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PubMed2005

The chloroplast genome of Phalaenopsis aphrodite (Orchidaceae): comparative analysis of evolutionary rate with that of grasses and its phylogenetic implications.

Whether the Amborella/Amborella-Nymphaeales or the grass lineage diverged first within the angiosperms has recently been debated. Central to this issue has been focused on the artifacts that might result from sampling only grasses within the monocots. We therefore sequenced the entire chloroplast genome (cpDNA) of Phalaenopsis aphrodite, Taiwan moth orchid. The cpDNA is a circular molecule of 148,964 bp with a comparatively short single-copy region (11,543 bp) due to the unusual loss and truncation/scattered deletion of certain ndh subunits. An open reading frame, orf91, located in the complementary strand of the rrn23 was reported for the first time. A comparison of nucleotide substitutions between P. aphrodite and the grasses indicates that only the plastid expression genes have a strong positive correlation between nonsynonymous (Ka) and synonymous (Ks) substitutions per site, providing evidence for a generation time effect, mainly across these genes. Among the intron-containing protein-coding genes of the sampled monocots, the Ks of the genes are significantly correlated to transitional substitutions of their introns. We compiled a concatenated 61 protein-coding gene alignment for the available 20 cpDNAs of vascular plants and analyzed the data set using Bayesian inference, maximum parsimony, and neighbor-joining (NJ) methods. The analyses yielded robust support for the Amborella/Amborella-Nymphaeales-basal hypothesis and for the orchid and grasses together being a monophyletic group nested within the remaining angiosperms. However, the NJ analysis using Ka, the first two codon positions, or amino acid sequences, respectively, supports the monocots-basal hypothesis. We demonstrated that these conflicting angiosperm phylogenies are most probably linked to the transitional sites at all codon positions, especially at the third one where the strong base-composition bias and saturation effect take place.

DNA, Chloroplast↗
PubMed2000

Functional role of residues corresponding to helical domain II (amino acids 35 to 46) of human immunodeficiency virus type 1 Vpr.

Vpr, encoded by the human immunodeficiency virus type 1 genome, contains 96 amino acids and is a multifunctional protein with features which include cell cycle arrest at G(2), nuclear localization, participation in transport of the preintegration complex, cation channel activity, oligomerization, and interaction with cellular proteins, in addition to its incorporation into the virus particles. Recently, structural studies based on nuclear magnetic resonance and circular dichroism spectroscopy showed that Vpr contains a helix (HI)-turn-helix (HII) core at the amino terminus and an amphipathic helix (HIII) in the middle region. Though the importance of helical domains HI and HIII has been defined with respect to Vpr functions, the role of helical domain HII is not known. To address this issue, we constructed a series of mutants in which the HII domain was altered by deletion, insertion, and/or substitution mutagenesis. To enable the detection of Vpr, the sequence corresponding to the Flag epitope (DYKDDDDK) was added, in frame, to the Vpr coding sequences. Mutants, expressed through the in vitro transcription/translation system and in cells, showed an altered migration corresponding to deletions in Vpr. Substitution mutational analysis of residues in HII showed reduced stability for VprW38S-FL, VprL42G-FL, and VprH45W-FL. An assay involving cotransfection of NLDeltaVpr proviral DNA and a Vpr expression plasmid was employed to analyze the virion incorporation property of Vpr. Mutant Vpr containing deletions and specific substitutions (VprW38S-FL, VprL39G-FL, VprL42G-FL, VprG43P-FL, and VprI46G-FL) exhibited a negative virion incorporation phenotype. Further, mutant Vpr-FL containing deletions also failed to associate with wild-type Vpr, indicating a possible defect in the oligomerization feature of Vpr. Subcellular localization studies indicated that mutants VprDelta35-50-H-FL, VprR36W-FL, VprL39G-FL, and VprI46G-FL exhibited both cytoplasmic and nuclear localization, unlike other mutants and control Vpr-FL. While wild-type Vpr registered cell cycle arrest at G(2), mutant Vpr showed an intermediary effect with the exception of VprDelta35-50 and VprDelta35-50-H. These results suggest that residues in the HII domain are essential for Vpr functions.

Amino Acid Sequence↗
PubMed1986

Sequence analysis and characterization of a maize gene encoding a high-sulfur zein protein of Mr 15,000.

