bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters.
Authors: Wilson P T , Gershoni J M , Hawrot E , Lentz T L
Published: May 2006

Proteolytic fragments of the alpha subunit of the acetylcholine receptor retain the ability to bind alpha-bungarotoxin following resolution by polyacrylamide gel electrophoresis and immobilization on protein transfers. The alpha subunit of the acetylcholine receptor of Torpedo electric organ was digested with four proteases: Staphylococcus aureus V-8 protease, papain, bromelain, and proteinase K. The proteolytic fragments resolved on 15% polyacrylamide gels were electrophoretically transferred onto positively charged nylon membrane filters. When incubated with 0.3 nM 125I-labeled alpha-bungarotoxin and autoradiographed, the transfers yielded patterns of labeled bands characteristic for each protease. The molecular masses of the fragments binding toxin ranged from 7 to 34 kDa, with major groupings in the 8-, 18-, and 28-kDa ranges. The apparent affinity of the fragments for alpha-bungarotoxin as determined from the IC50 value was 6.7 X 10(-8) M. The labeling of fragments with alpha-bungarotoxin could be inhibited by prior affinity alkylation of receptor-containing membranes with 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. These findings demonstrate that immobilized proteolytic fragments as small as 1/5 the size of the alpha subunit retain the structural characteristics necessary for binding alpha-bungarotoxin, although the toxin is bound to the fragments with lower affinity than to the native receptor. The effect of affinity ligand alkylation demonstrates that the alpha-bungarotoxin binding site detected on the proteolytic fragments is the same as the affinity-labeled acetylcholine binding site on the intact acetylcholine receptor.
Model calculations of the interaction of two parallel antiaromatic 4
n
π-electron systems
Authors: Böhm Michael C. , Bickert Peter , Hafner Klaus , Boekelheide V.
Published: May 2006

The nature of the interaction between decks of a pentalene dimer and an
s
-indacene dimer has been studied by semi-empirical MNDO/1 and MINDO/3 calculations for distances between decks of from 5 Å to 2 Å. In contradiction to qualitative predictions from a frontier orbital analysis, it is found that the 4
n
-4
n
π-electron interaction between decks for such dimers is destabilizing for distances exceeding about 2.5 Å.
Dirac operators coupled to vector potentials
Authors: Atiyah M. F. , Singer I. M.
Published: May 2006

Characteristic classes for the index of the Dirac family [unk]

A

are computed in terms of differential forms on the orbit space of vector potentials under gauge transformations. They represent obstructions to the existence of a covariant Dirac propagator. The first obstruction is related to a chiral anomaly.
Catabolite repression of chloroplast development in
Euglena
Authors: Monroy Antonio F. , Schwartzbach Steven D.
Published: May 2006

Two-dimensional gel electrophoresis resolved total cellular protein from
Euglena gracilis
Klebs var.
bacillaris
Cori into 640 polypeptides detectable by silver staining. The addition of 84 mM ethanol to dark-grown resting carbon-starved cells increased the relative amounts of 6 polypeptides and decreased the relative amounts of 3 polypeptides. The addition of 84 mM malate to resting cells increased the relative amounts of 3 of the ethanol-induced polypeptides, suggesting that the induction of these polypeptides represents a generalized response to the provision of a utilizable carbon source, a nutritional shift up, rather than a specific response to ethanol addition. Exposure of dark-grown resting
Euglena
to light increases the relative amounts of 79 polypeptides encoded by the nuclear as well as the chloroplast genome and decreases the relative amounts of 72 polypeptides. Ethanol but not malate specifically inhibited all of the light-dependent changes in polypeptide levels, indicating that chloroplast development in
Euglena
is a catabolite-sensitive process.
Cerebellum mediates modality-specific modulation of sensory responses of midbrain and forebrain in rat.
Authors: Crispino L , Bullock T H
Published: May 2006

Evidence of a sensory role of the cerebellum, mediating a modulation of effectiveness of afferent input at other parts of the brain, has been reported previously for certain sense modalities but has not been evaluated across several in a mammal. After a conditioning train of stimuli applied to the cerebellar surface in unanesthetized rats, diffuse flashes, acoustic clicks, and shocks to the sciatic nerve evoked multiunit and field potential responses that were recorded at three levels: midbrain, thalamus, and cerebral cortex. At a best interval between end of conditioning train (cerebellar) and test (sensory) stimuli, all three levels show modulation of the evoked responses, each in a specific direction (enhancement or depression), with a characteristic time course. Visual responses in the tectum are enhanced; those in the cortex are depressed. Tectal responses that have been nearly abolished by increasing background illumination are partially restored by the conditioning cerebellar train. Auditory brainstem responses (short latency, less than 10 ms, far-field waves I to III, attributed to medullary levels) are depressed; wave IV from the inferior colliculus is relatively enhanced at short intervals and is depressed at longer intervals. Somatosensory responses in thalamus and cortex are depressed. Lobulae V, VI, and VII of the vermis are more effective sites of stimulation than other areas tested. Most of the modulations are ascribed to central sites; a few are ascribed to peripheral sites.
Evidence for parallel actions of a molluscan neuropeptide and serotonin in mediating arousal in Aplysia.
Authors: Lloyd P E , Kupfermann I , Weiss K R
Published: May 2006

