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PubMed1999

Identification of an autoimmune enteropathy-related 75-kilodalton antigen.

BACKGROUND & AIMS: We have previously reported a 75-kilodalton autoantigen specific to X-linked autoimmune enteropathy (AIE) associated with tubulonephropathy. The aim of this study was to identify the autoantigen. METHODS: Complementary DNA (cDNA) clones were isolated by immunoscreening a human duodenal cDNA-expression library with serum from a patient with AIE. RESULTS: cDNA encoding the 75-kilodalton antigen (AIE-75) was identified. The composite nucleotide sequence of the cDNA for AIE-75 was 2214 base pairs long and encoded 552 amino acids. The genomic sequence of AIE-75 was found in Sequence DataBank, which consisted of 21 exons and was located on the chromosome 11p14.3. Recombinant AIE-75 specifically reacted with sera from 3 of 4 unrelated patients with AIE but not with 58 control sera. AIE-75 was predominantly distributed in the epithelial cells of the luminal surface and the upper half of the crypts of the intestine and in the proximal renal tubulus. Similarity searches revealed that the AIE-75 cDNA sequence was an authentic form of several colon cancer-related cDNAs of unknown function. The deduced amino acid sequence contained 3 conserved PSD-95/Dlg/ZO-1 (PDZ) domains. CONCLUSIONS: AIE-75 is a PDZ domain-containing protein expressed in the differentiated epithelial cells of the intestine and kidney and may be involved in protein-protein interaction. The identification of the autoantigen may prove useful in the approach to the pathogenesis of this poorly understood disease.

Amino Acid Sequence↗

PubMed2003

Profiles of PGH-alpha, GTH I-beta, and GTH II-beta mRNA transcript levels at different ovarian stages in the wild female Japanese eel Anguilla japonica.

The complete complementary DNA (cDNA) encoding pituitary gonadotropin II-beta subunit (GTH II-beta) of Japanese eel Anguilla japonica was cloned and sequenced, and the profiles of pituitary glycoprotein hormone alpha subunit (PGH-alpha), GTH I-beta, and GTH II-beta mRNA transcript levels at different stages of ovarian development before vitellogenesis in the wild females were investigated. The maturity of female eels was divided into four stages: juvenile, sub-adult, pre-silver, and silver stages based on ovarian development and skin color. The GTH II-beta cDNA was cloned by reverse transcription and polymerase chain reaction (RT-PCR) amplification from total pituitary RNA. The full length GTH II-beta cDNA was obtained using 5(')- and 3(')-rapid amplification of cDNA ends. The cloned eel GTH II-beta cDNA consists of 646 bp nucleotides, including 53 bp nucleotides of 5(')-untranslated region (UTR), 423 bp of open reading frame, and 170 bp nucleotides of 3(')-UTR followed by a poly(A) tail. It encodes a 140-amino acid precursor molecule of GTH II-beta subunit with a putative signal peptide of 24 amino acids and a mature peptide of 116 amino acids. RT-PCR analysis showed that the pituitary transcript levels of alpha subunit steadily increased during eel silvering. The expression of GTH I-beta and II-beta mRNA levels, however, varied in different ovarian developmental stages. The mRNA expression of both GTH I-beta and GTH II-beta were detectable in juvenile stage. The expression levels of GTH II-beta mRNA, but not GTH I-beta, were significantly increased in sub-adult stage. The transcript levels of GTH I-beta and II-beta subunits further increased in pre-silver and silver stages. We demonstrated for the first time that the differential transcription patterns of pituitary PGH-alpha, GTH I-beta, and GTH II-beta mRNAs occur during silvering of the wild female Japanese eels.

Amino Acid Sequence↗

PubMed2003

Trehalose metabolism genes in Caenorhabditis elegans and filarial nematodes.

The sugar trehalose is claimed to be important in the physiology of nematodes where it may function in sugar transport, energy storage and protection against environmental stresses. In this study we investigated the role of trehalose metabolism in nematodes, using Caenorhabditis elegans as a model, and also identified complementary DNA clones putatively encoding genes involved in trehalose pathways in filarial nematodes. In C. elegans two putative trehalose-6-phosphate synthase (tps) genes encode the enzymes that catalyse trehalose synthesis and five putative trehalase (tre) genes encode enzymes catalysing hydrolysis of the sugar. We showed by RT-PCR or Northern analysis that each of these genes is expressed as mRNA at all stages of the C. elegans life cycle. Database searches and sequencing of expressed sequence tag clones revealed that at least one tps gene and two tre genes are expressed in the filarial nematode Brugia malayi, while one tps gene and at least one tre gene were identified for Onchocerca volvulus. We used the feeding method of RNA interference in C. elegans to knock down temporarily the expression of each of the tps and tre genes. Semiquantitative RT-PCR analysis confirmed that expression of each gene was silenced by RNA interference. We did not observe an obvious phenotype for any of the genes silenced individually but gas-chromatographic analysis showed >90% decline in trehalose levels when both tps genes were targeted simultaneously. This decline in trehalose content did not affect viability or development of the nematodes.

