ABSTRACT
The purpose of this study was to develop the Complex Trauma Screener (CTS), a brief screener (seven items) of the ICD-11 trauma disorders that can be used in "quick-paced" facilities. We examined the factor structure of the CTS in two separate samples: civilian college students (N = 823) and military veterans (N = 130) who reported exposure to at least one traumatic event. Confirmatory factor analyses (CFAs) supported two highly-correlated factors (post-traumatic stress disorder [PTSD] and Disturbances in Self-Organization [DSO]) that loaded on the ICD-11-consistent items. The model fit indices indicated good to excellent model fit in both samples, and the internal consistencies for the scales were borderline to good (α = 0.68-0.86). Supplementary analyses supported the gender invariance of the CFA model in the civilian student sample, as well as convergent (with another trauma inventory) and discriminant validity (with borderline disorder features, depression, and mania) of the CTS in both samples. The CTS is, to our knowledge, the shortest instrument designed to measure the ICD-11 trauma disorders and is ideal for "fast-paced" facilities that have significant assessment time restraints. The CTS is, therefore, is a psychometrically-validated instrument that can help mental health professionals efficiently screen adults for ICD-11 trauma disorders.
Subject(s)
Stress Disorders, Post-Traumatic , Adult , Humans , Stress Disorders, Post-Traumatic/diagnosis , Stress Disorders, Post-Traumatic/psychology , International Classification of Diseases , Psychiatric Status Rating Scales , Personality , Factor Analysis, StatisticalABSTRACT
The size of the esophageal hiatus is clinically important for preserving the integrity of the lower esophageal sphincter mechanism. The purpose of this study was to systematically establish the mean hiatal surface area (HSA) for normal North American adults under physiologic conditions and assess the relationship between sex and age on HSA. Multi-Detector Computer Tomogram (MDCT) images of the esophageal hiatus in 119 healthy adult subjects (61 males and 58 females with an age range of 24-88 years) were retrospectively analyzed using the multi-planar reconstruction (MPR) technique to directly measure their hiatal length (long axis), width (short axis) and surface area at end inspiration. The mean HSA for males was 2.88 cm2, with a standard deviation of 0.74 cm2. The mean HSA for females was 2.51 cm2, with a standard deviation of 0.68 cm2. There was a statistically significant difference in HSA between males and females (p = 0.0053); however, there was no statistically significant difference between the HSA among different age groups (p = 0.8439). Similarly, significant differences between males and females were demonstrated in both the length (p = 0.0263) and width (p = 0.0311) measurements, but there was no evidence of an association of these parameters with age. For the first time, the normal size of the hiatus at end inspiration has been established noninvasively for a population of healthy adults from MDCT images.
Subject(s)
Gastroesophageal Reflux , Hernia, Hiatal , Adult , Aged , Aged, 80 and over , Female , Gastroesophageal Reflux/complications , Gastroesophageal Reflux/surgery , Hernia, Hiatal/complications , Hernia, Hiatal/surgery , Humans , Male , Middle Aged , Multidetector Computed Tomography/methods , North America , Retrospective Studies , Young AdultABSTRACT
We examined roles of loading and inflammation on forearm bones in a rat model of upper extremity overuse. Trabecular structure in distal radius and ulna was examined in three groups of young adult rats: 1) 5% food-restricted that underwent an initial training period of 10 min/day for 5 weeks to learn the repetitive task (TRHF); 2) rats that underwent the same training before performing a high repetition high force task, 2 hours/day for 12 weeks (HRHF); and 3) food-restricted only (FRC). Subsets were treated with oral ibuprofen (IBU). TRHF rats had increased trabecular bone volume and numbers, osteoblasts, and serum osteocalcin, indicative of bone adaptation. HRHF rats had constant muscle pulling forces, showed limited signs of bone adaptation, but many signs of bone resorption, including decreased trabecular bone volume and bone mineral density, increased osteoclasts and bone inflammatory cytokines, and reduced median nerve conduction velocity (15%). HRHF+IBU rats showed no trabecular resorptive changes, no increased osteoclasts or bone inflammatory cytokines, no nerve inflammation, preserved nerve conduction, and increased muscle voluntary pulling forces. Ibuprofen treatment preserved trabecular bone quality by reducing osteoclasts and bone inflammatory cytokines, and improving muscle pulling forces on bones as a result of reduced nerve inflammation.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Bone Resorption , Bone and Bones/drug effects , Cumulative Trauma Disorders/prevention & control , Ibuprofen/pharmacology , Animals , Bone and Bones/diagnostic imaging , Cumulative Trauma Disorders/complications , Disease Models, Animal , Female , Rats , Rats, Sprague-Dawley , Tomography, X-Ray ComputedABSTRACT
Cardiogenic fate maps are used to address questions on commitment, differentiation, morphogenesis and organogenesis of the heart. Recently, the accuracy of classical cardiogenic fate maps has been questioned, raising concerns about the conclusions drawn in studies based on these maps. We present accurate fate maps of the heart-forming region (HFR) in avian embryos and show that the putative cardiogenic molecular markers Bmp2 and Nkx2.5 do not govern the boundaries of the HFR as suggested in the literature. Moreover, this paper presents the first fate map of the HFR at stage 4 and addresses a void in the literature concerning rostrocaudal patterning of heart cells between stages 4 and 8.
