The mitochondrial genome sequence of the Tasmanian tiger (Thylacinus cynocephalus)

We report the first two complete mitochondrial genome sequences of the thylacine (Thylacinus cynocephalus), or so-called Tasmanian tiger, extinct since 1936. The thylacine's phylogenetic position within australidelphian marsupials has long been debated, and here we provide strong support for the thylacine's basal position in Dasyuromorphia, aided by mitochondrial genome sequence that we generated from the extant numbat (Myrmecobius fasciatus). Surprisingly, both of our thylacine sequences differ by 11%–15% from putative thylacine mitochondrial genes in GenBank, with one of our samples originating from a direct offspring of the previously sequenced individual. Our data sample each mitochondrial nucleotide an average of 50 times, thereby providing the first high-fidelity reference sequence for thylacine population genetics. Our two sequences differ in only five nucleotides out of 15,452, hinting at a very low genetic diversity shortly before extinction. Despite the samples’ heavy contamination with bacterial and human DNA and their temperate storage history, we estimate that as much as one-third of the total DNA in each sample is from the thylacine. The microbial content of the two thylacine samples was subjected to metagenomic analysis, and showed striking differences between a wild-captured individual and a born-in-captivity one. This study therefore adds to the growing evidence that extensive sequencing of museum collections is both feasible and desirable, and can yield complete genomes.     

The progression of the inflammation in established collagen-induced arthritis can be altered by treatments with immunological or pharmacological agents which inhibit T cell activities

Alterations in the progression of the inflammatory disease in mice with established collagen-induced arthritis (CIA) by in vivo treatments with either anti-T cell antibodies or an immunosuppressive drug, whose main targets of action are T cells, were studied. It was demonstrated that the in vivo administration of either monoclonal anti-L3T4, anti-Ly-2 or the combination of anti-L3T4 and anti-Ly-2 failed to modify the inflammatory disease after the arthritis had been initiated. Nevertheless, the progression of disease was decreased by treatments with rabbit anti-mouse lymphocyte sera (ALS) in mice with established CIA. While there was a substantial reduction in the proliferative responses to the T cell mitogen concanavalin A (Con A) in these ALS-treated mice, proliferation to a B cell mitogen, lipopolysaccharide, was unaffected. Furthermore, treatments with anti-Thy-1.2 monoclonal antibody (mAb) by itself did not alter the progression of the ongoing inflammatory disease, but combined treatments with both anti-Thy-1.2 and anti-L3T4 mAb prevented the further advancement of the arthritic disease. Although mice receiving either anti-Thy-1.2 alone or both anti-Thy-1.2 and anti-L3T4 exhibited complete absence of Thy-1+ cells within their inguinal lymph nodes, the proliferative responses to Con A by LN cells from mice treated with the combination of anti-Thy-1.2 and anti-L3T4 were drastically reduced compared to the responses of either the anti-Thy-1.2 or anti-L3T4-treated groups. Finally, daily treatments with cyclosporin A, an immunosuppressive drug which preferentially acts on T cells, were also effective in modifying the clinical course of the arthritic disease in mice with established CIA. These results suggest that immunocompetent cells which express the Thy-1 surface marker, most likely Thy-1+ T cells, may play a role in maintaining and perpetuating the inflammatory disease of established CIA.     

The complete sequence of the zebrafish (Danio rerio) mitochondrial genome and evolutionary patterns in vertebrate mitochondrial DNA.

We describe the complete sequence of the 16,596-nucleotide mitochondrial genome of the zebrafish (Danio rerio); contained are 13 protein genes, 22 tRNAs, 2 rRNAs, and a noncoding control region. Codon usage in protein genes is generally biased toward the available tRNA species but also reflects strand-specific nucleotide frequencies. For 19 of the 20 amino acids, the most frequently used codon ends in either A or C, with A preferred over C for fourfold degenerate codons (the lone exception was AUG: methionine). We show that rates of sequence evolution vary nearly as much within vertebrate classes as between them, yet nucleotide and amino acid composition show directional evolutionary trends, including marked differences between mammals and all other taxa. Birds showed similar compositional characteristics to the other nonmammalian taxa, indicating that the evolutionary trend in mammals is not solely due to metabolic rate and thermoregulatory factors. Complete mitochondrial genomes provide a large character base for phylogenetic analysis and may provide for robust estimates of phylogeny. Phylogenetic analysis of zebrafish and 35 other taxa based on all protein-coding genes produced trees largely, but not completely, consistent with conventional views of vertebrate evolution. It appears that even with such a large number of nucleotide characters (11,592), limited taxon sampling can lead to problems associated with extensive evolution on long phyletic branches.     

