bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and characterization of a Drosophila serotonin receptor that activates adenylate cyclase.
Authors: Witz P , Amlaiky N , Plassat J L , Maroteaux L , Borrelli E , Hen R
Published: May 2006

Using a strategy based on nucleotide sequence homology between genes encoding receptors that interact with guanine nucleotide-binding proteins, we have isolated Drosophila genomic and cDNA clones encoding a functional serotonin receptor (5HT-dro receptor). This protein is expressed predominantly in Drosophila heads and exhibits highest homology with the human 5HT1A receptor. The predicted structure of the 5HT-dro receptor reveals two unusual features: (i) eight putative transmembrane domains instead of the expected seven and (ii) a Gly-Ser repeat that is a potential glycosaminoglycan attachment site. When stably introduced into mouse NIH 3T3 cells, the 5HT-dro receptor activates adenylate cyclase in response to serotonin and is inhibited by serotonin receptor antagonists such as dihydroergocryptine. The 5HT-dro receptor or closely related receptors might be responsible for the serotonin-sensitive cyclase that has been suggested to play a role in learning and modulation of circadian rhythm in a number of invertebrate systems.
Alpha 6 beta 4 integrin heterodimer is a component of hemidesmosomes.
Authors: Stepp M A , Spurr-Michaud S , Tisdale A , Elwell J , Gipson I K
Published: May 2006

Antisera that recognize the alpha 6 and beta 4 subunits of integrins were found by immunoelectron microscopy to localize to hemidesmosomes in the basal cells of mouse corneal epithelium. Immunoprecipitation experiments using extracts of metabolically labeled corneal epithelial cells indicate that the primary alpha 6-subunit-containing integrin heterodimer present is alpha 6 beta 4 and not alpha 6 beta 1. Here we extend previous studies to report that by immunofluorescence microscopy the alpha 6 integrin subunit colocalizes with bullous pemphigoid antigen and type VII collagen in newly forming hemidesmosomes in the developing 17-day fetal rabbit eye. Neither the composition of the anchoring filaments, which span the region between the hemidesmosomal plaque and the lamina densa of basement membrane where the globular domain of type VII collagen is located, nor the extracellular ligand of alpha 6 beta 4 is known. Once anchoring filament proteins are identified, it will be of interest to determine whether any bind to alpha 6 beta 4.
Exon trapping: a genetic screen to identify candidate transcribed sequences in cloned mammalian genomic DNA.
Authors: Duyk G M , Kim S W , Myers R M , Cox D R
Published: May 2006

Identification and recovery of transcribed sequences from cloned mammalian genomic DNA remains an important problem in isolating genes on the basis of their chromosomal location. We have developed a strategy that facilitates the recovery of exons from random pieces of cloned genomic DNA. The basis of this "exon trapping" strategy is that, during a retroviral life cycle, genomic sequences of nonviral origin are correctly spliced and may be recovered as a cDNA copy of the introduced segment. By using this genetic assay for cis-acting sequences required for RNA splicing, we have screened approximately 20 kilobase pairs of cloned genomic DNA and have recovered all four predicted exons.
Human macrophage scavenger receptors: primary structure, expression, and localization in atherosclerotic lesions.
Authors: Matsumoto A , Naito M , Itakura H , Ikemoto S , Asaoka H , Hayakawa I , Kanamori H , Aburatani H , Takaku F , Suzuki H
Published: May 2006

Two types of cDNAs for human macrophage scavenger receptors were cloned from a cDNA library derived from the phorbol ester-treated human monocytic cell line THP-1. The type I and type II human scavenger receptors encoded by these cDNAs are homologous (73% and 71% amino acid identity) to their previously characterized bovine counterparts and consist of six domains: cytoplasmic (I), membrane-spanning (II), spacer (III), alpha-helical coiled-coil (IV), collagen-like (V), and a type-specific C-terminal (VI). The receptor gene is located on human chromosome 8. The human receptors expressed in CHO-K1 cells mediated endocytosis of modified low density lipoproteins. Two mRNAs, 4.0 and 3.2 kilobases, have been detected in human liver, placenta, and brain. Immunohistochemical studies using an anti-peptide antibody which recognizes human scavenger receptors indicated the presence of the scavenger receptors in the macrophages of lipid-rich atherosclerotic lesions, suggesting the involvement of scavenger receptors in atherogenesis.
v-Ha-ras transgene abrogates the initiation step in mouse skin tumorigenesis: effects of phorbol esters and retinoic acid.
Authors: Leder A , Kuo A , Cardiff R D , Sinn E , Leder P
Published: May 2006

Experimental carcinogenesis has led to a concept that defines two discrete stages in the development of skin tumors: (i) initiation, which is accomplished by using a mutagen that presumably activates a protooncogene, and (ii) promotion, which is a reversible process brought about most commonly by repeated application of phorbol esters. We have created a transgenic mouse strain that carries the activated v-Ha-ras oncogene fused to the promoter of the mouse embryonic alpha-like, zeta-globin gene. Unexpectedly, these animals developed papillomas at areas of epidermal abrasion and, because abrasion can also serve as a tumor-promoting event in mutagen-treated mouse skin, we tested these mice for their ability to respond to phorbol ester application. Within 6 weeks virtually all treated carrier mice had developed multiple papillomas, some of which went on to develop squamous cell carcinomas and, more frequently, underlying sarcomas. We conclude that the oncogene "preinitiates" carrier mice, replacing the initiation/mutagenesis step and immediately sensitizing them to the action of tumor promoters. In addition, treatment of the mice with retinoic acid dramatically delays, reduces, and often completely inhibits the appearance of promoter-induced papillomas. This strain has use in screening tumor promoters and for assessing antitumor and antiproliferative agents.
Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin.
Authors: Bierer B E , Mattila P S , Standaert R F , Herzenberg L A , Burakoff S J , Crabtree G , Schreiber S L
Published: May 2006

