Biomedical subjects

Peter Kristensen

Publications and source records attributed to Peter Kristensen.

8 recordsLinked to original sources

PubMed2003

A novel cell binding site in the coiled-coil domain of laminin involved in capillary morphogenesis.

Recently, we reported the isolation and characterization of an anti-laminin antibody that modulates the extracellular matrix-dependent morphogenesis of endothelial cells. Here we use this antibody to precisely map the binding site responsible for mediating this biologically important interaction. By using a phage display-assisted mapping strategy to preserve protein structure, we demonstrate for the first time that the coiled-coil region of laminin contains a cell binding site. The adhesion motif is formed by residues contributed by both alpha and gamma chains, and is located in the middle part of the rod-like portion in a highly flexible area, which corresponds to a protease-susceptible site. Based on this information, a peptide mimotope was used to characterize the cognate receptor. Although we can not rule out the implication of other receptors, our results demonstrate that the laminin helical rod active site interacts with alpha2beta1 integrin on the surface of endothelial cells. These findings provide new insight into the complex mechanisms regulating capillary morphogenesis.

Amino Acid Sequence↗

PubMed2003

Hypothalamic cocaine-amphetamine regulated transcript (CART) is regulated by glucocorticoids.

Cocaine-amphetamine-regulated transcript (CART) is one of the most abundantly expressed mRNAs in the rat hypothalamus. CART mRNA expression in the arcuate nucleus has been shown to be regulated by leptin, and CART peptides have been implicated in feeding behavior and in the regulation of the HPA-axis. To more fully understand the physiological regulation of CART gene expression, we have examined the effects of adrenalectomy and different types of glucocorticoid substitution (corticosterone and dexamethasone) on hypothalamic CART and POMC mRNA levels. In situ hybridization revealed a reduction in CART mRNA levels in both the hypothalamic paraventricular and arcuate nuclei in adrenalectomized rats, which was fully restored upon dexamethasone treatment but not by a subcutaneous 25% corticosterone pellet. Unlike CART mRNA levels hypothalamic POMC expression was unaltered by adrenenalectomy. The present results show that the CART gene is influenced by glucocorticoids, presumably via a GR dependent mechanism.

Adrenalectomy↗

PubMed2003

Identification of keratinocyte-specific markers using phage display and mass spectrometry.

Specific molecular markers for various normal and pathogenic cell states and cell types provide knowledge of basic biological systems and have a direct application in targeted therapy. We describe a proteomic method based on the combination of new and improved phage display antibody technologies and mass spectrometry that allows identification of cell type-specific protein markers. The most important features of the method are (i) reduction of experimental noise originating from background binding of phage particles and (ii) isolation of affinity binders after a single round of selection, which assures a high diversity of binders. The method demonstrates, for the first time, the ability to detect, identify, and analyze both secreted and membrane-associated extracellular proteins as well as a variety of different cellular structures including proteins and carbohydrates. The optimized phage display method was applied to analysis of human skin keratinocytes resulting in the isolation of a panel of antibodies. Fourteen of these antibodies were further characterized, half of which predominantly recognized keratinocytes in a screen of a range of different cell types. Three cognate keratinocyte antigens were subsequently identified by mass spectrometry as laminin-5, plectin, and fibronectin. The combination of phage display technology with mass spectrometry methods for protein identification is a general and promising approach for proteomic analysis of cell surface complexity.

Antibodies, Monoclonal↗

PubMed2002

Functional improvement of antibody fragments using a novel phage coat protein III fusion system.

Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.

Base Sequence↗

PubMed2002

Directed evolution of barnase stability using proteolytic selection.

We report the construction of a phage-displayed repertoire of mutants of the ribonuclease barnase from Bacillus amyloliquefaciens. The construction was guided by the natural variability between two closely related ribonucleases, barnase and binase from Bacillus intermedius. This repertoire was selected using a proteolytic selection method, allowing sorting of the library according to the resistance of the mutants toward proteolysis. Susceptibility toward proteolysis has been correlated with flexibility and unfolding, and is thus expected to yield mutants with increased thermal stability. Enrichment of barnase mutants with specific combinations of amino acid residues at four of the randomised positions was observed. Three of these enriched amino acid residues are present in neither barnase nor binase. For some of the mutations, the improvement in proteolytic stability does not lead to a pronounced improvement in thermodynamic stability, indicating that the factors governing the proteolytic stability in some cases may be different from those governing the thermodynamic stability, e.g. propensity to local unfolding.The results obtained add important knowledge to a novel use of phage display technology for selection of thermodynamically stable proteins. Only by carefully establishing the parameters that can be adjusted, and recognising the influence this will have on the outcome of selection, will it be possible to realise the powerful technique of proteolytic selection.

