bluuebunny/crossref_metadata_2025 huggingface dataset:
Early gene regulation by nerve growth factor in PC12 cells: induction of an interferon-related gene.
Authors: Tirone F , Shooter E M
Published: May 2006

Nerve growth factor (NGF) induces the chromaffin cell line PC12 to differentiate into cells with many of the properties of sympathetic neurons. We investigated the early differentiative phase and identified a gene, PC4, rapidly and transiently induced by NGF in PC12 cells. PC4 cDNA is homologous to the partial sequence of a putative mouse beta-interferon and encodes a protein related to a lymphokine, the rat gamma-interferon protein. Nonetheless, PC4 appears devoid of antiviral activity. PC4 is expressed in proliferating and differentiating tissues, such as amnion, placenta, and brain at embryonic day 13.5. The relationship of PC4 to interferons and lymphokines suggests that it could play a role in regulating gene activity in the pathways induced by NGF.
DNA bending is induced by a transcription factor that interacts with the human c-FOS and alpha-actin promoters.
Authors: Gustafson T A , Taylor A , Kedes L
Published: May 2006

Conserved sequence elements in the human cardiac and skeletal alpha-actin promoters that contain the CC(A + T-rich)6GG motif have been shown to regulate transcription of these genes. A similar sequence is found in the serum response element of the human c-FOS gene. In this study, we demonstrate that indistinguishable proteins bind to each of five CC(A + T-rich)6GG elements examined in the human cardiac and skeletal alpha-actin promoters and the c-FOS serum response element. Using electrophoretic techniques, we show that these factors induce a stable bend in the DNA upon binding, and the bend center is shown to coincide with the CC(A + T-rich)6GG element. In addition, the ability to bend DNA is retained by a small proteolytic fragment of the protein, suggesting that the DNA-binding domain of the protein is resistant to proteases and is sufficient to bend DNA.
High frequency of large spontaneous deletions of DNA in tumor-derived CHEF cells.
Authors: Kaden D A , Bardwell L , Newmark P , Anisowicz A , Skopek T R , Sager R
Published: May 2006

Spontaneous mutations arising at the HPRT locus were examined in 126 mutants recovered from a series of six CHEF-derived cell lines. Altered restriction fragment patterns were characterized by Southern blot hybridization, and gene expression by RNA blot hybridization. Point mutants and gene-expression mutants predominated in the control (nontumorigenic) 18-1D-3 cell line and in two tumor-derived lines, one of which (16-2 Tuk 4) displayed a mutator phenotype. In the other three lines, the majority of mutants had large partial or whole gene deletions. These results suggest that mutant enzymes in DNA replication or repair play an important role in neoplastic progression by causing extensive deletions in DNA, including excision of genes that encode tumor-suppressor functions, and deletion of regulatory sequences in protooncogenes.
Molecular heterogeneity of gamma delta T-cell antigen receptors expressed by CD4- CD8- T-cell clones from normal donors: both disulfide- and non-disulfide-linked receptors are delta TCS1+.
Authors: Seki H , Nanno M , Chen P F , Itoh K , Ioannides C , Good R A , Platsoucas C D
Published: May 2006

We investigated the molecular heterogeneity of gamma delta T-cell antigen receptors (TCR) expressed on T-cell clones generated from peripheral blood lymphocytes of normal donors. Extensive molecular heterogeneity was seen at the gamma-chain level and, to a lesser extent, at the delta-chain level. Both disulfide and non-disulfide gamma delta TCR were found and use different gamma chains with similar molecular masses (range, 41-43 kDa). In contrast, gamma chains of 55-60 kDa, which are expressed on T-cell lines derived from the peripheral blood of patients with immunodeficiency disorders, were not found on T-cell clones derived from the peripheral blood of normal donors. delta chains expressed on these T-cell clones had a molecular mass of 37 kDa and were either disulfide or nondisulfide linked. Significant delta-chain heterogeneity was identified in these clones using the anti-delta TCS1 and the anti-TCR delta 1 monoclonal antibodies. All clones tested were TCR delta 1+, whereas only 25% of the clones were delta TCS1+. The anti-delta TCS1 monoclonal antibody stained and immunoprecipitated both disulfide- and non-disulfide-linked gamma delta TCRs from different T-cell clones from normal donors.
Reversal of natural killing susceptibility in target cells expressing transfected class I HLA genes.
Authors: Storkus W J , Alexander J , Payne J A , Dawson J R , Cresswell P
Published: May 2006

A number of studies have suggested that resistance of target cells to natural killing (NK) may be correlated with their level of expression of major histocompatibility complex (MHC) class I antigens. To examine this hypothesis directly, a NK-sensitive class I-deficient human B-cell line was transfected with MHC class I genes. The expression of transfected HLA, but not H-2, class I gene products resulted in loss of susceptibility to human NK-mediated conjugation and cytolysis. Furthermore, this protection did not extend to cytotoxicity mediated by interleukin 2-stimulated human NK effector cells.
Ovarian steroid treatment blocks a postmenopausal increase in blood monocyte interleukin 1 release.
Authors: Pacifici R , Rifas L , McCracken R , Vered I , McMurtry C , Avioli L V , Peck W A
Published: May 2006

