ABSTRACT
Elevated peripheral proline is associated with psychiatric disorders, and there is evidence that proline is a neuromodulator. The proline dehydrogenase (PRODH) gene, which encodes the enzyme that catalyzes proline catabolism, maps to human chromosome 22q11.2, a region conferring risk of schizophrenia. In the Prodh-null mouse, an interaction between elevated peripheral proline and another 22q11.2 gene, catechol-O-methyltransferase (COMT), on neurotransmission and behavior has been reported. We explored the relationship between fasting plasma proline levels and COMT Val(158)Met genotype on symptoms (positive, negative and total) in schizophrenia patients. In an exploratory study we also examined symptom change in patients with bipolar disorder. There was a significant interaction between peripheral proline and COMT on negative symptoms in schizophrenia (P<0.0001, n=95). In COMT Val/Val patients, high proline was associated with low Scale for the Assessment of Negative Symptom (SANS) scores. In contrast, high proline was associated with high SANS scores in patients carrying a Met allele. The relationship between proline and COMT also appears to modify negative symptoms across psychiatric illness. In bipolar disorder, a significant interaction was also observed on negative-symptom change (P=0.007, n=43). Negative symptoms are intractable and largely unaddressed by current medications. These data indicate a significant interaction between peripheral proline and COMT genotype, influencing negative symptoms in schizophrenia and bipolar disorder. That high proline has converse effects on symptoms by COMT genotype, may have implications for therapeutic decisions.
Subject(s)
Bipolar Disorder/genetics , Catechol O-Methyltransferase/genetics , Proline/blood , Schizophrenia/genetics , Schizophrenic Psychology , Adult , Alleles , Bipolar Disorder/psychology , Female , Genetic Predisposition to Disease , Genotype , Humans , Male , Middle Aged , Polymorphism, Single NucleotideABSTRACT
Many children are identified as unaffected carriers for cystic fibrosis (CF) through newborn screening (NBS) programs. The aim of this study was to improve parental understanding of positive NBS results using an educational video in addition to genetic counseling. One hundred parents of infants identified as CF carriers through NBS were randomly assigned by household to either a genetic counseling only group or a genetic counseling and video group. All participants completed a knowledge-based questionnaire before, immediately after, and six weeks following genetic counseling. This included questions about resources accessed before and after the appointment. Seventy-two percent of participants accessed resources on their own prior to genetic counseling; these participants scored significantly higher on the pre-counseling questionnaire (p = 0.03). Post-counseling knowledge scores for both groups significantly improved after genetic counseling (p < 0.001). Post-counseling scores were significantly higher in the video group compared to the non-video group (p = 0.02). Knowledge was retained six weeks following genetic counseling. This study demonstrates the effectiveness of an educational video and reinforces the importance of genetic counseling following positive NBS results for CF.
Subject(s)
Cystic Fibrosis/diagnosis , Genetic Carrier Screening/methods , Neonatal Screening/psychology , Parents/education , Parents/psychology , Comprehension , Cystic Fibrosis/genetics , Female , Genetic Counseling/methods , Humans , Infant , Infant, Newborn , Male , Surveys and QuestionnairesABSTRACT
Microbial abundance is central to most investigations in microbial ecology, and its accurate measurement is a challenging task that has been significantly facilitated by the advent of molecular techniques over the last 20 years. Fluorescence in situ hybridization (FISH) is considered the gold standard of quantification techniques; however, it is expensive and offers low sample throughput, both of which limit its wider application. Quantitative PCR (qPCR) is an alternative that offers significantly higher throughput, and it is used extensively in molecular biology. The accuracy of qPCR can be compromised by biases in the DNA extraction and amplification steps. In this study, we compared the accuracy of these two established quantification techniques to measure the abundance of a key functional group in biological wastewater treatment systems, the ammonia-oxidizing bacteria (AOB), in samples from a time-series experiment monitoring a set of laboratory-scale reactors and a full-scale plant. For the qPCR analysis, we tested two different sets of AOB-specific primers, one targeting the 16SrRNA gene and one targeting the ammonia monooxygenase (amoA) gene. We found that there was a positive linear logarithmic relationship between FISH and the amoA gene-specific qPCR, where the data obtained from both techniques was equivalent at the order of magnitude level. The 16S rRNA gene-specific qPCR assay consistently underestimated AOB numbers.
