HgH Molecules Research Articles

Short stature explained by dimerization of human growth hormone induced by a p.C53S point mutation

A homozygous mutation in growth hormone 1 (GH1) was recently identified in an individual with growth failure. This mutation, c.705G>C, causes replacement of cysteine at position 53 of the 191-amino-acid sequence of 22 kDa human GH (hGH) with serine (p.C53S). This hGH molecule (hereafter referred to as GH-C53S) lacks the disulfide bond between p.Cys-53 and p.Cys-165, which is highly conserved among species. It has been reported previously that monomeric GH-C53S has reduced bioactivity compared with WT GH (GH-WT) because of its decreased ability to bind and activate the GH receptor in vitro In this study, we discovered that substitution of p.Cys-53 in hGH significantly increased formation of hGH dimers in pituitary cells. We expressed His-tagged hGH variants in the cytoplasm of genetically modified Rosetta-gami B DE3 Escherichia coli cells, facilitating high-yield production. We observed that the bioactivity of monomeric GH-C53S is 25.2% of that of GH-WT and that dimeric GH-C53S-His has no significant bioactivity in cell proliferation assays. We also found that the expression of GH-C53S in pituitary cells deviates from that of GH-WT. GH-C53S was exclusively stained in the Golgi apparatus, and no secretory granules formed for this variant, impairing its stimulated release. In summary, the unpaired Cys-165 in GH-C53S forms a disulfide bond linking two hGH molecules in pituitary cells. We conclude that the GH-C53S dimer is inactive and responsible for the growth failure in the affected individual.

Spin-orbit coupling in low-lying electronic states of mercury hydride

Ab initio calculations on the low-lying electronic states of mercury hydride (HgH) have been performed with the internally contracted multireference configuration interaction method. The potential energy curves (PECs) of the 10 Λ-S states as well as the 17 Ω states generated from the Λ-S states after considering the spin-orbit effect have been investigated. The spectroscopic constants of the bound states have been calculated. Based on the computed PECs, the Λ-S compositions of some low-lying states, as well as the transition dipole moments, the Franck–Condon factors and the radiative lifetimes for several vibronic transitions have been acquired. Our computed results should be valuable for further experimental research on the molecular electronic structure and properties of HgH molecules.

Large-scale preparation and in vitro characterization of biologically active human placental (20 and 22K) and pituitary (20K) growth hormones: Placental growth hormones have no lactogenic activity in humans

Expression plasmids containing DNA sequences optimized for expression in Escherichia coli were prepared encoding human pituitary (hGH-N 20K) and placental (hGH-V 20 and 22K) growth hormones. The proteins were expressed in bacteria, refolded and purified to homogeneity by anion-exchange chromatography on Q-Sepharose according to a unique protocol developed for each protein. The yields from 5 l of fermentation culture varied between 400 and 700 mg of electrophoretically pure, over 95% monomeric protein. Circular dichroism (CD) analysis revealed similarity of the purified hGHs’ secondary structure to that of the pituitary hGH-N 22K, except for hGH-V 20K, in which the α-helix content was lower. The purified proteins were stable as a 0.1% sterile solution held at pH 10–11 at 4 °C for at least one month. All three purified hGH molecules formed a 1:2 complex with hGH receptor extracellular domain (hGHR-ECD), similar to hGH-N 22K. Binding experiments using hGHR-ECD revealed that the differences between the two 22K variants or between the two 20K variants were not significant, except that hGH-V 20K exhibited slightly lower affinity. Somatogenic activity was tested in vitro using FDC-P1 cell lines. Whereas the bioactivity of 22K hGHs and hGH-N 20K in FDC-P1-9D11 cells stably transfected with hGHR was almost equal and two to threefold higher than that of hGH-V 20K, in FDC-P1 3B9 cells stably transfected with rabbit (rb) GHR, the bioactivity of both 20K analogues was significantly (five to ninefold) lower than that of the 22K hormones. The lactogenic activity measured in heterologous assays (Nb2-11C cells and Baf/3 cells stably transfected with the long form of rabbit prolactin receptor) revealed that the activity of hGH-N 20K was close to that of hGH-N 22K in the Baf/3 cells, but 4.5-fold lower in the Nb2 cells. The activity of hGH-V 22K was ninefold less in Nb2 cells and 55-fold less in Baf/3 cells, whereas hGH-V 20K had no lactogenic activity in either bioassay. In contrast, in a homologous lactogenic assay using Baf/3 LP cells stably transfected with hPRLR, the activity of both placental hGHs was nil and the activity of hGH-N 20K was 4.3-fold lower than that of hGH-N 22K. The latter finding raises the question of whether the lack of intrinsic lactogenic activity in the placental hGHs that dominate during pregnancy has any physiological relevance.

