Extensive fusion of haematopoietic cells with Purkinje neurons in response to chronic inflammation

Transplanted bone marrow-derived cells (BMDCs) have been reported to fuse with cells of diverse tissues, but the extremely low frequency of fusion has led to the view that such events are biologically insignificant. Nonetheless, in mice with a lethal recessive liver disease (tyrosinaemia), transplantation of wild-type BMDCs restored liver function by cell fusion and prevented death, indicating that cell fusion can have beneficial effects. Here we report that chronic inflammation resulting from severe dermatitis or autoimmune encephalitis leads to robust fusion of BMDCs with Purkinje neurons and formation of hundreds of binucleate heterokaryons per cerebellum, a 10-100-fold higher frequency than previously reported. Single haematopoietic stem-cell transplants showed that the fusogenic cell is from the haematopoietic lineage and parabiosis experiments revealed that fusion can occur without irradiation. Transplantation of rat bone marrow into mice led to activation of dormant rat Purkinje neuron-specific genes in BMDC nuclei after fusion with mouse Purkinje neurons, consistent with nuclear reprogramming. The precise neurological role of these heterokaryons awaits elucidation, but their frequency in brain after inflammation is clearly much higher than previously appreciated.     

The essential role of lipids in Alzheimer's disease

In the absence of efficient diagnostic and therapeutic tools, Alzheimer's disease (AD) is a major public health concern due to longer life expectancy in the Western countries. Although the precise cause of AD is still unknown, soluble β-amyloid (Aβ) oligomers are considered the proximate effectors of the synaptic injury and neuronal death occurring in the early stages of AD. Aβ oligomers may directly interact with the synaptic membrane, leading to impairment of synaptic functions and subsequent signalling pathways triggering neurodegeneration. Therefore, membrane structure and lipid status should be considered determinant factors in Aβ-oligomer-induced synaptic and cell injuries, and therefore AD progression. Numerous epidemiological studies have highlighted close relationships between AD incidence and dietary patterns. Among the nutritional factors involved, lipids significantly influence AD pathogenesis. It is likely that maintenance of adequate membrane lipid content could prevent the production of Aβ peptide as well as its deleterious effects upon its interaction with synaptic membrane, thereby protecting neurons from Aβ-induced neurodegeneration. As major constituents of neuronal lipids, n-3 polyunsaturated fatty acids are of particular interest in the prevention of AD valuable diet ingredients whose neuroprotective properties could be essential for designing preventive nutrition-based strategies. In this review, we discuss the functional relevance of neuronal membrane features with respect to susceptibility to Aβ oligomers and AD pathogenesis, as well as the prospective capacities of lipids to prevent or to delay the disease.     

Accurate intraocular pressure prediction from applanation response data using genetic algorithm and neural networks

The fact that Goldmann applanation tonometry does not accurately account for individual corneal elastic stiffness often leads to inaccuracy in the measurement of intraocular pressure (IOP). IOP should account not only for the effect of central corneal thickness (CCT) but should also account for other corneal biomechanical factors. A computational method for accurate and reliable determination of IOP is investigated with a modified applanation tonometer in this paper. The proposed method uses a combined genetic algorithm/neural network procedure to match the clinically measured applanation force-displacement history with that obtained from a nonlinear finite element simulation of applanation. An additional advantage of the proposed method is that it also provides the ability to determine CCT and material properties of the cornea from the same applanation response data. The performance of the proposed method has been demonstrated through a parametric study and via comparison with a well known clinical case. The proposed method is also shown to be computationally efficient, which is an important practical consideration for clinical application.     

The design and synthesis of a tricyclic single-nitrogen scaffold that serves as a 5-HT2C receptor agonist

5-HT2C agonists have shown efficacy in limiting food consumption and thus may serve as an important drug class in combating obesity. We describe the design and synthesis of a novel tricyclic single-nitrogen scaffold that was used to produce 5-HT2C agonists. SAR was developed around this chemotype and compounds were identified that were potent (Ki<15 nM) and selective relative to other 5-HT2 receptors. The most promising compound displayed a good pharmacokinetic profile in multiple animal species, and was efficacious in an acute feeding study in rats.     

Mitochondrial aberrations in mucolipidosis Type IV

Mucolipidosis type IV is a genetic lysosomal storage disease associated with degenerative processes in the brain, eye, and other tissues. Mucolipidosis type IV results from mutations in the gene MCOLN1, which codes for the TRP family ion channel, mucolipin 1. The connection between lysosomal dysfunction and degenerative processes in mucolipidosis type IV is unclear. Here we report that mucolipidosis type IV and several unrelated lysosomal storage diseases are associated with significant mitochondrial fragmentation and decreased mitochondrial Ca2+ buffering efficiency. The mitochondrial alterations observed in these lysosomal storage diseases are reproduced in control cells by treatment with lysosomal inhibitors and with the autophagy inhibitor 3-methyladenine. This suggests that inefficient autophagolysosomal recycling of mitochondria generates fragmented, effete mitochondria in mucolipidosis. Mitochondria accumulate that cannot properly buffer calcium fluxes in the cell. A decrease in mitochondrial Ca2+ buffering capacity in cells affected by these lysosomal storage diseases is associated with increased sensitivity to apoptosis induced by Ca2+-mobilizing agonists and executed via a caspase-8-dependent pathway. Deficient Ca2+ homeostasis may represent a common mechanism of degenerative cell death in several lysosomal storage diseases.     