In previous studies, we showed that blood monocyte elaboration of interleukin 1 (IL-1), a known stimulator of bone resorption, was higher in osteoporotic patients with rapid bone turnover than in those with slow turnover and in nonosteoporotic subjects. Since an acceleration of bone loss following menopause contributes to the risk of osteoporosis in women, we have studied the effects of menopause and ovarian steroid treatment on IL-1 release by monocytes obtained from nonosteoporotic and osteoporotic women. IL-1 activity in the monocyte culture medium derived from untreated postmenopausal women (nonosteoporotic and osteoporotic) was higher than in the medium derived from either untreated premenopausal or estrogen/progesterone-treated postmenopausal women. A significant negative correlation was found between IL-1 and years since menopause in both the healthy (r = -0.75; P less than 0.005) and the osteoporotic (r = -0.61; P less than 0.01) untreated postmenopausal women. The difference between the two slopes was significant at P less than 0.05. Premenopausal IL-1 levels were achieved within 8 years of menopause in the nonosteoporotic, but not in the osteoporotic, subjects in whom increases were evident as long as 15 years after menopause. IL-1 also correlated inversely with vertebral mineral density (r = -0.37; P less than 0.05), as measured by quantitative computed tomography. In prospective studies, treatment with estrogen/progesterone for 1 month caused a substantial highly significant decrease in IL-1 activity in each of three nonosteoporotic and five osteoporotic women, confirming the apparent effect of hormone therapy observed in the cross-sectional analysis. Although a cause-effect relationship has not been established, it is our hypothesis, based on these data, that alterations in IL-1 production may underlie the postmenopausal acceleration in bone loss and its inhibition by ovarian steroids. Persistent elevation of IL-1 secretion appears to be a feature of postmenopausal osteoporosis.
Selective infection of human CD4+ cells by simian immunodeficiency virus: productive infection associated with envelope glycoprotein-induced fusion.
Authors: Koenig S , Hirsch V M , Olmsted R A , Powell D , Maury W , Rabson A , Fauci A S , Purcell R H , Johnson P R
Published: May 2006

Simian immunodeficiency virus (SIV) and human immunodeficiency virus share the property of tropism for CD4-bearing cells. Infection is initiated by a high-affinity interaction between CD4 and conserved domains on the viral envelope glycoprotein. In this report, we demonstrated that SIV had a restricted host range among human CD4+ cells when compared with human immunodeficiency virus type 1 or type 2. This restricted tropism was associated with the inability of the SIV envelope glycoprotein to induce membrane fusion in cells not susceptible to productive exogenous infection by SIV. We conclude that the major route of SIV entry into CD4+ cells is by envelope-mediated direct fusion with the cell and that additional envelope-cell interactions after CD4 binding are required for productive infection.
In photosynthetic reaction centers, the free energy difference for electron transfer between quinones bound at the primary and secondary quinone-binding sites governs the observed secondary site specificity.
Authors: Giangiacomo K M , Dutton P L
Published: May 2006

The secondary quinone-binding site (QB site) of bacterial reaction centers from Rhodobacter sphaeroides is generally regarded to be highly specific for its native ubiquinone-10 molecule. We demonstrate here that this is a misconception rooted in the kinetic methods used to assay for occupancy of a quinone in the QB site. We show that observance of occupancy of the QB site, revealed by kinetic assay, is sensitive to the free-energy difference for electron transfer between the quinone at the primary quinone-binding site (QA site) and the QB site (-delta G0e-). For many of the compounds previously tested for binding at the QB site, the -delta G0e- between QA and QB is too small to permit detection of the functional quinone in the QB site. With an increased -delta G0e- achieved by replacing the native ubiquinone-10 at the QA site with lower-potential quinones or by testing higher-potential QB candidates, it is shown that the QB site binds and functions with the unsubstituted 1,4-benzoquinone, 1,4-naphthoquinone, and 9,10-phenanthraquinone, as well as with their various substituted forms. Moreover, quinones with the ortho-carbonyl configuration appear to function in a similar manner to quinones with the para-carbonyl configuration.
Medium-chain fatty acid binding to albumin and transfer to phospholipid bilayers.
Authors: Hamilton J A
Published: May 2006

Temperature-dependent (5-42 degrees C) 13C NMR spectra of albumin complexes with 90% isotopically substituted [1-13C]decanoic acids (3 mol of fatty acid per mol of albumin) showed a single peak at greater than 30 degrees C but three peaks at lower temperatures. The chemical-shift differences result from different ionic and/or hydrogen-bonding interactions between amino acid side chains and the fatty acid carboxyl carbon. Rapid exchange of fatty acid among binding sites obscures these sites at temperatures greater than 30 degrees C. Rate constants for exchange at 33 degrees C were 350 sec-1 for octanoate and 20 sec-1 for decanoate, corresponding to lifetimes in a binding site of 2.8 msec (octanoate) and 50 msec (decanoate). Temperature-dependent data for octanoate showed an activation energy of 2 kcal/mol for exchange. Spectra of albumin complexes with the 12-carbon saturated fatty acid, lauric acid, had several narrow laurate carboxyl peaks at 35 degrees C, indicating longer lifetimes (tau much greater than 66 msec) in the different binding sites. Fatty acid exchange between albumin and model membranes (phosphatidylcholine bilayers) occurred on a time scale comparable to that for exchange among albumin binding sites, following the order octanoate greater than decanoate greater than laurate. The equilibrium distribution of fatty acid between lipid bilayers and protein was measured directly from NMR spectra. Decreasing pH (8.0 to 5.5) increased the relative affinity of fatty acid for the lipid bilayer. The results predict that the relative affinity of octanoic acid for albumin and membranes will be similar to that of long-chain fatty acids (e.g., oleic acid), but the rate of equilibration will be approximately 10(4) faster for octanoic acid.
Molecular characterization of sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome.
Authors: Shelley C S , Remold-O'Donnell E , Davis A E , Bruns G A , Rosen F S , Carroll M C , Whitehead A S
Published: May 2006

