bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of homeobox-containing genes of the honeybee and Drosophila.
Authors: Walldorf U , Fleig R , Gehring W J
Published: May 2006

We report the isolation of seven homeobox-containing genes from the honeybee (Apis mellifera). Sequence analysis of all homeoboxes and some flanking sequences showed that six of seven genes are more than 90% identical to their corresponding Drosophila homologues within the homeobox and, with one exception, also in the flanking sequences. The homologues that were identified include three homeotic selector genes [Sex combs reduced (Scr), Antennapedia (Antp), and abdominal-A (abd-A); the two engrailed (en) genes; and the muscle segment homeobox (msh)]. Surprisingly, no homologue of the segmentation gene fushi tarazu was found in the honeybee. For the remaining bee gene, a Drosophila homologue is not known. This indicates that, with some exceptions, structurally homologous genes are involved in the control of bee and Drosophila development, although Hymenoptera differ significantly in their embryogenesis from Diptera and have evolved separately for some 250 million years.
Construction of human chromosome 21-specific yeast artificial chromosomes.
Authors: McCormick M K , Shero J H , Cheung M C , Kan Y W , Hieter P A , Antonarakis S E
Published: May 2006

Chromosome 21-specific yeast artificial chromosomes (YACs) have been constructed by a method that performs all steps in agarose, allowing size selection by pulsed-field gel electrophoresis and the use of nanogram to microgram quantities of DNA. The DNA sources used were hybrid cell line WAV-17, containing chromosome 21 as the only human chromosome and flow-sorted chromosome 21. The transformation efficiency of ligation products was similar to that obtained in aqueous transformations and yielded YACs with sizes ranging from 100 kilobases (kb) to greater than 1 megabase when polyamines were included in the transformation procedure. Twenty-five YACs containing human DNA have been obtained from a mouse-human hybrid, ranging in size from 200 to greater than 1000 kb, with an average size of 410 kb. Ten of these YACs were localized to subregions of chromosome 21 by hybridization of RNA probes (corresponding to the YAC ends recovered in Escherichia coli) to a panel of somatic cell hybrid DNA. Twenty-one human YACs, ranging in size from 100 to 500 kb, with an average size of 150 kb, were obtained from approximately equal to 50 ng of flow-sorted chromosome 21 DNA. Three were localized to subregions of chromosome 21. YACs will aid the construction of a physical map of human chromosome 21 and the study of disorders associated with chromosome 21 such as Alzheimer disease and Down syndrome.
Human Fc gamma RIII: cloning, expression, and identification of the chromosomal locus of two Fc receptors for IgG.
Authors: Peltz G A , Grundy H O , Lebo R V , Yssel H , Barsh G S , Moore K W
Published: May 2006

A cDNA clone encoding a human receptor for the Fc portion of IgG (Fc gamma R), Fc gamma RIII or CD16, was isolated from a human leukocyte library by a transient expression-immunoselection procedure. This cDNA (pGP5) encodes a 46-kDa phosphatidylinositol-linked cell surface protein with CD16 determinants and affinity for human IgG. The deduced protein sequence is most homologous to the murine receptor Fc gamma RII alpha, with slightly less homology to the human receptors Fc gamma RII and Fc epsilon RI. The cDNA hybridizes to a 2.2 kilobase mRNA in human leukocytes and a cloned human natural killer cell line. Fc gamma RIII is mapped to chromosome 1 by spot-blot analysis of sorted human chromosomes. Hybridization of Fc gamma RII and Fc gamma RIII probes to restriction digests of human genomic DNA separated by pulsed-field gel electrophoresis demonstrates physical linkage of the two genes within a maximum distance of 200 kilobases. The results identify a locus for at least two Fc gamma R genes on human chromosome 1.
Creation of H-2 class I epitopes using synthetic peptides: recognition by alloreactive cytotoxic T lymphocytes.
Authors: Olson C A , Williams L C , McLaughlin-Taylor E , McMillan M
Published: May 2006

A major role that cytotoxic T lymphocytes (CTLs) play in the immune response is the specific destruction of viral-infected cells and tissue from foreign grafts. Class I molecules encoded within the major histocompatibility complex are the target structures for these CTLs. Recently, viral-restricted CTLs have been shown to recognize viral peptides in association with class I molecules, while several studies with cloned alloreactive CTLs have indicated that these T cells can recognize class I-derived peptides in association with class I molecules. Together, these observations suggest that peptide binding is an important function of class I molecules. In this paper, we show that the recognition of a particular class I molecule by a bulk population of alloreactive CTLs can be altered by incubating with it a peptide derived from another class I molecule. Specifically, we used the hybrid D/Ldm1 molecule as a target structure together with the peptide Ld61-85, and we have shown that their associative recognition by Ld-specific CTLs depends on sequence and configuration of the peptide and is specific for Ld using a cold-target inhibition assay. Our results are discussed in light of three possible models for the target structure(s) that can be recognized by alloreactive CTLs and in terms of the role peptides may play during allorecognition.
Thrombus imaging in a primate model with antibodies specific for an external membrane protein of activated platelets.
Authors: Palabrica T M , Furie B C , Konstam M A , Aronovitz M J , Connolly R , Brockway B A , Ramberg K L , Furie B
Published: May 2006

