bluuebunny/crossref_metadata_2025 huggingface dataset:
Chloroplast DNA evidence for the origin of the genus
Heterogaura
from a species of
Clarkia
(Onagraceae)
Authors: Sytsma Kenneth J. , Gottlieb Leslie D.
Published: May 2006

Restriction-site variation in chloroplast DNA was examined in the morphologically distinct and monotypic genus
Heterogaura
and the related speciose genus
Clarkia
(Onagraceae), both native to California. Of the 605 restriction sites surveyed, a total of 119 mutations were identified. Of these, 55 were shared by at least two species and were used to construct a most parsimonious phylogenetic tree. This analysis, as well as one based on a distance metric, provided evidence that
Heterogaura
and
Clarkia dudleyana
, a member of a phylogenetically advanced section, share a more recent common ancestor than either does with any other species. The two species are more closely related than nearly all paris of
Clarkia
tested. The origin of
Heterogaura
from within another genus raises important questions about the adequacy of morphological data and suggests that the relationships of other well-known monotypic plant genera should be reinvestigated.
A partial genomic DNA clone for the alpha subunit of the mouse complement receptor type 3 and cellular adhesion molecule Mac-1.
Authors: Sastre L , Roman J M , Teplow D B , Dreyer W J , Gee C E , Larson R S , Roberts T M , Springer T A
Published: May 2006

A genomic clone coding for the alpha subunit of the mouse complement receptor type 3 and the cellular adhesion molecule Mac-1 has been isolated directly from a genomic library using synthetic oligonucleotide probes based on the amino-terminal amino acid sequence of the protein. The identity of the clone has been established by DNA sequencing and in vitro translation of hybrid-selected mRNA. The gene is present in a single copy in the murine genome. The region containing the amino-terminal exon has been sequenced. RNA gel blotting shows that the Mac-1 alpha-subunit mRNA is 6 kilobases in length. Mac-1 alpha-subunit mRNA is present in macrophages but not T lymphoma or L cells. During gamma interferon-stimulated maturation of the mouse premyelocytic cell line M1, Mac-1 alpha-subunit mRNA is induced. This corresponds with the tissue distribution of the Mac-1 alpha subunit, showing expression is regulated at least partially at the message level.
T-cell and mast cell lines respond to B-cell stimulatory factor 1.
Authors: Mosmann T R , Bond M W , Coffman R L , Ohara J , Paul W E
Published: May 2006

The murine lymphokine B-cell stimulatory factor 1 (BSF-1) has been described previously in terms of its action on B lymphocytes. We now provide evidence that BSF-1 is also responsible for two additional biological activities. The first of these is the stimulation or maintenance of a state of activation in mouse T-cell lines. The second activity is the increase in the proliferative rate of certain mast cell lines costimulated with interleukin 3. The T-cell and mast cell activities are mediated by purified BSF-1 and copurify with BSF-1 from supernatants of certain T-cell lines. Each of these activities is inhibited by monoclonal anti-BSF-1 but not by monoclonal anti-interleukin 2 antibody. The antibody inhibition results also indicate that BSF-1 is the major or only source of these two activities in the activated T-cell supernatants that we have tested.
Hemostatic mechanisms, independent of platelet aggregation, arrest gastric mucosal bleeding.
Authors: Whittle B J , Kauffman G L , Moncada S
Published: May 2006

Platelet adhesion, aggregation, and subsequent plug formation play a major role in the control of cutaneous and vascular hemostasis. Little is known, however, about the hemostatic processes in gastric mucosal tissue. A method for evaluating bleeding from a standard incision in the gastric mucosa of the rat, rabbit, and dog has therefore been developed. By using pharmacological agents that interfere with platelet aggregation and blood coagulation, the mechanism of gastric hemostasis has been compared to that in the vasculature, using the rat mesenteric artery. Intravenous infusion of prostacyclin (0.5 micrograms X kg-1 X min-1), which inhibits platelet aggregation directly, or administration of the thromboxane synthase inhibitor 1-benzylimidazole (50 mg X kg-1) significantly prolonged bleeding in the mesenteric artery yet failed to alter gastric mucosal bleeding. In contrast, a low dose of heparin (100 units X kg-1), which interferes with the clotting process, had no effect on mesenteric bleeding but substantially prolonged bleeding from the gastric mucosa. These findings suggest that, unlike in the skin or vasculature, platelet aggregation plays a minimal role in the initial hemostatic events in the gastric mucosa and that the arrest of gastric hemorrhage is brought about largely by processes primarily involving the coagulation system.
Mechanism of DNA polymerase I: exonuclease/polymerase activity switch and DNA sequence dependence of pyrophosphorolysis and misincorporation reactions.
Authors: Mizrahi V , Benkovic P , Benkovic S J
Published: May 2006

Mechanistic features of several processes involved in the idling-turnover reaction catalyzed by the large (Klenow) fragment of Escherichia coli DNA polymerase I have been established. The exonuclease----polymerase activity switch involved in the excision/incorporation mode of idling-turnover occurs without an intervening dissociation of the enzyme from its DNA substrate. Comparative studies on the pyrophosphorolysis kinetics of related DNA substrates indicate a significant dependence of the reaction rate upon the DNA sequence within the duplex region upstream of the primer-template junction. Finally, a gel electrophoretic analysis of the products of the idling-turnover reaction has provided direct evidence for an alternative DNA sequence-dependent misincorporation/excision pathway.
Leishmania and Trypanosoma surface glycoproteins have a common glycophospholipid membrane anchor.
Authors: Bordier C , Etges R J , Ward J , Turner M J , Cardoso de Almeida M L
Published: May 2006