Subject(s)
Bacteria/isolation & purification , In Situ Hybridization, Fluorescence/methods , Oxidoreductases/genetics , Polymerase Chain Reaction/methods , Sewage/microbiology , Ammonia/metabolism , Bacteria/enzymology , Bacteria/genetics , Betaproteobacteria/enzymology , Betaproteobacteria/genetics , Betaproteobacteria/isolation & purification , DNA, Bacterial/chemistry , DNA, Bacterial/isolation & purification , Linear Models , Oxidation-Reduction , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Species Specificity , Waste Disposal, FluidABSTRACT
We identified two families with an autosomal-recessive disorder manifested by severe enamel hypoplasia, delayed and failed tooth eruption, misshapen teeth, intrapulpal calcifications, and localized gingival hyperplasia. Genetic analyses identified novel FAM20A mutations associated with the disease phenotype in both families. The proband of Family 1 had an altered splice junction in Intron 1 (g.502011G>C; c.405-1G>C) and a missense mutation in Exon 8 (g.65094G>A; c.1207G>A; p.D403N). The missense mutation is notable because D(403) is strictly conserved among FAM20A homologues, and the corresponding defect in FAM20C caused osteosclerotic bone dysplasia and a loss of kinase activity. The proband at age 12 yrs tested negative for nephrocalcinosis. The proband and her affected father in Family 2 were homozygous for a single nucleotide deletion that altered a splice junction in Intron 10 (g.66622del; c.1361+4del). Minigene analyses demonstrated that this alteration precluded normal splicing. Immunohistochemistry (IHC) of mouse maxillary first molars localized FAM20A in secretory-stage ameloblasts, in odontoblasts, and in the eruption pathway. IHC of kidneys localized FAM20A in the renal tubules. We conclude that FAM20A is likely a secretory pathway kinase and that loss-of-function mutations cause pathology where its phosphorylations are necessary for normal development or homeostasis.
Subject(s)
Amelogenesis Imperfecta/genetics , Dental Enamel Proteins/genetics , Mutation/genetics , Nephrocalcinosis/genetics , Adenosine , Animals , Child , Child, Preschool , Cytosine , Dental Enamel Hypoplasia/genetics , Dental Pulp Calcification/genetics , Exons/genetics , Female , Follow-Up Studies , Genes, Recessive/genetics , Genetic Vectors/genetics , Gingival Hyperplasia/genetics , Guanine , HEK293 Cells , Homozygote , Humans , Introns/genetics , Male , Mice , Mutation, Missense/genetics , Phenotype , Polymorphism, Single Nucleotide/genetics , Sequence Deletion/genetics , Tooth Abnormalities/genetics , Tooth Eruption/geneticsABSTRACT
Elevated leukocyte cell numbers (leukocytosis), and monocytes in particular, promote atherosclerosis; however, how they become increased is poorly understood. Mice deficient in the adenosine triphosphate-binding cassette (ABC) transporters ABCA1 and ABCG1, which promote cholesterol efflux from macrophages and suppress atherosclerosis in hypercholesterolemic mice, displayed leukocytosis, a transplantable myeloproliferative disorder, and a dramatic expansion of the stem and progenitor cell population containing Lin(-)Sca-1(+)Kit+ (LSK) in the bone marrow. Transplantation of Abca1(-/-) Abcg1(-/-) bone marrow into apolipoprotein A-1 transgenic mice with elevated levels of high-density lipoprotein (HDL) suppressed the LSK population, reduced leukocytosis, reversed the myeloproliferative disorder, and accelerated atherosclerosis. The findings indicate that ABCA1, ABCG1, and HDL inhibit the proliferation of hematopoietic stem and multipotential progenitor cells and connect expansion of these populations with leukocytosis and accelerated atherosclerosis.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Atherosclerosis/physiopathology , Cholesterol/metabolism , Hematopoietic Stem Cells/physiology , Leukocytosis/physiopathology , Lipoproteins, HDL/metabolism , Lipoproteins/metabolism , Myeloid Progenitor Cells/physiology , ATP Binding Cassette Transporter 1 , ATP Binding Cassette Transporter, Subfamily G, Member 1 , ATP-Binding Cassette Transporters/genetics , Animals , Apolipoprotein A-I/genetics , Apolipoprotein A-I/metabolism , Atherosclerosis/metabolism , Atherosclerosis/therapy , Bone Marrow Transplantation , Cell Proliferation , Cells, Cultured , Hypercholesterolemia/metabolism , Leukocytosis/metabolism , Leukocytosis/therapy , Lipoproteins/genetics , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Multipotent Stem Cells/physiology , Myeloproliferative Disorders/metabolism , Myeloproliferative Disorders/physiopathology , Myeloproliferative Disorders/therapy , Phenotype , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Receptors, Interleukin-3/metabolism , Signal TransductionABSTRACT