The activated platelet is a potential target for the localization of thrombi in vivo since, after stimulation and secretion of granule contents, activated platelets are concentrated at sites of blood clot formation. In this study, we used antibodies specific for a membrane protein of activated platelets to detect experimental thrombi in an animal model. PADGEM (platelet activation-dependent granule-external membrane protein), a platelet alpha-granule membrane protein, is translocated to the plasma membrane during platelet activation and granule secretion. Since PADGEM is internal in unstimulated platelets, polyclonal anti-PADGEM and monoclonal KC4 antibodies do not bind to circulating resting platelets but do interact with activated platelets. Dacron graft material incubated with radiolabeled KC4 or anti-PADGEM antibodies in the presence of thrombin-activated platelet-rich plasma bound most of the antibody. Imaging experiments with 123I-labeled anti-PADGEM in baboons with an external arterial-venous Dacron shunt revealed rapid uptake in the thrombus induced by the Dacron graft; control experiments with 123I-labeled nonimmune IgG exhibited minimal uptake. Deep venous thrombi, formed by using percutaneous balloon catheters to stop blood flow in the femoral vein of baboons, were visualized with 123I-labeled anti-PADGEM. Thrombi were discernible against blood pool background activity without subtraction techniques within 1 hr. No target enhancement was seen with 123I-labeled nonimmune IgG. 123I-labeled anti-PADGEM cleared the blood pool with an initial half-disappearance time of 6 min and did not interfere with hemostasis. These results indicate that radioimmunoscintigraphy with anti-PADGEM antibodies can visualize thrombi in baboon models and is a promising technique for clinical thrombus detection in humans.
Skeletal muscle proteasome can degrade proteins in an ATP-dependent process that does not require ubiquitin.
Authors: Driscoll J , Goldberg A L
Published: May 2006

The proteasome (the multicatalytic endoproteinase complex) in mammalian tissues hydrolyzes proteins and several types of peptides. When this structure was isolated rapidly from rabbit skeletal muscle in the presence of glycerol, its various peptidase and protease activities showed a large reversible activation by physiological concentrations of ATP (Ka = 0.3-0.5 mM). Hydrolysis of succinyl-Leu-Leu-Val-Tyr-(4-methylcoumaryl-7-amide) was stimulated up to 12-fold by ATP, whereas degradation of casein and bovine serum albumin increased 4- to 7-fold. Neither ADP nor AMP had any effect. CTP, GTP, UTP, and the nonhydrolyzable analogs adenosine 5'-[beta,gamma-imino]triphosphate (AMPP[NH]P) and adenosine 5'-[alpha,beta-methylene]triphosphate (AMP[CH2]PP) increased peptide hydrolysis as well as ATP did. However, only ATP stimulated casein breakdown and only in the presence of Mg2+. Thus, nucleotide binding allows activation of the peptidase functions, but ATP hydrolysis seems necessary for enhanced degradation of proteins. The ATP effect on proteolysis was reversible and did not require ubiquitin. Sensitivity to ATP was labile, and with storage at 4 degrees C the enzyme became fully active in the absence of ATP or Mg2+. The ATP-activated form closely resembles the proteasome complex described previously, which did not show ATP dependence: both have molecular masses of 650 kDa, contain the same 8-10 subunits, and are precipitated by the same antibodies. A similar ATP-activated form was found in rabbit liver but not in rabbit reticulocytes. The proteasome seems to represent a ubiquitin-independent, ATP-stimulated proteolytic activity within nucleated mammalian cells.
Functional evidence for an auxin receptor at the plasmalemma of tobacco mesophyll protoplasts
Authors: Barbier-Brygoo Helene , Ephritikhine Genevieve , Klämbt Dieter , Ghislain Michel , Guern Jean
Published: May 2006

Tobacco mesophyll protoplasts were previously shown to respond to naphthaleneacetic acid by modifying their transmembrane potential difference. In the present work, evacuolated protoplasts were used to show that this response resides only at the plasmalemma. This electrical response was investigated by using polyclonal antibodies directed against plasma membrane antigens presumably involved in the reception and transduction of the auxin signal. An IgG fraction from an antiserum directed against the membrane auxin-binding protein from maize coleoptile completely inhibited the naphthaleneacetic acid-induced response of tobacco protoplasts. The suppression of the auxin-induced variation in the transmembrane potential difference by an IgG preparation directed against the plasmalemma ATPase from yeast demonstrated the involvement of the ATPase in the electrical response. Variation induced by fusicoccin in the transmembrane potential difference of tobacco protoplasts was unaffected by the anti-auxin-binding protein IgG fraction but was completely suppressed by the anti-ATPase IgG preparation. These results demonstrate the presence of a membrane receptor for auxin at the plasmalemma, the binding of the hormone to this receptor leading to the activation of the proton-pumping ATPase. They also show that at least the primary steps of activation by naphthaleneacetic acid are distinct from those of the fusicoccin-induced response.
T-cell receptor V alpha and C alpha alleles associated with multiple and myasthenia gravis.
Authors: Oksenberg J R , Sherritt M , Begovich A B , Erlich H A , Bernard C C , Cavalli-Sforza L L , Steinman L
Published: May 2006

