ABSTRACT
OBJECTIVE: To perform a preliminary study on the effects and safety of bilateral cingulotomy and anterior capsulotomy in patients with aggressive behavior. PATIENTS AND METHODS: Twenty-three psychiatric patients showing aggressive behavior refractory to conventional treatment were initially evaluated. The subjects were clinically selected using the Overt Aggression Scale (OAS) and the Global Assessment of Functioning Scale (GAF). Each case was carefully reviewed by the Ethics Committee of Mexico's General Hospital. Once selection criteria were met, stereotactic lesions were made using radiofrequency on the anterior limb of the internal capsule and supragenual cingulum. Statistical differences were evaluated with a Wilcoxon test at 6 months and at 4 years. RESULTS: Ten patients underwent surgery. Their OAS and GAF scores decreased after the procedure at the 6-month (p < 0.05) and at the 4-year (p = 0.068) follow-up. Four patients showed mild and transitory postsurgical complications (hyperphagia and somnolence). CONCLUSIONS: Bilateral anterior capsulotomy in combination with cingulotomy may reduce aggressive behavior and improve clinical evaluations. Very strict clinical and ethical evaluations were applied prior to considering patients for this treatment.
Subject(s)
Aggression , Gyrus Cinguli/surgery , Internal Capsule/surgery , Psychosurgery/methods , Adolescent , Adult , Female , Humans , Male , Middle Aged , Stereotaxic Techniques , Treatment OutcomeABSTRACT
BACKGROUND: Maternal metabolic demands change dramatically during the course of gestation and must be co-ordinated with the needs of the developing placenta and fetus. The liver is critically involved in metabolism and other important functions. However, maternal hepatic adjustments to pregnancy are poorly understood. AIM: The aim of the study was to evaluate the influences of pregnancy on the maternal liver growth and gene expression profile. METHODS: Holtzman Sprague-Dawley rats were mated and sacrificed at various stages of gestation and post-partum. The maternal livers were analysed in gravimetric response, DNA content by PicoGreen dsDNA quantitation reagent, hepatocyte ploidy by flow cytometry and hepatocyte proliferation by ki-67 immunostaining. Gene expression profiling of non-pregnant and gestation d18.5 maternal hepatic tissue was analysed using a DNA microarray approach and partially verified by northern blot or quantitative real-time PCR analysis. RESULTS: During pregnancy, the liver exhibited approximately an 80% increase in size, proportional to the increase in body weight of the pregnant animals. The pregnancy-induced hepatomegaly was a physiological event of liver growth manifested by increases in maternal hepatic DNA content and hepatocyte proliferation. Pregnancy did not affect hepatocyte polyploidization. Pregnancy-dependent changes in hepatic expression were noted for a number of genes, including those associated with cell proliferation, cytokine signalling, liver regeneration and metabolism. CONCLUSIONS: The metabolic demands of pregnancy cause marked adjustments in maternal liver physiology. Central to these adjustments are an expansion in hepatic capacity and changes in hepatic gene expression. Our findings provide insights into pregnancy-dependent hepatic adaptations.
Subject(s)
Adaptation, Physiological , Gene Expression Profiling , Liver/physiology , Pregnancy/genetics , Animals , Blotting, Northern , Cell Proliferation , DNA/metabolism , Female , Hepatocytes/cytology , Hepatocytes/metabolism , Ki-67 Antigen/metabolism , Liver/anatomy & histology , Liver/metabolism , Oligonucleotide Array Sequence Analysis , Pregnancy/physiology , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O-linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI-TOF/MS and ESI-MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high-performance anion-exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N-acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc(2), N-acetyl galactosamine(1), Gal(1)). After beta-elimination to release the oligosaccharide from glycosylated 24 kDa hGH, collision-induced dissociation of tryptic glycopeptide T6 indicated that there had been an O-linked oligosaccharide attached to Thr-60. The sequence and branching structure of the oligosaccharide were determined by ESI-MS/MS analysis of tryptic glycopeptide T6. The mucin-like O-oligosaccharide sequence linked to Thr-60 begins with N-acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high-affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH.
Subject(s)
Human Growth Hormone/chemistry , Mucins/chemistry , Oligosaccharides/chemistry , Amino Acid Sequence , Carbohydrate Sequence , Glycosylation , Human Growth Hormone/isolation & purification , Human Growth Hormone/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Molecular Weight , Protein Structure, Tertiary , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Threonine/chemistry , Trypsin/metabolismABSTRACT
BACKGROUND/AIMS: During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia-inducible factor-1alpha (HIF-1alpha), is critical for upregulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B and plasminogen activator inhibitor-1 (PAI-1) in the liver, during the development of fibrosis. What remains unknown is the cell type-specific regulation of these genes by HIF-1alpha in liver cell types. Accordingly, the hypothesis was tested that HIF-1alpha is activated in hypoxic hepatocytes and regulates the production of profibrotic mediators by these cells. METHODS: In this study, hepatocytes were isolated from the livers of control and HIF-1alpha- or HIF-1beta-deficient mice and exposed to hypoxia. RESULTS: Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF-1alpha and upregulated PAI-1, vascular endothelial cell growth factor and the vasoactive peptides adrenomedullin-1 (ADM-1) and ADM-2. In contrast, the levels of PDGF-A and PDGF-B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF-1alpha-deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia-regulated transcription factors, such as HIF-2alpha, may also regulate these genes. In support of this, HIF-2alpha was activated in hypoxic hepatocytes, and exposure of HIF-1beta-deficient hepatocytes to 1% oxygen completely prevented upregulation of PAI-1, vascular endothelial cell growth factor and ADM-1, suggesting that HIF-2alpha may also contribute to upregulation of these genes in hypoxic hepatocytes. CONCLUSIONS: Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes.
Subject(s)
Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Hepatocytes/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Liver Cirrhosis/metabolism , Adrenomedullin/genetics , Adrenomedullin/metabolism , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator/deficiency , Aryl Hydrocarbon Receptor Nuclear Translocator/genetics , Cell Hypoxia , Cell Survival , Cells, Cultured , Hepatocytes/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/deficiency , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Liver Cirrhosis/pathology , Mice , Mice, Knockout , Neuropeptides/genetics , Neuropeptides/metabolism , Plasminogen Activator Inhibitor 1/genetics , Plasminogen Activator Inhibitor 1/metabolism , Platelet-Derived Growth Factor/genetics , Platelet-Derived Growth Factor/metabolism , Proto-Oncogene Proteins c-sis/genetics , Proto-Oncogene Proteins c-sis/metabolism , RNA, Messenger/metabolism , Signal Transduction , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Maternal physiology changes dramatically during the course of gestation and lactation to meet the needs of the developing fetus and newborn. In the present study, we examined the influence of pregnancy and lactation on growth and erythroid gene expression patterns of the maternal spleen. Holtzman Sprague-Dawley rats and CD-1 mice were killed at various stages of gestation and post partum. We observed pregnancy dependent increases in spleen weight and spleen DNA content in both the rat and mouse. In the rat, spleen size was greatest at the end of pregnancy and regressed post partum. In contrast, mouse spleen size peaked by gestational Day 13 and regressed to its non-pregnant weight before parturition. Pregnancy dependent changes in the size of the spleen were primarily due to an increase in red pulp. Maternal spleen expression of erythroid-associated genes (erythroid Krüppel-like factor, erythroid 5-aminolevulinate synthase-2, beta-major globin) was influenced by pregnancy and lactation. A pregnancy dependent increase in erythroid progenitors was also observed. In summary, the demands of pregnancy and lactation cause marked adaptations in the maternal spleen. The maternal spleen increases in size and exhibits an expansion of the erythroid lineage.
Subject(s)
Gene Expression Regulation/physiology , Lactation/physiology , Pregnancy, Animal/physiology , Spleen/growth & development , 5-Aminolevulinate Synthetase/metabolism , Animals , Blotting, Northern , Female , Flow Cytometry , Kruppel-Like Transcription Factors/metabolism , Mice , Organ Size , Pregnancy , Rats , Rats, Sprague-Dawley , Species Specificity , Spleen/metabolismABSTRACT
Molecular mechanisms underlying the health disparity of prostate cancer (PCa) have not been fully determined. In this study, we applied bioinformatic approach to identify and validate dysregulated genes associated with tumor aggressiveness in African American (AA) compared to Caucasian American (CA) men with PCa. We retrieved and analyzed microarray data from 619 PCa patients, 412 AA and 207 CA, and we validated these genes in tumor tissues and cell lines by Real-Time PCR, Western blot, immunocytochemistry (ICC) and immunohistochemistry (IHC) analyses. We identified 362 differentially expressed genes in AA men and involved in regulating signaling pathways associated with tumor aggressiveness. In PCa tissues and cells, NKX3.1, APPL2, TPD52, LTC4S, ALDH1A3 and AMD1 transcripts were significantly upregulated (p < 0.05) compared to normal cells. IHC confirmed the overexpression of TPD52 (p = 0.0098) and LTC4S (p < 0.0005) in AA compared to CA men. ICC and Western blot analyses additionally corroborated this observation in PCa cells. These findings suggest that dysregulation of transcripts in PCa may drive the disparity of PCa outcomes and provide new insights into development of new therapeutic agents against aggressive tumors. More studies are warranted to investigate the clinical significance of these dysregulated genes in promoting the oncogenic pathways in AA men.
