Mitochondrial dysfunction in Huntington's disease: the bioenergetics of isolated and in situ mitochondria from transgenic mice

Mitochondrial dysfunction is believed to participate in Huntington's disease (HD) pathogenesis. Here we compare the bioenergetic behavior of forebrain mitochondria isolated from different transgenic HD mice (R6/2, YAC128 and Hdh150 knock-in) and wild-type littermates with the first determination of in situ respiratory parameters in intact HD striatal neurons. We assess the Ca2+-loading capacity of isolated mitochondria by steady Ca2+-infusion. Mitochondria from R6/2 mice (12-13 weeks) and 12 months YAC128, but not homozygous or heterozygous Hdh150 knock-in mice (15-17 weeks), exhibit increased Ca2+-loading capacity when compared with respective wild-type littermates. In situ mitochondria in intact striatal neurons show high respiratory control. Moreover, moderate expression of full-length mutant huntingtin (in Hdh150 knock-in heterozygotes) does not significantly impair mitochondrial respiration in unstimulated neurons. However, when challenged with energy-demanding stimuli (NMDA-receptor activation in pyruvate-based media to accentuate the mitochondria role in Ca2+-handling), Hdh150 neurons are more vulnerable to Ca2+-deregulation than neurons from their wild-type littermates. These results stress the importance of assessing HD mitochondrial function in the cellular context.     

Sexual selection and the evolution of sperm morphology in sharks

Post‐copulatory sexual selection, and sperm competition in particular, is a powerful selective force shaping the evolution of sperm morphology. Although mounting evidence suggests that post‐copulatory sexual selection influences the evolution of sperm morphology among species, recent evidence also suggests that sperm competition influences variation in sperm morphology at the intraspecific level. However, contradictory empirical results and limited taxonomic scope have led to difficulty in assessing the generality of sperm morphological responses to variation in the strength of sperm competition. Here, we use phylogenetically controlled analyses to explore the effects of sperm competition on sperm morphology and variance in sharks, a basal vertebrate group characterized by wide variation in rates of multiple mating by females, and consequently sperm competition risk. Our analyses reveal that shark species experiencing greater levels of sperm competition produce sperm with longer flagella and that sperm flagellum length is less variable in species under higher sperm competition risk. In contrast, neither the length of the sperm head and midpiece nor variation in sperm head and midpiece length was associated with sperm competition risk. Our findings demonstrate that selection influences both the inter‐ and intraspecific variation in sperm morphology and suggest that the flagellum is an important target of sexual selection in sharks. These findings provide important insight into patterns of selection on the ejaculate in a basal vertebrate lineage.     

Carriers of the Complex Allele HFE c.[187C>G;340+4T>C] Have Increased Risk of Iron Overload in S?o Miguel Island Population (Azores,Portugal)

Iron overload is associated with acquired and genetic conditions, the most common being hereditary hemochromatosis (HH) type-I, caused by HFE mutations. Here, we conducted a hospital-based case-control study of 41 patients from the São Miguel Island (Azores, Portugal), six belonging to a family with HH type-I pseudodominant inheritance, and 35 unrelated individuals fulfilling the biochemical criteria of iron overload compatible with HH type-I. For this purpose, we analyzed the most common HFE mutations– c.845G>A [p.Cys282Tyr], c.187C>G [p.His63Asp], and c.193A>T [p.Ser65Cys]. Results revealed that the family’s HH pseudodominant pattern is due to consanguineous marriage of HFE-c.845G>A carriers, and to marriage with a genetically unrelated spouse that is a -c.187G carrier. Regarding unrelated patients, six were homozygous for c.845A, and three were c.845A/c.187G compound heterozygous. We then performed sequencing of HFE exons 2, 4, 5 and their intron-flanking regions. No other mutations were observed, but we identified the -c.340+4C [IVS2+4C] splice variant in 26 (74.3%) patients. Functionally, the c.340+4C may generate alternative splicing by HFE exon 2 skipping and consequently, a protein missing the α1-domain essential for HFE/ transferrin receptor-1 interactions. Finally, we investigated HFE mutations configuration with iron overload by determining haplotypes and genotypic profiles. Results evidenced that carriers of HFE-c.187G allele also carry -c.340+4C, suggesting in-cis configuration. This data is corroborated by the association analysis where carriers of the complex allele HFE-c.[187C>G;340+4T>C] have an increased iron overload risk (RR = 2.08, 95% CI = 1.40−2.94, p<0.001). Therefore, homozygous for this complex allele are at risk of having iron overload because they will produce two altered proteins—the p.63Asp [c.187G], and the protein lacking 88 amino acids encoded by exon 2. In summary, we provide evidence that the complex allele HFE-c.[187C>G;340+4T>C] has a role, as genetic predisposition factor, on iron overload in the São Miguel population. Independent replication studies in other populations are needed to confirm this association.     

