A Bcl-2 transgene expressed in hepatocytes does not protect mice from fulminant liver destruction induced by Fas ligand

We compared the biological mechanism of cell death during hepatotoxicity induced by ligation of the Fas receptor in wild-type and liver-specific bcl-2 transgenic mice. Transgenic overexpression of Bcl-2 in mouse hepatocytes can prevent lethal hepatitis induced by agonistic anti-Fas antibodies. In contrast, Fas ligand (FasL)-induced death cannot be overcome in bcl-2 transgenic mice, indicating that anti-Fas antibodies do not reliably mimic the more physiological ligand. Different apoptotic parameters, viz. caspase activation, cytochrome c release and nuclear DNA degradation were analysed. No differences, however, could be observed between wild-type and bcl-2 transgenic mice after injection with a lethal dose of soluble FasL, indicating that apoptosis by FasL-dependent ligation is not modulated by Bcl-2 in vivo. These results demonstrate that the stimulus determines the outcome between type I mitochondria-independent apoptosis, in the case of FasL, or type II mitochondria-dependent and Bcl-2-inhibitable apoptosis, in the case of anti-Fas antibodies.     

Lack of effects of glycineB receptor ligands on the psychostimulant-induced discriminative stimuli in rats

To examine the role of glycineB receptors in the stimulus effects induced by psychostimulants, separate groups of rats were trained to discriminate amphetamine (AMPH; 1 mg/kg) from saline (SAL), or cocaine (COC; 10 mg/kg) from SAL, using a two-lever operant procedure. Substitution studies showed that neither 1-aminocyclopropanecarboxylic acid (ACPC; 200 mg/kg) nor 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-(H)quinolone (L-701,324; 3 mg/kg), being a partial agonist or an antagonist at glycineB receptors, respectively, generalized for the training drugs. Combination tests of glycineB ligands demonstrated that injection of a fixed dose of ACPC (200 mg/kg) or L-701,324 (3 mg/kg) together with different doses of AMPH or COC practically did not modify dose-response curves of the psychostimulants, nor did it affect their ED50 values. Our results indicate that glycineB receptors do not play a role in the discriminative effects of AMPH and COC.     

Matrix Metalloproteinases -8 and -9 and Tissue Inhibitor of Metalloproteinase-1 in Burn Patients. A Prospective Observational Study

IntroductionMatrix metalloproteinases (MMPs) -8 and -9 are released from neutrophils in acute inflammation and may contribute to permeability changes in burn injury. In retrospective studies on sepsis, levels of MMP-8, MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) differed from those of healthy controls, and TIMP-1 showed an association with outcome. Our objective was to investigate the relationship between these proteins and disease severity and outcome in burn patients.MethodsIn this prospective, observational, two-center study, we collected plasma samples from admission to day 21 post-burn, and burn blister fluid samples on admission. We compared MMP-8, -9, and TIMP-1 levels between TBSA<20% (N = 19) and TBSA>20% (N = 30) injured patients and healthy controls, and between 90-day survivors and non-survivors. MMP-8, -9, and TIMP-1 levels at 24-48 hours from injury, their maximal levels, and their time-adjusted means were compared between groups. Correlations with clinical parameters and the extent of burn were analyzed. MMP-8, -9, and TIMP-1 levels in burn blister fluids were also studied.ResultsPlasma MMP-8 and -9 were higher in patients than in healthy controls (P<0.001 and P = 0.016), but only MMP-8 differed between the TBSA<20% and TBSA>20% groups. MMP-8 and -9 were not associated with clinical severity or outcome measures. TIMP-1 differed significantly between patients and controls (P<0.001) and between TBSA<20% and TBSA>20% groups (P<0.002). TIMP-1 was associated with 90-day mortality and correlated with the extent of injury and clinical measures of disease severity. TIMP-1 may serve as a new biomarker in outcome prognostication of burn patients.     

Biochemical,Histopathological and Morphological Profiling of a Rat Model of Early Immune Stimulation: Relation to Psychopathology

Perinatal immune challenge leads to neurodevelopmental dysfunction, permanent immune dysregulation and abnormal behaviour, which have been shown to have translational validity to findings in human neuropsychiatric disorders (e.g. schizophrenia, mood and anxiety disorders, autism, Parkinson’s disease and Alzheimer’s disease). The aim of this animal study was to elucidate the influence of early immune stimulation triggered by systemic postnatal lipopolysaccharide administration on biochemical, histopathological and morphological measures, which may be relevant to the neurobiology of human psychopathology. In the present study of adult male Wistar rats we examined the brain and plasma levels of monoamines (dopamine, serotonin), their metabolites, the levels of the main excitatory and inhibitory neurotransmitters glutamate and γ-aminobutyric acid and the levels of tryptophan and its metabolites from the kynurenine catabolic pathway. Further, we focused on histopathological and morphological markers related to pathogenesis of brain diseases - glial cell activation, neurodegeneration, hippocampal volume reduction and dopaminergic synthesis in the substantia nigra. Our results show that early immune stimulation in adult animals alters the levels of neurotransmitters and their metabolites, activates the kynurenine pathway of tryptophan metabolism and leads to astrogliosis, hippocampal volume reduction and a decrease of tyrosine hydroxylase immunoreactivity in the substantia nigra. These findings support the crucial pathophysiological role of early immune stimulation in the above mentioned neuropsychiatric disorders.     

