bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence analysis of the core gene of 14 hepatitis C virus genotypes.
Authors: Bukh J , Purcell R H , Miller R H
Published: May 2006

We previously sequenced the 5' noncoding region of 44 isolates of hepatitis C virus (HCV), as well as the envelope 1 (E1) gene of 51 HCV isolates, and provided evidence for the existence of at least 6 major genetic groups consisting of at least 12 minor genotypes of HCV (i.e., genotypes I/1a, II/1b, III/2a, IV/2b, 2c, V/3a, 4a-4d, 5a, and 6a). We now report the complete nucleotide sequence of the putative core (C) gene of 52 HCV isolates that represent all of these 12 genotypes as well as two additional genotypes provisionally designated 4e and 4f that we identified in this study. The phylogenetic analysis of the C gene sequences was in agreement with that of the E1 gene sequences. A major division in the genetic distance was observed between HCV isolates of genotype 2 and those of the other genotypes in analysis of both the E1 and C genes. The C gene sequences of 9 genotypes have not been reported previously (i.e., genotypes 2c, 4a-4f, 5a, and 6a). Our analysis indicates that the C gene-based methods currently used to determine the HCV genotype, such as PCR with genotype-specific primers, should be revised in light of these data. We found that the predicted C gene was exactly 573 nt long in all 52 HCV isolates, with an N-terminal start codon and no in-frame stop codons. The nucleotide and predicted amino acid identities of the C gene sequences were in the range of 79.4-99.0% and 85.3-100%, respectively. Furthermore, we mapped universally conserved, as well as genotype-specific, nucleotide and deduced amino acid sequences of the C gene. The predicted C proteins of the different HCV genotypes shared the following features: (i) high content of proline residues, (ii) high content of arginine and lysine residues located primarily in three domains with 10 such residues invariant at positions 39-62, (iii) a cluster of 5 conserved tryptophan residues, (iv) two nuclear localization signals and a DNA-binding motif, (v) a potential phosphorylation site with a serine-proline motif, and (vi) three conserved hydrophilic domains that have been shown by others to contain immunogenic epitopes. Thus, we have extended analysis of the predicted C protein of HCV to all of the recognized genotypes, confirmed the existence of highly conserved regions of this important structural protein, and demonstrated that the genetic relatedness of HCV isolates is equivalent when analyzing the most conserved (i.e., C) and the most variable (i.e., E1) genes of the HCV genome.
Cytoplasmic transfer of the mtDNA nt 8993 T-->G (ATP6) point mutation associated with Leigh syndrome into mtDNA-less cells demonstrates cosegregation with a decrease in state III respiration and ADP/O ratio.
Authors: Trounce I , Neill S , Wallace D C
Published: May 2006

A point mutation in the mtDNA-encoded ATP6 gene (T-->G at nt 8993) associated with Leigh syndrome in two pedigrees was found to decrease ADP-stimulated (state III) respiration and the ratio of ADP molecules phosphorylated to oxygen atoms reduced (ADP/O ratio) but did not affect 2,4-dinitrophenol (DNP)-uncoupled respiration, suggesting a defective mitochondrial H(+)-translocating ATP synthase. Intact mitochondria isolated from patient and control lymphoblastoid cell lines were tested for state III, ADP-limited (state IV), and DNP-uncoupled respiration with various substrates. Mitochondria isolated from patient lymphoblasts harboring 95-100% of mtDNAs carrying the nt 8993 T-->G mutation showed state III respiration rates 26-50% lower than controls while having normal DNP-uncoupled rates. This resulted in state III/DNP ratios of 0.52-0.70 in patient mitochondria versus 0.88-0.97 in controls. The ADP/O ratio was also decreased 30-40% in patient mitochondria. Patient lymphoblasts heteroplasmic for the nt 8993 mutation were enucleated by using Percoll gradients and the cytoplasts were fused to mtDNA-deficient (rho 0) cells by electric shock. Cybrid clones homoplasmic for the wild-type nucleotide (T) at nt 8993 gave state III/DNP and ADP/O ratios similar to those of control cybrids, whereas cybrid clones homoplasmic for the mutant nucleotide (G) showed a 24-53% reduction in state III respiration, a state III/DNP ratio of 0.53-0.64, and a 30% decrease in the ADP/O ratio. Thus, the reduced state III respiration rates and ADP/O ratios are linked to the T-->G mutation at nt 8993.
Circular dichroism, molecular modeling, and serology indicate that the structural basis of antigenic variation in foot-and-mouth disease virus is alpha-helix formation.
Authors: France L L , Piatti P G , Newman J F , Toth I , Gibbons W A , Brown F
Published: May 2006

Seven antigenic variants obtained from a single field isolate of foot-and-mouth disease virus, serotype A12, differ only at residues 148 and 153 in the immunodominant loop of viral protein VP1. Synthetic peptides corresponding to the region 141-160 are highly immunogenic. UV circular dichroism shows that (i) in aqueous solution the peptides are nearly identical, but in 100% trifluoroethanol they display helix-forming properties which correlate well with their serological crossreactivities for anti-peptide sera, and (ii) these properties are insensitive to substitutions at position 153, except for proline, but are highly sensitive to substitutions at position 148. This pattern can be explained by the effects of these substitutions on the amphiphilic character and positions of helices postulated in the region 146-156. Molecular models indicate that residues 147, 148, 150, 151, 153-155, and 157 are most likely to interact with residues of the antibody paratopes. The data are consistent with the existence of an inverse gamma-turn around Pro-153, and a beta-turn at the cell-attachment site at residues 145-147.
Mitochondrial DNA sequence variation in human evolution and disease.
Authors: Wallace D C
Published: May 2006