Amino Acid Sequence↗

PubMed2003

New HLA-A, -B, and -C locus-specific primers for PCR amplification from cDNA: application in clinical immunology.

The individual cellular immune response to intracellular antigens is modeled by the highly polymorphic major histocompatibility complex (HLA) class I molecules. The epitopes presented and the T cell repertoire that recognizes them depend on the HLA constitution of the individual. Therefore, to monitor and to modify an individual's HLA class I-driven cellular immune response, it is necessary to know the HLA class I alleles of the person and the possible epitopes of the target antigen presented by those alleles. In particular, this is necessary in order to design peptide-based vaccines and immune therapies for the treatment of diseases caused by viruses, intracellular parasites or cancer, and to monitor the immune response during those treatments. We describe a new set of HLA-A, -B, and -C locus-specific primers for the polymerase chain reaction (PCR) amplification of the whole coding sequence of these genes from complementary DNA (cDNA). We describe their use for typing and for the production of a library of recombinant HLA class I genes. We discuss two downstream applications of this gene collection: production of soluble HLA molecules and discovery of new epitopes.

DNA Primers↗

PubMed1998

A highly sensitive and specific assay using a novel human growth hormone cDNA reporter gene regulated by the human interleukin-4 inducible germline epsilon transcript promoter.

We have successfully developed a highly sensitive and specific assay system for human interleukin-4 (IL-4) regulated gene expression. It is based on a human Jijoye cell line with the germline epsilon transcript promoter joined to the human growth hormone (hGH) cDNA. The germline epsilon transcript promoter is responsive to IL-4 and involved in immunoglobulin heavy chain class switching. We cloned hGH complementary DNA (cDNA) as the reporter gene instead of using conventional hGH genomic DNA which failed to generate any IL-4 inducible clone in human Jijoye cells. The two IL-4 inducible cell lines with the hGH cDNA reporter show high signal/noise ratio for IL-4-mediated induction (60-90 fold). The response to IL-4 is dose-dependent with ED50 of 10 pM. As expected, there is no response to other human cytokines and growth factors, as well as mouse IL-4. The mutant hIL-4 antagonist hIL-4.Y124D inhibits the induction mediated by native hIL-4. These IL-4 inducible cell lines provide a sensitive, specific assay system to study IL-4-regulated gene expression, and in particular the regulation of the germline epsilon promoter.

Animals↗

PubMed1983

Sequence-specific adenylations and deadenylations accompany changes in the translation of maternal messenger RNA after fertilization of Spisula oocytes.

A dramatic change in the pattern of protein synthesis occurs within ten minutes after fertilization of Spisula oocytes. This change is regulated entirely at the translational level. We have used DNA clones complementary to five translationally regulated messenger RNAs to follow shifts in mRNA utilization at fertilization and to characterize alterations in mRNA structure that accompany switches in translational activity in vivo. Four of the mRNAs studied are translationally inactive in the oocyte. After fertilization two of these mRNAs are completely recruited onto polysomes, and two are partially recruited. All four of these mRNAs have very short poly(A) tracts in the oocyte; after fertilization the poly(A) tails lengthen considerably. In contrast, a fifth mRNA, that encoding alpha-tubulin mRNA, is translated very efficiently in the oocyte and is rapidly lost from polysomes after fertilization. Essentially all alpha-tubulin mRNA in the oocyte is poly(A)+ and a large portion of this mRNA undergoes complete deadenylation after fertilization. These results reveal a striking relationship between changes in adenylation and translational activity in vivo. This correlation is not perfect, however. Evidence for and against a direct role for polyadenylation in regulating these translational changes is discussed. Changes in poly(A) tails are the only alterations in mRNA sizes that we have been able to detect. This indicates that, at least for the mRNAs studied here, translational activation is not due to extensive processing of larger translationally incompetent precursors. We have also isolated several complementary DNA clones to RNAs encoded by the mitochondrial genome. Surprisingly, the poly(A) tracts of at least two of the mitochondrial RNAs also lengthen in response to fertilization.

Animals↗

PubMed1999

Gene transfection of beta 2-adrenergic receptor into the normal rat heart enhances cardiac response to beta-adrenergic agonist.