The variant surface glycoprotein (VSG) of the African trypanosomes is the major membrane protein of the plasma membrane of the bloodstream stage of the parasite. It is anchored in the plasma membrane by a glycolipid covalently bound to the C-terminal amino acid of the protein. The VSG is released through the action of a phosphatidylinositol-specific phospholipase C that removes dimyristoylglycerol and exposes the carbohydrate antigenic determinant common to all VSGs. Promastigotes of Leishmania have a predominant surface glycoprotein, termed p63, that is anchored in the plasma membrane in a similar way. A water-soluble form of p63 can be generated through the action of phosphatidylinositol-specific phospholipase C from trypanosomes or from Bacillus cereus. Either treatment exposes on the Leishmania p63 an antigenic determinant recognized by antibody prepared against the trypanosomal crossreacting determinant. These findings indicate that p63 and VSG have a common membrane anchor and are structurally related.
Co-clustering of denatured hemoglobin with band 3: its role in binding of autoantibodies against band 3 to abnormal and aged erythrocytes.
Authors: Schlüter K , Drenckhahn D
Published: May 2006

Precipitates of hemoglobin, termed Heinz bodies, occur in a fraction of erythrocytes after removal of the spleen and are also observed in aged erythrocytes. This implies that precipitates of hemoglobin might play a particular role in senescent cell recognition. By using immunofluorescence microscopy, evidence is presented in splenectomized patients and in several patients with unstable (mutant) hemoglobins that membrane-attached Heinz bodies are associated with both clusters of the anion channel, band 3, and clusters of surface-bound immunoglobulins (IgG). In 75% of the cases of unstable hemoglobin, such as sickle cell anemia or hemoglobin Köln disease, the level of cell-bound IgG (measured by 125I-labeled staphylococcal protein A) was increased severalfold above the level found in healthy controls. Immunoblot analysis identified the major fraction of cell-bound IgG to be directed to band 3. These observations indicate copolymerization of denatured hemoglobin with the cytoplasmic domain of band 3, which may cause band 3 to form clusters. These clusters probably serve as thermodynamically favored binding sites for autoantibodies in serum, which promote elimination of the erythrocytes by the immune system. Thus, erythrocytes may be removed from circulation when hemoglobin begins to denature and the cells begin to fail in their main function of oxygen transport.
Synthesis of an O-glycosylated cell surface protein induced in yeast by α factor
Authors: Orlean P. , Ammer H. , Watzele M. , Tanner W.
Published: May 2006

A number of cell surface glycoproteins can be specifically and completely released from intact cells of
Saccharomyces cerevisiae
with 0.5% mercaptoethanol. Among these proteins is one with a molecular mass of 22 kDa, which is synthesized only in haploid a cells treated with the peptide mating pheromone α factor. This protein could be radiolabeled
in vivo
with [2-
3
H]mannose, [
14
C]phenylalanine, and [
35
S]sulfate. Its synthesis and export to the cell surface were not inhibited by tunicamycin. β-Elimination released almost all radioactivity from the [2-
3
H]mannose-labeled protein, 36% of its radioactivity being recovered subsequently as mannose and 43% as a dimannoside. Evidence is presented that the 22-kDa O-glycosylated protein is a mating-type specific a cell agglutinin.
2'-Phosphoadenylylation of eukaryotic proteins: a type of covalent modification.
Authors: Hilz H , Fanick W , Klapproth K
Published: May 2006

An enzymatic system in rat liver microsomal preparations has been detected that catalyzes the transfer of the 2'-phospho-AMP moiety from NADP to endogenous polypeptides; the major acceptor is a polypeptide of about 40 kDa (p40). Modification of the acceptor by 2'-phospho-AMP residues was deduced from the simultaneous transfer of 2'-[33P]phosphate and [3H]adenine residues from double-labeled NADP, while no incorporation of radioactivity into p40 was seen with NADP species labeled in the NMN moiety. The true substrate of this phosphoadenylylation reaction was 2'-phospho-ADP-ribose rather than NADP, because labeled phospho-ADP-ribose was as efficient as or more efficient than NADP in forming modified p40. Also, NADP was rapidly converted to phospho-ADP-ribose during incubation with microsomes. Furthermore, isonicotinic acid hydrazide, an inhibitor of NADP glycohydrolase, prevented phosphoadenylylation from NADP, but not from phospho-ADP-ribose, and glycohydrolase-resistant NADPH could not substitute for NADP. Transferase activity was found in liver and brain microsomes and, to a smaller extent, in the cytosol fractions. In Ehrlich ascites tumor cells, most of the activity resided in the cytosol, from which it could be partially purified. The apparent Km for phospho-ADP-ribose was about 2 X 10(-4) M, and the pH optimum was around 7. Divalent cations like Mg2+ and Mn2+ inhibited the reaction. In all compartmental preparations, activity was eliminated by heating or short treatment with alkali or acid. In submitochondrial particles from rat liver, a system with different characteristics led to the phosphoadenylylation of several endogenous polypeptides.
Molecular mechanical studies of d(CGTACG)2: complex of triostin A with the middle A - T base pairs in either Hoogsteen or Watson-Crick pairing.
Authors: Singh U C , Pattabiraman N , Langridge R , Kollman P A
Published: May 2006