Cloning of the orotidine 5′-phosphate decarboxylase (ODC) gene of Schwanniomyces occidentalis by complementation of the ura3 mutation in S. cerevisiae

Summary A DNA fragment containing the gene encoding orotidine 5-phosphate decarboxylase (ODC) from the yeast, Schwanniomyces occidentalis (formerly castellii) has been isolated from a genomic library constructed in the S. cerevisiae expression vector, pYcDE8. A recombinant plasmid, p2-lA, containing a 2.47 kb insert was shown to complement the ura3-52 mutation of several strains of S. cerevisiae. This DNA insert was shown to be from Schwanniomyces occidentalis by Southern hybridization analysis. A restriction enzyme cleavage map of the insert has been derived and the ODC gene localized to a 1.1 kb region by deletion analysis. In addition, we have demonstrated that expression of ODC is not dependent on the ADHI promoter carried on pYcDE8. This is the first report of the cloning of a gene from a member of the genus Schwanniomyces.     

The gene for the large (16S) ribosomal RNA from the Locusta migratoria mitochondrial genome

Summary The nucleotide sequence of a segment of the mtDNA molecule of the locust Locusta migratoria containing the complete large rRNA (16S) gene and some nucleotides in its vincinity has been determined. The gene contains 1314 nucleotides, comprising the smallest metazoan gene reported to date. The gene has a low content of GC (21%) and exhibits an extended sequence homology to the corresponding gene of the dipteran insect Drosophila yakuba, suggesting a comparable secondary structure. The gene structure is discussed in an evolutionary and functional context.     

The effect of antifungal treatment on the vaginal flora of women with vulvo-vaginal yeast infection with or without bacterial vaginosis

Antibacterial therapy may enhance the risk of symptomatic vulvo-vaginal candidosis in susceptible women. We addressed the question whether oral antifungal treatment for vulvo-vaginal candidosis also influences the bacterial vaginal microflora. One hundred and forty-two patients with a culture-proven acute episode of recurrent vulvo-vaginal candidosis (RVC) were treated with fuconazole according to the ReCiDiF regimen (induction dose of 600 mg orally per week followed by 200 mg per week) or with a single dose of 200 mg pramiconazole, a new potent oral triazole. At inclusion, 1 week and 1 month after the end of antifungal treatment, the bacterial microflora was assessed by microscopy of vaginal fluid to detect lactobacillary grades and bacterial vaginosis (BV). The presence of BV was studied in these patients with vulvo-vaginal candidosis after treatment with antifungal medication. At the start of oral antifungal treatment, 6.3% of women with Candida were co-infected with BV. Of the BV-negative women, 10 out of 133 (8%) developed BV after 1 week and after 1 month 8 of them (7%) were still BV-positive. Although no patients received antibacterial treatment at any moment of the study, 6 out of 9 (66%) of the women with Candida and BV at inclusion no longer had BV 1 week after antifungal treatment and 6 out of 7 (86%) lacked BV after 1 month. Treatment with antifungals may have a beneficial effect on women with concurrent BV, but does not prevent BV from occurring in BV-negative women with Candida vaginitis.     

The in vivo effect of acriflavine on mitochondrial functions in the petite negative yeast Hansenula saturnus

Summary In the petite negative yeast Hansenula saturnus, acriflavine determined a decrease of cell yield and of the total Q_O2,the disappearance of the cytochromes aa ₃ and b and the inhibition of in vivo mitochondrial protein synthesis without affecting the cell survival. The restriction enzymes analysis of mitDNA shows that no specific fragmentation occurred after acriflavine treatment.     

Subcortical band heterotopia in rare affected males can be caused by missense mutations in DCX (XLIS) or LIS1.

Subcortical band heterotopia (SBH) are bilateral and symmetric ribbons of gray matter found in the central white matter between the cortex and the ventricular surface, which comprises the less severe end of the lissencephaly (agyria-pachygyria-band) spectrum of malformations. Mutations in DCX (also known as XLIS ) have previously been described in females with SBH. We have now identified mutations in either the DCX or LIS1 gene in three of 11 boys studied, demonstrating for the first time that mutations of either DCX or LIS1 can cause SBH or mixed pachygyria-SBH (PCH-SBH) in males. All three changes detected are missense mutations, predicted to be of germline origin. They include a missense mutation in exon 4 of DCX in a boy with PCH-SBH (R78H), a different missense mutation in exon 4 of DCX in a boy with mild SBH and in his mildly affected mother (R89G) and a missense mutation in exon 6 of LIS1 in a boy with SBH (S169P). The missense mutations probably account for the less severe brain malformations, although other patients with missense mutations in the same exons have had diffuse lissencephaly. Therefore, it appears likely that the effect of the specific amino acid change on the protein determines the severity of the phenotype, with some mutations enabling residual protein function and allowing normal migration in a larger proportion of neurons. However, we expect that somatic mosaic mutations of both LIS1 and DCX will also prove to be an important mechanism in causing SBH in males.     

