic functions and is apparently affected by both genetic and environmental influences. We have employed a technique that allows us to estimate the level of unconstrained superhelical tension inside the cell. We study the formation of cruciform structures by alternating adenine-thymine sequences in plasmid DNA by in situ chemical probing. This structural transition is driven by superhelical torsion in the DNA and thus reports directly on the level of such tension in the cellular DNA. We observe that the effect of osmotic shock is an elevation of superhelical tension; quantitative comparison with changes in plasmid linking number indicates that the alteration in DNA topology is all unconstrained. We also show that the synthesis of defective topoisomerase leads to increased superhelical tension in plasmid DNA. These experiments demonstrate that the effect of environmental and genetic influences is felt directly at the level of torsional stress in the cellular DNA.
Genetic diversity and ecogeographical differentiation among ribosomal DNA alleles in wild and cultivated barley.
Authors: Saghai Maroof M A , Allard R W , Zhang Q F
Published: May 2006

DNA from 267 accessions of wild barley from ecologically diverse habitats in Israel and Iran and from 92 accessions of cultivated barley from throughout the world were assayed for the 20 ribosomal DNA (rDNA) spacer-length variants that have been identified in the barley species. These 20 spacer-length variants, which are detectable by Southern blot hybridization, serve as markers of rDNA alleles of two Mendelian loci, Rrn1 and Rrn2. All of the populations of wild barley studied were polymorphic for both loci. In wild barley allele 112 (Rrn1) and allele 107 (Rrn2) behaved as widely adapted wild-type alleles; in our sample of cultivated barley allele 112 also behaved as a wild-type allele but allele 104 was somewhat more frequent than allele 107 in Rrn2. A few other alleles were locally frequent in wild barley. However, most of the 20 alleles were infrequent or rare and such alleles were often associated as "hitchhikers" with one of the wild-type alleles in compound two-component alleles. Allelic and genotypic frequencies differed widely in different habitats in correlation with eight of nine factors of the physical environment. Discrete log-linear multivariate analyses revealed statistically significant associations among alleles of Rrn1 and Rrn2. It was concluded that natural selection acting differentially on various rDNA alleles plays a major role in the development and maintenance of observed patterns of molecular and genetic organization of rDNA variability.
Serotonergic control of the hippocampus via local inhibitory interneurons.
Authors: Freund T F , Gulyás A I , Acsády L , Görcs T , Tóth K
Published: May 2006

Information flow and processing in hippocampal neuronal networks is determined by a wide range of inhibitory mechanisms [e.g., feedforward or feedback, gamma-aminobutyrate (GABA) A or B receptor-mediated, perisomatic shunting, or distal dendritic inhibition], each subserving specialized functions. These forms of local inhibition are mediated by morphologically and neurochemically well-defined, mostly GABA-containing, interneurons, which control large populations of principal cells through their extensive axonal arborizations. These neurons can serve as ideal targets for subcortical pathways, such as those originating in the septum or raphe, which exercise a global control over hippocampal activity. This intriguing possibility prompted us to study whether the profound effect of the serotonergic raphe-hippocampal pathway is mediated by inhibitory interneurons or whether a direct diffuse action on the principal cells is dominant. We demonstrate that axons of this pathway form multiple synaptic contacts with hippocampal GABAergic interneurons. Interestingly, the serotonergic afferents selectively innervate the somata and dendritic trees of GABAergic neurons that contain the 28-kDa calcium-binding protein calbindin D28K, but never those that contain another calcium-binding protein, parvalbumin. These results show that the mechanism by which the serotonergic pathway may exert a powerful influence on hippocampal function involves the modulation of local inhibitory circuits. Furthermore, the selectivity in the choice of target GABAergic interneurons suggests a strong functional specialization among inhibitory circuits, as well as among the subcortical input pathways originating in the septum and raphe.
A 47-kDa human nuclear protein recognized by antikinetochore autoimmune sera is homologous with the protein encoded by RCC1, a gene implicated in onset of chromosome condensation.
Authors: Bischoff F R , Maier G , Tilz G , Ponstingl H
Published: May 2006