The neuropeptide designated SCPB (small cardioactive peptide B), the sequence of which has recently been determined, was found in the accessory radula closer muscle, a muscle involved in biting movements. The ganglia and nerves that innervate the accessory radula closer muscle also contain SCPB. At nanomolar concentrations, it enhances the contractions of the muscle. The effect of SCPB on the muscle resembles the effect of an identified serotonergic neuron that previously was shown to mediate behavioral effects that reflect a food arousal state in Aplysia. Like serotonin, SCPB enhances contractions by a postsynaptic action, which appears to involve an increase in cAMP levels in the muscle. Our findings suggest that parallel peptidergic and serotonergic pathways may mediate similar aspects of arousal in Aplysia.
Injected anti-sense RNAs specifically block messenger RNA translation in vivo.
Authors: Melton D A
Published: May 2006

As a test for a method to analyze gene function in embryogenesis, the translation of a specific mRNA has been blocked in vivo by microinjection of complementary (anti-sense) RNA. RNA complementary to globin mRNA was synthesized in vitro by transcription of an inverted globin cDNA clone. After injection into frog oocyte cytoplasm, the anti-sense globin RNA forms a hybrid with globin mRNA and selectively prevents its translation. Deletion mapping of the anti-sense RNA shows that the 5' region of the globin mRNA must be covered in order to block translation. This method may allow one to study the function of many genes for which DNA clones are available by preventing the expression of the endogenous gene as protein.
Interaction of selection and biased gene conversion in a multigene family.
Authors: Walsh J B
Published: May 2006

A model of the evolutionary dynamics of a multigene family in a finite population under the joint effects of selection and (possibly biased) gene conversion is analyzed. It is assumed that the loss or fixation of a polymorphism at any particular locus in the gene family occurs on a much faster time scale than the introduction of new alleles to a monomorphic locus by gene conversion. A general formula for the fixation of a new allele throughout a multigene family for a wide class of selection functions with biased gene conversion is given for this assumption. Analysis for the case of additive selection shows that (i) unless selection is extremely weak or bias is exceptionally strong, selection usually dominates the fixation dynamics, (ii) if selection is very weak, then even a slight conversion bias can greatly alter the fixation probabilities, and (iii) if both selection and conversion bias are sufficiently small, the substitution rate of new alleles throughout a multigene family is approximately the single locus mutation rate, the same result as for neutral alleles at a single-copy gene. Finally, I analyze a fairly general class of underdominant speciation models involving multigene families, concluding for these models under weak conversion that although the probability of fixation may be relatively high, the expected time to fixation is extremely long, so that speciation by "molecular drive" is unlikely. Furthermore, speciation occurs faster by fixing underdominant alleles of the same effect at single-copy genes than by fixing the same number of loci in a single multigene family under the joint effects of selection, conversion, and drift.
Phosphorylation of hydroxylysine residues in collagen synthesized by cultured aortic smooth muscle cells.
Authors: Urushizaki Y , Seifter S
Published: May 2006

O5-Phosphohydroxylysine was chemically synthesized and techniques were established for its identification by combined use of cation-exchange chromatography, thin-layer electrophoresis at pH 1.9 and 3.5, and thin-layer chromatography. Clean separation of phosphohydroxylysine from the other phospho amino acids, phosphoethanolamine, and phosphocholine was achieved. Conditions were also determined to permit hydrolysis of proteins in 2 M HCl without loss of the phosphono group of phosphohydroxylysine residues. Experiments were then performed showing that 32P was incorporated into the hydroxylysine residues of cell-associated collagens when cultured calf aorta medial smooth muscle cells were incubated with [32P]orthophosphate. In other experiments, the cells incorporated [3H]lysine into hydroxylysine residues of cell-associated collagen and then 32P into phosphohydroxylysine residues. The doubly labeled phosphohydroxylysine subsequently isolated showed nearly 1:1 stoichiometry with respect to incorporation of precursor lysine and phosphorus. Finally, in preliminary experiments done with a cell-free extract of the smooth muscle cells, 32P was transferred from [gamma-32P]ATP to hydroxylysine residues in several kinds of collagenous substrates. Thus, this work shows that smooth muscle cells have the capacity to phosphorylate hydroxylysine residues in their cell-associated collagens and provides preliminary evidence that a protein kinase is involved.
Cell cycle-dependent variations in the distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: subdivision of S phase.
Authors: Celis J E , Celis A
Published: May 2006

Immunofluorescence analysis of synchronously growing transformed human amnion cells (AMA) using autoantibodies specific for cyclin has revealed dramatic changes in the nuclear distribution of this protein during the S phase of the cell cycle. Cells in G1, G2, and mitosis exhibit weak staining with the antibody, while S-phase cells show variable patterns of staining in terms of both intensity and distribution of the antigen. Early in S phase, cyclin is localized throughout the nucleoplasm with the exception of the nucleoli. A similar, but stronger, staining pattern is observed as the cells progress through the S phase. At a later stage, before maximum DNA synthesis, cyclin redistributes to reveal a punctuated pattern with foci of staining throughout the nucleus. This pattern precedes a major change in the distribution of this protein, which is then detected in the nucleolus. At this stage, DNA synthesis is at or near a maximum. Thereafter, there are further changes in the distribution of this protein, with the pattern becoming punctuated and of decreasing intensity. All these staining patterns have also been detected in asynchronously growing normal human amnion cells (AF type), suggesting that the distribution of this protein is not a consequence of transformation. Analysis of cultured cells from several vertebrate species also revealed similar staining patterns. These results are consistent with the idea that cyclin is a central component of the pathway(s) leading to DNA replication and cell division.
Pertussis toxin inhibits chemotactic peptide-stimulated generation of inositol phosphates and lysosomal enzyme secretion in human leukemic (HL-60) cells.
Authors: Brandt S J , Dougherty R W , Lapetina E G , Niedel J E
Published: May 2006

