bluuebunny/crossref_metadata_2025 huggingface dataset:
RETRACTED: Heparin-binding growth factor 1 induces the formation of organoid neovascular structures in vivo.
Authors: Thompson J A , Haudenschild C C , Anderson K D , DiPietro J M , Anderson W F , Maciag T
Published: May 2006

One of the promises of modern molecular biology has been the opportunity to use genetically modified human cells in a patient to permanently restore inborn errors of metabolism. Although it has been possible to introduce genes into mammalian cells and to control their expression, it has proven difficult to introduce mammalian cells as carriers of the modified genetic information into hosts. The successful implantation of selective cells cannot be achieved without adequate vascular support, an essential step toward integration and reconstitution of a new biological function. Although a partial solution to this problem has been found by inducing specific site-directed neovessel formation using heparin-binding growth factor 1 (HBGF-1) adsorbed to a collagen matrix, these implants function for only a short period (weeks). We now report the formation of organoid neovascular structures using polytetrafluoroethylene fibers coated with collagen and HBGF-1 implanted in the peritoneal cavity of the rat. The organoid structures contained readily visible vascular lumina and nonvascular structures that resemble nerve tissue. It was also possible to demonstrate that the vascular system on the implant is continuous with the vascular tree of the host. This feature was used to demonstrate that the organoid structures are capable of sustaining the biological function of implanted normal rat hepatocytes over long periods of time (months) in the homozygous Gunn rat, thereby facilitating future applications involving the delivery of new genetic information.
Construction of a recombinant human parvovirus B19: adeno-associated virus 2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus.
Authors: Srivastava C H , Samulski R J , Lu L , Larsen S H , Srivastava A
Published: May 2006

To facilitate genetic analysis of the human pathogenic parvovirus B19, we constructed a hybrid B19 viral genome in which the defective B19 inverted terminal repeats were replaced with the full-length inverted terminal repeats from a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The hybrid AAV-B19 genome was rescued from a recombinant plasmid and then the DNA was replicated upon transfection into adenovirus 2-infected human KB cells in the presence of AAV genes coding for proteins required for AAV DNA replication (AAV-Rep proteins). In addition, in the presence of AAV genes coding for the viral capsid proteins (AAV-Cap proteins), the rescued/replicated hybrid AAV-B19 genomes were packed into mature AAV progeny virions, which were subsequently released into culture supernatants. The recombinant AAV-B19 progeny virions were infectious for normal human bone marrow cells and strongly suppressed erythropoiesis in vitro. The availability of an infectious recombinant B19 virus should facilitate the mutational analysis of the viral genome, which, in turn, may yield information on individual viral gene functions in B19-induced pathogenesis. The hybrid AAV-B19 genome may also prove to be a useful vector for gene transfer in human bone marrow cells.
Transformation of Tetrahymena thermophila with a mutated circular ribosomal DNA plasmid vector.
Authors: Yu G L , Blackburn E H
Published: May 2006

A circular plasmid containing a complete Tetrahymena thermophila rRNA gene (rDNA), with a tandem repeat of a 1.9-kilobase-pair segment encompassing the replication origin and the rRNA promoter, and a polylinker in the 3' nontranscribed spacer, was used to transform T. thermophila by microinjection. Most (20/21) stable transformants contained only recombinant linear palindromic rDNA molecules carrying rDNA sequences from both the donor plasmid and the recipient cell, as shown previously. However, in one transformant, the circular plasmid initially outreplicated the endogenous rDNA and was the major rDNA form for up to 65 generations. Stable circular replicons have not been reported previously in Tetrahymena. A single point mutation (+G) was identified in the repeated promoter of the plasmid maintained in this transformant. After recovery from the Tetrahymena transformant and recloning in Escherichia coli, the mutated circular plasmid again transformed Tetrahymena with stable maintenance of the circular rDNA plasmid. Transformants containing circular replicons were also obtained by using a similar plasmid from which the repeated promoter, but not the repeated replication origin, had been removed by BAL-31 deletion. We therefore propose that repeated rRNA promoters are deleterious in vivo in Tetrahymena, which normally lacks them. Transformants were obtained in 2-5 days compared with the 7-14 days required for transformation with unmutated rDNA plasmids by recombination. Similar results were obtained when a 550-base-pair segment containing the telomerase RNA gene of T. thermophila was inserted in the polylinker of the plasmid. We suggest that this plasmid is a useful vector system for transformation of Tetrahymena.
Inhibition of allorecognition by an H-2Kb-derived peptide is evidence for a T-cell binding region on a major histocompatibility complex molecule.
Authors: Schneck J , Munitz T , Coligan J E , Maloy W L , Margulies D H , Singer A
Published: May 2006