Polymorphic markers in genes encoding that alpha chain of the human T-cell receptor (TcR) have been detected by Southern blot analysis in Pss I digests. Polymorphic bands were observed at 6.3 and 2.0 kilobases (kb) with frequencies of 0.30 and 0.44, respectively, in the general population. Using the polymerase chain reaction (PCR) method, we amplified selected sequences derived from the full-length TcR alpha cDNA probe. These PCR products were used as specific probes to demonstrate that the 6.3-kb polymorphic fragment hybridizes to the variable (V)-region probe and the 2.0-kb fragment hybridizes to the constant (C)-region probe. Segregation of the polymorphic bands was analyzed in family studies. To look for associations between these markers and autoimmune diseases, we have studied the restriction fragment length polymorphism distribution of the Pss I markers in patients with multiple sclerosis, myasthenia gravis, and Graves disease. Significant differences in the frequency of the polymorphic V alpha and C alpha markers were identified between patients and healthy individuals.
Potential phytotoxicity associated with the use of soil urease inhibitors
Authors: Krogmeier Michael J. , McCarty Gregory W. , Bremner John M.
Published: May 2006

Recent work in our laboratory showed that the adverse effect of urea fertilizer on seed germination and seedling growth in soil is due to ammonia produced through hydrolysis of urea by soil urease (NH
2
CONH
2
+ H
2
O → 2NH
3
+ CO
2
) and can be eliminated by amending the fertilizer with a small amount of a urease inhibitor such as
N
-(
n
-butyl)thiophosphoric triamide or phenylphosphorodiamidate. Continuation of this work showed that these inhibitors can induce leaf-tip necrosis in plants. Research to account for this phytotoxicity indicated that it resulted from an accumulation of toxic amounts of urea in plants through inhibition of urease activity by
N
-(
n
-butyl)thiophosphoric triamide and phenylphosphorodiamidate. Support for this conclusion was provided by experiments showing that these urease inhibitors increased both leaf-tip necrosis and urea concentrations in wheat (
Triticum aestivum
L.) and sorghum [
Sorghum bicolor
(L.) Moench] plants grown in soils treated with urea and that the necrotic areas of such plants had a much higher concentration of urea than did the nonnecrotic areas. The potential of urease inhibitors for inducing phytotoxicity should not preclude their use to eliminate the adverse effects of urea fertilizers on seed germination and seedling growth in soil because the ammonia produced through hydrolysis of urea fertilizer by urease is much more detrimental to plant growth than is the urea accumulation induced by urease inhibitors.
Separate cis-regulatory elements confer expression of phosphoenolpyruvate carboxykinase (GTP) gene in different cell lines.
Authors: Benvenisty N , Nechushtan H , Cohen H , Reshef L
Published: May 2006

The gene encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) [PEPCK; GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32], a key enzyme in gluconeogenesis and glyceroneogenesis, is expressed in tissues that arise from different embryonal origins: the gluconeogenic liver arises from endoderm, whereas the gluconeogenic kidney cortex and glyceroneogenic adipose tissue arise from the mesoderm. To identify the cis-regulatory elements conferring the differential gene expression, PEPCK chimeric genes were transfected into two rat hepatoma cell lines (H4IIEC3 and HTC-M1.1) and mouse adipocytes (3T3F442A), which express the endogenous gene, and into myoblasts and preadipocytes, which do not express it. The results demonstrate that 597 base pairs of the 5' flanking region of the PEPCK gene are sufficient to confer cell-specific gene expression in the PEPCK-expressing hepatoma cells and adipocytes. However, different elements within this 597-base-pair region enhance the gene expression in the hepatoma cells (endoderm) and adipocytes (mesoderm). In the hepatocytes, expression is conferred by two elements--one 5' of position -362 and the other 3' of position -98 with respect to the transcription start site. The region in between these two elements (from -362 to -98), which seems to inhibit the gene expression in the hepatocytes, confers enhanced expression in the adipocytes. Moreover, the distal positive regulatory element of the hepatocytes seems to be orientation and PEPCK promoter dependent. In contrast, the positive regulatory element of the adipocytes seems to act as a more typical enhancer. These results suggest that separate cis-regulatory elements confer cell-specific expression of the PEPCK gene.
Molecular cloning and sequence of cDNA encoding the plasma membrane proton pump (H+-ATPase) of Arabidopsis thaliana.
Authors: Harper J F , Surowy T K , Sussman M R
Published: May 2006

In plants, the transport of solutes across the plasma membrane is driven by a proton pump (H+-ATPase) that produces an electric potential and pH gradient. We have isolated and sequenced a full-length cDNA clone that encodes this enzyme in Arabidopsis thaliana. The protein predicted from its nucleotide sequence encodes 959 amino acids and has a molecular mass of 104,207 Da. The plant protein shows structural features common to a family of cation-translocating ATPases found in the plasma membrane of prokaryotic and eukaryotic cells, with the greatest overall identity in amino acid sequence (36%) to the H+-ATPase observed in the plasma membrane of fungi. The structure predicted from a hydropathy plot contains at least eight transmembrane segments, with most of the protein (73%) extending into the cytoplasm and only 5% of the residues exposed on the external surface. Unique features of the plant enzyme include diverged sequences at the amino and carboxyl termini as well as greater hydrophilic character in three extracellular loops.
CD28 activation pathway regulates the production of multiple T-cell-derived lymphokines/cytokines.
Authors: Thompson C B , Lindsten T , Ledbetter J A , Kunkel S L , Young H A , Emerson S G , Leiden J M , June C H
Published: May 2006

