Growth hormone response to growth hormone-releasing peptide-2 in growth hormone-deficient Little mice

OBJECTIVE: To investigate a possible direct, growth hormone-releasing, hormone-independent action of a growth hormone secretagogue, GHRP-2, in pituitary somatotroph cells in the presence of inactive growth hormonereleasing hormone receptors. MATERIALS AND METHODS: The responses of serum growth hormone to acutely injected growth hormone-releasing P-2 in lit/litmice, which represent a model of GH deficiency arising frommutated growth hormone-releasing hormonereceptors, were compared to those observed in the heterozygous (lit/+) littermates and wild-type (+/+) C57BL/6J mice. RESULTS: After the administration of 10 mcg of growth hormone-releasing P-2 to lit/lit mice, a growth hormone release of 9.3±1.5 ng/ml was observed compared with 1.04±1.15 ng/ml in controls (p<0.001). In comparison, an intermediate growth hormone release of 34.5±9.7 ng/ml and a higher growth hormone release of 163±46 ng/ml were induced in the lit/+ mice and wild-type mice, respectively. Thus, GHRP-2 stimulated growth hormone in the lit/lit mice, and the release of growth hormone in vivo may be only partially dependent on growth hormone-releasing hormone. Additionally, the plasma leptin and ghrelin levels were evaluated in the lit/lit mice under basal and stimulated conditions. CONCLUSIONS: Here, we have demonstrated that lit/lit mice, which harbor a germline mutation in the Growth hormone-releasing hormone gene, maintain a limited but statistically significant growth hormone elevation after exogenous stimulation with GHRP-2. The present data probably reflect a direct, growth hormone-independent effect on Growth hormone S (ghrelin) stimulation in the remaining pituitary somatotrophs of little mice that is mediated by growth hormone S-R 1a.

Development of models and methods to simulate peptide-assisted nucleation and growth of calcium-minerals

In den vergangenen Jahren wurden einige bislang unbekannte Phänomene experimentell beobachtet, wie etwa die Existenz unterschiedlicher Prä-Nukleations-Strukturen. Diese haben zu einem neuen Verständnis von Prozessen, die auf molekularer Ebene während der Nukleation und dem Wachstum von Kristallen auftreten, beigetragen. Die Auswirkungen solcher Prä-Nukleations-Strukturen auf den Prozess der Biomineralisation sind noch nicht hinreichend verstanden. Die Mechanismen, mittels derer biomolekulare Modifikatoren, wie Peptide, mit Prä-Nukleations-Strukturen interagieren und somit den Nukleationsprozess von Mineralen beeinflussen könnten, sind vielfältig. Molekulare Simulationen sind zur Analyse der Formation von Prä-Nukleations-Strukturen in Anwesenheit von Modifikatoren gut geeignet. Die vorliegende Arbeit beschreibt einen Ansatz zur Analyse der Interaktion von Peptiden mit den in Lösung befindlichen Bestandteilen der entstehenden Kristalle mit Hilfe von Molekular-Dynamik Simulationen.rnUm informative Simulationen zu ermöglichen, wurde in einem ersten Schritt die Qualität bestehender Kraftfelder im Hinblick auf die Beschreibung von mit Calciumionen interagierenden Oligoglutamaten in wässrigen Lösungen untersucht. Es zeigte sich, dass große Unstimmigkeiten zwischen etablierten Kraftfeldern bestehen, und dass keines der untersuchten Kraftfelder eine realistische Beschreibung der Ionen-Paarung dieser komplexen Ionen widerspiegelte. Daher wurde eine Strategie zur Optimierung bestehender biomolekularer Kraftfelder in dieser Hinsicht entwickelt. Relativ geringe Veränderungen der auf die Ionen–Peptid van-der-Waals-Wechselwirkungen bezogenen Parameter reichten aus, um ein verlässliches Modell für das untersuchte System zu erzielen. rnDas umfassende Sampling des Phasenraumes der Systeme stellt aufgrund der zahlreichen Freiheitsgrade und der starken Interaktionen zwischen Calciumionen und Glutamat in Lösung eine besondere Herausforderung dar. Daher wurde die Methode der Biasing Potential Replica Exchange Molekular-Dynamik Simulationen im Hinblick auf das Sampling von Oligoglutamaten justiert und es erfolgte die Simulation von Peptiden verschiedener Kettenlängen in Anwesenheit von Calciumionen. Mit Hilfe der Sketch-Map Analyse konnten im Rahmen der Simulationen zahlreiche stabile Ionen-Peptid-Komplexe identifiziert werden, welche die Formation von Prä-Nukleations-Strukturen beeinflussen könnten. Abhängig von der Kettenlänge des Peptids weisen diese Komplexe charakteristische Abstände zwischen den Calciumionen auf. Diese ähneln einigen Abständen zwischen den Calciumionen in jenen Phasen von Calcium-Oxalat Kristallen, die in Anwesenheit von Oligoglutamaten gewachsen sind. Die Analogie der Abstände zwischen Calciumionen in gelösten Ionen-Peptid-Komplexen und in Calcium-Oxalat Kristallen könnte auf die Bedeutung von Ionen-Peptid-Komplexen im Prozess der Nukleation und des Wachstums von Biomineralen hindeuten und stellt einen möglichen Erklärungsansatz für die Fähigkeit von Oligoglutamaten zur Beeinflussung der Phase des sich formierenden Kristalls dar, die experimentell beobachtet wurde.

