bluuebunny/crossref_metadata_2025 huggingface dataset:
Helminth infection results in decreased virus-specific CD8+ cytotoxic T-cell and Th1 cytokine responses as well as delayed virus clearance.
Authors: Actor J K , Shirai M , Kullberg M C , Buller R M , Sher A , Berzofsky J A
Published: May 2006

During the time of egg deposition, schistosome-infected mice exhibit a downregulation in interleukin 2 and interferon gamma production toward parasite antigens, mitogens, and foreign nonparasite protein antigens. To determine whether this imbalance in cytokine response would impact on CD8+ cytotoxic T-lymphocyte (CTL) responses, as well as on immune clearance of viral infections, we challenged Schistosoma mansoni-infected BALB/c mice, when cytokine imbalance was prominent, with a recombinant vaccinia virus expressing human immunodeficiency virus type 1 gp160. In contrast to control vaccinia-infected animals, S. mansoni plus vaccinia-infected mice did not produce significant Th1 cytokine responses upon in vitro stimulation with recombinant gp120, consistent with previous results for nonparasite antigens. However, more striking was the downregulation of the virus-specific CTL response not previously studied. Spleen cells from vaccinia-infected control mice displayed strong CD8+ cytolytic activity against gp160-transfected fibroblasts and fibroblasts pulsed with a peptide (P18) representing a CTL epitope of gp160. In contrast, mice coinfected with S. mansoni and vaccinia manifested absent or markedly reduced in vitro CTL activity even in the presence of exogenous interleukin 2. To determine whether this immune dysregulation might impact on viral clearance, we measured virus titers in tissues as a function of time. Mice infected with vaccinia virus alone rapidly cleared the virus, whereas in animals coinfected with S. mansoni, viral clearance was delayed by as much as 3 weeks in the liver and by several days in the spleen and lungs. These observations suggest that helminth infection may influence immune responses to concurrent viral infections.
Differential patterns of DNA binding by myc and max proteins.
Authors: Prochownik E V , VanAntwerp M E
Published: May 2006

c-myc, N-myc, and L-myc genes are subject to highly variable degrees of tissue-specific regulation. Their aberrant expression has also been implicated in the pathogenesis of a variety of malignant tumors. The recently identified max protein dimerizes with c-myc to promote its sequence-specific DNA binding. max exists in two forms (long and short) that differ by virtue of a 9-amino acid insertion/deletion at the N terminus. We tested recombinant myc and max proteins for binding to six oligonucleotides containing related c-myc sites. Each myc protein, alone and in association with max proteins, manifested a unique pattern of DNA binding. Phosphorylation of both max proteins was observed when they were incubated in a rabbit reticulocyte lysate. This strongly affected DNA binding by max(long) but not by max(short). Our results point to the existence of specific DNA binding preferences for each of the myc proteins. The 9-amino acid segment that distinguishes max(long) from max(short) appears to serve a regulatory function that provides additional control over DNA sequence recognition.
Transferrin in a cockroach: molecular cloning, characterization, and suppression by juvenile hormone.
Authors: Jamroz R C , Gasdaska J R , Bradfield J Y , Law J H
Published: May 2006

In a study of juvenile hormone-regulated gene expression, we isolated an anonymous cDNA representing a message that was strongly suppressed by juvenile hormone in the fat body of the cockroach Blaberus discoidalis. The protein deduced from the cDNA sequence showed compelling resemblance in sequence to the transferrins, a superfamily of internally duplicated, 80-kDa iron-binding/transport proteins characterized from several vertebrates and, to date, one insect (the tobacco hornworm, Manduca sexta). We isolated a 78-kDa protein from cockroach hemolymph, verified its congruence with the cloned cDNA, and found that it did bind iron. The cockroach protein is a member of the transferrin superfamily based on several features, including 32-46% amino acid positional identity with transferrins whose sequences are known, internal homology, positioning of cysteine residues, and iron binding. Whereas the previously characterized insect transferrin binds one atom of iron per protein molecule, B. discoidalis transferrin binds two iron atoms as do the vertebrate transferrins. The diferric property of cockroach transferrin is consistent with presence of two sets of residues positioned appropriately for iron binding. Juvenile hormone suppressed transferrin mRNA levels drastically in the adult female cockroach.
The HNK-1 reactive sulfoglucuronyl glycolipids are ligands for L-selectin and P-selectin but not E-selectin.
Authors: Needham L K , Schnaar R L
Published: May 2006

E-selectin, L-selectin, and P-selectin are related cell adhesion molecules that bind via their lectin domains to sialyl Lewis x and related carbohydrate determinants. Reports have indicated that sulfated glycolipids and polysaccharides also bind selectins. To extend these findings, we compared binding of selectin-IgG chimeras to immobilized sulfated and sialylated glycosphingolipids. E-, L-, and P-selectin chimeras all bound to surfaces absorbed with 2,3-sialyl Lewis x glycolipid or sulfatide (galactosylceramide I3-sulfate) but not to surfaces adsorbed with control sulfated lipids (octadecyl sulfate, sphingosine sulfate). Notably, the L- and P-selectin chimeras but not E-selectin chimera bound to surfaces adsorbed with sulfoglucuronyl glycosphingolipids (SGNL lipids; e.g., IV3 glucuronylneolactotetraosylceramide V3-sulfate). These unusual lipids have been reported as antigenic determinants for monoclonal IgM antibodies produced in patients with neuropathy associated with paraproteinemia and react with the mouse monoclonal antibody HNK-1. Binding of L- and P-selectin chimeras to SGNL lipids was specifically inhibited by appropriate anti-selectin antibodies. While binding of all three selectin chimeras to sialyl Lewis x was blocked by removal of calcium, binding to SGNL lipid was only modestly reduced by EDTA. Chemically desulfated SGNL lipid retained binding activity for L- and P-selectin chimeras, while methyl esterification of the glucuronic acid eliminated binding. We conclude that SGNL lipids, unlike sialyl Lewis x and sulfatides, selectively support L- and P-selectin but not E-selectin chimera binding. The presence of SGNL lipids on brain microvascular endothelium (and other endothelia) may implicate these molecules in leukocyte trafficking to the nervous system and elsewhere.
Combined use of an immunotoxin and cyclosporine to prevent both activated and quiescent peripheral blood T cells from producing type 1 human immunodeficiency virus.
Authors: Bell K D , Ramilo O , Vitetta E S
Published: May 2006

