bluuebunny/crossref_metadata_2025 huggingface dataset:
A lag in intracellular degradation of mutant alpha 1-antitrypsin correlates with the liver disease phenotype in homozygous PiZZ alpha 1-antitrypsin deficiency.
Authors: Wu Y , Whitman I , Molmenti E , Moore K , Hippenmeyer P , Perlmutter D H
Published: May 2006

Liver injury in PiZZ alpha 1-antitrypsin (alpha 1-AT) deficiency probably results from toxic effects of the abnormal alpha 1-AT molecule accumulating within the ER of liver cells. However, only 12-15% of individuals with this same genotype develops liver disease. Therefore, we predicted that other genetic traits that determine the net intracellular accumulation of the mutant alpha 1-AT molecule would also determine susceptibility to liver disease. To address this prediction, we transduced skin fibroblasts from PiZZ individuals with liver disease or without liver disease with amphotropic recombinant retroviral particles designed for constitutive expression of the mutant alpha 1-AT Z gene. Human skin fibroblasts do not express the endogenous alpha 1-AT gene but presumably express other genes involved in postsynthetic processing of secretory proteins. The results show that expression of human alpha 1-AT gene was conferred on each fibroblast cell line. Compared to the same cell line transduced with the wild-type alpha 1-AT M gene, there was selective intracellular accumulation of the mutant alpha 1-AT Z protein in each case. However, there was a marked delay in degradation of the mutant alpha 1-AT Z protein after it accumulated in the fibroblasts from ZZ individuals with liver disease ("susceptible hosts") as compared to those without liver disease ("protected hosts"). Appropriate disease controls showed that the lag in degradation in susceptible hosts is specific for the combination of PiZZ phenotype and liver disease. Biochemical characteristics of alpha 1-AT Z degradation in the protected hosts were found to be similar to those of a common ER degradation pathway previously described in model experimental cell systems for T-cell receptor alpha subunits and asialoglycoprotein receptor subunits, therefore, raising the possibility that the lag in degradation in the susceptible host is a defect in this common ER degradation pathway. Thus, these data provide evidence that other genetic traits that affect the fate of the abnormal alpha 1-AT Z molecule, at least in part, determine susceptibility to liver disease. These data also validate a system for elucidating the biochemical/genetic characteristics of these traits and for examining the relevance to human disease of pathways for protein degradation in the ER.
Genetic variation detected by quantitative analysis of end-labeled genomic DNA fragments.
Authors: Asakawa J , Kuick R , Neel J V , Kodaira M , Satoh C , Hanash S M
Published: May 2006

The continuing efforts to evaluate specific human populations for altered germinal mutation rates would profit from more efficient and more specific approaches than those of the past. To this end, we have explored the potential usefulness of two-dimensional electrophoresis of DNA fragments obtained from restriction-enzyme-digested genomic DNA. This permits the analysis, on a single preparation, of approximately 2000 DNA fragments varying in size from 1.0 to 5.0 kb in the first dimension and from 0.3 to 2.0 kb in the second dimension. To enter into a genetic analysis, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requisite accuracy from spots that are the product of only one fragment, the coefficient of variation of spot intensity should be approximately < or = 0.12. At present, 482 of the spots in our preparations meet these standards. In an examination of preparations based on three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variation that segregated within families according to Mendelian principles. We have established the feasibility of cloning a variant fragment from such gels and establishing its nucleotide sequence. This technology should be highly efficient in monitoring for mutations resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.
Two yeast genes with similarity to TCP-1 are required for microtubule and actin function in vivo.
Authors: Chen X , Sullivan D S , Huffaker T C
Published: May 2006

We have isolated cold-sensitive mutations in two genes of the yeast Saccharomyces cerevisiae, BIN2 and BIN3, that cause aberrant chromosome segregation in vivo. BIN2 and BIN3 encode essential proteins that are similar to each other and to TCP-1. TCP-1 and TCP-1-like proteins are components of the eukaryotic cytoplasmic chaperonin that facilitates folding of tubulins and actin in vitro. Mutations in BIN2 and BIN3 cause defects in microtubule and actin assembly in vivo and confer supersensitivity to the microtubule-destabilizing drug benomyl. Overexpression of TCP1, BIN2, BIN3, or ANC2, a fourth member of the TCP-1 family in yeast, does not complement mutations in the other genes, indicating that the proteins have distinct functions. However, all double-mutant combinations are inviable; this synthetic lethality suggests that the proteins act in a common process. These results indicate that Bin2p and Bin3p are components of a yeast cytoplasmic chaperonin complex that is required for assembly of microtubules and actin in vivo.
Identification of soluble forms of the fibroblast growth factor receptor in blood.
Authors: Hanneken A , Ying W , Ling N , Baird A
Published: May 2006