Subject(s)
Body Patterning , Heart/embryology , Transcription Factors , Transforming Growth Factor beta , Xenopus Proteins , Animals , Biomarkers , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/genetics , Chick Embryo , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/genetics , Mesoderm , Myocardium/metabolism , Stem CellsABSTRACT
During chick embryogenesis, cells destined to form cardiac myocytes are located within the primitive streak at stage 3 in the same relative anterior-posterior distribution as in the prelooped heart. The most rostral cells contribute to the extreme anterior pole of the heart, the bulbus cordis, and the most caudal to the extreme posterior end, the sinoatrial region. After gastrulation, these cells commit to the myocyte lineage and, retaining their relative positions, migrate to the anterior lateral plate. From stages 5 to 10 they diversify into atrial and ventricular myocytes, with the former located posteriorly and the latter, anteriorly. To determine the effect of a change in the rostro-caudal position of these cells on their diversification, anterior lateral plate mesoderm and the underlying endoderm were cut and rotated 180 degrees along the longitudinal axis, at stages 4-8. The subsequent diversification of these precursor cells into atrial and ventricular myocytes was examined using lineage-specific markers. Our results showed that altering location along the longitudinal axis through stage 6 changed the normal fate of a precursor cell. The orientation of the overlying ectoderm did not alter normal morphogenesis or determination of fate.
Subject(s)
Heart/embryology , Muscle Fibers, Skeletal/cytology , Myocardium/cytology , Animals , Carbocyanines , Cell Differentiation/physiology , Chick Embryo , Ectoderm/cytology , Fluorescent Dyes , Heart Atria/cytology , Heart Atria/embryology , Heart Ventricles/cytology , Heart Ventricles/embryology , Mesoderm/cytology , RotationABSTRACT
We report on the cellular and molecular effects of acute and chronic hemodynamic overload on the fetal sheep heart. In one fetus of a twin gestation, the pulmonary artery was banded to create a condition of hemodynamic pressure overload in the right ventricle. The effects of this overload on the right ventricle (RV), left ventricle (LV), and intra-ventricular septum (IVS) of the heart were studied and compared to that in a control, unbanded twin fetus. At the cellular level, the histological data showed that both the size of the nuclei and the overall cell size of cardiac myocytes were increased after five days of banding; although, with one hour of banding no effects were detected at the cellular level. Based on prior studies on connexins which showed their involvement in differentiation, remodeling, and response to load we looked at their expression in control and experimental hearts. At the molecular level, changes in expression of connexin isoforms, the main gap junction protein in the heart, were observed after both one hour and five days of banding. Changes were observed in expression of connexins 40, 43, and 45. For connexin 43 there was a significant reduction confined to the right ventricle, in the chronically treated fetus, whereas, connexins 40 and 45 expression decreased after acute overload. These early molecular changes are significant because the "functional syncytium" of the myocardium is established through the gap junction connections. Alterations in connexin isoform expression affect the development, mechanical, and electrophysiological properties of the heart muscle. These changes may contribute to the ultimate result of continued hemodynamic stress on the right ventricle: heart failure.