CD28 is a 44-kDa glycoprotein expressed as a homodimer on the surface of a major subset of human T cells. Previous studies have demonstrated that the binding of monoclonal antibodies to the CD28 surface antigen can augment the proliferation of purified human T cells stimulated with suboptimal doses of mitogens or anti-T-cell receptor/CD3 complex antibodies. In this report, we show that CD28 stimulation augments T-cell immune responses by specifically inducing a 5- to 50-fold enhancement in the expression and secretion of interleukin 2, tumor necrosis factor type alpha, lymphotoxin, interferon gamma, and granulocyte-macrophage colony-stimulating factor in normal human T cells stimulated to proliferate by crosslinking of the T-cell receptor/CD3 complex. This CD28-mediated induction of lymphokine/cytokine gene expression occurred even in T cells stimulated with optimal concentrations of mitogens or anti-T-cell receptor/CD3 antibodies, although under these conditions CD28 activation failed to enhance the proliferative response. The activation pathway induced by stimulation of CD28 is distinct from other biochemical pathways that induce lymphokines/cytokines because CD28 stimulation can induce lymphokine/cytokine gene expression in the presence of the immunosuppressant cyclosporine. Together these data suggest that the CD28 cell surface molecule is part of a distinct activation pathway that specifically modulates the expression of multiple lymphokine/cytokine genes.
Concanavalin A selectively reduces desensitization of mammalian neuronal quisqualate receptors.
Authors: Mayer M L , Vyklicky L
Published: May 2006

A fast perfusion system was used to apply excitatory amino acids to embryonic hippocampal neurons grown in dissociated culture and voltage clamped in the whole-cell recording configuration. Responses to quisqualic acid and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA; a potent quisqualate-like agonist) showed rapid desensitization: at 100 microM the peak inward current declined to a plateau response on average 0.2 times the peak response (mean time constant, 30 ms). Responses to L-aspartic acid and N-methyl-D-aspartic acid also showed desensitization: at 100 microM, when recorded in Mg-free solution with 0.3 microM glycine, the peak inward current declined to a plateau value 0.5 times the peak, but with a time constant of desensitization (average, 248 ms) one order of magnitude slower than desensitization of responses to quisqualate. Responses to kainate and domoate (agonists at kainic acid receptors) did not show appreciable desensitization. Responses to L-glutamate and 5-Br-willardine (a potent non-NMDA receptor agonist), recorded in glycine-free solution with 1 mM Mg to suppress N-methyl-D-aspartic acid receptor activity, showed similar rapid desensitization to AMPA and quisqualate, but occurred with less depression of the peak current. The lectin concanavalin A (Con A) reduced desensitization at quisqualate receptors, with no effect on responses to kainate or N-methyl-D-aspartic acid. The effect of Con A developed slowly (average time constant at 2.5 microM, 250 s) but at steady state Con A increased the plateau current evoked by 100 microM quisqualate to 13 times control. Succinyl-Con A produced only a small reduction of desensitization to quisqualate, approximately 10% of that produced by native Con A. Con A did not change the decay time constant of fast excitatory synaptic currents evoked by stimulation of presynaptic neurons, although the peak synaptic current decreased after treatment with lectin. Con A was also without effect on the block of responses to kainate produced by coapplication of quisqualate.
Momentary alteration of the postsynaptic membrane during transmission of a single nerve impulse.
Authors: Dunant Y , Garcia-Segura L M , Muller D , Parducz A
Published: May 2006

Transmission of a nerve impulse at neuromuscular and other synapses is an extremely brief event. By using rapid-freezing and cryofracture techniques in the electric organ of Torpedo, synaptic transmission was found to be accompanied by significant changes affecting the postsynaptic membrane for a few milliseconds. In the replicas, the protoplasmic leaflet of this membrane was seen to contain intramembrane particles (IMPs) of two different forms, globular and elongated. Globular IMPs had a mean diameter of 8.8 nm; they were the most frequently found (80% in unstimulated specimens). Elongated IMPs had a major diameter of 17.9 nm, about twice that of globular IMPs. Transmission of a single nerve impulse was accompanied by a marked decrease in the number of globular IMPs and by an increase in the number of elongated IMPs, as if there were a coalescence of two adjacent round particles to form an elongated one. These changes started soon after the electrical stimulus and lasted for approximately 3 ms. IMPs in the postsynaptic protoplasmic face are thought to correspond to a certain proportion of nicotinic acetylcholine receptors that were extracted with this leaflet during the fracture process. The phenomenon described here reflects an abrupt change in the membrane, probably linked to activation of the acetylcholine nicotinic receptors.
Formation and action of 8-hydroxy-11,12-epoxy-5,9,14-icosatrienoic acid in Aplysia: a possible second messenger in neurons.
Authors: Piomelli D , Shapiro E , Zipkin R , Schwartz J H , Feinmark S J
Published: May 2006

In Aplysia neural tissue, the release and metabolism of arachidonic acid are stimulated by histamine or by activation of the identified L32 nerve cell circuit of the abdominal ganglion. Previously we found that histamine and intracellular stimulation of L32 cells, which are putatively histaminergic neurons, cause the production of 12-hydroxy-5,8,10,14-icosatetraenoic acid (12-HETE), a product of the 12-lipoxygenase pathway formed through 12-hydroperoxy-5,8,10,14-icosatetraenoic acid (12-HPETE). 12-HPETE, but not 12(S)-HETE, mimics the dual-action response of L14 ink motor neurons to histamine and stimulation of L32. 12-HPETE can also be further metabolized to 8-hydroxy-11,12-epoxy-5,9,14-icosatrienoic acid (8-HEpETE) which was identified by HPLC, enzymatic hydrolysis, and GC/MS. Production of 8-HEpETE is specific, as its positional isomer 10-hydroxy-11,12-epoxy-5,8,14-icosatrienoic acid is not formed after physiologic stimulation. 8-HEpETE can elicit the late component (hyperpolarization) of the dual-action response in L14 cells, suggesting that it may be a second messenger in Aplysia.
High molecular mass forms of basic fibroblast growth factor are initiated by alternative CUG codons.
Authors: Prats H , Kaghad M , Prats A C , Klagsbrun M , Lélias J M , Liauzun P , Chalon P , Tauber J P , Amalric F , Smith J A
Published: May 2006

