bluuebunny/crossref_metadata_2025 huggingface dataset:
Decrease in plasminogen activator correlates with synapse elimination during neonatal development of mouse skeletal muscle.
Authors: Hantaï D , Rao J S , Kahler C , Festoff B W
Published: May 2006

Previous studies have implicated proteases, acting extracellularly, in the mechanism of polyneuronal synapse elimination. Most studies have focused on mammalian, especially rodent, skeletal muscle, where retraction of subordinate nerve terminals occurs during a narrow time window 2-3 weeks after birth. To date no specific protease(s) has been detected that (i) coincides in time with maximal synapse elimination and (ii) is known to act extracellularly on specific extracellular matrix proteins. In previous studies of denervation in adult mouse muscle, rapid activation of urokinase-type plasminogen activator, a neutral serine protease, was detected. This enzyme, by activation of plasminogen to plasmin, specifically degrades matrix components such as fibronectin, type IV collagen, and laminin in muscle. We now present evidence for an initial increase and subsequent decrease in soluble urokinase-type PA--and, to a lesser extent, tissue PA--in developing muscle, suggesting postnatal developmental regulation of these enzymes during the period of maximal synapse elimination. Although considerably higher in specific activity, membrane-bound PA activity followed the wave of synapse elimination, possibly indicating a longer half-life of membrane-bound enzyme(s).
In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase.
Authors: Wild J R , Loughrey-Chen S J , Corder T S
Published: May 2006

The allosteric control of aspartate transcarbamoylase (ATCase, EC 2.1.3.2) of Escherichia coli involves feedback inhibition by both CTP and UTP rather than just CTP alone. It has been known that CTP functions as a heterotropic inhibitor of catalysis; however, the inhibition by CTP alone is incomplete (50-70% at various aspartate concentrations) and there is only a partial occupancy of the allosteric binding sites by CTP at saturating concentrations. The logic of these allosteric characteristics can now be understood in that UTP is a synergistic inhibitor of ATCase in the presence of CTP even though UTP has no independent effect at pH 7.0. When saturating concentrations of CTP are present, the concentration of substrate required for half-maximal activity (S0.5) of the native holoenzyme for aspartate increases from 5 to 11 mM. When CTP and UTP are both present, the aspartate requirement increases further (S0.5 = 17 mM). At aspartate concentrations less than 5 mM, the heterotropic inhibition of ATCase is 90-95% in the presence of both pyrimidine nucleotides. UTP does enhance the binding of CTP to the holoenzyme but the number of tight binding sites does not change (n = 3). The binding of UTP is stabilized in the presence of CTP although its binding characteristics are not as strong as those of CTP. The recent crystallographic studies of Kim et al. [Kim, H.K., Pan, Z., Honzatko, R.B., Ke, H.M. & Lipscomb, W.N. (1987) J. Mol. Biol. 196, 853-875] have described a structural asymmetry across the molecular two-fold axis that is consistent with these CTP/UTP interactions. The synergistic inhibition of ATCase by both CTP and UTP provides a satisfying logic for ensuring a balance of endogenous pyrimidine nucleotide pools.
Comparison of seismic and hydrodynamic yield determinations for the Soviet joint verification experiment of 1988
Authors: Sykes Lynn R. , Ekström Göran
Published: May 2006

Seismic magnitudes determined from surface and body waves for the Soviet underground nuclear explosion of September 14, 1988, are used to calculate the yield of that event from previously derived calibration curves. The yield obtained by combining the two seismic estimates is 113 kilotons, which is very close to those obtained by hydrodynamic measurements made on-site. This comparison substantiates previous conclusions about the sizes of past Soviet weapons tests and compliance with the Threshold Test Ban Treaty. The factor of uncertainty in the combined seismic yield is 1.28 at the 68% and 1.62 at the 95% confidence levels, demonstrating that accuracies considerably better than a factor of 2 can be obtained by combining seismic determinations of yield.
Plasmenylethanolamine is the major storage depot for arachidonic acid in rabbit vascular smooth muscle and is rapidly hydrolyzed after angiotensin II stimulation.
Authors: Ford D A , Gross R W
Published: May 2006

The present study demonstrates that rabbit aortic intimal smooth muscle cells contain the majority of their endogenous arachidonic acid mass in plasmenylethanolamine molecular species. To demonstrate the potential significance of these plasmenylethanolamines as substrates for the smooth muscle cell phospholipases that are activated during agonist stimulation, aortic rings were prelabeled with [3H]arachidonic acid and stimulated with angiotensin II. Although the specific activities of the choline and inositol glycerophospholipid pools were similar after the labeling interval, ethanolamine glycerophospholipids had a specific activity of only 20% of the specific activity of choline and inositol glycerophospholipids. Despite the marked disparity in the specific activities of these three phospholipid classes, angiotensin II stimulation resulted in similar fractional losses (35-41%) of [3H]arachidonic acid from vascular smooth muscle choline, ethanolamine, and inositol glycerophospholipid classes. Reverse-phase HPLC demonstrated that greater than 60% of the [3H]arachidonic acid released from ethanolamine glycerophospholipids after angiotensin II stimulation originated from plasmenylethanolamine molecular species. Taken together, the results demonstrate that the major phospholipid storage depot for arachidonic acid in vascular smooth muscle cells are plasmenylethanolamine molecular species which are important substrates for the phospholipase(s) that are activated during agonist stimulation.
Trans-activation by thyroid hormone receptors: functional parallels with steroid hormone receptors.
Authors: Thompson C C , Evans R M
Published: May 2006

