Ancient mitochondrial DNA and morphology elucidate an extinct island radiation of Indian Ocean giant tortoise (Cylindrapsis)

Ancient mitochondrial DNA sequences were used for investigating the evolution of an entire clade of extinct vertebrates, the endemic tortoises (Cylindraspis) of the Mascarene Islands in the Indian Ocean. Mitochondrial DNA corroborates morphological evidence that there were five species of tortoise with the following relationships: Cylindraspis triserrata ((Cylindraspis vosmaeri and Cylindraspis peltastes) (Cylindraspis inepta and Cylindraspis indica)). Phylogeny indicates that the ancestor of the group first colonized Mauritius where speciation produced C. triserrata and the ancestor of the other species including a second sympatric Mauritian form, C. inepta. A propagule derived from this lineage colonized Rodrigues 590 km to the east, where a second within-island speciation took place producing the sympatric C. vosmaeri and C. peltastes. A recent colonization of Réunion 150 km to the southwest produced C. indica. In the virtual absence of predators, the defensive features of the shells of Mascarene tortoises were largely dismantled, apparently in two stages. 'Saddlebacked' shells with high fronts evolved independently on all three islands. This and other features, such as a derived jaw structure and small body size, may be associated with niche differentiation in sympatric species and may represent a striking example of parallel differentiation in a large terrestrial vertebrate. The history of Mascarene tortoises contrasts with that of the Galápagos, where only a single species is present and surviving populations are genetically much more similar. However, they too show some reduction in anti-predator mechanisms and multiple development of populations with saddlebacked shells.

Characterization of mouse profilaggrin: Evidence for nuclear engulfment and translocation of the profilaggrin B-domain during epidermal differentiation

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N-terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal anti-body raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

Autosomal dominant hypocalcemia: A novel activating mutation (E604K) in the cysteine-rich domain of the calcium-sensing receptor

We report a novel activating mutation (E604K) of the calcium-sensing receptor in a family with autosomal dominant hypocalcemia. Whereas all affected individuals exhibited marked hypocalcemia, some cases with untreated hypocalcemia exhibited seizures in infancy, whereas others were largely asymptomatic from birth into adulthood. The missense mutation E604K (G2182A, GenBank accession no. U20759), which affects an amino acid residue in the C terminus of the cysteine-rich domain of the extracellular head, co-segregated with hypocalcemia in all seven individuals for whom DNA was available. Two unaffected, normocalcemic members of the family did not exhibit the mutation. The molecular impact of the mutation on two key components of the signaling response was assessed in HEK-293 cells transiently transfected with cDNA corresponding to either the wild-type calcium-sensing receptor or the E604K mutation derived by site-directed mutagenesis. There was a significant leftward shift in the concentration response curves for the effects of extracellular Ca2+ on both intracellular Ca2+ mobilization (determined by aequorin luminescence) and MAPK activity (determined by luciferase expression). The C terminus of the cysteine-rich domain of the extracellular head may normally act to suppress receptor activity in the presence of low extracellular Ca2+ concentrations.

Seroprevalence of hepatitis B virus infection in individuals with clinical evidence of hepatitis in Goiânia, Goiás: detection of viral DNA and determination of subtypes

The presence of serological markers for hepatitis B virus (HBsAg, anti-HBc IgM and Anti-HBc total) was investigated in the serum of 1,396 individuals who had clinical suspect of hepatitis. It was observed that 50.7% of the individuals were positive and, from the total of the studied individuals, 14.5% were positive for HBsAg. From these, 8.5% were also positive for anti-HBc IgM. The analysis in relation to gender showed a higher seroprevalence index among male individuals (p < 0.0001). It was observed the occurrence of subtypes adw2 (62.7%), ayw3 (23.5%), ayw2 (9.8%) and adw4 (3.9%). The viral DNA was detected in 61 (33.9%) HBsAg positive samples and in one sample positive only for anti-HBc total. These results indicate an important incidence of the HBV infection in this population, and reinforce previous studies regarding this virus in the central west region of Brazil.

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

Mediterranean populations of the lesser white-toothed shrew (Crocidura suaveolens group): an unexpected puzzle of Pleistocene survivors and prehistoric introductions.