Subject(s)
Black or African American/genetics , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/ethnology , Prostatic Neoplasms/genetics , Adult , Black or African American/statistics & numerical data , Cell Line, Tumor , Humans , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Prognosis , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/pathology , Signal Transduction/genetics , White People/genetics , White People/statistics & numerical dataABSTRACT
The prolactin (PRL) family of hormones/cytokines is involved in the maintenance of pregnancy and adaptations to physiological stressors. In this report, we identify and characterize a new member of the rat PRL family, examine the impact of maternal hypoxia on placental PRL family gene expression, and investigate maternal adaptive responses to hypoxia. Perusal of the PRL gene family locus in the rat genome resulted in the identification of a putative new member of the rat PRL family. The new member is closely related to the previously reported PRL-like protein-F (PLP-F) and has been named PLP-Fbeta and the originally characterized PLP-F, now termed PLP-Falpha. The two proteins exhibit structural similarities but possess distinct cell- and temporal-specific expression profiles. In vivo hypoxia stimulates placental PLP-Falpha and PLP-E mRNA expression in the rat and mouse, respectively. Rcho-1 trophoblast cells can differentiate into trophoblast giant cells, express PLP-Falpha, and exhibit enhanced PLP-Falpha mRNA levels when cultured under low oxygen tension (2%). Exposure to hypobaric hypoxia during latter part of pregnancy did not significantly impact the expression of PLP-Fbeta mRNA. Finally, exposure to hypobaric hypoxia during midpregnancy led to increased maternal red blood cells, hemoglobin concentrations, hematocrit, and increased concentrations of maternal splenic mRNAs for key proteins involved in hemoglobin synthesis, erythroid Krüppel-like factor, erythroid 5-aminolevulinate synthase-2, and beta-major globin. In summary, adaptive responses to maternal hypoxia include activation of placental PLP-Falpha/E gene expression, which may then participate in maternal hematological adjustments required for maintaining maternal and fetal oxygen delivery.
Subject(s)
Adaptation, Physiological , Cytokines/metabolism , Glycoproteins/metabolism , Hypoxia/physiopathology , Placental Hormones/metabolism , Pregnancy Complications/physiopathology , Pregnancy Proteins/metabolism , Amino Acid Sequence , Animals , Cell Line , Cytokines/genetics , Erythrocyte Count , Female , Gene Expression , Hematocrit , Hemoglobins/metabolism , Hypoxia/blood , Hypoxia/genetics , Hypoxia/metabolism , Male , Mice , Mice, Inbred Strains , Molecular Sequence Data , Oxygen/metabolism , Placental Hormones/blood , Placental Hormones/genetics , Pregnancy , Pregnancy Complications/blood , Pregnancy Complications/genetics , Pregnancy Complications/metabolism , Pregnancy Proteins/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/metabolism , Rats , Rats, Inbred Strains , Trophoblasts/metabolismABSTRACT
Although a 22-kDa human growth hormone (hGH) is the predicted protein product of the hGH-N gene, a pleiotropic collection of uncharacterized molecular weight and charge isoforms is also produced. Using chromatography and preparative SDS-PAGE under reducing conditions we isolated an unusually stable mercaptoethanol-resistant (MER) 45-kDa hGH. A 5-h incubation at 100 degrees C in the presence of 2-mercaptoethanol was required to convert approximately 90% of MER-45-kDa hGH into a 22-kDa hGH. Other reductants were not as effective in splitting MER-45-kDa hGH. After fracturing MER-45-kDa hGH, the 22-kDa hGH fragments would spontaneously reassociate if the reductant was removed; however, alkylation of cysteine residues prevented their reassociation. Identical amino acid sequences for the first six N-terminal residues were obtained for MER-45-kDa hGH and its 22-kDa hGH cleavage product. Structural identity of MER-45-kDa hGH and 22-kDa hGH was demonstrated by MALDI-TOF mass spectrometry of tryptic digests. MER-45-kDa hGH did not break up upon incubation with EDTA and EGTA. The significance of this work to our understanding of the structure of hGH isoforms is that it demonstrates that MER-45-kDa hGH is not a single chain polypeptide but is instead a homodimer of 22-kDa hGH monomers. The MER-45-kDa hGH dimer is held together by interchain disulfide bonds and not by divalent metal cation bridges. Additionally, MER-45-kDa hGH's interchain disulfide links are exceptionally resistant to reducing agents and thus confer extreme stability to the homodimer.
Subject(s)
Disulfides/chemistry , Human Growth Hormone/chemistry , Chelating Agents/pharmacology , Dimerization , Electrophoresis , Human Growth Hormone/isolation & purification , Humans , Mercaptoethanol/chemistry , Molecular Sequence Data , Molecular Weight , Protein Subunits/chemistry , Reducing Agents , Sequence Analysis, Protein , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Drugs account for about 20% of gynecomastia cases in men. As a number of factors can alter the estrogen:androgen ratio, several pathophysiologic mechanisms are associated with drugs causing this disorder. Antiandrogens, protease inhibitors, and nucleoside reverse transcriptase inhibitors are the most common drug causes of gynecomastia, whereas first-generation antipsychotics, spironolactone, verapamil, and cimetidine are less common causes. Other drugs have been reported rarely as causes. Treatment may involve switching to an alternative agent or may require surgery or irradiation if the causative agent cannot be discontinued. We reviewed the literature on drug-induced gynecomastia and provided another perspective by reviewing data from the United States Food and Drug Administration's Adverse Event Reporting System. Epidemiologic studies are needed to provide a more accurate description of the frequency of drug-induced gynecomastia.
Subject(s)
Drug-Related Side Effects and Adverse Reactions , Gynecomastia/chemically induced , Adverse Drug Reaction Reporting Systems , Androgen Antagonists/adverse effects , Androgens/metabolism , Estrogens/metabolism , Gynecomastia/physiopathology , Gynecomastia/therapy , Humans , Male , Protease Inhibitors/adverse effects , Reverse Transcriptase Inhibitors/adverse effectsABSTRACT
Pregnancy is characterized by physiological adjustments in the maternal compartment. In this investigation, the influence of pregnancy on maternal liver was examined in CD-1 mice. Dramatic changes were observed in the size of the maternal liver during pregnancy. Livers doubled in weight from the non-pregnant state to day 18 of pregnancy. The pregnancy-induced hepatomegaly was a physiological event of liver growth confirmed by DNA content increase and detection of hepatocyte hyperplasia and hypertrophy. Growth of the liver was initiated following implantation and peaked at parturition. The expression and/or activities of key genes known to regulate liver regeneration, a phenomenon of liver growth compensatory to liver mass loss, were investigated. The results showed that pregnancy-dependent liver growth was associated with interleukin (IL)-6, tumor necrosis factor α, c-Jun and IL-1ß, but independent of hepatocyte growth factor, fibroblast growth factor 1, tumor necrosis factor receptor 1, constitutive androstane receptor and pregnane X receptor. Furthermore, maternal liver growth was associated with the activation of hepatic signal transducer and activator of transcription 3, ß-catenin and epidermal growth factor receptor, but pregnancy did not activate hepatic c-Met. The findings suggest that the molecular mechanisms regulating pregnancy-induced liver growth and injury-induced liver regeneration exhibit overlapping features but are not identical. In summary, the liver of the mouse adapts to the demands of pregnancy via a dramatic growth response driven by hepatocyte proliferation and size increase.
Subject(s)
Liver/anatomy & histology , Pregnancy, Animal/physiology , Animals , Constitutive Androstane Receptor , Embryo Implantation/physiology , ErbB Receptors/metabolism , Female , Fibroblast Growth Factor 1/metabolism , Hepatocyte Growth Factor/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Liver/growth & development , Mice , Parturition/physiology , Pregnancy , Pregnane X Receptor , Proto-Oncogene Proteins c-jun/metabolism , Proto-Oncogene Proteins c-met/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/metabolism , Receptors, Tumor Necrosis Factor/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Tumor Necrosis Factor-alpha/metabolism , beta Catenin/metabolismABSTRACT
BACKGROUND: Human growth hormone (hGH) is a complex mixture of molecular isoforms. Gaps in our knowledge exist regarding the structures and biological significances of the uncharacterized hGH molecular variants. Mercaptoethanol-resistant 45-kDa human growth hormone (MER-45 kDa hGH) is an extraordinarily stable disulfide-linked hGH homodimer whose biological significance is unknown. OBJECTIVES: To elucidate the pharmacokinetic abilities of dimeric MER-45-kDa hGH to bind to GH and prolactin (PRL) receptors and to elucidate its abilities to stimulate cell proliferation in lactogen-induced and somatogen-induced in vitro cell proliferation bioassays. DESIGN: The binding of MER-45-kDa hGH to GH and PRL receptors was tested in radioreceptor assays (RRAs). Competitive displacements of [(125)I]-bovine GH from bovine liver membranes, [(125)I]-ovine PRL from lactating rabbit mammary gland membranes and [(125)I]-hGH from human IM-9 lymphocytes by unlabelled GHs, PRLs or dimeric MER-45-kDa hGH were evaluated. The abilities of dimeric MER-45-kDa hGH to stimulate proliferation of lactogen-responsive Nb2 lymphoma cells and to stimulate proliferation of somatogen-responsive T47-D human breast cancer cells were assessed by incubation of cells with GHs or PRLs and subsequently measuring growth using the MTS cell proliferation assay. RESULTS: Dimeric MER-45-kDa hGH, compared to monomeric hGH, had reduced binding affinities to both GH and prolactin receptors. In a bovine liver GH radioreceptor assay its ED(50) (197.5 pM) was 40.8% that of monomeric hGH. In a human IM-9 lymphocyte hGH RRA its ED(50) (2.96 nM) was 26.2% that of monomeric hGH. In a lactating rabbit mammary gland prolactin RRA its ED(50) (3.56 nM) was 16.8% that of a monomeric hGH. Dimeric MER-45-kDa hGH, compared to monomeric hGH, had a diminished capacity to stimulate proliferation of cells in vitro. In a dose-response relationship assessing proliferation of Nb2 lymphoma cells its ED(50) (191 pM) was 18.0% that of monomeric hGH. While monomeric hGH stimulated a 2.2-fold proliferation of T47-D human breast cancer cells above vehicle control, dimeric MER-45-kDa hGH was unable to stimulate the cells to proliferate and slightly inhibited their proliferation to 77.6% that of control. CONCLUSIONS: The topological arrangement of monomeric hGHs to form an unusually stable disulfide-linked dimer markedly diminishes hGH's binding affinities to both GH and PRL receptors and also drastically attenuates its ability to stimulate proliferation of cells in vitro.
Subject(s)
Cell Proliferation , Human Growth Hormone/chemistry , Human Growth Hormone/metabolism , Animals , Binding Sites , Cattle , Cell Line, Tumor , Female , Human Growth Hormone/genetics , Humans , Lymphocytes/metabolism , Prolactin/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Rabbits , Rats , Receptors, Prolactin/metabolismABSTRACT
The membrane-bound rat GH-R and an alternatively spliced isoform, the soluble rat GH-BP, are comprised of identical N-terminal GH-binding domains; however, their C-terminal sequences differ. Immunological reagents are needed to distinguish between the two isoforms in order to understand their respective roles in mediating the actions of GH. Accordingly, a tetravalent MAP dendrimer with four identical branches of a C-terminal peptide sequence of the rat GH-BP (GH-BP(263-279)) was synthesized and used as an immunogen in rabbits. Solid-phase peptide synthesis of four GH-BP(263-279) segments onto a tetravalent Lys(2)-Lys-beta-Ala-OH core peptide was carried out using Fmoc chemistry. The mass of the RP-HPLC-purified synthetic product, 8398 Da, determined by ESI-MS, was identical to expected mass. Three anti-rat GH-BP(263-279) MAP antisera, BETO-8039, BETO-8040, and BETO-8041, at dilutions of 10(-3), recognized both the rat GH-BP(263-279) MAP and recombinant mouse GH-BP with ED(50)s within a range of 5-10 fmol, but did not cross-react with BSA in dot blot analyses. BETO-8041 antisera (10(-3) dilution) recognized GH-BPs of rat serum and liver having M(r)s ranging from 35 to 130 kDa, but did not recognize full-length rat GH-Rs. The antisera also detected recombinant mouse GH-BPs. In summary, the tetravalent rat GH-BP(263-279) MAP dendrimer served as an effective immunogenic antigen in eliciting high titer antisera specific for the C-termini of both rat and mouse GH-BPs. The antisera will facilitate studies aimed at improving our understanding of the biology of GH-BPs.
Subject(s)
Antibodies/immunology , Dendrimers/chemistry , Peptides/chemistry , Protein Isoforms/metabolism , Receptors, Somatotropin/metabolism , Amino Acid Sequence , Animals , Cattle , Dendrimers/chemical synthesis , Female , Mice , Molecular Sequence Data , Peptides/immunology , Pregnancy , Protein Isoforms/chemistry , Protein Isoforms/genetics , Rabbits , Rats , Rats, Inbred F344 , Receptors, Somatotropin/chemistry , Receptors, Somatotropin/genetics , Sensitivity and SpecificityABSTRACT
The 40-60 pituitary human growth hormone (hGH) isoforms are so similar in their physico-chemical properties (charge, size, hydrophobicity) that the limited resolutions of chromatographic separation methodologies have not permitted most of them to be isolated. However, application of high-resolution preparative alkaline urea gradient PAGE has facilitated isolation of a disulfide-linked mercaptoethanol-resistant (MER) 45 kDa hGH dimer. Human pituitary extracts were separated by Sephadex G-100 chromatography under alkaline conditions. Pooled fractions containing MER-45 kDa hGH, as determined by SDS-PAGE, were then separated by Sephadex G-100 chromatography under acidic conditions followed by diethylaminoethyl (DEAE) anion-exchange chromatography. Pooled DEAE fractions containing MER-45 kDa hGH and other hGH isoforms were then separated by preparative electrophoresis in an alkaline polyacrylamide gradient (5-20%) slab gel containing 8 M urea into five distinct protein zones. One electroeluted zone contained pure MER-45 kDa hGH. The dimeric hGH isoform was immunoreactive at low concentrations (effective dose to produce 50% response (ED(50)) +/- S.E. = 58 +/- 5.00 pM) in a hGH radioimmunoassay, similar to that of standard monomeric hGH (ED(50) +/- S.E. = 22.93 +/- 3.90 pM), indicating that is was conformationally intact. Alkaline urea gradient PAGE is a valuable tool for preparative separation of structurally similar proteins such as isoforms of the hGH family.
Subject(s)
Disulfides/chemistry , Electrophoresis, Polyacrylamide Gel/methods , Human Growth Hormone/chemistry , Human Growth Hormone/isolation & purification , Urea/chemistry , Chromatography, Gel , Chromatography, Ion Exchange , Dimerization , Humans , Mercaptoethanol/chemistry , Molecular Weight , Pituitary Gland/chemistry , Protein Structure, TertiaryABSTRACT
A 2-D native-PAGE/SDS-PAGE method for detecting the subunit components of protein oligomers at low picomole sensitivity is presented. IgG was electrophoresed in a native acidic polyacrylamide gel in amounts ranging from 51 pmol to 60 fmol. Silver-staining (native fast silver stain, ammoniacal silver stain, permanganate silver stain), Coomassie-staining (R-250, G-250), metal ion-reverse-staining (zinc, copper), and fluorescent chromophore-staining (SYPRO Ruby) methods were used to visualize the IgG oligomers. The protein zones were then excised, separated by SDS-PAGE, and subunits visualized with a permanganate silver stain. The Coomassie R-250/permanganate silver-staining combination detected IgG subunits using 2 pmol of sample. Coomassie G-250 and native fast silver staining in the first-dimensional gel produced detectable subunits in the second-dimensional separation at 3 and 13 pmol, respectively. Staining with silver (ammoniacal, permanganate), copper, zinc, or SYPRO Ruby in the first-dimensional gel did not produce discernible subunits in the second-dimensional gels due to protein streaking or protein immobilization in the native gel. When using a 2-D native-PAGE/SDS-PAGE system, Coomassie staining of the first-dimensional native gel combined with permanganate silver staining of the second-dimensional denaturing gel provides the most sensitive method (2-3 pmol) for visualizing constituent subunits from their oligomeric assemblies.
Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Immunoglobulin G/analysis , Animals , Electrophoresis, Gel, Two-Dimensional/statistics & numerical data , Goats , Immunoglobulin G/chemistry , Protein Structure, Quaternary , Protein Subunits , Sensitivity and Specificity , Sodium Dodecyl Sulfate , Staining and Labeling/methodsABSTRACT
A method for separating proteins with a molecular mass difference of 2 kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24 kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2 kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24 kDa hGH. The 22 and 24 kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13-18% linear gradient gel, and a 15-10% linear inverted gradient gel. Fractions containing purified 24 kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2 kDa.