The effects of thyroid hormones are mediated through nuclear receptor proteins that modulate the transcription of specific genes in target cells. We previously isolated cDNAs encoding two different mammalian thyroid hormone receptors, one from human placenta (hTR beta) and the other from rat brain (rTR alpha), and showed that their in vitro translation products bind thyroid hormones with the characteritistic affinities of the native thyroid hormone receptor. We now demonstrate that both of the cloned receptors activate transcription from a thyroid hormone-responsive promoter in a hormone-dependent manner, with rTR alpha eliciting a greater response than hTR beta. The putative functional domains of the thyroid hormone receptors were examined by creating chimeric thyroid hormone/glucocorticoid receptors, producing receptors with hybrid functional properties. These experiments support the proposal that the thyroid hormone receptors are composed of interchangeable functional domains, and indicate that the mechanism of hormone-inducible gene regulation has been conserved in steroid and thyroid hormone receptors.
Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector.
Authors: Hantzopoulos P A , Sullenger B A , Ungers G , Gilboa E
Published: May 2006

This study describes a type of retroviral vector called double-copy (DC) vector that was designed to improve the expression of transduced genes. The unique feature of DC vectors is that the transduced gene is inserted within the U3 region of the 3' long terminal repeat (LTR). Consequently, in the infected cell the gene is duplicated and transferred to the 5' LTR. The important result is that in its new position the gene is placed outside the retroviral transcriptional unit, eliminating or at least reducing the negative effects of the retroviral transcriptional unit. The utility of the DC vector design was tested by using a 2.1-kilobase-pair (kbp)-long adenosine deaminase (ADA; EC 3.5.4.4) minigene that was inserted into the 3' LTR of the N2 retroviral vector, generating a 2.7-kbp-long chimeric LTR. DNA blot analysis was used to show that the chimeric LTR was faithfully duplicated in cells infected with the corresponding virus, generating two copies of the ADA minigene, one copy in each LTR. Insertion of the ADA minigene into the 3' LTR of the N2 vector led to a 10- to 20-fold increase in ADA transcripts and human ADA isozyme synthesized in NIH 3T3 cells as compared to cells harboring the same vector in which the ADA minigene was inserted between the two LTRs. A similar increase in ADA expression was observed in two human lymphoid cell lines tested, HUT 78 and Raji. These results are consistent with previous observations that upstream promoters exert an inhibitory effect on promoters placed downstream and bear out the predictions used in the design of DC vectors. The use of DC vectors may contribute to the solution of the problems encountered in expressing retrovirally transduced genes in cultured cells and, in particular, when introduced into the live animal.
A cytosolic protein binds to structural elements within the iron regulatory region of the transferrin receptor mRNA.
Authors: Koeller D M , Casey J L , Hentze M W , Gerhardt E M , Chan L N , Klausner R D , Harford J B
Published: May 2006

The level of mRNA encoding the transferrin receptor (TfR) is regulated by iron, and this regulation is mediated by a portion of the 3' untranslated region (UTR) of the TfR transcript. This portion of 3' UTR of the human TfR mRNA contains five RNA elements that have structural similarity to the iron-responsive element (IRE) found as a single copy in the 5' UTR of the mRNA for ferritin, whose translation is regulated by iron. Moreover, five very similar elements are also contained in the 3' UTR of the chicken TfR mRNA. Cytosolic extracts of human cell lines are shown by a gel shift assay involving RNase T1 protection to contain an IRE-binding protein capable of specific interaction with the human TfR 3' UTR. When the protecting protein is removed, the protected RNA can be digested with RNase T1 to yield oligoribonucleotide fragments characteristic of two of the IREs contained in the TfR 3' UTR. As judged by cross-competition experiments, the same IRE-binding protein interacts with the ferritin IRE. The apparent affinity of RNA sequence elements for the IRE-binding protein is shown to depend upon the sequence of the RNA. A comprehensive secondary structure for the regulatory region of the TfR mRNA is proposed based on the experimentally demonstrated presence of at least two IRE-like structural elements.
Molecular cloning and sequencing of a cDNA for plant calmodulin: signal-induced changes in the expression of calmodulin.
Authors: Jena P K , Reddy A S , Poovaiah B W
Published: May 2006

A cDNA clone (pPCM-1) for plant calmodulin was isolated by screening a potato stolon tip cDNA library with a chicken calmodulin cDNA. Nucleotide sequence analysis of pPCM-1 revealed that it contained 80 base pairs of 5' untranslated region, the entire coding region, and 376 base pairs of 3' untranslated region. Comparison of the nucleotide sequence of coding regions of potato and chicken calmodulin mRNA showed 78% homology. Comparison of the predicted amino acid sequence of potato calmodulin with other known calmodulin sequences indicated a high degree of homology with a few exceptions. Three changes in the amino acid sequence were found to be unique to the potato calmodulin sequence. In our earlier studies we showed the involvement of calcium and calmodulin in potato tuberization. The pPCM-1 clone was used as a probe to study the expression of calmodulin mRNA during tuberization and to monitor calmodulin mRNA level in various parts of the potato plant. Stolon tips showed the highest levels of calmodulin mRNA, suggesting a role for calmodulin in the tuberization process. In addition, pPCM-1 was used to investigate the effect of auxin and light on calmodulin gene expression in auxin-responsive strawberry fruit and light-responsive Merit corn roots, respectively. Both auxin and light signals were found to increase the level of mRNA for calmodulin. These results suggest that the altered calmodulin gene expression could be one of the molecular events involved in the signal transduction process in plants.
Specific inhibition of cell-surface T-cell receptor expression by antisense oligodeoxynucleotides and its effect on the production of an antigen-specific regulatory T-cell factor.
Authors: Zheng H , Sahai B M , Kilgannon P , Fotedar A , Green D R
Published: May 2006