We have purified three acidic (FGF-1) and basic (FGF-2) fibroblast growth factor binding proteins (FGF-BP1, FGF-BP2, and FGF-BP3) from human plasma and calf serum and demonstrate the presence of these circulating FGF-BPs in blood. Each are truncated forms of the high-affinity FGF receptor (FGFR-1). FGF-BP1 and FGF-BP2 have estimated molecular masses of 70-85 kDa and 55-60 kDa, respectively, and are detected by using 125I-labeled FGF-2 ligand blotting. Immunoblotting with four distinct antibodies to FGFR-1 reveals that FGF-BP1 and FGF-BP2 are immunologically and biochemically related to the extracellular domain of FGFR-1. Reverse-phase HPLC chromatography resolves FGF-BP2 into two proteins with estimated molecular masses of 55 kDa and 60 kDa. Protein sequencing of the amino terminus of FGF-BP2 and FGF-BP3 reveals identity with the extracellular domain of the two-IgG-loop form of human FGFR-1. The FGF-BPs do not require heparin to bind FGF-2 on affinity columns, but heparin does enhance their recovery from blood. These FGF-BPs may play an important physiological role in regulating the biological activity of FGF and the other members of the FGF family of growth factors.
Microglia in invertebrate ganglia.
Authors: Sonetti D , Ottaviani E , Bianchi F , Rodriguez M , Stefano M L , Scharrer B , Stefano G B
Published: May 2006

The results of this study lend strong support to the concept of the existence in insects and molluscs of a distinctive class of neuroglial cells comparable to vertebrate microglia. The evidence presented is as valid as that used in reference to the separate status of vertebrate microglia--i.e., the demonstration of a close structural and functional relationship of these cells with cells of the immune system. As in vertebrates, the excision of ganglia from three invertebrate species (the molluscs Planorbarius corneus and Mytilus edulis and the insect Leucophaea maderae) and their maintenance in incubation media led to an exodus of small cells and their accumulation in the culture dish. During this process, they underwent conformational changes from stellate to rounded, and then to more or less ameboid, comparable to those indicative of the process of activation in the animals' immunocytes. Functional characteristics which these translocated microglia-like cells share with immunocytes are motility, phagocytotic activity, and adherence to the culture dish. Furthermore, the two cells have certain biochemical features in common--e.g., the presence of certain cytokines and (at least in Planorbarius) that of corticotropin. An additional phenomenon of particular interest for the classification of microglial elements is their response to morphine. At 10(-6) M, this drug decreases not only the number of cells emerging from the excised ganglia but also the degree of their transformation to the "active" ameboid form. This dose-dependent and naloxone-sensitive effect of morphine on microglial cells parallels that on activated immunocytes of the same species. Corresponding results demonstrating an inhibitory effect of morphine on mobilized microglial cells of the frog Rana pipiens indicate that this relationship between the two cell types under consideration also exists in vertebrates. Binding and displacement experiments with membrane homogenates of microglial cells as well as immunocytes of Mytilus have shown that the effects of morphine on both cell types are mediated by the same special opiate receptor (mu 3).
STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells.
Authors: Small D , Levenstein M , Kim E , Carow C , Amin S , Rockwell P , Witte L , Burrow C , Ratajczak M Z , Gewirtz A M
Published: May 2006

We cloned the cDNA for stem cell tyrosine kinase 1 (STK-1), the human homolog of murine Flk-2/Flt-3, from a CD34+ hematopoietic stem cell-enriched library and investigated its expression in subsets of normal human bone marrow. The cDNA encodes a protein of 993 aa with 85% identity and 92% similarity to Flk-2/Flt-3. STK-1 is a member of the type III receptor tyrosine kinase family that includes KIT (steel factor receptor), FMS (colony-stimulating factor 1R), and platelet-derived growth factor receptor. STK-1 expression in human blood and marrow is restricted to CD34+ cells, a population greatly enriched for stem/progenitor cells. Anti-STK-1 antiserum recognizes polypeptides of 160 and 130 kDa in several STK-1-expressing cell lines and in 3T3 cells transfected with a STK-1 expression vector. Antisense oligonucleotides directed against STK-1 sequences inhibited hematopoietic colony formation, most strongly in long-term bone marrow cultures. These data suggest that STK-1 may function as a growth factor receptor on hematopoietic stem and/or progenitor cells.
Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(-/-) mice.
Authors: Clarke L L , Grubb B R , Yankaskas J R , Cotton C U , McKenzie A , Boucher R C
Published: May 2006

Although loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- channel function is common to all epithelia in cystic fibrosis (CF) patients, the severity of disease varies in different organs. We hypothesized that differences in disease severity in CF relate to the expression of an "alternative" plasma membrane Cl- conductance. In CF mice [Cftr(-/-); mice homozygous for Ser-489 to Xaa mutation], which do not express cAMP CFTR-mediated Cl- secretion, we surveyed organs that exhibit a range of disease severity for a Ca(2+)-mediated apical membrane epithelial Cl- conductance. This alternative conductance (Cl-a) was detected in epithelia of organs from CF mice that exhibit a mild disease phenotype (airway, pancreas) but not in epithelia with a severe phenotype (small, large intestine). We conclude that (i) there is an intracellular Ca(2+)-regulated Cl- conductance that is molecularly distinct from CFTR; and (ii) the level of expression of this alternative Cl- conductance in the epithelium is an important determinant of the severity of organ-level disease in CF.
G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets.
Authors: Offermanns S , Laugwitz K L , Spicher K , Schultz G
Published: May 2006

Using subtype-specific antisera, we were able to identify the recently described alpha subunits of G12 and G13 in platelet membranes as 43-kDa proteins. Activation of the thromboxane A2 and the thrombin receptors in platelet membranes led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha 12 and alpha 13, indicating that both receptors couple to G12 and G13. In addition, both activated receptors were demonstrated to couple to one or more members of the Gq family. In the absence of receptor agonists, incorporation of [alpha-32P]GTP azidoanilide into alpha 12 and alpha 13 was low over a long time period (up to 45 min) due to an obviously low basal nucleotide exchange rate, whereas an agonist-stimulated photolabeling of alpha 12 and alpha 13 could be observed after 4-8 min and reached a maximum after 30-45 min. Effective activation of G12 and G13 via the thromboxane A2 and the thrombin receptors was not dependent on the presence of GDP. Our results provide evidence that G12 and G13 play a functional role in transmembrane signal transduction and suggest that both proteins are involved in pathways leading to platelet activation.
Somatic diversification in the heavy chain variable region genes expressed by human autoantibodies bearing a lupus-associated nephritogenic anti-DNA idiotype.
Authors: Demaison C , Chastagner P , Thèze J , Zouali M
Published: May 2006

Monoclonal anti-DNA antibodies bearing a lupus nephritis-associated idiotype were derived from five patients with systemic lupus erythematosus (SLE). Genes encoding their heavy (H)-chain variable (VH) regions were cloned and sequenced. When compared with their closest VH germ-line gene relatives, these sequences exhibit a number of silent (S) and replacement (R) substitutions. The ratios of R/S mutations were much higher in the complementarity-determining regions (CDRs) of the antibodies than in the framework regions. Molecular amplification of genomic VH genes and Southern hybridization with somatic CDR2-specific oligonucleotide probes showed that the configuration of the VH genes corresponding to VH sequences in the nephritogenic antibodies is not present in the patient's own germ-line DNA, implying that the B-cell clones underwent somatic mutation in vivo. These findings, together with the characteristics of the diversity and junctional gene elements utilized to form the antibody, indicate that these autoantibodies have been driven through somatic selection processes reminiscent of those that govern antibody responses triggered by exogenous stimuli.
Evidence for RNA editing in mitochondria of all major groups of land plants except the Bryophyta.
Authors: Hiesel R , Combettes B , Brennicke A
Published: May 2006

RNA editing has been documented in mitochondria of higher plants, notably dicots and monocots. To determine the distribution of mitochondrial RNA editing in the plant kingdom, we have now undertaken a survey of evolutionarily distant plants. RNA editing occurs in all major groups of land plants except the Bryophyta, suggesting that this process is an ancient trait that was established before the radiation of kormophyte plants. No editing is observed in representatives of the green algae, suggesting that editing arose in early land plants after the split of the Bryophyta or has been lost selectively in both algae and mosses. In ferns several U-->C changes are observed, one of which eliminates a genomically encoded UAA termination codon and creates a functional open reading frame.
Insulin receptor substrate 1 is required for insulin-mediated mitogenic signal transduction.
Authors: Rose D W , Saltiel A R , Majumdar M , Decker S J , Olefsky J M
Published: May 2006

Insulin treatment of mammalian cells immediately stimulates the tyrosine phosphorylation of a cellular protein of 185 kDa referred to as pp185 or IRS-1 (insulin receptor substrate 1). The potential role of the IRS-1 protein in insulin signaling has been examined by microinjecting affinity-purified antibodies into living cells. Stably transfected Rat-1 fibroblasts, which overexpress the human insulin receptor, were microinjected and subsequently stimulated with insulin or other growth factors. Progression through the cell cycle was monitored by using a single-cell assay, which employs bromodeoxyuridine labeling of DNA and analysis with immunofluorescence microscopy. Microinjection of anti-IRS-1 antibody completely inhibited incorporation of bromodeoxyuridine into the nuclei of cells stimulated with insulin or insulin-like growth factor I but did not affect cells stimulated with serum or a variety of purified growth factors. These studies indicate that IRS-1 is a critical component of the insulin and insulin-like growth factor I signaling pathways, which lead to DNA synthesis and cell growth.
T-cell receptor gene homologs are present in the most primitive jawed vertebrates.
Authors: Rast J P , Litman G W
Published: May 2006

The phylogenetic origins of T-cell immunity and T-cell antigen receptor (TCR) genes have not been established. A PCR approach using short, minimally degenerate oligodeoxynucleotide primers complementing conserved variable region segments amplifies TCR-like products from the genomic DNA of Heterodontus francisci (horned shark), a representative phylogenetically primitive cartilaginous fish. One of these products has been used as a probe to screen a Heterodontus spleen cDNA library and a clone was identified that is most related at the nucleotide sequence and predicted peptide levels to higher vertebrate TCR beta-chain genes. Genomic analyses of the TCR homologs indicate that recombining variable and joining region segments as well as constant region exons are encoded by extensive gene families, organized in the multicluster form, characteristic of both the immunoglobulin heavy- and light-chain gene loci in the cartilaginous fishes. Greater numbers of homologous products were identified when a probe complementing the putative constant region of the TCR homolog was used to screen the same cDNA library. A high degree of intergenic variation is associated with the putative variable region segments of these isolates. Direct evidence is presented for TCR-like genes, which presumably are associated with T-cell function, at the earliest stages in the phylogenetic emergence of jawed vertebrates.
Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter.
Authors: Furth P A , St Onge L , Böger H , Gruss P , Gossen M , Kistner A , Bujard H , Hennighausen L
Published: May 2006