Two different populations of infected T cells are present in human immunodeficiency virus (HIV)-infected individuals: activated cells that produce virions and quiescent cells that harbor the viral genome but are unable to produce virus unless they are activated. Using an in vitro model of acute HIV infection, we have evaluated the effect of depleting activated T cells with an immunotoxin and subsequently inhibiting activation of quiescent T cells with an immunosuppressive agent. CD25 (Tac, p55), the alpha chain of the interleukin 2 receptor, is expressed on activated, but not quiescent, T cells. An anti-CD25-ricin A chain immunotoxin eliminated activated, CD25+ HIV-infected cells and, thereby, inhibited viral production by these cells. Subsequent addition of cyclosporine to the residual CD25- cells prevented their activation and thereby suppressed their ability to produce virus and to propagate the infection to uninfected T cells.
A beta-adrenergic receptor kinase-like enzyme is involved in olfactory signal termination.
Authors: Schleicher S , Boekhoff I , Arriza J , Lefkowitz R J , Breer H
Published: May 2006

We have previously shown that second-messenger-dependent kinases (cAMP-dependent kinase, protein kinase C) in the olfactory system are essential in terminating second-messenger signaling in response to odorants. We now document that subtype 2 of the beta-adrenergic receptor kinase (beta ARK) is also involved in this process. By using subtype-specific antibodies to beta ARK-1 and beta ARK-2, we show that beta ARK-2 is preferentially expressed in the olfactory epithelium in contrast to findings in most other tissues. Heparin, an inhibitor of beta ARK, as well as anti-beta ARK-2 antibodies, (i) completely prevents the rapid decline of second-messenger signals (desensitization) that follows odorant stimulation and (ii) strongly inhibits odorant-induced phosphorylation of olfactory ciliary proteins. In contrast, beta ARK-1 antibodies are without effect. Inhibitors of protein kinase A and protein kinase C also block odorant-induced desensitization and phosphorylation. These data suggest that a sequential interplay of second-messenger-dependent and receptor-specific kinases is functionally involved in olfactory desensitization.
Functional specificity of the homeodomain protein fushi tarazu: the role of DNA-binding specificity in vivo.
Authors: Schier A F , Gehring W J
Published: May 2006

The mechanisms determining the functional specificity of Drosophila homeodomain proteins are largely unknown. Here, the role of DNA-binding specificity for the in vivo function of the homeodomain protein fushi tarazu (ftz) is analyzed. We find that specific DNA binding is an important but not sufficient determinant of the functional specificity of ftz in vivo: The ftz DNA-binding specificity mutant ftzQ50K retains partial ftz wild-type activity in gene activation and phenotypic rescue assays. Furthermore, specificity mutations in a ftz-in vivo binding site only partially reduce enhancer activity as compared to null mutations of this site. Despite bicoid-like DNA-binding specificity ftzQ50K does not activate natural or artificial bcd target genes in the realms of ftz. These results are discussed in the light of recent observations on the mechanism of action of the yeast homeodomain protein alpha 2.
Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family.
Authors: Steele F R , Chader G J , Johnson L V , Tombran-Tink J
Published: May 2006

Cultured pigment epithelial cells of the fetal human retina secrete a protein, pigment epithelium-derived factor (PEDF), that induces a neuronal phenotype in cultured human retinoblastoma cells. Morphological changes include the induction of an extensive neurite meshwork and the establishment of corona-like cellular aggregates surrounding a central lumen. The differentiated cells also show increases in the expression of neuron-specific enolase and the 200-kDa neurofilament subunit. Amino acid and DNA sequence data demonstrate that PEDF belongs to the serine protease inhibitor (serpin) family. The PEDF gene contains a typical signal-peptide sequence, initiator methionine codon, and polyadenylylation signal and matches the size of other members of the serpin superfamily (e.g., alpha 1-antitrypsin). It lacks homology, however, at the putative serpin reactive center. Thus, PEDF could exert a paracrine effect in the embryonic retina, influencing neuronal differentiation by a mechanism that does not involve classic inhibition of serine protease activity.
Electrostatic interactions modulate the RNA-binding and transactivation specificities of the human immunodeficiency virus and simian immunodeficiency virus Tat proteins.
Authors: Tao J , Frankel A D
Published: May 2006