BACKGROUND: Beta-adrenergic receptor system has a major role in cardiac contraction. If the receptor can be increased by gene transfection by means of intracoronary infusion of beta 2-adrenergic receptor to the hearts in which the receptor is down-regulated, this maneuver may improve the cardiac function and may be applied as one therapeutic approach during cardiopulmonary bypass or percutaneous cardiopulmonary support. METHODS AND RESULTS: The beta 2-adrenergic receptor complementary DNA was transfected in vivo to the normal rat heart by intracoronary infusion by means of a hemagglutinating virus of Japan liposome method, and the transfected heart was transplanted into the abdomen of another rat. Four days after transfection, the sarcolemma of the cardiomyocytes was well labeled by immunohistochemical labeling. Expression of beta-adrenergic receptor in the heart was approximately 4 times greater than that in control hearts (134 +/- 42 vs 33 +/- 4 fmol/mg protein) according to a ligand binding assay. The cardiac response of the transfected heart to isoproterenol was shown to be enhanced in a Langendorff perfusion system: after isoproterenol, developed pressure and maximal derivative of the left ventricle were greater than in the control heart (200 +/- 12 vs 174 +/- 6 mm Hg and 4110 +/- 130 vs 3491 +/- 255 mm Hg/sec), and the minimal derivative of the left ventricle was markedly smaller (-3040 +/- 267 vs -2528 +/- 131 mm Hg/sec). CONCLUSIONS: These results indicate that expression of beta 2-adrenergic receptor was approximately 4 times greater than in normal rat hearts by gene transfection using a hemagglutinating virus of Japan liposome method, and the transfected hearts demonstrated marked enhancements in cardiac response to beta-agonist, suggesting that transfer of this gene by intracoronary infusion has potential as a novel approach to enhance cardiac function.

Adrenergic beta-Agonists↗

PubMed2003

A study on the localization and distribution of GnRH and its receptor in rat submaxillary glands by immunohistochemical, in situ hybridization and RT-PCR.

We investigated the rat submaxillary gland for the presence of GnRH and GnRH receptors, the localization and colocalization of GnRH, GnRH receptor and their mRNA, and studied the sequence of GnRH receptor complementary DNA (cDNA) by immunohistochemistry, in situ hybridization and RT-PCR. The results showed that GnRH and GnRH receptor immunoreactive materials were colocalized in the epithelial cells of the serous acinus and glandular duct. The GnRH and GnRH receptor mRNA hybridization signals were detected in the above cells. The sequence obtained from the RT-PCR product was identical to the published cDNA sequence of GnRH receptor in the rat pituitary. The results suggested that the rat submaxillary gland was capable of synthesizing GnRH and GnRH receptors. GnRH may be involved in the functional regulation of the submaxillary gland through autocrine or paracrine activity.

Animals↗

PubMed1993

Identification of a polymorphism in the human neurotensin receptor gene.

A complementary DNA (cDNA) clone encoding the neurotensin receptor was isolated from a human substantia nigra cDNA library. The deduced amino acid sequence of this clone was almost identical to that of a cDNA for this receptor cloned from a previously described HT29 human colonic adenocarcinoma cell line. We found three base changes between the previously reported HT29 cDNA clone and the current cDNA clone. We investigated these changes by using polymerase chain reactions to amplify these areas from various human samples. One of the differences, which resulted in an amino acid change at AA194 (a leucine in the HT29 sequence was a phenylalanine in the current sequence), was found in some, but not in all, human samples. This finding represents genetic variability in human neurotensin receptors, the first such report for a peptide receptor. Both of these receptors, however, when expressed separately in transfected cell lines, had similar affinities for neurotensin and some related peptides.

Amino Acid Sequence↗

PubMed1999

Cloning of a cDNA encoding a nerve growth factor precursor from the Agkistrodon halys Pallas.

Based on the high conservation in the 5' and 3' untranslated regions of NGF cDNAs, oligonucleotides complementary to all these known sequences were synthesized. By RT-PCR, we successfully isolated the complementary DNA encoding NGF precursor from the Agkistrodon halys Pallas (a Chinese snake strain). The nucleotide sequence which presents 90.5%, 88.6% and 63.4% homology to that of Krait Bungarus multicinctus, cobra and human NGF respectively, encoded a prepro-NGF molecule with 241 amino acids and a mature NGF molecule with 119 amino acids. The NGF cDNA inserts were subcloned into pCDNA3 expression vector and then transfected into COS-7 cells. The supernatant of the transfected cells turned out NGF biological activity as assayed by the survival rate of PC12 pheochromocytoma cells.

