Born again bone: tissue engineering for bone repair.

Destruction of bone tissue due to disease and inefficient bone healing after traumatic injury may be addressed by tissue engineering techniques. Growth factor, cytokine protein, and gene therapies will be developed, which, in conjunction with suitable carriers, will regenerate missing bone or help in cases of defective healing.

High-throughput liquid chromatography/mass spectrometry method for the determination of the chromatographic hydrophobicity index.

A fast gradient reversed-phase liquid chromatography (LC) method, using an acetonitrile gradient was developed to determine the chromatographic hydrophobicity index (CHI), as reported by Valco et al. (Anal. Chem.

The transcription factor Egr-1: a potential drug in wound healing and tissue repair.

In the United States, between 40 and 90 million hospital days are lost per year as a result of trauma and surgical procedures which result in the loss of functional tissue. This is estimated to cost the economy and healthcare providers in excess of US$ 500 billion, a figure that is increasing because of extending population lifespan. Tissue engineering and gene therapies are radical new treatments that are aimed at tissue regeneration ranging from dermal, osteal and occular repair to the replacement of failing tissue with entire biosynthetic organs.

Rapid method for the estimation of octanol/water partition coefficient (log P(oct)) from gradient RP-HPLC retention and a hydrogen bond acidity term (zetaalpha(2)(H)).

We propose a rapid method for the measurement of octanol/water partition coefficients (log P(oct)) via fast gradient reversed phase retention and the calculation of the hydrogen bond acidity of the compounds. The cycle time of the generic gradient HPLC method is 5 minutes. The general solvation equation obtained for the log Poct values and the fast gradient Chromatographic Hydrophobicity Indices with acetonitrile (CHI(ACN)) and methanol

Pharmacological characterisation of the human small conductance calcium-activated potassium channel hSK3 reveals sensitivity to tricyclic antidepressants and antipsychotic phenothiazines.

A stable CHO-K1 cell line was developed which expresses the human small conductance calcium-activated potassium channel hSK3. Immunofluorescence microscopy using an anti-SK3 antibody and radioligand binding using [(125)I]-apamin demonstrated the presence of hSK3 channel in the recombinant cell line. This cell line was utilised in a fluorescence assay using the membrane potential-sensitive dye DiBAC(4)(3) to functionally analyse and pharmacologically characterise this potassium channel.

Genomic organization of the human metabotropic glutamate receptor subtype 3.

In this study, the genomic organization of the human metabotropic glutamate receptor subtype 3 (mGluR3) gene has been determined. We have identified two transcription initiation sites and the polyadenylation signal by using 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE, respectively. The exon/intron organization of the human mGluR3 gene revealed the presence of 6 exons separated by 5 introns.

The discovery of a potent, intracellular, orally bioavailable, long duration inhibitor of human neutrophil elastase--GW311616A a development candidate.

The discovery of a potent intracellular inhibitor of human neutrophil elastase which is orally active and has a long duration of action is described. The pharmacodynamic and pharmacokinetic properties of a trans-lactam development candidate, GW311616A, are described.

Homing markers for atherosclerosis: applications for drug delivery, gene delivery and vascular imaging.

Endothelial dysfunction plays a major role in the pathogenesis of atherosclerosis. Pro-inflammatory cytokines such as interleukin-1 beta and tumour necrosis factor alpha activate endothelial cells changing their resting phenotype to become pro-adhesive, pro-thrombotic and pro-atherogenic. Phage display in vivo biopanning has been used to identify peptide sequences that home to diseased regions of the vessel wall in low density lipoprotein receptor (LDLr) knockout mice.

Distribution of the messenger RNA for the small conductance calcium-activated potassium channel SK3 in the adult rat brain and correlation with immunoreactivity.

Small conductance calcium-activated potassium channels are voltage independent potassium channels which modulate the firing patterns of neurons by activating the slow component of the afterhyperpolarization. The genes encoding a family of small conductance calcium-activated potassium channels have been cloned and up to now three known members have been described and named small conductance calcium-activated potassium channel type 1, small conductance calcium-activated potassium channel type 2 and small conductance calcium-activated potassium channel type 3; the distribution of their messenger RNA in the rat CNS has already been performed but only in a limited detail. The present study represents the first detailed analysis of small conductance calcium-activated potassium channel type 3 mRNA distribution in the adult rat brain and resulted in a strong to moderate expression of signal in medial habenular nucleus, substantia nigra compact part, suprachiasmatic nucleus, ventral tegmental area, lateral septum, dorsal raphe and locus coeruleus.

Studies on the effects of adenosine A3 receptor stimulation on human eosinophils isolated from non-asthmatic or asthmatic donors.