The binding of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine to its cell surface receptor rapidly elicits the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C to form the putative second messengers inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol. To investigate the possible role of a guanine nucleotide binding protein in transduction of this membrane signal, we examined the effects of pertussis toxin on chemotactic peptide-stimulated inositol phospholipid metabolism in differentiated HL-60 cells labeled with [3H]inositol. Pertussis toxin inhibited the chemotactic tripeptide-stimulated production of inositol mono-, bis-, and trisphosphates and secretion of N-acetyl-beta-D-glucosaminidase in a time- and concentration-dependent manner. Treatment with pertussis toxin did not alter the total incorporation or the distribution of [3H]inositol in inositol phospholipid. Chemotactic peptide receptor number was unchanged, although a slight decrease in binding affinity was observed. These findings suggest a role for a guanine nucleotide binding protein in coupling the chemotactic peptide receptor to phospholipase C.
Analysis of cDNA clones specific for human T cells and the alpha and beta chains of the T-cell receptor heterodimer from a human T-cell line.
Authors: Yanagi Y , Chan A , Chin B , Minden M , Mak T W
Published: May 2006

We report the isolation and characterization of 19 classes of nonrearranging T cell-specific cDNA clones and two cDNA clones encoding the alpha and beta chains of the T-cell antigen receptor from a human T-cell line, Jurkat. Results indicate that the human alpha-chain gene, like its beta-chain counterpart, undergoes somatic rearrangement in T cells. In addition, it shows sequence homology to its beta-chain counterpart and immunoglobulin, indicating that the human alpha chain is also a member of the immunoglobulin supergene family. Sequence comparison suggests that the alpha chain also may be composed of variable (V), diversity (D), joining (J), and constant (C) region gene segments. The protein deduced from the cDNA sequence has a molecular weight of 29,995 and possesses six potential N-glycosylation sites. The availability of alpha- and beta-chain genes of the T-cell receptor from the same T-cell line provides tools to study their possible roles in recognition of antigens and major histocompatibility complex products by the human T-cell receptor.
Isolation and characterization of an RNA-proteolipid complex associated with the malignant state in humans.
Authors: Wieczorek A J , Rhyner C , Block L H
Published: May 2006

An RNA-proteolipid complex was isolated from sera of patients with a variety of malignant disorders as well as from culture media of malignant cell lines. The complex, characterized by a relatively constant composition, contains 27S poly(A)+ RNA and Mr 1250 oligopeptide(s) and is rich in phospholipids and glycosphingolipids. Serum lipoproteins (low and high density) differ from this complex in density, chemical composition, and immunological reactivity. The complex was detected in 94 of 96 cases of malignancy tested but not in any of 58 patients with nonmalignant disorders or in 46 healthy individuals.
Generation of functional clonal cell lines from human bone marrow stroma.
Authors: Harigaya K , Handa H
Published: May 2006

Five clonal human bone marrow stromal cell lines were isolated from the adherent cell populations in long-term liquid cultures after transfection with the recombinant plasmid pSV3gpt. All the cell-line feeder layers and their conditioned media stimulated the proliferation of committed granulomonocytic stem cells (CFUc) from human bone marrow. The size and number of early erythroid stem cell (BFUe)-derived colonies were significantly increased when in the presence of 10% conditioned medium from the cell lines. Furthermore, a substantial number of mixed colonies with erythroid components were observed in the cultures in the presence of erythropoietin and conditioned medium. These findings suggest that the human bone marrow stromal cell lines obtained after transfection with pSV3gpt may be extremely useful in identifying the hematopoietic factors derived from the hematopoietic microenvironment and in analyzing their mechanism of action.
Alternative conformations in Escherichia coli 16S ribosomal RNA.
Authors: Klein B K , Staden A , Schlessinger D
Published: May 2006

Partially denatured 16S rRNA from 30S ribosomes shows features of secondary structure in electron microscopy that correspond to the well accepted secondary structure model derived from chemical modification and phylogenetic data. However, a very different conformation is seen in precursor 16S rRNA sequences contained within 30S pre-rRNA transcripts: the major 5'-terminal loop is absent, and several additional quite stable large loops, symmetrically placed in the molecule, are present. Features of the alternative structure are also seen in mature 16S rRNA from Escherichia coli and from two Bacillus species when heated in certain buffers. Microscopy thus reveals specific features of alternative conformations and their relative stabilities, suggesting a possible transition during ribosome formation.
Three-dimensional structure of ubiquitin at 2.8 A resolution.
Authors: Vijay-Kumar S , Bugg C E , Wilkinson K D , Cook W J
Published: May 2006