A 6.75-kilobase human hepatoma-derived basic fibroblast growth factor (bFGF) cDNA was cloned and sequenced. An amino-terminal sequence generated from a purified hepatoma bFGF was found to correspond to the nucleotide sequence and to begin 8 amino acids upstream from the putative methionine start codon thought to initiate a 154-amino acid bFGF translation product. This sequence suggests that a form of bFGF of at least 163 amino acids exists. The hepatoma cDNA was transcribed in vitro into RNA; in vitro translation of this RNA generated three forms of bFGF with molecular masses of 18, 21, and 22.5 kDa. By use of in vitro mutagenesis, it was found that the 22.5-kDa bFGF and possibly the 21-kDa form were initiated with CUG start codons. The 18-kDa bFGF was initiated with an AUG codon. By transfecting into COS cells human hepatoma bFGF cDNA and a construct from which the AUG initiator was eliminated, it was found that the higher molecular mass forms of bFGF were as biologically active as the 18-kDa form.
Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA.
Authors: Gibbs R A , Nguyen P N , McBride L J , Koepf S M , Caskey C T
Published: May 2006

The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. We have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. We also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN.
N-methyl-D-aspartate activates different channels than do kainate and quisqualate.
Authors: Lerma J , Kushner L , Zukin R S , Bennett M V
Published: May 2006

In the mammalian central nervous system, the excitatory amino acid transmitter L-glutamate activates three pharmacologically distinguishable receptors, the N-methyl-D-aspartate (NMDA), kainate, and quisqualate receptors. The present paper addresses the issue of whether these three receptors operate independent channels or whether they share channels that may have several conductance substates. The Xenopus oocyte provides a system for expression of exogenous mRNAs that permits detailed study of receptor structure and function. In oocytes injected with rat brain mRNA, NMDA has a stoichiometry of channel activation different from that for kainate and quisqualate. NMDA activates its own channels as indicated by simple summation or near-summation of currents evoked by NMDA with those evoked by quisqualate or kainate. Deviations from summation are ascribable to lack of selectivity in which an agonist at one receptor acts as a weak antagonist at another receptor. A further indication of separate channels is that block of NMDA channels by Mg2+ or phencyclidine has no effect on kainate or quisqualate responses evoked during the block. Interactions of kainate and quisqualate are more complex, but they can be explained by lack of complete specificity of these agonists for their own receptors.
On the origin and transmission of force in actomyosin subfragment 1.
Authors: Botts J , Thomason J F , Morales M F
Published: May 2006

A proximity map showing the three-dimensional arrangement of 12 chemically defined points in actomyosin subfragment 1 is developed and roughly correlated with published electron microscope reconstruction of others. Several additional points and topological relationships in the primary polypeptide chain folding are assimilated into this model. Certain crosslinkings and distance change observations are interpreted as indicators of transmission of force/displacement between the nucleotide-binding and an actin-binding site--i.e., as indications of how energy is transduced in this system.
Cachectin alters anterior pituitary hormone release by a direct action in vitro.
Authors: Milenkovic L , Rettori V , Snyder G D , Beutler B , McCann S M
Published: May 2006

Cachectin (tumor necrosis factor) is a powerful macrophage hormone released during infection, which circulates in blood to produce diverse effects in the organism. We examined the effect of cachectin on release of anterior pituitary hormones from either hemipituitaries or dispersed pituitary cells incubated in vitro. The action of cachectin on dispersed cells was demonstrable only after 2 hr of incubation. With this incubation time, the protein produced a dose-related stimulation of release of adrenocorticotropin (ACTH), growth hormone (GH), and thyrotropin (TSH), but not of prolactin (Prl), from both hemipituitaries and dispersed cells. The doses required for stimulation were low in the case of hemipituitaries, usually of the order of 10(-12) M, whereas they were higher by one or two orders of magnitude with the dispersed pituitary cells. This may be related either to loss of receptors for the protein during the dispersion procedure or to the fact that in the hemipituitary system cell interactions are facilitated because the cells are close to each other. In the dispersed cell system cachectin evoked a dose-related decrease in cyclic AMP content. Incubation with somatostatin lowered the cyclic AMP content of the cells and depressed GH output without altering output of TSH or Prl. When somatostatin and cachectin were incubated together with the cells, the suppression of cyclic AMP production was abolished; TSH and Prl release were stimulated, but the action of cachectin to stimulate GH release was blocked. The stimulation of Prl release by cachectin in the presence of somatostatin may be related to the elevation of cyclic AMP, a known stimulator of Prl release. The cyclooxygenase inhibitor indomethacin nearly completely blocked the stimulatory effect of cachectin on release of GH and TSH from dispersed pituitary cells but had only a slight and nonsignificant attenuating effect on its ACTH-releasing action. These results suggest that at least part of the stimulatory action of the peptide on pituitary hormone release is brought about by prostaglandins. The failure of indomethacin to block the release of ACTH induced by cachectin suggests that other mechanisms may be involved in the release of ACTH induced by this peptide. Since the concentrations of cachectin required to stimulate pituitary hormone release are similar to those that are encountered in plasma during infection, it is likely that this direct pituitary action has pathophysiological significance.
Multiple states of protein-DNA interaction in the assembly of transcription complexes on Saccharomyces cerevisiae 5S ribosomal RNA genes.
Authors: Braun B R , Riggs D L , Kassavetis G A , Geiduschek E P
Published: May 2006

