Daniel R. Matthews

THE RELATIONSHIP BETWEEN HEIGHT VELOCITY AND GROWTH HORMONE SECRETION IN SHORT PREPUBERTAL CHILDREN

We have performed 24 h growth hormone (GH) profiles in 50 short prepubertal children aged between 5.2 and 12‐9 years, growing with height velocity standard deviation scores (SDS) between 0.4 and ‐3.9. There was an asymptotic relationship between height velocity and spontaneous G H secretion described by the equation: height velocity SDS = A – B(e cx), where A B and C are constants and x is a measure of spontaneous G H secretion. We considered G H pulse amplitude to be the better description of spontaneous G H secretion as duration of the G H pulse (the time component of area under the curve) contributed little to the relationship between height velocity and area under the pulse. The distribution of G H secretion was continuous and there was no dividing point between G H insufficiency and sufficiency. Similar overlap was observed when the results of G H responses to insulin induced hypoglycaemia were considered; 14% of slowly growing children (height velocity S D S < –0‐8), had a response >15mU/l. Likewise serum I G F‐I concentrations could not clearly separate slowly growing children from normal individuals. We conclude that height velocity, which ultimately determines height achieved, is controlled predominately by G H pulse amplitude. The findings suggest that short normal children growing along or parallel to the third height centile could be made to grow faster by the administration of exogenous G H.

Fluorescence lifetime and polarization-resolved imaging in cell biology

Fluorescence lifetime imaging (FLIM) and fluorescence polarization imaging are complementary techniques that can be used to extract information about macromolecules from biological samples. Owing to the sensitivity of fluorescence to the physicochemical environment, and nanometer-scale interactions via Förster resonance energy transfer (FRET), FLIM has been implemented in many laboratories for numerous applications in the life sciences and beyond. This review seeks to provide a brief overview of some of the recent advances in the techniques and more pertinently their applications in cell and tissue imaging. The particular merits of polarization-resolved fluorescence measurements are highlighted, including the unique ability to elucidate the occurrence of homo-FRET.

Essential Role of hIST1 in Cytokinesis

The last steps of multivesicular body (MVB) formation, human immunodeficiency virus (HIV)-1 budding and cytokinesis require a functional endosomal sorting complex required for transport (ESCRT) machinery to facilitate topologically equivalent membrane fission events. Increased sodium tolerance (IST) 1, a new positive modulator of the ESCRT pathway, has been described recently, but an essential function of this highly conserved protein has not been identified. Here, we describe the previously uncharacterized KIAA0174 as the human homologue of IST1 (hIST1), and we report its conserved interaction with VPS4, CHMP1A/B, and LIP5. We also identify a microtubule interacting and transport (MIT) domain interacting motif (MIM) in hIST1 that is necessary for its interaction with VPS4, LIP5 and other MIT domain-containing proteins, namely, MITD1, AMSH, UBPY, and Spastin. Importantly, hIST1 is essential for cytokinesis in mammalian cells but not for HIV-1 budding, thus providing a novel mechanism of functional diversification of the ESCRT machinery. Last, we show that the hIST1 MIM activity is essential for cytokinesis, suggesting possible mechanisms to explain the role of hIST1 in the last step of mammalian cell division.

The interaction of growth hormone releasing hormone and somatostatin in the generation of a GH pulse in man

Summary. objective To study the regulation of the growth hormone (GH) response to growth hormone releasing hormone (GHRH) in the presence or absence of somatostatin pretreatment.

Biomedical Imaging: From Nano to Macro

Studying cellular protein-protein interactions in situ requires a technique such as fluorescence resonance energy transfer (FRET) which is sensitive on the nanometer scale. Observing FRET is significantly simplified if the fluorescence lifetime of the donor can be monitored. Results from live cells and tissue micro arrays are presented from an automated microscope incorporating time-domain TCSPC fluorescence lifetime imaging (FLIM). Novel hardware and software with a modular approach and scripting abilities allow us to work towards speed-optimized acquisition and ease of use to bring FLIM into the high-throughput regime.

Growth hormone secretion in children determined by time series analysis.