Proliferation and immunologic function of T lymphocytes are initiated by signals from the antigen receptor that are inhibited by the immunosuppressant FK506 but not by its structural analog, rapamycin. On the other hand, interleukin 2 (IL-2)-induced signals are blocked by rapamycin but not by FK506. Remarkably, these two drugs inhibit each other's actions, raising the possibility that both act by means of a common immunophilin (immunosuppressant binding protein). We find that the dissociation constant of rapamycin to the FK506 binding protein FKBP (Kd = 0.2 nM) is close to the dissociation constant of FK506 to FKBP (Kd = 0.4 nM) and to their effective biologic inhibitory concentrations. However, an excess of rapamycin is needed to revert FK506-mediated inhibition of IL-2 production, apoptosis, and transcriptional activation of NF-AT, a T-cell-specific transcription factor necessary for IL-2 gene activation. Similarly, an excess of FK506 is needed to revert rapamycin-mediated inhibition of IL-2-induced proliferation. The drug concentrations required for antagonism may be explained by the relative affinity of the drugs to, and by the abundance of, the immunophilin FKBP. FKBP has been shown to catalyze the interconversion of the cis- and trans-rotamers of the peptidyl-prolyl amide bond of peptide substrates; here we show that rapamycin, like FK506, is a potent inhibitor of the rotamase activity of FKBP (Ki = 0.2 nM). Neither FKBP binding nor inhibition of rotamase activity of FKBP alone is sufficient to explain the biologic actions of these drugs. Rather, these findings suggest that immunophilin bound to FK506 interferes with antigen receptor-induced signals, while rapamycin bound to the immunophilin interferes with IL-2-induced signals.
Convergent evolution of the red- and green-like visual pigment genes in fish, Astyanax fasciatus, and human.
Authors: Yokoyama R , Yokoyama S
Published: May 2006

We have isolated and sequenced genes from the blind cave fish, Astyanax fasciatus, that are homologous to the human red and green visual pigment genes. The data strongly suggest that, like human, these fish have one red-like visual pigment gene and multiple green-like visual pigment genes. By comparing the DNA sequences of the human and fish visual pigment genes and knowing their phylogenetic relationship, one can infer the direction of amino acid substitutions in the red and green visual pigments. The results indicate that the red pigments in human and fish evolved from the green pigment independently by identical amino acid substitutions in only a few key positions.
Direction selectivity of blowfly motion-sensitive neurons is computed in a two-stage process.
Authors: Borst A , Egelhaaf M
Published: May 2006

Direction selectivity of motion-sensitive neurons is generally thought to result from the nonlinear interaction between the signals derived from adjacent image points. Modeling of motion-sensitive networks, however, reveals that such elements may still respond to motion in a rather poor directionally selective way. Direction selectivity can be significantly enhanced if the nonlinear interaction is followed by another processing stage in which the signals of elements with opposite preferred directions are subtracted from each other. Our electrophysiological experiments in the fly visual system suggest that here direction selectivity is acquired in such a two-stage process.
Preferential binding of growth inhibitory prostaglandins by the target protein of a carcinogen.
Authors: Khan S H , Sorof S
Published: May 2006

Liver fatty acid binding protein (L-FABP) is the principal target protein of the hepatic carcinogen N-(2-fluorenyl)acetamide (2-acetylaminofluorene) in rat liver. In addition, the cyclopentenone prostaglandins (PG), PGA, PGJ2, and delta 12-PGJ2, inhibit the growth of many cell types in vitro. This report describes the preferential binding of the growth inhibitory prostaglandins by L-FABP and the reversible inhibition of thymidine incorporation into DNA by PGA2 and delta 12-PGJ2 in primary cultures of purified rat hepatocytes. As a model ligand, [3H]PGA1 bound to L-FABP specifically, reversibly, rapidly, and with high affinity. Its dissociation constants were 134 nM (high affinity) and 3.6 microM (low affinity). The high-affinity binding of [3H]PGA1 was 9- and approximately 13-fold more avid than the binding of the conventional fatty acid ligands, oleic acid and arachidonic acid, respectively. The abilities of different prostaglandins to compete with the high-affinity binding of [3H]PGA1 correlated with their growth inhibitory activities reported previously and here. The growth inhibitory cyclopentenone prostaglandins (PGA1, PGA2, delta 12-PGJ2, and PGJ2) were the best competitive ligands, intermediate competitors were the weak growth inhibitors PGE1 and PGD2, and the poorest competitors were PGE2 and PGF2 alpha, which stimulate rather than inhibit DNA synthesis in rat hepatocytes in primary culture. The in vitro actions of L-FABP are compatible with those of a specific and dissociable carrier of growth inhibitory prostaglandins in rat hepatocytes and suggest that the carcinogen may usurp the cellular machinery of the growth inhibitory prostaglandins.
Expression and chromosomal localization of a lymphocyte K+ channel gene.
Authors: Grissmer S , Dethlefs B , Wasmuth J J , Goldin A L , Gutman G A , Cahalan M D , Chandy K G
Published: May 2006