Promoters whose temporal activity can be directly manipulated in transgenic animals provide a tool for the study of gene functions in vivo. We have evaluated a tetracycline-responsive binary system for its ability to temporally control gene expression in transgenic mice. In this system, a tetracycline-controlled trans-activator protein (tTA), composed of the repressor of the tetracycline-resistance operon (tet from Escherichia coli transposon Tn10) and the activating domain of viral protein VP16 of herpes simplex virus, induces transcription from a minimal promoter (PhCMV*-1; see below) fused to seven tet operator sequences in the absence of tetracycline but not in its presence. Transgenic mice were generated that carried either a luciferase or a beta-galactosidase reporter gene under the control of PhCMV*-1 or a transgene containing the tTA coding sequence under the control of the human cytomegalovirus immediate early gene 1 (hCMV IE1) promoter/enhancer. Whereas little luciferase or beta-galactosidase activity was observed in tissues of mice carrying only the reporter genes, the presence of tTA in double-transgenic mice induced expression of the reporter genes up to several thousand-fold. This induction was abrogated to basal levels upon administration of tetracycline. These findings can be used, for example, to design dominant gain-of-function experiments in which temporal control of transgene expression is required.
Genetically modified keratinocytes transplanted to wounds reconstitute the epidermis.
Authors: Vogt P M , Thompson S , Andree C , Liu P , Breuing K , Hatzis D , Brown H , Mulligan R C , Eriksson E
Published: May 2006

Normal and retrovirally transfected keratinocyte suspensions expressing either the beta-galactosidase gene or the human growth hormone (hGH) gene were transplanted into chamber-enclosed skin full-thickness wounds of Yorkshire pigs. Immunostaining of sequential skin biopsies obtained for 4 weeks after transplantation showed survival of the transplanted keratinocytes as well as expression of beta-galactosidase. Transfected keratinocytes were first seen in the neodermal portions of the wounds, then in the regenerating basal epidermal layer, and finally in the terminally differentiating cells of the stratum spinosum. When keratinocytes transfected with the hGH gene were transplanted into similar wounds, hGH was detected for 10 days in wound fluid. In contrast, hGH was detected in vitro for 47 days. Wounds transplanted with either transfected or normal keratinocytes restored the epithelial barrier function significantly faster than nontransplanted controls (P < 0.05). The study confirms the successful transplantation of keratinocyte suspensions, their reconstitution of the epidermis, and their acceleration of repair. Further, this apparently normal incorporation of genetically engineered transplanted keratinocytes expressing either beta-galactosidase or hGH suggests the possibility of introducing other genes expressing therapeutic proteins into wounds to favorably affect healing. Wound fluid detection of the expressed peptide provided early demonstration of successful transfer of the hGH gene.
Transcriptional activation in vitro by the human immunodeficiency virus type 1 Tat protein: evidence for specific interaction with a coactivator(s).
Authors: Song C Z , Loewenstein P M , Green M
Published: May 2006

The Tat protein encoded by human immunodeficiency virus type 1 is a strong transcriptional activator of gene expression from the viral long terminal repeat and is essential for virus replication. We have investigated the molecular mechanism of Tat trans-activation by using a cell-free transcription system. We find that the trans-activation domain of Tat, amino acid residues 1-48 [Tat-(1-48)], can inhibit specifically--i.e., "squelch," transcriptional activation by full-length Tat [Tat-(1-86)]. Squelching depends upon the functional integrity of the Tat trans-activation domain because the mutant [Ala41]Tat-(1-48), which is defective in Tat trans-activation in vivo and in vitro, does not squelch in vitro Tat trans-activation. Inhibition is selective because Tat-activated transcription, but not Tat-independent transcription, is squelched. Preincubation experiments with Tat or Tat-(1-48) and nuclear extracts show that the trans-activation region of Tat can interact with cellular coactivator(s) required for Tat trans-activation and that this interaction can occur in the absence of the human immunodeficiency virus long terminal repeat promoter. Furthermore, the putative coactivator(s) mediating trans-activation by Tat differ from those mediating trans-activation by the acidic activator VP16, as shown by reciprocal squelching experiments in vitro. Our results suggest that specific cellular coactivator(s) are required for mediating activated transcription by human immunodeficiency virus type 1 Tat.
Modification of low density lipoprotein by advanced glycation end products contributes to the dyslipidemia of diabetes and renal insufficiency.
Authors: Bucala R , Makita Z , Vega G , Grundy S , Koschinsky T , Cerami A , Vlassara H
Published: May 2006