Formulation and implementation of the relativistic Fock-space coupled cluster method for molecules

An implementation of the relativistic multireference Fock-space coupled cluster method is presented which allows simultaneous calculation of potential surfaces for different oxidation states and electronic levels of a molecule, yielding values for spectroscopic constants and transition energies. The method is tested in pilot calculations on the I2 and HgH molecules, and is shown to give a good and balanced description of various electronic states and energies.

ELISA for Determination of Human Growth Hormone: Recognition of Helix 4 Epitopes.

Human growth hormone (hGH) signal transduction initiates with a receptor dimerization in which one molecule binds to the receptor through sites 1 and 2. A sandwich enzyme-linked immunosorbent assay was developed for quantifying hGH molecules that present helix 4 from binding site 1. For this, horse anti-rhGH antibodies were eluted by an immunoaffinity column constituted by sepharose-rhGH. These antibodies were purified through a second column with synthetic peptide correspondent to hGH helix 4, immobilized to sepharose, and used as capture antibodies. Those that did not recognize synthetic peptide were used as a marker antibody. The working range was of 1.95 to 31.25 ng/mL of hGH. The intra-assay coefficient of variation (CV) was between 4.53% and 6.33%, while the interassay CV was between 6.00% and 8.27%. The recovery range was between 96.0% to 103.8%. There was no cross-reactivity with human prolactin. These features show that our assay is an efficient method for the determination of hGH.

A comparison between two methacrylate cements as delivery systems for bioactive human growth hormone

Two methacrylate orthopaedic bone cements were compared as delivery systems for bioactive human growth hormone (hGH). These were London Hospital Bone Cement (LHBC) and a poly(methylmethacrylate) (PMMA) bone cement. A uniquely precise bioassay for hGH, named ESTA, was used. It was adapted to assess both the bioactivity of the hormone released from hGH loaded cements and also the in vitro cytotoxicity of the cements themselves. Eluates from both cements proved cytotoxic, this being most pronounced for LHBC. The cytotoxicity could be readily diluted out, and a lengthy exposure time (> 24 h) was required to kill the cells. Both cements released similar quantities of bioactive hGH, which were ∼0.6% of that originally incorporated. This could potentially provide significantly high local concentrations of the hormone to the tissues surrounding the implant. Approximately double quantities of hormone were released as measured by immunoassay, leading to significant decreases (p=<0.0001) in the B:I ratios of the released hormone. The effect was greatest for LHBC. Our results suggest that the structural integrity of the hGH molecules released from LHBC were the most compromised.

High molecular weight growth hormone (> 160 kD) in human serum characterized with monoclonal antibodies.

Human growth hormone (hGH) was analyzed by six monoclonal antibodies (Mabs) and a polyclonal antiserum (Pas) before and after molecular sieve chromatography of sera from healthy subjects. Their hGH levels were between < 0.2 and 0.4 ng/ml as determined with Pas. The six Mabs reacted with five distinct epitopes and bound to a hGH fragment corresponding to the amino acid sequence 15-125. Two of the Mabs showed reduced binding to 20-kD hGH. The binding of Mabs to dimeric forms of hGH varied. Human GH levels in unfractionated sera as determined with Mabs were < 3.1-390 ng/ml. After molecular sieve chromatography of the sera, one peak of hGH-immunoreactive material of high molecular weight (> 160 kD) and one at the elution volume of monomeric hGH were determined with Pas and Mabs. The major part of the high molecular weight hGH (> 160 kD) seemed to consist of 22-kD hGH molecules, since Pas and all Mabs detected the hGH immunoreactivity (> 160 kD) in a similar manner. This high molecular weight hGH (> 160 kD) was distinguishable from the identified, receptor-like hGH-binding protein in serum.

Reactions of H2, D2 with Hg(6 3P) or Hg(6 3P2): Product rotational state distributions

The initial rotational state distributions of the HgH(v=0) and HgD(v=0) molecules produced in the reactions of Hg(6 3P0) with H2, D2 have been determined. These distributions differ from the ones previously determined with Hg in the 6 3P1 state. This difference may be due to the decrease of the reaction exothermicity when Hg(6 3P1) is replaced by Hg(6 3P0). As for the reaction with Hg(6 3P2), it seems from a time analysis that there is some production of molecules in the higher rotational levels, nevertheless more work is necessary to conclude about the Hg(6 3P2) reactivity.