Germ-line and somatic mtDNA mutations are hypothesized to act together to shape our history and our health. Germ-line mtDNA mutations, both ancient and recent, have been associated with a variety of degenerative diseases. Mildly to moderately deleterious germ-line mutations, like neutral polymorphisms, have become established in the distant past through genetic drift but now may predispose certain individuals to late-onset degenerative diseases. As an example, a homoplasmic, Caucasian, tRNA(Gln) mutation at nucleotide pair (np) 4336 has been observed in 5% of Alzheimer disease and Parkinson disease patients and may contribute to the multifactorial etiology of these diseases. Moderately to severely deleterious germ-line mutations, on the other hand, appear repeatedly but are eliminated by selection. Hence, all extant mutations of this class are recent and associated with more devastating diseases of young adults and children. Representative of these mutations is a heteroplasmic mutation in MTND6 at np 14459 whose clinical presentations range from adult-onset blindness to pediatric dystonia and basal ganglial degeneration. To the inherited mutations are added somatic mtDNA mutations which accumulate in random arrays within stable tissues. These mutations provide a molecular clock that measures our age and may cause a progressive decline in tissue energy output that could precipitate the onset of degenerative diseases in individuals harboring inherited deleterious mutations.
Gene trees and hominoid phylogeny.
Authors: Ruvolo M , Pan D , Zehr S , Goldberg T , Disotell T R , von Dornum M
Published: May 2006

Here we present a DNA sequence study that incorporates intraspecific variation from all five genera of hominoids (apes and humans). Recently it has been claimed that using single individuals to analyze species' relationships might be misleading if within-species variation is great. Our results indicate that despite high intraspecific variation in mitochondrial cytochrome oxidase subunit II gene sequences of some hominoids, humans and chimpanzees are nonetheless significantly most closely related. We also report the observation that variation within the gorilla species exceeds that between common and pygmy chimpanzee species, a finding with implications for conservation. In contrast, humans are less mitochondrially diverse than lowland gorillas inhabiting western Africa.
Subcellular localization of the UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-mediated O-glycosylation reaction in the submaxillary gland.
Authors: Roth J , Wang Y , Eckhardt A E , Hill R L
Published: May 2006

Addition of N-acetylgalactosamine to threonine and serine is the first step in the synthesis of O-glycosidically linked oligosaccharides. A UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (EC 2.4.1.41) from porcine submaxillary glands was recently purified to electrophoretic homogeneity, and polyclonal antibodies against the purified transferase were raised. Immunoblots of porcine, bovine, and ovine submaxillary gland extracts with the anti-transferase antibodies gave a single band and the antibodies reacted equally well with the purified glycosylated and N-glycanase-treated transferase. Immunoelectron microscopic localization of the transferase was achieved in Lowicryl K4M thin sections and frozen-thawed thin sections of porcine and bovine submaxillary gland by using the protein A-gold technique. Specific gold particle labeling was observed in the cis Golgi apparatus and smooth-membraned vesicular structures in close topological relation with it. Labeling was undetectable in the rough endoplasmic reticulum, its transitional elements, and smooth-membraned structures close to them, the trans Golgi apparatus, mucin droplets, and the plasma membrane. The onset of labeling for peptide-bound GalNAc as detected with Vicia villosa isolectin G4 mirrored the transferase immunolocalization as directly shown by double labeling and extended into the trans Golgi apparatus and mucous droplets. Apomucin immunolabeling was found throughout the endoplasmic reticulum and the intermediate compartment and partially overlapped the region of transferase labeling in the Golgi apparatus as demonstrated by double immunolabeling. Thus, the initial step of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-mediated O-glycosylation in porcine and bovine submaxillary gland cells occurs in the cis Golgi apparatus. The possible involvement of the intermediate compartment remains to be clarified.
Computation with chaos: a paradigm for cortical activity.
Authors: Babloyantz A , Lourenço C
Published: May 2006

A device comprising two interconnected networks of oscillators exhibiting spatiotemporal chaos is considered. An external cue stabilizes input specific unstable periodic orbits of the first network, thus creating an "attentive" state. Only in this state is the device able to perform pattern discrimination and motion detection. We discuss the relevance of the procedure to the information processing of the brain.
Evidence that the vectorial competence of phlebotomine sand flies for different species of Leishmania is controlled by structural polymorphisms in the surface lipophosphoglycan.
Authors: Pimenta P F , Saraiva E M , Rowton E , Modi G B , Garraway L A , Beverley S M , Turco S J , Sacks D L
Published: May 2006