The transcriptional activating (Tat) proteins from human immunodeficiency virus and simian immunodeficiency virus are sequence-specific RNA-binding proteins. In human immunodeficiency virus Tat, a single arginine residue, flanked on each side by three to four basic amino acids, mediates specific binding to a bulge region in trans-acting responsive element (TAR) RNA. We have systematically mutated the flanking charged residues and found that, in addition to the position of the sequence-specific arginine, the particular arrangement of nonspecific electrostatic interactions is an important determinant of RNA-binding specificity and transactivation activity. These additional electrostatic contacts may help stabilize the structure of TAR RNA when bound to arginine. One critical electrostatic interaction, located two residues N-terminal to the arginine, is absent in the simian immunodeficiency virus Tat protein and accounts for the difference in promoter specificities of the human and simian immunodeficiency viral proteins.
Oscillatory flow in the cochlea visualized by a magnetic resonance imaging technique.
Authors: Denk W , Keolian R M , Ogawa S , Jelinski L W
Published: May 2006

We report a magnetic resonance imaging technique that directly measures motion of cochlear fluids. It uses oscillating magnetic field gradients phase-locked to an external stimulus to selectively visualize and quantify oscillatory fluid motion. It is not invasive, and it does not require optical line-of-sight access to the inner ear. It permits the detection of displacements far smaller than the spatial resolution. The method is demonstrated on a phantom and on living rats. It is projected to have applications for auditory research, for the visualization of vocal tract dynamics during speech and singing, and for determination of the spatial distribution of mechanical relaxations in materials.
Recombinant kinesin motor domain binds to beta-tubulin and decorates microtubules with a B surface lattice.
Authors: Song Y H , Mandelkow E
Published: May 2006

We have expressed the recombinant squid kinesin head domain in Escherichia coli and studied its interaction with microtubules. The head is active as a microtubule-stimulated ATPase and binds to microtubules, but it does not support microtubule gliding by itself. The head binds to both microtubules and depolymerized tubulin. In each case the zero-length crosslinker 1-ethyl-3-[3-dimethylamino)propyl] carbodiimide induces a bond specifically to beta- but not alpha-tubulin. The head decorates brain microtubules with an 8-nm axial spacing. Thus the stoichiometry is one kinesin head per tubulin dimer. The lattice is that of flagellar B-tubules, implying that reassembled microtubules are not symmetric. Moreover, the A- and B-tubules of intact flagellar outer doublets are both decorated with a B lattice. This suggests that the B lattice is a general property of microtubules.
Predicting the size of the T-cell receptor and antibody combining region from consideration of efficient self-nonself discrimination.
Authors: Percus J K , Percus O E , Perelson A S
Published: May 2006

The binding of antibody to antigen or T-cell receptor to major histocompatibility complex-peptide complex requires that portions of the two structures have complementary shapes that can closely approach each other. The question that we address here is how large should the complementary regions on the two structures be. The interacting regions are by necessity roughly the same size. To estimate the size (number of contact residues) of an optimal receptor combining region, we assume that the immune system over evolutionary time has been presented with a large random set of foreign molecules that occur on common pathogens, which it must recognize, and a smaller random set of self-antigens to which it must fail to respond. Evolutionarily, the receptors and the molecular groups that the immune system recognizes as epitopes are imagined to have coevolved to maximize the probability that this task is performed. The probability of a receptor matching a random antigen is estimated from this condition. Using a simple model for receptor-ligand interaction, we estimate that the optimal size binding region on immunoglobulin or T-cell receptors will contain about 15 contact residues, in agreement with experimental observation.
Defective pro alpha 2(I) collagen synthesis in a recessive mutation in mice: a model of human osteogenesis imperfecta.
Authors: Chipman S D , Sweet H O , McBride D J , Davisson M T , Marks S C , Shuldiner A R , Wenstrup R J , Rowe D W , Shapiro J R
Published: May 2006

Osteogenesis imperfecta (OI) is a heritable disorder of connective tissue associated with fractures, osteopenia, and short stature. OI results from mutations affecting the pro alpha 1 or pro alpha 2 gene of type I collagen. We describe a strain of mice with a nonlethal recessively inherited mutation (oim) that results in phenotypic and biochemical features that simulate moderate to severe human OI. The phenotype of homozygous oim mice includes skeletal fractures, limb deformities, generalized osteopenia, and small body size. Their femurs are smaller and demonstrate marked cortical thinning and fewer medullary trabeculae than those of wild-type mice. Breeding studies show the mutation is inherited in most crosses as a single recessive gene on chromosome 6, near the murine Cola-2 gene. Biochemical analysis of skin and bone, as well as isolated dermal fibroblast cultures, demonstrate that alpha 1(I) homotrimeric collagen accumulates in these tissues and is secreted by fibroblasts. Short labeling studies in fibroblasts demonstrate an absence of pro alpha 2(I) collagen chains. Nucleotide sequencing of the cDNA encoding the COOH-propeptide reveals a G deletion at pro alpha 2(I) nucleotide 3983; this results in an alteration of the sequence of the last 48 amino acids. The oim mouse will facilitate the study of type I collagen-related skeletal disease.
Adipose pyruvate carboxylase: amino acid sequence and domain structure deduced from cDNA sequencing.
Authors: Zhang J , Xia W L , Brew K , Ahmad F
Published: May 2006