We recently isolated a family of three closely related mouse K+ channel genes (MK1, MK2, and MK3) with coding regions contained in single uninterrupted exons. Here we have used patch-clamp recordings from Xenopus oocytes injected with mRNA to show that MK3 encodes a channel with biophysical and pharmacological properties indistinguishable from those of voltage-gated type n K+ channels in T cells. In addition, we used the polymerase chain reaction to demonstrate the presence of MK3 mRNA in T cells. These data suggest that MK3 may encode the T-cell voltage-gated type n K+ channel. We also show that MK3 and MK2 are localized on human chromosomes 13 and 12, respectively.
beta-Galactosidase containing a human immunodeficiency virus protease cleavage site is cleaved and inactivated by human immunodeficiency virus protease.
Authors: Baum E Z , Bebernitz G A , Gluzman Y
Published: May 2006

A "cleavage cassette" specifying a decapeptide human immunodeficiency virus (HIV) protease cleavage site was introduced into six different locations of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in Escherichia coli. Four of these constructs retained beta-galactosidase activity despite the insertion of the cleavage cassette. Of these four constructs, one was cleaved by HIV protease, resulting in the inactivation of beta-galactosidase both in vivo and in vitro. This cleavage was inhibited by pepstatin A, a known inhibitor of HIV protease. Thus, beta-galactosidase has been converted into an easily assayed substrate for HIV protease. An analogous construct of beta-galactosidase containing a polio protease cleavage site was cleaved likewise by polio protease, suggesting that this system may be generic for monitoring cleavage by a variety of proteases.
Loss of DNA-membrane interactions and cessation of DNA synthesis in myeloperoxidase-treated Escherichia coli.
Authors: Rosen H , Orman J , Rakita R M , Michel B R , VanDevanter D R
Published: May 2006

Neutrophils and monocytes employ a diverse array of antimicrobial effector systems to support their host defense functions. The mechanisms of action of most of these systems are incompletely understood. The present report indicates that microbicidal activity by a neutrophil-derived antimicrobial system, consisting of myeloperoxidase, enzymatically generated hydrogen peroxide, and chloride ion, is accompanied by prompt cessation of DNA synthesis in Escherichia coli, as determined by markedly reduced incorporation of [3H]thymidine into trichloracetic acid-precipitable material. Simultaneously, the myeloperoxidase system mediates a decline in the ability of E. coli membranes to bind hemimethylated DNA sequences containing the E. coli chromosomal origin of replication (oriC). Binding of oriC to the E. coli membrane is an essential element of orderly chromosomal DNA replication. Comparable early changes in DNA synthesis and DNA-membrane interactions were not observed with alternative oxidant or antibiotic-mediated microbicidal systems. It is proposed that oxidants generated by the myeloperoxidase system modify the E. coli membrane in such a fashion that oriC binding is markedly impaired. As a consequence chromosomal DNA replication is impaired and organisms can no longer replicate.
Cytochrome a1 of acetobacter aceti is a cytochrome ba functioning as ubiquinol oxidase.
Authors: Matsushita K , Shinagawa E , Adachi O , Ameyama M
Published: May 2006

Cytochrome a1 is a classic cytochrome that in the 1930s had already been detected in Acetobacter strains and in the 1950s was identified as a terminal oxidase. However, recent studies did not substantiate the previous observations. We have detected a cytochrome a1-like chromophore in Acetobacter aceti, which was purified and characterized in this study. The cytochrome was solubilized from membranes of the strain with octyl beta-D-glucopyranoside and was purified by single column chromatography. The purified cytochrome exhibited a broad alpha peak around 600-610 nm, which turned to a sharp peak at 589 nm in the presence of cyanide. Carbon monoxide difference spectra of the cytochrome indicated the presence of an alpha-type cytochrome. The cytochrome contained 1 mol each of hemes b and a and probably one copper ion. These results suggest that the cytochrome purified from A. aceti is the so-called cytochrome a1, and thus the existence of the classic cytochrome has been reconfirmed. The purified enzyme consisted of four polypeptides of 55, 35, 22, and 18 kDa, and it showed a sedimentation coefficient of 6.3 S in the native form. The enzyme had a high ubiquinol oxidase activity (140-160 mumol of ubiquinol-2 oxidized per min per mg of protein). When reconstituted into proteoliposomes, the cytochrome could generate an electrochemical proton gradient during oxidation of ubiquinol. Thus, cytochrome a1 of A. aceti has been shown to be a cytochrome ba terminal oxidase capable of generating an electrochemical proton gradient concomitant with ubiquinol oxidation.
Genetic and morphological analysis of a maize-teosinte F2 population: implications for the origin of maize.
Authors: Doebley J , Stec A , Wendel J , Edwards M
Published: May 2006

Genes controlling the dramatic morphological differences between maize and its presumed progenitor (teosinte) were investigated in a maize-teosinte F2 population through the use of molecular markers. Results indicate that the key traits differentiating maize and teosinte are each under multigenic control, although for some traits, such as the number of ranks of cupules, the data are consistent with a mode of inheritance that would involve a single major locus plus several modifiers. For other traits, such as the presence/absence of the pedicellate spikelet, the data indicate multigenic inheritance with no single locus having a dramatically larger effect than the others. Results also indicate that the tunicate locus (Tu) had no major role in the origin of maize, despite previous opinion that it was involved. The major loci affecting the morphological differences between maize and teosinte are located on the first four chromosomes. The data suggest that the differences between teosinte and maize involve, in part, developmental modifications that enable (i) primary lateral inflorescences, which are programmed to develop into tassels (male) in teosinte, to become ears (female) in maize, and (ii) the expression of male secondary sex traits on a female background in maize. Similar changes were likely involved in the origin of maize.
Oncogenic transformation by the tax gene of human T-cell leukemia virus type I in vitro.
Authors: Tanaka A , Takahashi C , Yamaoka S , Nosaka T , Maki M , Hatanaka M
Published: May 2006