Large-Scale Variations in Lumber Value Recovery of Yellow Birch and Sugar Maple in Quebec,Canada

Silvicultural restoration measures have been implemented in the northern hardwoods forests of southern Quebec, Canada, but their financial applicability is often hampered by the depleted state of the resource. To help identify sites most suited for the production of high quality timber, where the potential return on silvicultural investments should be the highest, this study assessed the impact of stand and site characteristics on timber quality in sugar maple (Acer saccharum Marsh.) and yellow birch (Betula alleghaniensis Britt.). For this purpose, lumber value recovery (LVR), an estimate of the summed value of boards contained in a unit volume of round wood, was used as an indicator of timber quality. Predictions of LVR were made for yellow birch and sugar maple trees contained in a network of more than 22000 temporary sample plots across the Province. Next, stand-level variables were selected and models to predict LVR were built using the boosted regression trees method. Finally, the occurrence of spatial clusters was verified by a hotspot analysis. Results showed that in both species LVR was positively correlated with the stand age and structural diversity index, and negatively correlated with the number of merchantable stems. Yellow birch had higher LVR in areas with shallower soils, whereas sugar maple had higher LVR in regions with deeper soils. The hotspot analysis indicated that clusters of high and low LVR exist across the province for both species. Although it remains uncertain to what extent the variability of LVR may result from variations in past management practices or in inherent site quality, we argue that efforts to produce high quality timber should be prioritized in sites where LVR is predicted to be the highest.     

Linking monitoring and modelling: can long-term datasets be used more effectively as a basis for large-scale prediction?

Data from long-term monitoring sites are vital for biogeochemical process understanding, and for model development. Implicitly or explicitly, information provided by both monitoring and modelling must be extrapolated in order to have wider scientific and policy utility. In many cases, large-scale modelling utilises little of the data available from long-term monitoring, instead relying on simplified models and limited, often highly uncertain, data for parameterisation. Here, we propose a new approach whereby outputs from model applications to long-term monitoring sites are upscaled to the wider landscape using a simple statistical method. For the 22 lakes and streams of the UK Acid Waters Monitoring Network (AWMN), standardised concentrations (Z scores) for Acid Neutralising Capacity (ANC), dissolved organic carbon, nitrate and sulphate show high temporal coherence among sites. This coherence permits annual mean solute concentrations at a new site to be predicted by back-transforming Z scores derived from observations or model applications at other sites. The approach requires limited observational data for the new site, such as annual mean estimates from two synoptic surveys. Several illustrative applications of the method suggest that it is effective at predicting long-term ANC change in upland surface waters, and may have wider application. Because it is possible to parameterise and constrain more sophisticated models with data from intensively monitored sites, the extrapolation of model outputs to policy relevant scales using this approach could provide a more robust, and less computationally demanding, alternative to the application of simple generalised models using extrapolated input data.     

Folding properties of the hepatitis B core as a carrier protein for vaccination research

The hepatitis B core (HBc) protein has been used successfully in numerous experiments as a carrier for heterologous peptides. Folding and capsid formation of the chimeric proteins is not always achieved easily. In silico analyses were performed to provide further comprehension of the feasibility for predicting successful capsid formation. In contrast to previous work, we show that common in silico predictions do not ensure assembly into particles. We included new considerations regarding capsid formation of HBc fusion proteins. Not only the primary sequence and the length of the inserts seem important, also the rigidity, the distance between the N and the C-terminus and the presence of cysteines, which could form disulphide bonds, could influence proper capsid formation. Furthermore, new conformational insights were formulated when linkers were added to create extra flexibility of the chimeric particles. Different hypotheses were suggested to clarify the obtained results. To this extent, the addition of glycine-rich linkers could lower high rigidity of the insert, removal of the strain of the core protein or ease interaction between the HBc and the insert. Finally, we observed specific changes in capsid formation properties when longer linkers were used. These findings have not been reported before in this and other virus-like particle carriers. In this study, we also propose a new high-yield purification protocol for fusion proteins to be used in vaccination experiments with the carrier protein or in comparative studies of particulate or non-particulate HBc fusion proteins.     

High expression and activity of ecto-5′-nucleotidase/CD73 in the male murine reproductive tract

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, steroidogenesis, and maintenance of fluid composition. Interestingly, adenosine might act as a key capacitative effector for mammalian spermatozoa to acquire the capacity for fertilisation. Extracellular nucleotide levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family regroups the most abundant and effective enzymes to hydrolyse ATP and ADP to AMP in physiological conditions. In the male reproductive tract three members of this family have been indentified: NTPDase1, NTPDase2 and NTPDase3 (Martín-Satué et al. in Histochem Cell Biol 131:615–628, 2009). The purpose of the present study was to characterize in the male reproductive tract the expression profile of the main enzyme responsible for the generation of adenosine from AMP, namely the ecto-5′-nucleotidase (CD73). The enzyme was identified by immunological techniques and by in situ enzymatic assays, including inhibition experiments with α,β-methylene-ADP, a specific CD73 inhibitor. High levels of ecto-5′-nucleotidase were detected in testes in association with both germinal and somatic cells, in smooth muscle cells throughout the tract, in secretory epithelia from exocrine glands, and remarkably, in principal cells of epididymis, where co-localization with NTPDase3 was found. The relevance of this co-expression on nucleotide hydrolysis in these cells directly involved in the control of sperm fluid composition was addressed biochemically. This study suggests close regulation of extracellular nucleoside and nucleotide levels in the genital tract by ecto-5′-nucleotidase that, in concurrence with NTPDases, may impact male fertility.