Computer graphics model building and molecular mechanical calculations have been carried out on d(CGTACG)2 and its bis-intercalation complexes with triostin A and an N-Me-Ala analogue of triostin A. Two conformations of the DNA have been considered both for the uncomplexed and for complexed nucleic acid: in one the central A - T base pairs are Watson-Crick base paired; in the other they are Hoogsteen base paired. The calculations offer a clear explanation why Hoogsteen base pairing is not favorable in isolated A + T-rich DNA and also suggest reasons why the bis-intercalation of triostin A might help stabilize the neighboring A - T base pairs into a Hoogsteen form. To our knowledge, this is the first study to use molecular mechanical and dynamical methods to investigate the bis-intercalator-DNA complex.
Human erythropoietin gene: high level expression in stably transfected mammalian cells and chromosome localization.
Authors: Powell J S , Berkner K L , Lebo R V , Adamson J W
Published: May 2006

The glycoprotein hormone erythropoietin plays a major role in regulating erythropoiesis and deficiencies of erythropoietin result in anemia. Detailed studies of the hormone and attempts at replacement therapy have been difficult due to the scarcity of purified material. We used a cloned human erythropoietin gene to develop stably transfected mammalian cell lines that secrete large amounts of the hormone with potent biological activity. These cell lines were produced by cotransfection of mammalian cells with a plasmid containing a selectable marker and plasmid constructions containing a cloned human erythropoietin gene inserted next to a strong promoter. The protein secreted by these cells stimulated the proliferation and differentiation of erythroid progenitor cells and, with increased selection, several of these cell lines secrete up to 80 mg of the protein per liter of supernatant. Hybridization analysis of DNA from human chromosomes isolated by high resolution dual laser sorting provides evidence that the gene for human erythropoietin is located on human chromosome 7.
Interferon enhances tryptophan metabolism by inducing pulmonary indoleamine 2,3-dioxygenase: its possible occurrence in cancer patients.
Authors: Yasui H , Takai K , Yoshida R , Hayaishi O
Published: May 2006

Human lungs bearing cancer (n = 27) exhibited up to an approximately 20-fold [on average approximately 5-fold (P less than 0.005)] increase in the enzyme activity that degrades tryptophan to form formylkynurenine, in comparison with lungs with benign lesions (blebs) (n = 7) taken as controls. On the basis of molecular and kinetic properties, this activity was ascribed to indoleamine 2,3-dioxygenase (IDO) [indoleamine:oxygen 2,3-oxidoreductase (decyclizing)]. In vitro studies with human lung slices revealed that human interferon gamma (IFN-gamma) induced the de novo synthesis of IDO dose dependently (10-10(4) units/ml), and at maximum the activity reached nearly 100 times that in the control lungs described above. Human IFN-alpha also served as an inducer, but it was two to three orders of magnitude less potent than IFN-gamma relative to the antiviral titers, suggesting that IFN-gamma is the main mediator of the IDO induction. IDO thus induced in slices avidly metabolized tryptophan in situ: Upon a 24-hr incubation of lung slices pretreated with varied doses of IFN-gamma (10-10(3) units/ml), up to 96% of the tryptophan in the slices was depleted and up to 70% of the tryptophan in the medium was converted, mainly to formylkynurenine, kynurenine, or both. The foregoing results suggest that an IFN-mediated induction of IDO also takes place in vivo in human lungs as a response to cancer, leading to metabolic consequences such as depletion of tryptophan and accumulation of (formyl)kynurenine, which may provide a unique host defense mechanism.
RNA polymerase specificity of mRNA production and enhancer action.
Authors: Lopata M A , Cleveland D W , Sollner-Webb B
Published: May 2006

To examine the RNA polymerase (EC 2.7.7.6) specificity of RNA maturation/utilization and transcriptional enhancement, we constructed a chimeric plasmid (pPolI-CAT) in which a promoter for mouse rRNA gene transcription was placed adjacent the coding sequences for chloramphenicol acetyltransferase (CAT; EC 2.3.1.28). A number of other constructs, including plasmids also containing a murine sarcoma virus enhancer or lacking any natural eukaryotic promoter sequences, were also prepared. In apparent agreement with earlier conclusions that an RNA polymerase I transcript can act as a messenger RNA, transient transfection of mouse L cells with pPolI-CAT yielded both high levels of transcription from the RNA polymerase I promoter and enzymatically active CAT protein. However, further examination revealed that CAT protein is not translated from RNA that begins at the normal rRNA transcription initiation site. Polysomal RNA is devoid of such RNA and instead consists of CAT-encoding transcripts that begin elsewhere in the mouse ribosomal DNA (rDNA) region. Since transcription of these aberrant RNAs is stimulated by the addition of a murine sarcoma virus enhancer segment, they are probably transcribed by RNA polymerase II. Transcripts that map to the authentic rRNA start site are not similarly enhanced. Moreover, unlike the RNAs deriving from the rRNA initiation site, these aberrant RNAs are more stable and the level of translatable CAT transcripts is suppressed by inclusion of larger segments of the rDNA promoter regions. Fortuitously initiated mRNAs are also formed in the absence of any natural eukaryotic promoter sequence. From these data we conclude that there is no evidence that normal RNA polymerase I transcription yields functional mRNA and that transcriptional enhancement appears to be RNA polymerase specific.
Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor.
Authors: Ny T , Sawdey M , Lawrence D , Millan J L , Loskutoff D J
Published: May 2006

