In vivo and in vitro development of mouse pancreatic β-cells in organotypic slices

Taking tissue slices of the embryonic and newborn pancreas is a novel approach for the study of the perinatal development of this gland. The aim of this study was to describe the morphology and physiology of in vivo and in vitro developing -cells. In addition, we wanted to lay a foundation for the functional analysis of other pancreatic cells, either alone or as part of an integrative pancreatic physiology approach. We used cytochemistry and light microscopy to detect specific markers and the whole-cell patch-clamp to assess the function of single -cells. The insulin signal in the embryonic -cells was condensed to a subcellular compartment and redistributed throughout the cytosol during the first 2 days after birth. The hormone distribution correlated well with the development of membrane excitability and hormone release competence in -cells. Endocrine cells survived in the organotypic tissue culture and maintained their physiological properties for weeks. We conclude that our preparation fulfills the criteria for a method of choice to characterize the function of developing pancreas in wild-type and genetically modified mice that die at birth. We suggest organotypic culture for in vitro studies of the development and regeneration of -cells.This work was supported by the European Commission (grant QLG1-CT-2001-02233 to TMR, AR and MR), the DFG Research Center for Molecular Physiology of the Brain (CMPB) and the Max-Planck Society (MR)     

Stereospecific activity of two glutamate analogs

Two glutamic acid analogs, (+)-(S)- and (-)-(R)-4-(2,2-diphenyl-1,3,2-oxazaborolidin-5-oxo)propionic acid ((+)-(S)- and (-)-(R)-Trujillon, respectively), were prepared. The stereospecific activity of their pharmacological properties was studied. The median convulsant dose (CD(50)) and median lethal dose (LD(50)) were analyzed in female Swiss Webster mice and their effects in vivo on unitary electrical activity in globus pallidus neurons were elucidated in male Wistar rats. Compounds were characterized by (1)H, (13)C, and (11)B nuclear magnetic resonance. The LD(50) of (+)-(S)-Trujillon was 449.08 mg/kg and it increased spontaneous motor activity, while with (-)-(R)-Trujillon there was no mortality up to 1,000 mg/kg and it decreased spontaneous motor activity. The CD(50) in experiments with (+)-(S)-Trujillon was 199.34 mg/kg. Unitary recording in globus pallidus neurons showed i.v. administration (+)-(S)-Trujillon (50 mg/kg) increased frequency 79.0 +/- 23.0% in relation to basal response. (-)-(R)-Trujillon and (+)-(S)-glutamate (50 mg/kg each) did not provoke changes in spontaneous basal firing. Local infusion of (+)-(S)-Trujillon (1 nMol) increased spontaneous firing in most neurons tested by 269.0 +/- 83.0% in relation to basal values. Intrapallidal infusion of (-)-(R)-Trujillon (1 nMol) and saline solution did not cause statistically significant changes in globus pallidus spiking. Results showed that (+)-(S)-Trujillon crosses the blood-brain barrier and has stereospecific activity.     

A survey of the year 2003 literature on applications of isothermal titration calorimetry

Over the last decade isothermal titration calorimetry (ITC) has developed from a specialist method which was largely restricted in its use to dedicated experts, to a major, commercially available tool in the arsenal directed at understanding molecular interactions. The number of those proficient in this field has multiplied dramatically, as has the range of experiments to which this method has been applied. This has led to an overwhelming amount of new data and novel applications to be assessed. With the increasing number of publications in this field comes a need to highlight works of interest and impact. In this overview of the literature we have attempted to draw attention to papers and issues for which both the experienced calorimetrist and the interested dilettante hopefully will share our enthusiasm.     

