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Human Growth Hormone (human + growth_hormone)
Kinds of Human Growth Hormone Selected AbstractsHUMAN GROWTH HORMONE: RESEARCH AND CLINICAL PRACTICEJOURNAL OF PAEDIATRICS AND CHILD HEALTH, Issue 2 2001Article first published online: 21 DEC 200 [source] Computational alanine scanning of the 1:1 human growth hormone,receptor complexJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 1 2002Shuanghong Huo Abstract The MM-PBSA (Molecular Mechanics,Poisson,Boltzmann surface area) method was applied to the human Growth Hormone (hGH) complexed with its receptor to assess both the validity and the limitations of the computational alanine scanning approach. A 400-ps dynamical trajectory of the fully solvated complex was simulated at 300 K in a 101 Å×81 Å×107 Å water box using periodic boundary conditions. Long-range electrostatic interactions were treated with the particle mesh Ewald (PME) summation method. Equally spaced snapshots along the trajectory were chosen to compute the binding free energy using a continuum solvation model to calculate the electrostatic desolvation free energy and a solvent-accessible surface area approach to treat the nonpolar solvation free energy. Computational alanine scanning was performed on the same set of snapshots by mutating the residues in the structural epitope of the hormone and the receptor to alanine and recomputing the ,Gbinding. To further investigate a particular structure, a 200-ps dynamical trajectory of an R43A hormone,receptor complex was simulated. By postprocessing a single trajectory of the wild-type complex, the average unsigned error of our calculated ,,Gbinding is ,1 kcal/mol for the alanine mutations of hydrophobic residues and polar/charged residues without buried salt bridges. When residues involved in buried salt bridges are mutated to alanine, it is demonstrated that a separate trajectory of the alanine mutant complex can lead to reasonable agreement with experimental results. Our approach can be extended to rapid screening of a variety of possible modifications to binding sites. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 15,27, 2002 [source] Legal issues arising out of blood testing for human growth hormoneDRUG TESTING AND ANALYSIS, Issue 9-10 2009Professor Matthew J. Mitten Copyright © 2009 John Wiley & Sons, Ltd. [source] Counterfeiting in performance- and image-enhancing drugsDRUG TESTING AND ANALYSIS, Issue 3 2009Michael R. Graham Abstract The current drastic escalation in obesity may be contributing to the exponential rise in drugs used for image enhancement. Drugs such as anabolic-androgenic steroids (AAS) are perceived as a viable method of achieving a perfect physique. They are also the most widely abused drugs in sport. The Internet has encouraged the abuse of expensive drugs, particularly human growth hormone (hGH), resulting in increased importation for personal use. The substantial increase in this market has opened up avenues for counterfeiting, estimated as a multi-million pound business. The acute adverse effects from contaminated vials may result in a variety of pathologies including communicable diseases. In 2007, in the UK, a series of intramuscular abscesses, requiring surgical treatment, led us to study samples obtained from the underground market. The analysis of 38 parenteral samples and 19 oral samples of tablets was performed by a World Anti-Doping Agency (WADA) accredited laboratory, in an attempt to establish the extent of available counterfeit products. Fifty-three per cent (20) of the injectable AAS esters and 21% (4) of the oral tablets were counterfeit. Culture and sensitivity revealed the presence of skin commensal organisms, which may have contributed to the development of the abscesses. Users of AAS and hGH for sport, including bodybuilding, are currently risking their health because of counterfeit and poorly controlled products. Copyright © 2009 John Wiley & Sons, Ltd. [source] Separation of proteins with a molecular mass difference of 2,kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: Application to the isolation of 24,kDa human growth hormoneELECTROPHORESIS, Issue 23 2005Juan J. Bustamante Abstract 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. [source] ELKS, a protein structurally related to the active zone protein CAST, is involved in Ca2+ -dependent exocytosis from PC12 cellsGENES TO CELLS, Issue 6 2006Eiji Inoue The active zone protein CAST binds directly to the other active zone proteins RIM, Bassoon and Piccolo, and it has been suggested that these protein,protein interactions play an important role in neurotransmitter release. To further elucidate the molecular mechanism, we attempted to examine the function of CAST using PC12 cells as a model system. Although PC12 cells do not express CAST, they do express ELKS, a protein structurally related to CAST. Endogenous and exogenously expressed ELKS, RIM2 and Bassoon were colocalized in punctate signals in PC12 cells. Over-expression of full-length ELKS resulted in a significant increase in stimulated exocytosis of human growth hormone (hGH) from PC12 cells, similar to the effect of full-length RIM2. This increase was not observed following over-expression of deletion constructs of ELKS that lacked either the last three amino acids (IWA) required for binding to RIM2 or a central region necessary for binding to Bassoon. Moreover, over-expression of the NH2 -terminal RIM2-binding domain of Munc13-1, which is known to inhibit the binding between RIM and Munc13-1, inhibited the stimulated increase in hGH secretion by full-length RIM2. Furthermore, this construct also inhibited the stimulated increase in hGH secretion induced by full-length ELKS. These results suggest that ELKS is involved in Ca2+ -dependent exocytosis from PC12 cells at least partly via the RIM2-Munc13-1 pathway. [source] Growth Hormone Administration and Exercise Effects on Muscle Fiber Type and Diameter in Moderately Frail Older PeopleJOURNAL OF AMERICAN GERIATRICS SOCIETY, Issue 7 2001James V. Hennessey MD OBJECTIVE: Reduced muscle mass and strength are characteristic findings of growth hormone deficiency (GHD) and aging. We evaluated measures of muscle strength, muscle fiber type, and cross sectional area in response to treatment with recombinant human growth hormone (rhGH) with or without a structured resistance exercise program in frail older subjects. DESIGN: Placebo-controlled, randomized, double blind trial. SETTING: Outpatient clinical research center at an urban university-affiliated teaching hospital. PARTICIPANTS: Thirty-one consenting older subjects (mean age 71.3 ± 4.5 years) recruited as a subset of a larger project evaluating rhGH and exercise in older people, who underwent 62 quadricep-muscle biopsies. INTERVENTION: Random assignment to a 6-month course of one of four protocols: rhGH administered subcutaneously daily at bedtime, rhGH and a structured resistance exercise program, structured resistance exercise with placebo injections, or placebo injections only. MEASUREMENTS: Muscle biopsy specimens were obtained from the vastus lateralis muscle. Isokinetic dynamometry strength tests were used to monitor individual progress and to adjust the weights used in the exercise program. Serum insulin-like growth factor-I (IGF-I) was measured and body composition was measured using a Hologic QDR 1000W dual X-ray densitometer. RESULTS: The administration of rhGH resulted in significant increase in circulating IGF-I levels in the individuals receiving rhGH treatment. Muscle strength increased significantly in both the rhGH/exercise (+55.6%, P = .0004) as well as the exercise alone (+47.8%, P = .0005) groups. There was a significant increase in the proportion of type 2 fibers between baseline and six months in the combined rhGH treated subjects versus those not receiving rhGH (P = .027). CONCLUSIONS: Our results are encouraging in that they suggest an effect of growth hormone on a specific aging-correlated deficit. IGF-I was increased by administrating rhGH and muscle strength was increased by exercise. The administration of rhGH to frail older individuals in this study resulted in significant changes in the proportions of fiber types. Whether changes in fiber cross-sectional area or absolute number occur with long-term growth hormone administration requires further study. [source] Growth Hormone Increases Bone Mineral Content in Postmenopausal Osteoporosis: A Randomized Placebo-Controlled TrialJOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2003Kerstin Landin-Wilhelmsen Abstract Eighty osteoporotic, postmenopausal women, 50,70 years of age, with ongoing estrogen therapy (HRT), were randomized to recombinant human growth hormone (GH), 1.0 U or 2.5 U/day, subcutaneous, versus placebo. This study was double-blinded and lasted for 18 months. The placebo group then stopped the injections, but both GH groups continued for a total of 3 years with GH and followed for 5 years. Calcium (750 mg) and vitamin D (400 U) were given to all patients. Bone mineral density and bone mineral content were measured with DXA. At 18 months, when the double-blind phase was terminated, total body bone mineral content was highest in the GH 2.5 U group (p = 0.04 vs. placebo). At 3 years, when GH was discontinued, total body and femoral neck bone mineral content had increased in both GH-treated groups (NS between groups). At 4-year follow-up, total body and lumbar spine bone mineral content increased 5% and 14%, respectively, for GH 2.5 U (p = 0.01 and p = 0.0006 vs. placebo). Femoral neck bone mineral density increased 5% and bone mineral content 13% for GH 2.5 U (p = 0.01 vs. GH 1.0 U). At 5-year follow-up, no differences in bone mineral density or bone mineral content were seen between groups. Bone markers showed increased turnover. Three fractures occurred in the GH 1.0 U group. No subjects dropped out. Side effects were rare. In conclusion, bone mineral content increased to 14% with GH treatment on top of HRT and calcium/vitamin D in postmenopausal women with osteoporosis. There seems to be a delayed, extended, and dose-dependent effect of GH on bone. Thus, GH could be used as an anabolic agent in osteoporosis. [source] Systemic Regulation of Distraction Osteogenesis: A Cascade of Biochemical Factors,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2002S. Weiss M.D. Abstract This study investigates the systemic biochemical regulation of fracture healing in distraction osteogenesis compared with rigid osteotomy in a prospective in vivo study in humans. To further clarify the influence of mechanical strain on the regulation of bone formation, bone growth factors (insulin-like growth factor [IGF] I, IGF binding protein [IGFBP] 3, transforming growth factor [TGF] ,1, and basic FGF [bFGF]), bone matrix degrading enzymes (matrix-metalloproteinases [MMPs] 1, 2, and 3), human growth hormone (hGH), and bone formation markers (ALP, bone-specific ALP [BAP], and osteocalcin [OC]) have been analyzed in serum samples from 10 patients in each group pre- and postoperatively. In the distraction group, a significant postoperative increase in MMP-1, bFGF, ALP, and BAP could be observed during the lengthening and the consolidation period when compared with the baseline levels. Osteotomy fracture healing without the traction stimulus failed to induce a corresponding increase in these factors. In addition, comparison of both groups revealed a significantly higher increase in TGF-,1, IGF-I, IGFBP-3, and hGH in the lengthening group during the distraction period, indicating key regulatory functions in mechanotransduction. The time courses of changes in MMP-1, bone growth factors (TGF-,1 and bFGF), and hGH, respectively, correlated significantly during the lengthening phase, indicating common regulatory pathways for these factors in distraction osteogenesis. Significant correlation between the osteoblastic marker BAP, TGF-,1, and bFGF suggests strain-activated osteoblastic cells as a major source of systemically increased bone growth factors during callus distraction. The systemic increase in bFGF and MMP-1 might reflect an increased local stimulation of angiogenesis during distraction osteogenesis. [source] Computational alanine scanning of the 1:1 human growth hormone,receptor complexJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 1 2002Shuanghong Huo Abstract The MM-PBSA (Molecular Mechanics,Poisson,Boltzmann surface area) method was applied to the human Growth Hormone (hGH) complexed with its receptor to assess both the validity and the limitations of the computational alanine scanning approach. A 400-ps dynamical trajectory of the fully solvated complex was simulated at 300 K in a 101 Å×81 Å×107 Å water box using periodic boundary conditions. Long-range electrostatic interactions were treated with the particle mesh Ewald (PME) summation method. Equally spaced snapshots along the trajectory were chosen to compute the binding free energy using a continuum solvation model to calculate the electrostatic desolvation free energy and a solvent-accessible surface area approach to treat the nonpolar solvation free energy. Computational alanine scanning was performed on the same set of snapshots by mutating the residues in the structural epitope of the hormone and the receptor to alanine and recomputing the ,Gbinding. To further investigate a particular structure, a 200-ps dynamical trajectory of an R43A hormone,receptor complex was simulated. By postprocessing a single trajectory of the wild-type complex, the average unsigned error of our calculated ,,Gbinding is ,1 kcal/mol for the alanine mutations of hydrophobic residues and polar/charged residues without buried salt bridges. When residues involved in buried salt bridges are mutated to alanine, it is demonstrated that a separate trajectory of the alanine mutant complex can lead to reasonable agreement with experimental results. Our approach can be extended to rapid screening of a variety of possible modifications to binding sites. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 15,27, 2002 [source] Characterization of N -palmitoylated human growth hormone by in situ liquid,liquid extraction and MALDI tandem mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2007Emmanuelle Sachon Abstract Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N, group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid,liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N -palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N -palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications. Copyright © 2007 John Wiley & Sons, Ltd. [source] Sustained low-dose growth hormone therapy optimizes bioactive insulin-like growth factor-I level and may enhance CD4 T-cell number in HIV infectionJOURNAL OF MEDICAL VIROLOGY, Issue 2 2010Ove Andersen Abstract High-dose recombinant human growth hormone (rhGH) (2,6,mg/day) regimes may facilitate T-cell restoration in patients infected with human immunodeficiency virus (HIV) on highly active antiretroviral therapy (HAART). However, high-dose rhGH regimens increase insulin-like growth factor-I (IGF-I) to supra-physiological levels associated with severe side effects. The present study investigated whether lower doses of rhGH may improve T-cell restoration in patients infected with HIV following an expedient response of total and bioactive (i.e., free) IGF-I. A previous 16-week pilot-study included six HIV-infected patients on stable HAART to receive rhGH 0.7,mg/day, which increased total (+117%, P,<,0.01) and free (+155%, P,<,0.01) IGF-I levels. The study was extended to examine whether continuous use of low-dose rhGH (0.7,mg/day until week 60; 0.4,mg/day from week 60 to week 140) would maintain expedient IGF-I levels and improve CD4 T-cell response. Total and free IGF-I increased at week 36 (+97%, P,<,0.01 and +125%, P,<,0.01, respectively) and week 60 (+77%, P,=,0.01 and +125%, P,<,0.01) compared to baseline levels (161,±,15 and 0.75,±,0.11,µg/L). CD4 T-cell number increased at week 36 (+15%, P,<,0.05) and week 60 (+31%, P,=,0.01) compared to baseline levels (456,±,55,cells/µL). Following rhGH dose reduction, total IGF-I and CD4 T-cell number remained increased at week 88 (+44%, P,=,0.01 and +33%, P,<,0.01) and week 140 (+46%, P,=,0.07 and +36%, P,=,0.02) compared to baseline levels. These data support the notion that low-dose rhGH regimens may increase expediently total and bioactive IGF-I and improve T-cell restoration in patients infected with HIV on HAART. J. Med. Virol. 82:197,205, 2010. © 2009 Wiley-Liss, Inc. [source] Solid-phase synthesis of cyclic analogues related to the hypoglycaemic peptide hGH[6,13]: Comparison of two i,i+4 lactam cyclization proceduresJOURNAL OF PEPTIDE SCIENCE, Issue 10 2001Vittoria Cavallaro Abstract The use of 1,3-diisopropylcarbodiimide (DIC) for the synthesis of cyclic analogues of the hypoglycaemic peptide fragment derived from the N -terminus of human growth hormone (hGH), namely hGH[6,13], is described. Different strategies were examined to achieve improved yields for the on resin side-chain to side-chain cyclization of the corresponding linear peptides containing reverse , - turn motifs. When compared with the more reactive Castro's reagent, the results confirm that DIC in the presence of HOBt can be successfully employed to minimize the formation of intermolecular oligomeric by-products associated with the preparation of cyclic hGH[6,14] peptide analogues based on an i,(i+4)Lys,Glu or Glu,Lys cyclization strategy. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd. [source] Immunogenicity of aggregates of recombinant human growth hormone in mouse modelsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2009Amber Haynes Fradkin Abstract Aggregation of recombinant therapeutic protein products is a concern due to their potential to induce immune responses. We examined the immunogenicity of protein aggregates in commercial formulations of recombinant human growth hormone produced by freeze-thawing or agitation, two stresses commonly encountered during manufacturing, shipping and handling of therapeutic protein products. In addition, we subjected each preparation to high-pressure treatment to reduce the size and concentration of aggregates present in the samples. Aggregates existing in a commercial formulation, as well as aggregates induced by freeze-thawing and agitation stresses enhanced immunogenicity in one or more mouse models. The use of high-pressure treatment to reduce size and concentrations of aggregates within recombinant human growth hormone formulations reduced their overall immunogenicity in agreement with the "immunon" hypothesis. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3247,3264, 2009 [source] Effect of dimethyl-,-cyclodextrin concentrations on the pulmonary delivery of recombinant human growth hormone dry powder in ratsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 12 2008Monireh Jalalipour Abstract The aim of this article is to prepare and characterize inhalable dry powders of recombinant human growth hormone (rhGH), and assess their efficacy for systemic delivery of the protein in rats. The powders were prepared by spray drying using dimethyl-,-cyclodextrin (DM,CD) at different molar ratios in the initial feeds. Size exclusive chromatography was performed in order to determine protecting effect of DM,CD on the rhGH aggregation during spray drying. By increasing the concentration of DM,CD, rhGH aggregation was decreased from 9.67 (in the absence of DM,CD) to 0.84% (using DM,CD at 1000 molar ratio in the spray solution). The aerosol performance of the spray dried (SD) powders was evaluated using Andersen cascade impactor. Fine particle fraction values of 53.49%, 33.40%, and 23.23% were obtained using DM,CD at 10, 100, and 1000 molar ratio, respectively. In vivo studies showed the absolute bioavailability of 25.38%, 76.52%, and 63.97% after intratracheal insufflation of the powders produced after spray drying of the solutions containing DM,CD at 10, 100, and 1000 molar ratio, respectively in rat. In conclusion, appropriate cyclodextrin concentration was achieved considering the protein aggregation and aerosol performance of the SD powders and the systemic absorption following administration through the rat lung. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5176,5185, 2008 [source] Thiomers in noninvasive polypeptide delivery: In vitro and in vivo characterization of a polycarbophil-cysteine/glutathione gel formulation for human growth hormoneJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2004Verena M. Leitner Abstract This study was aimed at investigating the potential of a new polycarbophil-cysteine (PCP-Cys)/glutathione (GSH) gel formulation to enhance the permeation of the model drug human growth hormone (hGH) across nasal mucosa in vitro and in vivo. The aqueous nasal gel contained PCP-Cys, GSH, and hGH in a final concentration of 0.3%, 0.5%, and 0.6% (m/v), respectively. In vitro permeation studies were performed in Ussing chambers on freshly excised bovine nasal mucosa using fluorescence-labeled dextran (molecular mass: 4.3 kDa; FD-4) and hGH (FITC-hGH). The release profile of FITC-hGH from the gel formulation and an unmodified PCP control formulation was determined. Furthermore, in vivo studies in rats were performed comparing the PCP-Cys/GSH/hGH gel with PCP/hGH control gel and physiological saline. The permeation of FD-4 and FITC-hGH across the nasal mucosa was improved two-fold and three-fold, respectively, in the presence of PCP-Cys/GSH. The PCP-Cys/GSH/hGH gel and the PCP/hGH control gel showed the same biphasic and matrix-controlled drug release. The nasal administration of the PCP-Cys/GSH/hGH gel formulation to rats resulted in a significantly increased and prolonged hGH plasma concentration,time profile versus unmodified PCP gel and physiological saline. According to these results, PCP-Cys gels might represent a promising new strategy for systemic nasal polypeptide delivery. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1682,1691, 2004 [source] Salivary gland delivery of pDNA-cationic lipoplexes elicits systemic immune responsesORAL DISEASES, Issue 6 2002V Sankar OBJECTIVE: To test the ability of two cationic lipoplexes, Vaxfectin and GAP-DLRIE/DOPE, to facilitate transfection and elicit immune responses from plasmid DNAs (pDNAs) after retrograde instillation into salivary glands. METHODS: Two pDNA expression vectors encoding either the influenza NP protein or human growth hormone (hGH) were complexed with the cationic lipid transfection reagents, GAP-DLRIE/DOPE or Vaxfectin, and delivered to the submandibular glands of rats. Samples from rats receiving the influenza NP protein pDNA and cationic lipoplexes were analyzed for anti-influenza NP-specific IgG1, IgG2a, and IgG2b in serum, and IgA in saliva, by an enzyme- linked immunosorbent assay (ELISA). Cytotoxic T-cell lymphocyte (CTL) assays were also performed. Transgene protein expression (hGH) was determined from extracts of submandibular glands of rats receiving hGH lipoplexes. RESULTS: Serum antibodies (IgG) against the NP protein developed and were highest in all rats vaccinated with GAP-DLRIE/DOPE or Vaxfectin. The major serum IgG subclass stimulated by this route of immunization was IgG2b, followed by IgG2a. CTL assay results showed statistically significantly higher percentage killing in the Vaxfectin group than controls (P < 0.05). No rats developed IgA antibodies to NP protein in saliva. Animals receiving plasmid encoding hGH and either lipoplex expressed significantly higher amounts of hGH compared with those receiving the hGH plasmid and water. Although hGH expression was higher in the animals receiving pDNA/Vaxfectin (, 30-fold > pDNA/water), the difference with those receiving pDNA/GAP-DLRIE/DOPE (,10-fold > pDNA/water) was not significant. CONCLUSIONS: Retrograde instillation of pDNA complexed with Vaxfectin into the salivary glands can stimulate cytotoxic and humoral responses to the influenza NP protein antigen. Optimization of the conditions required to stimulate humoral and secretory antibody formation may facilitate use of this tissue for specific clinical applications of pDNA immunization. [source] Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesisPROTEIN SCIENCE, Issue 9 2005Gábor Pál Abstract Combinatorial shotgun scanning mutagenesis was used to analyze two large, related protein binding sites to assess the specificity and importance of individual side chain contributions to binding affinity. The strategy allowed for cost-effective generation of a plethora of functional data. The ease of the technology promoted comprehensive investigations, in which the classic alanine-scanning approach was expanded with two additional strategies, serine- and homolog-scanning. Binding of human growth hormone (hGH) to the hGH receptor served as the model system. The entire high affinity receptor-binding sites (site 1) of wild-type hGH (hGHwt) and of an affinity-improved variant (hGHv) were investigated and the results were compared. The contributions that 35 residue positions make to binding were assessed on each hormone molecule by both serine- and homolog-scanning. The hormone molecules were displayed on the surfaces of bacteriophage, and the 35 positions were randomized simultaneously to allow equal starting frequencies of the wild-type residue and either serine or a homologous mutation in separate libraries. Functional selections for binding to the hGH receptor shifted the relative wild-type/mutant frequencies at each position to an extent characteristic of the functional importance of the side chain. Functional epitope maps were created and compared to previous maps obtained by alanine-scanning. Comparisons between the different scans provide insights into the affinity maturation process that produced hGHv. The serine and homolog-scanning results expand upon and complement the alanine-scanning results and provide additional data on the robustness of the high affinity receptor-binding site of hGH. [source] O -Glycosylated 