A pilot-scale primary maturation pond was spiked with (15)N-labelled ammonia ((15)NH(4)Cl) and (15)N-labelled nitrite (Na(15)NO(2)), in order to improve current understanding of the dynamics of inorganic nitrogen transformations and removal in WSP systems. Stable isotope analysis of delta(15)N showed that nitrification could be considered as an intermediate step in WSP, which is masked by simultaneous denitrification, under conditions of low algal activity. Molecular microbiology analysis showed that denitrification can be considered a feasible mechanism for permanent nitrogen removal in WSP, which may be supported either by ammonia-oxidising bacteria (AOB) or by methanotrophs, in addition to nitrite-oxidising bacteria (NOB). However, the relative supremacy of the denitrification process over other nitrogen removal mechanisms (e.g., biological uptake) depends upon phytoplanktonic activity.
Subject(s)
Nitrogen/metabolism , Phytoplankton/metabolism , Sewage/analysis , Waste Disposal, Fluid/methods , Water Pollutants, Chemical/isolation & purification , Water Purification/methods , Ammonia/chemistry , Bacteria/metabolism , Biomass , Eukaryota/metabolism , Nitrites/chemistry , Nitrogen/chemistry , Nitrogen Isotopes/chemistry , Seasons , Temperature , Water MicrobiologyABSTRACT
We present an algorithm, PyroNoise, that clusters the flowgrams of 454 pyrosequencing reads using a distance measure that models sequencing noise. This infers the true sequences in a collection of amplicons. We pyrosequenced a known mixture of microbial 16S rDNA sequences extracted from a lake and found that without noise reduction the number of operational taxonomic units is overestimated but using PyroNoise it can be accurately calculated.
Subject(s)
Bacteria/genetics , Genetic Variation , Sequence Analysis, DNA/methods , Algorithms , Models, Genetic , Polymerase Chain Reaction , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , SoftwareABSTRACT
T cells from the spleens of B(19)/B(19) and B(21)/B(21) chickens infected with MDV JM-16 strain were fractionated by flow cytometry at 4, 10, 21 days post infection (d.p.i.). The expression of cytokine and viral genes (meq and glycoprotein B (gB)) was measured by real-time RT-PCR. It was determined that CD4(+) and CD8(+) T cells had both become infected with Marek's disease virus (MDV) in both chicken lines. There was significantly higher expression of meq in CD4(+) T cells compared to CD8(+) T cells at 10 and 21 d.p.i. Furthermore, at 10 and 21 d.p.i., there was a tendency for higher expression of meq in both T cell subsets of B(19) chickens compared to those of B(21) chickens. There were temporal changes in the expression of cytokines, interferon (IFN)-gamma, interleukin (IL)-18, IL-6, and IL-10, in various T cell subsets. Among these changes, there was an increase in IL-10 expression in both T cell subsets at different time points, especially in the susceptible line at 10 and 21 d.p.i. Our results indicate that cytokines could be differentially induced by MDV in CD4(+) and CD8(+) T cell subsets and that IL-10 may play a role in the modulation of immune response to MDV. However, an association between cytokine gene expression in T cell subsets and resistance or susceptibility to MD was not established.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Chickens/genetics , Chickens/immunology , Cytokines/genetics , Mardivirus/immunology , Marek Disease/genetics , Marek Disease/immunology , Animals , Chickens/virology , Gene Expression , Genes, Viral , Interferon-gamma/genetics , Interleukin-10/genetics , Interleukin-18/genetics , Interleukin-4/genetics , Interleukin-6/genetics , Mardivirus/genetics , Mardivirus/pathogenicity , Species Specificity , Time FactorsABSTRACT
The bursa of Fabricius serves as an important tissue in the process of Marek's disease virus (MDV) pathogenesis, since B cells of the bursa harbor the cytolytic phase of MDV replication cycle. In the present study, host responses associated with MDV infection in the bursa of Fabricius of chickens were investigated. The expression of MDV phosphoprotein (pp)38 antigen, MDV glycoprotein (gB) and MDV viral interleukin (vIL)-8 transcripts was at the highest at 4 days post-infection (d.p.i.) and then showed a declining trend. On the contrary, the expression of meq (MDV EcoRI Q) gene as well as the viral genome load increased gradually until day 14 post-infection. The changes in viral parameters were associated with significantly higher infiltration of macrophages and T cell subsets, particularly CD4+ T cells into the bursa of Fabricius. Of the genes examined, the expression of interferon (IFN)-alpha, IFN-gamma genes and inducible nitric oxide synthase (iNOS) was significantly up-regulated in response to MDV infection in the bursa of Fabricius. The results suggest a role for these cells and cytokines in MDV-induced responses in the bursa of Fabricius.