Effects of Vasoactive Intestinal Peptide and Nerve Growth Factor on Rat Osteosarcoma Cells

Submitted in partial fulfillment of the requirements for a Certificate in Orthodontics, Dept. of Orthodontics, University of Connecticut Health Center, 1986

A peptide that inhibits hydroxyapatite growth is in an extended conformation on the crystal surface

Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solid-state NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constant-composition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two 13C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 ± 0.1 Å. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete α-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an α-helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.

A peptide derived from an extracellular domain selectively inhibits receptor internalization: Target sequences on insulin and insulin-like growth factor 1 receptors

Certain peptides derived from the α1 domain of the major histocompatibility class I antigen complex (MHC-I) inhibit receptor internalization, increasing the steady-state number of active receptors on the cell surface and thereby enhancing the sensitivity to hormones and other agonists. These peptides self-assemble, and they also bind to MHC-I at the same site from which they are derived, suggesting that they could bind to receptor sites with significant sequence similarity. Receptors affected by MHC-I peptides do, indeed, have such sequence similarity, as illustrated here by insulin receptor (IR) and insulin-like growth factor-1 receptor. A synthetic peptide with sequence identical to a certain extracellular receptor domain binds to that receptor in a ligand-dependent manner and inhibits receptor internalization. Moreover, each such peptide is selective for its cognate receptor. An antibody to the IR peptide not only binds to IR and competes with the peptide but also inhibits insulin-dependent internalization of IR. These observations, and binding studies with deletion mutants of IR, indicate that the sequence QILKELEESSF encoded by exon 10 plays a key role in IR internalization. Our results illustrate a principle for identifying receptor-specific sites of importance for receptor internalization, and for enhancing sensitivity to hormones and other agonists.

Oryza sativa PSK gene encodes a precursor of phytosulfokine-α, a sulfated peptide growth factor found in plants

Phytosulfokine-α [PSK-α, Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln], a sulfated mitogenic peptide found in plants, strongly promotes proliferation of plant cells in culture at very low concentrations. Oryza sativa PSK (OsPSK) cDNA encoding a PSK-α precursor has been isolated. The cDNA is 725 base pairs long, and the 89-aa product, preprophytosulfokine, has a 22-aa hydrophobic region that resembles a cleavable leader peptide at its NH2 terminus. The PSK-α sequence occurs only once within the precursor, close to the COOH terminus. [Ser4]PSK-α was secreted by transgenic rice Oc cells harboring a mutated OsPSK cDNA, suggesting proteolytic processing from the larger precursor, a feature commonly found in animal systems. Whereas PSK-α in conditioned medium with sense transgenic Oc cells was 1.6 times as concentrated as in the control case, antisense transgenic Oc cells produced less than 60% of the control level. Preprophytosulfokine mRNA was detected at an elevated constitutive level in rice Oc culture cells on RNA blot analysis. Although PSK-α molecules have never been identified in any intact plant, reverse transcription–PCR analysis demonstrated that OsPSK is expressed in rice seedlings, indicating that PSK-α may be important for plant cell proliferation both in vitro and in vivo. DNA blot analysis demonstrated that OsPSK homologs may occur in dicot as well as monocot plants.