We have used antisense oligodeoxynucleotides corresponding to genes encoding the variable (V) region of the T-cell receptor (TCR) alpha and beta chains (V alpha and V beta) to control TCR expression in T-cell hybridomas. Two hybridomas, A1.1 and B1.1, recognize a synthetic polypeptide antigen designated poly 18 (poly[Glu-Tyr-Lys-(Glu-Tyr-Ala)5]) together with I-Ad. We have found that TCR function (production of lymphokines in response to antigen) and T3 expression were removed after protease treatment of the cells and were fully recovered 48 hr later. However, when antisense oligodeoxynucleotides corresponding to the appropriate TCR V genes were present after protease treatment, little or no recovery of TCR function or T3 expression was observed. This effect was specific for the TCR V genes utilized by the T cell: antisense oligodeoxynucleotides corresponding to the TCR V regions of A1.1 had no effect on TCR expression in B1.1 and vice versa. Thus, antisense oligodeoxynucleotides can be used to temporarily block expression of a TCR gene in a T-cell hybridoma. This technique was then applied to a paradoxical phenomenon in A1.1 cells. We had observed previously that A1.1 releases an antigen-specific immunoregulatory activity that shows the same antigenic fine specificity as is displayed by the TCR of A1.1. We now report that antisense oligodeoxynucleotides corresponding to the A1.1 V alpha gene blocked the production of this soluble antigen-specific activity by the cell. Antisense oligodeoxynucleotides corresponding to A1.1 V beta, on the other hand, had no effect on the production of this antigen-specific activity. We discuss these observations in the context of recent findings on the nature of T cell-derived antigen-specific regulatory factors.
von Willebrand factor synthesized by endothelial cells from a patient with type IIB von Willebrand disease supports platelet adhesion normally but has an increased affinity for platelets.
Authors: de Groot P G , Federici A B , de Boer H C , d'Alessio P , Mannucci P M , Sixma J J
Published: May 2006

Endothelial cells were isolated from the umbilical vein of a patient with subtype IIB von Willebrand disease, and the biosynthesis and function of von Willebrand factor (vWF) synthesized by these cells were compared with those of vWF synthesized by endothelial cells from normal individuals. The patient's endothelial cells synthesized, stored, and secreted vWF indistinguishably from normal endothelial cells: it was synthesized as a prepolypeptide of Mr 270,000 and had a mature form of Mr 220,000; the full spectrum of multimers was found both inside the cells and in the culture medium; it was stored normally, in the Weibel-Palade bodies; and similar amounts of vWF were secreted into the medium and deposited in the extracellular matrix. In a perfusion set-up, the extracellular matrix from IIB cells supported platelet adhesion similarly to the matrix from normal cells. vWF secreted constitutively by IIB cells into the culture medium bound to platelets at concentrations of ristocetin lower than those necessary for vWF from normal cells. vWF stored in the Weibel-Palade bodies of type IIB cells was released upon stimulation with phorbol ester and bound almost completely to platelets even in the absence of ristocetin. Moreover, spontaneous platelet aggregation was induced by vWF synthesized by type IIB cells. These data support the hypothesis that the absence of highly multimeric forms of vWF in plasma of type IIB von Willebrand disease patients is due to specific removal of these multimers by platelets.
Regulation of cytochrome P-450b/e gene expression by a heme- and phenobarbitone-modulated transcription factor.
Authors: Rangarajan P N , Padmanaban G
Published: May 2006

The cloned DNA fragment of the cytochrome P-450b/e gene containing the upstream region from position -179 through part of the first exon is faithfully transcribed in freeze-thawed rat liver nuclei. Phenobarbitone treatment of the animal strikingly increases this transcription, and the increase is blocked by cycloheximide (protein synthesis inhibitor) or CoCl2 (heme biosynthetic inhibitor) treatment of animals. This picture correlates very well with the reported cytochrome P-450b/e mRNA levels in vivo and run-on transcription rates in vitro under these conditions. The upstream region (from position -179) was assessed for protein binding with nuclear extracts by nitrocellulose filter binding, gel retardation, DNase I treatment ("footprinting"), and Western blot analysis. Phenobarbitone treatment dramatically increases protein binding to the upstream region, an increase once again blocked by cycloheximide or CoCl2 treatments. Addition of heme in vitro to heme-deficient nuclei and nuclear extracts restores the induced levels of transcription and protein binding to the upstream fragment, respectively. Thus, drug-mediated synthesis and heme-modulated binding of a transcription factor(s) appear involved in the transcriptional activation of the cytochrome P-450b/e genes, and an 85-kDa protein may be a major factor in this regard.
Pseudomonas stutzeri N2O reductase contains CuA-type sites.
Authors: Scott R A , Zumft W G , Coyle C L , Dooley D M
Published: May 2006