The three-dimensional structure of ubiquitin has been determined at 2.8 A resolution. X-ray diffraction data for the native protein and derivatives were collected with an automated diffractometer. Phases were obtained by use of a single isomorphous mercuric acetate derivative. The molecule contains a pronounced hydrophobic core. Prominent secondary structural features include three and one-half turns of alpha-helix, a mixed beta-sheet that contains four strands, and seven reverse turns. The histidine, tyrosine, and two phenylalanine residues are located on the surface of the molecule.
A truncated immunoglobulin epsilon pseudogene is found in gorilla and man but not in chimpanzee.
Authors: Ueda S , Takenaka O , Honjo T
Published: May 2006

Molecular genetic analyses of the young pseudogenes of the immunoglobulin C epsilon genes were carried out to obtain qualitative evidence for the phylogenetic branching pattern of hominoid primates. We found that Old World monkeys had two C epsilon genes, one of which was processed. Among the hominoids examined only the gorilla and human genomes contained three C epsilon genes: an active, a truncated, and a processed gene. Other hominoids so far examined, including chimpanzee, contained two C epsilon genes: one active and the other processed. These results suggest that the processed C epsilon pseudogene was generated before the divergence between Old World monkeys and hominoids and that the gorilla is more closely related to man than the chimpanzee is, unless the chimpanzee has lost the C epsilon 2 gene after the divergence of this species.
Cross-reactive idiotypes and common antigen binding specificities expressed by a series of murine B-cell lymphomas: etiological implications.
Authors: Pennell C A , Arnold L W , Lutz P M , LoCascio N J , Willoughby P B , Haughton G
Published: May 2006

A series of 27 B-cell lymphomas (designated the CH series), induced in B10.H-2aH-4b p/Wts mice by intense adoptive immunization with sheep erythrocytes, was found to represent a subset of the total B-cell repertoire. This subset was characterized by expression of a limited number of Ig heavy chain variable regions, as evidenced by the presence of cross-reactive idiotypes and common antigen binding specificities. Twenty-one of the 27 CH lymphomas studied were classified into five groups, defined by a particular cross-reactive idiotype; four of these groups were linked in a single network. Seven of 16 idiotypes defined by absorption analysis were present on lymphomas bearing either kappa or lambda light chains and so were localized to the heavy chain variable region. The surface Ig on 14 CH lymphomas was found to be specific for epitopes on certain erythrocytes (bromelain-treated autologous erythrocytes, sheep, and chicken erythrocytes) or E. coli. We propose that the CH lymphomas represent the malignant counterparts of a subset of idiotypically related, normal B cells in B10.H-2aH-4b p/Wts mice. Perturbation of this idiotype network, by hyperimmunization with an antigen for which some of the members are specific (sheep erythrocytes), increases the risk for neoplasia. Possible mechanisms for this are discussed.
Functional organization of the cat's visual cortex after prenatal interruption of binocular interactions.
Authors: Shook B L , Maffei L , Chalupa L M
Published: May 2006

The functional consequences of interrupting in utero binocular interactions were studied by recording from single cells in area 17 of adult cats that had one eye removed at least 2 wk before birth. In these animals all cortical neurons could be driven by the remaining eye, and in tangential microelectrode penetrations, sequences of neurons containing a full 180-degree cycle of preferred orientations were encountered. Other response properties of cortical neurons in the prenatally enucleated animals were also normal with the notable exception that the dimensions of receptive fields were significantly smaller when compared with those of control animals. Our results indicate that orientation columns in the visual cortex can develop independently of ocular dominance columns, and they suggest that interruption of binocular interactions during prenatal development of the visual pathways may enhance the resolving power of the remaining eye.
Opposite-strand RNAs from the 5' flanking region of the mouse dihydrofolate reductase gene.
Authors: Farnham P J , Abrams J M , Schimke R T
Published: May 2006

We have characterized a transcription unit in the 5' flanking region of the mouse gene encoding dihydrofolate reductase (EC 1.5.1.3) that is oriented in the opposite direction to that of dihydrofolate reductase transcription. These opposite-strand RNAs are 180-240 nucleotides long, differing in length at their 5' ends. They are abundant in nuclear RNA and are not polyadenylylated. We suggest that the promoter region of the dihydrofolate reductase gene functions in a bidirectional manner to produce both these RNAs and dihydrofolate reductase mRNAs.
Neurochemical basis for the photic control of circadian rhythms and seasonal reproductive cycles: role for acetylcholine.
Authors: Earnest D J , Turek F W
Published: May 2006

A pharmacological approach was used to examine the role of acetylcholine in the photic control of circadian rhythms and seasonal reproductive cycles. The experimental protocol was designed to determine whether the administration of carbachol, a cholinergic agonist, could mimic the effects of brief light pulses on gonadal function and/or the circadian rhythm of wheel-running activity in golden hamsters. Intraventricular injections of carbachol, administered singularly at discrete phase points throughout the circadian cycle, induced phase-dependent shifts in the free-running rhythm of activity similar to those caused by a brief light exposure. Injections of carbachol once every 23.33 hr for 9 weeks entrained the activity rhythm and stimulated the neuroendocrine-gonadal axis in a manner similar to that observed after the presentation of 1-hr light pulses at this frequency. In contrast, the administration of carbachol once every 24 hr did not consistently provide an entraining signal for the activity rhythm and did not stimulate reproductive function. Importantly, the effects of carbachol on the seasonal reproductive response were correlated with the timing of the injections relative to the activity rhythm. These findings suggest that acetylcholine may play an important role in the mechanism by which light regulates circadian rhythms and seasonal reproductive cycles.
GTPase of bovine rod outer segments: the amino acid sequence of the alpha subunit as derived from the cDNA sequence.
Authors: Yatsunami K , Khorana H G
Published: May 2006