Trolox protection of myelin membrane in hydrogen peroxide-treated mature oligodendrocytes

Oligodendrocytes have the highest rate of metabolic activity in the brain and are highly vulnerable to oxidative stress. In this work we determined the protective effect of Trolox, a water-soluble analogue of vitamin E, and insulin, a peptide shown to be neuroprotective, in oligodendrocyte lesion induced by hydrogen peroxide (H₂O₂). Exposure of primary cultures of rat oligodendrocytes to H₂O₂ dose-dependently decreased their reducing capacity, as determined by the MTT assay. H₂O₂ (100 μM) had no effect on Bax levels, active-caspase-3, DNA fragmentation or lactate dehydrogenase (LDH) leakage. Nevertheless, under these conditions, H₂O₂ decreased the levels of myelin basic protein (MBP), used as a marker for oligodendrocyte myelin membrane. Treatment with insulin alone increased MBP levels, but no changes were observed in the presence of insulin plus H₂O₂. In contrast, incubation with Trolox completely prevented H₂O₂-induced decrease in MBP expression, suggesting that vitamin E analogues may prevent against oligodendrocyte oxidative damage.     

Adhesion of façade coating colonisers, as mediated by physico-chemical properties

The adhesion of Klebsormidium flaccidum, Stichococcus bacillaris and Chlorella cf. mirabilis, three strains of green microalgae isolated from biofilms on fa?ade coatings were investigated in a parallel plate flow chamber. The model surfaces tested were glass slides, and -CH(3) (mediated by octadecyltrichlorosilane [OTS] and hexamethyldisilazane [HMDZ] modification) and -NH(2) (aminopropyltriethoxysilane [APS] modification) terminated self-assembled monolayers. Algal physicochemical properties were evaluated by the microbial adhesion to solvents (MATS) assay and by contact angle measurements. The model surfaces were characterised by X-ray photoelectron spectroscopy analysis and by contact angle measurements. Predicted adhesion trends were then compared to in vitro measurements. The adhesion strength of the three algal strains followed the trend: APS > OTS > HMDZ > glass. The adhesion process thus seemed to be mediated by hydrophobic and electrostatic interactions, and was shown to be influenced by the algal culture age and the initial contact time.     

Phlebotomine (Diptera: Psychodidae) and leishmaniasis in Rio Grande do Norte State Brazil: anthropic environment responses

Natural environmental changes or those resulting from anthropic factors and their impact on infectious diseases have been evaluated in several studies. The objective of this work was to analyze the correlation between the anthropic environment, phlebotomine and leishmaniases in Rio Grande do Norte State, in Northeast Brazil. Information relative to the distribution of vector species in visceral and tegumentary leishmaniasis areas was associated to the record of cases notified by Public Health organs. The analysis suggests associations between the vector species and distribution of the disease with demographic and physionomic characteristics, disorderly growth in the metropolitan area, living conditions and environmental degradation of the Eastern Littoral, the principal area of notified visceral leishmaniasis cases.     