A peptide derived from the non-receptor-binding region of urokinase plasminogen activator inhibits glioblastoma growth and angiogenesis in vivo in combination with cisplatin

The urokinase plasminogen activator system is involved in angiogenesis and tumor growth of malignant gliomas, which are highly neovascularized and so may be amenable to antiangiogenic therapy. In this paper, we describe the activity of Å6, an octamer capped peptide derived from the non-receptor-binding region of urokinase plasminogen activator. Å6 inhibited human microvascular endothelial cell migration but had no effect on the proliferation of human microvascular endothelial cells or U87MG glioma cells in vitro. In contrast, Å6 or cisplatin (CDDP) alone suppressed subcutaneous tumor growth in vivo by 48% and 53%, respectively, and, more strikingly, the combination of Å6 plus CDDP inhibited tumor growth by 92%. Such combination treatment also greatly reduced the volume of intracranial tumor xenografts and increased survival of tumor-bearing animals when compared with CDDP or Å6 alone. Tumors from the combination treatment group had significantly reduced neovascularization, suggesting a mechanism involving Å6-mediated inhibition of endothelial cell motility, thereby eliciting vascular sensitivity to CDDP-mediated toxicity. These data suggest that the combination of an angiogenesis inhibitor that targets endothelial cells with a cytotoxic agent may be a useful therapeutic approach.

Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction.

Vaccination with synthetic peptides representing cytotoxic T lymphocyte (CTL) epitopes can lead to a protective CTL-mediated immunity against tumors or viruses. We now report that vaccination with a CTL epitope derived from the human adenovirus type 5 E1A-region (Ad5E1A234-243), which can serve as a target for tumor-eradicating CTL, enhances rather than inhibits the growth of Ad5E1A-expressing tumors. This adverse effect of peptide vaccination was rapidly evoked, required low doses of peptide (10 micrograms), and was achieved by a mode of peptide delivery that induces protective T-cell-mediated immunity in other models. Ad5E1A-specific CTL activity could no longer be isolated from mice after injection of Ad5E1A-peptide, indicating that tolerization of Ad5E1A-specific CTL activity causes the enhanced tumor outgrowth. In contrast to peptide vaccination, immunization with adenovirus, expressing Ad5E1A, induced Ad5E1A-specific immunity and prevented the outgrowth of Ad5E1A-expressing tumors. These results show that immunization with synthetic peptides can lead to the elimination of anti-tumor CTL responses. These findings are important for the design of safe peptide-based vaccines against tumors, allogeneic organ transplants, and T-cell-mediated autoimmune diseases.

The recombinant proregion of transforming growth factor beta1 (latency-associated peptide) inhibits active transforming growth factor beta1 in transgenic mice.

All three isoforms of transforming growth factors beta (TGF-betal, TGF-beta2, and TGF-beta3) are secreted as latent complexes and activated extracellularly, leading to the release of the mature cytokines from their noncovalently associated proregions, also known as latency-associated peptides (LAPs). The LAP region of TGF-beta1 was expressed in a baculovirus expression system and purified to homogeneity. In vitro assays of growth inhibition and gene induction mediated by TGF-beta3 demonstrate that recombinant TGF-beta1 LAP is a potent inhibitor of the activities of TGF-betal, -beta2, and -beta3. Effective dosages of LAP for 50% neutralization of TGF-beta activities range from 4.7- to 80-fold molar excess depending on the TGF-beta isoform and activity examined. Using 125I-labeled LAP, we show that the intraperitoneal application route is effective for systemic administration of LAP. Comparison of concentrations of LAP in tissues shows a homogenous pattern in most organs with the exception of heart and muscle, in which levels of LAP are 4- to 8-fold lower. In transgenic mice with elevated hepatic levels of bioactive TGF-betal, treatment with recombinant LAP completely reverses suppression of the early proliferative response induced by TGF-beta1 in remnant livers after partial hepatectomy. The results suggest that recombinant LAP is a potent inhibitor of bioactive TGF-beta both in vitro and in vivo, after intraperitoneal administration. Recombinant LAP should be a useful tool for novel approaches to study and therapeutically modulate pathophysiological processes mediated by TGF-beta3.

