Shugoshin Is Essential for Meiotic Prophase Checkpoints in C. elegans

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Intestinal microbiota and the immune system in metabolic diseases

The intestinal microbiota is comprised of millions of microorganisms that reside in the gastrointestinal tract and consistently interact with the host. Host factors such as diet and disease status affect the composition of the microbiota, while the microbiota itself produces metabolites that can further manipulate host physiology. Dysbiosis of the intestinal microbiota has been characterized in patients with certain metabolic diseases, some of which involve damage to the host intestinal epithelial barrier and alterations in the immune system. In this review, we will discuss the consequences of dietdependent bacterial dysbiosis in the gastrointestinal tract, and how the associated interaction with epithelial and immune cells impacts metabolic diseases.     

N-Heteroarylmethyl-5-hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylic acid a novel scaffold for the design of uncompetitive α-glucosidase inhibitors

The enzyme α-glucosidase has attracted interest owing to its involvement in the digestive process of carbohydrate, its role in intracellular glycoprotein trafficking, tumorigenesis and viral infection. In this study, several members of a new family of N-heteroarylmethyl substituted azasugars were synthesized and evaluated as α-glucosidase inhibitors. We systematically investigated the effect of different N-substituents as well as the role of hydroxyl and carboxylate moieties on the piperidine ring. The compounds N-heteroarylmethyl-5-hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylic acid emerged as potent α-glucosidase inhibitors. Unlike Acarbose and other clinically relevant α-glucosidase inhibitors, these compounds act through a reversible uncompetitive mechanism of inhibition which make them attractive candidates for drug development.     

Oleuropein Aglycone Protects Transgenic C. elegans Strains Expressing Aβ42 by Reducing Plaque Load and Motor Deficit

The presence of amyloid aggregates of the 42 amino acid peptide of amyloid beta (Aβ42) in the brain is the characteristic feature of Alzheimer’s disease (AD). Amyloid beta (Aβ deposition is also found in muscle fibers of individuals affected by inclusion body myositis (sIBM), a rare muscular degenerative disease affecting people over 50. Both conditions are presently lacking an effective therapeutic treatment. There is increasing evidence to suggest that natural polyphenols may prevent the formation of toxic amyloid aggregates; this applies also to oleuropein aglycone (OLE), the most abundant polyphenol in extra virgin olive oil, previously shown to hinder amylin and Aβ aggregation. Here we evaluated the ability of OLE to interfere with Aβ proteotoxicity in vivo by using the transgenic CL2006 and CL4176 strains of Caenorhabditis elegans, simplified models of AD and of sIBM, which express human Aβ in the cytoplasm of body wall muscle cells. OLE-fed CL2006 worms displayed reduced Aβ plaque deposition, less abundant toxic Aβ oligomers, remarkably decreased paralysis and increased lifespan with respect to untreated animals. A protective effect was also observed in CL4176 worms but only when OLE was administered before the induction of the Aβ transgene expression. These effects were specific, dose-related, and not mediated by the known polyphenolic anti-oxidant activity, suggesting that, in this model organism, OLE interferes with the Aβ aggregation skipping the appearance of toxic species, as already shown in vitro for Aβ42.     

Effect of the calcium buffer EGTA on the "hump" component of charge movement in skeletal muscle.

Three manifestations of excitation-contraction (E-C) coupling were measured in cut skeletal muscle fibers of the frog, voltage clamped in a double Vaseline gap: intramembrane charge movements, myoplasmic Ca2+ transients, and changes in optical transparency. Pulsing patterns in the presence of high [EGTA] intracellularly, shown by García et al. (1989. J. Gen. Physiol. 94:973-986) to deplete Ca2+ in the sarcoplasmic reticulum, were found to change the above manifestations. With an intracellular solution containing 15 mM EGTA and 0 Ca, 10-15 pulses (100 ms) to -20 mV at a frequency of 2 min-1 reduced the "hump" component of charge movement current. This effect was reversible by 5 min of rest. The same effect was obtained in 62.5 mM EGTA and 0 Ca by pulsing at 0.2 min-1. This effect was reversible by adding calcium to the EGTA solution, for a nominal [Ca2+]i of 200 nM, and was prevented by adding calcium to the EGTA solution before pulsing. The suppression of the hump was accompanied by elimination of the optical manifestations of E-C coupling. The current suppressed was found by subtraction and had the following properties: delayed onset, a peak at a variable interval (10-20 ms) into the pulse, a negative phase (inward current) after the peak, and a variable OFF transient that could be multi-phasic and carried less charge than the ON transient. In the previous paper (Csernoch et al., 1991. J. Gen. Physiol. 97:845-884) it was shown that several interventions suppress a similar component of charge movement current, identified with the "hump" or Q gamma current (I gamma). Based on the similarity to that component, the charge movement suppressed by the depletion protocols can also be identified with I gamma. The fact that I gamma is suppressed by Ca2+ depletion and the kinetic properties of the charge suppressed is inconsistent with the existence of separate sets of voltage sensors underlying the two components of charge movement, Q beta and Q gamma. This is explicable if Q gamma is a consequence of calcium release from the sarcoplasmic reticulum.     

