Luis S. Haro

Learning associated increase in heat shock cognate 70 mRNA and protein expression.

The Morris water maze is a task widely used to investigate cellular and molecular changes associated with spatial learning and memory. This task has both spatial and aversive (swimming related stress) components. It is possible that stress may influence cellular modifications observed after learning the Morris water maze spatial task. Heat shock proteins, also known as stress proteins, are up-regulated in response to thermal stress, trauma, or environmental insults. In the rat hippocampus, psychophysiological stress increases the levels of heat shock protein 70 (HSC70). In this study, we investigated whether the expression of the hsc70 gene is modulated in the hippocampus during learning of the Morris water maze task. Five groups of rats were trained in the Morris water maze task for varying amounts of time (either 1, 2, 3, 4, or 5 days). Training consisted of 10 trials/day in which the animals were given 60s to find a submerged platform. Rats were sacrificed 24h after their last training trial. Results showed a significant increase in hsc70 mRNA and protein levels in the hippocampal formation after two and three days of training, respectively. The increase in mRNA and protein was associated with learning but not stress because the increase was not observed in the yoked control animals. These findings suggest that cellular and molecular changes can occur independent of stress. Moreover, the results are the first to implicate hsc70 expression in spatial learning.

Extraordinarily stable disulfide-linked homodimer of human growth hormone

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°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 hGHs interchain disulfide links are exceptionally resistant to reducing agents and thus confer extreme stability to the homodimer.

Growth hormone responsive neural precursor cells reside within the adult mammalian brain

The detection of growth hormone (GH) and its receptor in germinal regions of the mammalian brain prompted our investigation of GH and its role in the regulation of endogenous neural precursor cell activity. Here we report that the addition of exogenous GH significantly increased the expansion rate in long-term neurosphere cultures derived from wild-type mice, while neurospheres derived from GH null mice exhibited a reduced expansion rate. We also detected a doubling in the frequency of large (i.e. stem cell-derived) colonies for up to 120 days following a 7-day intracerebroventricular infusion of GH suggesting the activation of endogenous stem cells. Moreover, gamma irradiation induced the ablation of normally quiescent stem cells in GH-infused mice, resulting in a decline in olfactory bulb neurogenesis. These results suggest that GH activates populations of resident stem and progenitor cells, and therefore may represent a novel therapeutic target for age-related neurodegeneration and associated cognitive decline.

Divalent metal cation chelators enhance chromatographic separation of structurally similar macromolecules: separation of human growth hormone isoforms

Human GH isoforms were separated by anion-exchange chromatography using a linear NaCl gradient in the presence and absence of EDTA and EGTA. SDS-PAGE showed that glycosylated 24-kDa hGH did not appreciably separate from other hGH variants in the absence of metal chelators. However, in the presence of metal chelators, glycosylated 24-kDa hGH separated from the bulk of the hGH isoforms. Human GH isoforms were also separated by size-exclusion chromatography in the presence and absence of metal chelators. Glycosylated 24-kDa hGH eluted with the bulk of the hGH isoforms in both separations. The inclusion of metal chelators in chromatographic buffers to alter the charge and/or size of proteins by stripping their metals may be a generally useful strategy in their fractionation.

O-Glycosylated 24 kDa human growth hormone has a mucin-like biantennary disialylated tetrasaccharide attached at Thr-60

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 β‐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.

Haptoglobin gene expression in human glioblastoma cell lines

Increases in cerebrospinal fluid (CSF) levels of the acute phase protein haptoglobin (Hp) occur in central nervous system (CNS) disorders such as Alzheimers disease. To establish if Hp CSF level increases can be associated with Hp expression in brain, reverse transcription-polymerase chain reaction (RT-PCR) experiments were conducted to determine if the Hp mRNA transcript is expressed in human glioblastoma cells. Furthermore, Western blots and immunoprecipitations were performed to elucidate if Hp protein is synthesized and secreted by human glioblastoma cells. The Hp mRNA (alpha2beta) transcript (1155 bp) was detected both in U-87MG and U-138MG cells, and was positively verified by nested PCR in which a part of the beta sequence (482 bp) was targeted for amplification. Despite the presence of Hp mRNA, Hp protein was not secreted by U-87MG cells as compared to the hepatoma cell line, HepG2, where Hp protein (approximately 46 kDa) was detected in the media. The results suggest the expression of Hp protein by glioblastoma cells is possible since the Hp mRNA transcript exist, but whether or not Hp mRNA is contained in a storage pool requiring a specific signal for translation or is transiently expressed remains to be uncovered in future studies.

MAP dendrimer elicits antibodies for detecting rat and mouse GH-binding proteins.

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‐BP263–279) was synthesized and used as an immunogen in rabbits. Solid‐phase peptide synthesis of four GH‐BP263–279 segments onto a tetravalent Lys2‐Lys‐β‐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‐BP263–279 MAP antisera, BETO‐8039, BETO‐8040, and BETO‐8041, at dilutions of 10−3, recognized both the rat GH‐BP263–279 MAP and recombinant mouse GH‐BP with ED50s 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 Mrs 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‐BP263–279 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. Copyright

Human Growth Hormone: 45-kDa Isoform with Extraordinarily Stable Interchain Disulfide Links has Attenuated Receptor-Binding and Cell-Proliferative Activities

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 hGHs binding affinities to both GH and PRL receptors and also drastically attenuates its ability to stimulate proliferation of cells in vitro.

Biochemistry and pharmacology of rabbit cardiac growth hormone (GH) receptors.

In this report we present the first in-depth description of the biochemical and pharmacological properties of rabbit cardiac GH receptors. The apparent M(r)s of the [125I]human (h) GH-receptor complexes were 380, 205, 90, 62, 52 and 38 kDa as demonstrated by an autoradiograph of affinity-labelled cardiac GH receptors separated under non-reducing conditions by SDS PAGE. The [125I]hGH-cardiac GH receptor complexes were disulfide-linked since the M(r)s of the complexes diminished to 170, 116, 97, 71, 45 and 38 kDa under reducing conditions, indicating the presence of multiple receptors, receptor-associated macromolecules or receptor and ligand in various ratios. The pharmacology of cardiac GH receptors is not typical of GH receptors present in other tissues. In radio receptor assays, both bovine GH and ovine prolactin were 50-fold and 100-fold less potent, respectively, than unlabelled hGH, in blocking the binding of [125I]hGH to cardiac binding sites and were, therefore, extremely weak antagonists. Similarly, neither bovine GH nor ovine prolactin blocked the [125I]hGH affinity-labelling of cardiac GH receptors compared to equivalent doses of unlabelled hGH. Parameters which characterize the kinetics for the association, dissociation and equilibrium binding of [125I]hGH to cardiac GH receptors were ascertained. Association kinetics for the binding of [125I]hGH to heart GH receptors exhibited a maximum specific binding at 17 h and 25 degrees C. The association of [125I]hGH to heart GH receptors was reversible with approximately 15 h required for half of the specifically bound [125I]hGH to dissociate. The coupling of [125I]hGH to heart GH receptors was optimum at pH 6 and the strength of the equilibrium binding, as measured by the ED50, was approximately 2 ng/ml. These data indicate that the cardiac GH receptors are pharmacologically distinct and that there is a M(r) heterogeneity in the [125I]hGH receptor complexes.