Subject(s)
Bursa of Fabricius/virology , Mardivirus/pathogenicity , Animals , Animals, Newborn , Antigens, Viral/metabolism , Base Sequence , Bursa of Fabricius/immunology , Bursa of Fabricius/pathology , Chickens , Cytokines/genetics , DNA Primers/genetics , Gene Expression , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Macrophages/pathology , Macrophages/virology , Mardivirus/genetics , Mardivirus/immunology , Mardivirus/physiology , Marek Disease/genetics , Marek Disease/immunology , Marek Disease/pathology , Marek Disease/virology , Nitric Oxide Synthase Type II/genetics , Phosphoproteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , T-Lymphocyte Subsets/pathology , T-Lymphocyte Subsets/virology , Virulence , Virus ReplicationABSTRACT
In Marek's disease virus infection, feather follicle epithelium (FFE) constitutes the site of formation of infectious virus particles and virus shedding. The objective of this study was to characterize cellular and cytokine responses as indicators of cell-mediated immune response in FFE and associated feather pulp following immunization against Marek's disease. Analysis of feather tips collected between 4 and 28 days post-immunization (d.p.i.) from chickens vaccinated post-hatch with either CVI988/Rispens or herpesvirus of turkeys revealed that replication of these vaccine viruses started at 7d.p.i., peaked by 21d.p.i., and subsequently, showed a declining trend. This pattern of viral replication, which led to viral genome accumulation in feather tips, was associated with infiltration of T cell subsets particularly CD8+ T cells into the feather pulp area and the expression of cytokine genes such as interferon-gamma, which is an indication of elicitation of cell-mediated immune responses at the site of virus shedding.
Subject(s)
Feathers/immunology , Marek Disease/prevention & control , Poultry Diseases/prevention & control , Viral Vaccines/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , Chickens , Cytokines/immunology , Cytokines/metabolism , Feathers/virology , Immunohistochemistry/veterinary , Marek Disease/immunology , Poultry Diseases/immunology , Poultry Diseases/virology , Reverse Transcriptase Polymerase Chain Reaction/veterinaryABSTRACT
Control measures are ineffective in curtailing Marek's disease virus (MDV) infection and replication in the feather follicle epithelium (FFE). Therefore, vaccinated birds which subsequently become infected with MDV, shed the virulent virus although they remain protected against disease. The present study investigated host responses generated against MDV infection in the feather. We observed that in parallel with an increase in viral genome load and viral replication in the feather, there was a gradual but progressive increase in infiltration of CD4+ and CD8+ T cells into the feather pulp of MDV-infected chickens, starting on day 4 and peaking by day 10 post-infection. Concomitant with infiltration of T cells, the expression of interleukin (IL)-18, IL-6, interferon (IFN)-gamma and major histocompatibility complex class I genes was significantly enhanced in the feather pulp of MDV-infected chickens. The finding that host responses are generated in the feather may be exploited for developing strategies to control MDV infection in the FFE, thus preventing horizontal virus transmission.