Subject(s)
Fetal Heart/pathology , Heart Ventricles/pathology , Hypertension, Pulmonary/pathology , Animals , Blotting, Northern , Connexins/genetics , Disease Models, Animal , Female , Fetal Heart/embryology , Fetal Heart/metabolism , Follow-Up Studies , Gestational Age , Heart Ventricles/embryology , Heart Ventricles/metabolism , Hypertension, Pulmonary/physiopathology , Organ Size , Pregnancy , Pulmonary Wedge Pressure , RNA/biosynthesis , SheepABSTRACT
The Myb gene family currently consists of three members, named A-, B- and c-myb. These genes encode nuclear proteins that bind DNA in a sequence-specific manner and function as regulators of transcription. In adult male mice, A-myb is expressed predominantly in male germ cells. In female mice, A-myb is expressed in breast ductal epithelium, mainly during pregnancy-induced ductal branching and alveolar development. We report here that mice homozygous for a germline mutation in A-myb develop to term but show defects in growth after birth and male infertility due to a block in spermatogenesis. Morphological examination of the testes of A-myb-/- males revealed that the germ cells enter meiotic prophase and arrest at pachytene. In adult homozygous null A-myb female mice, the breast epithelial compartment showed underdevelopment of breast tissue following pregnancy and the female mice were unable to nurse their newborn pups. These results demonstrate that A-myb plays a critical role in spermatogenesis and mammary gland development.
Subject(s)
Mammary Glands, Animal/physiology , Proto-Oncogene Proteins/physiology , Spermatogenesis/physiology , Testis/physiology , Trans-Activators/physiology , Animals , Female , Gene Expression , Gene Targeting , Germ-Line Mutation , Infertility, Male/genetics , Male , Mammary Glands, Animal/anatomy & histology , Mammary Glands, Animal/embryology , Mammary Glands, Animal/growth & development , Meiosis , Mice , Molecular Sequence Data , Pregnancy , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-myb , Spermatogenesis/genetics , Stem Cells , Testis/anatomy & histology , Testis/embryology , Testis/growth & development , Trans-Activators/geneticsABSTRACT
The cardiac muscle-specific enhancer-promoter of the slow/cardiac troponin C (cTnC) gene contains five protein binding regions, four of which bind cardiac-myocyte specific proteins. We screened a stage 11 chick embryo expression library with a double-stranded oligonucleotide probe consisting of one of these regions, CEF-1. One of the clones obtained was the chicken high mobility group protein, HMG-2. An electrophoretic gel mobility shift assay (EMSA) showed a specific binding interaction between the HMG-2 protein and the dsDNA CEF-1 probe. The cardiac-specific enhancer region of cTnC contains at least one possible HMG binding region and it is in the CEF-1 sequence overlapping a known GATA-4 binding site. Mutation of the nucleotide sequence of this HMG binding region diminishes its protein binding ability and markedly decreases its cardiac specific transcriptional activity. HMG-2 is a DNA bending protein that is predominantly found in the nucleus in proliferating cells and in the cytoplasm of terminally differentiated cells. It is an integral and stabilizing factor in the transcription activation nucleoprotein complex and is often described as an 'architectural transcription factor'. It markedly stimulates the transcription of many genes, often in association with tissue-specific transcription factors. We believe that the presence of HMG-2 in the enhancer-promoter binding protein complex of cTnC augments DNA bending and facilitates the DNA binding and interaction of other tissue-specific factors (e.g. GATA-4, which also binds to this region). This would result in increased transcription of the cTnC gene during the proliferation phase of embryonic cardiac myocyte development.
Subject(s)
Enhancer Elements, Genetic , High Mobility Group Proteins/metabolism , Myocardium/metabolism , Promoter Regions, Genetic , Troponin C/genetics , Animals , Base Sequence , Chick Embryo , DNA , DNA-Binding Proteins/metabolism , GATA4 Transcription Factor , Molecular Sequence Data , Transcription Factors/metabolismABSTRACT
A novel cDNA, pCMF1, which is expressed exclusively and transiently in the myogenic cells of the differentiating chicken heart was isolated and characterized. The full-length cDNA of pCMF1 has one open reading frame encoding 1538 predicted amino acids. While computer analysis predicts the presence of specific structural motifs, the overall sequence of pCMF1 is unique. The pattern of pCMF1 gene expression during heart formation was determined by whole-mount in situ hybridization. pCMF1 is transiently expressed within the myogenic cells of the primitive heart tube from stages 9 to 18 and is not detected in the heart or any other tissue thereafter. A replication-deficient retrovirus was used to mediate pCMF1 antisense expression in cardiogenic mesoderm. These analyses determined that the presence of pCMF1 antisense sequences disrupted myosin heavy chain expression during cardiac mesoderm differentiation. pCMF1 antisense had no effect on myosin heavy chain expression in differentiated cardiac myocytes. These data suggest a potential function for pCMF1 during cardiac myogenesis.