The class I and class II major histocompatibility complex (MHC) antigens are polymorphic cell-surface glycoproteins that present antigenic peptides to T lymphocytes in the generation of immune responses. While much is known about the recognition and processing of antigens, the nature of T-cell recognition sites on MHC molecules is poorly understood. Both structural and functional studies have suggested that the two major alpha-helical regions of the class I MHC molecule not only define the site for binding of antigenic peptide but also provide potential sites for interaction of the MHC molecule with the T-cell receptor. A peptide derived from one of these regions on the H-2Kb molecule, peptide Kb163-174, was previously shown to specifically inhibit the stimulation of an alloreactive T-cell hybridoma. To further investigate the role of this region in the recognition of H-2Kb, the effects of peptide Kb163-174 on allospecific T-cell lines and clones were studied. When peptide Kb163-174 was cocultured with either an H-2Kbm10 anti-H-2Kb cytotoxic T-lymphocyte (CTL) clone or a CTL line, this peptide inhibited lysis of H-2Kb targets. Pretreatment experiments showed that the blockade was due to interaction of the peptide with the effector T cells. Surprisingly, peptide Kb163-174 also inhibited lysis of H-2Kb targets by H-2Kbm1-, H-2Kbm3-, H-2Kbm6, and H-2Kbm8-anti-H-2Kb CTLs. These CTLs, which identify multiple antigenic sites on H-2Kb in the alpha 1 and alpha 2 domains, are not directed against amino acid residues 163-174 of H-2Kb. In addition, peptide Kb163-174 specifically blocked lysis of only H-2Kb and not H-2Ld targets by a single bulk CTL culture that was alloreactive on both H-2Kb and H-2Ld. These results indicate that peptide Kb163-174 interferes with T-cell receptor engagement of a contact site on the H-2Kb molecule. Thus, amino acid residues 163-174 define a site used by many alloreactive T cells to engage the H-2Kb molecule.
Inhibition of Ca2+/calmodulin-dependent protein kinase II by arachidonic acid and its metabolites.
Authors: Piomelli D , Wang J K , Sihra T S , Nairn A C , Czernik A J , Greengard P
Published: May 2006

A variety of evidence indicates that activation of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) in nerve terminals leads to enhanced neurotransmitter release. Arachidonic acid and its 12-lipoxygenase metabolite, 12-hydroperoxyeicosatetraenoic acid (12-HPETE), have been suggested to act as second messengers mediating presynaptic inhibition of neurotransmitter release. In the present study it was found that CaM-kinase II, purified from rat brain cortex, was inhibited both by arachidonic acid (IC50 = 24 microM) and by 12-HPETE (IC50 = 0.7 microM). Neither substance inhibited CaM-kinase I or III, protein kinase C, or the catalytic subunit of cAMP-dependent protein kinase. Specific inhibition of Ca2+/calmodulin-dependent protein phosphorylation by arachidonic acid was also demonstrated in intact synaptic terminals (synaptosomes) isolated from rat forebrain. These results suggest that arachidonate and its metabolites may modulate synaptic function through the inhibition of CaM-kinase II-dependent protein phosphorylation.
A second trans-spliced RNA leader sequence in the nematode Caenorhabditis elegans.
Authors: Huang X Y , Hirsh D
Published: May 2006

In the nematode Caenorhabditis elegans, the 22-nucleotide RNA sequence called the spliced leader (SL) is trans-spliced from the 100-nucleotide-long SL RNA to some mRNAs. We have identified a trans-spliced leader (SL2) whose sequence differs from that of the original spliced leader (SL1), although both are 22 nucleotides long. By primer-extension sequencing, SL2 but not SL1 was shown to be present at the 5' end of the mRNA encoded by one of the four glyceraldehyde-3-phosphate dehydrogenase genes. The other three glyceraldehyde-3-phosphate dehydrogenase genes encode mRNAs that have the SL1 but not the SL2 sequence at their 5' ends. Therefore, the trans-splicing process can discriminate the transfer of SL1 from that of SL2 in a gene-specific manner.
A family of receptor-linked protein tyrosine phosphatases in humans and Drosophila.
Authors: Streuli M , Krueger N X , Tsai A Y , Saito H
Published: May 2006

To understand the regulation of cell proliferation by tyrosine phosphorylation, characterization of protein tyrosine phosphatases (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) is essential. The human genes LCA (leukocyte common antigen) and LAR encode putative receptor-linked PTPases. By using consensus sequence probes, two additional receptor-linked PTPase genes, DLAR and DPTP, were isolated from Drosophila melanogaster. The extracellular segments of both DLAR and DPTP are composed of multiple immunoglobulin-like domains and fibronectin type III-like domains. The cytoplasmic region of DLAR and DPTP, as well as human LCA and LAR, are composed of two tandemly repeated PTPase domains. PTPase activities of immunoprecipitated LCA and LAR were demonstrated by measuring the release of phosphate from a 32P-labeled [Tyr(P)]peptide. Furthermore, the cytoplasmic domains of LCA, LAR, DLAR, and DPTP, expressed in Escherichia coli, have PTPase activity. Site-directed mutagenesis showed that a conserved cysteine residue is essential for PTPase activity.
Loss of heterozygosity suggests tumor suppressor gene responsible for primary hepatocellular carcinoma.
Authors: Buetow K H , Murray J C , Israel J L , London W T , Smith M , Kew M , Blanquet V , Brechot C , Redeker A , Govindarajah S
Published: May 2006