Phlebotomine vectors can in some instances transmit only certain species of Leishmania. Comparison of a large number of vector/parasite pairs revealed that species-specific differences in vectorial competence were in every case directly correlated with the ability of promastigotes to attach to the sand-fly midgut, the variable outcomes of which were controlled by structural polymorphisms in the surface lipophosphoglycan (LPG) of the parasite. The ability of Phlebotomus papatasi to transmit only Leishmania major could be attributed to the unique, highly substituted nature of L. major LPG that provides for multiple terminally exposed beta-linked galactose residues for binding. While the relatively unsubstituted LPGs of other Leishmania species were unable to mediate promastigote attachment to P. papatasi, they could mediate binding to midguts of Phlebotomus argentipes, which was found to be a potentially competent vector for every Leishmania species examined. The data suggest that at least some phlebotomine vectors differ with respect to the parasite recognition sites which they express and that midgut adhesion is a sufficiently critical component of vectorial competence as to provide the evolutionary drive for LPG structural polymorphisms.
Staphylococcal nuclease folding intermediate characterized by hydrogen exchange and NMR spectroscopy.
Authors: Jacobs M D , Fox R O
Published: May 2006

Pulsed hydrogen-deuterium exchange during refolding was used to probe the protection of backbone amide hydrogens from solvent exchange of the staphylococcal nuclease Pro117-->Gly variant. The extent of exchange for 39 residues was determined by two-dimensional proton NMR after refolding for 5 ms to 10 s. Three kinetic phases are inferred. Modest protection of amides in the early refolding intermediate composed of two beta-sheets formed by local sequence interactions was observed after a 5-ms refolding period. Protection factors were determined by varying the high pH labeling pulse after refolding for 100 ms. The intermediate state has modest, yet significant, protection for residues in the beta-sheets (protection factors of 10-60) and almost no protection in the alpha-helices (protection factors of < 10). The pattern of labeling is consistent with a role for beta-turns and beta-hairpins in the formation of the early intermediate.
Cloning and in situ localization of a brain-derived porin that constitutes a large-conductance anion channel in astrocytic plasma membranes.
Authors: Dermietzel R , Hwang T K , Buettner R , Hofer A , Dotzler E , Kremer M , Deutzmann R , Thinnes F P , Fishman G I , Spray D C
Published: May 2006

We have cloned a protein from bovine brain, brain-derived voltage-dependent anion channel 1 (BR1-VDAC), that is identical to a recently sequenced plasmalemmal-bound porin from human lymphocytes. mRNA hybridization indicates that BR1-VDAC is widely distributed throughout nervous and nonnervous tissues. In situ localization substantiated that the BR1-VDAC is associated with the plasmalemma of astrocytes. A monoclonal antibody that recognizes the N terminus of the BR1-VDAC protein completely blocks an astrocytic high-conductance anion channel that has electrophysiological similarities with the mitochondrial VDAC. Since the high-conductance anion channel in astrocytes has been shown to respond to hypoosmotic solutions, its molecular identification provides the basis for a better understanding of volume regulation in brain tissue.
Reconstitution of functional human single-stranded DNA-binding protein from individual subunits expressed by recombinant baculoviruses.
Authors: Stigger E , Dean F B , Hurwitz J , Lee S H
Published: May 2006

Human single-stranded DNA-binding protein (HSSB), also known as replication protein A, is composed of a 70-kDa single-stranded DNA-binding subunit (p70) and 34-kDa and 11-kDa (p34 and p11, respectively) subunits of unknown functions. We have examined interactions among the HSSB subunits in vivo by coinfecting insect cells with different combinations of recombinant baculoviruses encoding p70, p34, or p11. In vivo, coexpressed p34 and p11 subunits formed stable complexes, whereas neither p34 nor p11 formed stable complexes with p70. In cells coinfected with viruses expressing all three subunits, the stable heterotrimer formed, which, when purified, replaced HSSB isolated from HeLa cells in various assays, including simian virus 40 DNA replication in vitro. These data suggest that, in the assembly of functionally active HSSB, formation of the p34-p11 complex precedes p70 addition to the complex.
Development of transforming function during transduction of proto-ras into Harvey sarcoma virus.
Authors: Lang M , Treinies I , Duesberg P H , Kurth R , Cichutek K
Published: May 2006

Oncogenic retroviruses are generated by transduction of the coding region of a protooncogene and acquire genetic changes during subsequent replication. Critical genetic events which occurred during and after transduction of rat proto-ras-1Ha into Harvey sarcoma virus were identified by evaluating the transforming activity of plausible synthetic progenitor proviruses encompassing the complete proto-ras genomic region with or without various 5' deletions. All progenitor proviruses induced phenotypic transformation of mouse NIH 3T3 cells, although with a 5- to 10-fold lower frequency than Harvey sarcoma provirus. Although no tumor formation was observed in vivo after inoculation in the absence of helper murine retrovirus, both wild-type and progenitor viruses inoculated in the presence of helper virus induced tumors in newborn BALB/c mice. No critical alterations of the p21ras coding region and no deletion of 5' genomic elements were detected in a progenitor virus encompassing the complete proto-ras genomic region that had been isolated from tumors. However, one progenitor virus that included all proto-ras exons induced tumors with a decreased latency. This virus contained a mutation in codon 12 (glycine to valine), which had apparently been selected during tumorigenesis in vivo. During the genesis of Harvey sarcoma virus, critical steps conferring transforming function are therefore transduction of coding proto-ras exons and enhancement of their transforming function by specific amino acid changes in p21ras.
Monoclonal antibodies to nerve growth factor affect the postnatal development of the visual system.
Authors: Berardi N , Cellerino A , Domenici L , Fagiolini M , Pizzorusso T , Cattaneo A , Maffei L
Published: May 2006