The complete amino acid sequence of 3T3-L1 adipocyte pyruvate carboxylase (PC) [pyruvate:carbon-dioxide ligase (ADP-forming), EC 6.4.1.1] has been deduced from sequencing overlapping cDNA clones obtained from an adipocyte cDNA library constructed in the lambda Zap vector. The encoding mRNA for PC promoter contains 4067 nt, including a 3534-nt coding sequence and noncoding regions of 100 and 433 nt at the 5' and 3' ends, respectively. The biotinylated lysine of the encoded PC promoter (1178 amino acids with a calculated M(r) of apocarboxylase = 129,784) is located 35 residues from the COOH-terminal end and, as in most other biotin enzymes, is in the consensus sequence AMKM. The adipocyte PC is closely similar (53% identity) to the yeast enzyme and contains different segments that are homologous with regions from the biotin carboxylase component of Escherichia coli acetyl-CoA carboxylase, the keto acid-binding subunits of Propionibacterium shermanii oxaloacetate transcarboxylase and Klebsiella pneumoniae oxaloacetate decarboxylase, and to the biotin carboxyl-carrier protein of the bacterial biotin enzymes. In addition to the putative mitochondrial targeting signal, functional domains are readily identifiable in the sequence and are in the following order: biotin carboxylase-carboxyltransferase-biotin carboxyl-carrier protein, as proposed for yeast PC.
A single-amino acid substitution in a gamma-aminobutyric acid subtype A receptor locus is associated with cyclodiene insecticide resistance in Drosophila populations.
Authors: ffrench-Constant R H , Steichen J C , Rocheleau T A , Aronstein K , Roush R T
Published: May 2006

Resistance to cyclodiene insecticides, documented in at least 277 species, is perhaps the most common kind of resistance to any pesticide. By using cyclodiene resistance to localize the responsible gene, a gamma-aminobutyric acid type A receptor/chloride ion-channel gene was previously cloned and sequenced from an insecticide-susceptible Drosophila melanogaster strain. We now describe the molecular genetics of the resistance allele. A single-base-pair mutation, causing a single-amino acid substitution (Ala-->Ser) within the second membrane-spanning region of the channel, was found to be the only consistent difference between resistant and susceptible strains of D. melanogaster. Some resistant strains of Drosophila simulans show the same mutation, whereas others show an alternative single-base-pair mutation in the same codon, resulting in the substitution of a different amino acid (glycine). These constitute single-box-pair mutations in insects that confer high levels of resistance to insecticides. The presence of the resistance mutations was then tested in a much larger set of strains by the PCR and subsequent digestion with a diagnostic restriction endonuclease. Both resistance-associated mutations cause the loss of a Hae II site. This site was invariably present in 122 susceptible strains but absent in 58 resistant lines of the two species sampled from five continents. PCR/restriction endonuclease treatment was also used to examine linkage of an EcoRI polymorphism in a neighboring intron in D. melanogaster, which was found associated with resistance in all but 3 of 48 strains examined. These PCR-based techniques are widely applicable to examination of the uniqueness of different resistance alleles in widespread populations, the identification of resistance mechanisms in different species, and the determination of resistance frequencies in monitoring.
Photoreceptor differentiation of isolated retinal precursor cells includes the capacity for photomechanical responses.
Authors: Stenkamp D L , Adler R
Published: May 2006

Isolated retinal precursor cells, grown without pigment epithelial or glial cells and in the absence of intercellular contacts, develop a complex set of photoreceptor-specific properties, including polarized structural and molecular organization and opsin immunoreactivity. We report here that these isolated embryonic photoreceptors are also capable of responding to light. Sequential photography showed that 50% of the photoreceptors grown in a light cycle elongate when exposed to light and contract in response to darkness. A smaller population (20%) showed the opposite response. Responses of individual cells could be observed during several sequential light cycles and resemble photomechanical movements in vivo [Ali, M. A. (1971) Vision Res. 11, 1225-1288]. The differentiation program expressed by isolated precursor cells, therefore, includes the capacity for highly complex functional activities that require light sensitivity. These observations raise challenging questions regarding the nature of the chromophore and pigments that mediate light-regulated behaviors of cultured photoreceptors.
Purification to homogeneity and the N-terminal sequence of human leukotriene C4 synthase: a homodimeric glutathione S-transferase composed of 18-kDa subunits.
Authors: Nicholson D W , Ali A , Vaillancourt J P , Calaycay J R , Mumford R A , Zamboni R J , Ford-Hutchinson A W
Published: May 2006

Human leukotriene C4 (LTC4) synthase was purified > 25,000-fold to homogeneity from the monocytic leukemia cell line THP-1. Beginning with taurocholate-solubilized microsomal membranes, LTC4 synthase was chromatographically resolved by (i) anion exchange, (ii) affinity chromatography (through a resin of biotinylated LTC2 immobilized on streptavidin-agarose), and then (iii) gel filtration. The final preparation contained only an 18-kDa polypeptide. The molecular mass of the pure polypeptide was consistent with an 18-kDa polypeptide from THP-1 cell membranes that was specifically photolabeled by an LTC4 photoaffinity probe, 125I-labeled azido-LTC4. On calibrated gel-filtration columns, purified LTC4 synthase activity eluted at a volume corresponding to 39.2 +/- 3.3 kDa (n = 12). The sequence of the N-terminal 35 amino acids was determined and found to be a unique sequence composed predominantly of hydrophobic amino acids and containing a consensus sequence for protein kinase C phosphorylation. We therefore conclude that human LTC4 synthase is a glutathione S-transferase composed of an 18-kDa polypeptide that is enzymatically active as a homodimer and may be phosphoregulated in vivo.
Evaluation of potential models for imprinted and nonimprinted components of human chromosome 15q11-q13 syndromes by fine-structure homology mapping in the mouse.
Authors: Nicholls R D , Gottlieb W , Russell L B , Davda M , Horsthemke B , Rinchik E M
Published: May 2006