Primary hepatocellular carcinoma (PHC), epidemiologically associated with chronic hepatitis B virus (HBV) infection, has historically been felt to be caused by the activation or introduction of an oncogene. However, transforming sequences from human PHC have not been reproducibly isolated. In this paper, evidence is presented that suggests PHC may result instead from the loss of an anti-oncogene. Seven of 12 human primary liver tumors tested against a panel of restriction fragment length polymorphisms (RFLPs) demonstrated loss of constitutional heterozygosity for markers on chromosome 4. Tumor and nontumor liver tissue were typed for 11 chromosome 4 RFLPs. In addition, at least one RFLP on nine other chromosomes (1, 2, 6, 7, 9, 11, 13, 14, and 17) was tested for allelic loss. Seven of nine tumors constitutionally heterozygous for chromosome 4q markers showed allele loss in tumor tissue. Six of the seven samples were jointly informative for both 4p and 4q markers. Five of the six demonstrated loss for only 4q RFLPs. In one individual, in which two samples were taken from distant locations within the same tumor, both samples showed loss of the same alleles. Among the other chromosomes informative for allele loss, one tumor showed changes on 13q. No other changes were observed in RFLPs located on the eight other chromosomes tested. These results indicate that an anti-oncogene may be located on 4q and suggest a mechanism for PHC and other cancers seroepidemiologically related to virus infection. Liver cancer caused by chronic HBV infection or other environmental agents may be linked through genetic events responsible for the loss of a tumor suppressor locus (anti-oncogene) located on chromosome 4.
Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells.
Authors: Bodine D M , Karlsson S , Nienhuis A W
Published: May 2006

The effects of several hematopoietic growth factors on primitive murine bone marrow progenitor cells [colony-forming unit(s)-spleen (CFU-S)] have been investigated during culture for 2-6 days. Interleukin 3 (IL-3) was required for CFU-S survival in culture, and the combination of IL-3 and interleukin 6 (IL-6) increased the number of CFU-S in culture 10-fold over the number obtained with IL-3 alone. Stem cell function was measured by competitive repopulation; IL-3 was required, and IL-3 and IL-6 appear to act synergistically to enhance stem cell recovery from these cultures. These data appear to be relevant for retroviral-mediated gene transfer into stem and progenitor cells. Murine bone marrow cells were infected with a retrovirus containing the human beta-globin gene in the presence of various growth factors. Only 2 of 17 mice reconstituted with cells infected in the presence of IL-3 alone showed long-term expression of the human beta-globin gene (12 months), as opposed to 6 of 11 mice reconstituted with cells infected in the presence of IL-3 and IL-6. Medium conditioned by 5637 bladder carcinoma cells, a source of several hematopoietic growth factors, increased the frequency of infection of CFU-S but did not enhance stem cell infection or the repopulating potential of cultured bone marrow cells. Stem cells containing the human beta-globin provirus from these animals were shown to be capable of reconstituting secondary recipients in which the human beta-globin gene was expressed.
Gene flow by pollen into small populations: Data from experimental and natural stands of wild radish
Authors: Ellstrand Norman C. , Devlin B. , Marshall Diane L.
Published: May 2006

Gene flow can have an especially strong impact on the evolution of small populations. However, empirical studies on the actual rates and patterns of gene flow into small populations are few. Thus, we sought to measure gene flow into small populations of wild radish.
Raphanus sativus
. We found significant differences in gene flow receipt among experimental populations and within those populations over a season. A maximum-likelihood estimate revealed that almost all of the gene flow into these synthetic populations had its origin in relatively distant (>650 m), large natural populations rather than the proximal (255-400 m), small synthetic populations. We also estimated rates of interpopulation mating from simple paternity analysis of progeny produced by seven small (
ca.
50 plants) natural populations. Again, we found significant heterogeneity in gene flow receipt. Although these populations varied 10-fold in their range of isolation distances (100-1000 m), gene flow rates did not vary with distance. The magnitude of gene flow rates estimated in all but one population was great enough for gene flow to play an important role in the evolution of these small populations.
A retinoic acid-responsive element is present in the 5' flanking region of the laminin B1 gene.
Authors: Vasios G W , Gold J D , Petkovich M , Chambon P , Gudas L J
Published: May 2006

The retinoic acid (RA)-associated differentiation of murine F9 teratocarcinoma stem cells results in dramatic changes in gene expression. The cellular gene encoding the B1 subunit of the extracellular matrix protein laminin is transcriptionally activated by RA, and its transcription is further enhanced by N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) during the differentiation of F9 stem cells into extraembryonic parietal endoderm cells. We now report that expression vectors encoding the human RA receptors RAR-alpha, RAR-beta, and RAR-gamma can activate chloramphenicol acetyltransferase (CAT) expression from laminin B1 promoter/CAT expression vectors (e.g., p1.6LAMCAT) in RA-treated F9 cells, as measured in a transient transfection assay. Bt2cAMP does not further enhance the RA-associated increase in CAT activity. Through the use of deletion and mutation analyses, the RA-responsive element (RARE) of the murine laminin B1 gene has been defined as a 46-base-pair element between -477 and -432 of the laminin B1 5' flanking region. Insertion of a region of DNA containing this RARE in either orientation into a thymidine kinase promoter/CAT expression vector causes CAT expression to be activated 5- to 9-fold by the cotransfected human RAR-alpha or RAR-beta constructs in RA-treated F9 cells, and this RARE also functions in human HeLa cells. In contrast, this RARE in the p1.6LAMCAT vector does not activate CAT expression when cotransfected into F9 stem cells with the c-erbA gene in the presence of thyroid hormone. This suggests that the laminin B1 gene is activated by RA but not by thyroid hormone in vivo.
Negative regulation of the thyroid-stimulating hormone alpha gene by thyroid hormone: receptor interaction adjacent to the TATA box.
Authors: Chatterjee V K , Lee J K , Rentoumis A , Jameson J L
Published: May 2006