Spatiotemporal expression and functional implication of CXCL14 in the developing mice cerebellum

Cerebellar granule neurons migrate from the external granule cell layer (EGL) to the internal granule cell layer (IGL) during postnatal morphogenesis. This migration process through 4 different layers is a complex mechanism which is highly regulated by many secreted proteins. Although chemokines are well-known peptides that trigger cell migration, but with the exception of CXCL12, which is responsible for prenatal EGL formation, their functions have not been thoroughly studied in granule cell migration. In the present study, we examined cerebellar CXCL14 expression in neonatal and adult mice. CXCL14 mRNA was expressed at high levels in adult mouse cerebellum, but the protein was not detected. Nevertheless, Western blotting analysis revealed transient expression of CXCL14 in the cerebellum in early postnatal days (P1, P8), prior to the completion of granule cell migration. Looking at the distribution of CXCL14 by immunohistochemistry revealed a strong immune reactivity at the level of the Purkinje cell layer and molecular layer which was absent in the adult cerebellum. In functional assays, CXCL14 stimulated transwell migration of cultured granule cells and enhanced the spreading rate of neurons from EGL microexplants. Taken together, these results revealed the transient expression of CXCL14 by Purkinje cells in the developing cerebellum and demonstrate the ability of the chemokine to stimulate granule cell migration, suggesting that it must be involved in the postnatal maturation of the cerebellum.     

Expression of the alpha 1-proteinase inhibitor gene family during evolution of the genus Mus

alpha 1-Proteinase inhibitors (alpha 1-PIs) are members of the serpin superfamily of proteinase inhibitors, and are important in the maintenance of homeostasis in a wide variety of animal taxa. Previous studies have shown that in mice (genus Mus), evolution of alpha 1-PIs is characterized by gene amplification, region-specific concerted evolution, and rapid accumulation of amino acid substitutions. The latter occurs primarily in the reactive center, which is the region of the alpha 1-PI molecule that determines the inhibitor's specificity for target proteinases. The P1 residue within the reactive center, which is methionine in so-called orthodox alpha 1-PIs and an amino acid other than methionine in unorthodox alpha 1-PIs, is a primary determinant of inhibitor specificity. In the present study, we find that the expression of mRNAs encoding unorthodox alpha 1-PIs is polymorphic within Mus species, i.e., among individuals or inbred strains. This is in striking contrast to mRNAs that encode orthodox alpha 1-PIs, whose concentrations are relatively invariant. The intraspecies variations in mRNA expression represent polymorphisms in the structure of the alpha 1- PI gene family. The results, taken together with previously described aspects of alpha 1-PI evolution, indicate that the dissimilar levels of polymorphism exhibited by orthodox and unorthodox alpha 1-PIs, which likely have distinct physiological functions, may reflect different levels of selective constraint. The significance of this finding to the evolution of gene families is discussed.      

Affinity purification of novel bispecific antibodies recognising carcinoembryonic antigen and doxorubicin

We have developed a method which combines Protein A affinity chromatography and HPLC analytical and semi-preparative hydroxyapatite affinity chromatography to purify bispecific antibodies (BsMabs) from hybrid-hybridomas secreting antibodies recognising carcinoembryonic antigen (CEA) and the chemotherapeutic drug doxorubicin (Dox). Elution of the HPLC hydroxyapatite columns with a 60–360 mM phosphate buffer gradient was found to give better separation than elution with a 60–180 mM phosphate buffer gradient. Careful monitoring of HPLC fractions by enzyme linked immunosorbent assays for anti-CEA, anti-Dox and dual anti-CEA/anti-Dox activity, and pooling of fractions on the basis of these results, enabled the purification of novel BsMabs for use in in vitro and preclinical in vivo experiments.     

The involvement of the nif-associated ferredoxin-like genes fdxA and fdxN of Herbaspirillum seropedicae in nitrogen fixation

The pathway of electron transport to nitrogenase in the endophytic β-Proteobacterium Herbaspirillum seropedicae has not been characterized. We have generated mutants in two nif-associated genes encoding putative ferredoxins, fdxA and fdxN. The fdxA gene is part of the operon nifHDKENXorf1orf2fdxAnifQmodABC and is transcribed from the nifH promoter, as revealed by lacZ gene fusion. The fdxN gene is probably cotranscribed with the nifB gene. Mutational analysis suggests that the FdxA protein is essential for maximum nitrogenase activity, since the nitrogenase activity of the fdxA mutant strain was reduced to about 30% of that of the wild-type strain. In addition, the fdxA mutation had no effect on the nitrogenase switch-off in response to ammonium. Nitrogenase activity of a mutant strain lacking the fdxN gene was completely abolished. This phenotype was reverted by complementation with fdxN expressed under lacZ promoter control. The results suggest that the products of both the fdxA and fdxN genes are probably involved in electron transfer during nitrogen fixation.