An earlier study revealed the strong phylogeographical structure of the lesser white-toothed shrew (Crocidura suaveolens group) within the northern Palaearctic. Here, we aim to reconstruct the colonization history of Mediterranean islands and to clarify the biogeography and phylogeographical relationships of the poorly documented Middle East region with the northern Palaearctic. We performed analyses on 998-bp-long haplotypes of the mitochondrial cytochrome b gene of 143 samples collected around the Mediterranean basin, including islands and the Middle East. The analyses suggest that the Cypriot shrew belongs to the rare group of relict insular Pleistocene mammal taxa that have survived to the present day. In contrast, the Cretan, Corsican and Menorcan populations were independently introduced from the Middle East during the Holocene. The phylogeographical structure of this temperate Palaearctic species within the Middle East appears to be complex and rich in diversity, probably reflecting fragmentation of the area by numerous mountain chains. Four deeply divergent clades of the C. suaveolens group occur in the area, meaning that a hypothetical contact zone remains to be located in central western Iran.

Mitochondrial DNA sequence variation within the remnant populations of the endangered numbat (Marsupialia: Myrmecobiidae: Myrmecobius fasciatus).

The numbat has been reduced to two populations in Western Australia. To better understand the effects of range reduction on gene flow and genetic variation, and to address questions crucial for the species' management, we analysed mitochondrial DNA (mtDNA) sequences of free-ranging individuals and museum specimens. The results suggest recent connectivity between the remnant populations, although one of those may have lost significant amounts of genetic diversity during the recent population size reduction. We propose that for management purposes the remnant populations should be treated as a single historical lineage and that, subject to certain caveats, consideration should be given to population augmentation by translocation.

Population structure of the malaria vector Anopheles darlingi in Rondônia, Brazilian Amazon, based on mitochondrial DNA

Anopheles darlingi is the most important Brazilian malaria vector, with a widespread distribution in the Amazon forest. Effective strategies for vector control could be better developed through knowledge of its genetic structure and gene flow among populations, to assess the vector diversity and competence in transmitting Plasmodium. The aim of this study was to assess the genetic diversity of An. darlingi collected at four locations in Porto Velho, by sequencing a fragment of the ND4 mitochondrial gene. From 218 individual mosquitoes, we obtained 20 different haplotypes with a diversity index of 0.756, equivalent to that found in other neotropical anophelines. The analysis did not demonstrate significant population structure. However, haplotype diversity within some populations seems to be over-represented, suggesting the presence of sub-populations, but the presence of highly represented haplotypes complicates this analysis. There was no clear correlation among genetic and geographical distance and there were differences in relation to seasonality, which is important for malarial epidemiology.

Assessing protein stability of the dimeric DNA-binding domain of E2 human papillomavirus 18 with molecular dynamics

The objective of this study is to understand the structural flexibility and curvature of the E2 protein of human papillomavirus type 18 using molecular dynamics (6 ns). E2 is required for viral DNA replication and its disruption could be an anti-viral strategy. E2 is a dimer, with each monomer folding into a stable open-faced β-sandwich. We calculated the mobility of the E2 dimer and found that it was asymmetric. These different mobilities of E2 monomers suggest that drugs or vaccines could be targeted to the interface between the two monomers.

DNA multigene sequencing of topotypic specimens of the fascioliasis vector Lymnaea diaphana and phylogenetic analysis of the genus Pectinidens (Gastropoda)

Freshwater lymnaeid snails are crucial in defining transmission and epidemiology of fascioliasis. In South America, human endemic areas are related to high altitudes in Andean regions. The species Lymnaea diaphana has, however, been involved in low altitude areas of Chile, Argentina and Peru where human infection also occurs. Complete nuclear ribosomal DNA 18S, internal transcribed spacer (ITS)-2 and ITS-1 and fragments of mitochondrial DNA 16S and cytochrome c oxidase (cox)1 genes of L. diaphana specimens from its type locality offered 1,848, 495, 520, 424 and 672 bp long sequences. Comparisons with New and Old World Galba/Fossaria, Palaearctic stagnicolines, Nearctic stagnicolines, Old World Radix and Pseudosuccinea allowed to conclude that (i) L. diaphana shows sequences very different from all other lymnaeids, (ii) each marker allows its differentiation, except cox1 amino acid sequence, and (iii) L. diaphana is not a fossarine lymnaeid, but rather an archaic relict form derived from the oldest North American stagnicoline ancestors. Phylogeny and large genetic distances support the genus Pectinidens as the first stagnicoline representative in the southern hemisphere, including colonization of extreme world regions, as most southern Patagonia, long time ago. The phylogenetic link of L. diaphana with the stagnicoline group may give light to the aforementioned peculiar low altitude epidemiological scenario of fascioliasis.