Subject(s)
Feathers/immunology , Mardivirus/immunology , Marek Disease/immunology , Animals , Antigens, Viral/genetics , Base Sequence , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Chickens , DNA Primers/genetics , DNA, Viral/genetics , DNA, Viral/isolation & purification , Feathers/pathology , Feathers/virology , Gene Expression , Genes, MHC Class I , Genes, MHC Class II , Major Histocompatibility Complex , Mardivirus/genetics , Mardivirus/isolation & purification , Mardivirus/pathogenicity , Marek Disease/prevention & control , Marek Disease/transmission , Marek Disease/virology , Viral Envelope Proteins/geneticsABSTRACT
Chicken major histocompatibility complex (MHC) B21 and B19 haplotypes are associated with resistance and susceptibility to Marek's disease (MD), respectively. T-cell-mediated immune response is crucial in coordinating protection against Marek's disease virus (MDV) infection, but it has been difficult to identify and characterize antigen-specific T-cells. MHC class II tetramers and oligomers have been widely used for characterization of antigen-specific T-cells in the context of infectious and autoimmune diseases. Thus, the objective of this study was to synthesize chicken MHC class II oligomers of B21 and B19 haplotypes for the future identification of antigen-specific T-cells. To achieve this objective, full-length coding sequences of chicken MHC class II B21 and B19 molecules were amplified and the molecules were expressed as fusion proteins, carrying Fos and Jun leucine zipper (LZ), histidine-tag and biotin ligase recognition site sequences, using a baculovirus expression system. Recombinant MHC-II were loaded with self-peptides, which stabilized the heterodimer in SDS-PAGE and allowed the detection of these molecules in Western blots with a conformation-specific anti-chicken MHC class II antibody. Biotinylated MHC molecules were conjugated to streptavidin to form oligomers, which were resolved under the transmission electron microscope through immuno-gold labelling, thus confirming success of oligomerization. In conclusion, chicken MHC class II oligomers may be used in the future to study the antigen-specific CD4+ T-cell compartment.
Subject(s)
Baculoviridae , Histocompatibility Antigens Class II/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Animals , CD4-Positive T-Lymphocytes/immunology , Cell Line , Chickens/genetics , Genetic Predisposition to Disease/genetics , Haplotypes , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Leucine Zippers/genetics , Marek Disease/genetics , Marek Disease/immunology , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-jun/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SpodopteraSubject(s)
Evolution, Molecular , Galliformes/genetics , Genes, MHC Class II/genetics , Phylogeny , Polymorphism, Genetic , Amino Acid Substitution/genetics , Animals , Base Sequence , Cloning, Molecular , Cluster Analysis , DNA Primers , Molecular Sequence Data , Sequence Alignment , Sequence Analysis, DNAABSTRACT
Stromal cell derived factor-1, SDF-1, belongs to the CXC family of chemokines and has been identified in mammals, amphibians, and fish. This chemokine has a diverse array of functions in organogenesis, hematopoeisis, B cell development and recruitment of immune system cells. Here, we report the cloning of the chicken SDF-1 ortholog and examine its temporal and spatial expression. The chicken SDF-1 cDNA contained an open reading frame encoding a predicted protein of 89 amino acids, which shared 40-75% identity to SDF-1 protein in other species. Protein folding simulation predicted a tertiary structure very similar to that obtained for human SDF-1. Recombinant chicken SDF-1 was produced using a prokaryotic expression system and the recombinant protein was shown to be biologically active in a calcium flux assay. The SDF-1 gene was found to be expressed ubiquitously and constitutively in adult tissues and was present as early as the primitive streak stage of chicken embryos.