Exogenous supply of nerve growth factor (NGF) prevents the effects of monocular deprivation. This suggests that visual afferents may be competing for an endogenous neurotrophic factor, related to NGF, whose production by postsynaptic cells depends on the activity of afferent fibers. To test the hypothesis that endogenous NGF may play a role in the functional and anatomical development of the rat geniculo cortical system, the physiological action of NGF in the rat visual system was antagonized by using two independent monoclonal antibodies which neutralize NGF (alpha D11 and 4C8). To provide a continuous supply of antibodies during the period of visual cortical plasticity, alpha D11 or 4C8 antibody-producing hybridoma cells were implanted in the lateral ventricle of rats at postnatal day 15. This resulted in dramatic alterations of two of the most important parameters characterizing the functional development of the visual system, namely, visual acuity and binocularity of cortical neurons and in shrinkage of cells in the lateral geniculate nucleus. This demonstrates that the action of endogenous NGF is necessary for the normal functional and anatomical development of the geniculocortical system.
Foot-and-mouth disease virus particles contain replicase protein 3D.
Authors: Newman J F , Piatti P G , Gorman B M , Burrage T G , Ryan M D , Flint M , Brown F
Published: May 2006

An antibody against the Escherichia coli-expressed RNA polymerase of foot-and-mouth disease virus (FMDV) reacts with the virus in ELISA and radioimmunoprecipitation experiments and with a protein of the disrupted virus particle in an immunoblot analysis. Treatment of the virus with trypsin, which cleaves capsid protein VP1 and a 56-kDa polypeptide present in trace amount in the particles, reduces the level of the reaction in ELISA and radioimmunoprecipitation and eliminates the immunoblot reaction. Electron microscopy showed that only approximately 20% of the virus particles reacted with the anti-polymerase antibody, whereas most reacted with an antibody against the immunodominant G-H loop of the virus. In the presence of ammonium ions, the expressed polymerase degrades the RNA of the virus into molecules sedimenting at approximately 12 S, indicating that it can act as a hydrolytic as well as a polymerizing enzyme. Moreover, the RNA in trypsin-treated virus particles is degraded when incubated at 37 degrees C, suggesting that the cleaved 56-kDa protein still possesses hydrolytic activity. In addition, the anti-polymerase antibody, which inhibits the polymerase activity of the E. coli-expressed protein, also partially inhibits the hydrolytic activity of the previously described endonuclease of the virus particle, suggesting that this enzyme is identical with the polymerase or forms part of it.
Translation elongation factor Tu cleaved by a phage-exclusion system.
Authors: Yu Y T , Snyder L
Published: May 2006

Bacteriophage T4 multiples poorly in Escherichia coli strains carrying the defective prophage, e14; the e14 prophage contains the lit gene for late inhibitor of T4 in E. coli. The exclusion is caused by the interaction of the e14-encoded protein, Lit, with a short RNA or polypeptide sequence encoded by gol from within the major head protein gene of T4. The interaction between Lit and the gol product causes a severe inhibition of all translation and prevents the transcription of genes downstream of the gol site in the same transcription unit. However, it does not inhibit most transcription, nor does it inhibit replication or affect intracellular levels of ATP. Here we show that the interaction of gol with Lit causes the cleavage of translation elongation factor Tu (EF-Tu) in a region highly conserved from bacteria to humans. The depletion of EF-Tu is at least partly responsible for the inhibition of translation and the phage exclusion. The only other phage-exclusion system to be understood in any detail also attacks a highly conserved cellular component, suggesting that phage-exclusion systems may yield important reagents for studying cellular processes.
Major species differences between humans and rodents in the susceptibility to pancreatic beta-cell injury.
Authors: Eizirik D L , Pipeleers D G , Ling Z , Welsh N , Hellerström C , Andersson A
Published: May 2006

The ability of beta cells to endure assaults may be relevant in the development of insulin-dependent diabetes mellitus. This study examines the susceptibility of human pancreatic islets to agents that are cytotoxic for rodent beta cells--i.e., sodium nitroprusside (NP, a nitric oxide donor), streptozotocin (SZ), or alloxan. After 5-8 days in tissue culture, human or rodent islets were exposed for 14 h to NP (50-200 microM) or for 30 min to SZ or alloxan (1-3 mM). Glucose oxidation by human islets was not reduced by NP, but there was a dose-dependent inhibition in rat (40-90% inhibition; P < 0.001) and mouse (10-60% inhibition; P < 0.05) islet glucose oxidation. Glucose (16.7 mM)-induced insulin release by human islets was not impaired after a 30-min exposure to SZ or alloxan, at concentrations that inhibited insulin release from rat (30-80% inhibition; P < 0.001) or mouse (10-70% inhibition; P < 0.05) islets. The viability of human beta cells purified by flow cytometry was not affected by SZ or alloxan (5 mM), as judged 1 or 4 days after a 10-min exposure and subsequent culture; these conditions were cytotoxic for rat beta cells, with 65-95% (P < 0.01) dead beta cells after 4 days. Human islets transplanted under the kidney capsule of nude mice were not affected by in vivo alloxan exposure, as suggested by preserved graft morphology and insulin content, whereas the endogenous beta cells of the transplanted mice were severely damage (80% decrease in pancreatic insulin content and morphological signs of beta-cell destruction). Thus human beta cells are resistant to NP, SZ, or alloxan at concentrations that decrease survival and function of rat or mouse beta cells. These marked interspecies differences emphasize the relevance of repair and/or defense mechanisms in beta-cell destruction and raise the possibility that such differences may also be present among individuals of the same species.
Colony stimulating factor 1 is required for mammary gland development during pregnancy.
Authors: Pollard J W , Hennighausen L
Published: May 2006

