Arachidonic acid influences intracellular calcium handling in human osteoblasts

The effect of arachidonic acid (AA) on intracellular Ca(2+) concentration ([Ca(2+)]i) in human osteoblasts MG63 was studied. AA caused a concentration-dependent increase in [Ca(2+)]i, mainly due to inward Ca(2+) transport from extracellular environment. Moreover, AA in Ca(2+) -free medium produced a small, transient increase of [Ca(2+)]i, indicating that AA may also trigger Ca(2+) release from intracellular stores. Because the [Ca(2+)]i response to AA was inhibited by the cyclooxygenase (COX) inhibitor indomethacin, we tested the effect of prostaglandins (PGs), products of COX pathway. PGs E1 and E2 caused an increase in [Ca(2+)]i, which, however, was far lower than that obtained with AA. The [Ca(2+)]i response to AA was not inhibited by nifedipine, suggesting that AA did not activate a voltage-dependent Ca(2+) channel. Our results indicate that AA could modulate [Ca(2+)]i in MG63 human osteoblasts, where it may influence Ca(2+) transport across both plasma and endoplasmic membranes. Furthermore, they suggest that osteoblast activity may be modulated by AA.     

Diaryl substituted pyrrolidinones and pyrrolones as 5-HT2C inhibitors: synthesis and biological evaluation

Within the continuous quest for the discovery of novel compounds able to treat anxiety and depression, the generation of a pharmacophore model for 5-HT2C receptor antagonists and the discovery of a new class of potent and selective 5-HT2C molecules are reported.     

From pyrroles to pyrrolo[1,2-a]pyrazinones: a new class of mGluR1 antagonists

Exploiting the SAR of the known pyrrole derivatives, a new class of mGluR1 antagonists was developed through a cyclization of the C-2 position on the pyrrole N-1 nitrogen. The resulting pyrrolo[1,2-a]pyrazinones are potent and noncompetitive antagonists.     

Comparative proteomic profile of rat sciatic nerve and gastrocnemius muscle tissues in ageing by 2‐D DIGE

Ageing induces a progressive morphological change and functional decline in muscles and in nerves. Light and electron microscopy, 2‐D DIGE and MS, were applied to profile the qualitative and quantitative differences in the proteome and morphology of rat gastrocnemius muscle and sciatic nerve, in healthy 22‐month‐old rats. At muscle level, morphological changes are associated to fibre atrophy accompanied by myofibrillar loss and degeneration, disappearance of sarcomeres and sarcoplasmic reticulum dilatation, internal migration of nuclei, longitudinal fibre splitting, increment of subsarcolemmal mitochondria aggregates and increment of lipofuscin granules. Sciatic nerve shows myelin abnormalities like enfoldings, invaginations, onion bulbs, breakdowns and side axonal atrophy. Proteomic analysis identified changes correlated to morphological abnormalities in metabolic, contractile and cytoskeletal proteins, deregulation of iron homeostasis, change of Ca²⁺ balance and stress response proteins, accompanied by a deregulation of myelin membrane adhesion protein and proteins regulating the neuronal caliber. By comparing proteomic results from the two tissues, 16 protein isoforms showed the same up and down regulation trend suggesting that there are changes implying a general process which may act as a signal event of degeneration. Only β enolase and tropomyosin 1α were differentially expressed in the tissues.     

Thermodynamic and structural characterization of the macrochelates formed in the reactions of copper(II) and zinc(II) ions with peptides of histidine

Copper(II) complexes of the peptides Ac-HisSarHis-NH₂, Ac-HisSarHisSarHis-NH₂ and Ac-HisSarHisSarHisSarHis-NH₂ have been studied by potentiometric, UV-Vis, CD and EPR spectroscopic methods. Stability constants for the corresponding zinc(II) complexes have also been reported. The formation of M(II)-2N_im, M(II)-3N_im and M(II)-4N_im bonded macrochelates was suggested in the pH range 5-7. The macrochelates were, however, not stable enough to prevent metal ion hydrolysis in slightly alkaline solutions. In the case of copper(II) complexes, the metal ion promoted deprotonation and coordination of the amide groups of histidyl residues were also suggested. The stability constants of macrochelate complexes were compared to the literature data reported for the macrochelates of the other peptides of histidine. It was found that the thermodynamic stability of macrochelate species is largely influenced by the number and location of histidyl residues in the peptide backbone. The highest stability was obtained for the HXHYH-type sequences, while the distant arrangement of histidyl residues resulted in a significant reduction of the stability constants.     

