bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of the inositol trisphosphate receptor from smooth muscle.
Authors: Chadwick C C , Saito A , Fleischer S
Published: May 2006

The release of Ca2+ from internal stores is requisite to muscle contraction. In skeletal muscle and heart, the Ca2+ release channels (ryanodine receptor) of sarcoplasmic reticulum, involved in excitation-contraction coupling, have recently been isolated and characterized. In smooth muscle, inositol 1,4,5-trisphosphate (IP3) is believed to mobilize Ca2+ from internal stores and thereby modulate contraction. We describe the isolation of an IP3 receptor from smooth muscle. Bovine aorta smooth muscle microsomes were solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the IP3 receptor was purified by sucrose gradient centrifugation and column chromatography with heparin-agarose and wheat germ agglutinin-agarose. The purified receptor bound 2.7 +/- 0.18 nmol of IP3 per mg of protein with a Kd of 2.4 +/- 0.24 nM. That is, the purified receptor has been enriched about 1000-fold compared with the original microsomes, whereas the Kd for IP3 remains unchanged. The receptor is an oligomer of a single polypeptide with a Mr of 224,000 as determined by SDS/PAGE. Negative-staining electron microscopy reveals that the receptor is a large pinwheel-like structure having surface dimensions of approximately 250 X 250 A with fourfold symmetry. The IP3 receptor from smooth muscle is similar to the ryanodine receptor with regard to its large size and fourfold symmetry, albeit distinct with regard to appearance, protomer size, and ligand binding.
Concerted evolution of class I genes in the major histocompatibility complex of murine rodents.
Authors: Rada C , Lorenzi R , Powis S J , van den Bogaerde J , Parham P , Howard J C
Published: May 2006

Full-length cDNA sequences of two class I major histocompatibility complex molecules from the DA strain of Rattus norvegicus are reported. One codes for the classical class I restriction element RT1.Aa, which maps to the locus in the rat major histocompatibility complex homologous to H-2K in the mouse. The other probably codes for a soluble nonclassical class I molecule present in DA rat serum; a short deletion in the fifth exon implies that the translated product will terminate in the membrane-spanning region. These sequences have been compared with mouse classical class I sequences as well as with three published rat class I cDNA partial sequences. The results show, first, that "locus-specific" substitutions from the H-2K, H-2D, and H-2L data set are scrambled in the RT1.Aa molecule; a majority of these substitutions have H-2D/L-specific features. Second, the data show that the four rat sequences are strikingly similar to one another regardless of locus or haplotype of origin; they share a number of apparently species-specific features that distinguish them all from mouse classical class I sequences, which likewise share distinctive features of their own. The results suggest that segmental sequence exchange plays a major role in determining the evolution of sequence in class I major histocompatibility complex molecules.
Keratins as markers that distinguish normal and tumor-derived mammary epithelial cells.
Authors: Trask D K , Band V , Zajchowski D A , Yaswen P , Suh T , Sager R
Published: May 2006

Keratin 5 (K5) mRNA and protein are shown to be expressed in normal mammary epithelial cells in culture and are absent from tumor-derived cell lines. To extend these findings, the full complements of keratins in normal, immortalized, and tumor cells were compared. It is shown here that normal cells produce keratins K5, K6, K7, K14, and K17, whereas tumor cells produce mainly keratins K8, K18, and K19. In immortalized cells, which are preneoplastic or partially transformed, the levels of K5 mRNA and protein are lower than in normal cells, whereas the amount of K18 is increased. Thus, K5 is an important marker in the tumorigenic process, distinguishing normal from tumor cells, and decreased K5 expression correlates with tumorigenic progression.
Endothelin in brain: receptors, mitogenesis, and biosynthesis in glial cells.
Authors: MacCumber M W , Ross C A , Snyder S H
Published: May 2006

We have explored the cellular loci of endothelin (ET) actions and formation in the brain, using cerebellar mutant mice as well as primary and continuous cell cultures. A glial role is favored by several observations: (i) mutant mice lacking neuronal Purkinje cells display normal ET receptor binding and enhanced stimulation by ET of inositolphospholipid turnover; (ii) in weaver mice lacking neuronal granule cells, ET stimulation of inositolphospholipid turnover is not significantly diminished; (iii) C6 glioma cells and primary cultures of cerebellar astroglia exhibit substantial ET receptor binding and ET-induced stimulation of inositolphospholipid turnover; (iv) ET promotes mitogenesis of C6 glioma cells and primary cerebellar astroglia; and (v) primary cultures of cerebellar astroglia contain ET mRNA. ET also appears to have a neuronal role, since it stimulates inositolphospholipid turnover in primary cultures of cerebellar granule cells, and ET binding declines in granule cell-deficient mice. Thus, ET can be produced by glia and act upon both glia and neurons in a paracrine fashion.
Acute-phase reaction induces a specific complex between hepatic nuclear proteins and the interleukin 6 response element of the rat alpha 2-macroglobulin gene.
Authors: Hattori M , Abraham L J , Northemann W , Fey G H
Published: May 2006