Sialophorin (CD43) of leukocytes and platelets is a surface sialoglycoprotein that is phenotypically defective on lymphocytes of patients with the X chromosome-linked immunodeficiency Wiskott-Aldrich syndrome. Previous studies with monoclonal antibodies indicate that sialophorin is a component of a T-lymphocyte activation pathway. Here we describe the cDNA cloning and derived amino acid sequence of human sialophorin. The sequence predicts an integral membrane polypeptide with an N-terminal hydrophobic signal region followed by a mucin-like 235-residue extracellular region with a uniform distribution of 46 serine, 47 threonine, and 24 proline residues. This is followed by a 23-residue transmembrane region and a 123-residue C-terminal intracellular region. These latter regions have been highly conserved during evolution; the intracellular region contains a number of potential phosphorylation sites that might mediate transduction of activation signals. The chromosomal location of the sialophorin gene was determined and the implications of this assignment for the pathogenesis of the Wiskott-Aldrich syndrome are discussed.
Two motion perception mechanisms revealed through distance-driven reversal of apparent motion
Authors: Chubb Charles , Sperling George
Published: May 2006

We demonstrate two kinds of visual stimuli that exhibit motion in one direction when viewed from near and in the opposite direction from afar. These striking reversals occur because each kind of stimulus is constructed to simultaneously activate two different mechanisms: a short-range mechanism that computes motion from space-time correspondences in stimulus luminance and a long-range mechanism in which motion computations are performed, instead, on stimulus contrast that has been full-wave rectified (e.g., on the absolute value of contrast).
Protein fragments as probes in the study of protein folding mechanisms: differential effects of dihydrofolate reductase fragments on the refolding of the intact protein.
Authors: Hall J G , Frieden C
Published: May 2006

We describe an approach for investigating the protein folding process, using protein fragments as inhibitory probes of the refolding protein. The refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3), reversibly unfolded in 7 M urea, was monitored by the reappearance of enzyme activity after diluting the unfolded enzyme into low urea concentrations (less than or equal to 2 M) in the presence of substrates. Of eight protein fragments produced by limited proteolysis of the 159-residue enzyme, three isolated peptides--Ser-49/Glu-90, Ile-91/Glu-154, and Gln-102/Glu-154--were evaluated for their effects on the recovery of the refolding protein's enzymatic activity. By this criterion, 13 microM peptide Gln-102/Glu-154 inhibits the refolding of 0.015 microM enzyme by approximately 80%, while the related peptide, Ile-91/Glu-154, and peptide Ser-49/Glu-90 at the same concentration inhibit the recoverable activity of the refolding enzyme by less than or equal to 20%. None of these three peptides has any significant effect on the activity of the folded enzyme. Our results indicate that peptides may inhibit refolding differentially and that these effects may be extremely sensitive to fragment sequence and composition. We suggest that peptide specificity in the inhibition of protein folding may be exploited as a structural probe of protein folding mechanisms.
Sequence, tissue distribution, and differential expression of mRNA for a putative insulin-responsive glucose transporter in mouse 3T3-L1 adipocytes.
Authors: Kaestner K H , Christy R J , McLenithan J C , Braiterman L T , Cornelius P , Pekala P H , Lane M D
Published: May 2006

The cDNAs for two putative glucose transporters from mouse 3T3-L1 adipocytes were isolated and sequenced. One of these cDNAs encodes the murine homolog of the human hepG2/erythrocyte glucose transporter, termed GT1. GT1 mRNA is most abundant in mouse brain and is expressed in both 3T3-L1 preadipocytes and adipocytes. The other cDNA encodes a glucose transporter-like protein, termed GT2, that has a unique amino acid sequence and tissue distribution. GT2 cDNA encodes a protein with 63% amino acid sequence identity and a similar structural organization to GT1. GT2 mRNA is found at high levels in mouse skeletal muscle, heart, and adipose tissue, all of which exhibit insulin-stimulated glucose uptake. GT2 mRNA is absent from 3T3-L1 preadipocytes but is induced dramatically during differentiation into adipocytes. This increase in mRNA content correlates closely with the acquisition of insulin-stimulated glucose uptake. We propose that GT2 is an insulin-regulated glucose transporter.
Direct and sex-specific stimulation by sex steroids of creatine kinase activity and DNA synthesis in rat bone.
Authors: Sömjen D , Weisman Y , Harell A , Berger E , Kaye A M
Published: May 2006