Agkistrodon↗

PubMed2001

New molecular approaches for identifying novel targets, mechanisms, and biomarkers for prostate cancer chemopreventive agents.

Recently developed complementary DNA (cDNA) microarray technology allows simultaneous assessment of expression on many hundreds or thousands of genes simultaneously. This technology holds great promise for providing new insights into prostate carcinogenesis that will reveal new targets for preventive intervention strategies. In addition, this technology will deepen understanding of the means by which putative preventive compounds exert their effects, generating molecular genetic biomarkers of treatment efficacy. Several putative preventive agents are currently under investigation, and development of novel preventive strategies poses significant challenges. High throughput approaches, such as cDNA microarrays, will speed discovery and progress in prostate cancer chemoprevention.

Anticarcinogenic Agents↗

PubMed1995

Immunochemical characterization of recombinant and native tropomyosins as a new allergen from the house dust mite, Dermatophagoides farinae.

BACKGROUND: Two predominant mite species, Dermatophagoides farinae and Dermatophagoides pteronyssinus, are responsible for the immediate hypersensitivity reaction. Although a large number of antigens and allergens was detected in a whole mite culture extract, only the major allergens of Der I and II groups have been extensively studied. Much information on other important allergens remains to be accumulated. METHODS: A new allergen complementary DNA (cDNA) clone was isolated from a D. farinae protein cDNA library. The expression product of the cDNA and native tropomyosin from D. farinae were purified, characterized, and evaluated. RESULTS: An amino acid sequence deduced from the cDNA clone revealed significant homology with tropomyosins conserved in a wide range of animals. The amino acid sequences of two fragments obtained by degradation of the purified protein with cyanogen bromide coincided completely with the corresponding portions of the deduced amino acid sequence. The native tropomyosin reacted with specific IgE in the 31 sera tested at a high frequency (80.6%), comparable to that of Der f I (90.3%) and Der f II (74.2%). CONCLUSIONS: The cloned cDNA encodes tropomyosin. The high binding response of specific IgE antibodies to recombinant and native tropomyosins demonstrates that mite tropomyosin is an additional important allergen in house dust mite.

Allergens↗

PubMed1995

Detection of allergen-induced genes in peripheral blood mononuclear cells of patients with allergic asthma using subtractive hybridization.

When stimulated with mite antigens, peripheral blood mononuclear cells obtained from patients with mite allergy release eosinophil chemotactic factor, a type of protein. Complementary DNA libraries were formulated from both peripheral blood mononuclear cells stimulated with mite antigens and those not stimulated with mite antigens and underwent subtraction and differential screening. The authors obtained 13 genes for which expression had increased because of mite antigens. Of these, nine were mitochondrial genes; two, h-satellite DNA III; one, factor XIII; and one, ferritin heavy subunit. As a result of polymerase chain reaction on both cDNA libraries, IL-5 genes were amplified only from the cDNA library that had been stimulated with mite antigens.

Allergens↗

PubMed1996

Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen.

BACKGROUND: Pollen of grasses, such as Bermuda grass (Cynodon dactylon), represent a major cause of type I allergy. OBJECTIVE: In this report we attempted to clone and express a biologically active form of recombinant Cyn d 1, the major allergen of Bermuda grass pollen, in the yeast Pichia pastoris. METHODS: Clones encoding Cyn d 1 were isolated by screening a Bermuda grass pollen complementary DNA library with specific monoclonal antibodies and by polymerase chain reaction amplification. Recombinant Cyn d 1 was expressed in Escherichia coli and yeast. The expressed proteins were analyzed by Western blotting to assess binding to Cyn d 1-specific monoclonal antibodies and IgE from sera of patients allergic to Bermuda grass pollen. RESULTS: Two isoforms of Cyn d 1 were cloned. Recombinant Cyn d 1 expressed in bacteria bound two monoclonal antibodies raised against Cyn d 1 but was not recognized by IgE from sera of patients allergic to Bermuda grass pollen. Cyn d 1 expressed in yeast bound both the monoclonal antibodies and human IgE. CONCLUSION: An IgE-reactive Cyn d 1 was expressed in yeast but not in bacteria, suggesting that posttranslational modifications (e.g., glycosylation), which occur in eukaryotic cells such as yeast, are necessary for the production of a biologically active allergen.

Allergens↗

PubMed1996

IgE reactivity against a cross-reactive allergen in crustacea and mollusca: evidence for tropomyosin as the common allergen.