Subject(s)
Avian Proteins , Cell Differentiation/genetics , Gene Expression Regulation, Developmental , Heart/embryology , Muscle Proteins/genetics , Myocardium/cytology , Amino Acid Sequence , Animals , Antisense Elements (Genetics) , Cells, Cultured , Chick Embryo , Cloning, Molecular , DNA, Complementary , Fluorescent Antibody Technique , Histocytochemistry , In Situ Hybridization , Molecular Sequence Data , Muscle Proteins/chemistry , Myocardium/metabolism , Myosin Heavy Chains/biosynthesis , Myosin Heavy Chains/genetics , Protein Structure, Secondary , RNA Probes , Retroviridae/genetics , Transcription, GeneticABSTRACT
We recently reported the cloning and sequencing of the mouse A-myb proto-oncogene cDNA and the abundant expression of this mRNA primarily in the testis of adult mice. The A-myb mRNA is detectable by in situ hybridization specifically in the spermatogenic cells, and is downregulated during terminal differentiation. A low level of expression is observed in a few other tissues, including ovary, spleen and brain. We have extended those studies by examining A-myb and B-myb expression during testis development in the mouse. The A-myb and B-myb genes are both expressed in a cell- and stage-specific manner during testis development. The B-myb mRNA is expressed most highly in gonocytes of the fetal testis and in spermatogonia and early spermatocytes in the adult. B-myb expression decreases at day 18 post partum, coincident with the initial appearance of late pachytene spermatocytes. B-myb expression was also detectable in some interstitial cells of the fetal and adult testis. The A-myb mRNA was not detectable by in situ hybridization in fetal day 15.5 gonocytes but was detectable at a low abundance by RT-PCR in fetal and newborn mice. A-myb mRNA expression increased at post-natal day 10, when primary spermatocytes first appear. In the adult, the A-myb mRNA was expressed highly in a sub-population of spermatogonia and in primary spermatocytes, but was not detectable in spermatids. This expression of A-myb is consistent with the meiotic arrest that is observed in A-myb-deficient male mice. We conclude that B-myb may play a critical role in controlling the proliferation or differentiation of gonocytes and spermatogonia and possibly the somatic lineages as well, whereas A-myb is required for progression through the first meiotic prophase. These distinct roles for B-myb and A-myb during spermatogenesis may reflect distinct transactivation potentials of the two proteins. Further studies to determine the functions of A-myb and B-myb in the developing testis should improve our understanding of the molecular events associated with spermatogenesis and differentiation of the Sertoli and other somatic cell types of the testis.
Subject(s)
Cell Cycle Proteins , DNA-Binding Proteins/biosynthesis , Proto-Oncogene Proteins/biosynthesis , Spermatogenesis/physiology , Testis/growth & development , Testis/metabolism , Trans-Activators/biosynthesis , Transcription Factors/biosynthesis , Animals , Cell Division/physiology , DNA-Binding Proteins/genetics , Down-Regulation , Female , Gene Expression , Male , Meiosis/physiology , Mice , Mice, Inbred C57BL , Prophase/physiology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-myb , RNA, Messenger/genetics , RNA, Messenger/metabolism , Spermatocytes/cytology , Spermatocytes/metabolism , Spermatogonia/cytology , Spermatogonia/metabolism , Testis/embryology , Trans-Activators/genetics , Transcription Factors/geneticsABSTRACT
The myb gene family consists of three members, A-myb, B-myb and c-myb. The proteins encoded by these genes bind DNA in a sequence-specific manner and regulate transcription of target genes. In this communication, we report the nucleotide sequence of murine A-myb cDNA. This gene encodes a protein of 751 amino acids with an estimated molecular weight of 83 kDa. cDNA sequence analysis of multiple independent cDNA clones reveals the presence of alternatively spliced mRNAs that encode smaller proteins. Northern blot analysis of adult mouse tissue RNAs reveals A-myb expression predominantly in the testis, with very low levels of expression in the ovaries, spleen and brain. In situ hybridization analysis of adult mouse testis shows that this gene is expressed at high levels in type A spermatogonia (stem cells), and preleptotene and pachytene spermatocytes, with concomitant down-regulation of expression upon terminal differentiation of these cells into mature spermatozoa. This pattern of expression suggests that A-myb is involved in the control of proliferation and differentiation of spermatogonia. This potential function of A-myb in spermatogenesis is reminiscent of the role of c-myb in hematopoiesis.
Subject(s)
Oncogenes , Proto-Oncogene Proteins/genetics , RNA Splicing , Trans-Activators/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary , Down-Regulation , Humans , Male , Mice , Molecular Sequence Data , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins c-myb , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Spermatogenesis/genetics , Tissue Distribution , Trans-Activators/biosynthesisABSTRACT
The present study establishes the earliest time point for commitment of cardiac myocyte progenitors at gastrulation and determines the effects of bromodeoxyuridine (BrdU) on postgastrulated committed cardiac progenitor cells at a molecular level. Using immunochemical and reverse transcription/polymerase chain reaction assays for cardiac muscle-specific gene expression, we found that while both pre- and postgastrulated embryonic cells were capable of cardiogenic differentiation at high cell density, only postgastrulated cells exhibited the ability to differentiate at clonal density. These data indicate that while cardiac myocyte differentiation of pregastrulated cells can occur in vitro, cell-cell interactions are necessary for this to happen. Only gastrulated cardiac progenitor cells are able to differentiate in the absence of cell-cell interactions and are therefore both specified and committed. Next, by exposing postgastrulated committed cardiac progenitor cells from embryos at various stages to BrdU, we demonstrated that these cells from the lateral-plate mesoderm vary in their ability to differentiate into cardiac myocytes in vitro. Differentiation of cardiac myocyte progenitor cells from stages 4 and 5 was completely blocked by BrdU, whereas it was not blocked in cells from stages 7 and 8 and cells from stage 6 were varied in their reaction. Analysis of cardiac myogenesis in vivo revealed that cardiac progenitors acquire BrdU resistance as they migrate, postgastrulation, anteriorly along a rostrocaudal axis. The results from these two experiments suggest that while pregastrulated cells exhibit a limited ability to undergo cardiac myocyte differentiation, only postgastrulated anterior lateral-plate mesoderm contains committed cardiac myocyte progenitors and that these committed progenitors are not equivalent in their ability to differentiate.
Subject(s)
Heart/embryology , Animals , Bromodeoxyuridine/pharmacology , Cell Differentiation , Cells, Cultured , Chick Embryo , DNA Replication , Mesoderm/physiology , Stem Cells/physiologyABSTRACT
An antiserum (anti-H2) directed at the second helix of the helix-loop-helix (HLH) protein MyoD1 reacts with a protein expressed during avian cardiac myocyte differentiation. Indirect immunohistochemical whole mount staining with anti-H2 detected a protein expressed in stage 11 hearts, but not in hearts of older embryos. At the cellular level, this staining is confined to the nucleus of cardiac cells suggesting that these proteins may have DNA-binding abilities. Several proteins were immunoprecipitated by anti-H2 from stage 11 heart tissue. Protein extracts from similarly staged hearts, when incubated with the muscle-specific enhancer sequence of muscle creatinine kinase (MCK), gave a stage-specific band shift in electromobility shift assays (EMSA), and these protein-DNA complexes were recognized and supershifted by anti-H2. Incubation with a MCK sequence containing a mutated E box did not produce a shift. The specific shift was present as early as stage 6, remained through stage 13, and disappeared by stage 17. These data suggest the presence of at least one protein that is transiently expressed in the differentiating cardiac myocyte, that is immunochemically reactive with an antiserum raised against the second helix of MyoD1, and that binds to a muscle-specific DNA enhancer sequence.
Subject(s)
DNA-Binding Proteins/analysis , Heart/embryology , Myocardium/chemistry , Amino Acid Sequence , Animals , Binding Sites , Chick Embryo , DNA-Binding Proteins/isolation & purification , Enhancer Elements, Genetic , Gestational Age , Molecular Sequence Data , RabbitsABSTRACT
This article reviews what is known about the earliest stages of heart development focusing on the periods of commitment and differentiation of cardiac progenitor cells and their molecular regulation. The pathway from precursor to differentiated cardiac myocyte is crucial to forming a normal, functional heart. Congenital cardiac abnormalities are some of the most common, estimated at 5-8 per 1000 live births worldwide. These conditions affect mortality and morbidity of patients as infants, children, and adults. Knowledge of what steps are critical to normal heart development would lead to earlier diagnosis and possibly repair of these defects.