The Antigenic Epitopes of Human Grown Hormone as Mapped by Monoclonal Antibodies

The antigenic epitopes of human GH (hGH) have been investigated using monoclonal antibodies (Mabs) to hGH. A panel of 12 Mabs was incorporated into two-site binding assays in order to investigate the antigenic surface of hGH. Nine reaction patterns were observed. The Mabs were further characterized in terms of their cross-reactivity with human placental lactogen, human PRL, with recombinant hGH molecules (MetLeu8hGH, Met14hGH), and with fragments derived from enzymatic or chemical digestion of hGH. These studies provided information on the antigenic sites that are shared with human placental lactogen and on the N-terminal and C-terminal regions of the hGH molecule. Based upon these findings, we tentatively suggest the epitope model for hGH comprising at least 10 antigenic sites (epitopes) which may be grouped into five antigenic regions.

The rotational distribution in the mercury hydride molecules (X 2Σ+1/2;v″=0) produced in the reactions Hg(6 3P1)+H2, HD, or D2

Initial rotational distributions of the vibrationless HgH or HgD molecules produced in the exoergic reactions of Hg (3P1) with H2, D2, and HD have been determined. No isotope effects have been observed. In the case of the reaction with HD the ratio [HgH(v″=0)]/[HgD (v″=0)] as well as the summed over all v″ ratio [HgH]/[HgD] have been measured.

468 AMINO ACID RESIDUES 93–96 OF THE HUMAN GROWTH HORMONE MOLECULE ARE IMPORTANT FOR POLYCLONAL ANTISERUM RECOGNITION

The precise regions of the human growth hormone (hGH) molecule that mediate its biologic effects and immunoreactivity are not known. To address this issue, we have introduced mutations into the coding region of a cloned hGH gene and expressed the resulting mutants in a eukaryotic expression vector. Two clones with alterations in the DNA sequences encoding amino acids 93-96 within the hGH molecule were created by the insertion of Bam HI DNA linkers into the Mst II site. The hGH derived from these mutants had characteristics indistinguishable from those of wild type hGH in the NB-2 cell lactogenic assay, the IM-9 cell radioreceptor assay, and in the binding of four anti-hGH monoclonal antibodies. However, significant differences were observed between the mutants and standard human pituitary GH or hGH produced by the normal gene in the binding of polyclonal hGH antisera. Both mutants bound to the polyclonal antibody poorly but did not displace labeled standard hormone in a proportionate manner.These results suggest that amino acids 93-96 of hGH regulate the expression of antigenic determinants that are detected by polyclonal antiserum, but these determinants are distinct from those recognized by the four monoclonal antibodies that were tested. Moreover, hGH molecules with abnormal immunologic reactivity may retain normal biologic activities in certain assays.

ESR Spectroscopy and Chemical Bonding in CdCN and HgCN Molecules

The radical molecules CdCN and HgCN were trapped in rare gas matrices at 4°K and investigated via ESR spectroscopy. The g tensors and hyperfine structure constants (hfs) for interaction with 111Cd, 113Cd, 199Hg, and 201Hg were determined. These measurements indicate that CdCN and HgCN have 2Σ ground electronic states and predict the presence of 2IIr excited states near 2700 Å. The ionicity of these molecules are discussed based on an (ns)2S M+ limiting model and compared with previous results obtained for the more covalent CdH and HgH molecules.

Hyperfine Interaction, Chemical Bonding, and Isotope Effect in ZnH, CdH, and HgH Molecules

ESR spectra of ZnH, CdH, HgH, and HgD molecules in their ground Σ+2 states were measured while trapped in solid argon matrices at 4°K. g tensors and hyperfine structure (hfs) constants for interaction with H, D, 111,113Cd, and 199,201Hg nuclei were observed. Coefficients of approximate LCAO wavefunctions are derived from these magnetic parameters and correlated with chemical bonding trends in the series and with the limiting united-atom model. Replacing H by D in HgH causes an increase of spin density on Hg and a decrease in g∇. This is shown to be largely due to the small decrease in interatomic distance and is discussed relative to other spectroscopic observations.