BACKGROUND: Although cross-reactivity between mollusks and other crustaceans in shrimp-sensitive subjects has been reported, the mechanism of this allergenic cross-reactivity has not been studied in detail. OBJECTIVE: To investigate this cross-reactivity in vitro, we have taken advantage of a complementary DNA that expresses tropomyosin, the immunodominant shrimp allergen. METHODS: Serum IgE from nine patients with known anaphylaxis to shrimp and five normal volunteers were analyzed by immunoblotting against 13 distinct crustaceans and mollusks. As additional antigens, muscle preparations were isolated from grasshopper, cockroach, fruit fly, chicken, and mouse. RESULTS: Sera from all patients, but not control subjects, reacted specifically with a 38 kd protein in all crustaceans and mollusks studied. In addition, 8 of 9 sera from patients, but from none of the normal control subjects, recognized proteins of various other molecular weights among the mollusk extracts studied. The 38 kd protein was identified as tropomyosin and was shown to share immunodominant epitopes among all species of crustaceans and mollusks tested by specific absorption studies. Moreover, sera from all nine subjects with shrimp allergy demonstrated IgE reactivities against grasshopper, cockroach, and fruit fly but not chicken or murine muscle. CONCLUSION: The allergic epitopes on tropomyosin are conserved among invertebrates including not only shellfish but also insects. This latter observation suggests that persons sensitive to shrimp should undergo further study for potential cross-reactive inhalant or ingested insect sensitivity.

Allergens↗

PubMed1997

Mapping of IgE-binding epitopes on the recombinant major group I allergen of velvet grass pollen, rHol l 1.

BACKGROUND: New and more successful approaches to diagnosis and therapy of allergic diseases require a more subtle understanding of the structure and the epitopes on the allergen molecule. OBJECTIVE: This study was done to obtain more information on the structure and the IgE-binding epitopes of a major allergen of velvet grass pollen, Hol l 1. METHODS: We cloned Hol l 1 from a complementary DNA library and performed B-cell epitope mapping with 21 recombinant fragments expressed as fusion proteins in Escherichia coli. The fragments were analyzed by Western blotting with sera from 50 different patients. RESULTS: The patients' sera individually recognized at least four different IgE-binding regions (amino acids 1 to 27, 61 to 76, 84 to 105, and 158 to 240). According to their binding patterns with these epitopes, they were divided into five groups. Most sera (92%) bound to the C-terminal peptide (158 to 240), which consists of more than 80 amino acids, whereas there was virtually no binding to smaller fragments covering this region. In contrast to the C-terminal peptide, the IgE-binding peptides on the N terminus and on the middle region of the molecule were of a smaller size (15 to 30 amino acids). CONCLUSIONS: The major group I allergen of velvet grass bears at least four different IgE-binding epitopes, which were individually recognized by sera from different patients. The C terminus represents the major IgE-binding region and contains at least one discontinuous IgE-binding epitope, whereas the N terminus and middle region of Hol l 1 seem to contain continuous IgE-binding epitopes.

Allergens↗

PubMed1999

Comparison of specific and random priming in the reverse transcriptase polymerase chain reaction for genotyping group A rotaviruses.

This study describes an approach to the molecular typing of rotaviruses which requires only a single RNA extraction and reverse transcription (RT) reaction using random primers. Random-primed RT provides complementary DNA (cDNA) which can be used not only for G- and P-typing polymerase chain reactions (PCR), but also for the detection of other RNA viruses which may act as enteric pathogens. It is a sensitive and specific method that can detect 10 virus particles/ml of 10% faecal suspension provided the cDNA is amplified in a nested typing-PCR. Of 121 specimens positive for rotavirus by EM and analysed using this method, only 8% could not be G- or P-genotyped. The untyped samples were tested again performing the RT reaction with G- and P-specific primers, achieving a 5% increase in sensitivity. Comparing G-genotyping against G-serotyping, 92% were genotyped through random priming RT-PCR whereas only 64% were serotyped using G-serotype specific monoclonal antibodies (MAbs).

Aged↗

PubMed2001

Molecular cloning of rat transmembrane domain protein of 40 kDa regulated in adipocytes and its expression in H9c2 cells exposed to ischemic hypoxia and reoxygenation.

We clone a 1230 bp complementary DNA encoding rat transmembrane domain protein of 40 kDa regulated in adipocytes (TPRA40), an orphan receptor, by reverse transcription-polymerase chain reaction using H9c2 cells derived from embryonic rat heart. The deduced amino acid sequence of rat TPRA40 consists of 369 amino acids and has a longer carboxyl terminus than that of the mouse protein. The level of TPRA40 mRNA decreases significantly throughout ischemic hypoxia and reoxygenation.

Adipocytes↗