Biotechnological applications of bioluminescence and chemiluminescence

Recent progress in molecular biology has made available several biotechnological tools that take advantage of the high detectability and rapidity of bioluminescence and chemiluminescence spectroscopy. These developments provide inroads to in vitro and in vivo continuous monitoring of biological processes (e.g. gene expression, protein-protein interaction and disease progression), with clinical, diagnostic and drug discovery applications. Furthermore, combining luminescent enzymes or photoproteins with biospecific recognition elements at the genetic level has led to the development of ultrasensitive and selective bioanalytical tools, such as recombinant whole-cell biosensors, immunoassays and nucleic acid hybridization assays. The high detectability of the luminescence analytical signal makes it appropriate for miniaturized bioanalytical devices (e.g. microarrays, microfluidic devices and high-density-well microtiter plates) for the high-throughput screening of genes and proteins in small sample volumes.     

Average protein density is a molecular-weight-dependent function

The mass density of proteins is a relevant basic biophysical quantity. It is also a useful input parameter, for example, for three-dimensional structure determination by protein crystallography and studies of protein oligomers in solution by analytic ultracentrifugation. We have performed a critical analysis of published, theoretical, and experimental investigations about this issue and concluded that the average density of proteins is not a constant as often assumed. For proteins with a molecular weight below 20 kDa, the average density exhibits a positive deviation that increases for decreasing molecular weight. A simple molecular-weight-depending function is proposed that provides a more accurate estimate of the average protein density.     

Expression of the Stilbene Synthase (StSy) Gene from Grapevine in Transgenic White Poplar Results in High Accumulation of the Antioxidant Resveratrol Glucosides

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.     

New N-arylsulfonyl-N-alkoxyaminoacetohydroxamic acids as selective inhibitors of gelatinase A (MMP-2)

New N-arylsulfonyl-substituted alkoxyaminoaceto hydroxamic acid derivatives of types 8 and 10 designed as oxa-analogues of known sulfonamide-based MMPi of types 2 and 7 were synthesized and tested for their inhibitory activities on some matrix metalloproteinases. The combination of a biphenylsulfonamide group with oxyamino oxygen in the pharmacophoric central skeleton of sulfonamide-based MMPi obtained in the new sulfonamides 10 seems to be able to give selectivity for MMP-2 over MMP-1. The most potent derivative of this type, 10a, shows similar anti-invasive properties to the analogue reference drug CGS27023A, 2, in an in vitro model of invasion on matrigel, carried out on cellular lines of fibrosarcoma HT1080 (tumoural cells over-expressing MMP-2 and MMP-9).     

Structural study of four-stranded quadruplex structures containing 2'-deoxy-8-(propyn-1-yl)adenosine

In this paper, we report the NMR structural study of two quadruplex structures formed by truncations of the human telomeric sequence and containing a modified base, namely d(AprGGGT) and d(TAprGGGT), where Apr indicates 2'-deoxy-8-(propyn-1-yl)adenosines. Both oligonucleotides have been found to form 4-fold symmetric G-quadruplex structures with all strands parallel and equivalent to each other and characterized by higher thermal stabilities than the natural counterparts. The presence of the propynyl groups affects the conformations of the 5' edge of both quadruplexes in such a way to prevent the formation of one of the two possible H-bond patterns observed for a canonical A-tetrad. The increased thermal stabilities of the modified quadruplexes seem to be mostly due to a prevalent syn glycosidic conformation assumed by the Apr residues.     

Interference of plasmatic reduced glutathione and hemolysis on glutathione disulfide levels in human blood

The blood reduced glutathione (GSH)/GSH disulfide (GSSG) ratio is an index of the oxidant/antioxidant balance of the whole body. Nevertheless, data indicating GSH and GSSG physiological levels are still widely divergent, especially those on GSSG, probably due to its low concentration. Standardization in methodological protocols and sample manipulation could help to minimize these discrepancies. Therefore, we have investigated how plasma reduced GSH, which is rapidly oxidized after blood withdrawal, could alter the blood GSSG measurement if the sample is not suitably processed. We have observed that an increase in plasma GSH concentration, due to red blood cell hemolysis, is responsible for a significant overestimation of blood GSSG level. Our results show that, before performing blood GSSG determination, thiols have to be rapidly blocked, to avoid possible pitfalls in GSSG measurement, in particular when hemolysis is present.