Nitric oxide synthase-containing, peptide-containing, and acetylcholinesterase-positive nerves in the cat lower oesophagus

Summary The innervation of the cat lower oesophagus, including the lower oesophageal sphincter, was studied by enzyme histochemistry, immunohistochemistry, and confocal microscopy. In the lower oesophageal sphincter, and at a level 2 cm above it, no apparent differences were seen in the nerve distribution pattern. Among the nerve populations studied, acetylcholinesterase (AChE)-positive nerves were the most abundant in both these regions. The density of AChE-positive nerves was particularly marked in the circular muscle layer. A rich supply of nitric oxide synthase (NOS)-containing nerves was identified by using an antiserum against neuronal NOS, or by enzyme histochemical staining for NADPH diaphorase activity. Vasoactive intestinal peptide (VIP)-immunoreactive nerves had a similar distribution pattern as NOS-immunoreactive nerves, and nerves displaying immunoreactivity for NOS and VIP often showed profiles coinciding with AChE-positive nerves. As judged by confocal microscopy, immunoreactivities for helospectin, pituitary adenylate cyclase-activating peptide (PACAP) and VIP, to a large extent were found in the same nerves. At a level 7 cm above the lower oesophageal sphincter, the total nerve supply was less than in the sphincter itself and 2 cm above it. Immunoreactivity towards VIP, PACAP and helospectin was also found to co-exist with NOS and neuropeptide Y within the same nerve structures. It is concluded that there is an intricate innervation pattern in the feline lower oesophagus reflecting the complexity in the regulation of its motility.     

Impact of climate change factors on the clonal sedge Carex bigelown: implications for population growth and vegetative spread

Hypothesized life-cycle responses to climate change for the arctic, clonal perennial Carex bigelown are constructed using a range of earlier observations and experiments together with new information from monitoring and an environmental perturbation study These data suggest, that under current climate change scenarios, increases in CO₂, temperature and nutrient availability would promote growth in a qualitatively similar way The evidence suggests that both tiller size and daughter tiller production will increase, and be shifted towards production of phalanx tillers which have a greater propensity for flowering Furthermore, age at tillering as well as tiller life span may decrease, whereas survival of younger age classes might be higher Mathematical models using experimental data incorporating these hypotheses were used to a) integrate the various responses and to calculate the order of magnitude of changes in population growth rate (γ). and b) to explore the implications of responses in individual demographic parameters for population growth rate The models suggest that population growth rate following climate change might increase significantly, but not un-realistically so. with the younger, larger, guerilla ullers being the most important tiller categones in contributing to X The rate of vegetative spread is calculated to more than double, while cyclical trends in flowering and populauon growth are predicted to decrease substantially     

Studies on the differentiation capacity of neural epithelium cells in chick embryos

Cell and Tissue Research -     

Occludin is overexpressed in Alzheimer's disease and vascular dementia

The tight junctions (TJs) are key players in the control of blood-brain barrier (BBB) properties, the most complex TJs in the vascular system being found in the endothelial cells of brain capillaries. One of the main TJs proteins is occludin, which anchors plasma membranes of neighbour cells and is present in large amounts in the brain endothelia. Previous studies demonstrated that disruption of BBB in various pathological situations associates with changes in occludin expression, and this change could be responsible for malfunction of BBB. Therefore in this study, applying an immunohistochemical approach, we decided to explore the occludin expression in frontal cortex (FC) and basal ganglia in ageing control, Alzheimer's disease (AD), and vascular dementia (VD) brains, as far as all these pathologies associate microangiopathy and disruption of BBB. Strikingly, we found selected neurons, astrocytes and oligodendrocytes expressing occludin, in all cases studied. To estimate the number of occludin-expressing neurons, we applied a stereological approach with random systematic sampling and the unbiased optical fractionator method. We report here a significant increase in ratio of occludin-expressing neurons in FC and basal ganglia regions in both AD and VD as compared to ageing controls. Within the cerebral cortex, occludin was selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings could be important in unravelling new pathogenic pathways in dementia disorders and new functions of occludin and TJs.     