A lambda gt11 expression library containing cDNA inserts prepared from human placental mRNA was screened immunologically using an antibody probe developed against the beta-migrating plasminogen activator inhibitor (beta-PAI) purified from cultured bovine aortic endothelial cells. Thirty-four positive clones were isolated after screening 7 X 10(5) phages. Three clones (lambda 1.2, lambda 3, and lambda 9.2) were randomly picked and further characterized. These contained inserts 1.9, 3.0, and 1.9 kilobases (kb) long, respectively. Escherichia coli lysogenic for lambda 9.2, but not for lambda gt11, produced a fusion protein of 180 kDa that was recognized by affinity-purified antibodies against the bovine aortic endothelial cell beta-PAI and had beta-PAI activity when analyzed by reverse fibrin autography. The largest cDNA insert was sequenced and shown to be 2944 base pairs (bp) long. It has a large 3' untranslated region [1788 bp, excluding the poly(A) tail] and contains the entire coding region of the mature protein but lacks the initiation codon and part of the signal peptide coding region at the 5' terminus. The two clones carrying the 1.9-kb cDNA inserts were partially sequenced and shown to be identical to the 3.0-kb cDNA except that they were truncated, lacking much of the 3' untranslated region. Blot hybridization analysis of electrophoretically fractionated RNA from the human fibrosarcoma cell line HT-1080 was performed using the 3.0-kb cDNA as hybridization probe. Two distinct transcripts, 2.2 and 3.0 kb, were detected, suggesting that the 1.9-kb cDNA may have been copied from the shorter RNA transcript. The amino acid sequence deduced from the cDNA was aligned with the NH2-terminal sequence of the human beta-PAI. Based on this alignment, the mature human beta-PAI is 379 amino acids long and contains an NH2-terminal valine. The deduced amino acid sequence has extensive (30%) homology with alpha 1-antitrypsin and antithrombin III, indicating that the beta-PAI is a member of the serine proteinase inhibitor (serpin) superfamily.
Growth hormone-releasing factor stimulates proliferation of somatotrophs in vitro.
Authors: Billestrup N , Swanson L W , Vale W
Published: May 2006

The mitogenic effect of the hypothalamic peptides growth hormone-releasing factor (GRF) and somatostatin on cultured growth hormone (GH)-producing cells (somatotrophs) was studied. Using autoradiographic detection of [3H]thymidine uptake and immunocytochemical identification of GH-producing cells, we show that 5 nM GRF causes a 20-fold increase in the percentage of somatotrophs labeled with [3H]thymidine. The total number of somatotrophs in GRF-treated cultures was increased by 60%. Somatostatin had no measurable effect on the labeling index by itself, but it partly inhibited the GRF-induced increase in both the labeling index and the total number of cells. Forskolin caused an increase in both the percentage of somatotrophs with a [3H]thymidine-labeled nucleus and the somatotroph number similar to that caused by GRF. GH secretion as well as cellular GH content in the GRF- or forskolin-treated cells increased with culture time over the entire period, whereas secretion and content of GH gradually decreased in control or somatostatin-treated cultures during the entire culture period. These data suggest that GRF and somatostatin regulate the mitotic activity of GH-producing cells and that the effect of GRF is possibly mediated by cyclic AMP.
High rate of somatic point mutation in vitro in and near the variable-region segment of an immunoglobulin heavy chain gene.
Authors: Meyer J , Jäck H M , Ellis N , Wabl M
Published: May 2006

The "silent" allele at the immunoglobulin heavy-chain locus in the pre-B-lymphocyte line 18-81 contains a correctly assembled gene. However, an amber termination codon within the variable-region gene segment prematurely terminates translation into complete heavy chain. Revertants that do produce heavy chain are generated at a high rate, which is termed hypermutation. By DNA sequencing of subclones, we have confirmed that whenever mu chain is produced by the usually silent allele, a true reversion is found in the DNA. Mutations are not confined to the position of the amber termination codon but are also found at other sites in and near the variable-region gene segment.
Expression of T3 in association with a molecule distinct from the T-cell antigen receptor heterodimer.
Authors: Weiss A , Newton M , Crommie D
Published: May 2006

The T-cell antigen receptor consists of a disulfide-linked heterodimer (Ti) that is associated with another set of three nonpolymorphic, noncovalently linked peptides termed "T3." The cell surface expression of T3 has been thought to depend upon association with Ti. In this study, we demonstrate that T3 can be expressed in the absence of an associated Ti molecule on a T-cell leukemic line, PEER. Instead, on this cell line, T3 appears to be expressed in association with a 55- to 60-kDa glycoprotein that has a peptide backbone of 29 kDa. PEER fails to express Ti alpha-chain transcripts but does express Ti beta- and gamma-chain transcripts. Using a monoclonal antibody that reacts with nonpolymorphic epitopes expressed on Ti, WT31, we demonstrate that PEER fails to react with this antibody but does react with three independently derived anti-T3 antibodies. Moreover, a small subpopulation of T3-positive peripheral blood lymphocytes, like PEER, fails to express the antigenic determinants recognized by WT31. These results suggest that, on these normal lymphocytes, T3 may likewise be associated with a non-Ti molecule. The possibility that the 55- to 60-kDa molecule expressed on PEER, termed "Tp55-60," represents the protein product of the previously identified Ti gamma-chain gene is discussed.
Conservation of amino acid sequence of VP8 and cleavage region of 84-kDa outer capsid protein among rotaviruses recovered from asymptomatic neonatal infection.
Authors: Gorziglia M , Hoshino Y , Buckler-White A , Blumentals I , Glass R , Flores J , Kapikian A Z , Chanock R M
Published: May 2006