Interleukin 6 (IL-6) was established as a transcriptional inducer of the rat alpha 2-macroglobulin gene, a prototype liver acute-phase gene. Maximum induction occurred when the 5' flanking sequences of this gene (position -209 to -43) directed expression from the gene's own TATA box and transcription start site. Removal of the hexanucleotide CTGGGA (position -164 to -159) abolished 60-70% of the hormonal induction in FAO1 rat hepatoma cells. This hexanucleotide was defined as the IL-6 response element (IL-6-RE). The IL-6-RE is well conserved in the cytokine-responsive regions of other acute-phase genes and serves as a binding site for nuclear proteins. A characteristic DNA-protein complex (complex I) was formed with nuclear proteins from normal rat livers. A different, hormone-inducible complex (complex II) was assembled specifically with nuclear proteins from acute-phase rat livers or from IL-6-treated human Hep 3B hepatoma cells. Complex II was competitively inhibited by oligonucleotides representing the conserved IL-6-RE sequence from other acute-phase genes. Thus, the proteins building complex II likely participate in a general signal transduction mechanism mediating the transcriptional activation by IL-6 of several acute-phase genes.
Polyadenylylation signal of the mouse thymidylate synthase gene was created by insertion of an L1 repetitive element downstream of the open reading frame.
Authors: Harendza C J , Johnson L F
Published: May 2006

The mouse thymidylate synthase (TS; EC 2.1.1.45) mRNA is unusual in that the poly(A) tail is added at the translation stop codon. To determine the sequence requirements for 3' processing of this mRNA, we constructed TS minigenes with deletion and point mutations in potential regulatory sequences. The minigenes were transiently transfected into cultured cells and the effect on 3' processing was determined by S1 nuclease protection assays. These analyses revealed that at least two elements are required for efficient polyadenylylation at the stop codon. The first is an upstream AUUAAA sequence. When this was changed to AUCAAA, polyadenylylation at the stop codon was blocked. However, when it was changed to the canonical AAUAAA hexanucleotide, the amount of TS mRNA increased severalfold. The second element is a stretch of 14 consecutive uridylate residues 32 nucleotides downstream of the stop codon. This U-rich region is absent from the human TS gene, which explains why the human TS mRNA is not polyadenylylated at the stop codon even though the two genes are otherwise almost identical through this region. The most surprising observation was that the U-rich region corresponds to the 3' end of a 360-nucleotide mouse L1 repetitive element that was inserted in opposite orientation to the gene more than 5 million years ago. Thus the polyadenylylation signal of the present mouse TS gene was created by the transposition of a repetitive element downstream of a cryptic polyadenylylation signal.
Characterization of a mutation in a family with saposin B deficiency: a glycosylation site defect.
Authors: Kretz K A , Carson G S , Morimoto S , Kishimoto Y , Fluharty A L , O'Brien J S
Published: May 2006

Saposins are small, heat-stable glycoproteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. Saposins A, B, C, and D are derived by proteolytic processing from a single precursor protein named prosaposin. Saposin B, previously known as SAP-1 and sulfatide activator, stimulates the hydrolysis of a wide variety of substrates including cerebroside sulfate, GM1 ganglioside, and globotriaosylceramide by arylsulfatase A, acid beta-galactosidase, and alpha-galactosidase, respectively. Human saposin B deficiency, transmitted as an autosomal recessive trait, results in tissue accumulation of cerebroside sulfate and a clinical picture resembling metachromatic leukodystrophy (activator-deficient metachromatic leukodystrophy). We have examined transformed lymphoblasts from the initially reported saposin B-deficient patient and found normal amounts of saposins A, C, and D. After preparing first-strand cDNA from lymphoblast total RNA, we used the polymerase chain reaction to amplify the prosaposin cDNA. The patient's mRNA differed from the normal sequence by only one C----T transition in the 23rd codon of saposin B, resulting in a threonine to isoleucine amino acid substitution. An affected male sibling has the same mutation as the proband and their heterozygous mother carries both the normal and mutant sequences, providing additional evidence that this base change is the disease-causing mutation. This base change results in the replacement of a polar amino acid (threonine) with a nonpolar amino acid (isoleucine) and, more importantly, eliminates the glycosylation signal in this activator protein. One explanation for the deficiency of saposin B in this disease is that the mutation may increase the degradation of saposin B by exposing a potential proteolytic cleavage site (arginine) two amino acids to the amino-terminal side of the glycosylation site when the carbohydrate side chain is absent.
Use of site-directed mutagenesis to identify an upstream regulatory sequence of sodA gene of Escherichia coli K-12.
Authors: Naik S M , Hassan H M
Published: May 2006

Mn-containing superoxide dismutase (SodA; superoxide:superoxide oxidoreductase, EC 1.15.1.1) biosynthesis in Escherichia coli is regulated by several environmental stimuli. The DNA sequence of sodA shows the presence of a potential binding site for a regulatory protein(s) at the -35 region. To explore the possible role of this region in the regulation of sodA, we used oligonucleotide-directed site-specific mutagenesis to change the sequence of nucleotides -48 through -44 from 5'-GGCAT-3' to 5'-TTACG-3'. We studied the effect of this altered sequence on the expression of sodA. The data showed that the altered sequence resulted in the constitutive expression of the gene. Thus, E. coli harboring a plasmid containing the mutated sodA gene (pSNM6) were uninducible by paraquat in aerobiosis or by 2,2'-dipyridyl in aerobiosis or anaerobiosis. Furthermore, a multicopy plasmid containing the mutated sodA failed to titrate the repressor molecules present in an E. coli strain carrying the sodA-lacZ fusion. In contrast, multicopy plasmids containing the wild-type sodA gene were able to titrate the repressor protein and to cause the anaerobic induction of beta-galactosidase in this sodA-lacZ fusion strain. These results indicate that the region within and around the mutated sequence probably plays an important role in sodA regulation and that the mutation disrupts a sequence that interacts with the repressor.
Translation and a 42-nucleotide segment within the coding region of the mRNA encoded by the MAT alpha 1 gene are involved in promoting rapid mRNA decay in yeast.
Authors: Parker R , Jacobson A
Published: May 2006