The sequence of the 350 amino acids in the alpha subunit of GTPase of bovine rod outer segments has been determined. Enriched GTPase mRNA was used to prepare a cDNA library in the expression vector lambda gt11 and several overlapping cDNA clones corresponding to the alpha subunit of the GTPase were identified. The cDNA sequence determined contains 93 nucleotides upstream of the 5' end of the coding region, 1050 nucleotides that specify the amino acid sequence, and 45 nucleotides downstream from the 3' end. The previously described partial amino acid sequences and the sequences at the ADP-ribosylation sites for cholera and pertussis toxins are all confirmed and fitted into the present complete sequence. Homologies are found between the sequence of the alpha subunit and those of other guanine nucleotide-binding proteins, the ras proteins, peptide chain elongation factors EF-Tu and EF-G, and the initiation factor IF2.
Adenovirus VAI RNA prevents phosphorylation of the eukaryotic initiation factor 2 alpha subunit subsequent to infection.
Authors: Schneider R J , Safer B , Munemitsu S M , Samuel C E , Shenk T
Published: May 2006

The virus-associated VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficient translation of mRNAs late after infection. Deletion mutant dl331 fails to produce this RNA and, as a result, grows poorly. Three lines of evidence suggest that VAI RNA facilitates translation by preventing inactivation of the function of eukaryotic initiation factor 2 (eIF-2). First, the mutant's translational defect can be relieved by addition of eIF-2 or eIF-2B (GTP recycling factor). Second, extracts of mutant-infected cells exhibit enhanced protein P1/eIF-2 alpha subunit kinase activity. Third, dl331 can grow with nearly normal kinetics in cells that do not express the kinase.
Identification of an inhibitory region of the heat-stable protein inhibitor of the cAMP-dependent protein kinase.
Authors: Scott J D , Fischer E H , Demaille J G , Krebs E G
Published: May 2006

The present study was undertaken in order to identify the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKI) and to synthesize a peptide that could serve as a useful inhibitor of the enzyme. Digestion of purified PKI by mast cell proteinase II yielded a peptide fragment that retained inhibitory activity. A sequence of 20 amino acids of the peptide, (sequence in text) revealed the presence of a "pseudosubstrate site" (Arg-Arg-Asn-Ala-Ile) for the cAMP-dependent protein kinase in which alanine replaces the seryl or threonyl residue that is normally phosphorylated. Digestion of PKI with various other proteinases implicated the involvement of arginyl and hydrophobic residues as determinants for the inhibitory activity. The assumption that this region is part of the inhibitory site was confirmed by the synthesis of a corresponding duodecapeptide that displayed strong inhibitory activity. Inhibition by the peptide was competitive with a Ki of 0.8 microM as measured against a number of protein substrates. The sequence of this fragment bears a strong resemblance to the autophosphorylation site in the type II regulatory subunit of cAMP-dependent protein kinase, a region also postulated to interact with the catalytic subunit, and the analogous region of type I regulatory subunit. Neither intact PKI nor the synthetic peptide inhibit the cGMP-dependent protein kinase, phosphorylase kinase, myosin light-chain kinase, casein kinase II, or protein kinase C.
Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein
Authors: Chen Jychian , Varner Joseph E.
Published: May 2006

Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, we started by isolating poly(A)
+
RNA from carrot root tissue, and then translating the RNA
in vitro
, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor because: (
i
) it is rich in proline and poor in leucine, and (
ii
) the message appears to be more abundant when carrot tissue is wounded. From a cDNA library constructed with poly(A)
+
RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A)
+
RNA encoding this 33-kDa peptide. We isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pDC5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released
in vitro
translation indicate that the cDNA clone pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization and DNA sequence analysis indicate that pDC5 is a hybrid clone corresponding to two RNA species. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.
Predominant generation of 15-lipoxygenase metabolites of arachidonic acid by epithelial cells from human trachea.
Authors: Hunter J A , Finkbeiner W E , Nadel J A , Goetzl E J , Holtzman M J
Published: May 2006

Epithelial cells of 99% purity and 92% viability were isolated from human tracheas obtained post mortem, and the cellular pathways for lipoxygenation of arachidonic acid were examined in vitro. The lipoxygenase metabolites were identified by comparison with synthetic standards during reversed-phase and straight-phase high-pressure liquid chromatography, UV spectroscopy, and gas chromatography/mass spectrometry. Epithelial cells incubated without arachidonic acid failed to generate detectable quantities of metabolites, while cells incubated with arachidonic acid at 1-50 micrograms/mf for 1-30 min invariably generated predominantly 15-lipoxygenase products, including 15-hydroxyicosatetraenoic acid (15-HETE), four isomers of 8,15-dihydroxyicosatetraenoic acid (two 8,15-diHETES and two 8,15-leukotrienes), at least one isomer of 14,15-dihydroxyicosatetraenoic acid, and smaller amounts of 12-HETE and 8-HETE, but little or no detectable 5-HETE or 5,12-diHETEs. The capacity of epithelial cells from human pulmonary airway to selectively generate 15-lipoxygenase metabolites of arachidonic acid suggests a potential role for the products as mediators of airway epithelial function.
The sequence of a type II keratin gene expressed in human skin: conservation of structure among all intermediate filament genes.
Authors: Tyner A L , Eichman M J , Fuchs E
Published: May 2006