Within the past few years, rotavirus strains were recovered from four discrete prolonged outbreaks of infection in newborn nurseries in which affected infants failed to develop significant symptoms. The virus strains recovered from each outbreak belonged to a different human rotavirus serotype and thus each of the four human rotavirus serotypes was associated with asymptomatic infection of neonates. Marked conservation of sequence was observed among the fourth genes of the nursery rotavirus strains in a previous study using RNA X RNA hybridization, while a different conserved set of fourth gene sequences was identified among virulent human rotaviruses representing the four known serotypes. In the present study, this sequence dimorphism was further evaluated by comparing the sequence of the region of the fourth gene of virulent and asymptomatic human rotaviruses that codes for the VP8 protein, downstream cleavage sites, and the NH2 terminus of VP5. The corresponding sequences of a simian rotavirus were also determined. The fourth segment (+) strand RNA has a 5' conserved nontranslated sequence of nine nucleotides and encodes a VP8 protein of 240 amino acids in human rotavirus strains and 241 amino acids in simian rotavirus strains. Human and simian rotaviruses exhibit many similarities in this region of their genome, including identical NH2-terminal amino acid sequences, conservation of arginine at the two trypsin cleavage sites, and the position of a cysteine residue. Alignment of amino acid sequences of the VP8 protein, the downstream cleavage region, and the NH2 terminus of VP5 of asymptomatic and virulent human rotavirus strains indicates a high degree of homology (96% or more) among the asymptomatic viruses (serotypes 1, 2, 3, and 4), while homology between asymptomatic strains and virulent viruses is considerably less (68-72%). A high degree of conservation of amino acid sequence (92-97%) is also observed among three of the virulent strains (serotypes 1, 3, and 4). At 48 positions in the protein sequence of VP8, the cleavage region, and the NH2 terminus of VP5, an amino acid is conserved among asymptomatic rotaviruses, while a different amino acid is conserved among virulent rotaviruses. Notably, three of these differences are located within the cleavage region between VP8 and VP5. These findings suggest that the fourth genes of virulent and asymptomatic human rotavirus strains represent two lines of divergent evolution from a common ancestor. Also, it is possible that this sequence dimorphism may be responsible in part for the difference in virulence between these two groups of human rotaviruses.
Glycoconjugates as noninvasive probes of intrahepatic metabolism: pathways of glucose entry into compartmentalized hepatic UDP-glucose pools during glycogen accumulation.
Authors: Hellerstein M K , Greenblatt D J , Munro H N
Published: May 2006

Recent studies have questioned the efficiency with which administered glucose generates hepatic glycogen through the direct nonrecycling route compared with the indirect route from glucose recycled through glycolysis followed by gluconeogenesis. Using fasted and refed rats, we examined the relative access of infused [1-3H]- and [U-14C]glucose by way of these two pathways to liver glycogen and to hepatic glucuronic acid, the latter recovered from the urine as the glucuronide conjugated with administered acetaminophen. In fasted animals and during early refeeding, extensive dilution of administered [3H]- and [14C]glucose recovered in glycogen showed that 60-70% of the labeled glucose had undergone recycling by the indirect route. As refeeding progressed with substantial glycogen deposition, the contribution of the recycling pathway to glycogen and glucuronic acid diminished considerably. Thus, there is a shift in pathways of hepatic glucose utilization as liver glycogen accumulates. Consequently, the ratio of 3H/14C in glucuronic acid was closely correlated with the glycogen content of the liver at sacrifice, indicating that this ratio may prove useful as a noninvasive indicator of liver glycogen concentration. Since glycogen and glucuronic acid are derived by single reactions from UDP-glucose, they should show a common labeling pattern with 3H and 14C under various nutritional conditions. However, detailed analysis of their labeling patterns showed a striking divergence, implying that there must be compartmentation of the UDP-glucose pools leading to each of these end products, either because they are made in separate compartments within the same cell or because each is made in different hepatocyte populations. We favor the former explanation because galactose secreted in glycoproteins shows 3H and 14C labeling patterns similar to those of glucuronic acid conjugated with acetaminophen, and both of these conjugations occur in the endoplasmic reticulum of the liver, whereas most glycogen is present in the cytosol.
Isolation of an episomal yeast gene and replication origin as chromatin.
Authors: Pederson D S , Venkatesan M , Thoma F , Simpson R T
Published: May 2006

A multicopy yeast plasmid containing the TRP1 gene (coding for N-5'-phosphoribosylanthranilate isomerase) and ARS1 (autonomously replicating sequence 1) has been purified as chromatin. Electrophoretic analysis of nucleic acid and proteins and electron microscopy show that the plasmid chromatin is largely free of contaminants. Electron-microscopic and linking-number analyses indicate that the plasmid chromatin contains seven nucleosomes, as predicted by the indirect end-label analyses of Thoma, Bergman, and Simpson [J. Mol. Biol. (1984) 177, 715-733]. Indirect end label mapping of micrococcal nuclease cuts demonstrates that nucleosome positions and nuclease-sensitive regions are not altered by the purification. The plasmid chromatin behaves homogeneously with respect to its elution from nuclei, template activity, and intrinsic buoyant density. Taken together, these observations suggest that different copies of the TRP1ARS1 plasmid do not differ from each other grossly in chromatin structure. We discuss the potential for understanding eukaryotic gene regulation offered by the ability to isolate unique genes as chromatin.
Identification and purification of a bacterial ice-nucleation protein.
Authors: Wolber P K , Deininger C A , Southworth M W , Vandekerckhove J , van Montagu M , Warren G J
Published: May 2006