Thyroid-stimulating hormone alpha and beta subunit genes are negatively regulated by thyroid hormone at the transcriptional level. Transient gene expression studies were used to demonstrate that the erbA beta form of the thyroid hormone receptor mediates negative regulation of the alpha-subunit promoter in a hormone-dependent manner. In JEG-3 choriocarcinoma cells, which are deficient in thyroid hormone receptors, coexpression of erbA beta with alpha CAT reporter genes markedly suppressed alpha CAT expression after treatment with thyroid hormone, whereas a reporter gene containing a known positive thyroid response element was induced. Thus, a single form of thyroid hormone receptor mediates both positive and negative responses to thyroid hormone in this system. Transient expression analyses of alpha gene 5' flanking sequence deletion mutants localized the negative thyroid response element to the proximal region of the promoter between -100 and +4 base pairs. The location of the negative thyroid response element in the alpha gene is therefore distinct from that of previously identified regulatory elements including the tissue-specific upstream regulatory elements, the cAMP response elements, and the glucocorticoid response elements. Overlapping segments of the alpha promoter were examined for potential thyroid hormone receptor binding sites by using gel shift assays and biotinylated DNA-binding studies. A specific thyroid hormone receptor binding site was identified between -22 and -7 base pairs, which is immediately downstream from the TATA box. This region of the alpha promoter interacts with erbA beta receptor synthesized in vitro as well as with endogenous nuclear thyroid hormone receptors, and it competes for receptor binding to a known positive thyroid response element. These studies suggest a mechanism for negative regulation in which the thyroid hormone receptor interacts with the alpha gene promoter immediately downstream of the TATA box to inhibit transcription.
Antibacterial peptides from pig intestine: isolation of a mammalian cecropin.
Authors: Lee J Y , Boman A , Sun C X , Andersson M , Jörnvall H , Mutt V , Boman H G
Published: May 2006

Pig small intestine was used as starting material for a batchwise isolation of a peptide fraction enriched in antibacterial activities against Escherichia coli (anti-Ec factor) and against Bacillus megaterium (anti-Bm factor). Separation and further purification were by different types of chromatography. Sequence analysis showed the anti-Bm factor to be apparently similar to vasoactive intestinal peptide. The anti-Ec factor was found to have a 31-residue sequence that was cecropin-like. It was named cecropin P1 and its structure was confirmed by solid-phase synthesis. Synthetic cecropin P1 with and without C-terminal amide was assayed on eight different bacteria. Mobility comparison between synthetic and natural cecropin P1 indicates that the natural peptide has a free C-terminal carboxyl group.
Fidelity of DNA polymerases in DNA amplification.
Authors: Keohavong P , Thilly W G
Published: May 2006

Denaturing gradient gel electrophoresis (DGGE) was used to separate and isolate the products of DNA amplification by polymerase chain reaction (PCR). The strategy permitted direct enumeration and identification of point mutations created by T4, modified T7, Klenow fragment of polymerase I, and Thermus aquaticus (Taq) DNA polymerases. Incorrectly synthesized sequences were separated from the wild type by DGGE as mutant/wild-type heteroduplexes and the heteroduplex fraction was used to calculate the average error rate (mutations per base duplication). The error rate induced in the 104-base-pair low-temperature melting domain of exon 3 of the human hypoxanthine/guanine phosphoribosyltransferase (HPRT) gene was approximately 3.4 x 10(-5) for modified T7, 1.3 x 10(-4) for Klenow fragment, and 2.1 x 10(-4) for Taq polymerases after a 10(6)-fold amplification. The error rate for T4 DNA polymerase was not more than 3 x 10(-6) error per base duplication. The predominant mutations were sequenced and found to be transitions of G.C to A.T for T4 and modified T7 DNA polymerases, and A.T to G.C for Taq polymerase. Klenow fragment induced both possible transitions and deletions of 2 and 4 base pairs.
Endothelin-like pulmonary vasoconstrictor peptide release by alpha-thrombin.
Authors: Moon D G , Horgan M J , Andersen T T , Krystek S R , Fenton J W , Malik A B
Published: May 2006

The endothelial cells lining the vessel wall can modulate vasomotor tone by releasing vasoactive factors, such as endothelial-derived constricting factors. We observed that alpha-thrombin, but not catalytically inactivated alpha-thrombin, mediated the release of two pulmonary vasoconstrictor peptides into the venous effluent of guinea pig lungs. These peptides elicited a slow-onset, long-lasting pulmonary vasoconstriction similar to the effect of endothelin, an endothelial-derived 21-amino acid vasoconstrictor peptide previously isolated from cells in culture. One of the isolated peptides coelutes with endothelin upon reverse-phase HPLC with an acetonitrile gradient and has a molecular weight comparable to endothelin as determined by gel-permeation HPLC. The other vasoconstrictor peptide elutes earlier than endothelin on reverse-phase HPLC and exhibits a lower molecular weight. The studies show the release of endothelin-like pulmonary vasoconstrictor peptides in the intact lung by alpha-thrombin, a central regulatory enzyme in hemostasis.
CD4-Pseudomonas exotoxin hybrid protein blocks the spread of human immunodeficiency virus infection in vitro and is active against cells expressing the envelope glycoproteins from diverse primate immunodeficiency retroviruses.
Authors: Berger E A , Clouse K A , Chaudhary V K , Chakrabarti S , FitzGerald D J , Pastan I , Moss B
Published: May 2006