Objective: This study was designed to demonstrate the presence of adenosine A3 receptors on human peripheral blood eosinophils, and to investigate the effect of A3 receptor stimulation on eosinophil function.

Material: Eosinophils from either non-asthmatic or asthmatic donors.

Methods: Eosinophils were isolated from peripheral venous blood by discontinuous gradient centrifugation and negative immunoselection.

Resolution of positional isomers by capillary electrochromatography.

Electroseparations have been very successful in increasing efficiencies and reducing analysis times. The analytical technique originally applied to open tube capillaries (capillary electrophoresis) has been used as a basis to develop renewed interest in electrochromatography. This paper describes the use of capillary electrochromatography to separate two positional isomers and describes a comparison between gas chromatography (GC), capillary electrochromatography (CEC) and nano-HPLC.

Quantitative expression analysis of the small conductance calcium-activated potassium channels, SK1, SK2 and SK3, in human brain.

Small conductance calcium-activated potassium (SK) channels are important in controlling neuronal excitability and three SK channels have been identified to date. In the present study, we report the first quantitative analysis of SK1, SK2 and SK3 expression in human brain using TaqMan RT-PCR on a range of human brain and peripheral tissue samples. SK1 expression is restricted to the brain whereas SK2 and SK3 are more widely expressed.

Pyridoxal phosphate de-activation by pyrroline-5-carboxylic acid. Increased risk of vitamin B6 deficiency and seizures in hyperprolinemia type II.

We previously identified vitamin B6 deficiency in a child presenting with seizures whose primary diagnosis was the inherited disorder hyperprolinemia type II. This is an unrecognized association, which was not explained by diet or medication. We hypothesized that pyridoxal phosphate (vitamin B6 coenzyme) was de-activated by L-Delta(1)-pyrroline-5-carboxylic acid, the major intermediate that accumulates endogenously in hyperprolinemia type II.

Cloning and characterization of the human phosphoinositide-specific phospholipase C-beta 1 (PLC beta 1).

Phospholipase C-beta (PLC beta) catalyses the generation of inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (IP(2)), a key step in the intracellular transduction of a large number of extracellular signals, including neurotransmitters and hormones modulating diverse developmental and functional aspects of the mammalian central nervous system. Four mammalian isozymes are known (PLC beta 1-4), which differ in their function and expression patterns in vivo. We have characterized the human PLC beta 1 genomic locus (PLC beta 1), cloned two distinct PLC beta 1 cDNAs (PLC beta 1a and b) and analysed their respective expression patterns in a comprehensive panel of human tissues using quantitative TaqMan technology.

Studies on the interaction between TWEAK and the death receptor WSL-1/TRAMP (DR3).

WSL-1/TRAMP (DR3) is a member of the tumour necrosis factor (TNF) receptor superfamily which exhibits effects on NF-kappaB activation and apoptosis. TWEAK, a novel TNF-related molecule, has been proposed as the ligand for this receptor. Utilising both human and murine TWEAK ligand, it is shown that TWEAK and WSL-1/TRAMP do not interact in an in vitro binding assay and that TWEAK binds strongly to cells that do not express WSL-1/TRAMP on the cell surface.

Discovery of novel ansamycins possessing potent inhibitory activity in a cell-based oncostatin M signalling assay.

We describe the isolation and characterisation of novel non-benzoquinone ansamycin metabolites related to geldanamycin from a culture of Streptomyces sp. S6699. The compounds possess potent inhibitory activity in a cell-based assay measuring inhibition of oncostatin M signalling in a reporter cell line utilising a secreted placental alkaline phosphatase (sPAP) readout.

GABA(B) receptors function as heterodimers.

Our current understanding is that functional GABA(B) receptors exist as heterodimers of two related seven-transmembrane proteins, GABA(B)-R1 and GABA(B)-R2. GABA(B)-R1 requires GABA(B)-R2 to be expressed at the cell surface as a mature glycoprotein. Cloning of the GABA(B) receptor has failed to provide molecular evidence to support the existence of true receptor subtypes.

Rapid-gradient HPLC method for measuring drug interactions with immobilized artificial membrane: comparison with other lipophilicity measures.

A fast-gradient high-performance liquid chromatographic (HPLC) method has been suggested to characterize the interactions of drugs with an immobilized artificial membrane (IAM). With a set of standards, the gradient retention times can be converted to Chromatographic Hydrophobicity Index values referring to IAM chromatography (CHI(IAM)) that approximates an acetonitrile concentration with which the equal distribution of compound can be achieved between the mobile phase and IAM. The CHI(IAM) values are more suitable for interlaboratory comparison and for high throughput screening of new molecular entities than the log k(IAM) values (isocratic retention factor on IAM).

Analysis of the alpha4beta1 integrin-osteopontin interaction.

The integrin alpha4beta1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule.