Crowding alone cannot account for cosolute effect on amyloid aggregation

Amyloid fiber formation is a specific form of protein aggregation, often resulting from the misfolding of native proteins. Aimed at modeling the crowded environment of the cell, recent experiments showed a reduction in fibrillation halftimes for amyloid-forming peptides in the presence of cosolutes that are preferentially excluded from proteins and peptides. The effect of excluded cosolutes has previously been attributed to the large volume excluded by such inert cellular solutes, sometimes termed macromolecular crowding. Here, we studied a model peptide that can fold to a stable monomeric β-hairpin conformation, but under certain solution conditions aggregates in the form of amyloid fibrils. Using Circular Dichroism spectroscopy (CD), we found that, in the presence of polyols and polyethylene glycols acting as excluded cosolutes, the monomeric β-hairpin conformation was stabilized with respect to the unfolded state. Stabilization free energy was linear with cosolute concentration, and grew with molecular volume, as would also be predicted by crowding models. After initiating the aggregation process with a pH jump, fibrillation in the presence and absence of cosolutes was followed by ThT fluorescence, transmission electron microscopy, and CD spectroscopy. Polyols (glycerol and sorbitol) increased the lag time for fibril formation and elevated the amount of aggregated peptide at equilibrium, in a cosolute size and concentration dependent manner. However, fibrillation rates remained almost unaffected by a wide range of molecular weights of soluble polyethylene glycols. Our results highlight the importance of other forces beyond the excluded volume interactions responsible for crowding that may contribute to the cosolute effects acting on amyloid formation.     

Similar cation channels mediate protection from cerebellar exitotoxicity by exercise and inheritance

Exercise and inherited factors both affect recovery from stroke and head injury, but the underlying mechanisms and interconnections between them are yet unknown. Here, we report that similar cation channels mediate the protective effect of exercise and specific genetic background in a kainate injection model of cerebellar stroke. Microinjection to the cerebellum of the glutamatergic agonist, kainate, creates glutamatergic excito\xE2\x80\x90toxicity characteristic of focal stroke, head injury or alcoholism. Inherited protection and prior exercise were both accompanied by higher cerebellar expression levels of the Kir6.1 ATP-dependent potassium channel in adjacent Bergmann glia, and voltage-gated KVbeta2 and cyclic nucleotide-gated cation HCN1 channels in basket cells. Sedentary FVB/N and exercised C57BL/6 mice both expressed higher levels of these cation channels compared to sedentary C57BL/6 mice, and were both found to be less sensitive to glutamate toxicity. Moreover, blocking ATP-dependent potassium channels with Glibenclamide enhanced kainate-induced cell death in cerebellar slices from the resilient sedentary FVB/N mice. Furthermore, exercise increased the number of acetylcholinesterase-positive fibres in the molecular layer, reduced cerebellar cytokine levels and suppressed serum acetylcholinesterase activity, suggesting anti-inflammatory protection by enhanced cholinergic signalling. Our findings demonstrate for the first time that routine exercise and specific genetic backgrounds confer protection from cerebellar glutamatergic damages by similar molecular mechanisms, including elevated expression of cation channels. In addition, our findings highlight the involvement of the cholinergic anti-inflammatory pathway in insult-inducible cerebellar processes. These mechanisms are likely to play similar roles in other brain regions and injuries as well, opening new venues for targeted research efforts.     

Localization of voltage-sensitive calcium channels along developing neurites: their possible role in regulating neurite elongation

Calcium action potentials were extracellularly recorded from growth cones of differentiated N1E-115 neuroblastoma cells maintained in monolayer cultures. Extracellular recordings along the neurites suggest that voltage-activated Ca²⁺ channels are less abundant in the processes than in the growth cones. In order to investigate if Ca²⁺ entry into the growth cone plays a role in the regulation of neurite growth, we studied the morphological changes induced by experimental conditions which permit calcium entry. Cells were depolarized either by 30 mM potassium (for 10–60 min) or by stimulating the soma (for 20–120 min) with an intracellular electrode. Morphological changes in individual cells were followed by means of time-lapse video recordings. In more than 60% of the experiments, steady-state potassium depolarization induced a pronounced increase of 20–120% in the area of the growth cone. This was frequently associated with neurite elongation. However, such changes could not be detected in the presence of Cd²⁺ concentrations which block the Ca²⁺ channels. Similar results were obtained in the presence of 2 μM of the Ca²⁺ ionophore A-23187 or when the cells were repetitively stimulated (0.2 Hz) in a medium containing 10^?6M TTX and 15 mM TEA. Local microapplication, directly onto single growth cones, of a depolarizing solution containing 5 mM Ca²⁺ also led to similar observations. Scanning electron microscopy indicated that the depolarized growth cone membranes were flattened and contained markedly more rounded protuberances relative to control cultures. Our results indirectly suggest that Ca²⁺ entry might be a trigger in the process of neurite elongation.     