Human T-cell leukemia virus type I (HTLV-I) is a causative agent of adult T-cell leukemia (ATL). To elucidate the role of HTLV-I in leukemogenesis, we examined the biological activity of a defective HTLV-I provirus with the env-pX 3' long terminal repeat region cloned from leukemic cells of an ATL patient. Transfection experiments showed growth stimulation of NIH 3T3 cells--growing beyond the saturation density and growing in soft agar. Since the pX sequence is known to encode three proteins, Tax, Rex, and p21x, the biological activity of each pX gene was examined separately. The growth-stimulating activity was induced only by the tax gene in NIH 3T3 cells and Rat-1 cells. Furthermore, the tax gene induced tumorigenicity in nude mice when introduced into Rat-1 cells. Thus, a transcriptional transactivator gene of HTLV-I, tax, is clearly identified as a viral oncogene without a cellular homolog. The transforming activity of tax, possibly via a transcriptional deregulation of cell growth control, may play an important role in leukemogenesis of ATL in addition to its aberrant stimulation of the interleukin 2 system.
Herculin, a fourth member of the MyoD family of myogenic regulatory genes.
Authors: Miner J H , Wold B
Published: May 2006

We have identified and cloned herculin, a fourth mouse muscle regulatory gene. Comparison of its DNA and deduced amino acid sequences with those of the three known myogenic genes (MyoD, myogenin, and Myf-5) reveals scattered short spans with similarity to one or more of these genes and a long span with strong similarity to all three. This long span includes a sequence motif that is also present in proteins of the myc, achaete-scute, and immunoglobulin enhancer-binding families. The herculin gene is physically linked to the Myf-5 gene on the chromosome; only 8.5 kilobases separate their translational start sites. A putative 27-kDa protein is encoded by three exons contained within a 1.7-kilobase fragment of the herculin gene. When expressed under the control of the simian virus 40 early promoter, transfected herculin renders murine NIH 3T3 and C3H/10T1/2 fibroblasts myogenic. In doing so, it also activates expression of myogenin, MyoD, and endogenous herculin in NIH 3T3 recipients. In adult mice, herculin is expressed in skeletal muscle but is absent from smooth muscle, cardiac muscle, and all nonmuscle tissues assayed. Direct comparison of the four known myogenic regulators in adult muscle showed that herculin is expressed at a significantly higher level than is any of the others. This quantitative dominance suggests an important role in the establishment or maintenance of adult skeletal muscle.
The A3 allele of the HLA-DQA1 locus is associated with susceptibility to type 1 diabetes in Japanese.
Authors: Todd J A , Fukui Y , Kitagawa T , Sasazuki T
Published: May 2006

Analysis of the frequencies of class II HLA-DR and HLA-DQ alleles by serological and DNA typing in 49 Japanese patients with type 1 (insulin-dependent) diabetes and 31 Japanese controls indicates the following. (i) Susceptibility is more strongly associated with the HLA-DQ subregion than with the HLA-DR subregion. (ii) Of the class II alleles detected, the A3 allele of the DQA1 locus was the most strongly associated with disease. Ninety-six percent of the patients were positive for the A3 allele compared to 53% of the controls (P = 0.001; relative risk = 19.7; confidence limits = 3.72-188.64). (iii) The DQw8 allele of the DQB1 locus, which is associated with susceptibility to type 1 diabetes in Caucasians and Blacks, was not increased in frequency in Japanese patients (22%) versus controls (19%). (iv) Asp-57-encoding DQB1 alleles are associated with reduced susceptibility to type 1 diabetes in Caucasians. The major predisposing haplotypes in Japanese are DR4 and DR9. By DNA sequence analysis, both of these Japanese haplotypes have Asp-57-encoding DQB1 alleles. Oligonucleotide dot blot analysis showed that all, except 1, of the 49 Japanese patients and all of the 31 controls have at least one Asp-57-encoding DQB1 allele. In addition, 40% of the patients were homozygous for Asp-57-encoding DQB1 alleles versus 35% of the controls. The high frequencies of Asp-57-encoding DQB1 alleles in this ethnic group may account for the rarity of type 1 diabetes in Japan.
Human nonmuscle myosin heavy chain mRNA: generation of diversity through alternative polyadenylylation.
Authors: Saez C G , Myers J C , Shows T B , Leinwand L A
Published: May 2006

Myosin is a ubiquitous eukaryotic contractile protein that generates the force responsible for such diverse cellular movements as muscle contraction and cytokinesis. Although there have been numerous studies of sarcomeric myosin heavy chain (MHC) genes, no molecular clones have been reported that encode mammalian nonmuscle MHC. This study presents the molecular genetic characterization of a human nonmuscle MHC that is expressed in fibroblasts, endothelial cells, and macrophages. Human nonmuscle MHC amino acids are weakly homologous (33%) to sarcomeric MHC but are approximately 72% identical to smooth muscle MHC. In contrast to vertebrate sarcomeric MHCs, which generate diversity through the expression of members of a multigene family, an alternative polyadenylylation site is used in the nonmuscle MHC gene to generate multiple transcripts that encode the same protein. We have mapped this gene to chromosome 22. It is thus unlinked to either of the sarcomeric MHC gene clusters on human chromosomes 14 and 17.
Neoplastic transformation of immortalized human epidermal keratinocytes by ionizing radiation.
Authors: Thraves P , Salehi Z , Dritschilo A , Rhim J S
Published: May 2006