We report here the coding sequence of the gene for a 56-kDa type II keratin (designated K6b). Using a subclone specific for a unique 3' noncoding region of the encoded mRNA, we have shown that this gene is one of at least two 56-kDa keratin genes expressed in abundance in human epidermis. Segmenting the coding portion of this gene are eight introns, six of which are identically positioned with those of a distantly related type III intermediate filament gene (vimentin), and five of which are identically positioned with those of a distantly related type I gene (50-kDa keratin). These results indicate a common ancestral origin for all three classes of intermediate filament genes. All of the highly conserved intron positions are located within, but do not demarcate, the four central alpha-helical domains common to all intermediate filament polypeptides, suggesting that these genes were probably not created piecemeal by recombination-mediated linkage of separate structural domains as they presently are known.
cAMP-dependent phosphorylation of bovine lens alpha-crystallin.
Authors: Spector A , Chiesa R , Sredy J , Garner W
Published: May 2006

This communication reports that the A1 and B1 chains of bovine lens alpha-crystallin are phosphorylated. The conclusion is based on the following evidence: (i) When soluble preparations from lens cortex are incubated with [gamma-32P]ATP, a cAMP-dependent labeling of a high molecular weight protein is obtained. (ii) After NaDodSO4/PAGE, the label is found in two bands with Mr 22,000 and 20,000, corresponding to the B and A chains of alpha-crystallin, respectively. (iii) Isoelectric focusing indicates that the radioactivity is almost exclusively in bands with pI values of 5.58 and 6.70, corresponding to the A1 and B1 chains, respectively. (iv) Similar results are obtained in experiments of [32P]orthophosphate incorporation in lens organ culture. (v) Analyses of the digested protein indicate the label is exclusively in phosphoserine. (vi) 31P NMR analyses of native, proteolytically digested, and urea-treated alpha-crystallin gives a chemical shift of 4.6 ppm relative to 85% H3PO4 at pH 7.4, suggesting that the phosphate is covalently bound to a serine in the protein. An abundance of approximately one phosphate per four or five monomer units was found. (vii) Similar results were obtained by chemical analyses of independently prepared alpha-crystallin samples. The results are consistent with the view that the A1 and B1 chains arise as result of the phosphorylation of directly synthesized A2 and B2 polypeptides. It is suggested that this metabolically controlled phosphorylation may be associated with the terminal differentiation of the lens epithelial cell and the intracellular organization of the lens fiber cell.
Molecular comparison of alpha 1- and alpha 2-adrenergic receptors suggests that these proteins are structurally related "isoreceptors".
Authors: Shreeve S M , Fraser C M , Venter J C
Published: May 2006

The structures of human platelet alpha 2-adrenergic receptors and rat liver alpha 1-adrenergic receptors were compared by utilizing isoelectric focusing, NaDodSO4/PAGE, and monoclonal antibody crossreactivity. Digitonin-solubilized alpha 1- and alpha 2-adrenergic receptors have an identical isoelectric point of 4.6. Under reducing conditions in NaDodSO4/polyacrylamide gels, the alpha 1-adrenergic receptor has an apparent molecular mass of 85 kDa. Similarly, the alpha 2-adrenergic receptor, which had been affinity-labeled with [3H]phenoxybenzamine and partially purified by isoelectric focusing or photoaffinity-labeled with p-[3,5-3H]azidoclonidine, was also found to have an apparent molecular mass of 85 kDa. One hybridoma, developed from a fusion between SP2/O myeloma cells and splenic lymphocytes from BALB/c mice immunized with human platelet alpha 2-adrenergic receptors, secreted a monoclonal antibody (alpha 2-116p) against the ligand binding site of alpha 2-adrenergic but not alpha 1-adrenergic receptors. In contrast, three monoclonal antibodies raised against the alpha 1-receptor polypeptide backbone but not the ligand binding site were found to specifically immunoprecipitate human platelet alpha 2-adrenergic receptors. These data suggest that the alpha 1- and alpha 2-adrenergic receptors are "isoreceptors," sharing immunogenic and, by implication, structural determinants that most likely evolved as a result of gene duplication.
Inhibition of phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate and dihydrofolic acid polyglutamates.
Authors: Allegra C J , Drake J C , Jolivet J , Chabner B A
Published: May 2006

We report the enhanced inhibitory potency of methotrexate (MTX) polyglutamates and dihydrofolate pentaglutamate on the catalytic activity of phosphoribosylaminoimidazolecarboxamide (AICAR) transformylase purified from MCF-7 human breast cancer cells. In the present work, MTX (4-amino-10-methylpteroylglutamic acid) and dihydrofolate, both monoglutamates, were found to be weak competitive inhibitors of AICAR transformylase with Kis of 143 and 63 microM, respectively, and their inhibitory capacity was largely unaffected by the glutamated state of the folate cosubstrate. In contrast, MTX polyglutamates were found to be potent competitive inhibitors, with an approximately 10-fold increase in inhibitory potency with the addition of each glutamate group up to four (i.e., the pentaglutamate derivative). MTX tetra-and pentaglutamates were the most potent, with equivalent Kis of 5.6 X 10(-8) M or 2500-fold more potent than MTX. Dihydrofolate pentaglutamate was as potent an inhibitor as MTX pentaglutamate, with a Ki of 4.3 X 10(-8) M. The potent inhibitory effects demonstrated by the polyglutamate compounds when tested against the folate monoglutamate substrate were sharply curtailed when folate pentaglutamate was used as the substrate. MTX and dihydrofolate pentaglutamates were only 7- and 25-fold more potent than their monoglutamate counterparts under these conditions. A model depicting these complex interactions is postulated. These findings have significant implications regarding the mechanism of action of MTX.
Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes.
Authors: Stoppelli M P , Corti A , Soffientini A , Cassani G , Blasi F , Assoian R K
Published: May 2006

