bluuebunny/crossref_metadata_2025 huggingface dataset:
The murine c-fgr gene product associated with Ly6C and p70 integral membrane protein is expressed in cells of a monocyte/macrophage lineage.
Authors: Hatakeyama S , Iwabuchi K , Ogasawara K , Good R A , Onoé K
Published: May 2006

The c-fgr gene is a member of the Src family of protooncogene tyrosine kinases. A monoclonal antibody (2H2) that recognizes the specific region of the N-terminal domain of the murine c-fgr gene product (Fgr) has been established. With an immune complex kinase assay in a monocytic leukemia cell line, 2H2 monoclonal antibody was shown to precipitate a 59-kDa protein that corresponds in molecular mass to murine Fgr. Fgr was expressed highly in lymph nodes, slightly in spleen and peripheral blood leukocytes, and barely in the thymus and was not detected in bone marrow. In the presence of a mild detergent, Fgr was coimmunoprecipitated with a 70-kDa protein (p70) or with p70 plus several other molecules that were expressed on the cell-surface membrane of macrophage tumor cell lines PU5-1.8 and J774.1, respectively. By contrast, Fgr was not coimmunoprecipitated with a low-affinity receptor for the Fc portion of IgG that is associated with human Fgr. The molecule was also coimmunoprecipitated with the Ly6C molecule from a macrophage cell line (J774.1) that showed protein-tyrosine kinase activity. Peptide mapping revealed that this kinase activity was derived from Fgr. The similarity of relationship between this intramembrane p70 and/or Ly6C and cytoplasmic Fgr to relationships previously reported between T-cell antigen receptor complex, including CD4 and CD8 coreceptors, and Lck or Fyn in T cells and between surface IgM and Lyn or Blk in B cells, suggests that the Fgr and p70 or Ly6C are, indeed, associated with each other and in the murine system may be responsible for recognition of extracellular substances (either cellular or noncellular) and for signal transduction in cells of monocyte/macrophage lineage.
Synthetic peptide ligands of the antigen binding receptor induce programmed cell death in a human B-cell lymphoma.
Authors: Renschler M F , Bhatt R R , Dower W J , Levy R
Published: May 2006

Peptide ligands for the antigen binding site of the surface immunoglobulin receptor of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8- and 12-amino acid peptides. Corresponding synthetic peptides bound specifically to the antigen binding site of this immunoglobulin receptor and blocked the binding of an anti-idiotype antibody. The ligands, when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro with an IC50 between 40 and 200 nM. This effect was associated with specific stimulation of intracellular protein tyrosine phosphorylation.
Molecular basis of splotch and Waardenburg Pax-3 mutations.
Authors: Chalepakis G , Goulding M , Read A , Strachan T , Gruss P
Published: May 2006

Pax genes control certain aspects of development, as mutations result in (semi)dominant defects apparent during embryogenesis. Pax-3 has been associated with the mouse mutant splotch (Sp) and the human Waardenburg syndrome type 1 (WS1). We have examined the molecular basis of splotch and WS1 by studying the effect of mutations on DNA binding, using a defined target sequence. Pax-3 contains two different types of functional DNA-binding domains, a paired domain and a homeodomain. Mutational analysis of Pax-3 reveals different modes of DNA binding depending on the presence of these domains. A segment of Pax-3 located between the two DNA-binding domains, including a conserved octapeptide, participates in protein homodimerization. Pax-3 mutations found in splotch alleles and WS1 individuals change DNA binding and, in the case of a protein product of the Sp allele, dimerization. These findings were taken as a basis to define the molecular nature of the mutants.
In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity.
Authors: Barbas C F , Hu D , Dunlop N , Sawyer L , Cababa D , Hendry R M , Nara P L , Burton D R
Published: May 2006

A method is described that allows for the improvement of antibody affinity. This method, termed complementary-determining region (CDR) walking, does not require structural information on either antibody or antigen. Complementary-determining regions are targeted for random mutagenesis followed by selection for fitness, in this case increased binding affinity, by the phage-display approach. The current study targets a human CD4-binding-site anti-gp120 antibody that is potently and broadly neutralizing. Evolution of affinity of this antibody demonstrates in this case that affinity can be increased while reactivity to variants of human immunodeficiency virus type 1 is broadened. The neutralizing ability of this antibody is improved, as assayed with laboratory and primary clinical isolates of human immunodeficiency virus type 1. The ability to produce human antibodies of exceptional affinity and broad neutralizing ability has implications for the therapeutic and prophylactic application of antibodies for human immunodeficiency virus type 1 infection.
The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B.
Authors: Moon I S , Apperson M L , Kennedy M B
Published: May 2006