Atherosclerosis develops rapidly in patients with diabetes or renal insufficiency. Plasma lipoprotein profiles are frequently abnormal in these conditions and reflect an elevation in the level of the apoprotein B (ApoB)-containing components very low density lipoprotein (VLDL) and low density lipoprotein (LDL). High levels of circulating advanced glycation end products (AGEs) also occur in diabetes and end-stage renal disease (ESRD). These products arise from glucose-derived Amadori products and include AGE-modified peptides (AGE-peptides) which result from the catabolism of AGE-modified tissue proteins. AGE-peptides have been shown to crosslink protein amino groups and to accumulate in plasma as a consequence of renal insufficiency. To address potential mechanisms for the dyslipidemia of diabetes and ESRD, we investigated the possibility that circulating AGEs react directly with plasma lipoproteins to prevent their recognition by tissue LDL receptors. AGE-specific ELISA showed a significantly increased level of AGE-modified LDL in the plasma of diabetic or ESRD patients compared with normal controls. AGE-LDL formed readily in vitro when native LDL was incubated with either synthetic AGE-peptides or AGE-peptides isolated directly from patient plasma. LDL which had been modified by AGE-peptides in vitro to the same level of modification as that present in the plasma of diabetics with renal insufficiency exhibited markedly impaired clearance kinetics when injected into transgenic mice expressing the human LDL receptor. These data indicate that AGE modification significantly impairs LDL-receptor-mediated clearance mechanisms and may contribute to elevated LDL levels in patients with diabetes or renal insufficiency. This hypothesis was further supported by the observation that the administration of the advanced glycation inhibitor aminoguanidine to diabetic patients decreased circulating LDL levels by 28%.
Intradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses.
Authors: Raz E , Carson D A , Parker S E , Parr T B , Abai A M , Aichinger G , Gromkowski S H , Singh M , Lew D , Yankauckas M A
Published: May 2006

The skin and mucous membranes are the anatomical sites were most viruses are first encountered by the immune system. Previous experiments have suggested that striated muscle cells are unique among mammalian cell types in their capacity to take up and express free DNA in the absence of a viral vector or physical carrier. However, we have found that mice injected into the superficial skin with free (naked) plasmid DNA encoding the influenza nucleoprotein gene had discrete foci of epidermal and dermal cells, including cells with dendritic morphology, that contained immunoreactive nucleoprotein antigen. A single intradermal administration of 0.3-15 micrograms of free plasmid DNA induced anti-nucleoprotein-specific antibody and cytotoxic T lymphocytes that persisted for at least 68-70 weeks after vaccination. Intradermal gene administration induced higher antibody titers than did direct gene injection into skeletal muscle and did not cause local inflammation or necrosis. Compared with control animals, the gene-injected mice were resistant to challenge with a heterologous strain of influenza virus. These results indicate that the cells of the skin can take up and express free foreign DNA and induce cellular and humoral immune responses against the encoded protein. We suggest that DNA uptake by the skin-associated lymphoid tissues may play a role in the induction of cytotoxic T cells against viruses and other intracellular pathogens.
Role of interferon alpha/beta receptor chain 1 in the structure and transmembrane signaling of the interferon alpha/beta receptor complex.
Authors: Constantinescu S N , Croze E , Wang C , Murti A , Basu L , Mullersman J E , Pfeffer L M
Published: May 2006

A previously cloned cDNA encodes one subunit of the human interferon alpha/beta receptor (IFN alpha R), denoted IFN alpha R1. To study the expression and signaling of IFN alpha R1, we used monoclonal antibodies (mAbs) generated against the baculovirus-expressed ectodomain of IFN alpha R1. Immunoprecipitation and immunoblotting of lysates from a variety of human cell lines showed that IFN alpha R1 has an apparent molecular mass of 135 kDa. Binding analysis with 125I-labeled mAb demonstrated high levels of cell surface expression of IFN alpha R1 in human cells and in mouse cells transfected with IFN alpha R1 cDNA, whereas no cross-reactivity was observed in control mouse L929 cells expressing only the endogenous mouse receptor. The subunit was rapidly down-regulated by IFN alpha (80% decrease within 2 hr) and degraded upon internalization. The IFN alpha R1 chain appeared to be constitutively associated with the 115-kDa subunit of the IFN alpha/beta receptor, since the mAbs coprecipitated this protein. IFN alpha/beta treatment induced tyrosine phosphorylation of IFN alpha R1 within 1 min, with kinetics paralleling that of the IFN-activated protein-tyrosine kinases Jak1 and Tyk2. Ligand-induced tyrosine phosphorylation of IFN alpha R1 was blocked by the kinase inhibitors genistein or staurosporine. Although IFN alpha R1 cDNA-transfected mouse cells expressed high levels of this subunit when compared with empty vector-transfected cells the number of binding sites for human IFN alpha (50-75 sites per cell) was not increased. Human IFN alpha induced the expression of a mouse IFN alpha/beta-responsive gene (the 204 gene) in mouse L929 cells transfected with the IFN alpha R1 cDNA, but not in mock-transfected cells. These results suggest that the IFN alpha R1 subunit acts as a species-specific signal transduction component of the IFN alpha/beta receptor complex.
Fluctuation of the number of particles deposited on a flat surface by a random sequential adsorption mechanism.
Authors: Senger B , Schaaf P , Voegel J C , Wojtaszczyk P , Reiss H
Published: May 2006