Subject(s)
Chemokines, CXC/genetics , Chick Embryo/immunology , Chickens/immunology , Amino Acid Sequence , Animals , Chemokine CXCL12 , Chemokines, CXC/chemistry , Chemokines, CXC/metabolism , Cloning, Molecular , Humans , Molecular Sequence Data , RNA, Messenger/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
BACKGROUND: and aims: In neonates the gastrointestinal tract is exposed to food and bacterial antigens at a time when the gut mucosal immune system has not developed the ability to induce oral tolerance. This increases the risk for an inappropriate immune response to oral antigens. Transforming growth factor beta (TGF-beta) is an immunoregulatory cytokine present in high concentration in maternal milk. Interleukin 18 (IL-18) is a cytokine that mediates early immune events, and drives T cell development. We assessed the role of TGF-beta in mediating mucosal immune development and specifically the effect on endogenous IL-18. METHODS: Rat pups were randomly assigned to the following groups, naturally suckled, maternal milk via cannula, and formula fed with and without physiological levels of TGF-beta2. A comparison of the immune response profile was then carried out. Cytokine profiles, dendritic cell, intestinal mast cell, and eosinophil numbers were assessed. RESULTS: We show that feeding formula deficient in TGF-beta2 resulted in accumulated IL-18 protein release from intestinal epithelial cells and IL-18 mRNA up regulation. A proinflammatory cytokine profile resulted in the gut, along with increased numbers of activated dendritic cells, eosinophils, and mast cells. Supplementation of the formula with TGF-beta2 down regulated the proinflammatory cytokine mRNA as well as the number of activated lymphocytes, eosinophils, mast cells, CD80, and CD86 positive dendritic cells. CONCLUSION: The data suggests an important role for maternal milk, in regulating immune responses after exposure to food antigens, which might otherwise induce deleterious immune responses in the intestine of suckling neonates. This regulation is potentially mediated by milk TGF-beta2, as well as endogenous IL-18.
Subject(s)
Interleukin-18/immunology , Intestinal Mucosa/immunology , Milk/immunology , Transforming Growth Factor beta/immunology , Animals , Animals, Suckling , Antigens, CD/immunology , Blotting, Western/methods , Cell Count/methods , Dendritic Cells/immunology , Down-Regulation/immunology , Eosinophils/immunology , Female , Fluorescent Antibody Technique/methods , Ileum/immunology , Interleukin-18/analysis , Intestine, Small/immunology , Lymphocyte Activation/immunology , Mast Cells/immunology , RNA, Messenger/analysis , Rats , Rats, Wistar , Transforming Growth Factor beta/analysis , Up-Regulation/immunologyABSTRACT
BACKGROUND: Ussing chambers are commonly utilized for in vitro investigations into gastrointestinal permeability. However, their sensitivity and applicability to the small intestine have not been well characterized. METHODS: In order to investigate the effects of experimentally induced damage and the relative contribution of the mucosa and muscularis externa layers to transmural permeability in the small intestine, stomach and colon, normal rat intestinal tissues were mounted in Ussing chambers with or without removal of the muscularis externa or mucosal layers. Gastric tissues were damaged in vivo by exposure to indomethacin (100 mg kg(-1)), while ileal tissues were damaged in vitro by 0.4 M NaCl. Tissue damage was assessed histologically, while permeability parameters included conductance (G), potential difference (PD) and mucosal to serosal flux of horseradish peroxidase (HRP). RESULTS: Damage localized to the tissue edges (edge damage) accounted for 25%-50% of the exposed epithelial length in the ileum, while less than 20% of stomach and colon epithelium was affected by edge damage. In the damaged stomach, a 20% reduction in epithelialization was accompanied by increases in G (P < 0.001) and HRP (P < 0.01) flux. Removal of the muscularis externa did not affect mucosal viability in the undamaged ileum or colon although HRP flux in the colon, but not ileum, was increased (P < 0.01). Removal of the ileal mucosa produced increases in G and HRP flux, while PD was maintained. CONCLUSION: We conclude that the Ussing chamber technique is suitable for application to studies of gastric and colonic permeability in rats. However, owing to the prevalence and extent of edge damage in the small intestine, we would caution against the use of this technique for permeability studies in this region of the gastrointestinal tract in the rat.
Subject(s)
Colon/metabolism , Diffusion Chambers, Culture , Gastric Mucosa/metabolism , Intestinal Mucosa/metabolism , Animals , Colon/drug effects , Disease Models, Animal , Gastric Mucosa/drug effects , In Vitro Techniques , Indomethacin/pharmacology , Intestinal Mucosa/drug effects , Intestines/drug effects , Male , Permeability , Rats , Rats, Wistar , Reproducibility of Results , Sensitivity and Specificity , Sodium Chloride/pharmacology , Stomach/drug effectsABSTRACT
During early postnatal development, the intestine is highly responsive to LR(3)IGF-I administration but refractory to IGF-I, in contrast to the mature intestine. Given that LR(3)IGF-I is an IGF-I analog that binds poorly to IGF binding proteins, the response of the intestine is likely to reflect regulation of IGF-I bioactivity by IGF binding proteins. This study measures the delivery of exogenous IGF-I peptides to the intestine in preweaning (d-19) and adult rats to determine whether a correlation exists with the potency advantage of LR(3)IGF-I in the intestine during postnatal development. IGF-I or LR(3)IGF-I (2.6 microg/kg) was spiked with corresponding (125)I-labeled peptide (10 x 10(6) cpm) and administered iv as a bolus (n = 5-6/group) with blood and tissue samples collected 5 and 10 min post injection. In both age groups, the levels of (125)I-IGF-I retained in the blood at both 5 and 10 min were higher than the levels of (125)I-LR(3)IGF-I, consistent with the slower clearance rate for the native peptide. In the gastrointestinal tract, the levels of (125)I-LR(3)IGF-I per gram of tissue were 37-50% higher than (125)I-IGF-I. Surprisingly, there was little difference in the relative delivery of LR(3)IGF-I to IGF-I to the intestine, across developmental age. Although bolus iv-injected LR(3)IGF-I was cleared more rapidly from the circulation than IGF-I and was subsequently delivered to the intestine in higher amounts than the native peptide, the ratio of LR(3)IGF-I to IGF-I in gut tissues was approximately 2:1 in both age groups. Hence, selective delivery to the gut is unlikely to explain the markedly higher potency of (125)I-LR(3)IGF-I in stimulating growth of the preweaning vs. adult intestine.
Subject(s)
Aging/metabolism , Animals, Suckling/metabolism , Insulin-Like Growth Factor I/analogs & derivatives , Insulin-Like Growth Factor I/pharmacokinetics , Intestinal Mucosa/metabolism , Animals , Biological Availability , Female , Humans , Insulin-Like Growth Factor Binding Proteins/metabolism , Insulin-Like Growth Factor I/metabolism , Peptide Fragments/blood , Rats , Rats, WistarABSTRACT
In contrast to the adult gut, the immature intestine is refractory to subcutaneously infused insulin-like growth factor I (IGF-I). IGF binding protein (IGFBP) mRNA expression was characterized in intestinal tissues from 6-, 19-, and 90-day-old rats to determine if changes in local expression could account for this age-related change in IGF-I potency. For all age groups, IGFBP-3 to -6, but not IGFBP-1 or -2, were detected by Northern blot analysis. IGFBP-3, -4, and -5 were more intensely expressed in the 6-day-old rat intestine compared with weanling or adult tissue. In contrast, IGFBP-6 expression peaked at the time of weaning. In situ hybridization showed IGFBP-3 to -6 expression was confined to cells of the lamina propria and submucosa and also in the muscularis layer for IGFBP-5. Furthermore, the pattern of IGFBP-5 localization in the intestine changed with development. The findings indicate that the expression of IGFBP-3 to -6 is higher in the immature intestine compared with the adult intestine, suggesting locally produced IGFBPs may inhibit systemically derived IGF-I action in the intestine. Therefore, changes to local IGFBP expression may contribute to the varying response of the rat intestine to IGF-I peptides during postnatal development.