The postsynaptic density (PSD) is a specialization of the submembranous cytoskeleton that is visible in the electron microscope on the cytoplasmic face of the postsynaptic membrane. A subcellular fraction enriched in structures with the morphology of PSDs contains signal-transduction molecules thought to regulate receptor localization and function in the central nervous system. We have purified a prominent tyrosine-phosphorylated glycoprotein of apparent molecular mass 180 kDa, termed PSD-gp180, that is highly enriched in the rat forebrain PSD fraction. The sequences of four tryptic peptides generated from the protein reveal that it is the 2B subunit of the N-methyl-D-aspartate (NMDA) type glutamate receptor. We have confirmed the identity of PSD-gp180 by showing that it reacts with antibodies raised against a unique fragment of the 2B subunit of the NMDA receptor. We also show that the 2B subunit is the most prominently tyrosine-phosphorylated protein in the PSD fraction based upon recognition by an anti-phosphotyrosine antibody. Two types of NMDA receptor subunits have been identified by molecular cloning [Nakanishi, S. (1992) Science 258, 597-603]. The single type 1 subunit is expressed throughout the brain and is necessary for formation of the receptor channel. The four type 2 subunits (2A, 2B, 2C, and 2D) are expressed in discrete brain regions, contain unusually long unique C termini, and confer distinct kinetic properties on NMDA receptors that contain them. Our findings suggest that, in the forebrain, NMDA receptor subunit 2B may serve to anchor NMDA receptors at the postsynaptic membrane through its interaction with the PSD. The prominent presence of tyrosine phosphate further suggests that the NMDA receptor may be regulated by tyrosine phosphorylation or that it may participate in signaling through tyrosine phosphorylation and through its ion channel.
Dual inhibition of angiotensin-converting enzyme and neutral endopeptidase by the orally active inhibitor mixanpril: a potential therapeutic approach in hypertension.
Authors: Fournié-Zaluski M C , Gonzalez W , Turcaud S , Pham I , Roques B P , Michel J B
Published: May 2006

In the treatment of cardiovascular disease, it could be of therapeutic interest to associate the hypotensive effects due to the inhibition of angiotensin II formation with the diuretic and natriuretic responses induced by the protection of the endogenous atrial natriuretic peptide (ANP). Investigation of this hypothesis requires an orally active compound able to simultaneously inhibit angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP), which is involved in renal ANP metabolism. Such compounds have been rationally designed by taking into account the structural characteristics of the active site of both peptidases. Among them, RB 105, N-[(2S,3R)-2-mercaptomethyl-1-oxo-3-phenylbutyl]-(S)-alanine, inhibited NEP and ACE with Ki values of 1.7 +/- 0.3 nM and 4.2 +/- 0.5 nM, respectively. Intravenous infusion of RB 105 in conscious spontaneously hypertensive rats prevented the pressor response to exogenous angiotensin I and potentiated the natriuretic response to ANP. Infusion of RB 105, at 2.5, 5, 10, 25, and 50 mg/kg per hr decreased blood pressure dose-dependently in conscious catheterized spontaneously hypertensive rats and increased diuresis and natriuresis. Infusion of RB 105 as a bolus of 25 mg/kg followed by 25 mg/kg per hr similarly decreased blood pressure and increased natriuresis in three different models of hypertension (renovascular, deoxycorticosterone acetate-salt, and spontaneously hypertensive rats). Mixanpril, a lipophilic prodrug of RB 105 (ED50 values when given orally to mice, 0.7 mg/kg for NEP; 7 mg/kg for ACE), elicited dose-dependent hypotensive effects of long duration in spontaneously hypertensive rats after oral administration [-37 mmHg for 50 mg/kg twice a day (1 mmHg = 133 Pa) and is therefore the first dual NEP/ACE inhibitor potentially useful for clinical investigations.
Thalidomide is an inhibitor of angiogenesis.
Authors: D'Amato R J , Loughnan M S , Flynn E , Folkman J
Published: May 2006

Thalidomide is a potent teratogen causing dysmelia (stunted limb growth) in humans. We have demonstrated that orally administered thalidomide is an inhibitor of angiogenesis induced by basic fibroblast growth factor in a rabbit cornea micropocket assay. Experiments including the analysis of thalidomide analogs revealed that the antiangiogenic activity correlated with the teratogenicity but not with the sedative or the mild immunosuppressive properties of thalidomide. Electron microscopic examination of the corneal neovascularization of thalidomide-treated rabbits revealed specific ultrastructural changes similar to those seen in the deformed limb bud vasculature of thalidomide-treated embryos. These experiments shed light on the mechanism of thalidomide's teratogenicity and hold promise for the potential use of thalidomide as an orally administered drug for the treatment of many diverse diseases dependent on angiogenesis.
Gene transfer in vivo: sustained expression and regulation of genes introduced into the liver by receptor-targeted uptake.
Authors: Perales J C , Ferkol T , Beegen H , Ratnoff O D , Hanson R W
Published: May 2006

Receptor-mediated gene transfer has been used to introduce genes into tissues of animals in vivo. The genes introduced by this approach have been transiently expressed at low levels in animal tissues. High levels of expression, for longer periods, have been attained by the induction of cell division (i.e., partial hepatectomy) or disruption of lysosomal degradation of the DNA. We have studied the correlation of specific structural features on the DNA/ligand complexes with their ability to efficiently introduce DNA into the livers of intact animals. A chimeric gene containing the phosphoenolpyruvate carboxykinase gene promoter (nucleotides -460 to +73) linked to the structural gene for human factor IX (PEPCK-hFIX gene) was condensed with galactosylated poly(L-lysine) by titration with NaCl, resulting in complexes of defined size (10-12 nm in diameter) and shape. The PEPCK-hFIX gene complex was injected into the caudal vena cava of adult rats and the conjugated DNA was specifically targeted to the livers of the animals; no detectable DNA was noted in other tissues. The plasmid containing the PEPCK-hFIX gene was found as an episome in the livers of the rats 32 days after injection of the DNA complex. Human factor IX DNA, mRNA, and functional protein were detected up to 140 days after administration of the DNA complex (the duration of the experiment). Transcription from the PEPCK promoter could be induced over the entire course of the experiment by feeding the rats a high-protein, carbohydrate-free diet. We conclude that the structure of the DNA/ligand complexes is of key importance for the successful introduction of genes into the tissues of animals by receptor-mediated endocytosis.
"Latent" inhibitory connections become functional during activity-dependent plasticity.
Authors: Charpier S , Behrends J C , Triller A , Faber D S , Korn H
Published: May 2006