ABSTRACT
Molecular markers for specific cell lineages would be useful in studies of cellular differentiation. To isolate such markers monoclonal antibodies (MoABs) were raised against plasma membranes isolated from gastrulating Xenopus embryos. Those antibodies that recognized subsets of cells within the embryo were selected by indirect immunofluorescence. The analysis of eight such MoAbs is presented. Western blot analysis showed that all but one MoAb recognized a complex pattern of glycoconjugates associated with glycoproteins. All the antigens recognized by the MoAbs were maternal in origin and displayed similar spatial patterns of pregastrular expression. This pattern of immunoreactivity at the apical surface was inherited passively during cleavage by the resulting superficial blastomeres suggesting that ectodermal specific markers of maternal origin are pre-localized to the cortical ooplasm in mature oocytes. We suggest that these maternal components may be specific glycosyl transferases. Three different patterns of expression were observed during gastrulation as exemplified by MoAbs 1F10C1, 3A4D1, and 6F10B6. MoAb 6F10B6 was specific for both neural and non-neural epithelium. MoAb 3A4D1 was specific for non-neural epidermis. MoAb 1F10C1 appeared to recognize a protein epitope on an extracellular component expressed by the superficial and involuting epithelial cells. The pattern of expression for the 1F10C1 antigen suggests that it may play a role in facilitating the movement of the involuting cells during gastrulation.
Subject(s)
Antigens, Surface/genetics , Gastrula/immunology , Gene Expression Regulation , Xenopus laevis/genetics , Animals , Antibodies, Monoclonal , Fluorescent Antibody Technique , Humans , Immunization , Immunoblotting , Mice , Mice, Inbred BALB C , Xenopus laevis/embryology , Xenopus laevis/immunologyABSTRACT
We have raised monoclonal antibodies (Mabs) to myosin heavy chain isoforms (MHCs) that have specific patterns of temporal expression during the development of quail pectoral muscle and that are expressed in very restricted, tissue-specific patterns in adult birds. We find that an early embryonic, a perinatal, and an adult-specific, fast myosin heavy chain are co-expressed at different levels in the pectoral muscle of 8-12 day quail embryos. The early embryonic MHC disappears from the pectoral muscle at approximately 14 days in ovo, whereas the perinatal MHC persists until 26 days post-hatching. The adult-specific MHC accumulates preferentially and eventually completely replaces the other isoforms. These Mabs cross-react with the homologous isoforms of the chick and detect a similar pattern of MHC expression in the pectoral muscle of developing chicks. Although the early embryonic and perinatal MHC isoforms recognized by our Mabs are expressed in the pectoral muscle only during distinct developmental stages, our Mabs also recognize MHC isoforms present in the heart and extraocular muscle of adult quail. Immunofingerprinting using Staphylococcus aureus protease V8 suggests that the early embryonic and perinatal MHC isoforms that we see are strongly homologous with the adult ventricular and extraocular muscle isoforms, respectively. These observations suggest that at least three distinct MHC isoforms, which are normally expressed in adult muscles, are co-expressed during the early development of the pectoral muscle in birds. In this respect, the pattern of expression of the MHCs recognized by our Mabs in developing, fast muscle is very similar to the patterns described for other muscle contractile proteins.
Subject(s)
Myosins/genetics , Pectoralis Muscles/embryology , Quail/genetics , Age Factors , Animals , Antibodies, Monoclonal , Blotting, Western , Chromatography, Affinity , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Gene Expression , In Vitro Techniques , Myosins/immunology , Organ Specificity , Pectoralis Muscles/enzymology , Peptide MappingABSTRACT
Chronic consumption of ethanol by pregnant and nonpregnant guinea pigs for 8 weeks at doses of 1.2 or 1.6 g/kg body weight twice daily affected pregnancy outcome and changed the pharmacokinetics of ethanol elimination. Ethanol treatment as compared to that of isocaloric sucrose decreased maternal weight, and decreased both the litter size and the number of liveborn offspring. Total litter weight was significantly decreased with the low ethanol dose (12% alcohol-derived energy). During pregnancy, low and high doses produced peak blood ethanol concentrations (BEC) of 89 +/- 8 mg/dl (mean +/- SE) and 125 +/- 6 mg/dl, respectively. At the high dose, peak BEC decreased dramatically (about 30%) in both pregnant and nonpregnant animals from treatment weeks 0 to 4; thereafter peak BEC remained depressed up to 8 weeks of treatment, which occurred with a concomitant increased volume of ethanol distribution. With both doses, rates of ethanol elimination and Michaelis-Menten's Vm values were significantly lower among pregnant as compared with nonpregnant guinea pigs during 8 weeks of treatment. These data suggest that the guinea pigs can be a valuable animal model to study the effects of low ethanol doses on fetal growth, the adaptation of peak BEC with duration of treatment and the lower rate of ethanol elimination in pregnancy.