N2O reductase (N2O----N2) is the terminal enzyme in the energy-conserving denitrification pathway of soil and marine denitrifying bacteria. The protein is composed of two identical subunits and contains eight copper ions per enzyme molecule. The magnetic circular dichroism spectrum of resting (oxidized) N2O reductase is strikingly similar to the magnetic circular dichroism spectrum of the CuA site in mammalian cytochrome c oxidase [Greenwood, C., Hull, B. C., Barber, D., Eglinton, D. G. & Thomson, A. J. (1983) Biochem. J. 215, 303-316] and is unlike the magnetic circular dichroism spectra of all other biological copper chromophores obtained to date. Sulfur (or chlorine) scatterers are required to fit the copper extended x-ray absorption fine structure data of both the oxidized and reduced forms of N2O reductase. Satisfactory fits require a Cu-N or Cu-O [denoted Cu-(N, O)] interaction at 2.0 A, a Cu-(S, Cl) interaction at 2.3 A and an additional Cu(S, Cl) interaction at approximately 2.6 A (oxidized) or approximately 2.7 A (reduced). Approximately eight sulfur ions (per eight copper ions) at approximately 2.3 A are required to fit the extended x-ray absorption fine structure data for both the oxidized and reduced N2O reductase. The 2.3-A Cu-(S, Cl) distance is nearly identical to that previously determined for the CuA site in cytochrome c oxidase. A 2.6-2.7 A Cu-(S, Cl) interaction is also present in resting and fully reduced cytochrome c oxidase. Comparison of the N2O reductase sequence, determined by translating the structural NosZ gene, with cytochrome c oxidase subunit II sequences from several sources indicates that a Gly-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Ser-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-His stretch is highly conserved. This sequence contains three of the probable ligands (two cysteines and one histidine) in a CuA-type site. Collectively these data establish that Pseudomonas stutzeri N2O reductase contains CuA-type sites.
Regulation of viral expression of human immunodeficiency virus in vitro by an antisense phosphorothioate oligodeoxynucleotide against rev (art/trs) in chronically infected cells.
Authors: Matsukura M , Zon G , Shinozuka K , Robert-Guroff M , Shimada T , Stein C A , Mitsuya H , Wong-Staal F , Cohen J S , Broder S
Published: May 2006

In this report, we demonstrate the sequence-specific suppression of viral expression in T cells chronically infected with human immunodeficiency virus 1 (HIV-1), using antisense phosphorothioate oligodeoxynucleotides. As a target for antisense intervention, we used the HIV-1 gene rev, which is essential for viral replication and regulates the expression of virion proteins, in part, by affecting the splicing of the viral mRNA. A phosphorothioate oligomer complementary to the initiation sequence of HIV-1 rev had a significant and selective inhibitory effect on the production of several viral proteins in chronically HIV-1-infected T cells and drastically reduced the unspliced (genomic) viral mRNA transcripts, with relative sparing of smaller (spliced) transcripts. By contrast, the antisense sequence with unmodified normal phosphodiester linkages as well as phosphorothioate oligomers containing sense, random, homopolymeric sequences, or antisense sequence with N3-methylthymidine residues did not have an inhibitory effect on viral expression. Thus, sequence specificity and nuclease resistance were critical for the anti-viral-gene regulatory effect of the antisense molecules. The altered HIV-1 mRNA profile induced by the antisense phosphorothioate oligomer suggests that the mechanism for the inhibition of viral expression is due to an interference with the regulatory gene, rev, by translation arrest.
The Drosophila gene coding for the alpha subunit of a stimulatory G protein is preferentially expressed in the nervous system.
Authors: Quan F , Wolfgang W J , Forte M A
Published: May 2006

In mammals, the alpha subunit of the stimulatory guanine nucleotide-binding protein (Gs alpha) functions to couple a variety of extracellular membrane receptors to adenylate cyclase. Activation of Gs alpha results in the stimulation of adenylate cyclase and an increase in the second messenger cAMP. A 1.7-kilobase cDNA has been identified and characterized from Drosophila that codes for a protein 71% identical to bovine Gs alpha. The similarity is most striking in the regions thought to be responsible for the interactions with receptors and effectors, suggesting that the basic components of this signal-transduction pathway have been conserved through evolution. RNA blot hybridization and DNA sequence analysis suggest that a single transcript, expressed predominantly in the head, is present in Drosophila. In situ hybridization studies indicate that the Drosophila Gs alpha transcript is localized primarily in the cells of the central nervous system and in the eyes.
Molecular cloning of the human eosinophil-derived neurotoxin: a member of the ribonuclease gene family.
Authors: Rosenberg H F , Tenen D G , Ackerman S J
Published: May 2006