Simultaneous pre- and postsynaptic recordings from identified glycinergic inhibitory interneurons and the Mauthner cell showed that 25% of the afferents produced no or extremely small postsynaptic responses. Morphological determination of the number of contacts made by these cells on the Mauthner cell revealed a connectivity similar to that of functional neurons which always produce clear inhibitory postsynaptic potentials, suggesting that most of the endings, made by weak interneurons are silent. Intraaxonal injection of 4-aminopyridine or Ca2+ greatly enhanced transmission at functional connections but did not modify those which were ineffective. However, after eighth nerve tetanic stimuli, transmission at the weak connections was unmasked or enhanced for prolonged periods and was twice as likely to be potentiated, with a 6-fold greater mean enhancement than the potent ones. This result provides additional support for long-term potentiation of inhibitory synapses. Furthermore, weakly functional junctions represent a "reserve" pool which can be critical for the expression of plasticity within a network, and, consequently, for setting the threshold of reflex activities such as the escape reaction mediated by the Mauthner cell.
Increased cell surface expression and enhanced folding in the endoplasmic reticulum of a mutant erythropoietin receptor.
Authors: Hilton D J , Watowich S S , Murray P J , Lodish H F
Published: May 2006

In both transfected and normal hematopoietic cells, the majority of newly made erythropoietin receptor (EPO-R) subunits are retained in the endoplasmic reticulum (ER), destined for degradation. Only a small fraction exit the ER and are competent to bind EPO, suggesting that the EPO-R folds inefficiently. The EPO-R contains a 5-amino acid motif, WSXWS, in the extracellular domain that is conserved among members of the cytokine receptor family. We describe a mutant EPO-R with a change in the middle residue of this motif, A234E, that is transported from the ER more efficiently than the wild-type (wt) receptor and is expressed in elevated numbers at the cell surface. This mutant polypeptide is processed more efficiently in the ER than its wt counterpart, suggesting that it folds better than the wt EPO-R. Inefficient folding and processing of the wt EPO-R in the ER may be one mechanism for controlling the number of plasma membrane receptors.
Virus-mediated or transgenic suppression of a G-protein alpha subunit and attenuation of fungal virulence.
Authors: Choi G H , Chen B , Nuss D L
Published: May 2006

Strains of the chestnut blight fungus Cryphonectria parasitica harboring RNA viruses of the genus Hypovirus exhibit significantly reduced levels of virulence (called hypovirulence). The accumulation of a heterotrimeric GTP-binding protein (G protein) alpha subunit of the Gi class was found to be reduced in hypovirus-containing C. parasitica strains. Transgenic cosuppression, a phenomenon frequently observed in transgenic plants, reduced the accumulation of this alpha subunit in virus-free fungal strains. Significantly, the resulting transgenic fungal strains were also hypovirulent. These results indicate a crucial role for G-protein-linked signal transduction in fungal pathogenesis and suggest a molecular basis for virus-mediated attenuation of fungal virulence.
Chemical ecology: a view from the pharmaceutical industry.
Authors: Caporale L H
Published: May 2006

Biological diversity reflects an underlying molecular diversity. The molecules found in nature may be regarded as solutions to challenges that have been confronted and overcome during molecular evolution. As our understanding of these solutions deepens, the efficiency with which we can discover and/or design new treatments for human disease grows. Nature assists our drug discovery efforts in a variety of ways. Some compounds synthesized by microorganisms and plants are used directly as drugs. Human genetic variations that predispose to (or protect against) certain diseases may point to important drug targets. Organisms that manipulate molecules within us to their benefit also may help us to recognize key biochemical control points. Drug design efforts are expedited by knowledge of the biochemistry of a target. To supplement this knowledge, we screen compounds from sources selected to maximize molecular diversity. Organisms known to manipulate biochemical pathways of other organisms can be sources of particular interest. By using high throughput assays, pharmaceutical companies can rapidly scan the contents of tens of thousands of extracts of microorganisms, plants, and insects. A screen may be designed to search for compounds that affect the activity of an individual targeted human receptor, enzyme, or ion channel, or the screen might be designed to capture compounds that affect any step in a targeted metabolic or biochemical signaling pathway. While a natural product discovered by such a screen will itself only rarely become a drug (its potency, selectivity, bioavailability, and/or stability may be inadequate), it may suggest a type of structure that would interact with the target, serving as a point of departure for a medicinal chemistry effort--i.e., it may be a "lead." It is still beyond our capability to design, routinely, such lead structures, based simply upon knowledge of the structure of our target. However, if a drug discovery target contains regions of structure homologous to that in other proteins, structures known to interact with those proteins may prove useful as leads for a medicinal chemistry effort. The specificity of a lead for a target may be optimized by directing structural variation to specificity-determining sites and away from those sites required for interaction with conserved features of the targeted protein structure. Strategies that facilitate recognition and exploration of sites at which variation is most likely to generate a novel function increase the efficiency with which useful molecules can be created.
The importance of intramolecular ion pairing in intermediate filaments.
Authors: Letai A , Fuchs E
Published: May 2006

Nuclear and cytoskeletal networks of 10-nm intermediate filaments (IFs) are probably ubiquitous in multicellular eukaryotes. They likely play a role in maintaining the mechanical integrity of a cell. With the exception of the nuclear lamins, IF proteins can form IFs in vitro in the absence of cofactors or associated proteins. Below we present data suggesting that the large alpha-helical "rod" domains of IF proteins are stabilized by large numbers (up to 50) of intra-helical ion pairs formed by residues of opposite charge situated four residues apart. These many ion pairs, sometimes involving up to 30% of the residues within a coiled-coil IF segment, can potentially contribute as much as 10-25 kcal/mol (1 kcal = 4.18 kJ) to the stability of a single alpha-helical rod. Such stabilization is likely to play a major role in the chemical and physical stability of IF networks in vitro and in vivo. An investigation of other coiled-coil proteins shows that selection for intrahelical ion pairing is not simply a property intrinsic to coiled-coil proteins. Rather, there is a correlation between the degree to which there is selection for intrahelical ion pairs and the extent to which a coiled-coil protein participates in highly ordered multimolecular interactions--e.g., as in IFs and myosin thick filaments. The propensity of putative ion pairs in some IF proteins--e.g., epidermal keratins--suggests that an underlying structural stability at the level of the monomer may play an important role in the extraordinary stability of dimers and higher ordered structures in cytoplasmic IFs.
Low major histocompatibility complex class II diversity in European and North American moose.
Authors: Mikko S , Andersson L
Published: May 2006

Major histocompatibility complex (MHC) genes encode cell surface proteins whose function is to bind and present intracellularly processed peptides to T lymphocytes of the immune system. Extensive MHC diversity has been documented in many species and is maintained by some form of balancing selection. We report here that both European and North American populations of moose (Alces alces) exhibit very low levels of genetic diversity at an expressed MHC class II DRB locus. The observed polymorphism was restricted to six amino acid substitutions, all in the peptide binding site, and four of these were shared between continents. The data imply that the moose have lost MHC diversity in a population bottleneck, prior to the divergence of the Old and New World subspecies. Sequence analysis of mtDNA showed that the two subspecies diverged at least 100,000 years ago. Thus, viable moose populations with very restricted MHC diversity have been maintained for a long period of time. Both positive selection for polymorphism and intraexonic recombination have contributed to the generation of MHC diversity after the putative bottleneck.
Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7.
Authors: Orlic D , Anderson S , Biesecker L G , Sorrentino B P , Bodine D M
Published: May 2006

Pluripotent hematopoietic stem cells (PHSCs) were highly enriched from mouse bone marrow by counterflow centrifugal elutriation, lineage subtraction, and fluorescence-activated cell sorting based on high c-kit receptor expression (c-kitBR). We used reverse transcriptase polymerase chain reaction to assay the c-kitBR subset and the subsets expressing low (c-kitDULL) and no (c-kitNEG) c-kit receptor for expression of mRNA encoding hematopoietic growth factor receptors and transcription factors. The c-kitBR cells had approximately 3.5-fold more c-kit mRNA than unfractionated bone marrow cells. The c-kitDULL cells had 47-58% of the c-kit mRNA found in c-kitBR cells and the c-kitNEG cells had 4-9% of the c-kit mRNA present in c-kitBR cells. By comparing mRNA levels in c-kitBR cells (enriched for PHSCs) with those of unfractionated bone marrow, we demonstrated that c-kitBR cells contained low or undetectable levels of mRNA for c-fms, granulocyte colony-stimulating factor receptor, interleukin 5 receptor (IL-5R), and IL-7R. These same cells had moderate levels of mRNA for erythropoietin receptor, IL-3R subunits IL-3R alpha (SUT-1), AIC-2A, and AIC-2B, IL-6R and its partner gp-130, and the transcription factor GATA-1 and high levels of mRNA for transcription factors GATA-2, p45 NF-E2, and c-myb. We conclude from these findings that PHSCs are programmed to interact with stem cell factor, IL-3, and IL-6 but not with granulocyte or macrophage colony-stimulating factor. These findings also indicate that GATA-2, p45 NF-E2, and c-myb activities may be involved in PHSC maintenance or proliferation.
Pax-6 is essential for lens-specific expression of zeta-crystallin.
Authors: Richardson J , Cvekl A , Wistow G
Published: May 2006

Pax-6 is essential for normal eye development and has been implicated as a "master gene" for lens formation in embryogenesis. Guinea pig zeta-crystallin, a taxon-specific enzyme crystallin, achieves high expression specifically in lens through use of an alternative promoter. Here we show that Pax-6 binds a site in this promoter, which is essential for lens-specific expression. Lens and lens-derived cells exhibit a tissue-specific pattern of alternative splicing of Pax-6 transcripts and Pax-6 is expressed in adult lenses and cells that support zeta-crystallin expression. These results suggest that zeta-crystallin is a natural target gene for Pax-6 and that this Pax family member has a direct role in the continuing expression of tissue-specific genes.
Ectopic expression of the agouti gene in transgenic mice causes obesity, features of type II diabetes, and yellow fur.
Authors: Klebig M L , Wilkinson J E , Geisler J G , Woychik R P
Published: May 2006