24,kDa human growth hormone has a mucin-like biantennary disialylated tetrasaccharide attached at Thr-60PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2009Juan J. Bustamante Abstract 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 (NeuAc2, N -acetyl galactosamine1, Gal1). After ,-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. [source] A proteomic approach combining MS and bioinformatic analysis for the detection and identification of biomarkers of administration of exogenous human growth hormone in humansPROTEOMICS - CLINICAL APPLICATIONS, Issue 8 2009Joshua Boateng Abstract An integrated MS-based proteomic approach is described that combines MALDI-MS and LC-MS with artificial neural networks for the identification of protein and peptide biomarkers associated with recombinant human growth hormone (rhGH) administration. Serum from exercised males administered with rhGH or placebo was analysed using ELISA to determine insulin-like growth factor-I concentrations. Diluted serum from rhGH- and placebo-treated subjects was analysed for protein biomarkers by MALDI-MS, whereas LC-MS was used to analyse tryptically digested ACN-depleted serum extracts for peptide biomarkers. Ion intensities and m/z values were used as inputs to artificial neural networks to classify samples into rhGH- and placebo-treated groups. Six protein ions (MALDI-MS) correctly classified 96% of samples into their respective groups, with a sensitivity of 91% (20 of 22 rhGH treated) and specificity of 100% (24 of 24 controls). Six peptide ions (LC-MS) were also identified and correctly classified 93% of samples with a sensitivity of 90% (19 of 21 rhGH treated) and a specificity of 95% (20 of 21 controls). The peptide biomarker ion with the highest significance was sequenced using LC-MS/MS and database searching and found to be associated with leucine-rich ,-2-glycoprotein. [source] Effect of growth hormone on testicular dysfunction induced by methotrexate in ratsANDROLOGIA, Issue 2 2009H. Serati Nouri Summary Methotrexate (MTX) is a chemotherapeutic agent causing defective oogenesis and spermatogenesis. This study was performed to assess the role of human growth hormone (GH) on testis recovery after treatment with MTX. Forty male Wistar rats were selected and randomly divided into four groups (n = 10): control (vehicle), GH group (0.3 mg kg,1 GH for 28 days, IP), MTX group (MTX 1 mg kg,1 week,1 for 4 weeks, IP) and GH/MTX group (0.3 mg kg,1 GH for 28 day plus 1 mg kg,1 week,1 MTX for 4 weeks, IP). On days 14 and 28, five rats from each group were killed, testes of rats of all groups were removed, spermatozoa were collected from epididymis and then prepared for analysis. MTX caused significant increase in interstitial tissue and capsular thickness and decrease of testicular and body weight (P < 0.05). Moreover, it caused significant decline in seminiferous tubule diameter and epithelium thickness (P < 0.05). There was no obvious change in morphometrical parameters between MTX/GH and control groups. In MTX group, sperm parameters decreased significantly (P < 0.05). Administration of GH plus MTX reduced the effects of MTX on sperm parameters and testosterone concentration. These results suggested that GH had a protective effect on almost all destructive effects caused by MTX in rat testes and thus improved sperm parameters. [source] Crystallization of recombinant human growth hormone at elevated pressures: Pressure effects on PEG-induced volume exclusion interactionsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Ryan L. Crisman Abstract Crystallization of recombinant human growth hormone (rhGH) at elevated pressures was investigated in the presence of 6,000 molecular weight poly(ethylene glycol; PEG-6000). Crystallization of rhGH at atmospheric pressure occurred at a protein concentration of 15,mg/mL in 6% PEG-6000. Crystallization did not occur in the same solutions at 250,MPa. In contrast, at a pressure of 250,MPa in the presence of 8% PEG-6000, rhGH readily crystallized from solutions containing 35,mg/mL rhGH, whereas amorphous precipitate formed in the same solutions at atmospheric pressure. Osmotic virial coefficients were determined from static light scattering measurements and combined with a hard-sphere activity coefficient model to predict rhGH activity coefficients as a function of pressure and PEG concentration. Predicted activity coefficients quantitatively matched those determined from equilibrium solubility measurements. The ability to adjust the thermodynamic non-ideality with pressure provides a valuable tool to study protein crystallization in addition to providing a methodology for obtaining crystals at elevated pressures. Biotechnol. Bioeng. 