The problem of the fluctuations of the number n of particles adsorbed on surfaces through a random sequential adsorption process is discussed. Attention is paid, in particular, to the effect of the size of the adsorbing surfaces upon the variance sigma2(n) of this number. On the basis of computer simulations, it is shown that sigma2(n) is not proportional to the area a of the surface but can be written as a sum of three contributions, which are proportional to a, a1/2, and a0. A theoretical estimate based on the relation between the radial distribution function g(r) and the fluctuation is presented and provides a basis for these findings. This analysis is of general validity and can, in particular, also be applied to the equilibrium case (in the absence of a phase transition) and to the ballistic deposition process.
Site-specific integration by adeno-associated virus is directed by a cellular DNA sequence.
Authors: Giraud C , Winocour E , Berns K I
Published: May 2006

Different regions of an 8.2-kb cloned DNA segment containing the target for adeno-associated virus (AAV) integration in human chromosome 19q13-3-qter (AAVS1 locus) were subcloned in an Epstein-Barr virus-based shuttle vector and propagated as episomes in a derivative of the 293 human embryonic kidney cell line. Preferential recombination with an infecting AAV genome was assessed by measuring the frequency of recombinants among the shuttle vectors recovered in Escherichia coli. The signals which direct recombination with the AAV genome were localized to a 510-nt region at the 5' end of the 8.2-kb AAVS1 DNA. Hence, the results indicate that site-specific integration of AAV is directed by a specific DNA sequence on human chromosome 19. An unusual degree of DNA heterogeneity in the recovered vector was also associated with the 510 nt at the 5' end of AAVS1 DNA, suggesting that the AAV chromosomal integration locus may be involved in genomic instability.
Purification of a Ran-interacting protein that is required for protein import into the nucleus.
Authors: Moore M S , Blobel G
Published: May 2006

Previously we reported the isolation of two cytosolic fractions (A and B) from Xenopus ovary that are required sequentially to support protein import into the nuclei of digitonin-permeabilized cells. Fraction A is required for recognition of the nuclear localization sequence and targeting to the nuclear envelope, whereas fraction B is required for the subsequent translocation of the bound substrate into the nucleus. The first protein required for fraction B activity to be purified was the small GTPase Ran (ras-related nuclear protein). Here we report the purification of the second (and final) protein required for fraction B activity. By SDS/PAGE, the purified protein appeared as a single band with an apparent molecular mass of 10 kDa, but the native protein fractionated upon gel filtration chromatography with an apparent size of 30 kDa. Peptide sequence analysis indicated that the purified protein was highly homologous to a previously identified human protein of unknown function called placental protein 15 (pp15) and to the predicted protein product of a yeast open reading frame from Saccharomyces cerevisiae.
Endothelial cell implantation and survival within experimental gliomas.
Authors: Lal B , Indurti R R , Couraud P O , Goldstein G W , Laterra J
Published: May 2006

The delivery of therapeutic genes to primary brain neoplasms opens new opportunities for treating these frequently fatal tumors. Efficient gene delivery to tissues remains an important obstacle to therapy, and this problem has unique characteristics in brain tumors due to the blood-brain and blood-tumor barriers. The presence of endothelial mitogens and vessel proliferation within solid tumors suggests that genetically modified endothelial cells might efficiently transplant to brain tumors. Rat brain endothelial cells immortalized with the adenovirus E1A gene and further modified to express the beta-galactosidase reporter were examined for their ability to survive implantation to experimental rat gliomas. Rats received 9L, F98, or C6 glioma cells in combination with endothelial cells intracranially to caudate/putamen or subcutaneously to flank. Implanted endothelial cells were identified by beta-galactosidase histochemistry or by polymerase chain reaction in all tumors up to 35 days postimplantation, the latest time examined. Implanted endothelial cells appeared to cooperate in tumor vessel formation and expressed the brain-specific endothelial glucose transporter type 1 as identified by immunohistochemistry. The proliferation of implanted endothelial cells was supported by their increased number within tumors between postimplantation days 14 and 21 (P = 0.015) and by their expression of the proliferation antigen Ki67. These findings establish that genetically modified endothelial cells can be stably engrafted to growing gliomas and suggest that endothelial cell implantation may provide a means of delivering therapeutic genes to brain neoplasms and other solid tumors. In addition, endothelial implantation to brain may be useful for defining mechanisms of brain-specific endothelial differentiation.
The Ada protein acts as both a positive and a negative modulator of Escherichia coli's response to methylating agents.
Authors: Saget B M , Walker G C
Published: May 2006

The adaptive response of Escherichia coli protects the cells against the toxic and mutagenic effects of certain alkylating agents. The major effector molecule regulating this response is the 39-kDa Ada protein, which functions as both a DNA repair protein and a transcriptional activator. Ada removes methyl groups from phosphotriester and O6-methylguanine lesions in DNA, irreversibly transferring them to cysteine residues at positions 69 and 321, respectively. When methylated at Cys-69, Ada is converted into a potent activator of ada and alkA transcription and binds to a sequence (Ada box) present in both promoters. We have found that physiologically relevant higher concentrations of unmethylated Ada are able to inhibit the activation of ada transcription by methylated Ada, both in vitro and in vivo. In contrast, the same concentrations of unmethylated Ada do not inhibit the activation of alkA transcription by methylated Ada, either in vitro or in vivo. Deletion of the carboxyl-terminal 67 amino acids of Ada abolished the ability of the unmethylated form of the protein to inhibit activation of ada transcription but not the ability of the methylated form to activate ada or alkA transcription. Our results suggest that the Ada protein plays a pivotal role in the negative modulation of its own synthesis and therefore in the down-regulation of the adaptive response. Elements present in the carboxyl terminus of Ada appear to be necessary for this negative regulatory function.
Molecular structure and chromosomal mapping of the human homolog of the agouti gene.
Authors: Kwon H Y , Bultman S J , Löffler C , Chen W J , Furdon P J , Powell J G , Usala A L , Wilkison W , Hansmann I , Woychik R P
Published: May 2006

