Respiratory Complex I in Brain Development and Genetic Disease

A study is presented on the expression and activity of complex I, as well as of other complexes of the respiratory chain, in the course of brain development and inherited encephalopathies. Investigations on mouse hippocampal cells show that differentiation of these cells both in vivo and in cell cultures is associated with the expression of a functional complex I, whose activity markedly increases with respect to that of complexes III and IV. Data are presented on genetic defects of complex I in six children with inborn encephalopathy associated with isolated deficiency of the complex. Mutations have been identified in nuclear and mitochondrial genes coding for subunits of the complex. Different mutations were found in the nuclear NDUFS4 gene coding for the 18 kD (IP, AQDQ) subunit of complex I. All the NDUFS4 mutations resulted in impairment of the assembly of a functional complex. The observations presented provide evidence showing a critical role of complex I in differentiation and functional activity of brain cells.     

Yessotoxin, a shellfish biotoxin, is a potent inducer of the permeability transition in isolated mitochondria and intact cells

The diarrhetic poisoning by bivalve molluscs, diarrhetic shellfish poisoning, is due to consumption of mussels containing biotoxins produced by some Dinoflagellate species. Toxic effects of yessotoxin (YTX) include morphological alterations of mitochondria from heart and liver but the biochemical basis for these alterations is completely unknown. This paper demonstrates that YTX is a very powerful compound that opens the permeability transition pore (PTP) of the inner mitochondrial membrane of rat liver mitochondria at nanomolar concentrations. The effect requires the presence of a permissive level of calcium, by itself incapable of opening the pore. The direct effect of YTX on PTP is further confirmed by the inhibition exerted by cyclosporin A (CsA) that is known as a powerful inhibitor of PTP opening. Moreover, YTX induces membrane depolarization as shown by the quenching of tetramethylrhodamine methyl ester (TMRM), also prevented by the addition of CsA. YTX caused PTP opening in Morris Hepatoma 1C1 cells, as shown by the occurrence of CsA-sensitive depolarization within minutes of the addition of submicromolar concentrations of the toxin. These results provide a biochemical basis for the mitochondrial alterations observed in the course of intoxication with YTX, offering the first clue into the pathogenesis of diseases caused by YTX, and providing a novel tool to study the PTP in situ.     

High frequency of TNF alleles -238A and -376A in individuals from northern Sardinia

The G to A single nucleotide polymorphisms (SNPs), at position -376, -308 and -238 in the promoter of the tumor necrosis factor alpha (TNF) gene, have been independently correlated with numerous diseases. Alleles TNF(-376A) and TNF(-238A) are normally found throughout the world with very low frequencies. We investigated the frequency of these SNPs in Sicilian subjects hospitalized after traumatic brain injury and in three groups of subjects from northern Sardinia: healthy subjects and individuals with multiple sclerosis or ischemic stroke. While no significant difference was found between healthy and disease subjects, the frequency of TNF(-376A) and TNF(-238A) was elevated up to 10 times in Sardinia compared to Sicily and other populations throughout the world. These elevated frequencies may be the result of genetic drift or of selective pressure on TNF itself or on neighboring genes, including the HLA. Malaria, endemic to Sardinia until the end of the 1940s, and the bubonic plague, are among the possible causes of selection. These findings indicate that Sardinia is an ideal location to further elucidate the correlation between TNF or HLA polymorphisms and diseases, including multiple sclerosis and type-I diabetes, present with an unusually high frequency and co-morbidity in Sardinia.     

Evidence of aromatase localization in cytoplasmic droplet of human immature ejaculated spermatozoa

Lytochrome P450 aromatase is a microsomal enzyme catalyzing the conversion of androgens to estrogens. P450arom expression has been demonstrated in testicular and epididymal sperm cells of several species but very limited data have been reported about maturating human germ cells. In this study, human spermatozoa with cytoplasmic droplet anomaly have been utilized to investigate aromatase immunolocalization in the immature germ cells of human ejaculate. Immunodetection has utilized a polyclonal antiserum as primary antibody, a biotinylated IgG as secondary antibody and then the avidin-biotin-peroxidase complex amplification followed by the diaminobenzidine staining. A strong immunoreaction was observed in the cytoplasmic droplets retained around the midpiece of immature spermatozoa and also in the descending droplets of late maturing sperm, while the other cellular components were unstained. Therefore, this investigation has demonstrated, for the first time, aromatase immunolocalization in residual cytoplasm of human ejaculated sperm, suggesting cytoplasmic droplets as possible estrogen biosynthesis sites during human sperm differentiation.     

Signal amplification by combining two advanced immunohistochemical techniques

The immunohistochemical techniques known as EnVision trade mark + System (EVS) and Mirror Image Complementary Antibody (MICA) were recently introduced into laboratory practice because of their high sensitivity. In this paper these techniques were compared and their sequences combined to obtain a new method possibly more sensitive than the original ones. The immunohistochemical staining employing the avidin-biotin complex (ABC), largely used as routine, was adopted as a term of comparison. Samples from the small and large intestine of pigs and sheep were fixed in Bouin and embedded in Paraplast. The primary antibodies utilized were directed against the neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP) and chromogranin A (Cr A). Targets of these antibodies were nerve structures of the intestinal wall, as well as endocrine cells scattered in the mucosa of the bowel, defined neuroendocrine cells or paraneurons. The EVS method appeared as slightly superior to the MICA method regarding sensitivity of detection. The EVS/MICA (combined) method resulted four/eight times more effective than the original techniques regarding sensitivity of detection and staining intensity, both at low and high dilutions of the primary antibodies. Of these latter, immunopositive structures were still clearly identifiable, at a dilution of 1:256,000. Such efficiency could be explained by the high number of revealing molecules of peroxidase contained in the new sequence. The application of the combined method is recommended when a small quantity of tissue antigens needs to be detected immunohistochemically.     

Preparation and characterization of geodin. A betagamma-crystallin-type protein from a sponge

Geodin is a protein encoded by a sponge gene homologous to genes from the betagamma-crystallins superfamily. The interest for this crystallin-type protein stems from the phylogenesis of porifera, commonly called sponges, the earliest divergence event in the history of metazoans. Here we report the preparation of geodin as a recombinant protein from Escherichia coli, its characterization through physico-chemical analyses, and a model of its 3D structure based on homology modelling. Geodin is a monomeric protein of about 18 kDa, with an all-beta structure, as all other crystallins in the superfamily, but more prone to unfold in the presence of chemical denaturants, when compared with other homologues from the superfamily. Its thermal unfolding, studied by far- and near-CD, and by calorimetry, is described by a two-state model. Geodin appears to be structurally similar in many respects to the bacterial protein S crystallin, with which it also shares a significant, albeit more modest stabilizing effect exerted by calcium ions. These results suggest that the crystallin-type structural scaffold, employed in the evolution of bacteria and moulds, was successfully recruited very early in the evolution of metazoa.     

Poly(hydroxyalkanoate) synthase genotype and PHA production of Pseudomonas corrugata and P. mediterranea

A collection of Pseudomonas corrugata and P. mediterranea strains, two closely related species, was evaluated for the presence and variability of pha loci. Using PCR methods that specifically amplify segments of medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) synthase genes, we demonstrated the presence of phaC1 and phaC2 in all P. mediterranea strains tested and in six out of 56 strains of P. corrugata screened. The remaining 50 strains of P. corrugata yielded only the phaC2 subgene fragment on detection by a combined PCR-restriction endonuclease analysis method or a semi-nested PCR-amplification approach. A Southern hybridization study on a representative strain from this group, however, indicated the presence of the phaC1 gene. Nucleic acid sequences of the subgene phaC fragments of the representative strains from the three groups showed an overall similarity ranging from 95% to 100%. The major repeat-unit monomers of the mcl-PHAs isolated from these selected strains are -hydroxyoctanoate (33–47 mol%) and -hydroxydecanoate (26–36 mol%). These results differentiate for the first time the strains of P. corrugata into two pha-distinguishable groups. This study also documents for the first time the production of mcl-PHA in P. mediterranea.     

Antioxidants induce different phenotypes by a distinct modulation of signal transduction

Antioxidants are known to exert a preventive activity against degenerative diseases. Here, we investigated the mechanism of action of three antioxidants: resveratrol, which causes differentiation of HL-60 cells, and hydroxytyrosol and pyrrolidine dithiocarbamate which, in the same model system, activate apoptosis. The expression profile of hydroxytyrosol-treated cells showed the up-regulation of several genes, including c-jun and egr1. Pyrrolidine dithiocarbamate activates both genes, while resveratrol increases uniquely egr1. A selective modulation of signalling pathway explained this finding. All antioxidants up-regulate Erk1/2, while only hydroxytyrosol and pyrrolidine dithiocarbamate activate c-Jun N-terminal kinase (JNK). Since JNK induces apoptosis by Bcl-2 phosphorylation, we investigated this event. Bcl-2 phosphorylation was increased by hydroxytyrosol and pyrrolidine dithiocarbamate and not by resveratrol. Our results indicate that the different phenotypical effects of antioxidants correlate with modulation of selective transduction pathways.