Amino Acid Sequence↗

PubMed2002

Cocaine-amphetamine regulated transcript (CART) expression is not regulated by amphetamine.

Cocaine-amphetamine-regulated transcript (CART) is one of the most abundantly expressed mRNAs in the rat hypothalamus. Nevertheless, CART was identified from striatal extracts as a transcript induced acutely by cocaine or amphetamine treatment. In the hypothalamus, CART mRNA expression has been shown to be regulated by leptin, and CART peptides have been implicated in feeding behavior and in the regulation of the HPA-axis. In the present experiments we have re-examined the effects of amphetamine on CART expression in the forebrain and in the hypothalamus by the use of in situ hybridization. Although we used the same amphetamine treatment paradigm as that originally reported we failed to demonstrate any regulation of CART mRNA levels by amphetamine in either the forebrain or in the hypothalamus. The present results question the acronym chosen for this predominantly hyopthalamic neuropeptide.

Amphetamine↗

PubMed2002

Ups and downs for neuropeptides in body weight homeostasis: pharmacological potential of cocaine amphetamine regulated transcript and pre-proglucagon-derived peptides.

Although most humans experience an underlying upwards drift of the body-weight set-point, body weight appears tightly regulated throughout life. The present review describes the structural basis of the adipostat and hypothesise, which components may constitute available targets for pharmacotherapy of excess body weight. Hypothalamic neurones constitute the major components of the body weight homeostasis maintaining device. Together with neurones of the nucleus of the solitary tract, neurones of the hypothalamic arcuate nucleus constitute the sensory components of the adipostat. The arcuate nucleus neurones respond to circulating levels of leptin and insulin, both of which reflect the levels of energy stored as triacylglycerol in adipocytes. The arcuate nucleus projects heavily to the hypothalamic paraventricular nucleus. Neurones of the hypothalamic paraventricular nucleus are hypothesised to constitute, at least partly, the adipostat motor pattern generator, which upon stimulation activates either net anabolic or catabolic physiological responses. The overall sensitivity of the adipostat is influenced by gain setting neurones hypothesised to be located in the dorsomedial hypothalamic nucleus and lateral hypothalamic area. Cocaine amphetamine regulated transcript (CART) peptides and pre-proglucagon derived peptides, glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) are catabolic neurotransmitters synthesised in neurones of the arcuate nucleus and the nucleus of the solitary tract, respectively. The present review summarises the available evidence that both families of peptides constitute endogenous transmitters mediating satiety and touch upon potential pharmacological exploitation of this knowledge.

Adipose Tissue↗

PubMed2002

Selection of large diversities of antiidiotypic antibody fragments by phage display.

Antiidiotypic antibodies (Ab2) are needed as tools for a better understanding of molecular mimicry and the immunological network, and for many potential applications in the biomedical and pharmaceutical field. Antiidiotypic antibodies mimicking carbohydrate or conformational epitopes (Ab2beta) are of considerable interest as surrogate immunogens for cancer vaccination. However, it has so far been difficult and tedious to produce Ab2s to a given antigen. Here we describe a fast and reliable technique for generating large diversities of antiidiotypic single chain antibody fragments from non-immunized phagemid libraries using phage display. Key elements are a specific elution with the original antigen followed by trypsin treatment of the eluted phages in combination with the protease sensitive helperphage KM13. This novel method was compared with various conventional selection and elution methods, including, specific elution with or without trypsin treatment, elution with glycine at pH 2.2 with or without trypsin treatment, and elution by trypsin treatment only. The results clearly show that specific elution in combination with trypsin treatment of the eluted phages is by far superior to the other conventional methods, enabling for the first time the generation of a large variety of Ab2s after only two to three rounds of selection, thereby maintaining maximum diversity. We obtained 28 to 88 antiidiotypes out of 96 tested clones after two to three rounds of selection with a diversity of 55-90 %. This was achieved for two carbohydrate (di-, and tetrasaccharides) and one conformational protein epitope using two large naïve libraries and their corresponding monoclonal Ab1. The antiidiotypic nature of the selected scFv-phages was verified by ELISA and immunocytochemistry inhibition experiments.

Amino Acid Sequence↗