Ultrastructure of islet ghrelin cells in the human fetus

Ghrelin is a peptide hormone predominantly produced in the stomach. Ghrelin expression has also been reported in other tissues including the pancreas. We have reported that ghrelin cells constitute a novel endocrine cell type in the human and the developing rat islets. The cells are most numerous pre- and neonatally and, in humans, constitute 10% of all islet cells from mid-gestation to birth. Since gastric ghrelin expression is low before birth, the islets may be the main source of circulating ghrelin during this time. In the present investigation, we have performed an ultrastructural analysis of pancreatic ghrelin cells in human fetuses by using transmission electron microscopy and immunogold labelling. In addition, morphometrical analysis of secretory granules size was performed. Our data provide evidence for the unique ultrastructural features of ghrelin cells versus other islet cells. Notably, the secretory granules of ghrelin cells were of small size with a mean dense-core diameter of 110 nm. We conclude that ghrelin cells constitute a novel islet cell type, distinct from the previously hormonally characterised islet cell types.This work was supported by grants from the Swedish Medical Research Council (Project No. 4499), the Royal Physiographic Society and the Novo Nordic, Påhlsson and Gyllenstiernska Krapperup Foundations     

First synthesis of a pentadeuterated 3'-hydroxystanozolol--an internal standard in doping analysis

The first synthesis of 16,16,20,20,20-pentadeuterio-3'-hydroxystanozolol (8) in 26% yield over nine steps is described using moderately priced starting materials and economic amounts of reagents. Compound 8 can be used as an internal standard in screening procedures for anabolic steroids as well as for the quantification of stanozolol metabolites via mass spectrometric techniques, such as LC-MS or gas chromatography-mass spectrometry (GC-MS).     

Acclimation to ozone affects host/pathogen interaction and competitiveness for nitrogen in juvenile Fagus sylvatica and Picea abies trees infected with Phytophthora citricola

In a two-year phytotron study, juvenile trees of European beech (Fagus sylvatica) and Norway spruce (Picea abies) were grown in mixture under ambient and twice ambient ozone (O3) and infected with the root pathogen Phytophthora citricola. We investigated the influence of O3 on the trees' susceptibility to the root pathogen and assessed, through a 15N-labelling experiment, the impact of both treatments (O3 exposure and infection) on belowground competitiveness. The hypotheses tested were that: (1) both P. citricola and O3 reduce the belowground competitiveness (in view of N acquisition), and (2) that susceptibility to P. citricola infection is reduced through acclimation to enhanced O3 exposure. Belowground competitiveness was quantified via cost/benefit relationships, i.e., the ratio of structural investment in roots relative to their uptake of 15N. Beech had a lower biomass acquisition and captured less 15N under enhanced O3 and P. citricola infection alone than spruce, whereas the latter species appeared to profit from the lower resource acquisition of beech in these treatments. Nevertheless, in the combined treatment, susceptibility to P. citricola of spruce was increased, while beech growth and 15N uptake were not further reduced below the levels found under the single treatments. Potential trade-offs between stress defence, growth performance, and associated nitrogen status are discussed for trees affected through O3 and/or pathogen infection. With respect to growth performance, it is concluded that O3 enhances susceptibility to the pathogen in spruce, but apparently raises the defence capacity in beech..     

Stereoselective synthesis of a ketohexofuranose from an aldohexopyranose by a [6+1-1] strategy

Ozonolysis of 2-acetoxymethyl-1,5-anhydro-3,4,6-tri-O-benzyl-2-deoxy-D-arabino-hex-1-enitol gave 1-O-acetyl-3,4,6-tri-O-benzyl-4-O-formyl-D-arabino-hex-2-ulose (5). Subsequent hydrolysis and acetylation of 5 provided 1,2-di-O-acetyl-3,4,6-tri-O-benzyl-D-fructofuranose 6 in excellent yield. This methodology allows specific deuteration at C-1 of a protected D-fructofuranose derivative. This approach therefore could serve as [6+1-1] formulation for hexose series inter-conversion, that is, aldohexopyranose to ketohexofuranose.     

Imaging of primary human hepatocytes performed with micron-sized iron oxide particles and clinical magnetic resonance tomography

Transplantation of primary human hepatocytes is a promising approach in certain liver diseases. For the visualization of the hepa-tocytes during and following cell application and the ability of a timely response to potential complications, a non-invasive modality for imaging the transplanted cells has to be established. The aim of this study was to label primary human hepatocytes with micron-sized iron oxide particles (MPIOs), enabling the detection of cells by clinical magnetic resonance imaging (MRI). Primary human hepatocytes isolated from 13 different donors were used for the labelling experiments. Following the dose-finding studies, hepatocytes were incubated with 30 particles/cell for 4 hrs in an adhesion culture. Particle incorporation was investigated via light, fluorescence and electron microscopy, and labelled cells were fixed and analysed in an agarose suspension by a 3.0 Tesla MR scanner. The hepatocytes were enzymatically resuspended and analysed during a 5-day reculture period for viability, total protein, enzyme leakage (aspartate aminotransferase [AST], lactate dehydrogenase [LDH]) and metabolic activity (urea, albumin). A mean uptake of 18 particles/cell could be observed, and the primary human hepatocytes were clearly detectable by MR instrumentation. The particle load was not affected by resuspension and showed no alternations during the culture period. Compared to control groups, labelling and resuspension had no adverse effects on the viability, enzyme leakage and metabolic activity of the human hepatocytes. The feasibility of preparing MPIO-labelled primary human hepatocytes detectable by clinical MR equipment was shown in vitro. MPIO-labelled cells could serve for basic research and quality control in the clinical setting of human hepatocyte transplantation.     