Prader-Willi and Angelman syndromes are complex neurobehavioral contiguous gene syndromes whose expression depends on the unmasking of genomic imprinting for different genetic loci in human chromosome 15q11-q13. The homologous chromosomal region in the mouse genome has been fine-mapped by using interspecific (Mus spretus) crosses and overlapping, radiation-induced deletions to evaluate potential animal models for both imprinted and nonimprinted components of these syndromes. Four evolutionarily conserved sequences from human 15q11-q13, including two cDNAs from fetal brain (DN10, D15S12h; DN34, D15S9h-1), a microdissected clone (MN7; D15F37S1h) expressed in mouse brain, and the gene for the beta 3 subunit of the gamma-aminobutyric acid type A receptor (Gabrb3), were mapped in mouse chromosome 7 by analysis of deletions at the pink-eyed dilution (p) locus. Three of these loci are deleted in pre- and postnatally lethal p-locus mutations, which extend up to 5.5 +/- 1.7 centimorgans (cM) proximal to p; D15S9h-1, which maps 1.1 +/- 0.8 cM distal to p and is the mouse homolog of the human gene D15S9 (which shows a DNA methylation imprint), is not deleted in any of the p-locus deletion series. A transcript from the Gabrb3 gene, but not the transcript detected by MN7 at the D15F37S1h locus, is expressed in mice homozygous for the p6H deletion, which have an abnormal neurological phenotype. Furthermore, the Gabrb3 transcript is expressed equally well from the maternal or paternal chromosome 7 and, therefore, its expression is not imprinted in mouse brain. Deletions at the mouse p locus should serve as intermediate genetic reagents and models with which to analyze the genetics and etiology of individual components of human 15q11-q13 disorders.
A widely expressed human protein-tyrosine phosphatase containing src homology 2 domains.
Authors: Ahmad S , Banville D , Zhao Z , Fischer E H , Shen S H
Published: May 2006

A cDNA encoding a nontransmembrane protein-tyrosine phosphatase (PTP; EC 3.1.3.48), termed PTP2C, was isolated from a human umbilical cord cDNA library. The enzyme contains a single phosphatase domain and two adjacent copies of the src homology 2 (SH2) domain at its amino terminus. A variant of PTP2C (PTP2Ci) which has four extra amino acid residues within the catalytic domain has been identified also. PTP2C is widely expressed in human tissues and is particularly abundant in heart, brain, and skeletal muscle. The catalytic domain of PTP2C was expressed as a recombinant enzyme in Escherichia coli and purified to near homogeneity by two chromatographic steps. The recombinant enzyme was totally specific toward phosphotyrosine residues. The structural similarity between PTP2C and the previously described PTP1C suggests the existence of a subfamily of SH2-containing PTPs; these may play an important role in signal transduction through interaction of their SH2 domains with phosphotyrosine-containing proteins.
Phosphorylation at threonine-235 by a ras-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6.
Authors: Nakajima T , Kinoshita S , Sasagawa T , Sasaki K , Naruto M , Kishimoto T , Akira S
Published: May 2006

NF-IL6, a member of the basic leucine zipper (bZIP) family transcription factors, is involved in expression of inducible genes involved in immune and inflammatory responses. We observed that coexpression of oncogenic p21ras stimulated the transactivating activity of NF-IL6 and induced phosphorylation of Thr-235 located just N-terminal to the DNA binding domain of NF-IL6. Recently, mitogen-activated protein (MAP) kinases have been shown to be implicated in the cellular response to activated ras. Purified MAP kinases specifically phosphorylated Thr-235 of NF-IL6 in vitro. Mutation of Thr-235 abolished the ras-dependent activation of NF-IL6. From these results, we conclude that NF-IL6 is regulated through phosphorylation by MAP kinases in response to activated ras.
Identification of Ras farnesyltransferase inhibitors by microbial screening.
Authors: Hara M , Akasaka K , Akinaga S , Okabe M , Nakano H , Gomez R , Wood D , Uh M , Tamanoi F
Published: May 2006

A microbial screen using a yeast strain with conditional deficiency in the GPA1 gene was carried out to search for inhibitors of protein farnesyltransferase (PFT). A strain of Streptomyces was found to produce active compounds named UCF1-A, UCF1-B, and UCF1-C. Structural determination of these compounds revealed that UCF1-C is identical to the known antibiotic, manumycin, whereas UCF1-A and UCF1-B are structurally related to manumycin. All three UCF1 compounds suppress the lethality of gpa1 disruption, with UCF1-C exhibiting the strongest activity. UCF1 inhibits yeast as well as rat brain PFT. Fifty percent inhibition of yeast PFT activity is observed with 5 microM UCF1-C. Kinetic analyses of the inhibition suggest that UCF1-C acts as a competitive inhibitor of PFT with respect to farnesyl pyrophosphate, exhibiting a Ki of 1.2 microM, whereas the same compound appears to act as a noncompetitive inhibitor of PFT with respect to the farnesyl acceptor, the Ras protein. UCF1-C shows significant activity to inhibit the growth of Ki-ras-transformed fibrosarcoma, raising the possibility of its use as an antitumor drug.
Disruption of the transthyretin gene results in mice with depressed levels of plasma retinol and thyroid hormone.
Authors: Episkopou V , Maeda S , Nishiguchi S , Shimada K , Gaitanaris G A , Gottesman M E , Robertson E J
Published: May 2006

Transthyretin (TTR) is thought to play a major role in vitamin A metabolism and thyroid hormone transport in mammals. To investigate the physiological role of the TTR protein in development of the embryo and in the adult, we used gene targeting techniques to generate a null mutation at the mouse ttr locus. The resultant mutant animals are phenotypically normal, viable, and fertile. However, levels of serum retinol, retinol-binding protein, and thyroid hormone are significantly depressed in the mutant animals. These observations demonstrate that the TTR protein maintains normal levels of these metabolites in the circulating plasma.
Inducible operation of the erythropoietin 3' enhancer in multiple cell lines: evidence for a widespread oxygen-sensing mechanism.
Authors: Maxwell P H , Pugh C W , Ratcliffe P J
Published: May 2006

Adaptive responses to hypoxia occur in many biological systems. A well-characterized example is the hypoxic induction of the synthesis of erythropoietin, a hormone which regulates erythropoiesis and hence blood oxygen content. The restricted expression of the erythropoietin gene in subsets of cells within kidney and liver has suggested that this specific oxygen-sensing mechanism is restricted to specialized cells in those organs. Using transient transfection of reporter genes coupled to a transcriptional enhancer lying 3' to the erythropoietin gene, we show that an oxygen-sensing system similar, or identical, to that controlling erythropoietin expression is wide-spread in mammalian cells. The extensive distribution of this sensing mechanism contrasts with the restricted expression of erythropoietin, suggesting that it mediates other adaptive responses to hypoxia.
Identification of a direct interaction between interleukin 2 and the p64 interleukin 2 receptor gamma chain.
Authors: Voss S D , Leary T P , Sondel P M , Robb R J
Published: May 2006

The interleukin 2 receptor (IL-2R) consists of at least two subunits, alpha and beta, both of which can bind interleukin 2 (IL-2). Recent studies have demonstrated the existence of a third subunit, a 64-kDa molecule termed IL-2R gamma chain, and have suggested that gamma chain functions to regulate the rate of IL-2 dissociation from the receptor. In the present report we have addressed whether the gamma chain modulates IL-2R affinity by contributing contact sites for IL-2 binding. Using reagents that allow the IL-2R complex to be immunoprecipitated through the IL-2 molecule itself, we demonstrate the existence of a stable IL-2-IL-2R gamma-chain complex. These studies thus establish that the IL-2R gamma chain directly contributes to the IL-2-binding site, consistent with the hypothesis that gamma chain influences IL-2R affinity through its direct interaction with IL-2.
Stearoyl-acyl carrier protein delta 9 desaturase from Ricinus communis is a diiron-oxo protein.
Authors: Fox B G , Shanklin J , Somerville C , Münck E
Published: May 2006

A gene encoding stearoyl-acyl carrier protein delta 9 desaturase (EC 1.14.99.6) from castor was expressed in Escherichia coli. The purified catalytically active enzyme contained four atoms of iron per homodimer. The desaturase was studied in two oxidation states with Mössbauer spectroscopy in applied fields up to 6.0 T. These studies show conclusively that the oxidized enzyme contains two (identical) clusters consisting of a pair of antiferromagnetically coupled (J > 60 cm-1, H = JS1.S2) Fe3+ sites. The diferric cluster exhibited absorption bands from 300 to 355 nm; addition of azide elicited a charge transfer band at 450 nm. In the presence of dithionite, the clusters were reduced to the diferrous state. Addition of stearoyl-CoA and O2 returned the clusters to the diferric state. These properties are consistent with assigning the desaturase to the class of O2-activating proteins containing diiron-oxo clusters, most notably ribonucleotide reductase and methane monooxygenase hydroxylase. Comparison of the primary structures for these three catalytically diverse proteins revealed a conserved pair of the amino acid sequence -(Asp/Glu)-Glu-Xaa-Arg-His- separated by approximately 100 amino acids. Since each of these proteins can catalyze O2-dependent cleavage of unactivated C--H bonds, we propose that these amino acid sequences represent a biological motif used for the creation of reactive catalytic intermediates. Thus, eukaryotic fatty acid desaturation may proceed via enzymatic generation of a high-valent iron-oxo species derived from the diiron cluster.
Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning, expression, and chromosomal localization.
Authors: Ramamoorthy S , Bauman A L , Moore K R , Han H , Yang-Feng T , Chang A S , Ganapathy V , Blakely R D
Published: May 2006

A Na(+)- and Cl(-)-coupled serotonin (5-hydroxytryptamine, 5HT) transporter is expressed on human neuronal, platelet, placental, and pulmonary membranes. The brain 5HT transporter appears to be a principal site of action of therapeutic antidepressants and may mediate behavioral and/or toxic effects of cocaine and amphetamines. Oligonucleotides derived from consensus transporter sequences were used to identify human placental cDNAs highly related to the rat brain 5HT carrier. Transfection of one of these cDNAs into HeLa cells yields a high-affinity (Km = 463 nM), Na(+)- and Cl(-)-dependent 5HT transport activity which can be blocked by selective 5HT transport inhibitors, including paroxetine, fluoxetine, and imipramine, and which is antagonized by cocaine and amphetamine. Sequence analysis reveals a 630-amino acid open reading frame bearing 92% identity to the cloned rat brain 5HT transporter, with identical predicted topological features and conserved sites for posttranslational modifications. Unlike the rodent, where a single mRNA appears to encode 5HT transporters, multiple hybridizing RNAs are observed in human placenta and lung. Somatic cell hybrid and in situ hybridization studies are consistent, however, with a single gene encoding the human 5HT transporter, localized to chromosome 17q11.1-17q12.
Molecular basis of genetic polymorphism in major histocompatibility complex-linked proteasome gene (Lmp-2).
Authors: Zhou P , Cao H , Smart M , David C
Published: May 2006