Mid childhood growth has been studied in 26 short children (18M;8F) aged between 5·2 and 11·9 years growing with height velocity standard deviation score (SDS) between 0 and — 0·8 and 24 short children (17M;7F) growing with height velocity SDS >0·8. Twenty‐four hour GH profiles were analysed by an iterative method of pulse detection and subjected to time series analysis to determine dominant periodicity within the data arrays. Children aged >7 years displayed no dominant periodicity but after this age a periodicity of 200 min emerged. In the whole sample, differences between growth rate could be explained entirely by pulse amplitude. Nevertheless, the normal decline in height velocity over this age period occurred despite a significant shift in periodicity and an increase in GH pulse amplitude. This modulation of childhood growth by GH pulse amplitude perisisted into puberty and the pubertal growth spurt of 14 tall girls was shown to be amplitude modulated with the periodicity unchanged. We conclude that mid childhood and pubertal growth is GH pulse amplitude modulated with a periodicity of approximately 200 min.

THE HALF-LIFE OF EXOGENOUS GROWTH HORMONE AFTER SUPPRESSION OF ENDOGENOUS GROWTH HORMONE SECRETION WITH SOMATOSTATIN

We have estimated the half‐life of serum growth hormone (GH) in six subjects on 14 occasions following an intravenous bolus injection of either 50 or 500 mU of biosynthetic human growth hormone (B‐hGH) while endogenous GH secretion was suppressed by a continuous infusion of somatostatin. The disappearance curve of serum GH was mono‐exponential and the mean half‐life was 8.9 min (SD 1.5). This is less than previously reported and has important implications for the performance of GH profiles, which should be performed with 10–15 min sampling intervals, and the calculation of pituitary GH secretion rates.

Fluorescence lifetime spectroscopy and imaging of nano-engineered glucose sensor microcapsules based on glucose/galactose-binding protein

We aimed to develop microsensors for eventual glucose monitoring in diabetes, based on fluorescence lifetime changes in glucose/galactose-binding protein (GBP) labelled with the environmentally sensitive fluorophore dye, badan. A mutant of GBP was labelled with badan near the binding site, the protein adsorbed to microparticles of CaCO(3) as templates and encapsulated in alternating nano-layers of poly-L-lysine and heparin. We used fluorescence lifetime imaging (FLIM) with two-photon excitation and time-correlated single-photon counting to visualize the lifetime changes in the capsules. Addition of glucose increased the mean lifetime of GBP-badan by a maximum of approximately 2 ns. Analysis of fluorescence decay curves was consistent with two GBP states, a short-lifetime component (approximately 0.8 ns), likely representing the open form of the protein with no bound glucose, and a long-lifetime component (approximately 3.1 ns) representing the closed form with bound glucose and where the lobes of GBP have closed round the dye creating a more hydrophobic environment. FLIM demonstrated that increasing glucose increased the fractional proportion of the long-lifetime component. We conclude that fluorescence lifetime-based glucose sensing using GBP encapsulated with nano-engineered layer-by-layer films is a glucose monitoring technology suitable for development in diabetes management.

Reproducibility of 24-hour serum growth hormone profiles in man

objective To study the reproducibility of 24‐h serum growth hormone (GH) concentration profiles in adults. DESIGN 24‐h serum GH concentrations were constructed by drawing blood samples at 20‐min Intervals. Four study occasions over a period of 1 year were chosen to assess the reproducibility

How Forster Resonance Energy Transfer Imaging Improves the Understanding of Protein Interaction Networks in Cancer Biology

Herein we discuss how FRET imaging can contribute at various stages to delineate the function of the proteome. Therefore, we briefly describe FRET imaging techniques, the selection of suitable FRET pairs and potential caveats. Furthermore, we discuss state-of-the-art FRET-based screening approaches (underpinned by protein interaction network analysis using computational biology) and preclinical intravital FRET-imaging techniques that can be used for functional validation of candidate hits (nodes and edges) from the network screen, as well as measurement of the efficacy of perturbing these nodes/edges by short hairpin RNA (shRNA) and/or small molecule-based approaches.