We have isolated a gene coding for a sulfur-rich zein protein from a maize genomic library. The nucleotide sequence of this gene predicts a protein composed of 180 amino acids, including a 20-amino acid signal peptide. As is true of other zeins, there are no intervening sequences in the gene. Comparison of the nucleotide sequence of this gene with that of a homologous cDNA clone revealed only a single difference resulting in a valine/alanine substitution. The Mr 15,000 zein contains no repetitive nucleotide sequences and shows no homology with genes encoding the Mr 22,000 and 19,000 zeins. Circular dichroism analysis of the Mr 15,000 zein protein revealed that it is composed primarily of beta and turn structures. This gene has a short region of nucleotide sequence homology to the cysteine-rich domain of the Mr 27,000 zein, as well as the cysteine-containing barley B-hordein.

Amino Acid Sequence↗
PubMed2002

Complete genome sequence, taxonomic assignment, and comparative analysis of the untranslated regions of the Modoc virus, a flavivirus with no known vector.

We recently developed a model for flavivirus infection in mice and hamsters using the Modoc virus (MODV), a flavivirus with no known vector (P. Leyssen, A. Van Lommel, C. Drosten, H. Schmitz, E. De Clercq, and J. Neyts, 2001, Virology 279, 27-37). We now present the coding and noncoding sequence of MODV. The Modoc virus genome was determined to be 10,600 nucleotides in length with a single open reading frame extending from nucleotides 110 to 10,234, encoding 3374 amino acids. The deduced gene order of the single open reading frame is C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5, which is exactly the same as that of the mosquito- and tick-borne flaviviruses. It is flanked by a 5'- and 3'-untranslated region (UTR) of 109 and 366 nucleotides, respectively. Alignment of the MODV amino acid sequence with that of 20 other flaviviruses revealed several regions with high sequence similarity corresponding to functionally important domains (e.g., the serine protease/helicase/NTPase of NS3 and the methyltransferase/RNA-dependent RNA polymerase of NS5) and conserved sites for proteolytic cleavage by viral and cellular proteases. Phylogenetic analysis of the entire coding region confirmed the classification of MODV within the flaviviruses with no known vector, which is in agreement with previous findings based on partial NS5 sequences. A detailed comparative analysis of the putative folding patterns of the 5'- and 3'-UTR of MODV and of the tick- and mosquito-borne viruses was carried out. Structural elements in the 5'- and 3' UTR of MODV that are preserved among vector-borne flaviviruses were noted and so were structural elements distinguishing the MODV UTRs from mosquito-borne and tick-borne flaviviruses. Also the putative secondary structure of circularized MODV RNA is presented.

3' Untranslated Regions↗
PubMedDate not supplied

HU and IHF: similarities and differences. In Escherichia coli, the lack of HU is not compensated for by IHF.

HU is one of the most abundant DNA binding proteins in Escherichia coli. Like the histones, HU is able to condense DNA in vitro and to introduce negative super-coiling in covalently closed circular, relaxed DNA molecules in the presence of topoisomerase I. HU is well conserved in all prokaryotes but surprisingly, it is also homologous to another E. coli DNA-binding protein, IHF. Contrary to HU, IHF shows sequence specificity and is much less abundant that HU. Both are heterodimers and all four polypeptide chains probably arose from a common ancestor. The question we raised was whether IHF could supply the main functions of HU in its absence. The answer seems to be negative for the following reasons. We did not observe any significant regulation of expression of the genes coding for HU by IHF, or vice-versa. The structures that these two proteins form with double-stranded or single-stranded DNA are completely different. Finally, overexpression of IHF does not relieve the growth defects observed in HU-less mutants. However, it can be speculated from our results that even if the two proteins are not equivalent and cannot replace one another, both could stimulate (or inhibit) some specific protein-DNA interactions or affect the DNA-binding properties of one another.

Bacterial Proteins↗
PubMed1984

The Drosophila mitochondrial genome.