We have isolated a 725-base-pair cDNA clone for human eosinophil-derived neurotoxin (EDN). EDN is a distinct cationic protein of the eosinophil's large specific granule known primarily for its ability to induce ataxia, paralysis, and central nervous system cellular degeneration in experimental animals (Gordon phenomenon). The open reading frame encodes a 134-amino acid mature polypeptide with a molecular mass of 15.5 kDa and a 27-residue amino-terminal hydrophobic leader sequence. The sequence of the mature polypeptide is identical to that reported for human urinary ribonuclease [Beintema, J. J., Hofsteenge, J., Iwama, M., Morita, T., Ohgi, K., Irie, M., Sugiyama, R. H., Schieven, G. L., Dekker, C. A. & Glitz, D. G. (1988) Biochemistry 27, 4530-4538] and to the amino-terminal sequence of human liver ribonuclease [Sorrentino, S., Tucker, G. K. & Glitz, D. G. (1988) J. Biol. Chem. 263, 16125-16131]; the cDNA encodes a tryptophan in position 7, which was previously unidentified in the amino acid sequences of EDN or the urinary and liver ribonucleases. Both EDN and the related granule protein, eosinophil cationic protein, have ribonucleolytic activity; sequence similarities among EDN, eosinophil cationic protein, ribonucleases from liver, urine, and pancreas, and angiogenin define a ribonuclease multigene family. mRNA encoding EDN was detected in uninduced HL-60 cells and was up-regulated in cells induced toward eosinophilic differentiation with B-cell growth factor 2/interleukin 5 and toward neutrophilic differentiation with dimethyl sulfoxide. EDN mRNA was detected in mature neutrophils even though EDN-like neurotoxic activity is not found in neutrophil extracts. These results suggest that neutrophils contain a protein that is closely related or identical to EDN.
Extracellular ATP induces the release of calcium from intracellular stores without the activation of protein kinase C in Swiss 3T6 mouse fibroblasts.
Authors: Gonzalez F A , Rozengurt E , Heppel L A
Published: May 2006

Exposure of Swiss 3T6 mouse fibroblasts to extracellular ATP stimulated the formation of inositol phosphates and mobilized intracellular calcium. The mobilization of intracellular calcium was verified by imaging of fura-2 fluorescence in individual cells and by monitoring the efflux of 45Ca2+ from preloaded cells. However, we found no activation of protein kinase C as measured by phosphorylation of an 80-kDa acidic protein and by transmodulation of the receptor for epidermal growth factor. A careful examination of the kinetics of the phosphorylation reaction (from 30 sec to 10 min) revealed no activation of protein kinase C by extracellular ATP at any time. The lack of activation of protein kinase C was demonstrated even when a concentration of ATP 10-fold higher than that required to give a strong Ca2+ signal was used. Extracellular ATP did not inhibit protein kinase C activation by fetal bovine serum, platelet-derived growth factor, or phorbol esters. The effects of ATP were also produced by UTP but not by ADP, AMP, or adenosine. These findings demonstrate that it is possible to induce the mobilization of intracellular calcium by an inositol phosphate-mediated pathway without the activation of protein kinase C.
Relative contribution of "determinant selection" and "holes in the T-cell repertoire" to T-cell responses.
Authors: Schaeffer E B , Sette A , Johnson D L , Bekoff M C , Smith J A , Grey H M , Buus S
Published: May 2006

Using BALB/c and CBA/J mice, the I-region associated (Ia) binding capacity and T-cell immunogenicity of a panel of 14 overlapping peptides that span the entire sequence of the protein staphylococcal nuclease (Nase) was examined to evaluate major histocompatibility gene complex (MHC) control of T-cell responses. Ia binding and Ia-restricted T-cell immunogenicity could be determined for a total of 54 peptide-MHC combinations. Only 30% of the 54 instances examined involved detectable Ia binding, but they represented almost all (12 of 13) of the immune responses found. However, binding to Ia was not sufficient to ensure T-cell immunogenicity, since only 70% of the binding events were productive--i.e., were associated with an immune response. Thus, Ia molecules have the expected characteristics of a highly permissive capacity for antigen interaction that allows them to function as restriction elements for a large universe of antigens. On the other hand, since the Ia molecules cannot distinguish between self and non-self, not all antigen-Ia interactions would be permitted to elicit a T-cell response. It appears that both Ia binding ("determinant selection") and T-cell repertoire act in concert to define the immune response status of an individual toward any particular T-cell epitope.
Altered growth of a human neuroendocrine carcinoma line after transfection of a major histocompatibility complex class I gene.
Authors: Sunday M E , Isselbacher K J , Gattoni-Celli S , Willett C G
Published: May 2006

The major histocompatibility complex (MHC) class I molecules are known to serve as recognition elements for cytotoxic T cells in mediating the rejection of transplanted tumors. We demonstrate that MHC molecules may have nonimmune functions in modulating tumor cell growth in addition to their classical role in antitumor immunity. A human neuroendocrine carcinoma cell line, COLO 320, with low levels of endogenous class I expression was transfected with the murine H-2Ld gene. Eleven independent stable clones were established, four containing only pRSV-neo and seven also containing varying copy numbers of the transfected Ld gene. The ability of the different clones to grow as colonies in soft agar correlated strongly with the relative amounts of Ld antigen expression (r = 0.89; P less than 0.001). There was a weaker correlation between increased clonogenic ability and higher levels of Ld mRNA (r = 0.67; P less than 0.05). There was no correlation between clonogenic ability and relative expression of amplified c-myc gene or of integrated pRSV-neo. Furthermore, in nude mice, Ld antigen expression was associated with increased formation of metastatic lung colonies 6 weeks after intravenous injection of 10(5) cells. These observations are consistent with the concept that MHC class I antigens may have a role in modulating the growth potential of certain tumor cells independent of their involvement in immune responses.
Deletions of bases in one strand of duplex DNA, in contrast to single-base mismatches, produce highly kinked molecules: possible relevance to the folding of single-stranded nucleic acids.
Authors: Hsieh C H , Griffith J D
Published: May 2006