Three-dimensional structure of recombinant human osteogenic protein 1: structural paradigm for the transforming growth factor beta superfamily.

We report the three-dimensional structure of osteogenic protein 1 (OP-1, also known as bone morphogenetic protein 7) to 2.8-A resolution. OP-1 is a member of the transforming growth factor beta (TGF-beta) superfamily of proteins and is able to induce new bone formation in vivo. Members of this superfamily share sequence similarity in their C-terminal regions and are implicated in embryonic development and adult tissue repair. Our crystal structure makes possible the structural comparison between two members of the TGF-beta superfamily. We find that although there is limited sequence identity between OP-1 and TGF-beta 2, they share a common polypeptide fold. These results establish a basis for proposing the OP-1/TGF-beta 2 fold as the primary structural motif for the TGF-beta superfamily as a whole. Detailed comparison of the OP-1 and TGF-beta 2 structures has revealed striking differences that provide insights into how these growth factors interact with their receptors.

The specificity of the transforming growth factor beta receptor kinases determined by a spatially addressable peptide library.

Type I and II receptors for the transforming growth factor beta (TGF-beta) are transmembrane serine/threonine kinases that are essential for TGF-beta signaling. However, little is known about their in vivo substrates or signal transduction pathways. To determine the substrate specificity of these kinases, we developed combinatorial peptide libraries synthesized on a hydrophilic matrix that is easily accessible to proteins in aqueous solutions. When we subjected these libraries to phosphorylation by the cAMP-dependent protein kinase, we obtained the optimal peptide sequence RRXS (I/L/V), in perfect agreement with the substrate sequence deduced from mutagenesis and crystal structure analyses. By using the same libraries, we showed that the optimal substrate peptide for both the type I and II TGF-beta receptors was KKKKKK(S/T)XXX. Since the two kinases are thought to play different roles in intracellular signal transduction, it was a surprise to find that they have almost identical substrate specificity. Our method is direct, sensitive, and simple and provides information about the kinase specificity for all the amino acid residues at each position.

Thermodynamic parameters for the helix-coil transition of oligopeptides: molecular dynamics simulation with the peptide growth method.

The helix-coil transition equilibrium of polypeptides in aqueous solution was studied by molecular dynamics simulation. The peptide growth simulation method was introduced to generate dynamic models of polypeptide chains in a statistical (random) coil or an alpha-helical conformation. The key element of this method is to build up a polypeptide chain during the course of a molecular transformation simulation, successively adding whole amino acid residues to the chain in a predefined conformation state (e.g., alpha-helical or statistical coil). Thus, oligopeptides of the same length and composition, but having different conformations, can be incrementally grown from a common precursor, and their relative conformational free energies can be calculated as the difference between the free energies for growing the individual peptides. This affords a straightforward calculation of the Zimm-Bragg sigma and s parameters for helix initiation and helix growth. The calculated sigma and s parameters for the polyalanine alpha-helix are in reasonable agreement with the experimental measurements. The peptide growth simulation method is an effective way to study quantitatively the thermodynamics of local protein folding.

A nerve growth factor peptide retards seizure development and inhibits neuronal sprouting in a rat model of epilepsy.

Kindling, an animal model of epilepsy wherein seizures are induced by subcortical electrical stimulation, results in the upregulation of neurotrophin mRNA and protein in the adult rat forebrain and causes mossy fiber sprouting in the hippocampus. Intraventricular infusion of a synthetic peptide mimic of a nerve growth factor domain that interferes with the binding of neurotrophins to their receptors resulted in significant retardation of kindling and inhibition of mossy fiber sprouting. These findings suggest a critical role for neurotrophins in both kindling and kindling-induced synaptic reorganization.