The purified amino-terminal fragment (ATF) of human urokinase plasminogen activator (residues 1-135), which is not required for activation of plasminogen, binds with high affinity to specific plasma membrane receptors on U937 monocytes. Intact urokinase efficiently competes for 125I-labeled ATF binding; 50% competition occurs with 1 nM urokinase. A large part of receptor-bound urokinase remains on the cell surface for at least 2 hr at 37 degrees C. Differentiation of U937 monocytes into macrophage-like cells specifically increases ATF binding 10- to 20-fold. These results suggest an important role for urokinase in monocyte/macrophage biology: the native enzyme binds to the cells with the amino-terminal domain; the catalytic, carboxyl-terminal domain remains exposed on the cell surface to stimulate localized proteolysis and facilitate cell migration.
Isolation and functional analysis of a human 70,000-dalton heat shock protein gene segment.
Authors: Voellmy R , Ahmed A , Schiller P , Bromley P , Rungger D
Published: May 2006

A human 70-kDa heat shock protein (hsp70) gene segment has been isolated. The segment contains 3.15 kilobase pairs (kbp) of 5' nontranscribed sequence, an RNA leader of 119 bp, and a protein-coding region of 741 bp. The human protein sequence shows a high degree of homology to hsp70 sequences from other species. Expression experiments in Xenopus oocytes and mammalian cells indicate that a region that includes only 105 bp of 5' nontranscribed sequence contains all elements required for the efficient heat-controlled expression of the human gene. Two adjacent identical sequence elements, which are partly homologous to the Drosophila "heat shock consensus" sequence, are located 57 to 76 bp upstream from the capping site. Interestingly, the capping site itself is flanked by inverted repeat sequences.
Self-degradation of heat shock proteins.
Authors: Mitchell H K , Petersen N S , Buzin C H
Published: May 2006

The 70-kDa heat shock protein of Drosophila decays in vivo at a much faster rate than other abundantly labeled proteins. Degradation also occurs in vitro, even during electrophoresis. It appears that this degradation is not mediated by a general protease and that the 70-kDa heat shock protein has a slow proteolytic action upon itself. Heat-induced proteins in CHO cells and a mouse cell line also degrade spontaneously in vitro, as do certain non-heat shock proteins from Drosophila tissues as well as the cell lines.
Purification of the opiate receptor of NG108-15 neuroblastoma-glioma hybrid cells.
Authors: Simonds W F , Burke T R , Rice K C , Jacobson A E , Klee W A
Published: May 2006

Opiate receptors from NG108-15 neuroblastoma-glioma hybrid cell membranes were purified to apparent homogeneity in a form covalently labeled with the opiate affinity ligand 3-methylfentanylisothiocyanate (super-FIT). The purification procedure consists of five steps: quantitative labeling of opiate receptors in membranes with super-FIT; solubilization in a lubrol/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate mixture; adsorption to and elution from a wheat germ agglutinin-Sepharose column; immunoaffinity chromatography on columns of immobilized anti-fentanyl; preparative gel electrophoresis in the presence of NaDodSO4. The protein was purified approximately 30,000-fold in 3% overall yield to a state in which there is 1 mol of super-FIT bound per mol of protein, corresponding to 21,000 pmol per mg of protein, the theoretically expected specific activity. The protein is glycosylated and migrates on NaDodSO4 gel electrophoresis with a Mr near 58,000. It has a strong tendency to dimerize, even in the presence of denaturing detergents, and it exists primarily as an oligomer in nondenaturing detergents.
Human class I alcohol dehydrogenases catalyze the oxidation of glycols in the metabolism of norepinephrine.
Authors: Mårdh G , Luehr C A , Vallee B L
Published: May 2006

Investigations of the function of human liver alcohol dehydrogenase (ADH) in norepinephrine metabolism have revealed that class I ADH catalyzes the oxidation of the intermediary alcohols 4-hydroxy-3-methoxyphenyl glycol (HMPG) and 3,4-dihydroxyphenyl glycol (DHPG) in vitro. The kcat/Km values for the individual homogeneous class I isozymes are generally in the range from 2.0 to 10 mM-1 X min-1, slightly lower than those obtained for ethanol oxidation, 16-66 mM-1 X min-1, but considerably higher than those obtained for ethylene glycol oxidation, 0.23-1.5 mM-1 X min-1. Importantly, HMPG and DHPG are not substrates for the class II or class III ADHs. 4-Methylpyrazole and 1,10-phenanthroline inhibit the class I ADH-catalyzed oxidation of HMPG, DHPG, and ethanol with inhibition constants of 75-90 nM and 19-22 microM, respectively, indicating that these substrates interact at the same catalytic site of ADH. Moreover, ethanol inhibits the oxidation of HMPG. The competition of ethanol with HMPG for ADH provides a basis for the in vivo changes observed in norepinephrine metabolism after acute ethanol intake. Any assessment of norepinephrine function through the study of metabolites in peripheral body fluid must include monitoring the oxidation of HMPG by ADH.
Purification and characterization of caldesmon77: a calmodulin-binding protein that interacts with actin filaments from bovine adrenal medulla.
Authors: Sobue K , Tanaka T , Kanda K , Ashino N , Kakiuchi S
Published: May 2006