A direct in vitro effect of 17 beta-estradiol (E2) was demonstrated on bone and cartilage cell energy metabolism. Sex-specific stimulation by E2 and testosterone was shown in diaphyseal bone of weanling rats. E2 (30 nM) caused, within 24 hr, a 70-200% increase in creatine kinase (CK; ATP:creatine N-phosphotransferase, EC 2.7.3.2) specific activity in ROS 17/2.8 rat osteogenic sarcoma cells, MC3T3-E1 mouse calvaria-derived cells, and rat fetal calvaria cells, and a 40% increase in rat epiphyseal cartilage cells. Stimulation of CK activity by E2 was dose and time dependent: in ROS 17/2.8 cells, a highly significant increase was found at 3 nM E2 and a greater than 100% increase in CK activity was found 1 hr after E2 administration. In female 20-day-old Wistar-derived rats, E2 (5 micrograms per rat) increased CK activity in diaphyseal bone by 82% within 1 hr of i.p. injection, with a maximal increase of 200% after 24 hr; neither the weakly estrogenic agonist 17 alpha-estradiol, testosterone, nor progesterone showed this effect. Conversely, in male rat diaphyseal bone, testosterone or dihydrotestosterone increased CK activity after 24 hr by approximately 100%, while E2 was ineffective. In epiphyseal cartilage, both E2 and testosterone increased CK activity. Stimulation of CK activity by sex hormones was paralleled by significant increases in [3H]thymidine incorporation into DNA. Therefore, it is possible that direct sex-specific actions of gonadal steroids may contribute to stimulating bone growth and maintaining balanced bone turnover.
Role of oligosaccharides in the processing and maturation of envelope glycoproteins of human immunodeficiency virus type 1.
Authors: Pal R , Hoke G M , Sarngadharan M G
Published: May 2006

The processing and maturation of envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) were studied in infected cells treated with inhibitors of oligosaccharide processing. In MOLT-3 cells chronically infected with HIV-1 (strain HTLV-IIIB), tunicamycin severely inhibited the glycosylation of envelope proteins. Deoxynojirimycin, an inhibitor of glucosidase I in the rough endoplasmic reticulum, inhibited the proteolytic processing of gp160, whereas no such effect was noted with either deoxymannojirimycin or swainsonine, inhibitors of mannosidase I and II, respectively, in the Golgi complex. The processed gp120 and gp41 synthesized in the presence of deoxymannojirimycin were found to contain mannose-rich oligosaccharide cores as evidenced by their susceptibility to endoglycosidase H digestion. The formation of syncytia normally observed when CEM cells are cocultured with HIV-1-infected cells was markedly inhibited in the presence of deoxynojirimycin, but such inhibition was not observed in cells treated with deoxymannojirimycin or swainsonine. The infectivity of virions released from MOLT-3/HTLV-IIIB cells treated with deoxynojirimycin or deoxymannojirimycin was significantly lower than the infectivity of virions released from untreated cells. On the other hand, treatment with swainsonine did not affect the infectivity of the progeny virus. These results suggest that the proteolytic processing of gp160 takes place in infected cells when the glycoprotein has mannose-rich oligosaccharide structures. Trimming of glucose residues and the primary trimming of mannose residues are necessary for the release of infectious virus.
Selectivity of the cleavage/attachment site of phosphatidylinositol-glycan-anchored membrane proteins determined by site-specific mutagenesis at Asp-484 of placental alkaline phosphatase.
Authors: Micanovic R , Gerber L D , Berger J , Kodukula K , Udenfriend S
Published: May 2006

Many proteins are now known to be anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety that is attached to their COOH termini. Placental alkaline phosphatase (PLAP) has been used as a model for investigating mechanisms involved in the COOH-terminal processing of PI-G-tailed proteins. The COOH-terminal domain of pre-pro-PLAP provides a signal for processing during which a largely hydrophobic 29-residue COOH-terminal peptide is removed, and the PI-G moiety is added to the newly exposed Asp-484 terminus. This cleavage/attachment site was subjected to an almost saturation mutagenesis, and the enzymatic activities, COOH-terminal processing, and cellular localizations of the various mutant PLAP forms were determined. Substitution of Asp-484 by glycine, alanine, cysteine, asparagine, or serine (category I) resulted in PI-G-tailed and enzymatically active proteins. However, not all category I mutant proteins were PI-G tailed to the same extent. Pre-pro-PLAP with other substituents at position 484 (threonine, proline, methionine, valine, leucine, tyrosine, tryptophan, lysine, glutamic acid, and glutamine; category II) were expressed, as well as the category I amino acids, but there was little or no processing to the PI-G-tailed form, and this latter group exhibited very low enzyme activity. The bulk of the PLAP protein produced by category II mutants and some produced by category I mutants were sequestered within the cell, apparently in the endoplasmic reticulum (ER). Most likely, certain amino acids at residue 484 are preferred because they yield better substrates for the putative "transamidating" enzyme. In transfected COS cells, at least, posttranslational PI-G-tail processing does not go to completion even for preferred substrates. Apparently PI-G tailing is a requisite for transport from the ER and for PLAP enzyme activity. Proteins that are not transamidated are apparently retained in the ER in an inactive conformation.
The anterior cingulate cortex mediates processing selection in the Stroop attentional conflict paradigm.
Authors: Pardo J V , Pardo P J , Janer K W , Raichle M E
Published: May 2006