2010;107:663,672. © 2010 Wiley Periodicals, Inc. [source] Plant-produced human growth hormone shows biological activity in a rat modelBIOTECHNOLOGY PROGRESS, Issue 2 2009Shailaja Rabindran Abstract Plants have been shown to be efficient systems for expressing a wide range of recombinant proteins from various origins. Here, using a plant virus-based expression vector to produce human growth hormone (hGH) in Nicotiana benthamiana plants, we demonstrate, for the first time, that the plant-produced hGH (pphGH) is biologically active in a hypophysectomized rat model. We observed an average weight gain of ,17 g per animal in a group of 10 animals that were injected subcutaneously with pphGH with 60 ,g/dose for 10 days. With the increasing demand for hGH, accompanied with the need to make this recombinant protein available to a wider population at a more reasonable cost, plants provide a feasible alternative to current production platforms. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Expression system for recombinant human growth hormone production from Bacillus subtilisBIOTECHNOLOGY PROGRESS, Issue 1 2009Tunçer H. Özdamar Abstract We demonstrate for the first time, an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hGH) from Bacillus subtilis. A hybrid-gene of two DNA fragments, i.e., signal (pre- ) DNA sequence of B. licheniformis serine alkaline protease gene (subC) and cDNA encoding hGH, were cloned into pMK4 and expressed under deg -promoter in B. subtilis. Recombinant-hGH (rhGH) produced by B. subtilis carrying pMK4::pre(subC)::hGH was secreted. N-terminal sequence and mass spectrometry analyses of rhGH confirm the mature hGH sequence, and indicate that the signal peptide was properly processed by B. subtilis signal-peptidase. The highest rhGH concentration was obtained at t = 32 h as CrhGH = 70 mg L,1 with a product yield on substrate YrhGH/S = 9 g kg,1, in a glucose based defined medium. Fermentation characteristics and influence of hGH gene on the rhGH production were investigated by comparing B. subtilis carrying pMK4::pre(subC)::hGH with that of carrying merely pMK4. Excreted organic-acid concentrations were higher by B. subtilis carrying pMK4::pre(subC)::hGH, whereas excreted amino-acid concentrations were higher by B. subtilis carrying pMK4. The approach developed is expected to be applicable to the design of expression systems for heterologous protein production from Bacillus species. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Heterologous Protein Production from the Inducible MET25 Promoter in Saccharomyces cerevisiaeBIOTECHNOLOGY PROGRESS, Issue 2 2005Steven P. Solow Heterologous protein production late in Saccharomyces cerevisiae fermentations is often desirable because it may help avoid the unintentional selection of more rapidly growing, non-protein-expressing cells or allow for the expression of toxic proteins. Here, we describe the use of the MET25 promoter for the production of human serum albumin (HSA) and HSA-fusion proteins in S. cerevisiae. In media lacking methionine, the MET25 promoter yielded high expression levels of HSA and HSA fused to human glucagon, human growth hormone, human interferon ,, and human interleukin-2. More importantly, we have shown that this system can be used to delay heterologous protein production until late log phase of the growth of the culture and does not require the addition of an exogenous inducer. [source] Growth hormone therapy,established uses in short childrenACTA PAEDIATRICA, Issue 2006W. Frederick Schwenk II Abstract Since the first reported efficacious use of human growth hormone in 1958, numerous children have been treated with this hormone. This review discusses the five indications for use of human growth hormone in children that have been approved to date by the United States Food and Drug Administration. Conclusion: Further, long-term studies will be needed to address the optimal use of this hormone in each of these conditions. [source] | ||||||||||||||||