We previously described an unusual recombinant protein, designated CD4(178)-PE40, containing the gp120 binding region of human CD4 linked to active regions of Pseudomonas exotoxin A. The ability of this molecule to selectively inhibit protein synthesis in cells expressing the surface envelope glycoprotein of human immunodeficiency virus (HIV) suggested this molecule may be useful in treating infected individuals. To further evaluate its therapeutic potential, several in vitro properties of this hybrid toxin were examined. CD4(178)-PE40 was found to be an extremely potent cytotoxic agent, selectively killing HIV-infected cells with IC50 values around 100 pM. In a coculture system employing mixtures of HIV-infected and -uninfected cells, the hybrid toxin inhibited spread of the infection, as judged by a delay in HIV-induced cell killing and a dramatic suppression of free virus production. Experiments with control recombinant proteins indicated that this protective effect was primarily due to selective killing of the HIV-infected cells, rather than to a simple blocking effect of the CD4 moiety of the hybrid toxin. Using recombinant vaccinia viruses as expression vectors, we found the hybrid toxin to be active against cells expressing the envelope glycoproteins of divergent isolates of HIV-1, as well as HIV-2 and simian immunodeficiency virus. These results provide further support for the therapeutic potential of CD4(178)-PE40 in the treatment of HIV-infected individuals.
Overexpression of src family gene for tyrosine-kinase p59fyn in CD4-CD8- T cells of mice with a lymphoproliferative disorder.
Authors: Katagiri T , Urakawa K , Yamanashi Y , Semba K , Takahashi T , Toyoshima K , Yamamoto T , Kano K
Published: May 2006

Overexpression of a src family gene, lck, has been associated with differentiation of the murine thymic lymphoma line LSTRA. Recent findings by several groups strongly suggest a functional role for the gene product p56lck protein-tyrosine kinase (PTK) in the activation of normal T cells. A single recessive gene, lpr or gld, induces a lymphoproliferative disorder concomitant with autoimmune disease in mice. In this study, a 10-fold elevated activity of PTK encoded by fyn, another src family gene, was demonstrated in CD4-CD8- T cells in mutant mice. The increased PTK activity was consistent with overexpression of fyn mRNA. The elevated fyn mRNA expression appeared to be a characteristic of CD4-CD8- T cells, since it was not observed in normal T cells at any stage of differentiation. The fact that fyn mRNA expression was markedly induced in normal T cells by mitogenic stimulation with anti-T3 epsilon antiserum supports the possibility that p59fyn PTK is a signal-generating molecule in T cells. Thus, our findings provide insight into the physiological role for a src gene family kinase in T-cell development and contribute to a better understanding of the molecular mechanisms of disease-inducing recessive genes.
Identification of cell surface receptors for the 86-kilodalton glycoprotein of human cytomegalovirus.
Authors: Keay S , Merigan T C , Rasmussen L
Published: May 2006

Cell surface receptors for the 86-kDa glycoprotein (gp86) of human cytomegalovirus (HCMV) were identified by using two monoclonal anti-idiotype antibodies that bear the internal image of gp86. These antibodies bound to cells permissive for HCMV infection by both ELISA and immunofluorescence assay and inhibited HCMV plaque formation in human embryonic lung (HEL) cells. Immunoblot analysis showed specific binding of both internal image anti-idiotype antibodies as well as gp86 to an HEL cell membrane protein with an approximate molecular mass of 92.5 kDa. In addition, immunoprecipitation of radiolabeled membrane and cell surface proteins from human foreskin tissue, human foreskin fibroblasts, or HEL cells showed specific binding of anti-idiotype antibody predominantly to the 92.5-kDa protein.
The gene SCL is expressed during early hematopoiesis and encodes a differentiation-related DNA-binding motif.
Authors: Begley C G , Aplan P D , Denning S M , Haynes B F , Waldmann T A , Kirsch I R
Published: May 2006

We have identified the human gene, SCL. We discovered this gene because of its involvement in a chromosomal translocation associated with the occurrence of a stem cell leukemia manifesting myeloid and lymphoid differentiation capabilities. Here we report the sequence of a cDNA for the normal SCL transcript, as well as for an aberrant fusion transcript produced in the leukemic cells. Although different at their 3' untranslated regions, both cDNAs predict a protein with primary amino acid sequence homology to the previously described amphipathic helix-loop-helix DNA binding and dimerization motif of the Ly1-1, myc, MyoD, immunoglobulin enhancer binding, daughterless, and achaete-scute families of genes. For these cDNAs, at least two different 5' ends are predicted, both of which retain this putative DNA binding domain and predict proteins in the range of 20-30 kDa. SCL mRNA is observed in "early" hematopoietic tissues. Taken together, these studies lead to the speculation that SCL plays a role in differentiation and/or commitment events during hematopoiesis.
Molecular and functional characterization of amylin, a peptide associated with type 2 diabetes mellitus.
Authors: Roberts A N , Leighton B , Todd J A , Cockburn D , Schofield P N , Sutton R , Holt S , Boyd Y , Day A J , Foot E A
Published: May 2006