The JNK binding domain of islet-brain 1 inhibits IL-1 induced JNK activity and apoptosis but not the transcription of key proapoptotic or protective genes in insulin-secreting cell lines.

The stress-activated protein kinase c-Jun NH2-terminal kinase (JNK) is a central signal for interleukin-1beta (IL-1beta)-induced apoptosis in insulin-producing beta-cells. The cell-permeable peptide inhibitor of JNK (JNKI1), that introduces the JNK binding domain (JBD) of the scaffold protein islet-brain 1 (IB1) inside cells, effectively prevents beta-cell death caused by this cytokine. To define the molecular targets of JNK involved in cytokine-induced beta-cell apoptosis we investigated whether JNKI1 or stable expression of JBD affected the expression of selected pro- and anti-apoptotic genes induced in rat (RIN-5AH-T2B) and mouse (betaTC3) insulinoma cells exposed to IL-1beta. Inhibition of JNK significantly reduced phosphorylation of the specific JNK substrate c-Jun (p&lt;0.05), IL-1beta-induced apoptosis (p&lt;0.001), and IL-1beta-mediated c-fos gene expression. However, neither JNKI1 nor JBD did influence IL-1beta-induced NO synthesis or iNOS expression or the transcription of the genes encoding mitochondrial manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase rho (GSTrho), heat shock protein (HSP) 70, IL-1beta-converting enzyme (ICE), caspase-3, apoptosis-inducing factor (AIF), Bcl-2 or Bcl-xL. We suggest that the anti-apoptotic effect of JNK inhibition by JBD is independent of the transcription of major pro- and anti-apoptotic genes, but may be exerted at the translational or posttranslational level.

Molecular phylogenetics of shrews (Mammalia: Soricidae) reveal timing of transcontinental colonizations.

We sequenced 2167 base pairs (bp) of mitochondrial DNA cytochrome b and 16S, and 1390 bp of nuclear genes BRCA1 and ApoB in shrews taxa (Eulipotyphla, family Soricidae). The aim was to study the relationships at higher taxonomic levels within this family, and in particular the position of difficult clades such as Anourosorex and Myosorex. The data confirmed two monophyletic subfamilies, Soricinae and Crocidurinae. In the former, the tribes Anourosoricini, Blarinini, Nectogalini, Notiosoricini, and Soricini were supported. The latter was formed by the tribes Myosoricini and Crocidurini. The genus Suncus appeared to be paraphyletic and included Sylvisorex. We further suggest a biogeographical hypothesis, which shows that North America was colonized by three independent lineages of Soricinae during middle Miocene. Our hypothesis is congruent with the first fossil records for these taxa. Using molecular dating, the first exchanges between Africa and Eurasia occurred during the middle Miocene. The last one took place in the Late Miocene, with the dispersion of the genus Crocidura through the old world.

Forensic identification of urine samples: a comparison between nuclear and mitochondrial DNA markers.

Urine samples from 20 male volunteers of European Caucasian origin were stored at 4 degrees C over a 4-month period in order to compare the identification potential of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) markers. The amount of nDNA recovered from urines dramatically declined over time. Consequently, nDNA likelihood ratios (LRs) greater than 1,000 were obtained for 100, 70 and 55% of the urines analysed after 6, 60 and 120 days, respectively. For the mtDNA, HVI and HVII sequences were obtained for all samples tested, whatever the period considered. Nevertheless, the highest mtDNA LR of 435 was relatively low compared to its nDNA equivalent. Indeed, LRs obtained with only three nDNA loci could easily exceed this value and are quite easier to obtain. Overall, the joint use of nDNA and mtDNA markers enabled the 20 urine samples to be identified, even after the 4-month period.