Four genes, closely linked to major histocompatibility complex (MHC) class II genes, have been identified in humans, mice, and rats and are thought to be involved in the generation and transport of endogenous immunogenic peptides for the MHC class I antigen-processing pathway. The Tap-1 and Tap-2 genes presumably encode a heterodimeric protein complex responsible for transporting endogenous immunogenic peptides to the lumen of the endoplasmic reticulum. The Lmp-2 and Lmp-7 gene products are two subunits of the large cytosolic proteasome complex possibly involved in generation of endogenous peptides. To study the genetic polymorphism of the Lmp-2 gene, we used a published cDNA sequence as a consensus sequence and PCR-amplified, cloned, and sequenced the Lmp-2 gene from 12 inbred mouse strains. We found three amino acid variants, LMP-2d, LMP-2b, and LMP-2q, which partially correlated with restriction fragment length polymorphism variants identified with Southern blots. Allelic polymorphism of the Lmp-2 gene may be involved in peptide selection, leading to autoimmune disease susceptibility.
Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14.
Authors: Pugin J , Schürer-Maly C C , Leturcq D , Moriarty A , Ulevitch R J , Tobias P S
Published: May 2006

Myeloid cell activation by lipopolysaccharides (LPS) involves two proteins, plasma LPS-binding protein (LBP) and cell-membrane CD14. Cell membrane CD14, anchored by a glycerophosphatidylinositol tail, is the cellular receptor for LPS-LBP complexes. Another form of CD14, without the lipid tail, circulates as a soluble plasma protein. In this work we show that soluble CD14 (sCD14) is required for activation of endothelial and epithelial cells by LPS. We propose that LPS-LBP complexes transfer LPS to sCD14, and the LPS-sCD14 complexes then bind to a cellular receptor. Support for this pathway comes from experiments in which LBP and CD14 in normal human serum are blocked by specific antibodies, experiments in which serum is replaced by purified LBP and sCD14, and experiments in which specific binding of [3H]LPS to epithelial cells is quantitated.
Autoreactive epitopes defined by diabetes-associated human monoclonal antibodies are localized in the middle and C-terminal domains of the smaller form of glutamate decarboxylase.
Authors: Richter W , Shi Y , Baekkeskov S
Published: May 2006

The gamma-aminobutyrate-synthesizing enzyme glutamate decarboxylase (GAD; L-glutamate 1-carboxy-lyase, EC 4.1.1.15) is a major target of autoantibodies associated with both early and late stages of pancreatic beta-cell destruction and development of type 1 diabetes. We have used five monoclonal anti-islet-cell antibodies (MICAs 1,2,3,4, and 6) derived from a newly diagnosed diabetic patient to probe the autoimmune epitopes in the enzyme. All the MICAs specifically recognized the smaller GAD protein, GAD65, and did not recognize the nonallelic GAD67 protein. A series of N-terminal, C-terminal, and internal deletion mutants, as well as protein footprinting, were used to identify the target regions in GAD65. Immunoprecipitation revealed two major native epitope areas in the GAD65 molecule. The first, defined by MICAs 1 and 3, is destroyed by deleting 41 amino acids at the C terminus but is also dependent on intact amino acids 244-295. This epitope (or epitopes) may span both middle and C-terminal domains of the protein. The second conformational epitope region, defined by MICAs 4 and 6, is dependent on intact amino acids 245-295 but is not affected by deletion of 110 amino acids at the C terminus and is therefore confined to domain(s) in the middle of the molecule. MICA 2 recognizes a linear epitope close to the C terminus. Thus, the N-terminal domain of GAD65, which differs most significantly from GAD67, does not harbor the MICA epitopes. Rather subtle amino acid differences in the middle and C-terminal domains define the GAD65-specific autoimmune epitopes. Analysis of sera from 10 type 1 diabetic patients suggests that MICAs 1, 3, 4, and 6 represent a common epitope recognition in this disease, whereas the MICA 2 epitope is rare. Furthermore, autoantibodies in some sera are restricted to the MICA 1/3 epitope, suggesting that this epitope may represent a single dominant epitope in the early phases of beta-cell autoimmunity.
Antibody-promoted dimerization bypasses the regulation of DNA binding by the heme domain of the yeast transcriptional activator HAP1.
Authors: Zhang L , Bermingham-McDonogh O , Turcotte B , Guarente L
Published: May 2006

The yeast transcriptional activator HAP1 contains a DNA-binding domain homologous to the zinc finger of GAL4 and an adjacent regulatory domain that blocks DNA binding in the absence of the inducer heme. We show that short HAP1 fragments containing the zinc finger are unable to bind to DNA but can be rescued by antibody to the HAP1 zinc finger. These fragments are missing a coiled-coil sequence similar to that within the dimerization domain of GAL4 and dimerization domains of myosin heavy chain. We surmise that the antibody promotes DNA binding by bringing together two monomers. Interestingly, the antibody will also promote DNA binding of a larger HAP1 fragment containing the DNA-binding and the heme-regulatory domains. This suggests that the regulatory domain acts by preventing dimerization of HAP1 in the absence of heme. Consistent with this view is an in vivo assay that also reveals that heme promotes HAP1 dimerization in yeast cells.
Synergistic activation of transcription by Escherichia coli cAMP receptor protein.
Authors: Joung J K , Le L U , Hochschild A
Published: May 2006