Quenching of Hg*(3P1) Atoms by Hydrogen Molecules Investigated by Phase-Space Theory

Phase-space theory is used to investigate product distribution and internal energy distribution among various products in the mercury-sensitized decomposition of hydrogen molecules. Calculated quantum yields at room temperature for H atoms, HgH, and undissociated hydrogen molecules are 0.52, 0.16, and 0.58, respectively. The H-atom yield is much lower than the possible maximum of 2.0 but agrees well with an experimental value of about 0.5. The majority of undissociated hydrogen molecules is produced in vibrationally excited states. In mercury-sensitized decompositions, these vibrationally hot H2 molecules may undergo reactions which were, in the past, attributed solely to H-atom reactions.

Primary Step in the Mercury Photo-Sensitized Decomposition of Propane and of Hydrogen

An attempt has been made to obtain resonance emission of HgH bands in propane-mercury mixtures illuminated with λ2537. It is concluded that if HgH is formed in the primary step in the photo-sensitized decomposition of paraffins, it must possess excess energy and largely dissociate immediately. Further experiments with mercury-hydrogen mixtures illuminated with λ2537 have also been made, and the mechanism of the production of excited HgH molecules is discussed.

The Mechanism of Formation of Mercury Hydride and Mercury Deuteride. Optical Excitation of Cadmium Deuteride

An attempt has been made to secure resonance excitation of HgH by illuminating (1) a mixture of mercury, hydrogen and nitrogen, and (2) a mixture of mercury and water vapor, with the light from a mercury and hydrogen discharge. This discharge emitted a fairly intense HgH spectrum. Weak HgH band radiation was observed which was similar to that secured when the same mixtures were illuminated with a discharge tube which did not emit the HgH radiation. Inability to secure resonance is evidence that the weak band radiation is due to HgH molecules formed in excited states which dissociate after emission. Experimental evidence is presented which indicates that the formation process is Hg63P1+H→HgH2π1/2, 3/2+Δ,where Δ is an energy parameter. Essentially similar results were secured for HgD. Resonance excitation of CdD was observed when a mixture of cadmium and deuterium was illuminated with a cadmium and deuterium discharge.

Transfer of Rotational Energy in Molecular Collisions II. Exchange of Energy in Collisions Between Unexcited HgH and N2 Molecules

It has been found that the intensity distribution in the HgH 4017 band excited by sensitized fluorescence in the mixture Hg+H2+N2 is influenced by the intensity of the primary light which excites the fluorescence. This observation may be interpreted as evidence of a very slow exchange of vibrational energy in collisions between N2 and HgH molecules in their normal electronic states. Two other possible causes of the phenomenon—self-reversal and reactions involving high energy Hg atoms formed by stepwise excitation—are ruled out by additional experiments.

Transfer of Rotational Energy in Molecular Collisions I. Elementary Processes Which Lead to Abnormal Rotation of the HgH Molecule

By the application of photographic photometry, the intensity distribution within the HgH 4017 band excited by mercury-sensitized fluorescence has been studied as a function of the pressure of nitrogen and the pressure of water vapor present in the fluorescence tube. From the effect which the foreign gases exert on the observed distribution of molecules among rotational levels, conclusions are drawn regarding the transfers of energy which occur in collisions of the HgH molecule with metastable Hg atoms and with normal H2O and N2 molecules. The proportion of excited HgH molecules with rotational energy greater than 0.2 volt greatly exceeds the thermal value under all circumstances; the excess rotational energy has its source in the two following processes. In a collision between an unexcited HgH molecule and a metastable Hg atom, the excitation energy of the latter is sometimes taken up as both rotational and electronic energy of the HgH molecule. In a collision between a normal N2 molecule and a highly excited HgH molecule, part of the electronic and vibrational energy of the latter is converted into rotational energy.

On the Persistence of Molecular Rotation and Vibration in Collision

The present paper attempts to explain some observations on the exchange of vibrational and rotational energy of molecules on the basis of the laws of impact, taking into account the postulate of quantisation. It is pointed out that when one of the bodies involved in the impact is much lighter than the other the exchange of energy becomes improbable. The persistence of rotation of HgH molecules in an excess of nitrogen, suggested by Beutler and Rabinowitsch, is discussed. A related phenomenon is found in some chemical activations produced by ${\mathrm{H}}_{2}$ molecules but not by any other molecule. The resonance rule for the probability of impacts of the second kind needs some modification for the exchange of vibrational and rotational energy between molecules. The transfer of vibration and rotation from the excited iodine molecule is discussed. In electron impact, the rotation of the molecule does not change, but the vibration does. This is interpreted on the basis of a hypothesis introduced by Franck and Jordan. Within ordinary gases, ${\mathrm{N}}_{2}$ or ${\mathrm{H}}_{2}$ or ${\mathrm{I}}_{2}$, no persistence of vibration or rotation should be expected.