The protein product of a gene (inaZ) responsible for ice nucleation by Pseudomonas syringae S203 has been identified and purified after overexpression in Escherichia coli. The amino acid composition and the N-terminal sequence of the purified, denatured protein corresponded well with that predicted from the sequence of the inaZ gene. The product of inaZ was also found to be the major component in preparations of ice-nucleating, proteinaceous particles, obtained after extraction with and gel filtration in a mixture of urea and the nondenaturing detergent octyl beta-D-thioglucopyranoside. The activity of these preparations in the absence of added lipid implies that the protein participates directly in the nucleation process.
Mechanosensitivity of cultured ciliated cells from the mammalian respiratory tract: implications for the regulation of mucociliary transport.
Authors: Sanderson M J , Dirksen E R
Published: May 2006

Mechanical stimulation of the cell surface or cilia of cultured ciliated epithelial cells derived from the rabbit tracheal mucosa resulted in a transitory ciliary beat frequency increase of 20% or more. This response was composed of a lag, rise, and recovery phase. The duration of the response, but not the maximal frequency, was increased by stronger stimulation. The ability of these ciliated cells to respond to stimulation depended on culture age; generally, cultures younger than 4 days were insensitive. The frequency response was transmitted to adjacent and more distal cells in all directions. The lag phase but not the rise time of the adjacent cell response was extended. These ciliary responses were lost when extracellular Ca2+ was removed. Replacement of Ca2+ resulted in a restoration of mechanosensitivity. In 1 mM verapamil and Ca2+ the frequency response was also lost. These results suggest that the frequency response is dependent on Ca2+ influx (although its intracellular action is unknown) and imply that mucociliary clearance is a localized self-regulating process.
Tumor necrosis factor type alpha stimulates human endothelial cells to produce granulocyte/macrophage colony-stimulating factor.
Authors: Broudy V C , Kaushansky K , Segal G M , Harlan J M , Adamson J W
Published: May 2006

Tumor necrosis factor type alpha (TNF-alpha) is produced by monocytes and has been purified, sequenced, and cloned from the HL-60 cell line. Soluble products of monocytes stimulate endothelial cells to release multilineage hematopoietic colony-stimulating activity. To determine whether TNF-alpha could stimulate endothelial cells to produce these activities, we added recombinant human TNF-alpha to cultured human umbilical vein endothelial cells. Untreated endothelial cell conditioned medium and TNF-alpha-stimulated endothelial cell conditioned medium were tested for hematopoietic colony stimulating activity in colony-forming assays in methylcellulose. TNF-alpha stimulated growth factor production by endothelial cells. Fifth-passage human endothelial cells and multiply-passaged bovine aortic endothelial cells responded similarly to first-passage endothelial cells, indicating that the action of TNF-alpha on endothelial cells is direct and not due to contaminating lymphocytes or monocytes present in the first-passage cultures. To investigate the molecular basis for these findings, polyadenylylated RNA was prepared from the TNF-alpha-stimulated endothelial cells and probed for granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor mRNA. Granulocyte-macrophage colony-stimulating factor, but not granulocyte colony-stimulating factor, message was detected. This finding suggests that at least some of the hematopoietic colony-stimulating activity released by the TNF-alpha-stimulated endothelial cells is granulocyte-macrophage colony-stimulating factor. These results demonstrate that a purified monocyte product can stimulate endothelial cells to produce the multilineage growth factor granulocyte-macrophage colony-stimulating factor and extend the role of this immunoregulatory protein to the regulation of hematopoiesis in vitro.
Analysis by immunocytochemistry and in situ hybridization of renin and its mRNA in kidney, testis, adrenal, and pituitary of the rat.
Authors: Deschepper C F , Mellon S H , Cumin F , Baxter J D , Ganong W F
Published: May 2006

Renin gene expression in cells and tissues of the rat was examined by in situ hybridization histochemistry and immunocytochemistry. By using a mouse cDNA probe, hybridization histochemistry revealed renin mRNA in the renal juxtaglomerular cells, testicular Leydig cells, adrenal zona glomerulosa cells, the intermediate lobe of the pituitary, and scattered cells of the anterior lobe of the pituitary. With four separate antisera to mouse submaxillary renin, there was immunoreactivity in the renal juxtaglomerular cells. However, only one of the antisera stained the Leydig cells, a second stained the adrenal zona glomerulosa, a third stained the intermediate lobe of the pituitary, and a fourth stained scattered cells of the anterior lobe of the pituitary that were identified as gonadotrophs. The variations with the different antisera in detecting extrarenal renin are unexplained but could imply that posttranslational proteolysis or glycosylation of preprorenin varies in different tissues with consequent variations in immunoreactivity. The finding of renin mRNA and renin-like immunoreactivity in these tissues supports the notion that these tissues are sites for production of renin.
Variable stoichiometry of phosphate-linked anion exchange in Streptococcus lactis: implications for the mechanism of sugar phosphate transport by bacteria.
Authors: Ambudkar S V , Sonna L A , Maloney P C
Published: May 2006