Mice that carry the lethal yellow (Ay) or viable yellow (Avy) mutation, two dominant mutations of the agouti (a) gene in mouse chromosome 2, exhibit a phenotype that includes yellow fur, marked obesity, a form of type II diabetes associated with insulin resistance, and an increased susceptibility to tumor development. Molecular analyses of these and several other dominant "obese yellow" a-locus mutations suggested that ectopic expression of the normal agouti protein gives rise to this complex pleiotropic phenotype. We have now tested this hypothesis directly by generating transgenic mice that ectopically express an agouti cDNA clone encoding the normal agouti protein in all tissues examined. Transgenic mice of both sexes have yellow fur, become obese, and develop hyperinsulinemia. In addition, male transgenic mice develop hyperglycemia by 12-20 weeks of age. These results demonstrate conclusively that the ectopic agouti expression is responsible for most, if not all, of the phenotypic traits of the dominant, obese yellow mutants.
Aod1, the immunoregulatory locus controlling abrogation of tolerance in neonatal thymectomy-induced autoimmune ovarian dysgenesis, maps to mouse chromosome 16.
Authors: Wardell B B , Michael S D , Tung K S , Todd J A , Blankenhorn E P , McEntee K , Sudweeks J D , Hansen W K , Meeker N D , Griffith J S
Published: May 2006

Mice thymectomized at three days of age (D3Tx) develop during adulthood a variety of organ-specific autoimmune diseases, including autoimmune ovarian dysgenesis (AOD). The phenotypic spectrum of AOD is characterized by the development of anti-ovarian autoantibodies, oophoritis, and atrophy. The D3Tx model of AOD is unique in that disease induction depends exclusively on perturbation of the normal developing immune system, is T-cell-mediated, and is strain specific. For example, D3Tx A/J mice are highly susceptible to AOD, whereas C57BL/6J mice are resistant. After D3Tx, self ovarian antigens, expressed at physiological levels, trigger an autoimmune response capable of eliciting disease. The D3Tx model provides, therefore, the opportunity to focus on the mechanisms of self-tolerance that are relevant to disease pathogenesis. Previous studies indicate that the principal mechanisms involved in AOD susceptibility are genetically controlled and govern developmental processes associated with the induction and maintenance of peripheral tolerance. We report here the mapping of the Aod1 locus to mouse chromosome 16 within a region encoding several loci of immunologic relevance, including scid, Igl1, VpreB, Igll, Igl1r, Mtv6 (Mls-3), Ly-7, Ifnar, and Ifgt.
Human steroidogenic acute regulatory protein: functional activity in COS-1 cells, tissue-specific expression, and mapping of the structural gene to 8p11.2 and a pseudogene to chromosome 13.
Authors: Sugawara T , Holt J A , Driscoll D , Strauss J F , Lin D , Miller W L , Patterson D , Clancy K P , Hart I M , Clark B J
Published: May 2006

Steroidogenic acute regulatory protein (StAR) appears to mediate the rapid increase in pregnenolone synthesis stimulated by tropic hormones. cDNAs encoding StAR were isolated from a human adrenal cortex library. Human StAR, coexpressed in COS-1 cells with cytochrome P450scc and adrenodoxin, increased pregnenolone synthesis > 4-fold. A major StAR transcript of 1.6 kb and less abundant transcripts of 4.4 and 7.5 kb were detected in ovary and testis. Kidney had a lower amount of the 1.6-kb message. StAR mRNA was not detected in other tissues including placenta. Treatment of granulosa cells with 8-bromo-adenosine 3',5'-cyclic monophosphate for 24 hr increased StAR mRNA 3-fold or more. The structural gene encoding StAR was mapped using somatic cell hybrid mapping panels to chromosome 8p. Fluorescence in situ hybridization placed the StAR locus in the region 8p11.2. A StAR pseudogene was mapped to chromosome 13. We conclude that StAR expression is restricted to tissues that carry out mitochondrial sterol oxidations subject to acute regulation by cAMP and that StAR mRNA levels are regulated by cAMP.
Mimics of the binding sites of opioid receptors obtained by molecular imprinting of enkephalin and morphine.
Authors: Andersson L I , Müller R , Vlatakis G , Mosbach K
Published: May 2006