The 37-amino acid peptide called amylin is a major component of the islet amyloid deposited in the pancreases of persons with type 2 diabetes mellitus. We report the isolation of a partial cDNA clone and a phage lambda genomic clone of the coding region of the amylin gene. The DNA sequence encodes a protein sequence identical to that of amylin isolated from the amyloid found in the diabetic pancreas and shows that amylin is likely to be synthesized as a precursor peptide, now named proamylin. We have demonstrated that the amylin gene is present on chromosome 12 and that it is probably transcribed in the islets of Langerhans. The sequences of the genes for amylin and the calcitonin gene-related peptides (CGRPs) show strong similarity, especially over their 5' coding regions, where both peptides have a conserved intramolecular disulfide bridge, and also over their 3' coding regions, where the presence of a glycine codon strongly suggests that the carboxyl-terminal residue of amylin, like that of CGRP, is amidated. To examine the functional relevance of these posttranslational modifications, the biological activity of amylin synthesized with or without the disulfide bridge and/or amidation was measured. It was found that both features are necessary for full biological activity, thereby confirming the functional importance of those regions of the molecule whose sequences are conserved at both protein and genetic levels.
Visualization of crystallin droplets associated with cold cataract formation in young intact rat lens.
Authors: Lo W K
Published: May 2006

The structure, distribution, and nature of scattering elements associated with cold cataract formation in the young rat lens were studied in situ using light and electron microscopy and ImmunoGold electron microscopy. A large accumulation of spherical droplets, ranging from approximately 1.5 microns to 10 microns in diameter, were found in the lens nucleus in cold cataracts induced at 22 degrees C or 4 degrees C in TC-199 culture medium. Many droplets of all sizes were associated with the cell membranes. A cooled and then rewarmed lens was found to lose its opacity and subsequently no droplets were observed, indicating that there was a good correlation between the onset of opacification and the formation of droplets. Electron microscopy showed that droplets were composed of homogeneous electron-dense aggregates without limiting membranes. ImmunoGold study revealed that alpha-, beta-, and gamma-crystallins were all present within each droplet. This study demonstrates that extensive accumulation of the crystallin droplets in the lens nucleus is the contributing factor for the light scattering and opacification of the cold cataract in the young intact rat lens.
Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis.
Authors: Gentz R , Chen C H , Rosen C A
Published: May 2006

Expression of the human immunodeficiency virus tat-encoded protein (Tat) is required for virus replication. A genetic approach was used to facilitate the purification of biologically active Tat. A recombinant Tat protein containing a stretch of six histidine residues and a protease cleavage site was engineered and purified to greater than 95% homogeneity in a single step by immobilized metal-ion chromatography with a special affinity resin that has selectivity for proteins with neighboring histidine residues. A modified scrape loading method for introduction of protein into cell monolayers was used to demonstrate that the purified Tat retained biological activity. Tat function was completely blocked in the presence of transcription inhibitors, which demonstrates the requirement of ongoing mRNA synthesis for trans-activation. These studies indicate that the mechanism of trans-activation is unlikely to involve a direct action of Tat on mRNA stability, transport, or translation and provides the basis for a rapid assay that can be used to identify inhibitors of trans-activation. The methods described herein should be useful for the functional analysis of other proteins that do not confer activity through a receptor-mediated pathway.
Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase.
Authors: Metraux J P , Burkhart W , Moyer M , Dincher S , Middlesteadt W , Williams S , Payne G , Carnes M , Ryals J
Published: May 2006

An extracellular, acidic chitinase was purified to homogeneity from tobacco necrosis virus-infected leaves of Cucumis sativis. The amino acid sequences of the intact protein and of peptides isolated following endoproteinase Lys-C digestion, cyanogen bromide cleavage, and trypsin digestion were determined. Oligonucleotide probes derived from this sequence were used to isolate a cDNA clone encoding this protein. No significant homology was found between this chitinase and either the basic chitinase isolated from bean or tobacco or the chitinase isolated from Serratia marcescens; however, strong homology was found between the cucumber chitinase and a lysozyme/chitinase from Parthenocissus quinquifolia. The induction of the protein by tobacco necrosis virus infection or salicylate was found to be at the level of RNA accumulation. Genomic Southern analysis indicates that a single gene in the cucumber genome encodes this protein.
Thrombin treatment induces rapid changes in tyrosine phosphorylation in platelets.
Authors: Golden A , Brugge J S
Published: May 2006

We previously demonstrated that platelets express high levels of the tyrosine protein kinase pp60c-src. By a quantitative immunoblot assay, it is shown in this report that pp60c-src represents 0.2-0.4% of total platelet protein. The expression of high levels of pp60c-src in platelets correlated with high levels of total cell phosphotyrosine. Unstimulated platelets were shown to possess numerous phosphotyrosine-containing proteins by immunoblot analysis using antibodies that specifically recognize phosphotyrosine residues. To examine whether the pattern of phosphotyrosine-containing proteins changes upon platelet activation, lysates from thrombin- and phorbol ester-treated platelets were subjected to immunoblot analysis. Novel phosphotyrosine-containing proteins were detected within seconds following platelet stimulation. These results suggest that tyrosine phosphorylation, perhaps mediated by pp60c-src, may be involved in events associated with platelet activation.
A plasmid-encoded regulatory gene, rns, required for expression of the CS1 and CS2 adhesins of enterotoxigenic Escherichia coli.
Authors: Caron J , Coffield L M , Scott J R
Published: May 2006