Multiple stages of protein-DNA interaction in the assembly of RNA polymerase III transcription complexes on a Saccharomyces cerevisiae 5S rRNA gene have been distinguished by DNase I "footprinting" and gel retardation. Transcription factor IIIA interacts with approximately 35 base pairs of the internal promoter region. Transcription factors IIIC and IIIB incrementally extend the interaction along the 5S gene, if, and only if, transcription factor IIIA is also bound. Complexes assembled from the complete set of purified transcription factors or from a complete transcription system extend over the entire transcription unit together with almost 50 base pairs of 5' flanking sequence.
Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli.
Authors: Butt T R , Jonnalagadda S , Monia B P , Sternberg E J , Marsh J A , Stadel J M , Ecker D J , Crooke S T
Published: May 2006

Despite the availability of efficient transcription and translation signals, some heterologous gene products are not adequately expressed when introduced into prokaryotes and eukaryotes. An expression system has been established in Escherichia coli to increase the yield of cloned gene products, where the C terminus of ubiquitin was fused to the N terminus of unstable or poorly expressed proteins. Fusion of ubiquitin to yeast metallothionein or to the alpha subunit of the adenylate cyclase-stimulatory GTP-binding protein increased the yield from undetectable to 20% of the total cellular protein. A ubiquitin-N alpha-protein hydrolase has been partially purified from rabbit reticulocytes; this enzyme faithfully cleaves the junction peptide bound between the C-terminal Gly-76 of ubiquitin and the fusion protein. The increased yield of cloned gene products is very likely due to increased stability and/or more efficient translation of the fusion proteins. Possible mechanisms for the augmentation of ubiquitin fusion-protein expression in prokaryotes and eukaryotes are discussed.
Complete amino acid sequence of rat brain hexokinase, deduced from the cloned cDNA, and proposed structure of a mammalian hexokinase.
Authors: Schwab D A , Wilson J E
Published: May 2006

The complete amino acid sequence for the type I isozyme of hexokinase from rat brain has been deduced from the nucleotide sequence of cloned cDNA. The nucleotide sequence of 91 bases in the 5' untranslated region as well as that of the entire 3' untranslated region preceding the poly(A) sequence have also been determined. The N- and C-terminal halves of brain hexokinase show extensive sequence similarity to each other and to yeast hexokinase. These results provide direct support for the proposal that the mammalian hexokinases of approximately 100 kDa have evolved by a process of duplication and fusion of a gene encoding an ancestral hexokinase similar to the yeast enzyme of approximately 50 kDa. Taking this similarity in sequence to indicate basic similarity in structure between the N- and C-terminal regions of brain hexokinase and the yeast enzyme, a proposed structure for the mammalian hexokinase has been developed by fusing two molecules of yeast hexokinase, whose structure has previously been determined by x-ray crystallographic studies. Various features of the model are shown to be consistent with experimental observations bearing on the structure of the brain enzyme.
Involvement of the proteasome in various degradative processes in mammalian cells.
Authors: Matthews W , Driscoll J , Tanaka k , Ichihara A , Goldberg A L
Published: May 2006

Eukaryotic cells contain a 700-kDa proteolytic complex (the "proteasome" or multicatalytic endopeptidase complex), whose role in intracellular protein breakdown is unclear. It has been suggested that the proteasome functions in the rapid degradation of oxidant-damaged proteins and in the ATP-dependent proteolytic pathway. To test these possibilities, oxidant-damaged hemoglobin and albumin were produced by treating hemoglobin and albumin with phenylhydrazine, with hydroxyl radicals, or with both hydroxyl and superoxide radicals. After oxidant damage, these proteins were degraded more rapidly in erythrocyte extracts and also by the purified proteasome. However, complete removal of proteasomes from these extracts by immunoprecipitation (or inhibitors of its proteolytic activity) did not reduce the breakdown of oxidant-damaged hemoglobin and decreased degradation of hydroxyl- and superoxide-treated proteins by only 30-40%. Thus, erythrocytes must contain another proteolytic system for degradation of oxidant-damaged proteins. In contrast, immunoprecipitation of proteasomes with polyclonal or monoclonal antibodies prevented the ATP/ubiquitin-dependent degradation of lysozyme and also blocked the ATP-stimulated degradation of ubiquitin-conjugated lysozyme in reticulocyte and skeletal muscle extracts. These data indicate a critical role of the proteasome in the degradation of ubiquitin-conjugated proteins and suggest that the proteasome is associated with or is a component of the larger ubiquitin-conjugate-degrading enzyme complex.
Illegitimate transcription: transcription of any gene in any cell type.
Authors: Chelly J , Concordet J P , Kaplan J C , Kahn A
Published: May 2006