The study of colony stimulating factor 1 (CSF-1), a homodimeric serum growth factor that regulates mononuclear phagocytes and is involved in maternal-fetal interactions during pregnancy, was dramatically enhanced by the observation that the recessive mutation osteopetrosis, op, is an inactivating mutation in the CSF-1-encoding gene. Homozygous mutant (op/op) mice completely lack CSF-1, are osteopetrotic consequent to a deficiency in osteoclasts, have severely reduced numbers of macrophages, and have reduced fertility evident at the pre- and postimplantation stages of pregnancy. We show here that op/op females have a lactational defect, and consequently, although some are able to produce offspring, few nurture any pups and none feeds a full litter. This lactational defect is due to incomplete mammary gland ductal growth during pregnancy, a precocious development of the lobulo-alveolar system, and despite expression of milk proteins, a failure to switch to a lactational state. These data show that CSF-1 has a role in the development of the mammary gland during pregnancy.
Conditional lethality of a yeast strain expressing human RHOA in place of RHO1.
Authors: Qadota H , Anraku Y , Botstein D , Ohya Y
Published: May 2006

The yeast RHO1 GTPase, which has 72% amino acid sequence identity with its human counterpart, RHOA, is essential for growth, although the reason has not been investigated. We report here that yeast strains that rely solely on expression of human RHOA in place of RHO1 are able to grow at 23 degrees C but grow neither at 37 degrees C nor in the presence of 300 mM CaCl2 even at 23 degrees C. Measurements of steady-state protein levels indicate that inability to grow at the restrictive temperature is not due to instability of the protein. Homolog scanning with the two GTPases identified a small, 27-residue region of RHO1 which, when substituted into RHOA, confers full function in yeast. This region corresponds to the alpha 3-helix loop 7 region of RAS; the same region was reported to determine specificity of function between GTPases of the RAB family, Sec4p and Ypt1p. By examining the phenotype of RHOA substitution strains at nonpermissive temperature, we found evidence suggesting that the normal function of RHO1 is to maintain osmotic integrity.
Flavin reductase: sequence of cDNA from bovine liver and tissue distribution.
Authors: Quandt K S , Hultquist D E
Published: May 2006

Flavin reductase catalyzes electron transfer from reduced pyridine nucleotides to methylene blue or riboflavin, and this catalysis is the basis of the therapeutic use of methylene blue or riboflavin in the treatment of methemoglobinemia. A cDNA for a mammalian flavin reductase has been isolated and sequenced. Degenerate oligonucleotides, with sequences based on amino acid sequences of peptides derived from bovine erythrocyte flavin reductase, were used as primers in PCR to selectively amplify a partial cDNA that encodes the bovine reductase. The template used in the PCR was first strand cDNA synthesized from bovine liver total RNA using oligo(dT) primers. A PCR product was used as a specific probe to screen a bovine liver cDNA library. The sequence determined from two overlapping clones contains an open reading frame of 621 nucleotides and encodes 206 amino acids. The amino acid sequence deduced from the bovine liver flavin reductase cDNA matches the amino acid sequences determined for erythrocyte reductase-derived peptides, and the predicted molecular mass of 22,001 Da for the liver reductase agrees well with the molecular mass of 21,994 Da determined for the erythrocyte reductase by electrospray mass spectrometry. The amino acid sequence at the N terminus of the reductase has homology to sequences of pyridine nucleotide-dependent enzymes, and the predicted secondary structure, beta alpha beta, resembles the common nucleotide-binding structural motif. RNA blot analysis indicates a single 1-kilobase reductase transcript in human heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle.
Purification and sequence of rat oxyntomodulin.
Authors: Collie N L , Walsh J H , Wong H C , Shively J E , Davis M T , Lee T D , Reeve J R
Published: May 2006

Structural information about rat enteroglucagon, intestinal peptides containing the pancreatic glucagon sequence, has been based previously on cDNA, immunologic, and chromatographic data. Our interests in testing the physiological actions of synthetic enteroglucagon peptides in rats required that we identify precisely the forms present in vivo. From knowledge of the proglucagon gene sequence, we synthesized an enteroglucagon C-terminal octapeptide common to both proposed enteroglucagon forms, glicentin and oxyntomodulin, but sharing no sequence overlap with glucagon. We then developed a radioimmunoassay using antibodies raised against the octapeptide that was specific for enteroglucagon peptides without cross-reacting with glucagon. Rat intestine was extracted, and one presumptive enteroglucagon form was purified by following the enteroglucagon C-terminal octapeptide-like immunoreactivity through several HPLC purification steps. Structural characterization of the material by amino acid composition, microsequence, and mass spectral analyses identified the peptide as rat oxyntomodulin. The 37-residue peptide consists of pancreatic glucagon plus the C-terminal extension, Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala. This now permits synthesis of an unambiguous duplicate of endogenous rat oxyntomodulin for physiological studies.
Cloning, expression, and in situ localization of rat intestinal cGMP-dependent protein kinase II.
Authors: Jarchau T , Häusler C , Markert T , Pöhler D , Vanderkerckhove J , De Jonge H R , Lohmann S M , Walter U
Published: May 2006