In yeast, the mRNA encoded by the MAT alpha 1 gene is unstable (t1/2 = 5 min) and the mRNAs encoded by the ACT1 gene (t1/2 = 30 min) and the PGK1 gene (t1/2 = 45 min) are stable. To understand the RNA structural features that dictate mRNA decay rates in yeast, we have constructed PGK1/MAT alpha 1 and ACT1/MAT alpha 1 gene fusions and analyzed the decay rates of the resultant chimeric transcripts. Fusion of a MAT alpha 1 segment containing 73% of the coding region and the 3' untranslated region to either of the stable genes is sufficient to cause rapid decay of the chimeric mRNAs (t1/2 = 6-7.5 min). Sequences required for this rapid decay are not found in the MAT alpha 1 3' untranslated region but are located within a 42-nucleotide segment of the coding region that has a high content (8 out of 14) of rare codons. Introduction of a translational stop codon upstream of this region stabilizes the hybrid mRNAs, indicating that the rapid decay promoted by these sequences is dependent on ribosomal translocation.
Solution conformations of the B-loop fragments of human transforming growth factor alpha and epidermal growth factor by 1H nuclear magnetic resonance and restrained molecular dynamics.
Authors: Han K H , Syi J L , Brooks B R , Ferretti J A
Published: May 2006

A restrained molecular dynamics simulation approach that explicitly includes the effect of the surrounding solvent molecules is applied to the NMR determination of the conformations of the B-loop fragments of human transforming growth factor alpha and epidermal growth factor. Backbone interproton distance restraints are obtained by using two-dimensional rotating frame nuclear Overhauser effect spectroscopy (ROESY). The simulations are carried out both in "vacuum" and in "water." The results are discussed in terms of the energetics, agreement with the NMR distances, and the flexibility of the peptides.
Jet-propelled escape in the squid Loligo opalescens: concerted control by giant and non-giant motor axon pathways.
Authors: Otis T S , Gilly W F
Published: May 2006

Recordings of stellar nerve activity were made during escape responses in living squid. Short-latency activation of the giant axons is triggered by light-flash stimulation that elicits a stereotyped startle-escape response and powerful jet. Many other types of stimuli produce a highly variable, delayed-escape response with strong jetting primarily controlled by a small axon motor pathway. In such cases, activation of the giant axons is not necessary for a vigorous escape jet. When they are utilized, the giant axons are not activated until well after the non-giant system initiates the escape response, and excitation is critically timed to boost the rise in intramantle pressure. Squid thus show at least two escape modes in which the giant axons can contribute in different ways to the control of a highly flexible behavior.
Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations.
Authors: Schild D , Brake A J , Kiefer M C , Young D , Barr P J
Published: May 2006

Functional complementation of mutations in the yeast Saccharomyces cerevisiae has been used to clone three multifunctional human genes involved in de novo purine biosynthesis. A HepG2 cDNA library constructed in a yeast expression vector was used to transform yeast strains with mutations in adenine biosynthetic genes. Clones were isolated that complement mutations in the yeast ADE2, ADE3, and ADE8 genes. The cDNA that complemented the ade8 (phosphoribosylglycinamide formyltransferase, GART) mutation, also complemented the ade5 (phosphoribosylglycinamide synthetase) and ade7 [phosphoribosylaminoimidazole synthetase (AIRS; also known as PAIS)] mutations, indicating that it is the human trifunctional GART gene. Supporting data include homology between the AIRS and GART domains of this gene and the published sequence of these domains from other organisms, and localization of the cloned gene to human chromosome 21, where the GART gene has been shown to map. The cDNA that complemented ade2 (phosphoribosylaminoimidazole carboxylase) also complemented ade1 (phosphoribosylaminoimidazole succinocarboxamide synthetase), supporting earlier data suggesting that in some organisms these functions are part of a bifunctional protein. The cDNA that complemented ade3 (formyltetrahydrofolate synthetase) is different from the recently isolated human cDNA encoding this enzyme and instead appears to encode a related mitochondrial enzyme.
Molecular basis of differential resistance to cycloguanil and pyrimethamine in Plasmodium falciparum malaria.
Authors: Peterson D S , Milhous W K , Wellems T E
Published: May 2006