Acid ceramidase but not acid sphingomyelinase is required for tumor necrosis factor-{alpha}-induced PGE2 production

Sphingolipids are well established effectors of signal transduction downstream of the tumor necrosis factor (TNF) receptor. In a previous study, we showed that the sphingosine kinase/sphingosine 1-phosphate (S1P) pathway couples TNF receptor to induction of the cyclooxygenase 2 gene and prostaglandin synthesis (Pettus, B. J., Bielawski, J., Porcelli, A. M., Reames, D. L., Johnson, K. R., Morrow, J., Chalfant, C. E., Obeid, L. M., and Hannun, Y. A. (2003) FASEB J. 17, 1411-1421). In this study, the requirement for acid sphingomyelinase and sphingomyelin metabolites in the TNFalpha/prostaglandin E(2) (PGE(2)) pathway was investigated. The amphiphilic compound desipramine, a frequently employed inhibitor of acid sphingomyelinase (ASMase), blocked PGE(2) production. However, the action of desipramine was independent of its action on ASMase, since neither genetic loss of ASMase (Niemann-Pick fibroblasts) nor knockdown of ASMase using RNA interference affected TNFalpha-induced PGE(2) synthesis. Further investigations revealed that desipramine down-regulated acid ceramidase (AC), but not sphingosine kinase, at the protein level. This resulted in a time-dependent drop in sphingosine and S1P levels. Moreover, exogenous administration of either sphingosine or S1P rescued PGE(2) biosynthesis after desipramine treatment. Interestingly, knockdown of endogenous AC by RNA interference attenuated cyclooxygenase 2 induction by TNFalpha and subsequent PGE(2) biosynthesis. Taken together, these results define a novel role for AC in the TNFalpha/PGE(2) pathway. In addition, the results of this study warrant careful reconsideration of desipramine as a specific inhibitor for ASMase.     

Mutations in the SPARC-related modular calcium-binding protein 1 gene, SMOC1, cause waardenburg anophthalmia syndrome

Waardenburg anophthalmia syndrome, also known as microphthalmia with limb anomalies, ophthalmoacromelic syndrome, and anophthalmia-syndactyly, is a rare autosomal-recessive developmental disorder that has been mapped to 10p11.23. Here we show that this disease is heterogeneous by reporting on a consanguineous family, not linked to the 10p11.23 locus, whose two affected children have a homozygous mutation in SMOC1. Knockdown experiments of the zebrafish smoc1 revealed that smoc1 is important in eye development and that it is expressed in many organs, including brain and somites.     

Zinc inhibition of bacterial cytochrome bc(1) reveals the role of cytochrome b E295 in proton release at the Q(o) site

The cytochrome (cyt) bc(1) complex (cyt bc(1)) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc(1) is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn(2+)-induced inhibition of Rhodobacter capsulatus cyt bc(1) using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn(2+) binding mechanism and its inhibitory effect on cyt bc(1) function. Analogous studies were conducted with a mutant of cyt b, E295, a residue previously proposed to bind Zn(2+) on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 to valine, a noncoordinating residue, results in a decrease in Zn(2+) binding affinity. The kinetic study showed that wild-type cyt bc(1) and its E295V mutant have similar levels of apparent K(m) values for decylbenzohydroquinone as a substrate (4.9 ± 0.2 and 3.1 ± 0.4 μM, respectively), whereas their K(I) values for Zn(2+) are 8.3 and 38.5 μM, respectively. The calorimetry-based K(D) values for the high-affinity site of cyt bc(1) are on the same order of magnitude as the K(I) values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn(2+), whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn(2+). Our overall results indicate that the proton-active E295 residue near the Q(o) site of cyt bc(1) can bind directly to Zn(2+), resulting in a decrease in the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to electron transfer at the Q(o) site of cyt bc(1).     

Synthetic adjuvants for vaccine formulations: evaluation of new phytol derivatives in induction and persistence of specific immune response

Terpenoids are ubiquitous natural compounds that have been shown to improve vaccine efficacy as adjuvants. To gain an understanding of the structural features important for adjuvanticity, we studied compounds derived from a diterpene phytol and assessed their efficacy. In a previous report, we showed that phytol and one of its derivatives, PHIS-01 (a phytol-derived immunostimulant, phytanol), are excellent adjuvants. To determine the effects of varying the polar terminus of PHIS-01, we designed amine and mannose-terminated phytol derivatives (PHIS-02 and PHIS-03, respectively). We studied their relative efficacy as emulsions with soluble proteins, ovalbumin, and a hapten-protein conjugate phthalate-KLH. Immunological parameters evaluated consisted of specific antibody responses in terms of titers, specificities and isotype profiles, T cell involvement and cytokine production. Our results indicate that these new isoprenoids were safe adjuvants with the ability to significantly augment immunogen-specific IgG1 and IgG2a antibody responses. Moreover, there was no adverse phthalate cross-reactive anti-DNA response. Interestingly, PHIS-01 and PHIS-03 influenced differentially T-helper polarization. We also observed that these compounds modulated the immune response through apoptotic/necrotic effects on target tumor cells using murine lymphomas. Finally, unlike squalene and several other terpenoids reported to date, these phytol derivatives did not appear arthritogenic in murine models.