A 32-base-pair (bp) DNA duplex with deletions in one strand, and thus extra bases in the opposing strand, was ligated head-to-tail to produce linear and circular multimers. The electrophoretic mobility of the linear multimers was analyzed in polyacrylamide gels and the size of the circular DNA was determined by electron microscopy. A 1-base deletion produced a marked retardation in the mobility of the linear multimers coincident with the formation of a population of multimeric circles of a smaller average size than the deletionless 32-mer; 2-, 3-, or 4-base deletions at the same site produced proportionately greater effects. Two 1-base deletions separated by 10 bp on the same strand produced a greater reduction in mobility than a 1-base deletion, whereas two 1-base deletions spaced by 5 bp on the same strand yielded a molecule that behaved more like the deletionless DNA. We conclude that deletions of 1-4 bases at a single site on duplex DNA produce molecules that behave as if they contain sharp bends or kinks. In contrast, single mismatches in the 32-bp duplex produced no abnormality in behavior relative to normally base-paired DNA in the gel mobility and electron microscopic assays. The possible role of such structures in organizing the three-dimensional folding of single-stranded nucleic acids is considered.
Identification of critical regions in mouse granulocyte-macrophage colony-stimulating factor by scanning-deletion analysis.
Authors: Shanafelt A B , Kastelein R A
Published: May 2006

Structure-function relationships for mouse granulocyte-macrophage colony-stimulating factor were examined by generating a series of small deletions scanning the entire length of the molecule. Deletions of three amino acids were introduced at intervals of five amino acids by site-directed mutagenesis of the mature mouse granulocyte-macrophage colony-stimulating factor gene. The mutant proteins were expressed in Escherichia coli and assayed for biological activity. This procedure identified four regions critical to activity. These critical regions were further delineated by additional three-amino acid deletion mutants. Larger deletions at each terminus were also made, as well as changes of specific amino acid residues. The four critical regions span amino acid residues 18-22, 34-41, 52-61, and 94-115. The disulfide bridge between Cys-51 and Cys-93 was also shown to be essential for activity, whereas that between Cys-85 and Cys-118 could be removed without loss of activity. The possible structural and/or functional roles of the critical regions are discussed.
Studies on the DNA elongation inhibitor and its proliferating cell nuclear antigen-dependent control in simian virus 40 DNA replication in vitro.
Authors: Lee S H , Kwong A D , Ishimi Y , Hurwitz J
Published: May 2006

A 120-kDa protein that blocks DNA termini has been purified from extracts of HeLa cells. This protein inhibits the action of a number of enzymes that catalyze reactions involving the 5' and 3' ends of DNA (DNA ligase, 3' and 5' exonucleases, and DNA polymerase alpha). The 120-kDa protein blocks the synthesis of long DNA chains that are normally formed during simian virus 40 DNA replication, causing the accumulation of small DNA fragments. The effects of this protein can be reversed by the addition of proliferating cell nuclear antigen and other protein fractions (activators).
Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.
Authors: Tapprich W E , Goss D J , Dahlberg A E
Published: May 2006

A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia coli rrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affinity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis.
"Bundle blot" purification and initial protein characterization of hair cell stereocilia.
Authors: Shepherd G M , Barres B A , Corey D P
Published: May 2006

Stereocilia were isolated from bullfrog (Rana catesbeiana) saccular hair cells by nitrocellulose adhesion. The high purity and high yield of the preparation were demonstrated by microscopy. SDS/PAGE of stereociliary proteins resolved 12-15 major bands. Actin, previously identified as a component of the stereociliary core, was identified in purified stereocilia as a band comigrating with authentic actin and by phalloidin labeling of intact isolated stereocilia. Fimbrin was identified in immunoblots of purified stereocilia. The most abundant other proteins migrated at 11, 14, 16-19, 27, and 36 kDa. Demembranated stereociliary cores consisted primarily of protein bands corresponding to actin and fimbrin and several proteins ranging from 43 to 63 kDa. Because the adaptation mechanism in hair cells is calcium-sensitive and seems localized to stereocilia, we sought evidence for calcium-binding proteins in stereocilia. Calmodulin and calbindin antibodies labeled stereocilia in intact cells. A protein band in purified stereocilia exhibited a Ca2+-dependent shift in electrophoretic mobility identical to that of authentic calmodulin, and the 27-kDa band may represent calbindin. These biochemical data demonstrate that stereocilia consist of a relatively small set of proteins. Most of these, including those involved in transduction and adaptation, are as yet uncharacterized. The availability of purified stereocilia should prove useful in further studies of structure-function relationships in these mechanically sensitive organelles.
A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence.
Authors: Miller S I , Kukral A M , Mekalanos J J
Published: May 2006