The mitochondrial genome of Drosophila yakuba is a circular DNA molecule of 16019 nucleotide pairs. The sequence contains the genes for two rRNA molecules, 22 tRNA molecules, five known polypeptides (cytochrome b, cytochrome c oxidase subunits I, II, III and ATPase subunit 6) and eight unidentified polypeptides (URF1, 2, 3, 4L, 4, 5, 6 and A6L). Between the tRNA(ile) and small rRNA genes there occurs a sequence of 1077 nucleotides that is 92.8 per cent A + T and lacks reading frames greater than 123 nucleotides. Replication of the molecule originates in this A + T-rich region and proceeds toward the small rRNA gene. Non-coding nucleotides between genes are either absent or occur in low numbers (1 to 31). A sequence equivalent in size and secondary structure potential to the sequence associated with the initiation of second strand synthesis in mammalian mtDNA is missing in Drosophila mtDNA. While the genes found in D. yakuba and mammalian mtDNAs are the same, the relative arrangement of many of these genes differs considerably in the two molecules. The proportions of the two strands of the D. yakuba molecule which serve as template for transcription of genes are approximately equal. This contrasts with the situation in mammalian mtDNAs where all genes except those for URF6 and eight tRNAs are transcribed from one strand. The dihydrouridine and T psi C loops of D. yakuba mt-tRNA genes are highly variable in size, and among these genes there is a general deficiency of nucleotides which are highly conserved in prokaryotic and eukaryotic nuclear-coded tRNAs. The D. yakuba tRNA(AGYser) gene is unusual in that an eleven nucleotide loop replaces the dihydrouridine arm. D. yakuba mitochondrial polypeptide genes utilize 59 sense codons. However, 93.8 per cent of all codons used end in A or T. Unique variations occur in the Drosophila mitochondrial genetic code. AGA appears to specify serine rather than arginine as in the standard code, or termination as in the mammalian mitochondrial code. The Drosophila COI gene lacks a standard translation initiation codon, and may utilize a four nucleotide codon ATAA for that purpose. As in other metazoan mitochondria, TGA and ATA specify tryptophan and methionine, respectively. As a tRNA with an anticodon (TCT) specific for AGA codons does not appear to be encoded in D. yakuba mtDNA, it seems likely that the GCU anticodon of the D. yakuba tRNA which recognizes AGY (serine) codons can also recognize AGA.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1996

The mitochondrial DNA of Allomyces macrogynus: the complete genomic sequence from an ancestral fungus.

We have determined the complete nucleotide sequence of the circular mitochondrial DNA (mtDNA) of the chytridiomycete fungus, Allomyces macrogynus (57,473 bp; A + T content 60.5%). The identified genes that are typical for most fungal mitochondria include those for the large (rnl) and small subunit (rns) ribosomal RNAs, a complete set of 25 tRNAs, three ATPase subunits (atp6, atp8 and atp9), apocytochrome b(cob), three subunits of the cytochrome oxidase complex (cox1, cox2 and cox3), and seven subunits of the NADH dehydrogenase complex (nad1, nad2, nad3, nad4, nad4L, nad5 and nad6). A total of 28 introns of both groups are found, some of which contain open reading frames (ORFs) coding for potential endonucleases (group I) or reverse-transcriptases (group II). All mitochondrial genes are transcribed from the same DNA strand, as is the case in many other eufungi. Particular features of the A. macrogynus mtDNA include: (1) the first documented case of a fungal mitochondrial ribosomal protein gene (rps3) that is clearly identified by similarity with bacterial homologues; (2) four unique ORFs; (3) the presence of an insert in the atp6 gene that may have been acquired by interspecific transfer; (4) more than 67 short, highly structured and conserved DNA elements inserted in intergenic spacers, introns, and variable regions of the rnl and rns genes: these elements are unusually G + C rich; (5) rRNA structures that resemble more closely those of eubacteria than their counterparts in other fungal mitochondria. The high degree of conservation of the A. macrogynus mitochondrial rRNA secondary structures, the existence of a mitochondrial rps3 gene (common to protist but unique in fungal mtDNAs), and phylogenetic relationships inferred from highly conserved protein genes, demonstrate consistently the ancestral character of this fungal mitochondrial genome.

Amino Acid Sequence↗
PubMed1997

Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit immunohistochemistry.