The agouti (a) locus in mouse chromosome 2 normally regulates coat color pigmentation. The mouse agouti gene was recently cloned and shown to encode a distinctive 131-amino acid protein with a consensus signal peptide. Here we describe the cloning of the human homolog of the mouse agouti gene using an interspecies DNA-hybridization approach. Sequence analysis revealed that the coding region of the human agouti gene is 85% identical to the mouse gene and has the potential to encode a protein of 132 amino acids with a consensus signal peptide. Chromosomal assignment using somatic-cell-hybrid mapping panels and fluorescence in situ hybridization demonstrated that the human agouti gene maps to chromosome band 20q11.2. This result revealed that the human agouti gene is closely linked to several traits, including a locus called MODY (for maturity onset diabetes of the young) and another region that is associated with the development of myeloid leukemia. Initial expression studies with RNA from several adult human tissues showed that the human agouti gene is expressed in adipose tissue and testis.
Antigenic mimicry of natural L-peptides with retro-inverso-peptidomimetics.
Authors: Guichard G , Benkirane N , Zeder-Lutz G , van Regenmortel M H , Briand J P , Muller S
Published: May 2006

Three analogues of the model peptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 were synthesized, and their antigenicity, immunogenicity, and resistance to trypsin were compared to those of the natural L-peptide. The three analogues correspond to the D-enantiomer, containing only D-residues, and two retro-peptides containing NH-CO bonds instead of natural peptide bonds. The chirality of each residue was maintained in the retro-peptide and inverted in the retro-inverso-peptide. Antibodies to the four peptide analogues were produced by injecting BALB/c mice with peptides covalently coupled to small unilamellar liposomes containing monophosphoryl lipid A. Each of the four peptide analogues induced IgG antibodies of various subclasses. The IgG3 antibodies reacted similarly with the four analogues, whereas antibodies of the IgG1, IgG2a, and IgG2b isotypes showed strong conformational preferences for certain peptides. The retro-inverso-peptide IRGERA mimicked the structure and antigenic activity of the natural L-peptide but not of the D- and retro-peptides, whereas the retro-peptide IRGERA mimicked the D-peptide but not the L- and retro-inverso-peptides. The equilibrium affinity constants (Ka) of three monoclonal antibodies generated against the L- and D-peptides with respect to the four peptide analogues were measured in a biosensor system. Large differences in Ka values were observed when each monoclonal antibody was tested with respect to the four peptides. The use of retro-inverso-peptides to replace natural L-peptides is likely to find many applications in immunodiagnosis and as potential synthetic vaccines.
Molecular cloning of a functional human galanin receptor.
Authors: Habert-Ortoli E , Amiranoff B , Loquet I , Laburthe M , Mayaux J F
Published: May 2006

The ubiquitous neuropeptide galanin controls numerous functions such as endocrine secretions, intestinal motility, and behavioral activities. These regulatory effects of galanin are mediated through the interaction with specific membrane receptors and involve the pertussis toxin-sensitive guanine nucleotide binding proteins Gi/Go as transducing elements. We report here the isolation of a cDNA coding for a human galanin receptor from a Bowes melanoma cell line cDNA expression library, by using a radioligand binding strategy. The nucleotide sequence of the cloned receptor reveals an open reading frame encoding a 349-amino acid protein with seven putative hydrophobic transmembrane domains and significant homology with members of the guanine nucleotide binding protein-coupled neuropeptide receptor family. The cloned receptor expressed in COS cells specifically binds human, porcine, and rat galanin with high affinity (Kd in the nanomolar range) and mediates the galanin inhibition of adenylate cyclase. A 2.8-kb galanin receptor transcript was identified in several human tissues. Cloning of this galanin receptor should enhance our knowledge of its distribution, structure, and function in human physiology and pathophysiology.
Two additional glutaredoxins exist in Escherichia coli: glutaredoxin 3 is a hydrogen donor for ribonucleotide reductase in a thioredoxin/glutaredoxin 1 double mutant.
Authors: Aslund F , Ehn B , Miranda-Vizuete A , Pueyo C , Holmgren A
Published: May 2006