The cDNA for a membrane-associated cGMP-dependent protein kinase (cGK II) was cloned from rat intestine using reverse transcriptase PCR and oligonucleotide primers encoding two conserved motifs of known cGMP-dependent protein kinases and subsequently by screening a rat intestine cDNA library. A full-length clone encodes a protein of 761 amino acids with an estimated size of 87 kDa. Sequences of eight peptides from purified pig intestinal mucosa cGK II were found in the derived amino acid sequence of this clone, identifying it as rat intestinal cGK II. Phylogenetic analysis showed that rat intestinal cGK II is less related to mammalian cGK I than to the Drosophila DG1 gene product and most closely related to a recently cloned mouse brain CGKII isoform. Like several other cGK sequences, that of cGK II contained a leucine/isoleucine heptad repeat motif that has been implicated in dimer formation in cGK I. Expression of cGK II cDNA in HEK 293 cells followed by subcellular fractionation revealed cGK II localization in the cell particulate fraction, consistent with the membrane association of endogenous rat cGK II. On Northern blots, the major cGK II poly(A) RNA form was 4.8 kb, with minor forms of 6.2 and 3.1 kb. The cGK II RNA was highly expressed in rat intestinal mucosa and was 20 times less abundant in rat brain and kidney. The localization of endogenous cGK II RNA in rat small intestine was shown by in situ hybridization to be in villous epithelial cells and to some extent in crypt cells.
Defective human interleukin 2 receptor gamma chain in an atypical X chromosome-linked severe combined immunodeficiency with peripheral T cells.
Authors: DiSanto J P , Rieux-Laucat F , Dautry-Varsat A , Fischer A , de Saint Basile G
Published: May 2006

X chromosome-linked severe combined immunodeficiency disease (SCIDX1) is characterized by the absence of T-cell and natural killer cell development and results from molecular mutations of the interleukin 2 receptor (IL-2R) gamma chain. The IL-2R gamma chain is a common component of the IL-2, IL-4, and IL-7 receptor systems, which may explain the severe immunophenotype in SCIDX1. We have previously described an atypical SCIDX1 syndrome demonstrating poorly functioning peripheral T cells, which we hypothesized to represent a variant allele at the SCIDX1 locus. We now demonstrate that a splice site mutation in the IL-2R gamma gene is responsible for this atypical SCIDX1. Aberrant RNA splicing resulted in the generation of two IL-2R gamma transcripts: an abundant, nonfunctional isoform containing a small intronic insertion and a second functional isoform with a single amino acid substitution present in limited amounts. Radiolabeled IL-2 binding studies revealed a 5-fold decreased level of expression of functional high-affinity IL-2Rs, which correlated with the quantity of full-length IL-2R gamma transcripts. Further analysis of the T-cell antigen receptor beta-chain repertoire of the patient's T cells demonstrated oligoclonality in multiple V beta families, thus strongly suggesting that the defect in the IL-2R gamma chain generated a limited number of peripheral T-cell clones. This atypical SCIDX1 patient demonstrates that certain IL-2R gamma chain abnormalities can also result in partial immunodeficiency phenotypes, potentially through differential effects on the IL-2, IL-4, or IL-7 receptor systems.
Transcriptional activity of transcription factor IIE is dependent on zinc binding.
Authors: Maxon M E , Tjian R
Published: May 2006

The functions of individual basal transcription factors during the formation of an initiation complex by RNA polymerase II remain largely unknown. Transcription factor IIE (TFIIE) has recently been shown to bind to multiple targets in the initiation complex. To assess the role of zinc binding in basal transcription, we have mutated the predicted zinc-finger domain of human TFIIE. Atomic absorption spectroscopy using purified recombinant proteins revealed that the large subunit, TFIIE-56, is indeed a zinc-binding protein. Mutation of a cysteine residue in the putative zinc-finger domain abolished zinc binding. Moreover, mutant TFIIE-56 failed to support reconstituted basal transcription in vitro, suggesting that zinc binding is required for TFIIE function. However, gel-filtration experiments and protein affinity experiments suggest that mutant TFIIE-56 forms a stable heterotetramer with the small subunit, TFIIE-34, that is similar to wild type. Interestingly, gel mobility shift experiments reveal that loss of transcriptional activity by mutant TFIIE is correlated with its inability to stably assemble into the transcription complex. These findings establish that zinc binding by TFIIE may help form a specific structure that is required for stable entry into the transcription complex.
Molecular cloning reveals isoforms of bovine alpha 1-antichymotrypsin.
Authors: Hwang S R , Kohn A B , Hook V Y
Published: May 2006