Interstitial cells of Cajal (ICC) of various morphologies have been described in the gastrointestinal (GI) tracts of mammals. Different classes of ICC are likely to have different functional roles. ICC of the mouse GI tract have been shown to express c-kit, a proto-oncogene that codes for a receptor tyrosine kinase. We have studied the distribution of ICC within the guinea pig GI tract using antibodies to c-Kit protein and immunohistochemical techniques. c-Kit-like immunoreactivity revealed at least 6 types of ICC: (1) intramuscular ICC (IC-IM1) that lie within the muscle layers of the esophagus, stomach, and cecum, (2) ICC within the myenteric plexus region (IC-MY1) in the corpus, antrum, small intestine, and colon, (3) ICC that populate the deep muscular plexus of the small intestine (IC-DMP), (4) ICC at the submucosal surface of the circular muscle layer in the colon (IC-SM), (5) stellate ICC that are closely associated with the myenteric plexus (IC-MY2) and orientated toward the longitudinal muscle layer in the colon, and (6) branching intramuscular ICC (IC-IM2) in the proximal colon within the circular and longitudinal muscle layers. c-Kit immunohistochemistry appears to be an excellent and selective technique for labeling ICC of the guinea-pig GI tract. Labeling of these cells at the light-microscopic level provides an opportunity for characterizing the distribution, density, organization, and relationship between ICC and other cell types.

Animals↗
PubMed1999

Calcium-dependent immunoglobulin E recognition of the apo- and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phl p 7.

Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind-pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF-hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen-specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7-homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium-bound and apo-rPhl p 7 indicated that differences in IgE recognition may be due to calcium-induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation-dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.

Allergens↗
PubMed1984

Transcription of episomal papillomavirus DNA in human condylomata acuminata and Buschke-Löwenstein tumours.

Condylomata acuminata and Buschke-Löwenstein tumours were analysed for the presence of human papillomavirus (HPV) transcripts. HPV DNA and RNA sequences were present in all 13 samples investigated. Ten contained HPV6 and three harboured HPV11. The HPV genomes were found exclusively as extrachromosomal circular molecules. In six biopsy specimens, viral RNA transcripts were not detectable by Northern blot analysis but could be demonstrated in dot blots. From seven HPV6-containing samples it was possible to obtain sufficient amounts of undegraded mRNA. We have found consistently one major species (1.4 kb). Less prominent species of 1.7, 1.85, 2.7 and 3.2 kb, respectively, were also detected. The 3' ends of the HPV6 mRNAs were located between nucleotides 3917 and 4441 in the putative early region and between nucleotides 7232 and 7696 in the putative late region. The arrangement of the 3' termini and the adjacent coding areas within the HPV6 genome show that the RNA species are transcribed from one DNA strand.

Base Sequence↗
PubMedDate not supplied

On the probability of a common origin for tRNA and 5S rRNA.

As part of a continuing study on the origin of genetic coding and the process of protein synthesis, we have compared sequences of a large number of transfer RNAs and several 5S ribosomal RNAs using automated routines. Transfer RNAs were found to exhibit a high degree of matching with 5S rRNAs. These matches are considered to be indicative of sequence homology, reflecting common ancestry of the two molecules. Other possible explanations for the matches (convergence, transfection) are discussed and found to be highly implausible. Matches are also found between 5S rRNA and the introns of yeast precursor tRNAs. Many matches extend from before the 3' end to after the 5' end of circularized 5S rRNA sequences. Data is presented which indicates a tandemly duplicated or circular molecule could have served as the precursor to both 5S rRNA and tRNA. A derivative of this molecule may have functioned as a universal translator before the evolution of the highly specialized tRNAs.

Animals↗
PubMed1981

[Inhibition of hepatitis B virus associated DNA polymerase by antiviral agents: in vitro studies with clinical implications (author's transl)].

The complete hepatitis B virus (Dane particle) contains a circular doublestranded DNA with single stranded regions and an endogenous DNA polymerase. The HBV associated DNA polymerase closes the single stranded regions of the HBV-DNA in the presence of triphosphatase and a detergent. The DNA polymerase reaction can be inhibition by antiviral substances that exhibit different mode of actions: intercalating agents, phosphonoformiate and the triphosphates of arabinofuranosyladenine and arabinofuranosylcytosine. The value of these in vitro test for the therapy of HBsAg positive chronic liver disease is limited by the fact that is remains so far unclear whether the HBV associated DNA polymerase is a virus- or a host-coded enzyme, and whether virus- or host-coded enzymes are involved in HBV-DNA synthesis in vivo.

Antiviral Agents↗
PubMedDate not supplied

The minimal cellular genome of mycoplasma.