Rat brain gamma-secretase activity is highly influenced by detergents

Gamma-secretase is important for the development of Alzheimer's disease, since it is a crucial enzyme for the generation of the pathogenic amyloid beta-peptide (Abeta). Most data on gamma-secretase is derived from studies in cell lines overexpressing gamma-secretase components or amyloid precursor protein (APP), and since gamma-secretase is a transmembrane protein complex, detergents have been frequently used to facilitate the studies. However, no extensive comparison of the influence of different detergents at different concentrations on gamma-secretase activity in preparations from brain has been made. Here, we establish the optimal conditions for gamma-secretase activity in rat brain, using an activity assay detecting endogenous production of the APP intracellular domain, which is generated when gamma-secretase cleaves the APP C-terminal fragments. We performed a subcellular fractionation and noted the highest gamma-secretase activity in the 100000g pellet and that the optimal pH was around 7. We found that gamma-secretase was active for at least 16 h at 37 degrees C and that the endogenous substrate levels were sufficient for activity measurements. The highest activity was obtained in 0.4% CHAPSO, which is slightly below the critical micelle concentration (0.5%) for this detergent, but the complex was not solubilized efficiently at this concentration. On the other hand, 1% CHAPSO solubilized a substantial amount of the gamma-secretase components, but the activity was low. The activity was fully restored by diluting the sample to 0.4% CHAPSO. Therefore, using 1% CHAPSO for solubilization and subsequently diluting the sample to 0.4% is an appropriate procedure for obtaining a soluble, highly active gamma-secretase from rat brain.     

Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue

DNA polymerases discriminate between correct and incorrect nucleotide substrates during a "nonchemical" step that precedes phosphodiester bond formation in the enzymatic cycle of nucleotide incorporation. Despite the importance of this process in polymerase fidelity, the precise nature of the molecular events involved remains unknown. Here we report a fluorescence resonance energy transfer (FRET) system that monitors conformational changes of a polymerase-DNA complex during selection and binding of nucleotide substrates. This system utilizes the fluorescent base analogue 1,3-diaza-2-oxophenothiazine (tC) as the FRET donor and Alexa-555 (A555) as the acceptor. The tC donor was incorporated within a model DNA primer/template in place of a normal base, adjacent to the primer 3' terminus, while the A555 acceptor was attached to an engineered cysteine residue (C751) located in the fingers subdomain of the Klenow fragment (KF) polymerase. The FRET efficiency increased significantly following binding of a correct nucleotide substrate to the KF-DNA complex, showing that the fingers had closed over the active site. Fluorescence anisotropy titrations utilizing tC as a reporter indicated that the DNA was more tightly bound by the polymerase under these conditions, consistent with the formation of a closed ternary complex. The rate of the nucleotide-induced conformational transition, measured in stopped-flow FRET experiments, closely matched the rate of correct nucleotide incorporation, measured in rapid quench-flow experiments, indicating that the conformational change was the rate-limiting step in the overall cycle of nucleotide incorporation for the labeled KF-DNA system. Taken together, these results indicate that the FRET system can be used to probe enzyme conformational changes that are linked to the biochemical function of DNA polymerase.     

VEGF reduces astrogliosis and preserves neuromuscular junctions in ALS transgenic mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting from motor neuron loss in the spinal cord and brain stem. In the present study, we found that systemic administration of recombinant vascular endothelial growth factor (VEGF) significantly diminished astrogliosis and increased the number of neuromuscular junctions in a Cu/Zn superoxide dismutase (SOD1) transgenic mouse model of ALS. Our results thus demonstrate a novel regulatory role of VEGF on astrocytes and are suggestive of protective effects of VEGF both in the peripheral and central nervous system in the SOD1 transgenic mouse model. These findings warrant further evaluation of the mechanism(s) of regulatory effects of VEGF on neuronal and non-neuronal cells, and the relation of these events to motor neuron degeneration and the onset and progression of ALS.     

A short-term dietary supplementation with high doses of vitamin E increases NK cell cytolytic activity in advanced colorectal cancer patients

Cancer patients with advanced disease display signs of immune suppression, which constitute a major obstacle for effective immunotherapy. Both T cells and NK cells are affected by a multitude of mechanisms of which the generation of reactive oxygen species is of major importance. Therefore, we hypothesized that two weeks of high-dose treatment with the anti-oxidant vitamin E may enhance NK cell function in cancer patients by protecting from oxidative stress. Seven patients with colorectal cancer (Dukes stage C and D) received a daily dose of 750 mg of vitamin E during a period of two weeks and the function, phenotype and receptor expression of NK cells were analyzed. The short-term vitamin E treatment significantly improved NK cell cytolytic activity in six out of the seven patients analyzed. The increased NK cell activity in patients’ PBMC was not due to increased numbers of NK cells or an increase in the proportion of the CD56^dim NK cell subpopulation. Furthermore, neither an increased perforin expression nor an enhanced ability of NK cells to produce IFN-γ was observed as a result of vitamin E treatment. Finally, vitamin E treatment was associated with a minor, but consistent, induction of NKG2D expression in all patients analyzed. In conclusion, this pilot study demonstrates that vitamin E may boost NK cell function in patients with colorectal cancer. Further studies are warranted to explore the potential of vitamin E as an adjuvant for immunotherapy against cancer and to determine the underlying mechanism(s) behind vitamin E induced NK cell activation.