We have determined that Salmonella typhimurium strains with mutations in the positive regulatory locus phoP are markedly attenuated in virulence for BALB/c mice. The DNA sequence for the phoP locus indicates that it is composed of two genes present in an operon, termed phoP and phoQ. The deduced amino acid sequence of the phoP and phoQ gene products are highly similar to other members of bacterial two-component transcriptional regulators that respond to environmental stimuli. S. typhimurium strains with transposon insertions that create transcriptional and translational gene fusions that require phoP and phoQ for expression have been isolated and have different chromosomal locations, indicating that this system is a regulon. One of these fusion strains, containing a mutation in a gene termed pagC, has a virulence defect. Other strains, including those containing mutations in the phoN gene, encoding an acid phosphatase, have wild-type virulence. Strains with pagC, phoP, or phoQ mutations have decreased survival in cultured mouse macrophages. When used as live vaccines in mice, strains with phoP or phoQ mutations afford partial protection to subsequent challenge by wild-type S. typhimurium.
Genomic DNA differences between pathogenic and nonpathogenic Entamoeba histolytica.
Authors: Tannich E , Horstmann R D , Knobloch J , Arnold H H
Published: May 2006

cDNA libraries were constructed from pathogenic (HM-1:IMSS) and nonpathogenic (SAW 1734) isolates of Entamoeba histolytica. A cDNA clone (cEH-P1) specific for pathogenic amoebae was identified by screening with a pool of sera from patients with invasive amoebiasis that had been absorbed with nonpathogenic amoebae. This clone was used for the identification of a homologous clone (cEH-NP1) in the cDNA from nonpathogenic amoebae. Sequence analysis and comparison of the predicted amino acid sequences for both clones disclosed 12% evolutionary divergence in structure. Hybridization of both cDNA probes to genomic DNA from four pathogenic and five nonpathogenic E. histolytica isolates revealed two distinct Southern blot patterns, one characteristic for pathogenic amoebae and the other for nonpathogenic amoebae. Further, the complex pattern of restriction fragments hybridizing to an actin cDNA probe was also different between pathogenic and nonpathogenic isolates but was conserved within each group of amoebae. The results indicate that pathogenic isolates of E. histolytica are genetically distinct from nonpathogenic isolates.
Reconstitution of an inwardly rectifying potassium channel from the basolateral membranes of Necturus enterocytes into planar lipid bilayers.
Authors: Costantin J , Alcalen S , de Souza Otero A , Dubinsky W P , Schultz S G
Published: May 2006

Basolateral membrane vesicles from Necturus enterocytes, highly (greater than 20-fold) enriched in Na+,K+-ATPase, were reconstituted into planar lipid bilayers. The principal channel activity observed is selective for K+ over Na+ and Cl-. This K+ channel is blocked by Ba2+ and Leiurus quinquestriatus venom but is not affected by Ca2+ over the range of 10(-3) to less than 10(-7) M and is not inhibited by charybdotoxin. L. quinquestriatus venom also markedly reduces the conductance of the basolateral membrane of intact villus cells of Necturus small intestine. The open-time probability (Po) of the channel displays a voltage-dependence characteristic of an "inward rectifier"; i.e., the channel inactivates when the basolateral membrane is depolarized and Po increases with increasing hyperpolarization of that barrier. Assuming that similar properties prevail under physiological conditions, this characteristic could provide, in part, an explanation for the parallelism between Na+-pump and K+-leak activities of the basolateral membrane observed in this epithelium. Thus, an increase in rheogenic Na+-pump activity at the basolateral membrane would hyperpolarize that barrier and, in turn, increase the open time of this K+ channel.
cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family.
Authors: Cool D E , Tonks N K , Charbonneau H , Walsh K A , Fischer E H , Krebs E G
Published: May 2006

A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). One positive clone was isolated and the nucleotide sequence was determined. It contained 1305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3' untranslated end, although a poly(A)+ tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues (Mr, 48,400). This was supported by the synthesis of a Mr 48,000 protein in an in vitro reticulocyte lysate translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low Mr PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes.
Imaging of single uncoated DNA molecules by scanning tunneling microscopy.
Authors: Keller D , Bustamante C , Keller R W
Published: May 2006

Scanning tunneling microscope images of DNA molecules absorbed onto highly oriented pyrolytic graphite have been obtained. Three methods of deposition and sample preparation have been utilized. In the first method, a highly concentrated solution of DNA is sonicated, and a drop is deposited on freshly cleaved graphite. Under these conditions, the molecules tend to align in a parallel fashion, forming liquid-crystalline phases. In the second method, a solution of DNA is deposited directly on the graphite surface without sonication. In this case, ammonium acetate, a volatile salt, is used to decrease the amount of the residual salt crystals left after drying. In the third method, a solution containing lysed phage particles and DNA is adsorbed onto a graphite surface. The molecules are seen either isolated or in small bundles. The values of height, periodicity, and thickness observed and the handedness of the molecules are consistent with those expected for DNA. In all cases, the molecules were identified by their characteristic periodic structure and because, at higher magnification, no graphite-like structure was detectable on the surface of the molecules. Often the DNA molecules appear to adsorb in areas of the graphite that have many steps and defects. A mechanism that explains the magnitude of the tunneling currents measured in DNA is proposed. This mechanism, in turn, suggests a general method by which large insulating molecules can be rendered conductive.
Lung surfactant apoprotein SP-A (26-36 kDa) binds with high affinity to isolated alveolar type II cells.
Authors: Wright J R , Borchelt J D , Hawgood S
Published: May 2006