Phosphate/2-deoxyglucose 6-phosphate antiport in Streptococcus lactis showed an exchange stoichiometry that varied over a 2-fold range when assay pH was shifted between pH 8.2 and pH 5.2. At pH 7.0 and above, 2 mol of phosphate moved per mol of sugar phosphate; at pH 6.1 the ratio was 1.5:1, while at pH 5.2 the overall stoichiometry fell to 1.1:1. This pattern was not affected by valinomycin in potassium-based media, nor could variable stoichiometry be attributed to altered hydrolysis of the sugar phosphate substrate. In kinetic studies at pH 7.0 or pH 5.2, sugar 6-phosphate was a competitive inhibitor of phosphate transport, indicating operation of a single system. Parallel tests showed that the affinity of antiport for its sugar 6-phosphate substrate was insensitive to pH in this range. Overall, such results suggest a neutral exchange that has specificity for monovalent phosphate but that selects randomly among the available mono- and divalent sugar 6-phosphates. A simple model that shows this behavior suggests a mechanistic role for anion exchange in bacterial transport of sugar phosphate or other organic anions.
Antiketogenic and hypoglycemic effects of aminocarnitine and acylaminocarnitines.
Authors: Jenkins D L , Griffith O W
Published: May 2006

DL-Aminocarnitine (DL-3-amino-4-trimethylaminobutyrate) is a potent, noncovalent inhibitor of carnitine palmitoyltransferase (palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21). Here we show that decanoyl-DL-aminocarnitine and palmitoyl-DL-aminocarnitine inhibit carnitine palmitoyltransferase in vitro about 7-fold and 100-fold more effectively than does aminocarnitine. Aminocarnitine and its decanoyl and palmitoyl derivatives are active in vivo following oral or parenteral administration and, at doses of 0.3 mmol/kg or less, inhibit the oxidation of [14C]palmitate to 14CO2 by 45-70% in mice. Larger doses do not significantly increase the extent of inhibition, a finding suggesting that substantial carnitine palmitoyltransferase-independent long-chain fatty acid oxidation may occur in vivo. Small doses of aminocarnitine and palmitoylaminocarnitine prevent the development of ketoacidemia in fasted, normal mice and reverse the ketoacidemia observed in diabetic mice. Aminocarnitine has a strong hypoglycemic effect in fasted diabetic mice; a single dose (0.3 mmol/kg) normalizes plasma glucose levels within 4-8 hr and remains effective for at least 12 hr.
Cloning and sequence analysis of cDNA encoding urotensin I precursor.
Authors: Ishida I , Ichikawa T , Deguchi T
Published: May 2006

The primary structure of the precursor of urotensin I, a neuropeptide hormone from the caudal neurosecretory system of the carp Cyprinus carpio, has been determined by analyzing the nucleotide sequence of cloned DNA complementary to the mRNA encoding it. The precursor consists of 145 amino acid residues and the carboxyl terminus represents the 41-amino acid sequence of urotensin I, preceded by Lys-Arg and followed by Gly-Lys. Sequence homology as well as similar organization of the precursors of urotensin I and mammalian corticotropin-releasing factors suggest that they are evolutionarily related. RNA transfer blot analysis indicates that mRNA encoding the precursor of urotensin I is present only in the spinal cord and not in the brain, intestine, liver, or kidney of the carp.
Light-scattering study of depolymerization kinetics of sickle hemoglobin polymers inside single erythrocytes.
Authors: Peetermans J , Nishio I , Ohnishi S T , Tanaka T
Published: May 2006

The time course of intracellular depolymerization of hemoglobin S aggregates is directly observed by using microscope laser light-scattering spectroscopy in single sickle erythrocytes upon slow reoxygenation. From the correlation functions of the light intensity scattered from a single cell, we determine the average diffusion coefficient as well as the fraction of hemoglobin aggregates that have intracellular mobility. The oxygen saturation of the hemoglobin of the same cell is measured by single-cell absorption spectrophotometry. Combining the results obtained with these techniques and information on the cellular morphology, we propose a model for the depolymerization process of hemoglobin inside single erythrocytes as they transform from the fully sickled to the normal biconcave shape upon reoxygenation.
Loci affecting flagellar assembly and function map to an unusual linkage group in Chlamydomonas reinhardtii.
Authors: Ramanis Z , Luck D J
Published: May 2006

The uni1 mutant of Chlamydomonas reinhardtii lacks one of the paired flagella seen in wild-type cells. The missing flagellum is cis to the eye spot and at this site the basal body is incomplete, lacking a transition zone. Together with nine other loci affecting flagellar assembly and two loci affecting flagellar function uni1 defines an approximately equal to 100-centimorgan linkage group with a circular map. Measurements of gene-centromere distances on the uni linkage group are consistent with the ordering of loci determined by recombination analysis and place the centromere near pf29, a motility-defective mutant. In well-synchronized meiotic cycles, recombination frequencies between loci on the uni linkage group but not loci on other linkage groups show striking temperature sensitivity during a 1- to 2-hr period 5 days before meiosis.
Histamine and chondroitin sulfate E proteoglycan released by cultured human colonic mucosa: indication for possible presence of E mast cells.
Authors: Eliakim R , Gilead L , Ligumsky M , Okon E , Rachmilewitz D , Razin E
Published: May 2006