Subject(s)
Gene Expression , Insulin-Like Growth Factor Binding Proteins/genetics , Intestine, Small/growth & development , Aging , Animals , Blotting, Northern , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Female , In Situ Hybridization , Insulin-Like Growth Factor Binding Protein 1/analysis , Insulin-Like Growth Factor Binding Protein 1/genetics , Insulin-Like Growth Factor Binding Protein 2/analysis , Insulin-Like Growth Factor Binding Protein 2/genetics , Insulin-Like Growth Factor Binding Protein 3/analysis , Insulin-Like Growth Factor Binding Protein 3/genetics , Insulin-Like Growth Factor Binding Protein 4/analysis , Insulin-Like Growth Factor Binding Protein 4/genetics , Insulin-Like Growth Factor Binding Protein 5/analysis , Insulin-Like Growth Factor Binding Protein 5/genetics , Insulin-Like Growth Factor Binding Protein 6/analysis , Insulin-Like Growth Factor Binding Protein 6/genetics , Insulin-Like Growth Factor Binding Proteins/analysis , Insulin-Like Growth Factor Binding Proteins/blood , Intestine, Small/chemistry , Male , RNA, Messenger/analysis , Rats , Rats, WistarABSTRACT
RBP16 is a mitochondrial Y-box protein from the parasitic protozoan Trypanosoma brucei that binds guide RNAs and ribosomal RNAs. It is comprised of an N-terminal cold-shock domain and a C-terminal domain rich in glycine and arginine residues, resembling the RGG RNA-binding motif. Arginine residues found within RGG domains are frequently asymmetrically dimethylated by a class of enzymes termed protein arginine methyltransferases (PRMTs). As Arg-93 of RBP16 exists in the context of a preferred sequence for asymmetric arginine dimethylation (G/FGGRGGG/F), we investigated whether modified arginines are present in native RBP16 by MALDI-TOF and post-source decay analyses. These analyses confirmed that Arg-93 is dimethylated. In addition, Arg-78 exists as an unmodified or as a monomethylated derivative, and Arg-85 is present in forms corresponding to the unmodified, di-, and tri-methylated state. While Arg-93 is apparently constitutively dimethylated, the methylation of Arg-78 and Arg-85 is mutually exclusive. Furthermore, whole cell extracts from procyclic form T. brucei are able to methylate bacterially expressed RBP16 (rRBP16), as well as endogenous proteins, in the presence of S-adenosyl-L-[methyl-3H]methionine. While assays of mitochondrial extracts suggest a small amount of PRMT may also be present in this subcellular compartment, the majority of trypanosome PRMT activity is extramitochondrial. We show that rRBP16 is methylated in trypanosome extracts through the action of a type I methyltransferase as well as serving as a substrate for heterologous mammalian type I PRMTs. In addition, we demonstrate the presence of type II PRMT activity in trypanosome cell extracts. These results suggest that protein arginine methylation is a common posttranslational modification in trypanosomes, and that it may regulate the function of RBP16.
Subject(s)
Arginine/metabolism , Mitochondria/metabolism , Protein Processing, Post-Translational , Protozoan Proteins , RNA, Guide, Kinetoplastida/metabolism , RNA-Binding Proteins/metabolism , Trypanosoma brucei brucei/metabolism , Animals , Mass Spectrometry , Methylation , Protein-Arginine N-Methyltransferases/metabolism , RNA Editing , RNA, Guide, Kinetoplastida/genetics , RNA-Binding Proteins/genetics , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/growth & developmentABSTRACT
Transforming growth factor-beta (TGF-beta) and insulin-like growth factor (IGF-I) can attenuate drug-induced cell death in epithelial cells. Since milk whey contains a mixture of these and other growth factors, we evaluated mitogenic bovine whey extract (MBWE) for protective activity against chemotherapy drug damage in cultured epithelial cells (mink lung, MvlLu). Etoposide and vinblastine reduced cell survival by up to 90%. This was attenuated by the addition of MBWE before and during drug exposure, but not following drug removal. MBWE was compared with individual growth factors known to be present in the mixture. IGF-I and platelet-derived growth factor were ineffective, whereas TGF-beta2 induced growth inhibition and cell survival, with a maximum response at 3 ng/ml. TGF-beta2 bioactivity was also demonstrated by showing that acidification of MBWE (A-MBWE), to activate TGF-beta2, enhanced its growth inhibitory and chemoprotective activities 60- and 12-fold, respectively. However, MBWE contained additional protective factors. When TGF-beta2 and the MBWE preparations were compared, on the basis of growth inhibition equivalents, MBWE protected cells against drug toxicity at concentrations an order of magnitude lower than with TGF-beta2 or A-MBWE. Immunoneutralization of the TGF-beta present in MBWE and A-MBWE eliminated all growth inhibitory activity but not all cell survival activity. We conclude that the MBWE preparations are cytoprotective against two chemotherapy drugs when added before and during drug exposure. TGF-beta contributes to this activity, but the extracts contain other factors that promote the survival of epithelial cells after chemotherapy drug exposure.