Molecular imprinting of morphine and the endogenous neuropeptide [Leu5]enkephalin (Leu-enkephalin) in methacrylic acid-ethylene glycol dimethacrylate copolymers is described. Such molecular imprints possess the capacity to mimic the binding activity of opioid receptors. The recognition properties of the resultant imprints were analyzed by radioactive ligand binding analysis. We demonstrate that imprinted polymers also show high binding affinity and selectivity in aqueous buffers. This is a major breakthrough for molecular imprinting technology, since the binding reaction occurs under conditions relevant to biological systems. The antimorphine imprints showed high binding affinity for morphine, with Kd values as low as 10(-7) M, and levels of selectivity similar to those of antibodies. Preparation of imprints against Leu-enkephalin was greatly facilitated by the use of the anilide derivative rather than the free peptide as the print molecule, due to improved solubility in the polymerization mixture. Free Leu-enkephalin was efficiently recognized by this polymer (Kd values as low as 10(-7) M were observed). Four tetra- and pentapeptides, with unrelated amino acid sequences, were not bound. The imprints showed only weak affinity for two D-amino acid-containing analogues of Leu-enkephalin. Enantioselective recognition of the L-enantiomer of phenylalanylglycine anilide, a truncated analogue of the N-terminal end of enkephalin, was observed.
A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA.
Authors: Chen L , Frankel A D
Published: May 2006

A 17-amino acid arginine-rich peptide from the bovine immunodeficiency virus Tat protein has been shown to bind with high affinity and specificity to bovine immunodeficiency virus transactivation response element (TAR) RNA, making contacts in the RNA major groove near a bulge. We show that, as in other peptide-RNA complexes, arginine and threonine side chains make important contributions to binding but, unexpectedly, that one isoleucine and three glycine residues also are critical. The isoleucine side chain may intercalate into a hydrophobic pocket in the RNA. Glycine residues may allow the peptide to bind deeply within the RNA major groove and may help determine the conformation of the peptide. Similar features have been observed in protein-DNA and drug-DNA complexes in the DNA minor groove, including hydrophobic interactions and binding deep within the groove, suggesting that the major groove of RNA and minor groove of DNA may share some common recognition features.
Presence of antibodies to different subunits of replication protein A in autoimmune sera.
Authors: Garcia-Lozano R , Gonzalez-Escribano F , Sanchez-Roman J , Wichmann I , Nuñez-Roldan A
Published: May 2006

A human cDNA expression library was used to investigate the nature of molecules recognized by serum from a patient with Sjögren syndrome that exhibits a mixed immunofluorescence pattern and reacts with multiple components on an immunoblot. The data demonstrated that this serum contains IgG antibodies specific for the 70- and 32-kDa subunits of replication protein A (RPA; RPA-70 and RPA-32, respectively), a highly conserved multisubunit DNA binding protein. Affinity purification of serum autoantibodies demonstrated a complete lack of cross-reactivity between RPA-70 and RPA-32, suggesting a direct participation of the native protein complex in the autoimmune response in this patient. Purified anti-RPA-70 and anti-RPA-32 antibodies labeled nuclear and cytoplasmic components in an immunofluorescence assay, suggesting that RPA is present in both cellular compartments. Additional sera from 55 patients with different autoimmune conditions were screened against purified RPA-70 and RPA-32 recombinant proteins. One of these 55 sera was positive and reacted with only RPA-32. Twenty sera from healthy control individuals did not react with RPA. These results show that RPA is a target for autoantibodies in human autoimmune diseases, although its precise frequency, occurrence in other autoimmune diseases, and pathological significance remain to be fully elucidated.
Isolation of a yeast protein kinase that is activated by the protein encoded by SRP1 (Srp1p) and phosphorylates Srp1p complexed with nuclear localization signal peptides.
Authors: Azuma Y , Tabb M M , Vu L , Nomura M
Published: May 2006

Srp1p, the protein encoded by SRP1 of Saccharomyces cerevisiae, is a nuclear-pore-associated protein. Its Xenopus homolog, importin, was recently shown to be an essential component required for nuclear localization signal (NLS)-dependent binding of karyophilic proteins to the nuclear envelope [Gorlich, D., Prehn, S., Laskey, R. A. & Hartman, E. (1994) Cell 79, 767-778]. We have discovered a protein kinase whose activity is stimulated by Srp1p (Srp1p fused to glutathione S-transferase and expressed in Escherichia coli) and is detected by phosphorylation of Srp1p and of a 36-kDa protein, a component of the protein kinase complex. The enzyme, called Srp1p kinase, is a protein-serine kinase and was found in extracts in two related complexes of approximately 180 kDa and 220 kDa. The second complex, when purified, contained four protein components including the 36-kDa protein. We observed that, upon purification of the kinase, phosphorylation of Srp1p became very weak, while activation of phosphorylation of the 36-kDa protein by Srp1p remained unaltered. Significantly, NLS peptides and the nuclear proteins we have tested greatly stimulated phosphorylation of Srp1p, suggesting that Srp1p, complexed with karyophilic proteins carrying an NLS, is the in vivo substrate of this protein kinase.
Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist.
Authors: Tavernier J , Tuypens T , Verhee A , Plaetinck G , Devos R , Van der Heyden J , Guisez Y , Oefner C
Published: May 2006

A detailed structure-function analysis of human interleukin 5 (hIL5) has been performed. The hIL5 receptor is composed of two different polypeptide chains, the alpha and beta subunits. The alpha subunit alone is sufficient for ligand binding, but association with the beta subunit leads to a 2- to 3-fold increase in binding affinity. The beta chain is shared with the receptors for IL3 and granulocyte/macrophage-colony-stimulating factor--hence the descriptor beta C (C for common). All hIL5 mutants were analyzed in a solid-phase binding assay for hIL5R alpha interaction and in a proliferation assay using IL5-dependent cell lines for receptor-complex activation. Most residues affecting binding to the receptor alpha subunit were clustered in a loop connecting beta-strand 1 and helix B (mutants H38A, K39A, and H41A), in beta-strand 2 (E89A and R91A; weaker effect for E90A) and close to the C terminus (T109A, E110A, W111S, and I112A). Mutations at one position, E13 (Glu13), caused a reduced activation of the hIL5 receptor complex. In the case of E13Q, only 0.05% bioactivity was detected on a hIL5-responsive subclone of the mouse promyelocytic cell line FDC-P1. Moreover, on hIL5-responsive TF1 cells, the same mutant was completely inactive and proved to have antagonistic properties. Interactions of this mutant with both receptor subunits were nevertheless indistinguishable from those of nonmutated hIL5 by crosslinking and Scatchard plot analysis of transfected COS-1 cells.
Chimeric tumor necrosis factor receptors with constitutive signaling activity.
Authors: Bazzoni F , Alejos E , Beutler B
Published: May 2006

Many hormone and cytokine receptors are crosslinked by their specific ligands, and multimerization is an essential step leading to the generation of a signal. In the case of the tumor necrosis factor (TNF) receptors (TNF-Rs), antibody-induced crosslinking is sufficient to trigger a cytolytic effect. However, the quaternary structural requirements for signaling--i.e., the formation of dimers, trimers, or higher-order multimers--have remained obscure. Moreover, it has not been clear whether the 55-kDa or 75-kDa TNF-R is responsible for initiation of cytolysis. We reasoned that an obligate receptor dimer, targeted to the plasma membrane, might continuously signal the presence of TNF despite the actual absence of the ligand. Such a molecule, inserted into an appropriate vector, could be used to project receptor-specific "TNF-like" activity to specific cells and tissues in vivo. Accordingly, we constructed sequences encoding chimeric receptors in which the extracellular domain of the mouse erythropoietin receptor (Epo-R) was fused to the "stem," transmembrane domain, and cytoplasmic domain of the two mouse TNF-Rs. Thus, the Epo-R group was used to drive dimerization of the TNF-R cytoplasmic domain. These chimeric proteins were well expressed in a variety of cell lines and bound erythropoietin at the cell surface. Both the 55-kDa and the 75-kDa Epo/TNF-R chimeras exerted a constitutive cytotoxic effect detected by cotransfection or clonogenic assay. Thus, despite the lack of structural homology between the cytoplasmic domains of the two TNF-Rs, a similar signaling endpoint was observed. Moreover, dimerization (rather than trimerization or higher-order multimerization) was sufficient for elicitation of a biological response.
Reciprocal modulations between p53 and Tat of human immunodeficiency virus type 1.
Authors: Li C J , Wang C , Friedman D J , Pardee A B
Published: May 2006

Infection by human immunodeficiency virus type 1 (HIV-1) causes acquired immunodeficiency syndrome (AIDS) after a long clinical latency. This disease is associated with a spectrum of cancers. Here we report that wild-type p53 is a potent suppressor of Tat, a major transactivator of HIV-1. Reciprocally, Tat inhibits the transcription of p53. Downregulation of p53 by upregulated tat may be important for the establishment of productive viral infection in a cell and also may be involved in the development of AIDS-related malignancies.
Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta chain but is not essential for the proliferative signal transmission.
Authors: Fujii H , Nakagawa Y , Schindler U , Kawahara A , Mori H , Gouilleux F , Groner B , Ihle J N , Minami Y , Miyazaki T
Published: May 2006

The high-affinity interleukin 2 (IL-2) receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two members of the Janus kinase family, Jak1 and Jak3, are associated with IL-2R beta c and IL-2R gamma c, respectively, and they are activated upon IL-2 stimulation. The cytokine-mediated Jak kinase activation usually results in the activation of a family of latent transcription factors termed Stat (signal transducer and activator of transcription) proteins. Recently, the IL-2-induced Stat protein was purified from human lymphocytes and found to be the homologue of sheep Stat5/mammary gland factor. We demonstrate that the human Stat5 is activated by IL-2 and that Jak3 is required for the efficient activation. The cytoplasmic region of the IL-2R beta c chain required for activation of Stat5 is mapped within the carboxyl-terminal 147 amino acids. On the other hand, this region is not essential for IL-2-induced cell proliferation.
A simple test of the vicarious trial-and-error hypothesis of hippocampal function.
Authors: Hu D , Amsel A
Published: May 2006

Vicarious trial-and-error (VTE) is a term that Muenzinger and Tolman used to describe the rat's conflict-like behavior before responding to choice. Recently, VTE was proposed as a mechanism alternative to the concept of "cognitive map" in accounts of hippocampal function. That is, many phenomena of impaired learning and memory related to hippocampal interventions may be explained by behavioral first principles: reduced conflicting, incipient, pre-choice tendencies to approach and avoid. The nonspatial black-white discrimination learning and VTE behavior of the rat were investigated. Hippocampal-lesioned and sham-lesioned animals were trained for 25 days (20 trials per day) starting at 60 days of age. Each movement of the head from one discriminative stimulus to the other was counted as a VTE instance. Lesioned rats had fewer VTEs than sham controls, and the former learned much more slowly or never learned. After learning, VTE frequency declined. Male and female rats showed no significant differences in VTE behavior or discrimination learning.
Cortical correlate of pattern backward masking.
Authors: Kovács G , Vogels R , Orban G A
Published: May 2006