Proguanil and pyrimethamine are antifolate drugs with distinct chemical structures that are used commonly in the prophylaxis and treatment of Plasmodium falciparum malaria. Clinical reports and field studies have suggested that some parasites refractory to proguanil can be treated with pyrimethamine, and vice versa. Analysis of the P. falciparum dihydrofolate reductase (DHFR) from different parasites reveals the structural basis for differential susceptibility to these antifolate drugs. Parasites harboring a pair of point mutations from Ala-16 to Val-16 and from Ser-108 to Thr-108 are resistant to cycloguanil (the active metabolite of proguanil) but not to pyrimethamine. A single Asn-108 mutation, on the other hand, confers resistance to pyrimethamine with only a moderate decrease in susceptibility to cycloguanil. Significant cross-resistance to both drugs occurs in parasites having mutations that include Ser-108----Asn-108 and Ile-164----Leu-164. These results reflect the distinct structures of pyrimethamine and cycloguanil and suggest fine differences in binding within the active site cavity of DHFR. Alternative inhibitors, used alone or in combination, may be effective against some strains of cycloguanil- or pyrimethamine-resistant malaria.
Positive and negative regulatory elements of chicken vitellogenin II gene characterized by in vitro transcription competition assays in a homologous system.
Authors: Vaccaro M , Pawlak A , Jost J P
Published: May 2006

A homologous in vitro transcription system was developed in which the cloned chicken vitellogenin II gene is faithfully transcribed by extracts prepared from chicken liver nuclei. The use of template deleted of its upstream region resulted in poor transcriptional efficiency, as did the use of extracts prepared from rooster liver, in which the gene is silent. The influence of individual cis elements was determined by transcription competition analysis. Oligonucleotides covering greater than 500 base pairs of the promoter region were used as competitor DNA in the in vitro reactions. Competition with an oligonucleotide covering part of the expression-specific DNase I hypersensitivity site B2, which contains a demethylation site, mCpG, at nucleotide position + 10, increased transcription of the gene, suggesting the binding of a repressor to this region. The enhancement of transcription was even more pronounced when the same oligonucleotide was methylated at the corresponding + 10 cytosine. Competition with oligonucleotides covering the TATA box, or the estrogen response element half-palindromic motif (GGTCA) at nucleotide positions -198 to -194, resulted in a large decrease in vitellogenin gene transcription, indicating that strongly activating factors bind to these regions. Competing oligonucleotides covering other GGTCA-containing motifs situated further upstream at nucleotide positions -292 to -288, -367 to -351, and -626 to -614 were increasingly less effective in inhibiting transcription. The results indicate that factors other than the estrogen receptor are involved in transcriptional activation of the vitellogenin II gene.
Cerebellar degeneration-related antigen: a highly conserved neuroectodermal marker mapped to chromosomes X in human and mouse.
Authors: Chen Y T , Rettig W J , Yenamandra A K , Kozak C A , Chaganti R S , Posner J B , Old L J
Published: May 2006

Cerebellar degeneration-related antigen (designated CDR34) was previously cloned by antibody screening of a cDNA library and was shown to be one of the target molecules recognized by autoantibodies in patients with paraneoplastic cerebellar degeneration. This molecule is distinctive in that it contains a tandem hexapeptide repetitive structure, presumably the basis for its high immunogenicity. In this study, we cloned the human CDR34 gene and proved that the entire repetitive sequence is encoded by a single exon without introns. We also showed that the nucleotide repeats are preserved only in the protein-coding sequences, suggesting evolutionary constraint in this region of the gene. Corresponding mouse cDNA clones were also isolated, which encoded a larger molecule with very similar hexapeptide repeating units. Comparison of the human and mouse repeats revealed a highly conserved Glu-Asp core in each unit, implicating the functional significance of this motif. Chromosomal mapping by somatic cell hybrid analysis mapped CDR34 to both human and mouse chromosomes X, and in situ hybridization further assigned CDR34 to human Xq24-q27.
Normal diploid human and rodent cells lack a detectable frequency of gene amplification.
Authors: Tlsty T D
Published: May 2006

Gene amplification is frequently observed in tumors and transformed cell lines. This phenomenon is known to contribute to the generation of drug-resistant tumor cells and quantitation of the event is believed to have prognostic value in several types of neoplasias. To date, most studies of gene amplification have used immortalized cell lines and biopsied tumor samples. In this study I examine the amplification potentials of primary diploid cells, both human and rodent, and quantitatively compare them to the amplification potentials of their transformed counterparts. I have used a strictly defined protocol (i.e., selection at a stringency of 9 X LD50) to measure amplification potential at two loci, the gene for the multifunctional protein containing activities for carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase (CAD) and the gene for dihydrofolate reductase (DHFR). I find that the frequency of amplification in transformed cells is at least four orders of magnitude greater than that in normal cells. Out of 29 cell populations studied, the 7 diploid normal cell populations exhibited no detectable amplification frequency (limit of detection at 10(-8) whereas the 22 transformed cell lines demonstrated amplification frequencies between 10(-3) and 10(-7). These results demonstrate that a dramatic difference exists between primary diploid cell populations and immortalized cell populations in their ability to amplify genomic sequences and suggests a significant difference in genetic stability between these two cell types.
Chimeric opioid peptides: tools for identifying opioid receptor types.
Authors: Xie G X , Miyajima A , Yokota T , Arai K , Goldstein A
Published: May 2006

We synthesized several chimeric peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the kappa opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affinity and type selectivity characteristic of its N-terminal sequence. The common C-terminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surface or membrane preparation, these peptides could still bind specifically to the monoclonal antibody. These chimeric peptides should be useful for isolating mu, delta, and kappa opioid receptors and for identifying opioid receptors on transfected cells in expression cloning procedures. The general approach using chimeric peptides should be applicable to other peptide receptors.
Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues.
Authors: Tashiro K , Hagiya M , Nishizawa T , Seki T , Shimonishi M , Shimizu S , Nakamura T
Published: May 2006