Caldesmon150, a protein composed of the Mr 150,000/147,000 doublet, alternately binds to calmodulin and actin filaments in a Ca2+-dependent "flip-flop" fashion. In all fibroblast cell lines examined, we also found a Mr 77,000 protein that crossreacts with anti-caldesmon150 antibody by using an immunoprecipitation technique [Owada, M.K., Hakura, A., Iida, K., Yahara, I., Sobue, K. & Kakiuchi, S. (1984) Proc. Natl. Acad. Sci. USA 81, 3133-3137]. In this report, we examine the tissue distribution of caldesmon by the method of immunoblotting, using caldesmon-specific antibody. Both caldesmon150 and caldesmon77 show widespread distribution in the tissues examined. Caldesmon77 is more widely distributed than caldesmon150, and we have purified caldesmon77 from bovine adrenal medulla. Its molecular weight estimated by NaDodSO4/polyacrylamide gel electrophoresis was 77,000, and a tetramer of this polypeptide may constitute the native molecule (Mr, 300,000). Caldesmon77 possesses a number of features in common with caldesmon150, including flip-flop binding to calmodulin and actin filaments depending on the concentration of Ca2+ and crossreactivity with caldesmon150-specific antibody. Analysis of caldesmon77-F actin interaction by sedimentation and electrophoresis revealed that 0.5 mg of caldesmon77 bound to 1 mg of F actin. This indicated that the molar ratio between caldesmon77 (tetramer) and actin monomer was calculated to be 1:12-14. In addition, caldesmon77 regulated the actin-myosin interaction in Ca2+-sensitive actomyosin obtained from adrenal medulla. These results suggest that caldesmon77 might be a ubiquitous actin-linked regulator of nonmuscle contractile processes, including those in adrenal medulla.
Primary structure of the soybean nodulin-35 gene encoding uricase II localized in the peroxisomes of uninfected cells of nodules
Authors: Nguyen T. , Zelechowska M. , Foster V. , Bergmann H. , Verma D. P. S.
Published: May 2006

Nodulin-35 (N-35), a subunit of nodule-specific uricase (uricase II) of soybean (
Glycine max
), is shown to be preferentially synthesized on free polysomes during nodule development and is localized in peroxisomes of the uninfected cells of this tissue. A cDNA clone, isolated by using mRNA from immunoprecipitated polysomes, revealed the primary structure of this protein with a molecular mass of 35,100. That this clone represents N-35 was confirmed by comparing the deduced amino acid sequence with the partial sequence of a CNBr-cleaved peptide of purified N-35. Southern blot hybridizations with genomic DNA suggest that there are several
Eco
RI fragments containing N-35 sequences. Three of these sequences were isolated from a genomic library of soybean. Nucleotide sequence analysis showed that the complete gene extends almost 5000 base pairs on two
Eco
RI fragments and the coding region (309 codons) is interrupted by seven introns ranging in size from 154 to 1341 base pairs. Lack of a signal sequence and its translation on free polysomes suggest that N-35 is posttranslationally transported to the peroxisomes. Furthermore, there is no cross-hybridization of N-35 cDNA with RNA from young (3- to 4-day) roots and leaves, indicating that the observed “uricase” activity in these tissues is due to the product of a different gene.
A population genetic framework for the study of invasive diseases caused by serotype b strains of Haemophilus influenzae.
Authors: Musser J M , Granoff D M , Pattison P E , Selander R K
Published: May 2006

One hundred seventy-seven isolates of serotype b Haemophilus influenzae recovered largely from children with invasive disease in the United States were characterized by the electrophoretic mobilities of 16 metabolic enzymes, the NaDodSO4/PAGE pattern of outer-membrane proteins (OMP), and biotype. Thirty-two distinctive multilocus genotypes (electrophoretic types, ETs) were distinguished on the basis of allele profiles at the enzyme loci. Twenty-eight OMP types and five biotypes were identified, but only 55 distinctive combinations of ET, OMP type, and biotype were represented. The strong nonrandom associations of characters and the recovery of isolates with identical properties in widely separated geographic regions and over a 40-year period suggest that the population structure of H. influenzae is basically clonal. Examination of nonserotype b isolates indicated that clones of serotype b are a restricted subset of the genotypes in the species as a whole. Currently, most of the invasive H. influenzae disease in the United States is caused by serotype b strains of two related ETs, and, more specifically, much of it is attributable to two subclones marked by OMP type. There is evidence that the frequency of the ET-1/OMP 1H/biotype I subclone has increased dramatically in the United States since the 1939-1954 period. The hypothesis that populations of H. influenzae are subject to marked temporal variation in clonal composition is supported by evidence of major differences in the genetic structure of populations in the United States and the Netherlands.