Efforts to investigate the progression of events that cause human cells to become neoplastic in response to ionizing radiation have been aided by the development of tissue culture systems of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus type 12 and simian virus 40 have been transformed by exposure to x-ray irradiation. Such transformants showed morphological alterations, formed colonies in soft agar, and induced carcinomas when transplanted into nude mice, whereas primary human epidermal keratinocytes exposed to radiation in this manner failed to show any evidence of transformation. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of a DNA tumor virus and radiation, indicating a multistep process for radiation-induced neoplastic conversion. This in vitro system may be useful as a tool for dissecting the process of radiation-induced neoplastic transformation of human epithelial cells and for detecting previously unreported human oncogenes.
Suppression of meiosis of male germ cells by an antagonist of luteinizing hormone-releasing hormone.
Authors: Szende B , Redding T W , Schally A V
Published: May 2006

Male nude mice were implanted with osmotic minipumps releasing 50 micrograms of a potent antagonist of luteinizing hormone-releasing hormone (LH-RH) per day [N-Ac-[D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-RH] (SB-75) [Nal(2), 3-(2-naphthyl)alanine; Phe(pCl), 4-chlorophenylalanine; Pal(3), 3-(3-pyridyl)alanine; Cit, citrulline], or they were treated with s.c. injections of SB-75 (25 micrograms twice a day). Another group of nude mice received an injection of microcapsules of the agonist [D-Trp6]LH-RH liberating 25 micrograms/day. One month after the initiation of treatment, the testicular weights were significantly reduced and the blood testosterone values were at castration levels in all treated groups. Histologically, only the testicles of the mice treated with SB-75 released from minipumps showed a significant decrease of meiosis. The most advanced forms of germ cells were spermatogonia in 26%, spermatocytes in 17%, and round spermatids in 35% of the seminiferous tubules. Only 22% of the tubules contained elongated spermatids. The suppression of meiotic activity by this modern LH-RH antagonist can possibly be used for the development of methods for male contraception and for the protection of germ cells against the damage caused by cytotoxic drugs and x-radiation.
Two independent retrons with highly diverse reverse transcriptases in Myxococcus xanthus.
Authors: Inouye S , Herzer P J , Inouye M
Published: May 2006

A reverse transcriptase (RT) was recently found in Myxococcus xanthus, a Gram-negative soil bacterium. This RT has been shown to be associated with a chromosomal region designated a retron responsible for the synthesis of a peculiar extrachromosomal DNA called msDNA (multicopy single-stranded DNA). We demonstrate that M. xanthus contains two independent, unlinked retrons, one for the synthesis of msDNA-Mx162 and the other for msDNA-Mx65. The structural analysis of the retron for msDNA-Mx65 revealed that the coding regions for msdRNA (msr) and msDNA (msd), and an open reading frame (ORF) downstream of msr are arranged in the same manner as found for the Mx162 retron. The ORF encodes a polypeptide of 427 amino acid residues. The amino-terminal domain (residues 1-138) shows no striking similarity to these proteins presently available in the data bases including the msDNA-Mx162 ORF, while the sequence from residues 139-394 can be aligned with various known RT sequences and has 47% identity with the RT domain of the msDNA-Mx162 ORF. On the basis of these findings, possible origins of two highly diverse retrons on the M. xanthus chromosome are discussed.
Erythrocyte metallothionein as an index of zinc status in humans.
Authors: Grider A , Bailey L B , Cousins R J
Published: May 2006

Metallothionein concentrations in erythrocyte lysates derived from human subjects were measured by an ELISA procedure. IgG obtained from serum of sheep injected with human metallothionein 1 was used in this competitive assay. Subjects were fed a semipurified zinc-deficient diet (0.7 mg of zinc per kg of diet) for an 8-day depletion period after 3 days of acclimation. Fasting plasma zinc concentrations were reduced approximately 7%. Metallothionein in the erythrocyte lysates was significantly decreased to 59% of the initial level by the end of the depletion period. Supplementation of these depleted subjects with zinc (50 mg) did not increase erythrocyte metallothionein levels within 24 hr. Daily supplementation of control subjects with zinc (50 mg/day) increased erythrocyte metallothionein to a 7-fold maximum within 7 days. These levels were reduced by 61% within 14 days after zinc supplementation was terminated. Incubation of rat [35S]metallothionein with human erythrocyte lysate showed a time-dependent increase in 35S soluble in 20% trichloroacetic acid, indicating degradation of the labeled protein, presumably via protease activity in the lysate. It is proposed that zinc supplementation induces erythrocyte metallothionein during erythropoiesis and that low zinc intake decreases synthesis and/or accelerates degradation of the protein in reticulocytes/erythrocytes. Metallothionein levels in erythrocytes may provide a useful index upon which to assess zinc status in humans.
A dual laminin/collagen receptor acts in peripheral nerve regeneration.
Authors: Toyota B , Carbonetto S , David S
Published: May 2006