An association between the release of histamine and chondroitin sulfate E proteoglycan (PG) was demonstrated in human colonic mucosa (HCM). Colonic biopsy samples incorporated [35S]sulfate (2.7 X 10(6) +/- 188 X 10(3) cpm/mg of wet tissue; mean +/- SEM, n = 5) into PG, which was partially released into the culture medium during the incubation period. Ascending thin-layer chromatography of the released 35S-labeled PG after its digestion by chondroitin ABC lyase (chondroitinase, EC 4.2.2.4) followed by autoradiography yielded three products that migrated in the position of monosulfated disaccharides of N-acetylgalactosamine 4-sulfate and N-acetylgalactosamine 6-sulfate and of an oversulfated disaccharide possessing N-acetylgalactosamine 4,6-disulfate. Cultured colonic mucosa released 23.6 +/- 3.7 ng of histamine per mg of wet tissue (mean +/- SEM, n = 16) without any specific trigger. Comparison by linear regression analysis of the release of histamine and chondroitin [35S]sulfate E PG revealed a correlation coefficient (r) of 0.7 (n = 16; P less than 0.005). Histological examination of the colonic biopsies revealed the presence of many mast cells in various degrees of degranulation in the mucosa and submucosa, most of which were found in the submucosa. Incubation of the HCM biopsies in the presence of anti-human IgE revealed 58% +/- 12% (mean +/- SEM, n = 3) enhancement in the release of chondroitin [35S]sulfate E PG and 64% +/- 10% (mean +/- SEM, n = 4) of histamine release. The above correlation, the observation that most of the mast cells showed various degrees of degranulation, and the lack of heparin synthesis as opposed to the synthesis and immunological release of chondroitin sulfate E strongly suggest that the E mast cell exists in the human colon.
Tightly bound nuclear progesterone receptor is not phosphorylated in primary chick oviduct cultures.
Authors: Garcia T , Jung-Testas I , Baulieu E E
Published: May 2006

Oviduct cells from estradiol-treated chicks were grown in primary culture. After 3-5 days of culture in medium containing estradiol, 90% of the cellular progesterone binding sites were detected in the cytosol. After exposure to [3H]progesterone at 37 degrees C, 80% of the progesterone binding sites were found in nuclear fractions. Progesterone receptor phosphorylation was assessed after incubating the cells with [32P]orthophosphate. Receptor components were immunoprecipitated with a specific polyclonal antibody (IgG-G3) and analyzed by NaDodSO4/PAGE and autoradiography. In the cytosol, constant amounts of 32P-labeled 110-kDa subunit (the B subunit, one of the progesterone-binding components of the receptor) and of the non-steroid-binding heat shock protein hsp90 were found, whether cells had been exposed to progesterone or not. No 32P-labeled 79-kDa subunit (the A subunit, another progesterone-binding subunit) was detected. Various procedures were used to solubilize nuclear progesterone receptor (0.5 M KCl, micrococcal nuclease, NaDodSO4), and in no case was 32P-labeled B subunit detected in the extracts. However, nonradioactive B subunit was detected by immunoblot in a nuclear KCl extract of progesterone-treated cells. These results suggest that the fraction of the B subunit that becomes strongly attached to nuclear structures is not phosphorylated upon exposure of cells to progesterone.
On the spin trapping and ESR detection of oxygen-derived radicals generated inside cells.
Authors: Samuni A , Carmichael A J , Russo A , Mitchell J B , Riesz P
Published: May 2006

Recently several attempts to identify oxygen-derived radicals in whole cells by spin trapping and electron spin resonance have been reported by using 5,5-dimethyl-1-pyrroline-N-oxide as the spin trap. In the present study, the feasibility of this method is examined. Chinese hamster V79 cells and human erythrocytes served as the test systems, while OH radicals were generated by gamma radiolysis. Several spin traps were used to scavange the radicals and a distinction between exo- and endocellular ESR observable species was achieved using tri(oxalato) chromiate(III) as a line broadening agent. To distinguish between exo- and endocellular sites of radical formation, we studied the effects of high molecular weight scavengers (polyethylene glycols), which do not enter the cell. Various possible obstacles associated with trapping and detecting the radicals inside the cells were examined. The results indicate that the primary radicals react with the spin traps. However, these spin adducts decayed within the cells. Cellularly induced decay of 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl radical presented the major difficulty in detecting the endogenous radicals, and potential experimental approaches to overcome this difficulty are discussed.
Mismatch correction catalyzed by cell-free extracts of Saccharomyces cerevisiae.
Authors: Muster-Nassal C , Kolodner R
Published: May 2006

Heteroduplex DNA substrates containing a 4- or 7-base-pair insertion/deletion mismatch or each of the eight possible single-base-pair mismatches were constructed. Extracts of mitotic Saccharomyces cerevisiae cells catalyzed the correction of mismatched nucleotides in a reaction that required Mg2+ and had a partial requirement for ATP and the four dNTPs. The insertion/deletion mismatches and the A X C and G X T mismatches were repaired efficiently, while the six other single-base-pair mismatches were repaired poorly or at undetectable rates. Mismatch correction was accompanied by the specific incorporation of less than 20 nucleotides at or near the site of the repaired mismatch.
all-trans-Retinal stimulates superoxide release and phospholipase C activity in neutrophils without significantly blocking protein kinase C.
Authors: Lochner J E , Badwey J A , Horn W , Karnovsky M L
Published: May 2006

all-trans-Retinal was previously shown to stimulate high levels of superoxide release by guinea pig neutrophils. When the cells, previously labeled with [3H]inositol, are treated with all-trans-retinal, they exhibit a decrease in the levels of [3H]inositol phospholipids and an increase in the accumulation of [3H]inositol phosphates. The maximal accumulation of inositol phosphates and the optimal rate of superoxide release occurred together at approximately 7 min after stimulation. The levels of [3H]inositol phosphates accumulated were comparable to those observed when the cells were stimulated with a chemotactic peptide. In direct measurements, using concentrations that stimulate intact cells maximally, all-trans-retinal was found not to inhibit protein kinase C from the cytosol of neutrophils significantly. This contrasts with the situation with this kinase obtained from other sources. These observations represent additional effects of vitamin A on cells.