The mycoplasmas are the smallest and simplest self-replicating organisms, being built of a plasma membrane, ribosomes, and a circular double-stranded DNA molecule-the typical prokaryotic genome. The idea of using mycoplasmas as models for defining in molecular terms the entire machinery of a living cell was raised by Morowitz in 1984. The goal has been to prove the dogma of the completeness of molecular biology, that is, that the logic of life is finite, relatively simple and subject to full exploration. The recent complete sequencing of the genome of the human pathogen Mycoplasma genitalium brings us much closer to achieving this goal. The M. genitalium genome is only 580 kb long and contains only 470 predicted coding sequences(genes), as compared with 1727 in Haemophilus influenzae and about 4000 in E. coli. Thus, M. genitalium is apparently the simplest organism capable of independent life with a minimal set of genes. The drastic economization in genetic information must be associated with the parasitic mode of life of the mycoplasmas. Mycoplasmas evolved by reductive evolution from Gram-positive bacteria with low G + C genomes. During evolution the mycoplasmas have lost the cell wall and many biosynthetic systems involved in synthesis of macromolecule building blocks provided by their host. Thus, the M. genitalium genome carries only one gene involved in amino acid biosynthesis, and very few genes for vitamin and nucleic acid precursors; the lack of genes involved in fatty acid biosynthesis, leads to dependence on exogenous fatty acids, enabling the introduction of controlled variations in membrane acyl chains and the use of mycoplasmas as models in studying membrane fluidity. Moreover, the dependence of mycoplasmas on exogenous cholesterol for growth was exploited to show the role of cholesterol as a buffer of membrane fluidity. The mycoplasma genome carries the minimal set of energy metabolism genes, being content with a restricted supply of ATP needed for their parasitic mode of life. Being limited by a single permeability barrier enabled the saving of a considerable number of transport system genes. Nevertheless, these minimal organisms were shown to carry all the essential genes needed for DNA replication, transcription and translation, but even here gene saving is expressed in a minimal number of rRNA and tRNA genes. A genomic price had been paid to maintain parasitism, so that a significant number of mycoplasmal genes is devoted to adhesins, attachment organelles and variable membrane surface antigens directed towards evasion of the host immune system.

Genome, Bacterial↗
PubMed1995

Cloning of the gene encoding DNA binding protein HU from Bacillus stearothermophilus and its expression in Escherichia coli.

The gene (hbst) encoding the DNA binding protein HU from Bacillus stearothermophilus was cloned, with the Bacillus subtilis HU gene (hbsu) as a hybridization probe. The nucleotide sequence, which contains a ribosome binding site, a transcriptional termination signal, as well as the coding region, was analyzed by the dideoxy chain-termination method. The deduced amino acid sequence of an open reading frame was perfectly matched with that of B. stearothermophilus HU (BstHU) determined by the protein chemical methods [M. Kimura and K. S. Wilson, J. Biol. Chem., 258, 4007-4011 (1983)]. The gene, hbst, was overexpressed using the expression vector pET-5a in Escherichia coli, and the recombinant HU protein (r-BstHU) was purified to be homogeneity by heparin-agarose column chromatography followed by ion-exchange column chromatography on S-Sepharose. The recombinant protein thus obtained had a circular dichroism spectrum identical to that of the authentic protein and bound to DNA to the same extent as the authentic protein.

Amino Acid Sequence↗
PubMed2000

The genome of Entamoeba histolytica.

Estimation of genome size of Entamoeba histolytica by different methods has failed to give comparable values due to the inherent complexities of the organism, such as the uncertain level of ploidy, presence of multinucleated cells and a poorly demarcated cell division cycle. The genome of E. histolytica has a low G+C content (22.4%), and is composed of both linear chromosomes and a number of circular plasmid-like molecules. The rRNA genes are located exclusively on some of the circular DNAs. Karyotype analysis by pulsed field gel electrophoresis suggests the presence of 14 conserved linkage groups and an extensive size variation between homologous chromosomes from different isolates. Several repeat families have been identified, some of which have been shown to be present in all the electrophoretically separated chromosomes. The typical nucleosomal structure has not been demonstrated, though most of the histone genes have been identified. Most Entamoeba genes lack introns, have short 3' and 5' untranslated regions, and are tightly packed. Promoter analysis revealed the presence of three conserved motifs and several upstream regulatory elements. Unlike typical eukaryotes, the transcription of protein coding genes is alpha-amanitin resistant. Expressed Sequence Tag analysis has identified a group of highly abundant polyadenylated RNAs which are unlikely to be translated. The Expressed Sequence Tag approach has also helped identify several important genes which encode proteins that may be involved in different biochemical pathways, signal transduction mechanisms and organellar functions.