Regional cerebral blood flow, an index of local neuronal activity, was measured using positron emission tomography (PET) during the performance of the classic Stroop color/word task in eight healthy right-handed subjects. In the first condition of this paradigm, subjects name the color of the words presented on a video monitor. All the words are the color names congruent to the color presented (e.g., the noun "red" displayed in red color). In the second condition, subjects also name the color of the words presented on the monitor. However, during these trials all words are color names incongruent to the color presented (e.g., the noun "red" displayed in green color). The difference in brain activity between these two conditions (i.e., incongruent minus congruent) could reveal brain systems involved in the attentionally mediated resolution of the conflict between the habitual response of reading words vs. the task demands of naming the color of the words--i.e., the Stroop interference effect. The most robust responses occurred in the anterior cingulate cortex. Other responses noted were in the left premotor cortex, left postcentral cortex, left putamen, supplementary motor area, right superior temporal gyrus, and bilateral peristriate cortices. These data provide support for the role of the anterior cingulate cortex in attentional processing through the selection and recruitment of processing centers appropriate for task execution. Furthermore, the extensive distributed network of activated regions suggests that the Stroop interference effect cannot be explained simply in terms of stimulus encoding or response interference.
Human gene transfer: characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man.
Authors: Kasid A , Morecki S , Aebersold P , Cornetta K , Culver K , Freeman S , Director E , Lotze M T , Blaese R M , Anderson W F
Published: May 2006

Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible, we transduced human TILs with the bacterial gene for neomycin-resistance (NeoR) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact NeoR gene integration and expressed high levels of neomycin phosphotransferase activity. The NeoR gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for beta- and gamma-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the beta and gamma chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors (alpha and beta) and interleukin 2 receptor P55 but not for interleukin 1 beta, granulocyte/macrophage colony-stimulating factor, interleukin 6, and interferon gamma when grown with high-dose interleukin 2 without subsequent activation with mitogen or specific antigen. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The NeoR gene-transduced TILs could thus be used to follow the trafficking and survival of TILs in vivo, and clinical protocols using these transduced TILs in cancer patients have begun. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs.
Regulation of glutathione S-transferase Ya subunit gene expression: identification of a unique xenobiotic-responsive element controlling inducible expression by planar aromatic compounds.
Authors: Rushmore T H , King R G , Paulson K E , Pickett C B
Published: May 2006

We have identified a region in the 5' flanking sequence of the glutathione S-transferase (RX:glutathione R-transferase, EC 2.5.1.18) Ya subunit gene that contains a unique xenobiotic-responsive element (XRE). The regulatory region spans nucleotides -722 to -682 of the 5' flanking sequence and is responsible for part of the basal level as well as inducible expression of the Ya subunit gene by planar aromatic compounds such as beta-naphthoflavone (beta-NF) and 3-methyl-cholanthrene. The DNA sequence of this region (beta-NF-responsive element) is distinct from the DNA sequence of the XRE found in the cytochrome P-450 IA1 gene. In addition to the region containing the beta-NF-responsive element, two other regulatory regions of the Ya subunit gene have been identified. One region spans nucleotides -867 to -857 and has a DNA sequence with identity to the hepatocyte nuclear factor 1 recognition motif found in several liver-specific genes. The second region spans nucleotides -908 to -899 and contains a DNA sequence with identity to the XRE found in the cytochrome P-450 IA1 gene. The XRE sequence also contributes to part of the responsiveness of the Ya subunit gene to planar aromatic compounds. Our data suggest that regulation of gene expression by planar aromatic compounds can be mediated by a DNA sequence that is distinct from the XRE sequence.
Chromosomal protein HMG-14 gene maps to the Down syndrome region of human chromosome 21 and is overexpressed in mouse trisomy 16.
Authors: Pash J , Popescu N , Matocha M , Rapoport S , Bustin M
Published: May 2006

The gene for human high-mobility-group (HMG) chromosomal protein HMG-14 is located in region 21q22.3, a region associated with the pathogenesis of Down syndrome, one of the most prevalent human birth defects. The expression of this gene is analyzed in mouse embryos that are trisomic in chromosome 16 and are considered to be an animal model for Down syndrome. RNA blot-hybridization analysis and detailed analysis of HMG-14 protein levels indicate that mouse trisomy 16 embryos have approximately 1.5 times more HMG-14 mRNA and protein than their normal littermates, suggesting a direct gene dosage effect. The HMG-14 gene may be an additional marker for the Down syndrome. Chromosomal protein HMG-14 is a nucleosomal binding protein that may confer distinct properties to the chromatin structure of transcriptionally active genes and therefore may be a contributing factor in the etiology of the syndrome.
Molecular basis for dominantly inherited inclusion body beta-thalassemia.
Authors: Thein S L , Hesketh C , Taylor P , Temperley I J , Hutchinson R M , Old J M , Wood W G , Clegg J B , Weatherall D J
Published: May 2006