The primary structure of rat hepatocyte growth factor (HGF) was elucidated by determining the base sequence of the complementary DNA (cDNA) of HGF. The cDNA for rat HGF was isolated by screening a liver cDNA library with oligonucleotides based on the partial N-terminal amino acid sequence of the beta subunit of purified rat HGF. HGF is encoded in an mRNA of about 6 kilobases. Both alpha and beta subunits of HGF are specified in a single open reading frame for a 728-amino acid protein with a calculated molecular weight of 82,904. The N-terminal part of HGF has a signal sequence and a prosequence with 30 and 25 amino acid residues, respectively. The mature heterodimer structure is derived proteolytically from this single pre-pro precursor polypeptide. The calculated molecular weights of the alpha and beta subunits are 50,664 and 25,883, respectively, and each subunit has two potential N-linked glycosylation sites. The amino acid sequence of HGF is 38% identical with that of plasminogen. The alpha subunit of HGF contains four "kringle" structures, and the beta subunit has 37% amino acid identity with the serine protease domain of plasmin. Northern blot analysis revealed that HGF mRNA was expressed in rat various tissues, including the liver, kidney, lung, and brain.
Atomic-resolution structure of the cellulose synthase regulator cyclic diguanylic acid.
Authors: Egli M , Gessner R V , Williams L D , Quigley G J , van der Marel G A , van Boom J H , Rich A , Frederick C A
Published: May 2006

Cyclic diguanylic acid acts as a regulator for cellulose synthase activity in the bacterium Acetobacter xylinum. We report the x-ray crystal structure of the regulator at atomic resolution. The structure contains two independent molecules that adopt almost identical conformations. The two molecules form self-intercalated units that are stacked on each other. Two different G.G base-pairing modes occur between the stacks. The more stable one has two or possibly three hydrogen bonds between two guanines and is related to the type of hydrogen bonding that is believed to exist between G-rich strands at the ends of chromosomes.
A frequent tyrosinase gene mutation in classic, tyrosinase-negative (type IA) oculocutaneous albinism.
Authors: Giebel L B , Strunk K M , King R A , Hanifin J M , Spritz R A
Published: May 2006

We have identified a tyrosinase gene mutation in several patients with classic, tyrosinase-negative (type IA) oculocutaneous albinism. This mutation, which results in a proline----leucine substitution at codon 81 of the tyrosinase polypeptide (EC 1.14.18.1), was observed in 20% (6 of 30) of oculocutaneous albinism alleles from independent probands, but it was not observed in any normal individuals. This mutation thus appears to be a frequent cause of tyrosinase-negative oculocutaneous albinism.
Isolation of fetal DNA from nucleated erythrocytes in maternal blood.
Authors: Bianchi D W , Flint A F , Pizzimenti M F , Knoll J H , Latt S A
Published: May 2006

Fetal nucleated cells within maternal blood represent a potential source of fetal genes obtainable by venipuncture. We used monoclonal antibody against the transferrin receptor (TfR) to identify nucleated erythrocytes in the peripheral blood of pregnant women. Candidate fetal cells from 19 pregnancies were isolated by flow sorting at 12 1/2-17 weeks gestation. The DNA in these cells was amplified for a 222-base-pair (bp) sequence present on the short arm of the Y chromosome as proof that the cells were derived from the fetus. The amplified DNA was compared with standardized DNA concentrations; 0.1-1 ng of fetal DNA was obtained in the 20-ml maternal samples. In 7/19 cases, a 222-bp band of amplified DNA was detected, consistent with the presence of male DNA in the isolated cells; 6/7 of these were confirmed as male pregnancies by karyotyping amniocytes. In the case of the female fetus, DNA prepared from samples at 32 weeks of gestation and cord blood at delivery also showed the presence of the Y chromosomal sequence, suggesting Y sequence mosaicism or translocation. In 10/12 cases where the 222-bp band was absent, the fetuses were female. Thus, we were successful in detecting the Y chromosomal sequence in 75% of the male-bearing pregnancies, demonstrating that it is possible to isolate fetal gene sequences from cells in maternal blood. Further refinement in methodology should increase sensitivity and facilitate noninvasive screening for fetal gene mutations.
Transcriptional stimulation of the delta 1-crystallin gene by insulin-like growth factor I and insulin requires DNA cis elements in chicken.
Authors: Alemany J , Borras T , de Pablo F
Published: May 2006

Insulin-like growth factor I (IGF-I) and insulin regulate expression of the endogenous delta 1-crystallin gene in embryonic lens cells that express receptors for both peptides. To further analyze the transcriptional component of this hormonal effect, transient transfections of lens cells were prepared with DNA constructs containing deletions of the delta 1-crystallin promoter and the chloramphenicol acetyltransferase reporter gene. A 77-nucleotide DNA segment of the delta 1-crystallin promoter from nucleotide positions-120 to -43 confers sensitivity to insulin and IGF-I. The hormonal effect is dose-dependent, and maximal stimulation of promoter activity (2- to 2.5-fold induction) is obtained with 10(-8) M IGF-I and 10(-7) M insulin. Mobility-shift DNA-binding analysis shows specific binding of nuclear protein(s) to the delta 1-crystallin promoter DNA between positions -120 and +23, which appears to be regulated by IGF-I. An SP1-binding motif is involved in this DNA-protein interaction. The bivalent IgG fraction of an anti-insulin receptor antiserum (B-10), known to mimic insulin action in other systems, stimulates promoter activity to the same extent as insulin.
Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites.
Authors: Lenz J , Okenquist S A , LoSardo J E , Hamilton K K , Doetsch P W
Published: May 2006

Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites. A binding assay based on the use of beta-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins.
Distribution of saposin proteins (sphingolipid activator proteins) in lysosomal storage and other diseases.
Authors: Morimoto S , Yamamoto Y , O'Brien J S , Kishimoto Y
Published: May 2006

Saposins (A, B, C, and D) are small glycoproteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. Concentrations of these saposins in brain, liver, and spleen from normal humans as well as patients with lysosomal storage disease were determined. A quantitative HPLC method was used for saposin A, C, and D and a stimulation assay was used for saposin B. In normal tissues, saposin D was the most abundant of the four saposins. Massive accumulations of saposins, especially saposin A (about 80-fold increase over normal), were found in brain of patients with Tay-Sachs disease or infantile Sandhoff disease. In spleen of adult patients with Gaucher disease, saposin A and D accumulations (60- and 17-fold, respectively, over normal) were higher than that of saposin C (about 16-fold over normal). Similar massive accumulations of saposins A and D were found in liver of patients with fucosidosis (about 70- and 20-fold, respectively, over normal). Saposin D was the primary saposin stored in the liver of a patient with Niemann-Pick disease (about 30-fold over normal). Moderate increases of saposins B and D were found in a patient with GM1 gangliosidosis. Normal or near normal levels of all saposins were found in patients with Krabbe disease, metachromatic leukodystrophy, Fabry disease, adrenoleukodystrophy, I-cell disease, mucopolysaccharidosis types 2 and 3B, or Jansky-Bielschowsky disease. The implications of the storage of saposins in these diseases are discussed.
Isolation and characterization of fra-2, an additional member of the fos gene family.
Authors: Nishina H , Sato H , Suzuki T , Sato M , Iba H
Published: May 2006

Antiserum raised against a Fos peptide (amino acids Lys-127 to Arg-140 of chicken c-Fos) recognizes a 46-kDa Fos-related protein in cell lysates of growth-stimulated chicken embryo fibroblasts. Induction of the 46-kDa protein is transient but is slightly prolonged relative to c-Fos following growth stimulation. Using a mixed oligonucleotide probe encoding the peptide antigen, we have cloned the chicken genomic locus that encodes this protein and have determined its gene structure. This locus consists of four exons, each of which has some homology with the corresponding exons of the chicken c-fos gene, and it expresses a 6-kilobase mRNA after growth stimulation. The deduced amino acid sequence of the gene (323 amino acids) contains a "leucine zipper" and includes five distinct regions that exhibit strong sequence homology to the recently identified fos-related antigens Fra-1 (rat) and FosB (mouse) as well as c-Fos. Since the other regions of the gene have little homology to any of these three proteins, this gene was named "fra-2." When the fra-2 gene was overexpressed by an avian retrovirus vector, it caused transformation of chicken embryo fibroblasts. The fra-2 gene product formed a complex in cells with the c-Jun protein, indicating that c-fos and fra-2 share biological and biochemical functions.
NG-methyl-L-arginine inhibits tumor necrosis factor-induced hypotension: implications for the involvement of nitric oxide.
Authors: Kilbourn R G , Gross S S , Jubran A , Adams J , Griffith O W , Levi R , Lodato R F
Published: May 2006

Clinical assessment of the activity of tumor necrosis factor (TNF) against human cancer has been limited by a dose-dependent cardiovascular toxicity, most frequently hypotension. TNF is also thought to mediate the vascular collapse resulting from bacterial endotoxin. The present studies address the mechanism by which TNF causes hypotension and provide evidence for elevated production of nitric oxide, a potent vasodilator initially characterized as endothelium-derived relaxing factor. Nitric oxide is synthesized by several cell types, including endothelial cells and macrophages, from the guanidino nitrogen of L-arginine; the enzymatic pathway is competitively inhibited by NG-methyl-L-arginine. We found that hypotension induced in pentobarbital-anesthetized dogs by TNF (10 micrograms/kg, i.v., resulting in a fall in mean systemic arterial pressure from 124.7 +/- 7 to 62.0 +/- 22.9 mmHg; 1 mmHg = 133 Pa) was completely reversed within 2 min following administration of NG-methyl-L-arginine (4.4 mg/kg, i.v.). In contrast, NG-methyl-L-arginine failed to reverse the hypotensive response to an equivalent depressor dose of nitroglycerin, a compound that acts by forming nitric oxide by a nonenzymatic, arginine-independent mechanism. The effect of NG-methyl-L-arginine on TNF-induced hypotension was antagonized, and the hypotension restored, by administration of excess L-arginine (100 mg/kg, i.v.). Our findings suggest that excessive nitric oxide production mediates the hypotensive effect of TNF.
The Ams (altered mRNA stability) protein and ribonuclease E are encoded by the same structural gene of Escherichia coli.
Authors: Babitzke P , Kushner S R
Published: May 2006