Animals↗
PubMed2001

Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle.

In eukaryotic cells, freshly synthesized messenger RNA (pre-mRNA) contains stretches of non-coding RNA that must be excised before the RNA can be translated into protein. Their removal is catalysed by the spliceosome, a large complex formed when a number of small nuclear ribonucleoprotein particles (snRNPs) bind sequentially to the pre-mRNA. The first snRNP to bind is called U1; other snRNPs (U2, U4/U6 and U5) follow. Here we describe the three-dimensional structure of human U1 snRNP, determined by single-particle electron cryomicroscopy at 10 A resolution. The reconstruction reveals a doughnut-shaped central element that accommodates the seven Sm proteins common to all snRNPs, supporting a proposed model of circular Sm protein arrangement. By taking earlier biochemical results into account, we were able to assign the remaining density of the map to the other known components of U1 snRNP, deriving a structural model that describes the three-dimensional arrangement of proteins and RNA in U1 snRNP.

Cryoelectron Microscopy↗
PubMed1983

[Replication and expression of plasmid pBR 322 during discontinuous culture of a stringent and relaxed Escherichia coli strain].

The E. coli strains CP78 and CP79 carrying the plasmid pBR 322 display similar growth kinetics in discontinuous culture. During limitation of amino acids the stringent strain CP78 is able to synthesize guanosine-5'diphosphate-3'-diphosphate (ppGpp) and guanosine-5'-triphosphate-3' diphosphate, but the relaxed strain can not produce highly phosphorylated guanosine nucleotides. During the logarithmic phase of growth both strains contain similar amounts of plasmid DNA. During amino acid starvation plasmid DNA is amplified in the relaxed strain only, whereas in the stringent strain the plasmid content per cell remains constant. In stationary phase cells of CP78 a higher activity of plasmid-coded beta-lactamase than in CP79 cells was detected. Furthermore, remarkable differences between both strains were observed in the composition of proteins derived from the periplasmic fraction and separated by polyacrylamide gel electrophoresis. Our results might indicate a negative control of pBR 322 DNA replication by ppGpp during amino acid starvation.

Amino Acids↗
PubMed2005

Serum from achalasia patients alters neurochemical coding in the myenteric plexus and nitric oxide mediated motor response in normal human fundus.

BACKGROUND AND AIMS: Achalasia is a disease of unknown aetiology. An immune mechanism has been suggested on the basis of previous morphological observations. The objective of this study was to test whether the serum of achalasia patients could reproduce the phenotype and functional changes that occur with disease progression in an ex vivo human model. METHODS: Specimens of normal human fundus were maintained in culture in the presence of serum from patients with achalasia, gastro-oesophageal reflux disease (GORD), or healthy subjects (controls). Immunohistochemical detection of choline acetyltransferase (ChAT), neurone specific enolase (NSE), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), and substance P was carried out in whole mounts of gastric fundus myenteric plexus. In addition, the effects of achalasia serum on electrical field stimulation (EFS) induced contractions were measured in circular muscle preparations. RESULTS: Serum from achalasia patients did not affect the number of myenteric neurones. Tissues incubated with serum from achalasia patients showed a decrease in the proportion of NOS (-26% of NSE positive neurones; p=0.016) and VIP (-54%; p=0.09) neurones, and a concomitant increase in ChAT neurones (+16%; p<0.001) compared with controls. In contrast, GORD serum did not modify the phenotype of myenteric neurones. Area under the curve of EFS induced relaxations (abolished by N-nitro-L-arginine methyl ester) was significantly decreased following incubation with serum from achalasia patients compared with controls (-7.6 (2.6) v -14.5 (5.0); p=0.036). CONCLUSIONS: Serum from achalasia patients can induce phenotypic and functional changes which reproduce the characteristics of the disease. Further identification of putative seric factors and mechanisms involved could lead to the development of novel diagnostic and/or therapeutic strategies in achalasia.

Adult↗