Activation of gene expression in eukaryotes generally involves the action of multiple transcription factors that function synergistically when bound near a particular target gene. Such effects have been suggested to occur because multiple activators can interact simultaneously with one or more components of the basal transcription machinery. In prokaryotes, examples of synergistic effects on transcription are much more limited and can often be explained by cooperative DNA binding. Here we show that the Escherichia coli cAMP receptor protein (CRP) functions synergistically to activate transcription from a derivative of the lac promoter that bears a second CRP-binding site upstream of the natural binding site. We present evidence indicating that cooperative DNA binding of two CRP dimers does not account for the magnitude of the observed cooperative activation. We suggest, instead, that the two dimers stimulate transcription directly by contacting two distinct surfaces of RNA polymerase simultaneously. Thus, synergistic activation by CRP may provide a relatively simple model for examining the molecular basis of such effects in higher organisms.
T-type calcium channels in adrenal glomerulosa cells: GTP-dependent modulation by angiotensin II.
Authors: McCarthy R T , Isales C , Rasmussen H
Published: May 2006

With the use of whole-cell and single-channel current recordings, we have examined in more detail the site of action of angiotensin II (AII) on multiple populations of voltage-gated calcium channels in bovine adrenal glomerulosa cells. AII (10 nM) enhances whole-cell T-type calcium channel current and increases the activity of single T-type calcium channels in cell-attached patch recordings. The AII-induced enhancement of whole-cell calcium channel currents is dependent on the presence of internal GTP and can be inhibited by the competitive AII-receptor antagonist saralasin (1 microM). These results show that AII augments the T-type calcium channel current in bovine adrenal glomerulosa cells.
Sp-1 binds promoter elements regulated by the RB protein and Sp-1-mediated transcription is stimulated by RB coexpression.
Authors: Udvadia A J , Rogers K T , Higgins P D , Murata Y , Martin K H , Humphrey P A , Horowitz J M
Published: May 2006

The retinoblastoma (RB) protein is implicated in transcriptional regulation of at least five cellular genes, including c-fos, c-myc, and transforming growth factor beta 1. Cotransfection of RB and truncated promoter constructs has defined a discrete element (retinoblastoma control element; RCE) within the promoters of each of these genes as being necessary for RB-mediated transcription control. Previously, we have shown that RCEs form protein-DNA complexes in vitro with three heretofore unidentified nuclear proteins and mutation of their DNA-binding site within the c-fos RCE results in an abrogation of RCE-dependent transcription in vivo. Here, we demonstrate that one of the nuclear proteins that binds the c-fos, c-myc, and transforming growth factor beta 1 RCEs in vitro is Sp-1 and that Sp-1 stimulates RCE-dependent transcription in vivo. Moreover, we show that Sp-1-mediated transcription is stimulated by the transient coexpression of RB protein. We conclude from these observations that RB may regulate transcription in part by virtue of its ability to functionally interact with Sp-1.
Acquired multicellular-mediated resistance to alkylating agents in cancer.
Authors: Kobayashi H , Man S , Graham C H , Kapitain S J , Teicher B A , Kerbel R S
Published: May 2006

EMT-6 murine mammary tumor sublines highly resistant to cyclophosphamide, cis-diamminedichloro-platinum(II), or N,N',N"-triethylenethiophosphoramide were generated in vivo by sequential treatment of tumor-bearing mice with the respective drugs. Previous studies demonstrated the drug-resistant phenotypes of the sublines were not expressed in vitro when the cells were grown as monolayer cultures. We now show that expression of drug resistance--including patterns of cross-drug resistance observed in vivo--can be fully recapitulated in vitro when the cells are grown under in vivo-like, three-dimensional conditions--namely, as multicellular tumor spheroids. Moreover, the spheroids generated from all of the drug-resistant sublines manifested a much more compact structure. Immediate drug-sensitivity testing of single cells released by trypsin treatment from compact drug-resistant spheroids revealed that such cells lost much of their drug-resistant properties. The results suggest a possible mechanism of acquired drug resistance in tumors based on the response of a cell population (i.e., multicellular or tissue resistance) as opposed to classic (uni)cellular resistance mechanisms.
A regulatory cascade hypothesis for mammalian sex determination: SRY represses a negative regulator of male development.
Authors: McElreavey K , Vilain E , Abbas N , Herskowitz I , Fellous M
Published: May 2006

The mammalian Y chromosome carries the SRY gene, which determines testis formation. Here we review data on individuals who are XX but exhibit male characteristics: some have SRY; others do not. We have analyzed three families containing more than one such individual and show that these individuals lack SRY. Pedigree analysis leads to the hypothesis that they carry recessive mutations (in a gene termed Z) that allow expression of male characteristics. We propose that wild-type Z product is a negative regulator of male sex determination and is functional in wild-type females. In males, SRY product represses or otherwise negatively regulates Z and thereby allows male sex determination. This hypothesis can also explain other types of sex reversal in mammals, in particular, XY females containing SRY. Some of these individuals may have mutations at the Z locus rendering them insensitive to SRY. Recessive mutations (such as the polled mutation of goats) leading to sex reversal are known in a variety of animals and might be used to map and ultimately clone the human Z gene.