Thioredoxin (Trx) and glutaredoxin (Grx1) are hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis. The viability of a double mutant lacking both Trx and Grx1 implies the presence of a third, unknown hydrogen donor. This paper reports the purification and characterization of two proteins with glutaredoxin activity (using hydroxyethyl disulfide as a substrate) from an Escherichia coli mutant lacking Trx and Grx1 (delta trxA, grx::kan). Affinity chromatography was used to bind glutaredoxin on a glutathione-containing thiol-Sepharose column. The molecular weight of Grx2, 27,000, was atypical for glutaredoxins, whereas Grx3 had a molecular weight of 10,000. Amino acid sequence analysis revealed novel structures with putative active sites typical of glutaredoxins: Cys-Pro-Tyr-Cys. The proteins are therefore referred to as Grx2 and Grx3. The low hydrogen donor activity for ribonucleotide reductase in the crude extract was recovered in the purification of Grx3, whereas Grx2 was inactive. As a hydrogen donor for E. coli ribonucleotide reductase, Grx3 showed approximately the same Km value (0.35 microM) as Grx1, whereas its Vmax value was only 5% that of Grx1. The combination of the Grx3 hydrogen donor activity and a 25-fold induction of ribonucleotide reductase activity in a delta trxA, grx double mutant provides an explanation for its viability and deoxyribonucleotide biosynthesis. The physiological functions of Grx2 and Grx3 remain to be determined.
Group I introns are inherited through common ancestry in the nuclear-encoded rRNA of Zygnematales (Charophyceae).
Authors: Bhattacharya D , Surek B , Rüsing M , Damberger S , Melkonian M
Published: May 2006

Group I introns are found in organellar genomes, in the genomes of eubacteria and phages, and in nuclear-encoded rRNAs. The origin and distribution of nuclear-encoded rRNA group I introns are not understood. To elucidate their evolutionary relationships, we analyzed diverse nuclear-encoded small-subunit rRNA group I introns including nine sequences from the green-algal order Zygnematales (Charophyceae). Phylogenetic analyses of group I introns and rRNA coding regions suggest that lateral transfers have occurred in the evolutionary history of group I introns and that, after transfer, some of these elements may form stable components of the host-cell nuclear genomes. The Zygnematales introns, which share a common insertion site (position 1506 relative to the Escherichia coli small-subunit rRNA), form one subfamily of group I introns that has, after its origin, been inherited through common ancestry. Since the first Zygnematales appear in the middle Devonian within the fossil record, the "1506" group I intron presumably has been a stable component of the Zygnematales small-subunit rRNA coding region for 350-400 million years.
Visual form discrimination from color or motion cues: functional anatomy by positron emission tomography.
Authors: Gulyás B , Heywood C A , Popplewell D A , Roland P E , Cowey A
Published: May 2006

To explore the extent to which various cortical functional pathways are involved in processing and analyzing different types of information that yield the same perceptual entity, we mapped anatomical structures in the human brain participating in the discrimination of visual forms mediated either by motion or color cues. Changes in regional cerebral blood flow were measured in 10 young male volunteers with positron emission tomography and with [15O]butanol. During the measurements, the subjects performed four visual discrimination tasks (form-from-motion, motion alone, form-from-color, and color alone discrimination). The individual regional cerebral blood flow images were standardized in shape and size with the help of a computerized brain atlas. Subtraction images were determined and averaged across data from all subjects. The resulting images were analyzed for statistically significant changes between specific and reference tasks. The discrimination of form by means of motion cues activated functional fields bilaterally in the inferior and lateral occipital gyri, in the lingual, anterior cingulate, middle frontal and orbitofrontal gyri, and in the left fusiform and right inferior temporal gyri. Form discrimination by color cues resulted in activation bilaterally in the inferior temporal, lateral occipital, and orbitofrontal gyri, the left precuneus and intraparietal sulcus, and the right precentral gyrus. The regions engaged in the two kinds of form discrimination did not overlap, demonstrating that differences in visual forms mediated by color or motion cues are processed and analyzed by disparate networks of functional fields in human cerebral cortex.
Targeted disruption of the Hexa gene results in mice with biochemical and pathologic features of Tay-Sachs disease.
Authors: Yamanaka S , Johnson M D , Grinberg A , Westphal H , Crawley J N , Taniike M , Suzuki K , Proia R L
Published: May 2006

Tay-Sachs disease, the prototype of the GM2 gangliosidoses, is a catastrophic neurodegenerative disorder of infancy. The disease is caused by mutations in the HEXA gene resulting in an absence of the lysosomal enzyme, beta-hexosaminidase A. As a consequence of the enzyme deficiency, GM2 ganglioside accumulates progressively, beginning early in fetal life, to excessive amounts in the central nervous system. Rapid mental and motor deterioration starting in the first year of life leads to death by 2-4 years of age. Through the targeted disruption of the mouse Hexa gene in embryonic stem cells, we have produced mice with biochemical and neuropathologic features of Tay-Sachs disease. The mutant mice displayed < 1% of normal beta-hexosaminidase A activity and accumulated GM2 ganglioside in their central nervous system in an age-dependent manner. The accumulated ganglioside was stored in neurons as membranous cytoplasmic bodies characteristically found in the neurons of Tay-Sachs disease patients. At 3-5 months of age, the mutant mice showed no apparent defects in motor or memory function. These beta-hexosaminidase A-deficient mice should be useful for devising strategies to introduce functional enzyme and genes into the central nervous system. This model may also be valuable for studying the biochemical and pathologic changes occurring during the course of the disease.