The perception of a briefly presented shape is strongly impaired when it is followed by another pattern, a phenomenon called backward masking. We found that the vast majority of a sample of shape-selective neurons in the macaque inferior temporal cortex respond selectively to backward-masked shapes, although these shapes could not be discriminated by human and monkey subjects. However, this selective response was brief, since it was either interrupted by the mask or overridden by a response to the mask itself. We show that reliable discrimination of briefly presented shapes by single neurons depends on the temporal integration of the response. Presentation of the mask, however, reduces the number of spikes available for integration, explaining backward masking. These results also provide direct neurophysiological evidence for the "interruption theory" of backward masking.
Mitochondrial DNA replication but no nuclear DNA replication during development of Dictyostelium.
Authors: Shaulsky G , Loomis W F
Published: May 2006

Dictyostelium discoideum cells initiate development when nutrients are depleted. DNA synthesis decreases rapidly thereafter but resumes during late aggregation, only in prespore cells. This observation has been previously interpreted as indicating progression of prespore cells through the cell cycle during development. We show that developmental DNA replication occurs only in mitochondria and not in nuclei. We also show that the prestalk morphogen known as differentiation-inducing factor 1 can inhibit mitochondrial respiration. A model is proposed for cell type divergence, based on competition to become prespores, that involves mitochondrial replication in prespore cells and reduction of mitochondrial activity in prestalk cells.
Adaptive mutations in Escherichia coli as a model for the multiple mutational origins of tumors.
Authors: Hall B G
Published: May 2006

The cells in most tumors are found to carry multiple mutations; however, based upon mutation rates determined by fluctuation tests, the frequency of such multiple mutations should be so low that tumors are never detected within human populations. Fluctuation tests, which determine the cell-division-dependent mutation rate per cell generation in growing cells, may not be appropriate for estimating mutation rates in nondividing or very slowly dividing cells. Recent studies of time-dependent, "adaptive" mutations in nondividing populations of microorganisms suggest that similar measurements may be more appropriate to understanding the mutation origins of tumors. Here I use the ebgR and ebgA genes of Escherichia coli to measure adaptive mutation rates where multiple mutations are required for rapid growth. Mutations in either ebgA or ebgR allow very slow growth on lactulose (4-O-beta-D-galactosyl-D-fructose), with doubling times of 3.2 and 17.3 days, respectively. However, when both mutations are present, cells can grow rapidly with doubling times of 2.7 hr. I show that during prolonged (28-day) selection for growth on lactulose, the number of lactulose-utilizing mutants that accumulate is 40,000 times greater than can be accounted for on the basis of mutation rates measured by fluctuation tests, but is entirely consistent with the time-dependent adaptive mutation rates measured under the same conditions of prolonged selection.
Elevated expression of H type GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase is associated with human colon adenocarcinoma progression.
Authors: Sun J , Thurin J , Cooper H S , Wang P , Mackiewicz M , Steplewski Z , Blaszczyk-Thurin M
Published: May 2006

GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase (EC 2.4.1.69) is a key enzyme in the biosynthesis of fucosylated type 1 and 2 lactoseries structures, such as Lewis b and the H type 2 and Lewis Y, respectively, that are accumulated in colon adenocarcinoma. Analysis of the mRNA transcript level for the human H gene-encoded beta-D-galactoside alpha-2-L-fucosyltransferase revealed 40- and 340-fold increases in the mRNA levels in all adenocarcinomas and tumor cell lines, respectively, compared to normal colon mucosa where a low level of mRNA transcript was detected. A variable increase in mRNA transcript levels was observed in 50% of adenomatous polyps. Nucleotide sequence analysis of the protein coding region of the cDNAs derived from normal colon, adenoma, and colon adenocarcinoma revealed 100% homology, suggesting that there are no tumor-associated allelic variations within the H beta-D-galactoside alpha-2-L-fucosyltransferase cDNA. These results suggest that beta-D-galactoside alpha-2-L-fucosyltransferase expression highly correlates with malignant progression of colon adenocarcinoma.
Evidence from molecular systematics for decreased avian diversification in the pleistocene Epoch.
Authors: Zink R M , Slowinski J B
Published: May 2006

Pleistocene glaciations have been suggested as major events influencing speciation rates in vertebrates. Avian paleontological studies suggest that most extant species evolved in the Pleistocene Epoch and that species' durations decreased through the Pleistocene because of heightened speciation rates. Molecular systematic studies provide another data base for testing these predictions. In particular, rates of diversification can be determined from molecular phylogenetic trees. For example, an increasing rate of speciation (but constant extinction) requires shorter intervals between successive speciation events on a phylogenetic tree. Examination of the cumulative distribution of reconstructed speciation events in mtDNA phylogenies of 11 avian genera, however, reveals longer intervals between successive speciation events as the present time is approached, suggesting a decrease in net diversification rate through the Pleistocene Epoch. Thus, molecular systematic studies do not indicate a pulse of Pleistocene diversification in passerine birds but suggest, instead, that diversification rates were lower in the Pleistocene than for the preceding period. Documented habitat shifts likely led to the decreased rate of diversification, although from molecular evidence we cannot discern whether speciation rates decreased or extinction rates increased.