Subject(s)
Ethanol/pharmacokinetics , Fetal Alcohol Spectrum Disorders/blood , Maternal-Fetal Exchange , Animals , Dose-Response Relationship, Drug , Ethanol/administration & dosage , Female , Guinea Pigs , Litter Size/drug effects , PregnancyABSTRACT
The spatial segregation of informational molecules in the unfertilized egg and embryo has been hypothesized to be a necessary phenomenon for the normal progression of development leading to the determination of cellular phenotypes. This study describes the selection of a monoclonal antibody (Mab: 2G6) that identifies an antigen (Ag: 2G6) which is localized in the germinal vesicle of oocytes and has a discrete pattern of inheritance during embryogenesis. The antigen displayed biochemical and physical characteristics very similar to nucleoplasmin, which is the histone-binding and nucleosome-assembly protein previously described. Immunoblot analysis with purified oocyte nucleoplasmin confirmed this relationship. Indirect immunofluorescence was used to study the temporal expression and spatial distribution of nucleoplasmin. From early cleavage stages through gastrulation, it is preferentially localized in nuclei of blastomeres at the animal pole. By tadpole stages, it was detected only in nuclei of postmitotic cells of the central nervous system and in nuclei of striated muscle. It was not detected in adult tissues. Western blot analysis during embryogenesis revealed at least five immunologically related polypeptides that displayed distinct patterns of expression during development. The different species observed most likely represent different levels of phosphorylation of nucleoplasmin. The more acidic forms, known to be more active in nucleosome assembly, were present during cleavage stages. Analysis of labelled oocyte proteins by two-dimensional immunoblots and autoradiography revealed that synthesis of nucleoplasmin was first detected in stage-2 oocytes, reached 60% maximum levels at stage 3, peaked at stage 4 and was undetectable in stage-6 oocytes. The amount of nucleoplasmin message present does not follow a similar pattern during oogenesis. These results suggest that the message undergoes pronounced changes in translational efficiency during oogenesis. A comparative immunoblot analysis using proteins from a variety of adult tissues revealed that, whereas the polyclonal antisera against amphibian vitellogenic oocyte nucleoplasmin recognized several different, tissue-specific polypeptides, two different monoclonal antibodies (Mab: b7-1D1, Mab: 2G6) failed to recognize any of the adult tissues tested. We conclude that nucleoplasmin is a family of closely related proteins with distinct embryonic and adult members.
Subject(s)
Nuclear Proteins/analysis , Phosphoproteins , Xenopus laevis/embryology , Animals , Antibodies, Monoclonal , Antigens/analysis , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Nucleoplasmins , OogenesisABSTRACT
Human eosinophils were purified to greater than 92% using 16-30% metrizamide gradients, and these cells cultured for up to 72 h in vitro to label sulfated glycosaminoglycans. Over 90% of the sulfated glycosaminoglycan-containing material was extracted in 4 M guanidine HCl and had a hydrodynamic size similar to a glycosaminoglycan marker with an approximate average molecular weight of 60,000. Treatment of this salt-extracted 35S-labeled glycosaminoglycan-containing material with 0.5 M NaOH resulted in a change in mass to approx. 20,000 daltons, suggesting that the larger molecules were proteoglycans with side chains with an approximate molecular weight of 20,000. These salt extracted presumptive 35S-labeled proteoglycans were protease insensitive and behaved in a highly charged fashion on DEAE-cellulose. The composition of 35S-labeled glycosaminoglycans from human eosinophils as identified using selected polysaccharides was 70-81% chondroitin 4-sulfate, 9-12% chondroitin 6-sulfate, and 5-12% dermatan sulfate. The predominance of chondroitin 4-sulfate in human eosinophils is similar to the predominance of chondroitin 4-sulfate in human neutrophils and human platelets.