Differentiation of myoblasts and CNS cells grown either separately or as co-cultures on microcarriers

Dispersed neuronal and muscular elements from fetal or neonatal origin, can organize and mature in culture when grown on positively charged cylindrical microcarriers (MCS), to a stage which simulatein vivo maturation. Cells arrange themselves on the MCS to form aggregates which remain floating in the nutrient medium. In such a tridimensional organization, the neuronal tissue is capable of regenerating a network of nerve fibers which establish synapse interconnections and undergo myelination. Oligodendrocytes organize on MCS in a tridimensional pattern and produce extensive myelin-like membranes. Myoblasts in MC-cultures fuse into polynucleated myotubes which become striated and contract spontaneously. Creatine kinase and acetylcholine receptor (AChR) are formed during myogenesis in similar quantities in MC-cultures and in monolayers. When both neuronal and muscle tissues are prepared from the same fetus (autologous nerve-muscle co-cultures) and are cultured on MCS, they interconnect to form neuro-muscular junctions. Cells from both tissues, exhibit better differentiation, for longer periods in MC-cultures than they do in monolayers. The floating functional entities are easy to sample and can be harvested for ultrastructural, immunocytochemical and biochemical analysis. In addition, MC-cultures can be used as a good tool for the study of acute and chronic exposures to toxicological agents, as well as for implantation into demyelinated, injured or dystrophic tissues. In this case the MCS in the implanted entities will serve as identifiable markers.     

Design,synthesis and evaluation of novel 5,6-dimethoxy-1-oxo-2,3-dihydro-1H-2-indenyl-3,4-substituted phenyl methanone analogues

In present investigation, a series of substituted phenyl-5,6-dimethoxy-1-oxo-2,3-dihydro-1H-2-indenylmethanone analogues were synthesized and were tested for their potential for treating AD disease. All the newly synthesized compounds were showing moderate to high AChE inhibitory activities, with compound 5,6-dimethoxy-1-oxo-2,3-dihydro-1H-2-indenyl-3,4,5-trimethoxyphenylmethanone (5f) produced significant activities with 2.7 ± 0.01 μmol/L.     

The Role of Culture/Ethnicity in Communicating with Cancer Patients About Mental Health Distress and Suicidality

Culture, Medicine, and Psychiatry - To explore the role of culture in communicating with cancer patients about mental health distress and suicidality. The Grounded Theory method of data collection...     

A single nucleotide change in the 5' noncoding region of Sindbis virus confers neurovirulence in rats

Two pairs of Sindbis virus (SV) variants that differ in their neuroinvasive and neurovirulent traits in mice have been isolated. Recently, we mapped the genetic determinants responsible for neuroinvasiveness in weanling mice. Here, we extend this study to newborn and adult rats and to rat neuronal cultures. Remarkably, certain aspects of the pathogenesis of these strains in rats were found to be quite distinct from the mouse model. Suckling rats were susceptible to all four isolates, and replication in the brain was observed after both intraperitoneal and intracranial (i.c.) inoculation. None of the isolates was neuroinvasive in adult rats, although all replicated after i.c. inoculation. For the isolate pair that was highly neurovirulent in mice, SVN and SVNI, only SVNI caused death after i.c. inoculation of adult rats. Similarly, only SVNI was cytotoxic for primary cultures of mature neurons. The genetic determinants responsible for the pathogenic properties of SVNI were mapped to the E2 glycoprotein and the 5' noncoding region (5'NCR). Substitution of two amino acids in SVN E2 with the corresponding residues of SVNI (Met-190 and Lys-260) led to paralysis in 3- and 5-week-old rats. More dramatically, a single substitution in the 5'NCR of SVN (G at position 8) transformed the virus into a lethal pathogen for 3-week-old rats like SVNI. In 5-week-old rats, however, this recombinant was attenuated relative to SVNI by 2 orders of magnitude. Combination of the E2 and 5'NCR determinants resulted in a recombinant with virulence properties indistinguishable from those of SVNI. These data indicate that the 5'NCR and E2 play an instrumental role in determining the age-dependent pathogenic properties of SV in rats.     

Action of steroid hormones on growth and differentiation of CNS and spinal cord organotypic cultures

Summary During the prenatal period, gonadal steroid environment induces dramatic sexually dimorphic changes in the nervous system.We have usedin vitro methods to study the mechanism and timing of hormonal influences on neuronal sprouting and myelination during the prenatal period.Organotypic cultures of hypothalamus and lumbar spinal cord (SC) slices from rat fetuses were grown on plasma clot or in hyaluronic acid and exposed to estogen (17 estradiol) and testosterone (T) during cultivation. Both steroid hormones were active: 17 estradiol enhanced sprouting of hypothalamic neuronal fibers and increased the amount of synapses. In SC cultures T induced regeneration of thick nerve processes and an early onset of myelination, mainly of peripheral myelin.