Several autoimmune sera from patients with Raynaud phenomenon decorated mammalian kinetochores and bound to a 47-kDa protein on immunoblots of nuclear lysates. Antibody affinity-purified from immunoblots of the 47-kDa band recognized kinetochores, but due to crossreaction with an 18-kDa protein, localization remains elusive. We used one of these sera to purify the antigen from HeLa cells synchronized in mitosis as a noncovalent complex with a 25-kDa protein. The antigen was released from DNA by intercalation with 25 mM chloroquine. Ion-exchange chromatography yielded the pure complex with an apparent molecular size of 68 kDa, which was separated into its components by gel filtration in 6 M guanidinium chloride. Upon two-dimensional gel electrophoresis the 47-kDa protein gave two main spots of pI 6.6 and 6.7, respectively. Posttranslational modification is indicated by additional antigenic spots, by lack of a free alpha-amino group, and by chromatographic behavior of peptides on reversed-phase chromatography. The amino acid sequence for 205 residues of the 47-kDa protein has been established. This sequence is highly homologous with the translated reading frame of RCC1, a gene reportedly involved in regulating onset of mammalian chromosome condensation.
Mouse transgenes in human cells detect specific base substitutions.
Authors: Schaff D A , Jarrett R A , Dlouhy S R , Ponniah S , Stockelman M , Stambrook P J , Tischfield J A
Published: May 2006

We describe a system of transgenic human cell lines that detects and identifies specific point mutations at defined positions within a gene. The target transgenome is a mouse adenine phosphoribosyltransferase (APRT) gene rendered nonfunctional by introduction of a substitution at either of two bases that comprise a splice acceptor site. Reversion at a mutated site results in the expression of wild-type mouse APRT and consequent growth of APRT+ transgenic cell colonies. Site-specific reversion to wild-type sequence is confirmed by regeneration of a previously destroyed diagnostic Pst I site. Two independent cell clones, each with mutant transgenomes bearing an A----G transition, exhibited an up to 7500-fold, dose-dependent induction of reversion following treatment with ethyl methanesulfonate. Treatment of these clones with 2-aminopurine resulted in no induction of revertants. In contrast, another transgenic cell clone, bearing a G----A transition, reverted as a consequence of 2-aminopurine, but not ethyl methanesulfonate, treatment. These data confirm for human cells the proposed mechanisms of action of these mutagens and provide evidence for the utility of our site-specific reversion method for mutagenesis studies.
Tat-responsive region RNA of human immunodeficiency virus 1 can prevent activation of the double-stranded-RNA-activated protein kinase.
Authors: Gunnery S , Rice A P , Robertson H D , Mathews M B
Published: May 2006

Transcription from the human immunodeficiency virus type 1 promoter gives rise to short cytoplasmic transcripts of approximately 60 nucleotides as well as to longer mRNAs. These RNAs contain the Tat-responsive region sequence, which is capable of assuming a stem-loop structure and has been implicated in the regulation of both transcription and translation. It has been reported that Tat-responsive region RNA inhibits translation in vitro through activation of an interferon-induced protein kinase, the double-stranded-RNA-activated inhibitor, which phosphorylates eukaryotic initiation factor 2. We show that the activation property is due to double-stranded RNA that often contaminates RNA synthesized in vitro using bacteriophage RNA polymerases. After purification, high concentrations of Tat-responsive region RNA inhibit the activation of double-stranded RNA-activated inhibitor, suggesting that it may serve to protect human immunodeficiency virus type 1 infection from a cellular defense mechanism.
Three different mRNAs encoding human granulocyte colony-stimulating factor receptor.
Authors: Fukunaga R , Seto Y , Mizushima S , Nagata S
Published: May 2006

Three cDNAs for the human granulocyte colony-stimulating factor (G-CSF) receptor were isolated from the cDNA libraries of human U937 leukemia cells and placenta by using a murine G-CSF receptor cDNA as the probe. The human G-CSF receptor containing 813 amino acids had a marked homology (62.5%) with its murine counterpart and consisted of extracellular, transmembrane, and cytoplasmic domains. The WSXWS motif found in members of the newly identified growth factor receptor family was also present in the extracellular domain of the human G-CSF receptor. Expression of the cloned cDNA in monkey COS cells gave rise to a protein that could specifically bind G-CSF with a high affinity (Kd, 550 pM). Two other classes of the human G-CSF receptor were also identified, one of which had a deletion of the transmembrane domain and seemed to encode a secreted, soluble receptor. The third class of the G-CSF receptor contained a 27-amino acid insertion in the cytoplasmic domain and was highly expressed in placenta.
Differential intron loss and endosymbiotic transfer of chloroplast glyceraldehyde-3-phosphate dehydrogenase genes to the nucleus.
Authors: Liaud M F , Zhang D X , Cerff R
Published: May 2006

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is composed of two different subunits, GAPA and GAPB, which are encoded in the nucleus by two related genes of eubacterial origin. In the present work the genes encoding chloroplast GAPA and GAPB from pea have been cloned and sequenced. The gene for GAPB is split by eight introns. Two introns interrupt the region encoding the transit peptide and six are found within the region encoding the mature subunit, four of which are in identical or similar positions relative to genes for cytosolic GAPDH of eukaryotic organisms. As opposed to this, the gene encoding pea GAPA has only two introns in the region encoding the mature subunit. These findings strongly support the "intron early" hypothesis and suggest that the low number of introns in the gene for chloroplast GAPA is due to differential loss of introns during the streamlining period of the chloroplast genome following the GAPB/GAPA separation. We deduce from this that eubacteria and chloroplasts contained GT-AG introns until relatively recently and that the duplication event leading to the genes encoding GAPB and GAPA and their respective transit peptides occurred in the chloroplast progenitor prior to the successive transfer and functional reintegration of these genes into the nuclear environment. These conclusions imply that GAPA/GAPB transit peptides are of eubacterial origin.
Fuel selection in rufous hummingbirds: ecological implications of metabolic biochemistry.
Authors: Suarez R K , Lighton J R , Moyes C D , Brown G S , Gass C L , Hochachka P W
Published: May 2006

Hummingbirds in flight display the highest rates of aerobic metabolism known among vertebrates. Their flight muscles possess sufficient maximal activities of hexokinase and carnitine palmitoyltransferase to allow the exclusive use of either glucose or long-chain fatty acids as metabolic fuels during flight. Respiratory quotients (RQ = VCO2/VO2) indicate that fatty acid oxidation serves as the primary energy source in fasted resting birds, while subsequent foraging occurs with a rapid shift towards the use of carbohydrate as the metabolic fuel. We suggest that hummingbirds building up fat deposits in preparation for migration behave as carbohydrate maximizers (or fat minimizers) with respect to the metabolic fuels selected to power foraging flight.
Postsynaptic Hebbian and non-Hebbian long-term potentiation of synaptic efficacy in the entorhinal cortex in slices and in the isolated adult guinea pig brain.
Authors: Alonso A , de Curtis M , Llinás R
Published: May 2006

Long-term potentiation (LTP) was investigated in the mammalian entorhinal cortex by using two in vitro preparations, the isolated brain and the entorhinal cortex slice. Hebbian and non-Hebbian types of LTP appear to be present in layer II entorhinal cortex cells as demonstrated using two protocols: (i) tetanic stimulation of the piriform-entorhinal cortex afferent pathway to generate homosynaptic potentiation and (ii) postsynaptic subthreshold rhythmic membrane potential manipulation not paired to presynaptic activation, which gives rise to non-Hebbian LTP. The induction and the expression of both types of LTP were found to be dependent on activation of N-methyl-D-aspartate receptors as shown by their sensitivity to the receptor agonist D-2-amino-5-phosphonovalerate. This is in contrast to LTP in the hippocampus [Zalutsky, R. A. & Nicoll, R. A. (1990) Science 248, 1619-1624], where LTP is expressed by quisqualate receptors. Since, in the entorhinal cortex, LTP is linked to a selective increase of the N-methyl-D-aspartate-receptor-mediated synaptic responses, this enhancement is most likely due to postsynaptic factors.
Evidence for coiled-coil dimer formation by an Epstein-Barr virus transactivator that lacks a heptad repeat of leucine residues.
Authors: Flemington E , Speck S H
Published: May 2006

Two regions of the Epstein-Barr virus (EBV) BZLF1 gene product, ZEBRA, share sequence homology with c-Fos, one of which corresponds to the DNA binding domain of c-Fos. ZEBRA does not, however, contain the heptad repeat of leucines present in the dimerization domains of leucine zipper proteins. Here it is shown that ZEBRA binds its recognition sites as a homodimer and that the region adjacent to the basic DNA binding domain is essential for dimerization. This region contains a 4-3 repeat of predominantly hydrophobic residues, which is precisely in register with the hydrophobic heptad repeat present in the leucine zipper proteins with respect to the basic DNA binding domain. A mutational analysis of ZEBRA supports a model for dimerization involving a coiled-coil interaction. These results indicate that a heptad repeat of leucines is not a structural requirement for formation of coiled-coil dimers by transcription factors.
Structure and differential expression of two genes encoding carbonic anhydrase in Chlamydomonas reinhardtii.
Authors: Fujiwara S , Fukuzawa H , Tachiki A , Miyachi S
Published: May 2006

Two copies of structurally related genes (CAH1 and CAH2) for carbonic anhydrase (EC 4.2.1.1) were found to be tandemly clustered on the Chlamydomonas reinhardtii genome. The previously isolated cDNA clones for carbonic anhydrase polypeptides were derived from the upstream gene, CAH1, which has 10 introns in its coding region. The downstream gene, CAH2, also has 10 introns, at positions identical to those of CAH1. Although amino acid sequences deduced from the two genes showed 91.8% identity, partial sequences of the authentic enzyme isolated from air-induced cells were identical only to those of the CAH1 product. Northern hybridization using gene-specific probes showed that the level of 2.0-kilobase CAH1 mRNA increased in response to a decrease in CO2 concentration in the presence of light. The CAH1 mRNA did not accumulate when CO2 was lowered in the dark. In contrast, the level of 2.0-kilobase CAH2 mRNA decreased in response to lowering of CO2 and increased upon transfer to the high-CO2 condition in light. The decrease of CAH2 mRNA under the low-CO2 condition was not observed in the dark. The fully induced mRNA level was much higher for CAH1 than for CAH2. These results indicate that CAH1 is a gene coding for the major periplasmic carbonic anhydrase whose level of transcript is rapidly induced under the low-CO2 condition in the presence of light, and that CAH2 may encode another periplasmic isozyme, which is made under the high-CO2 condition.
Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42.
Authors: Shinjo K , Koland J G , Hart M J , Narasimhan V , Johnson D I , Evans T , Cerione R A
Published: May 2006

We have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated Gp (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human Gp protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins (approximately 50% identical), and the rac proteins (approximately 70% identical). The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell-division-cycle protein CDC42. The human placental gene, which we designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein.
Uv-visible spectroscopy of bacteriorhodopsin mutants: substitution of Arg-82, Asp-85, Tyr-185, and Asp-212 results in abnormal light-dark adaptation.
Authors: Duñach M , Marti T , Khorana H G , Rothschild K J
Published: May 2006

The light-dark adaptation reactions of a set of bacteriorhodopsin (bR) mutants that affect function and color of the chromophore were examined by using visible absorption spectroscopy. The absorbance spectra of the mutants Arg-82 in equilibrium Ala (Gln), Asp-85 in equilibrium Ala (Asn, Glu), Tyr-185 in equilibrium Phe, and Asp-212 in equilibrium Ala (Asn, Glu) were measured at different pH values during and after illumination. None of these mutants exhibited a normal dark-light adaptation, which in wild-type bR causes a red shift of the visible absorption maximum from 558 nm (dark-adapted bR) to 568 nm (light-adapted bR). Instead a reversible light reaction occurs in the Asp-85 and Asp-212 mutants from a blue form with lambda max near 600 nm to a pink form with lambda max near 480 nm. This light-induced shift explains the appearance of a reversed light adaptation previously observed for the Asp-212 mutants. In the case of the Tyr-185 and Arg-82 mutants, light causes a purple-to-blue transformation similar to the effect of lowering the pH. However, the blue forms observed in these mutants are not identical to those formed by acid titration or deionization of wild-type bR. It is suggested that in all of these mutants, the chromophore has lost the ability to undergo the normal 13-cis, 15-syn to all-trans, 15-anti light-driven isomerization, which occurs in native bR. Instead these mutants may have as stable forms all-trans,syn and 13-cis,anti chromophores, which are not allowed in native bR, except transiently.
The role of an autoantigen, histidyl-tRNA synthetase, in the induction and maintenance of autoimmunity.
Authors: Miller F W , Waite K A , Biswas T , Plotz P H
Published: May 2006

Patients with systemic autoimmune diseases make specific autoantibodies that are directed against self structures. According to one view, these autoantibodies arise as a result of an immune response to foreign antigens such as infectious agents that share, by molecular mimicry, common structures with host proteins. An alternative view is that the target autoantigen itself initiates, selects, and sustains autoantibody synthesis. We show here that anti-Jo-1 autoantibodies directed against histidyl-tRNA synthetase in the human autoimmune muscle disease polymyositis undergo, in addition to spectrotype broadening and class switching, the sine qua non of an immune response to the target antigen--affinity maturation to that antigen. We demonstrate further that these autoantibodies, unlike anti-synthetase antibodies induced in mice immunized with heterologous antigen, bind only nonlinear epitopes on the native human synthetase that remain exposed when the enzyme is complexed to tRNA(His). These data suggest that the native target autoantigen itself has played a direct role in selecting and sustaining the autoantibody response and sharply restrict the time and the way in which a molecular mimic might act to provoke autoantibodies.
Evidence that down-regulation of beta-cell glucose transporters in non-insulin-dependent diabetes may be the cause of diabetic hyperglycemia.
Authors: Orci L , Ravazzola M , Baetens D , Inman L , Amherdt M , Peterson R G , Newgard C B , Johnson J H , Unger R H
Published: May 2006

Non-insulin-dependent diabetes mellitus (NIDDM) is attributed to a failure of pancreatic beta cells to maintain insulin secretion at a level sufficient to compensate for underlying insulin resistance. In the ZDF rat, a model of NIDDM that closely resembles the human syndrome, we have previously reported profound underexpression of GLUT-2, the high-Km facilitative glucose transporter expressed by beta cells of normal animals. Here we report that islets of diabetic rats exhibit a marked decrease in the volume of GLUT-2-positive beta cells and a reduction at the electron-microscopic level in the number of GLUT-2-immunoreactive sites per unit of beta-cell plasma membrane. The deficiency of GLUT-2 cannot be induced in normal beta cells by in vivo or in vitro exposure to high levels of glucose nor can it be prevented in beta cells of prediabetic ZDF rats by elimination of hyperglycemia. We conclude that this dearth of immunodetectable GLUT-2 in NIDDM is not secondary to hyperglycemia and therefore that it may well play a causal role in the development of hyperglycemia.
Isolation and structure of an arrestin gene from Drosophila.
Authors: Smith D P , Shieh B H , Zuker C S
Published: May 2006

A Drosophila gene encoding a homologue of vertebrate arrestin was isolated by subtractive hybridization and identified as a member of a set of genes that are preferentially expressed in the visual system. This gene encodes a 364-amino acid protein that displays greater than 40% amino acid sequence identity with human and bovine arrestin. Interestingly, the Drosophila homologue lacks the C-terminal sequences that were postulated to interact with rhodopsin during the quenching of the phototransduction cascade in the vertebrate visual response. These findings are discussed in terms of invertebrate phototransduction. The Drosophila gene was mapped cytogenetically to chromosomal position 36D1-2, near the ninaD locus. However, the arrestin gene does not appear to be the ninaD locus, as sequence analysis of three ethylmethane sulfate-induced ninaD mutant alleles reveals no alteration in amino acid sequence.