The in vitro and in vivo analysis of the ribonuclease E-deficient (rne-) and the altered mRNA stability protein-deficient (ams-) strains of Escherichia coli has demonstrated that they carry mutations in the same structural gene. Strains encoding either thermolabile RNase E (rne-3071) or Ams protein (ams-1) are defective in both rRNA processing and mRNA turnover. Immediately after a shift to the nonpermissive temperature, the chemical decay rate of bulk mRNA is slowed 2- to 3-fold, and within 70 min, precursors to 5S rRNA begin to accumulate. In addition, all of the phenotypes associated with either the rne-3071 or the ams-1 alleles were complemented by a recombinant plasmid carrying ams+. When taken together with previous genetic studies, these results suggest that the role of ribonuclease E in mRNA turnover involves endonucleolytic cleavages at the proposed ACAG(A/U)AUUUG consensus sequence.
Lack of correlation between DNA methylation and transcriptional inactivation: the chicken lysozyme gene.
Authors: Wölfl S , Schräder M , Wittig B
Published: May 2006

We have analyzed the methylation state of all nine CpG sites in the transcription start region (-420 to +250 base pairs) of the chicken lysozyme gene by genomic sequencing. One of these sites, at -81, lies within the promoter, seven are clustered within the first exon, and the last is in the first intron. Five cell types and tissues have been investigated to study the relationship between methylation level and gene expression. For each cell type used, the majority of CpG sites showed a similar level of methylation. Of two gene-nonexpressing tissues, erythrocytes are hypomethylated, whereas liver is methylated at most of its CpG sites. For gene-expressing tissues, oviduct is completely unmethylated, whereas HD-11 culture cells are methylated. Thus no correlation is observed between degree of CpG methylation and level of expression of the lysozyme gene. The observed methylation patterns are discussed in terms of possible features of the local chromatin structure.
Pancreatic tumor pathogenesis reflects the causative genetic lesion.
Authors: Sandgren E P , Quaife C J , Paulovich A G , Palmiter R D , Brinster R L
Published: May 2006

Transgenic mice in which c-myc expression is targeted to pancreatic acinar cells develop mixed acinar/ductal pancreatic adenocarcinomas between 2 and 7 months of age. This contrasts with the effect on pancreas of the simian virus 40 tumor antigen or activated ras, which in adult mice causes lesions composed exclusively of acinar-like cells. Furthermore, during an early stage of myc-induced pathology, transformed acinar-derived cells appear within islets, suggesting that islet hormones may influence the progression of these exocrine pancreatic tumors. These findings demonstrate that the initial oncogenic alteration can influence the pattern of subsequent tumor pathogenesis and, given that human exocrine pancreatic tumors are predominantly ductal adenocarcinomas, support the suggestion that transformed acinar cells may contribute to the genesis of this serious disease in man.
The multifunctional protein OBF1 is phosphorylated at serine and threonine residues in Saccharomyces cerevisiae.
Authors: Francesconi S C , Eisenberg S
Published: May 2006

We have purified a DNA replication enhancer-binding protein, OBF1, from yeast cells grown in a medium containing 32P-labeled orthophosphate. The purified 32P-labeled protein comigrated on polyacrylamide gels with OBF1 bands identified by immunoblotting with anti-OBF1 antibodies. Furthermore, trypsin treatment of the 32P-labeled OBF1 revealed several phosphorylated peptides, suggesting that OBF1 is multiply phosphorylated in vivo. Incubation of phosphorylated peptides with calf intestinal phosphatase liberated the radiolabel as free phosphate, indicating a phosphoester linkage. Acid hydrolysis of the tryptic peptides revealed 32P-label label comigrating with phosphoserine; some of it, however, was also identified as phosphothreonine. Using anti-OBF1 antibodies, we cloned the OBF1 gene from a lambda gt11 yeast expression library. The DNA sequence of the isolated gene and its over-expression in yeast indicated that OBF1 is identical to ABF-1 and BAF1 proteins, believed to have a role in transcriptional repression and activation. Therefore, we suggest that OBF1 is a multifunctional protein, acting in transcription and replication, and that these activities are regulated by phosphorylation.
Correction of a human beta S-globin gene by gene targeting.
Authors: Shesely E G , Kim H S , Shehee W R , Papayannopoulou T , Smithies O , Popovich B W
Published: May 2006

As a step toward using gene targeting for gene therapy, we have corrected a human beta S-globin gene to the normal beta A allele by homologous recombination in the mouse-human hybrid cell line BSM. BSM is derived from a mouse erythroleukemia cell line and carries a single human chromosome 11 with the beta S-globin allele. A beta A-globin targeting construct containing a unique oligomer and a neomycin-resistance gene was electroporated into the BSM cells, which were then placed under G418 selection. Then 126 resulting pools containing a total of approximately 29,000 G418-resistant clones were screened by PCR for the presence of a targeted recombinant: 3 positive pools were identified. A targeted clone was isolated by replating one of the positive pools into smaller pools and rescreening by PCR, followed by dilution cloning. Southern blot analysis demonstrated that the isolated clone had been targeted as planned. The correction of the beta S allele to beta A was confirmed both by allele-specific PCR and by allele-specific antibodies. Expression studies comparing the uninduced and induced RNA levels in unmodified BSM cells and in the targeted clone showed no significant alteration in the ability of the targeted clone to undergo induction, despite the potentially disrupting presence of a transcriptionally active neomycin gene 5' to the human beta A-globin gene. Thus gene targeting can correct a beta S allele to beta A, and the use of a selectable helper gene need not significantly interfere with the induction of the corrected gene.
Initial synaptic efficacy influences induction and expression of long-term changes in transmission.
Authors: Yang X D , Faber D S
Published: May 2006