Comparison of bovine alpha 1-antichymotrypsin (ACT) protease inhibitor with that in human was achieved by cloning a nearly full-length bovine ACT cDNA of 1.5 kb, obtained by screening a bovine liver cDNA library with the human liver ACT cDNA. The deduced primary sequence indicated that the 1456-bp bovine ACT cDNA encodes a protein of 416 amino acids that contains the predicted full-length ACT with a 26-residue NH2-terminal signal sequence. Overall, the primary sequence of bovine ACT possesses a high degree of homology (55%) with human ACT; both bovine and human ACTs share common sequences in the reactive-site domains. Importantly, the reactive site of bovine ACT possesses serine as the predicted P1 position (residue at the NH2-terminal side of the cleaved peptide bond) of the reactive site, whereas human ACT contains leucine in the P1 position. Interestingly, further evidence for heterogeneity in P1 residues was provided by a second partial 0.9-kb bovine liver ACT cDNA clone (pHHK11) that contains isoleucine as P1 residue and shares only partial homology (68%) with the deduced primary sequence of the full-length bovine liver ACT cDNA clone (pHHK12). These findings suggest that isoforms of ACT in bovine liver vary in reactive-site P1 residues; the P1 position of the reactive site is often involved in protease inhibitor specificity. Consistent with the hypothesis of ACT isoforms was the demonstration of multiple copies of the bovine ACT gene by genomic blots.
Ribozyme-mediated suppression of Moloney murine leukemia virus and human immunodeficiency virus type I replication in permissive cell lines.
Authors: Sun L Q , Warrilow D , Wang L , Witherington C , Macpherson J , Symonds G
Published: May 2006

Several hammerhead ribozymes targeted to different sites within the retroviral packaging (psi) sequences of the Moloney murine leukemia virus (Mo-MLV) and the human immunodeficiency virus type 1 (HIV-1) were designed and shown to cleave target RNA in vitro at the chosen sites. The engineered ribozymes, as well as antisense sequence complementary to the Mo-MLV psi packaging region, were cloned into the 3' untranslated region of the neomycin-resistance gene (neo). This was coupled to the simian virus 40 early promoter within the pSV2neo vector. For the ribozymes against the Mo-MLV psi site, the constructs were transfected into Mo-MLV-infected and virus-producing mouse NIH 3T3 cells. With the exception of one of the single ribozymes (the one least effective in cutting target RNA in vitro), all of the constructs effectively (70-80%) suppressed retrovirus production. These results demonstrate a direct correlation between in vitro cleavage and in vivo ribozyme-mediated virus suppression. In addition, a ribozyme targeted to the HIV-1 psi packaging site was engineered into the same vector and transfected into the human T-cell line SupT1. The transfectants were cloned and then challenged with HIV-1. When compared to vector-transfected control cells, a significant reduction in HIV-1 production was observed as measured by p24 and syncytia formation assays. This study demonstrates a feasible approach to the suppression of retrovirus replication by targeting the psi packaging site with hammerhead ribozymes.
Identification of a mammary transforming gene (MAT1) associated with mouse mammary carcinogenesis.
Authors: Bera T K , Guzman R C , Miyamoto S , Panda D K , Sasaki M , Hanyu K , Enami J , Nandi S
Published: May 2006

We have developed an efficient in vitro transformation system using N-methyl-N-nitrosourea that allows us to study the role of hormones and growth factors in mouse mammary tumorigenesis. Utilizing this system, we reported earlier that mammary tumors induced in vitro with N-methyl-N-nitrosourea in the presence of mammogenic hormones (progesterone and prolactin) contain predominately an activated c-Ki-ras protooncogene with a G35 --> A35 transitional mutation in the 12th codon. Mammary tumors induced in the presence of another mitogen, lithium (Li), do not have a mutation in the c-Ki-ras protooncogene. By using an expression cloning system, a plasmid clone containing a 1.75-kb cDNA insert has been isolated from this group of tumors. Nucleic acid sequence analysis of the insert reveals that it has a short open reading frame of 61 amino acids and that it does not have sequence homology with any known gene. The gene, designated MAT1, can neoplastically transform NIH 3T3 cells and also the mammary epithelial cell line TM3. Expression of this gene occurs in normal mouse tissues including mammary gland and is overexpressed in the original mammary tumors as indicated by Northern blot analysis. In vitro transcription and translation of the clone shows a protein product of 6000 Da, which agrees with the predicted open reading frame.
Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein.
Authors: Sedegah M , Hedstrom R , Hobart P , Hoffman S L
Published: May 2006

Immunization with irradiated sporozoites protects animals and humans against malaria, and the circumsporozoite protein is a target of this protective immunity. We now report that adjuvant-free intramuscular injection of mice with plasmid DNA encoding the Plasmodium yoelii circumsporozoite protein induced higher levels of antibodies and cytotoxic T lymphocytes against the P. yoelii circumsporozoite protein than did immunization with irradiated sporozoites. Mice immunized with this vaccine had an 86% reduction in liver-stage parasite burden after challenge with 5 x 10(5) sporozoites (> 10(5) median infectious doses). Eighteen (68%) of 28 mice that received two or three doses of vaccine were protected against challenge with 10(2) sporozoites, and the protection was dependent on CD8+ T cells. These studies demonstrate the utility of plasmid DNA immunization against a nonviral infection. By obviating the requirement for peptide synthesis, expression and purification of recombinant proteins, and adjuvants, this method of immunization provides an important alternative for rapid identification of protective B- and T-cell epitopes and for construction of vaccines to prevent malaria and other infectious diseases.
Antisense oligonucleotides adsorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice.
Authors: Schwab G , Chavany C , Duroux I , Goubin G , Lebeau J , Hélène C , Saison-Behmoaras T
Published: May 2006