To be virulent, enterotoxigenic Escherichia coli (ETEC) must produce a toxin and a pilus-like structure that mediates specific attachment to host tissue. Expression of two of these specific adherence structures, CS1 and CS2, requires the presence of a plasmid in an ETEC strain of a particular serotype and biotype. We show here that this plasmid does not contain the structural gene for a pilin protein, as previously believed. Instead we have identified a plasmid-encoded gene called rns that is required for expression of CS1 or CS2 colonization factor antigens and for adhesion. The rns gene, defined by two separately isolated insertion mutations, produces a 26-kDa protein when transcribed and translated in vitro. At the protein level the rns gene product is homologous to AraC, a positive regulator of the arabinose operon of enteric bacteria, and to RhaR and RhaS, which regulate the rhamnose operon of E. coli. The homology of the Rns protein to AraC is localized to regions that are believed to bind to DNA. Moreover, the sequence of one of these homologous regions is consistent with a DNA binding helix-turn-helix motif. The average G + C content of E. coli DNA is 50%; yet the rns gene contains only 28% G + C, suggesting that it was acquired from some other organism.
Isolation of two alleles of the b locus of Ustilago maydis.
Authors: Kronstad J W , Leong S A
Published: May 2006

The basidiomycete Ustilago maydis is the causative agent of the disease corn smut. To be pathogenic, strains of U. maydis must be heterozygous for a locus called "b," which appears to control both pathogenicity and sexual development. Two alleles of the b locus of U. maydis were isolated by complementation and hybridization. The clones have the specificities of b1 and b2 alleles as demonstrated by their effects on the colony morphology and pathogenicity of haploid and diploid strains upon transformation. For example, nonpathogenic haploid and diploid strains of U. maydis carrying b2 alleles became pathogenic when transformed with the cloned b1 allele. Furthermore, an a2 b2 haploid strain could be transformed to an a2 b1 genotype by gene replacement using a DNA fragment containing a b1 allele and a selectable marker. The isolation of b alleles represents an important step toward understanding the control of dicaryon formation, dicaryon maintenance, and pathogenicity in U. maydis.
Molecular patterns of X chromosome-linked color vision genes among 134 men of European ancestry.
Authors: Drummond-Borg M , Deeb S S , Motulsky A G
Published: May 2006

We used Southern blot hybridization to study X chromosome-linked color vision genes encoding the apoproteins of red and green visual pigments in 134 unselected Caucasian men. One hundred and thirteen individuals (84.3%) had a normal arrangement of their color vision pigment genes. All had one red pigment gene; the number of green pigment genes ranged from one to five with a mode of two. The frequency of molecular genotypes indicative of normal color vision (84.3%) was significantly lower than had been observed in previous studies of color vision phenotypes. Color vision defects can be due to deletions of red or green pigment genes or due to formation of hybrid genes comprising portions of both red and green pigment genes [Nathans, J., Piantanida, T.P., Eddy, R.L., Shows, T.B., Jr., & Hogness, D.S. (1986) Science 232, 203-210]. Characteristic anomalous patterns were seen in 15 (11.2%) individuals: 7 (5.2%) had patterns characteristic of deuteranomaly (mild defect in green color perception), 2 (1.5%) had patterns characteristic of deuteranopia (severe defect in green color perception), and 6 (4.5%) had protan patterns (the red perception defects protanomaly and protanopia cannot be differentiated by current molecular methods). Previously undescribed hybrid gene patterns consisting of both green and red pigment gene fragments in addition to normal red and green genes were observed in another 6 individuals (4.5%). Only 2 of these patterns were considered as deuteranomalous. Thus, DNA testing detected anomalous color vision pigment genes at a higher frequency than expected from phenotypic color vision tests. Some color vision gene arrays associated with hybrid genes are likely to mediate normal color vision.
Identification of a phorbol ester-repressible v-src-inducible gene.
Authors: Simmons D L , Levy D B , Yannoni Y , Erikson R L
Published: May 2006

Chicken embryo fibroblasts (CEF) infected with a temperature-sensitive Rous sarcoma virus (RSV) mutant, tsNY72-4, express a set of pp60v-src-induced RNAs soon after shift to the permissive temperature. By subtractive and differential screening, we have cloned 12 of these sequences, 2 of which were c-fos and krox-24. Serum induced all the v-src-inducible genes tested, suggesting that these genes serve roles in normal cell division and are not specific to transformation per se. Significantly, however, v-src produced prolonged, and in some cases kinetically complex, patterns of induction compared to serum. For most of the clones, phorbol 12-tetradecanoate 13-acetate (TPA) induced mRNAs with kinetics similar to that of serum. However, one clone (CEF-4) was expressed in a biphasic manner. Another (CEF-10) was repressed by TPA at 1 hr, after which this mRNA was permanently induced. The pattern of repression-induction of CEF-10 mRNA is the inverse of protein kinase C (PKC) activity in the cell, suggesting that PKC actively represses this gene. In vivo expression of CEF-10 mRNA is restricted predominantly to the lung. A full-length CEF-10 cDNA encodes a 41-kDa protein that has an amino-terminal signal peptide for secretion, contains a markedly high number of cysteine residues, and shows no sequence similarity to known proteins.
Sequential expression and cooperative interaction of c-Ha-ras and c-erbB genes in in vivo chemical carcinogenesis.
Authors: Husain Z , Fei Y B , Roy S , Solt D B , Polverini P J , Biswas D K
Published: May 2006