A regeneration chamber was created in vivo by suturing a synthetic tube sealed at its distal end onto the proximal stump of a severed rat sciatic nerve. Nerves regenerated into tubes coated with laminin at a rate of 0.33 mm/day after a lag of about 2 days. At 25 days, regenerating nerves had extended 23% farther into laminin-coated tubes as compared with uncoated ones. Monoclonal antibody 3A3, which functionally interferes with a dual laminin/collagen receptor, inhibited nerve regeneration into laminin-coated tubes by 32%. In contrast, monoclonal antibody JG22, which inhibits chicken matrix receptors, had no significant effect on regeneration. Immunohistochemical studies of teased adult rat sciatic nerves indicate that 3A3 bound to Schwann cells and possibly to axons. In other studies, the heterodimeric, laminin/collagen receptor recognized by 3A3 has been shown to be a member of the integrin superfamily of adhesive receptors. These data provide evidence that an integrin receptor functions in nerve regeneration in vivo.
Angiopathic consequences of saturating the plasma scavenger system for actin.
Authors: Haddad J G , Harper K D , Guoth M , Pietra G G , Sanger J W
Published: May 2006

Two plasma proteins, vitamin D-binding protein (actin monomer sequestrant) and gelsolin (actin polymer severing), have been found in association with actin in plasma from ill humans and during experimental injury. In vitro, these are the only plasma proteins that display a high affinity for actin. We infused increasing amounts of globular actin intravenously to rats to evaluate its disposition in plasma and tissues. Intravascular filament formation, microthrombi, and endothelial injury were observed, especially in the pulmonary circulation. These pathological changes were not observed when the globular actin in the infusate had been preincubated with the vitamin D-binding protein in vitro. Complexes of actin with both proteins were found in the plasma, suggesting a saturable, plasma actin-binding system in vivo. Our findings suggest that in vivo saturation of these proteins' actin-binding capacities may serve as a paradigm for pulmonary vascular disorders seen during widespread tissue trauma and cell lysis.
Insertion in the mRNA of a metachromatic leukodystrophy patient with sphingolipid activator protein-1 deficiency.
Authors: Zhang X L , Rafi M A , DeGala G , Wenger D A
Published: May 2006

The lysosomal catabolism of sulfatide requires arylsulfatase A and a specific sphingolipid activator protein, SAP-1. While most patients with metachromatic leukodystrophy have mutations in the gene for arylsulfatase A, some patients have deficient SAP-1, as determined by immunological techniques. We now describe the molecular findings in a patient who died at 22 years of age with SAP-1 deficiency. The DNA polymerase chain reaction was used to amplify regions of cDNA which were subcloned in M13 phage DNA and sequenced by the dideoxy chain-termination method. The patient was found to have a 33-base-pair insertion between nucleotides 777 and 778 (numbered from the A of the ATG initiation codon). No other changes were found in the coding sequence of the cDNA from this patient. At the site of the insertion some normal people have an additional 9 base pairs, which correspond to the last 9 nucleotides at the 3' end of the insertion. The cDNAs from the second-cousin parents were amplified and sequenced, and in both two alleles were identified, one with the 33-base-pair insertion and one with no insertion. Two brothers were found to have only the normal alleles and a sister was found to have the 33-base-pair insertion and a normal allele. The findings confirm studies performed on leukocyte extracts demonstrating normal antigen levels in the two brothers and a lower level in the sister. The presence of 11 additional amino acids in the coding region of mature SAP-1 in this patient causes significant changes in the hydropathy profile compatible with the previous findings at the protein level.
Minimal model for signal-induced Ca2+ oscillations and for their frequency encoding through protein phosphorylation.
Authors: Goldbeter A , Dupont G , Berridge M J
Published: May 2006

In a variety of cells, hormonal or neurotransmitter signals elicit a train of intracellular Ca2+ spikes. The analysis of a minimal model based on Ca2(+)-induced Ca2+ release from intracellular stores shows how sustained oscillations of cytosolic Ca2+ may develop as a result of a rise in inositol 1,4,5-trisphosphate (InsP3) triggered by external stimulation. This rise elicits the release of a certain amount of Ca2+ from an InsP3-sensitive intracellular store. The subsequent rise in cytosolic Ca2+ in turn triggers the release of Ca2+ from a second store insensitive to InsP3. In contrast to the model proposed by Meyer and Stryer [Meyer, T. & Stryer, L. (1988) Proc. Natl. Acad. Sci. USA 85, 5051-5055], the present model, which contains only two variables, predicts the occurrence of periodic Ca2+ spikes in the absence of InsP3 oscillations. Such results indicate that repetitive Ca2+ spikes evoked by external stimuli do not necessarily require the concomitant, periodic variation of InsP3. The model is closely related to that proposed by Kuba and Takeshita [Kuba, K. & Takeshita, S. (1981) J. Theor. Biol. 93, 1009-1031] for Ca2+ oscillations in sympathetic neurones, based on Ca2(+)-induced Ca2+ release. We extend their results by showing the minimal conditions in which the latter process gives rise to periodic behavior and take into account the role of the rise in InsP3 caused by external stimulation. The analysis further shows how signal-induced Ca2+ oscillations might be effectively encoded in terms of their frequency through the phosphorylation of a cellular substrate by a protein kinase activated by cytosolic Ca2+.
A highly immunogenic tumor transfected with a murine transforming growth factor type beta 1 cDNA escapes immune surveillance.
Authors: Torre-Amione G , Beauchamp R D , Koeppen H , Park B H , Schreiber H , Moses H L , Rowley D A
Published: May 2006

A highly immunogenic C3H-derived UV-induced tumor was cotransfected with a murine transforming growth factor type beta 1 (TGF-beta 1) cDNA and a neomycin-resistance gene. Stable clones were isolated and used in vitro and in vivo to determine the effects of endogenously produced TGF-beta on cytolytic T-lymphocyte (CTL) responses. Tumor cells producing TGF-beta, though retaining expression for class I major histocompatibility complex molecules and the tumor-specific antigen, did not stimulate primary CTL responses in vitro and were not effective in vivo for directly stimulating primary CTL or in priming for CTL responses. Furthermore, TGF-beta-producing tumors grew progressively in transiently immunosuppressed mice without losing the tumor antigen; thus, TGF-beta produced by tumors may promote escape from immune surveillance.
myc products induce the expression of catecholaminergic traits in quail neural crest-derived cells.
Authors: Fauquet M , Stehelin D , Saule S
Published: May 2006

The avian myelocytomatosis virus strain MC29 v-myc oncogene transforms a wide panel of avian cells in vitro and either blocks or maintains differentiation, depending on the cell type. In the present work, we have investigated the effect of this oncogene on the differentiation of early embryonic cells, neural crest cells, grown in vitro. We report that the MC29 v-myc gene product induces a strong cellular proliferation of 2-day quail neural crest with the appearance of catecholaminergic traits. Other v-myc as well as the c-myc gene products also trigger this phenotype. Retroviruses carrying some other oncogenes do not elicit this phenotypic expression, although they activate cell multiplication. Thus, our results indicate that myc gene products induce (directly or indirectly) a differentiated phenotype in a subpopulation of neural crest cells.
Reversible and irreversible labeling and autoradiographic localization of the cerebral histamine H2 receptor using [125I]iodinated probes.
Authors: Ruat M , Traiffort E , Bouthenet M L , Schwartz J C , Hirschfeld J , Buschauer A , Schunack W
Published: May 2006

Iodoaminopotentidine (I-APT)--i.e., N-[2-(4-amino-3-iodobenzamido)ethyl]-N'-cyano-N''-(3-[3- (1-piperidinylmethyl)phenoxy]propyl)guanidine--represents one of the most potent H2-receptor antagonists known so far. In membranes of guinea pig brain 125I-APT bound reversibly, selectively, and with high affinity (Kd = 0.3 nM) to a homogeneous population of sites unambiguously identified as H2 receptors by inhibition studies conducted with a large panel of antagonists. 125I-APT binding was also inhibited by histamine, and the effect was modulated by a guanyl nucleotide, which is consistent with the association of the H2 receptor with a guanine nucleotide binding regulatory protein. The low nonspecific binding of 125I-APT generated high contrast autoradiographic pictures in brain sections and established the precise distribution of H2 receptors. Their highly heterogeneous distribution and laminated pattern in some areas--e.g., cerebral and hippocampal cortices--suggest their major association with neuronal elements. These localizations were more consistent than those of H1 receptors with the distribution of histaminergic projections, indicating that H2 receptors mediate a larger number of postsynaptic actions of histamine--e.g., in striatum. Colocalizations of H1 and H2 receptors in some areas account for their known synergistic interactions in cAMP formation induced by histamine. The distribution of 125I-APT binding sites did not strictly parallel that of the H2-receptor-linked adenylate cyclase activity, which may reflect heterogeneity among H2 receptors. After UV irradiation and SDS/PAGE analysis, [125I]iodoazidopotentidine (125I-AZPT), a photoaffinity probe derived from 125I-APT, was covalently incorporated in several peptides, among which the labeling of two peptides of 59 and 32 kDa was prevented by H2 antagonists, suggesting that they correspond to H2-receptor binding peptides or proteolysis products of the latter. These probes should be useful for sensitive radioassays, localization, purification, and molecular studies of the H2 receptor, which were previously impracticable.
Amplified RNA synthesized from limited quantities of heterogeneous cDNA.
Authors: Van Gelder R N , von Zastrow M E , Yool A , Dement W C , Barchas J D , Eberwine J H
Published: May 2006

The heterogeneity of neural gene expression and the spatially limited expression of many low-abundance messenger RNAs in the brain has made cloning and analysis of such messages difficult. To generate amounts of nucleic acids sufficient for use in standard cloning strategies, we have devised a method for producing amplified heterogeneous populations of RNA from limited quantities of cDNA. Whole cerebellar RNA was primed with a synthetic oligonucleotide containing the T7 RNA polymerase promoter sequence 5' to a polythymidylate region. After second-strand cDNA synthesis, T7 RNA polymerase was used to generate amplified antisense RNA (aRNA). Up to 80-fold molar amplification has been achieved from nanogram quantities of cDNA. The amplified material is similar in size distribution to the parent cDNA and shows sequence heterogeneity as assessed by Southern and Northern blot analysis. Specific messages for moderate-abundance mRNAs for actin and guanine nucleotide-binding protein (G-protein) alpha subunits have been detected in the amplified material. By using in situ transcription to generate cDNA, sequences for cyclophilin have been detected in aRNA derived from single cerebellar tissue sections. cDNA derived from a single cerebellar Purkinje cell also has been amplified and yields material that hybridizes to cognate whole RNA and mRNA but not to Escherichia coli RNA.
Substrate sequence effects on "hammerhead" RNA catalytic efficiency.
Authors: Fedor M J , Uhlenbeck O C
Published: May 2006

The "hammerhead" RNA self-cleaving domain can be assembled from two RNA molecules: a large (approximately 34 nucleotide) ribozyme RNA containing most of the catalytically essential nucleotides and a small (approximately 13 nucleotide) substrate RNA containing the cleavage site. Four such hammerheads that contained identical catalytic core sequences but differed in the base composition of the helices that are involved in substrate binding had been reported to vary in cleavage rates by more than 70-fold under similar reaction conditions. Steady-state kinetic analyses reveal that kcat values are nearly the same for these hammerheads but Km values vary nearly 60-fold. The substrates for reactions having high Km values form aggregates that are virtually nonreactive. These observations demonstrate that the secondary structure of substrate RNA can be a major determinant of hammerhead catalytic efficiency.
Therapeutic response to intravenous infusions of glucocerebrosidase in a patient with Gaucher disease.
Authors: Barton N W , Furbish F S , Murray G J , Garfield M , Brady R O
Published: May 2006

Enzyme replacement has been under consideration as a therapeutic strategy for patients with Gaucher disease for more than two decades. Previous studies indicated that single injections of purified glucocerebrosidase reduced the amount of storage material in the liver. It was important to determine whether administration of exogenous enzyme on a regular basis would be of clinical benefit. We report here that weekly i.v. infusions of a macrophage-targeted preparation of human placental glucocerebrosidase in a child with type 1 Gaucher disease increased hemoglobin from 6.9 +/- 0.8 g/dl (+/- 1 SD) to 10.2 +/- 0.4 g/dl (+/- 1 SD) over a 20-week period. The platelet count also increased from a pretreatment value of 30,000 +/- 7000/mm3 (+/- 1 SD) to 54,000 +/- 11,000/mm3 (+/- 1 SD). Phagocytic activity in the spleen decreased during the period of enzyme administration, and there was radiographic evidence of skeletal improvement. These observations document objective clinical responses to enzyme supplementation in a patient with a sphingolipid storage disorder.
Cannabinoid receptor localization in brain.
Authors: Herkenham M , Lynn A B , Little M D , Johnson M R , Melvin L S , de Costa B R , Rice K C
Published: May 2006

[3H]CP 55,940, a radiolabeled synthetic cannabinoid, which is 10-100 times more potent in vivo than delta 9-tetrahydrocannabinol, was used to characterize and localize a specific cannabinoid receptor in brain sections. The potencies of a series of natural and synthetic cannabinoids as competitors of [3H]CP 55,940 binding correlated closely with their relative potencies in several biological assays, suggesting that the receptor characterized in our in vitro assay is the same receptor that mediates behavioral and pharmacological effects of cannabinoids, including human subjective experience. Autoradiography of cannabinoid receptors in brain sections from several mammalian species, including human, reveals a unique and conserved distribution; binding is most dense in outflow nuclei of the basal ganglia--the substantia nigra pars reticulata and globus pallidus--and in the hippocampus and cerebellum. Generally high densities in forebrain and cerebellum implicate roles for cannabinoids in cognition and movement. Sparse densities in lower brainstem areas controlling cardiovascular and respiratory functions may explain why high doses of delta 9-tetrahydrocannabinol are not lethal.
Localization of the MEN1 gene to a small region within chromosome 11q13 by deletion mapping in tumors.
Authors: Byström C , Larsson C , Blomberg C , Sandelin K , Falkmer U , Skogseid B , Oberg K , Werner S , Nordenskjöld M
Published: May 2006

The gene for multiple endocrine neoplasia type 1 (MEN1), an inherited predisposition to neuroendocrine neoplasm of the parathyroid glands, the pancreatic islet parenchyma, and the anterior pituitary gland, was recently mapped to chromosome 11q13 based on genetic linkage in families. We now show that the pathogenesis of MEN1-associated parathyroid lesions involves unmasking of a recessive mutation at the disease locus and that sporadic primary hyperparathyroidism shares the same mechanisms. By examination of allele losses in MEN1-associated lesions, we could define deletions of chromosome 11 and map the MEN1 locus to a small region within chromosome band 11q13, telomeric to the PYGM locus. In contrast, a low incidence of deletions involving the MEN1 gene was found in sporadic pituitary adenomas.
Nonsense mutation in the glycoprotein Ib alpha coding sequence associated with Bernard-Soulier syndrome.
Authors: Ware J , Russell S R , Vicente V , Scharf R E , Tomer A , McMillan R , Ruggeri Z M
Published: May 2006

Three distinct gene products, the alpha and beta chains of glycoprotein (GP) Ib and GP IX, constitute the platelet membrane GP Ib-IX complex, a receptor for von Willebrand factor and thrombin involved in platelet adhesion and aggregation. Defective function of the GP Ib-IX complex is the hallmark of a rare congenital bleeding disorder of still undefined pathogenesis, the Bernard-Soulier syndrome. We have analyzed the molecular basis of this disease in one patient in whom immunoblotting of solubilized platelets demonstrated absence of normal GP Ib alpha but presence of a smaller immunoreactive species. The truncated polypeptide was also present, along with normal protein, in platelets from the patient's mother and two of his four children. Genetic characterization identified a nucleotide transition changing the Trp-343 codon (TGG) to a nonsense codon (TGA). Such a mutation explains the origin of the smaller GP Ib alpha, which by lacking half of the sequence on the carboxyl-terminal side, including the trans-membrane domain, cannot be properly inserted in the platelet membrane. Both normal and mutant codons were found in the patient, suggesting that he is a compound heterozygote with a still unidentified defect in the other GP Ib alpha allele. Nonsense mutation and truncated GP Ib alpha polypeptide were found to cosegregate in four individuals through three generations and were associated with either Bernard-Soulier syndrome or carrier state phenotype. The molecular abnormality demonstrated in this family provides evidence that defective synthesis of GP Ib alpha alters the membrane expression of the GP Ib-IX complex and may be responsible for Bernard-Soulier syndrome.