Long-term depression (LTD) of glutamatergic and electrotonic transmission can be induced at mixed synapses between eighth nerve fibers and the goldfish Mauthner (M) cell in vivo, by pairing weak presynaptic tetani with postsynaptic inhibition. This LTD can be reversed by stronger tetani that produce long-term potentiation (LTP). Moreover, the depression is more likely to occur and tends to last longer when the initial synaptic efficacy is high--that is, if the synaptic strength is first potentiated. In addition, when synaptic efficacy is initially elevated, a weak tetanization that usually results in a gradually developing potentiation instead produces no change in chemical transmission and even a depression of electrotonic coupling. Thus, the modifications in synaptic transmission caused by a certain tetanizing protocol depend upon the history of synaptic efficacy. This last concept provides an experimental basis for theoretical models concerned with pre- and postsynaptic contributions to the regulation of synaptic plasticity.
Sequence-specific DNA binding by Myc proteins.
Authors: Kerkhoff E , Bister K , Klempnauer K H
Published: May 2006

Myc proteins have a tripartite carboxyl-terminal domain containing specific amino acid sequence motifs: a basic motif, a helix-loop-helix motif, and a leucine heptad repeat. Similar sequence motifs have been identified in several eukaryotic transcription factors and were shown to facilitate protein-DNA and protein-protein interactions. By using recombinant v-Myc proteins obtained by bacterial expression of full-length or partially deleted avian v-myc alleles, the functional relevance of these sequence motifs for Myc protein oligomerization and for DNA binding was investigated. All recombinant v-Myc proteins that have retained the carboxyl-terminal domain dimerize and specifically bind to double-stranded DNA containing the palindromic core sequence CACGTG. This and a closely related DNA sequence element have been defined previously as part of the binding sites for human transcription factors USF and TFE3, which specifically bind to the adenovirus major late promoter or the muE3 motif within the immunoglobulin heavy-chain enhancer, respectively. It is shown that a 61-amino-acid peptide sequence containing only the bipartite basic motif/helix-loop-helix domain of Myc is necessary and sufficient for dimerization and sequence-specific DNA binding of v-Myc recombinant proteins.
Sites within the complement C3b/C4b receptor important for the specificity of ligand binding.
Authors: Krych M , Hourcade D , Atkinson J P
Published: May 2006

Cysteine-rich repeated units of 40-70 amino acids are building blocks of many mammalian proteins, including 12 proteins of the complement system. Human complement arranged motifs, designated short consensus repeats (SCRs), which constitute the entire extracellular portion of this protein. Klickstein et al. [Klickstein, L. B., Bartow, T. J., Miletic, V., Rabson, L. D., Smith, J. A. & Fearon, D. T. (1988) J. Exp. Med. 168, 1699-1717 (abstr.)] localized a C4b binding domain to SCR-1 and/or SCR-2 and a C3b binding domain to SCR-8 and/or SCR-9. These SCRs bind different ligands, although SCR-1 and SCR-8 are 55% homologous and SCR-2 and SCR-9 are 70% homologous. To examine if one or two SCRs are required for ligand binding and to define sites within the SCRs that determine specificity of binding, mutagenesis analysis of a truncated, secreted form of CR1, called CR1-4 by Hourcade et al. [Hourcade, D., Meisner, D. R., Atkinson, J. P. & Holers, V. M. (1988) J. Exp. Med. 168, 1255-1270], was undertaken. The latter, composed of the first eight and one-half amino-terminal SCRs of CR1, efficiently bound C4b but not iC3. SCR-1 and SCR-2 were necessary for this interaction. Analysis of the mutant CR1-4 proteins, in which amino acids in SCR-1 and SCR-2 were substituted a few at a time with the homologous amino acids of SCR-8 and SCR-9, led to the identification of one amino acid in SCR-1 and three amino acids in SCR-2 important for C4b binding. Furthermore, five amino acids at the end of SCR-9, if placed in the homologous positions of SCR-2, conferred iC3 binding and are likely essential for ligand binding activity of SCR-8 and SCR-9. This iC3 binding occurred only if SCR-1 was present, indicating that two contiguous SCRs are necessary for this interaction. These results provide identification of amino acids within SCRs that are important for ligand binding.
Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces.
Authors: Kang A S , Barbas C F , Janda K D , Benkovic S J , Lerner R A
Published: May 2006

We describe a method based on a phagemid vector with helper phage rescue for the construction and rapid analysis of combinatorial antibody Fab libraries. This approach should allow the generation and selection of many monoclonal antibodies. Antibody genes are expressed in concert with phage morphogenesis, thereby allowing incorporation of functional Fab molecules along the surface of filamentous phage. The power of the method depends upon the linkage of recognition and replication functions and is not limited to antibody molecules.