Structures and Functions of Calcium Channel β Subunits

Calcium channel subunits have profound effects on how ₁ subunits perform. In this article we summarize our present knowledge of the primary structures of subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with ₁ subunits, the effects of subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by subunits, such as long-lasting prepulse facilitation of _1C (absent in _1E) and inhibition by G protein dimer of _1E, absent in _1C. One subunit, a brain 2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of ₁ in oocytes requires a subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca²⁺ channels to account for ₁ alone channels seen in cells with limited subunit expression. In one model, dissociates from the mature ₁ after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of would then have subunit composition ₁. In the other model, the chaperoning remains associated with the mature channel and ₁ alone channels would in fact be ₁ channels. Upon co-expression of high levels of the regulated channels would have composition [₁].     

Molecular control of mitochondrial calcium uptake

The recently identified Mitochondrial Calcium Uniporter (MCU) is the protein of the inner mitochondrial membrane responsible for Ca²⁺ uptake into the matrix, which plays a role in the control of cellular signaling, aerobic metabolism and apoptosis. At least two properties of mitochondrial calcium signaling are well defined: (i) mitochondrial Ca²⁺ uptake varies greatly among different cells and tissues, and (ii) channel opening is strongly affected by extramitochondrial Ca²⁺ concentration, with low activity at resting and high capacity after cellular stimulation. It is now becoming clear that these features of the mitochondrial Ca²⁺ uptake machinery are not embedded in the MCU protein itself, but are rather due to the contribution of several MCU interactors. The list of the components of the MCU complex is indeed rapidly growing, thus revealing an unexpected complexity that highlights the pleiotropic role of mitochondrial calcium signaling.     

Neurophysiological Age Differences During Task-Performance in a Stereoscopic Virtual Environment

In today’s society, there is an increasing number of workplaces in virtual environments (VE). But, there are only a few reports dealing with occupational health issues or age effects. The question arises how VR generally interferes with cognitive processes. This interference might have relevant implications for workability and work-efficiency in virtual environments. Event-related potentials are known to reflect different stages of stimulus reception, evaluation, and response. We have established an electroencephalographic (EEG) monitoring, focussing on event-related potentials (N100; mismatch negativity, i.e., MMN) to obtain access to attention dependent and pre-attentive processing of sensory stimuli applied in VE. The MMN is known to be correlated with the ability of subjects to react to an unexpected event. The aim of the present study was to investigate cognitive responses to distracting auditory stimuli in two different age groups in a virtual environment (VE) and in a real environment (“real reality”), and to compare characteristic neurophysiological response patterns. Data show that stimulus detection as given by the N100 amplitude and latency does not differ in both age groups and task conditions. In contrast, the pre-attentive processing as given by the MMN is altered in the VR such as the non-VR condition in an age-related manner. A relevant finding of the present study was that the age related differences seen in the non-VR condition were not strengthened in VR.     

Uneven Distribution and Size of Rabbit Lens Capsule Proteoglycans

To document the ultrastructural distribution of lens capsule proteoglycans, rabbit lens capsules were fixed and stained overnight in 50mM sodium acetate, pH 5.6, containing 2.5% glutaraldehyde, 0.2% Cuprolinic Blue and 0.2M MgCl2. They were rinsed, stained with 1% aqueous sodium tungstate, embedded in Epon, sectioned (60nm), and examined with an electron microscope at 60kV.Proteoglycan–Cuprolinic Blue complexes mainly appeared as networks of small electron-dense filaments throughout the posterior and anterior capsules. The posterior capsule was a single layer with a network of small proteoglycan filaments gradually decreasing in size from the humoral side (90×10nm) to the lenticular side (30×8nm). The humoral side of the anterior capsule had a thin lamina (400nm) containing large (180×40nm), very electron-dense proteoglycan–Cuprolinic Blue complexes plus small proteoglycans. Below this lamina, the complexes were only seen as filaments slightly smaller than those in the corresponding area of the posterior capsule.Cuprolinic Blue binding of the anterior and posterior lens capsules revealed differences in the size and distribution of their sulphated proteoglycans which do not correspond to the patterns of their immunoreactivity with anti-heparan sulphate proteoglycan. The humoral lamina in the anterior capsules, with large proteoglycan structures, might be a distinct structural and functional compartment.     

Stabilisation of alpha-helices by site-directed mutagenesis reveals the importance of secondary structure in the transition state for acylphosphatase folding

The effects of stabilising mutations on the folding process of common-type acylphosphatase have been investigated. The mutations were designed to increase the helical propensity of the regions of the polypeptide chain corresponding to the two alpha-helices of the native protein. Various synthetic peptides incorporating the designed mutations were produced and their helical content estimated by circular dichroism. The most substantial increase in helical content is found for the peptide carrying five mutations in the second alpha-helix. Acylphosphatase variants containing the corresponding mutations display, to different extents, enhanced conformational stabilities as indicated by equilibrium urea denaturation experiments monitored by changes of intrinsic fluorescence. All the protein variants studied here refold with apparent two-state kinetics. Mutations in the first alpha-helix are responsible for a small increase in the refolding rate, accompanied by a marked decrease in the unfolding rate. On the other hand, multiple mutations in the second helix result in a considerable increase in the refolding rate without any significant effect on the unfolding rate. Addition of trifluoroethanol was found to accelerate the folding of the acylphosphatase variants, the extent of the acceleration being inversely proportional to the intrinsic rate of folding of the corresponding mutant. The trifluoroethanol-induced acceleration is far less marked for those variants whose alpha-helical structure is efficiently stabilised by amino acid replacements. This observation suggests that trifluoroethanol acts in a similar manner to the stabilising mutations in promoting native-like secondary structure. Analysis of the kinetic data indicates that the second helix is fully consolidated in the transition state for folding of acylphosphatase, whereas the first helix is only partially formed. These data suggest that the second helix is an important element in the folding process of the protein.