Analysis of the molecular basis of dominantly inherited beta-thalassemia in four families has revealed different mutations involving exon 3 of the beta-globin gene. It is suggested that the phenotypic difference between this condition and the more common recessive forms of beta-thalassemia lies mainly in the length and stability of the abnormal translation products that are synthesized and, in particular, whether they are capable of binding heme and producing aggregations that are relatively resistant to proteolytic degradation.
Epidermal growth factor or transforming growth factor alpha is required for kidney tubulogenesis in matrigel cultures in serum-free medium.
Authors: Taub M , Wang Y , Szczesny T M , Kleinman H K
Published: May 2006

The ability of matrigel, a reconstituted basement membrane gel, to induce the differentiation of baby mouse kidney cells has been examined in a hormonally defined serum-free medium. Primary cultures of baby mouse kidney cells were observed to form tubules over a time interval of 1-2 weeks in matrigel. Electron microscopic studies showed that tubules with lumens were present, and the tubule morphology was similar to that of the collecting duct. When using matrigel from which the growth factors had been removed, tubule formation no longer occurred, unless the medium was further supplemented with epidermal growth factor (10 ng/ml). Transforming growth factor alpha stimulated tubule formation as effectively as epidermal growth factor, whereas transforming growth factor beta had an inhibitory effect on tubule formation. These data suggest that both an extracellular matrix and specific growth factors may regulate kidney differentiation during development.
A soluble, high-affinity, interleukin-4-binding protein is present in the biological fluids of mice.
Authors: Fernandez-Botran R , Vitetta E S
Published: May 2006

Cytokines such as interleukin 4 (IL-4) play a key role in the regulation of immune responses, but little is known about how their multiple activities are regulated in vivo. In this report, we demonstrated that an IL-4-binding protein (IL-4BP) is constitutively present in the biological fluids of mice (serum, ascites, fluid, and urine). Binding of 125I-labeled IL-4 to the IL-4BP is specific and saturable and can be inhibited by an excess of unlabeled IL-4 but not IL-2. The IL-4BP binds IL-4 with an affinity similar to that reported for the cellular IL-4 receptor (Kd approximately 7 x 10(-11) M) and has a molecular mass of 30-40 kDa and pI values of 3.6-4.8. IL-4BP-containing biological fluids or purified IL-4BP competitively inhibit the binding of 125I-labeled IL-4 to mouse T or B cells and inhibit the biological activity of IL-4 but not IL-2. The serum levels of IL-4BP in severe combined immunodeficiency (SCID) mice are lower than those of normal mice. The above findings suggest that IL-4BP plays an important immunoregulatory role in vivo.
Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma.
Authors: Humphrey P A , Wong A J , Vogelstein B , Zalutsky M R , Fuller G N , Archer G E , Friedman H S , Kwatra M M , Bigner S H , Bigner D D
Published: May 2006

We have investigated human gliomas that amplify and rearrange the epidermal growth factor receptor gene, with generation of an in-frame deletion mutation of 802 nucleotides in the external domain. This in-frame deletion mutation generates a local amino acid sequence at the fusion junction of what normally were distant polypeptide sequences in the intact epidermal growth factor receptor. This 14-amino acid peptide was chemically synthesized, coupled to keyhole limpet hemocyanin, and used as an immunogen in rabbits. The elicited antibody reacted specifically with the fusion peptide in ELISA. The anti-fusion junction peptide antibody was purified by passage of the antiserum over a peptide affinity column with acidic elution. The purified antibody selectively bound the glioma deletion mutant as compared to the intact epidermal growth factor receptor as assessed by immunocytochemistry, immunofluorescence, immunoprecipitation with gel electrophoresis, and binding experiments using radioiodinated antibody. These data indicate that it is feasible to generate site-specific anti-peptide antibodies that are highly selective for mutant proteins in human tumors. The anti-peptide antibody described here, and other mutation site-specific antibodies, should be ideal candidates for tumor immunoimaging and immunotherapy.
Protection of mice against tumor growth by immunization with an oncogene-encoded growth factor.
Authors: Talarico D , Ittmann M , Balsari A , Delli-Bovi P , Basch R S , Basilico C
Published: May 2006

The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family that is secreted and transforms cells through a mechanism of autocrine cell proliferation. K-fgf-transformed cells are highly tumorigenic in immunocompetent allogeneic and syngeneic animals. BALB/c mice were immunized with a bacterial fusion protein consisting of a portion of the MS2 polymerase and of the human K-FGF precursor lacking only the first 4 amino acids or with a recombinant protein corresponding to the mature, secreted form of K-FGF (176 amino acids). They were then challenged with syngeneic K-fgf- or H-ras-transformed cells. Vaccinated animals exhibited a significant degree of protection against tumor induction, which was specific for K-fgf-transformed cells and correlated with the ability of the immunized mice to produce high titers of anti-K-FGF antibodies. Thus immunization with a single oncogene product can protect animals against tumor cells expressing this oncogene.
Excised patches of plasma membrane from vertebrate rod outer segments retain a functional phototransduction enzymatic cascade.
Authors: Ertel E A
Published: May 2006

Ion channels in excised patches of plasma membrane are generally considered to be isolated from any intracellular regulation mechanisms. For example, in excised patches of vertebrate rod outer segment plasma membrane, the cGMP-activated cation channels have traditionally been studied in room light because the enzyme cascade linking photon absorption to channel closure was assumed to be inoperative. To investigate the possibility that, in fact, such excised patches retain a functional phototransduction enzymatic cascade, this same preparation was studied in darkness. Patches excised in the dark were found to retain the light sensitivity of their cGMP-induced conductance and the ability to synthesize cGMP. In the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a nonhydrolyzable GTP analog, light suppresses the cGMP-induced conductance irreversibly. Furthermore, inhibitors of phosphodiesterase activity reduce light sensitivity, whereas activated phosphodiesterase or activated transducin does not directly affect the channels. These results (i) establish that excised patches from rod outer segment retain functional phototransduction enzymes, (ii) support the classical view that channel opening is modulated by phosphodiesterase-mediated cGMP hydrolysis, and, most surprisingly, (iii) demonstrate that diffusion in excised patches is so restricted that local enzymes can induce variations in the concentration of small molecules. The indication that excised patches are not as simple as usually surmised opens the possibility of using them to study other intracellular transduction mechanisms.
Genetic analysis of the Kirsten-ras-revertant 1 gene: potentiation of its tumor suppressor activity by specific point mutations.
Authors: Kitayama H , Matsuzaki T , Ikawa Y , Noda M
Published: May 2006

Kirsten-ras-revertant 1 (Krev-1) cDNA encodes a ras-related protein and exhibits an activity of inducing flat revertants at certain frequencies (2-5% of total transfectants) when introduced into a v-K-ras-transformed mouse NIH 3T3 cell line, DT. Toward understanding the mechanism of action of Krev-1 protein, we constructed a series of point mutants of Krev-1 cDNA and tested their biological activities in DT cells and HT1080 human fibrosarcoma cells harboring the activated N-ras gene. Substitutions of the amino acid residues in the putative guanine nucleotide-binding regions (Asp17 and Asn116), in the putative effector-binding domain (residue 38), at the putative acylation site (Cys181), and at the unique Thr61 all decreased the transformation suppressor activity. On the other hand, substitutions such as Gly12 to Val12 and Gln63 to Glu63 were found to significantly increase the transformation suppressor/tumor suppressor activity of Krev-1. These findings are consistent with the idea that Krev-1 protein is regulated like many other G proteins by the guanine triphosphate/guanine diphosphate-exchange mechanism probably in response to certain negative growth-regulatory signals.
Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.
Authors: Reid L H , Gregg R G , Smithies O , Koller B H
Published: May 2006

We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from the HPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene.
Serum calcium and vitamin D regulate 1,25-dihydroxyvitamin D3 receptor concentration in rat kidney in vivo.
Authors: Sandgren M E , DeLuca H F
Published: May 2006

The effects of vitamin D status, serum calcium, and serum phosphorus levels on 1,25-dihydroxyvitamin D receptor levels in kidney were investigated. Weanling rats were fed for 4 weeks on a diet with various levels of calcium and phosphorus with or without vitamin D. The 1,25-dihydroxyvitamin D3 receptor concentration in kidney was determined by an immunoradiometric assay. In the absence of vitamin D, total receptor concentration is increased 2-fold by an increase in serum calcium concentration. At normal serum calcium levels, the administration of vitamin D resulted in a 5-fold increase in receptor concentration. In hypocalcemic animals, however, vitamin D did not change receptor levels. Serum phosphorus levels could not be linked to any changes in 1,25-dihydroxyvitamin D3 receptor concentration. This study demonstrates that serum calcium levels and vitamin D regulate 1,25-dihydroxyvitamin D3 receptor concentration in vivo in kidney. On the other hand, vitamin D is unable to exert control of receptor levels in kidney under hypocalcemic conditions.
Location of phosphotyrosine-containing proteins by immunocytochemistry in the rat forebrain corresponds to the distribution of the insulin receptor.
Authors: Moss A M , Unger J W , Moxley R T , Livingston J N
Published: May 2006

Cellular regulation by certain growth factor receptors and protooncogene products involves tyrosine kinase activity with the resultant tyrosine phosphorylation of protein substrates. In the present report we describe the distribution of phosphotyrosine-containing material detected by immunocytochemistry (ICC) in the rat forebrain. Specificity of the affinity-purified antibody against phosphotyrosine used in the ICC technique was demonstrated by the ability of phosphotyrosine and p-nitrophenyl phosphate but not phosphoserine, phosphothreonine, or L-tyrosine to inhibit the immunostaining reaction. With ICC, relatively high amounts of phosphotyrosine-positive material were observed in neurons in specific structures that included the supraoptic, paraventricular, and arcuate nuclei; the median eminence; medial habenula; subfornical organ; and piriform cortex. Moderate to high amounts were present in the cerebral cortical layers II-IV and in the pyramidal cell layer of the hippocampus. Small to moderate amounts were detected in a few other locations. Glial elements showed minimal staining. Other areas of the rat forebrain failed to react with this antibody. Importantly, the distribution of the areas positive for phosphotyrosine agreed to a remarkable extent with the distribution of the brain insulin receptor, which itself has tyrosine kinase activity. These findings suggest a relationship between the insulin receptor and the increased phosphotyrosine content of these neurons and support the concept that the brain insulin receptor is active in vivo.
Inhibition of protein synthesis blocks long-term enhancement of generator potentials produced by one-trial in vivo conditioning in Hermissenda.
Authors: Crow T , Forrester J
Published: May 2006

A one-trial in vivo conditioning procedure produces short- and long-term cellular changes that can be detected in identified sensory neurons of the pathway mediating the conditioned stimulus. The memory of the associative experience in the conditioned stimulus pathway is expressed by short- and long-term enhancement of light-evoked generator potentials recorded from identified lateral and medial type B photoreceptors within the eyes of Hermissenda. To identify mechanisms of the induction and expression of short- and long-term enhancement in identified photoreceptors, we have investigated the effects of inhibiting protein synthesis during the application of the one-trial in vivo conditioning procedure. Anisomycin (1 microM) present during and after the conditioning trial blocked long-term enhancement without affecting the induction or expression of short-term enhancement. Application of a control compound, deacetylanisomycin (1 microM), or delaying the application of anisomycin until 1 hr after the conditioning trial did not block either long- or short-term enhancement. These results indicate that synthesis of proteins during or shortly after training may be a critical step in the formation of long-term memory of the associative experience.
Direct electrophoretic detection of the allelic state of single DNA molecules in human sperm by using the polymerase chain reaction.
Authors: Li H , Cui X , Arnheim N
Published: May 2006

We have developed a procedure that allows the detection of polymerase chain reaction (PCR) products derived from a single target DNA molecule in a human sperm without using radioactive probes. With this method, three genetic loci present in a single sperm can be amplified simultaneously. The amplification procedure is specific as well as efficient and permits detection of the PCR product by ethidium bromide staining after polyacrylamide gel electrophoresis. When allele-specific PCR primers that differ in length are used, the size of the PCR products of different alleles also vary in length, allowing the allelic state at each locus to be determined electrophoretically. Studies on individual sperm by using this procedure should facilitate the measurement of genetic recombination in humans over small physical distances. The ability to directly analyze the allelic state of PCR products from one cell rapidly and simply will also be useful for the prenatal diagnosis of genetic disease, especially in the analysis of single blastomeres taken from in vitro fertilized eggs prior to implantation.
Minisatellite linkage maps in the mouse by cross-hybridization with human probes containing tandem repeats.
Authors: Julier C , de Gouyon B , Georges M , Guénet J L , Nakamura Y , Avner P , Lathrop G M
Published: May 2006

Tests of 29 human variable number of tandem repeat probes in inbred mouse lines showed that 80% (23/29) cross-hybridize, and 48% (14/29) produce multiple band, minisatellite polymorphisms (fingerprint patterns). Mini-satellite-type polymorphisms detected by 11 probes were characterized in eight different strains; on average, 240 polymorphic differences were detected between pairs of strains. Reproducible fingerprint patterns permit the study of the segregation of the minisatellite polymorphisms in experimentally designed crosses. As an example, we constructed primary minisatellite genetic linkage maps containing 346 polymorphic bands, distributed in 101 groups of closely linked systems, from genotypes on a recombinant inbred panel (C57BL/6J X DBA/2J); 38 of the groups were assigned by linkage to 15 autosomal chromosomes. The minisatellite genetic maps of C57BL/6J and DBA/2J can be applied in other linkage studies involving these strains.
Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2.
Authors: Bischoff J R , Friedman P N , Marshak D R , Prives C , Beach D
Published: May 2006

The human anti-oncoprotein p53 is shown to be a substrate of cdc2. The primary site of phosphorylation is serine-315. Serine-315 is phosphorylated by both p60-cdc2 and cyclin B-cdc2 enzymes. The phosphorylation of p53 is cell cycle-dependent. The abundance of p53 also oscillates during the cell cycle. The protein is largely absent from cells that have just completed division but accumulates in cells during G1 phase. Phosphorylation by cdc2 might regulate the antiproliferative activity of p53.
TolC, an Escherichia coli outer membrane protein required for hemolysin secretion.
Authors: Wandersman C , Delepelaire P
Published: May 2006

Secretion of Escherichia coli alpha-hemolysin into the medium does not require the cleavage of an N-terminal signal peptide. The specific secretion apparatus was shown to consist of two proteins, HlyB and HlyD, both located in the inner membrane and encoded by genes contiguous to the hemolysin structural gene (hlyA). It was proposed that these two proteins constitute a membrane-bound translocator for hemolysin [Mackman, N., Nicaud, J. M., Gray, L. & Holland, I. B. (1986) Curr. Top. Microbiol. Immunol. 125, 159-181]. We show here that an E. coli outer membrane protein, the TolC protein, encoded by a gene not located in the hly cluster, is specifically required for hemolysin secretion. This result suggests that an outer membrane protein might be a component of the secretion apparatus allowing a specific interaction between the inner and the outer membrane.