The level of expression of several cellular protooncogenes is examined at different stages of 7,12-dimethylbenzanthracene (DMBA)-induced tumor development in hamster buccal pouch epithelium (HBPE). Results presented demonstrate overexpression of c-Ha-ras gene at a very early stage of tumor development, and this elevated level of expression of the gene persists throughout the tumorigenesis process. The expression of the cellular protooncogene c-erbB, on the other hand, can be detected only after 8-10 weeks of DMBA treatment of the tissue and increases with the progression of the disease. The overexpression of c-erbB gene can be correlated with the stage of extensive proliferation and subsequent invasion of the HBPE cells into the underlying connective tissue. This sequential pattern of stage-specific expression of the two cellular protooncogenes can be observed in (i) treated tissues, (ii) stage-representative cultured cells, and (iii) NIH 3T3 transformants derived with DNA from HBPE cells. The low-level expression of c-myc and c-sis genes detected in control tissues remains unaffected, while c-fos gene activity cannot be detected at any stage of tumor development. The overexpression of c-Ha-ras gene alone in HBPE cells derived from tissues treated for 5 weeks (DM5) is not sufficient to induce tumors in athymic mice, whereas expression of c-Ha-ras and c-erbB genes at later stages of tumor development (DM10 and HCPC cells) induce histopathologically defined epithelial cell carcinoma in athymic mice within 2-3 weeks. The sequential overexpression of c-Ha-ras and c-erbB genes in a stage-specific manner and their cooperative interaction in the DMBA-induced in vivo oral carcinogenesis have been demonstrated.
Effect of recombinant factor VIIa on the hemostatic defect in dogs with hemophilia A, hemophilia B, and von Willebrand disease.
Authors: Brinkhous K M , Hedner U , Garris J B , Diness V , Read M S
Published: May 2006

Recombinant factor VIIa (rF.VIIa) is a two-chain procoagulant enzyme (Mr, approximately 50,000) active only when complexed with tissue factor in the extrinsic clotting system. We administered human rF.VIIa to hemophilic and von Willebrand disease (vWD) dogs to determine its hemostatic effectiveness and survival in the circulation. Hemophilia A dogs lacking factor VIII demonstrated an immediate increase in plasma rF. VIIa and prompt stoppage of hemorrhage at bleeding time (BT) sites. In seven studies in two dogs, the range of dose of rF. VIIa was 50-220 micrograms/kg, with an apparent 7- to 11-fold increase in plasma factor VII and a mean recovery in plasma of 34%. The t1/2 was 2.8 +/- 0.5 hr. The BT was normalized except in an animal given the minimum dose. In four studies in two hemophilia B dogs lacking factor IX, BT was normalized. The elevation in plasma factor VII was by a factor of 8-30, with a mean recovery of rF.VIIa in plasma of 44%. In two studies in a homozygous vWD dog lacking von Willebrand factor, which is needed for platelet function, BT was not corrected even though large doses of rF. VIIa were given. The human rF. VIIa protein was immunogenic for dogs. These studies indicate that factor VIIa corrects the hemostatic defect in dogs with hemophilia A and B, diseases primarily of the intrinsic clotting system, but does not correct the hemostatic defect in vWD.
Dopamine turnover and glutathione oxidation: implications for Parkinson disease.
Authors: Spina M B , Cohen G
Published: May 2006

Parkinson disease is characterized by a major loss (approximately 80% or more) of dopaminergic nigrostriatal neurons and by an increased turnover of neurotransmitter by surviving neurons of the nigrostriatal tract. In theory, increased turnover of dopamine should be associated with an oxidative stress derived from increased production of hydrogen peroxide. The peroxide is formed during the oxidative deamination of dopamine by monoamine oxidase. In experiments with mice, increased presynaptic turnover of dopamine was evoked by injection of reserpine, which interferes with the storage of dopamine in synaptic vesicles. Loss of dopamine and formation of deaminated metabolites were accompanied by a significant rise (87.8%) in the level of oxidized glutathione in brain. This change was observed in the striatum, which is richly innervated by dopamine terminals, but not in the frontal cortex, which receives a much sparser innervation by catecholamine nerve terminals. The rise in oxidized glutathione was seen even though dopamine terminals constitute only 1% or less of the mass of the striatum. Clorgyline, an inhibitor of monoamine oxidase type A, blocked the formation of oxidized glutathione. These observations confirm that a selective increase in neurotransmitter turnover within nigrostriatal nerve terminals can evoke a change in cellular redox status. We suggest that an oxidative stress may play a role in the natural history of Parkinson disease.
Regulation of duodenal bicarbonate secretion during stress by corticotropin-releasing factor and beta-endorphin.
Authors: Lenz H J
Published: May 2006

Proximal duodenal mucosal bicarbonate secretion is an important factor in the pathogenesis of duodenal ulcer disease. To examine the central nervous system regulation of duodenal bicarbonate secretion, an animal model was developed that allowed cerebroventricular and intravenous injections as well as collection of duodenal perfusates in awake, freely moving rats. The hypothalamic peptide corticotropin-releasing factor (CRF) and stress (physical restraint) significantly stimulated duodenal bicarbonate secretion. These responses were abolished by pretreatment of the animals with the CRF receptor antagonist alpha-helical CRF-(9-41), hypophysectomy, and naloxone. In contrast, blockade of autonomic efferents by surgical and pharmacological means did not prevent the stimulatory effects of stress and CRF. Intravenous, but not cerebroventricular, administration of beta-endorphin that produced plasma concentrations of beta-endorphin that were similar to those produced by exogenous CRF and stress significantly stimulated duodenal bicarbonate secretion. These results indicate that endogenous CRF released during stress and exogenously administered CRF stimulate duodenal bicarbonate secretion by release of beta-endorphin from the pituitary, thus, demonstrating a functional hypothalamus-pituitary-gut axis.