Pleiotropic effects of spastin on neurite growth depending on expression levels

Hereditary spastic paraplegia (HSP) is characterized by weakness and spasticity of the lower limbs, owing to degeneration of corticospinal axons. The most common form is due to heterozygous mutations in the SPG4 gene, encoding spastin, a microtubule (MT)-severing protein. Here, we show that neurite growth in immortalized and primary neurons responds in pleiotropic ways to changes in spastin levels. Spastin depletion alters the development of primary hippocampal neurons leading to abnormal neuron morphology, dystrophic neurites, and axonal growth defects. By live imaging with End-Binding Protein 3-Fluorescent Green Protein (EB3-GFP), a MT plus-end tracking protein, we ascertained that the assembly rate of MTs is reduced when spastin is down-regulated. Spastin over-expression at high levels strongly suppresses neurite maintenance, while slight spastin up-regulation using an endogenous promoter enhances neurite branching and elongation. Spastin severing activity is exerted preferentially on stable acetylated and detyrosinated MTs. We further show that SPG4 nonsense or splice site mutations found in hereditary spastic paraplegia patients result in reduced spastin levels, supporting haploinsufficiency as the molecular cause of the disease. Our study reveals that SPG4 is a dosage-sensitive gene, and broadens the understanding of the role of spastin in neurite growth and MT dynamics.     

The morphology of gills of Haliotis tuberculata (Linnaeus, 1758)

Manganaro, M., Laurà, R., Guerrera, M.C., Lanteri, G., Zaccone, D. and Marino, F. 2011. The morphology of gills of Haliotis tuberculata (Linnaeus, 1758). —Acta Zoologica (Stockholm) 93 : 436–443. Although the morphology of abalone gills has been studied by some authors, up to date no data are available about the gills of Haliotis tuberculata. This study was carried out, by light and electron microscopy, on 10 wild adult H. tuberculata. Gills lamellae produce an undulated surface increasing the area in contact with water. At the level of skeletal rods, we observed a joint‐like structure that allows a checked movement. The left ctenidium is always decidedly larger than the right, probably because of the enormous size of the shell muscle. The cilia permit oxygenated water that leaves the afferent border and is thrust away at the tips of the lamellae by the extremely long cilia. Ciliary movement may take part in sweeping mucous secretions to capture extraneous particles and remove them from the gills. Three types of mucous cells are distributed along the epithelium of the afferent and efferent zones of the gill filament. They seem to play a role in the cleansing of gills in coordination with the muscle contraction and ciliary movement. The presence of microvilli on particular cells reflects their role associated with the absorption of substances from the environment. A haemolymphatic vessel is located in the central zone of the gill filament. The backbone of the haemolymphatic vessel is a chitino‐like structure, which gives support to the gills.     

Cowchock Syndrome Is Associated with a Mutation in Apoptosis-Inducing Factor

Cowchock syndrome (CMTX4) is a slowly progressive X-linked recessive disorder with axonal neuropathy, deafness, and cognitive impairment. The disease locus was previously mapped to an 11 cM region at chromosome X: q24-q26. Exome sequencing of an affected individual from the originally described family identified a missense change c.1478A>T (p.Glu493Val) in AIFM1, the gene encoding apoptosis-inducing factor (AIF) mitochondrion-associated 1. The change is at a highly conserved residue and cosegregated with the phenotype in the family. AIF is an FAD-dependent NADH oxidase that is imported into mitochondria. With apoptotic insults, a N-terminal transmembrane linker is cleaved off, producing a soluble fragment that is released into the cytosol and then transported into the nucleus, where it triggers caspase-independent apoptosis. Another AIFM1 mutation that predicts p.Arg201del has recently been associated with severe mitochondrial encephalomyopathy in two infants by impairing oxidative phosphorylation. The c.1478A>T (p.Glu493Val) mutation found in the family reported here alters the redox properties of the AIF protein and results in increased cell death via apoptosis, without affecting the activity of the respiratory chain complexes. Our findings expand the spectrum of AIF-related disease and provide insight into the effects of AIFM1 mutations.     

l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria

As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M.