Ras oncogenes owe their transforming properties to single point mutations in the sequence coding for the active site of the p21 protein. These mutations lead to changes in cellular proliferation and induce tumorigenic properties. Point mutations represent a well-defined target for antisense oligonucleotides that can specifically suppress the translation of the targeted mutant mRNA. We show that the stability and cellular disponibility of antisense oligonucleotides can be markedly improved by adsorption to polyalkylcyanoacrylate nanoparticles. Nanoparticle-adsorbed antisense oligonucleotides directed to a point mutation (G-->U) in codon 12 of the Ha-ras mRNA selectively inhibited the proliferation of cells expressing the point-mutated Ha-ras gene at a concentration 100 times lower than free oligonucleotides. In addition they markedly inhibited Ha-ras-dependent tumor growth in nude mice after subcutaneous injection. These experiments show that inhibition of ras oncogenes by antisense oligonucleotides can block tumor development even though ras oncogenic activation might be an early event in tumor progression.
Suppression of mammalian 5' splice-site defects by U1 small nuclear RNAs from a distance.
Authors: Cohen J B , Snow J E , Spencer S D , Levinson A D
Published: May 2006

One of the earliest events in the process of intron removal from mRNA precursors is the establishment of a base-pairing interaction between U1 small nuclear (sn) RNA and the 5' splice site. Mutations at the 5' splice site that prevent splicing can often be suppressed by coexpression of U1 snRNAs with compensatory changes, but in yeast, accurate splicing is not restored when the universally conserved first intron base is changed. In our mammalian system as well, such a mutation could not be suppressed, but the complementary U1 caused aberrant splicing 12 bases downstream. This result is reminiscent of observations in yeast that aberrant 5' splice sites can be activated by U1 snRNA from a distance. Using a rapid, qualitative protein expression assay, we provide evidence that 5' splice-site mutations can be suppressed in mammalian cells by U1 snRNAs with complementarity to a range of sequences upstream or downstream of the site. Our approach uncouples in vivo the commitment-activation step of mammalian splicing from the process of 5' splice-site definition and as such will facilitate the genetic characterization of both.
Purification and characterization of a kinase specific for the serine- and arginine-rich pre-mRNA splicing factors.
Authors: Gui J F , Tronchère H , Chandler S D , Fu X D
Published: May 2006

Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.
Protein-tyrosine phosphorylation regulates apoptosis in human eosinophils and neutrophils.
Authors: Yousefi S , Green D R , Blaser K , Simon H U
Published: May 2006

Early signaling events that control the process of programmed cell death are largely unknown. Tyrosine phosphorylation plays a major role in transmembrane signal transduction through most cell surface receptors. Granulocyte/macrophage colony-stimulating factor (GM-CSF), a cytokine released by activated T cells, has been shown to increase tyrosine phosphorylation in several cells and to inhibit granulocyte cell death in vitro. In this study, we demonstrate that the effect of GM-CSF on granulocyte cell death can be blocked by the tyrosine kinase inhibitor genistein, suggesting that increases in tyrosine phosphorylation are essential to inhibit cell death. To analyze the role of tyrosine phosphorylation for the regulation of granulocyte cell death more precisely, we increased levels of tyrosine phosphorylation using the protein-tyrosine phosphatase inhibitor phenylarsine oxide (PAO). Similar to GM-CSF, treatment of the cells with PAO was followed by high increases in tyrosine phosphorylation and inhibition of programmed cell death in human eosinophils and neutrophils. Strikingly, at low concentrations of the inhibitor and low induction of tyrosine phosphorylation, acceleration of apoptosis was observed. Genistein and herbimycin A reversed the effects of PAO on tyrosine phosphorylation and granulocyte apoptosis. These results suggest that programmed eosinophil and neutrophil death is regulated by early events of signal transduction pathways such as tyrosine phosphorylation.
Isolation of an allele of reeler by insertional mutagenesis.
Authors: Miao G G , Smeyne R J , D'Arcangelo G , Copeland N G , Jenkins N A , Morgan J I , Curran T
Published: May 2006

Reeler (rl) is an autosomal recessive mutation that affects migration of postmitotic neurons in the mouse central nervous system. The reeler (rl/rl) mouse displays a disruption of laminar structures in both the cerebellum and the forebrain and it exhibits tremors, dystonia, and ataxia. The molecular basis of the reeler phenotype is unknown because the gene involved has not yet been identified. We report here the isolation and characterization of an allele of rl, reelertransgene (rltg). This allele was generated by the fortuitous insertion of a transgene, supfos (sf), into the mouse rl locus. Crosses between rl/+ and rltg/+ mice yielded offspring that exhibited the reeler phenotype, indicating that rl and rltg are allelic. We cloned the genomic sequences flanking the transgene insertion site from the rltg/rltg mouse genome. Chromosomal mapping studies revealed that the 5' flanking cellular sequence maps to a locus, D5Gmr1, that lies in a region of mouse chromosome 5 that also contains the rl locus. Southern blot analysis using a probe derived from the D5Gmr1 locus revealed no gross structural rearrangement in the rl locus. Thus, unlike the two rl alleles described previously, rltg provides a molecular probe that can now be used to identify and isolate the rl gene.