Pulmonary surfactant is synthesized and secreted by alveolar type II cells. These cells recycle surfactant lipids by an internalization process that is enhanced in vitro by the surfactant proteins with molecular masses of 26-36 kDa (SP-A). SP-A also inhibits the secretion of lipid by type II cells. These results suggest that SP-A may play a role in feedback regulation of surfactant pool size and are consistent with the hypothesis that the type II cell surface has receptors for SP-A. The goal of this study is to characterize the binding of radioiodinated SP-A to isolated rat type II cells. Binding of SP-A to type II cells at 4 degrees C has a K1/2 of approximately 5 X 10(-10) M, is saturable, and is inhibited by excess unlabeled SP-A. Binding is dependent on calcium and is reduced by heat treatment of SP-A. The binding of a proteolytic fragment of SP-A that is produced by collagenase treatment is reduced by excess unlabeled SP-A. The binding of the fragment to macrophages and lung fibroblasts is not inhibited by excess unlabeled SP-A. Trypsinization of the type II cell surface reduces the binding of both intact SP-A and the collagenase-resistant fragment. These results show that SP-A binds to type II cells with high affinity and suggest that these cells have receptors that recognize the carboxyl-terminal domain of SP-A.
Evidence for diploidy in metacyclic forms of African trypanosomes.
Authors: Kooy R F , Hirumi H , Moloo S K , Nantulya V M , Dukes P , Van der Linden P M , Duijndam W A , Janse C J , Overdulve J P
Published: May 2006

The DNA contents of bloodstream form trypanosomes (life cycle stages circulating in the blood of the vertebrate host) of four African Trypanosoma species and of metacyclic forms (the life cycle stage that is injected into the vertebrate by the tsetse fly during its bite) of the same four species were measured by cytofluorometry of individual cells or nuclei. The results showed unambiguously that the metacyclic forms cannot be considered to be products of meiosis containing only half of the DNA of bloodstream forms, in contrast to what was previously reported for Trypanosoma brucei [Zampetti-Bosseler, F., Schweizer, J., Pays, E., Jenni, L. & Steinert, M. (1986) Proc. Natl. Acad. Sci. USA 83, 6063-6064] during an attempt to localize the gametes in the life cycle after experimental evidence of sexual gene exchange in this parasite was reported.
ATP-dependent directional movement of rat synaptic vesicles injected into the presynaptic terminal of squid giant synapse.
Authors: Llinás R , Sugimori M , Lin J W , Leopold P L , Brady S T
Published: May 2006

The question as to whether synaptic vesicles prepared from vertebrate brain can be transported to the active zones of the squid giant synapse was studied by using a combined optical and electrophysiological approach. In order to visualize the behavior of the vertebrate synaptic vesicles in situ, synaptic vesicles isolated from rat brain were labeled with a fluorescent dye (Texas red) and injected into the presynaptic terminal of the squid giant synapse. The pattern of fluorescence that would result from passive diffusion was determined by coinjection of an unconjugated fluorescent dye (fluorescein). The patterns obtained with fluorescent synaptic vesicles were strikingly different from that obtained by simple diffusion of fluorescein. Although the fluorescein diffused freely in both directions, the vesicles moved preferentially into the terminal--i.e., toward the release sites--at a rate of 0.5 microns/sec. The final distribution of the injected fluorescent synaptic vesicles displayed a discrete localization that suggested a distribution coincident with the active zones of the presynaptic terminal. Like fast axonal transport, but unlike fluorescein movements in the terminal, the vesicle movement was energy dependent, since the addition of 2,4-dinitrophenol blocked the redistribution of vesicles completely. In addition, reduction of extracellular calcium concentration reversibly blocked vesicular movement as well. In conclusion, mammalian synaptic vesicles retain the cytoplasmic surface components necessary for translocation, sorting, and targeting to the proper locations by the native machinery of the squid giant synapse.
Xenopus homolog of the mos protooncogene transforms mammalian fibroblasts and induces maturation of Xenopus oocytes.
Authors: Freeman R S , Pickham K M , Kanki J P , Lee B A , Pena S V , Donoghue D J
Published: May 2006

The oncogene v-mos transforms mammalian fibroblasts and encodes a serine/threonine protein kinase. Expression of the c-mos protooncogene is most abundant in germ cells, suggesting a normal role for c-mos in meiosis. Here we describe the isolation of cDNA clones containing the complete coding region of the Xenopus laevis homolog of c-mos (mosxe). The mosxe gene is transforming when introduced into murine NIH 3T3 cells, and transformation is abrogated by a lysine-to-arginine mutation in the canonical ATP-binding site. Microinjection of in vitro transcribed mosxe RNA into prophase-arrested Xenopus oocytes causes a resumption of meiosis, leading to germinal vesicle breakdown and oocyte maturation. Oocyte maturation was not observed after microinjection of in vitro transcribed mosxe RNA encoding the lysine-to-arginine mutation. These results demonstrate that the mosxe-encoded protein can induce progression through the cell cycle for both meiotic and mitotic cells and that this property is dependent on the presumptive ATP-binding domain in the protein kinase.