A cross-species quantitative proteomic study of salt adaptation in a halotolerant environmental isolate using 15N metabolic labelling

We examine differential protein expression in Euhalothece sp. BAA001, an extremely halotolerant and unsequenced cyanobacterium, under adaptation to low (0% w/v), medium (3% w/v), high (6% w/v) and very high (9% w/v) salt concentrations using cross-species protein identification tools. We combine stable isotope labelling with 1-D SDS-PAGE, and MASCOT protein identification software with MS-driven BLAST searches, to produce an accurate method for protein identification and quantitation. The use of metabolic labelling to improve the confidence in identification of proteins in cross-species proteomics is demonstrated. Three hundred and eighty-three unique proteins were identified, and 72 were deemed to be differentially expressed (average CV for quantitations was 0.10 +/- 0.08), belonging to 24 functional groups. Responses to low salt as well as high salt are discussed in terms of adaptation and evidence shows that Euhalothece cells display 'stress' responses in nonsaline conditions as well as higher salt environments.     

Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration

BACKGROUND INFORMATION: Electron microscopic immunolabelling of ultrathin thawed cryo-sections, according to the method of Tokuyasu, is widely used as a very sensitive high-resolution localization technique. Its main advantages are that antigens remain in a hydrated environment prior to immunolabelling, and that antigen accessibility is improved compared with resin section labelling. However, the quality of structural appearance and antigenicity depends highly on the limitations of the initial conventional chemical fixation step, such as slow diffusion and selective reaction/cross-linking of fixative molecules. RESULTS AND CONCLUSIONS: Cryofixation, instead of conventional chemical fixation, followed by freeze-substitution/chemical fixation, rehydration and further processing for Tokuyasu cryo-sectioning leads to an improved preservation of both ultrastructure and antigenicity. This is especially true for tissues which are difficult to preserve by conventional chemical fixation at ambient temperatures, such as plant material, Drosophila embryos or nematode tissue. In particular labile and highly dynamic structures (for example, microtubules and Golgi apparatus) are remarkably better preserved. These improvements are also valid for light microscopic applications.     

Light enhanced amino acid uptake by dominant bacterioplankton groups in surface waters of the Atlantic Ocean

(35)S-Methionine and (3)H-leucine bioassay tracer experiments were conducted on two meridional transatlantic cruises to assess whether dominant planktonic microorganisms use visible sunlight to enhance uptake of these organic molecules at ambient concentrations. The two numerically dominant groups of oceanic bacterioplankton were Prochlorococcus cyanobacteria and bacteria with low nucleic acid (LNA) content, comprising 60% SAR11-related cells. The results of flow cytometric sorting of labelled bacterioplankton cells showed that when incubated in the light, Prochlorococcus and LNA bacteria increased their uptake of amino acids on average by 50% and 23%, respectively, compared with those incubated in the dark. Amino acid uptake of Synechococcus cyanobacteria was also enhanced by visible light, but bacteria with high nucleic acid content showed no light stimulation. Additionally, differential uptake of the two amino acids by the Prochlorococcus and LNA cells was observed. The populations of these two types of cells on average completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community, suggesting a plausible way of harnessing light energy for selectively transporting scarce nutrients that could explain the numerical dominance of these groups in situ.     

Developments in cell biology for quantitative immunoelectron microscopy based on thin sections: a review

Quantitative immunoelectron microscopy uses ultrathin sections and gold particle labelling to determine distributions of molecules across cell compartments. Here, we review a portfolio of new methods for comparing labelling distributions between different compartments in one study group (method 1) and between the same compartments in two or more groups (method 2). Specimen samples are selected unbiasedly and then observed and expected distributions of gold particles are estimated and compared by appropriate statistical procedures. The methods can be used to analyse gold label distributed between volume-occupying (organelle) and surface-occupying (membrane) compartments, but in method 1, membranes must be treated as organelles. With method 1, gold counts are combined with stereological estimators of compartment size to determine labelling density (LD). For volume-occupiers, LD can be expressed simply as golds per test point and, for surface-occupiers, as golds per test line intersection. Expected distributions are generated by randomly assigning gold particles to compartments and expressing observed/expected counts as a relative labelling index (RLI). Preferentially-labelled compartments are identified from their RLI values and by Chi-squared analysis of observed and expected distributions. For method 2, the raw gold particle counts distributed between compartments are simply compared across groups by contingency table and Chi-squared analysis. This identifies the main compartments responsible for the differences between group distributions. Finally, we discuss labelling efficiency (the number of gold particles per target molecule) and describe how it can be estimated for volume- or surface-occupiers by combining stereological data with biochemical determinations.