Using in vitro amplification of cDNA by the polymerase chain reaction, we have detected spliced transcripts of various tissue-specific genes (genes for anti-Müllerian hormone, beta-globin, aldolase A, and factor VIIIc) in human nonspecific cells, such as fibroblasts, hepatoma cells, and lymphoblasts. In rats, erythroid- and liver-type pyruvate kinase transcripts were also detected in brain, lung, and muscle. The abundance of these "illegitimate" transcripts is very low; yet, their existence and the possibility of amplifying them by the cDNA polymerase chain reaction provide a powerful tool to analyze pathological transcripts of any tissue-specific gene by using any accessible cell.
Characterization of vacuolar and extracellular β(1,3)-glucanases of tobacco: Evidence for a strictly compartmentalized plant defense system
Authors: Bulcke M. Van Den , Bauw G. , Castresana C. , Van Montagu M. , Vandekerckhove J.
Published: May 2006

β(1,3)-Glucanases are a class of hormonally and developmentally regulated plant hydrolytic enzymes, which are also induced upon pathogen infection, suggesting a role of these hydrolases in the defense response to pathogens. We have purified several β(1,3)-glucanases present in tobacco leaves from control plants and from plants treated with salicylic acid or infected with
Pseudomonas syringae
and studied in detail the subcellular localization of the different isoforms. Partial protein sequence analysis demonstrated that each of the different isoforms had a unique amino acid sequence and was therefore encoded by a different gene. We have also demonstrated that two of these isoforms, similar to the cytokinin/auxin-regulated isoforms previously isolated from tobacco cell suspensions, are located in the central vacuole. Upon salicylic acid treatment or
P. syringae
infection, three secreted isoforms are induced, belonging to the so-called pathogenesis-related proteins. These pathogenesis-related β(1,3)-glucanases are all distinct from each other and also different from the vacuolar isoforms. We demonstrate that the vacuolar isoforms are not secreted to the extracellular spaces of the plant following pathogen infection, suggesting that any function they play in the plant defense response is restricted to an intracellularly coordinated defense process.
Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque.
Authors: Wilcox J N , Smith K M , Schwartz S M , Gordon D
Published: May 2006

Tissue factor (TF)-producing cells were identified in normal human vessels and atherosclerotic plaques by in situ hybridization and immunohistochemistry using a specific riboprobe for TF mRNA and a polyclonal antibody directed against human TF protein. TF mRNA and protein were absent from endothelial cells lining normal internal mammary artery and saphenous vein samples. In normal vessels TF was found to be synthesized in scattered cells present in the tunica media as well as fibroblast-like adventitial cells surrounding vessels. Atherosclerotic plaques contained many cells synthesizing TF mRNA and protein. Macrophages present as foam cells and monocytes adjacent to the cholesterol clefts contained TF mRNA and protein, as did mesenchymal-appearing intimal cells. Significant TF protein staining was found deposited in the extracellular matrix surrounding mRNA-positive cells adjacent to the cholesterol clefts and within the necrotic cores. These results suggest that deposition of TF protein in the matrix of the necrotic core of the atherosclerotic plaque may contribute to the hyperthrombotic state of human atherosclerotic vessels.
Induction of megakaryocytic differentiation and modulation of protein kinase gene expression by site-selective cAMP analogs in K-562 human leukemic cells.
Authors: Tortora G , Clair T , Katsaros D , Ally S , Colamonici O , Neckers L M , Tagliaferri P , Jahnsen T , Robins R K , Cho-Chung Y S
Published: May 2006

Two classes (site 1- and site 2-selective) of cAMP analogs, which either alone or in combination demonstrate a preference for binding to type II rather than type I cAMP-dependent protein kinase isozyme, potently inhibit growth in a spectrum of human cancer cell lines in culture. Treatment of K-562 human leukemic cells for 3 days with 30 and 10 microM 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP) (site 1-selective) resulted in 60% and 20% growth inhibition, respectively (with over 90% viability). N6-Benzyl-cAMP (site 2-selective) (30 microM) treatment resulted in 20% growth inhibition by day 3. When 8-Cl-cAMP (10 microM) and N6-benzyl-cAMP (30 microM) were both added, growth was almost completely arrested. The growth inhibition was accompanied by megakaryocytic differentiation in K-562 cells. The untreated control cells expressed little or no detectable levels of glycoprotein IIb-IIIa surface antigen complex. 8-Cl-cAMP (30 microM) treatment for 3 days substantially increased the antigen expression, while N6-benzyl-cAMP caused little or no change in the antigen expression. When cells were treated with 8-Cl-cAMP in combination with N6-benzyl-cAMP, antigen expression was synergistically enhanced, and cells demonstrated megakaryocyte morphology. By Northern blotting, we examined the mRNA levels of the type I and type II protein kinase regulatory subunits (RI alpha and RII beta), the catalytic subunit, and c-myc during 8-Cl-cAMP treatment. The steady-state level of RII beta cAMP receptor mRNA sharply increased within 1 hr of treatment and remained elevated for 3 days, while that of the RI alpha receptor markedly decreased to below control level within 6 hr and remained low during treatment. However, 8-Cl-cAMP did not affect the mRNA level of the catalytic subunit. 8-Cl-cAMP treatment also brought about a rapid decrease in c-myc mRNA. Thus, differential regulation of cAMP receptor genes is an early event in cAMP-induced differentiation and growth control of K-562 leukemia cells.
Characterization of a cDNA encoding a 34-kDa Purkinje neuron protein recognized by sera from patients with paraneoplastic cerebellar degeneration.
Authors: Furneaux H M , Dropcho E J , Barbut D , Chen Y T , Rosenblum M K , Old L J , Posner J B
Published: May 2006

Paraneoplastic cerebellar degeneration is a neurological disorder of unknown cause occurring in patients with an identified or occult cancer. An autoimmune etiology is likely since autoantibodies directed against the Purkinje cells of the cerebellum have been found in the serum and cerebrospinal fluid of some patients. Two Purkinje cell-specific antigens are recognized by these autoantibodies, a major antigen of 62 kDa (CDR 62, cerebellar degeneration-related 62-kDa protein) and a minor antigen of 34 kDa (CDR 34). Our previous studies have described the isolation and characterization of a human cerebellar cDNA that encodes an epitope recognized by sera from patients with paraneoplastic cerebellar degeneration. We have now established by two independent methods that this gene is uniquely expressed in Purkinje cells of the cerebellum and corresponds to the minor antigen CDR 34. This antigen is also expressed in tumor tissue from a patient with paraneoplastic cerebellar degeneration.
Isolation of human immunodeficiency virus-related simian immunodeficiency viruses from African green monkeys.
Authors: Kraus G , Werner A , Baier M , Binniger D , Ferdinand F J , Norley S , Kurth R
Published: May 2006

We have isolated lentivirus strains that are related to the human immunodeficiency virus (HIV) from African green monkeys (Cercopithecus aethiops; AGM). Although immunologically related, these SIVagm are clearly distinct from other simian immunodeficiency virus (SIV) isolates, including isolates from Macaca mulatta (SIVmac) or even from other AGM. The SIVagm strains described in this communication grow well in a limited number of human T-lymphoma lines. Virus density, morphology, and reverse transcriptase activity are characteristic of the lentivirus group. SIVagm exhibits the following pattern of major virus proteins: p18, p28, gp45, p64, gp140. They appear to bind to the target cell via the CD4 or its primate analogue. Four virus isolates have already been molecularly cloned for detailed genomic analysis and within this SIV agm group they exhibit the genomic variability that is typical of lentiviruses. AGMs infected with this virus apparently remain healthy and therefore SIVagm not only provides a virus model for vaccine studies but also allows investigation of the defense mechanisms (immunological and others) that keep the AGMs healthy. Furthermore, precise genomic analysis of these and other SIV strains will lead to a better understanding of the evolution and pathogenicity of human lentiviruses like HIV.
Isolation and characterization of an olfactory mutant in Drosophila with a chemically specific defect.
Authors: Helfand S L , Carlson J R
Published: May 2006

A Drosophila mutant was isolated and shown to exhibit defective response to the chemical odorant benzaldehyde in two distinctly different behavioral assays. The defect exhibited chemical specificity: response to three other chemicals was normal. The mutant also showed abnormalities in pigmentation and fertility. Genetic mapping and complementation analysis provide evidence that the olfactory, pigmentation, and fertility defects arise as a result of a lesion at the pentagon locus. The specificity of the olfactory defect suggests the possibility that the mutation may define a molecule required in reception, transduction, or processing of a specific subset of chemical information in the olfactory system.
Monitoring cytosolic free magnesium in cultured chicken heart cells by use of the fluorescent indicator Furaptra.
Authors: Murphy E , Freudenrich C C , Levy L A , London R E , Lieberman M
Published: May 2006

Cytosolic free magnesium concentration [Mg2+]i and its regulation were studied in cultured embryonic chicken heart cells by use of the fluorescent indicator 2-[2-(5-carboxy)oxazole]-5-hydroxy-6-aminobenzofuran-N,N,O-triacet ic acid (Furaptra). The intracellular location of Furaptra was confirmed by its complete release from cells upon addition of saponin. The basal [Mg2+]i, which averaged 0.48 +/- 0.03 mM (n = 31), increased 3-fold on perfusion with sodium-free solution. This increase could not simply be attributed to intracellular sodium-extracellular magnesium exchange because a similar increase in [Mg2+]i occurred with magnesium-free, sodium-free perfusion. Furthermore, the increase in [Mg2+]i was largely attenuated when calcium was removed from the sodium-free perfusate. Thus, a substantial part of the increase in [Mg2+]i that occurs upon sodium-free perfusion is dependent on an increase in cytosolic free calcium (intracellular sodium-extracellular calcium exchange). The data suggest that [Mg2+]i is altered by calcium, most likely due to a competition for intracellular binding sites.
Regulation of the function of eukaryotic DNA topoisomerase I: topological conditions for inactivity.
Authors: Camilloni G , Di Martino E , Di Mauro E , Caserta M
Published: May 2006

The effects of supercoiling on the cleavage reaction by eukaryotic DNA topoisomerases I (wheat germ, chicken erythrocyte, and calf thymus) have been analyzed on DNA fragments (0.96 and 2.3 kilobases) encompassing an immunoglobulin kappa light-chain promoter. In one topological condition of the substrate, the absolutely relaxed state, cleavage was found to be impeded. This finding defines the topology-dependent step of the eukaryotic DNA topoisomerase I reaction and shows that for the cleavage reaction topology is more critical than sequence effects. These findings suggest a simple model for the regulation of the DNA topoisomerase I reaction based on topological factors, which may explain the regulatory function of the enzyme in in vivo eukaryotic transcription.