The curly-tail (ct) mouse,an animal model of neural tube defects,displays altered homocysteine metabolism without folate responsiveness or a defect in Mthfr

Maternal mild hyperhomocysteinemia is associated with increased risk for bearing children with neural tube defects (NTD). Folate intake corrects hyperhomocysteinemia and prevents up to 70% of NTD. The curly-tail (ct) mouse, an animal model for NTD, has been suggested to display features that closely resemble the human defect. We therefore investigated folate metabolism in ct mice. On control and folate-/choline-deficient diets, ct mice exhibited higher plasma homocysteine levels than control C57Bl/6 mice. This increase was associated with increased liver S-Adenosylhomocysteine and decreased S-adenosylmethionine:S-adenosylhomocysteine (SAM/SAH) ratios. Since the ct locus maps in close proximity to the gene for methylenetetrahydrofolate reductase (Mthfr), a modifier of homocysteine levels in man, we also assayed Mthfr activity and sequenced the 5(') regulatory region; these experiments suggested that Mthfr is not defective in the ct strain. Finally, we examined the influence of dietary folate on NTD incidence in the ct strain, but did not identify significant differences among the four diets used in the study. Our work suggests that altered homocysteine metabolism may contribute to the pathogenetic mechanism of the ct defect, but, unlike human NTD, nutritional or genetic deficiencies in folate metabolism do not appear to play a significant direct role.     

The human neutrophil FcRIII (CD16) on the plasma membrane can be replenished from an intracellular source.

The internalization of peroxidase-antiperoxidase (PAP) immune complexes by human neutrophil granulocytes was studied at an ultrastructural level. PAP initially bound to the plasma membrane at 4 degrees C accumulated in endosomes within 5 min of internalization. By that time, all of the ligand bound to the plasma membrane had been removed from the cell surface and the cells were able to bind newly added PAP. Pre-embedding labelling of FcRIII on these cells showed that this receptor was present on the cell surface, indicating involvement of FcRIII in the rebinding of PAP in neutrophils. The origin of FcRIII present on the plasma membrane after Fc receptor-mediated internalization of PAP was investigated in another series of experiments. Incubation of the cells with pronase eliminated the epitope on the Fc receptor recognized by anti-FcRIII. After the pronase treatment hardly any Fc receptors were detected on the plasma membrane. However, incubation of the cells for only 5 min in a protease-free medium after the pronase treatment led to an abundance of FcRIII on the plasma membrane of the neutrophils. These findings support the hypothesis that FcRIII on the plasma membrane of human neutrophil granulocytes is replenished from an internal source of free Fc receptors and suggest that at least some of the receptors present on the cell surface after the binding and internalization of PAP originate from this source in the cytoplasm.     

Inhibitory effect of PGE(2) on the killing of Paracoccidioides brasiliensis by human monocytes can be reversed by cellular activation with cytokines

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a deep mycosis endemic in Latin America. Studies to elucidate the host-parasite relationship in this mycosis have demonstrated that non-activated phagocytes fail to kill the etiologic agent. Investigations of human monocytes have shown that the lack of fungicidal activity is partially associated with the capacity of a high-virulence strain to induce PGE(2) release by these cells. This eicosanoid inhibits production of TNF-α, the cytokine involved in cell activation for release of H(2)O(2), the fungicidal metabolite. Cell priming with IFN-γ was shown to partially reverse this inhibitory effect. In this study, we asked whether monocyte challenge with a low-virulence strain of this fungus would also result in PGE(2) release and consequently inhibition of antifungal activities. We also assessed whether PGE(2,) besides inhibiting production of TNF-α, a monocyte-activating cytokine, also affects IL-10. The latter, in contrast to TNF-α is a monocyte-suppressing cytokine. Finally, we evaluated whether priming cells with other cytokines, namely TNF-α and GM-CSF, could be more effective than IFN-γ in reversing the PGE(2) inhibitory effect. The results revealed that the less virulent P. brasiliensis strain also induces human monocytes to release PGE(2). However, the inhibitory effect of PGE(2) was less pronounced when cells were challenged with this strain than with the more virulent one. It was also demonstrated that PGE(2), while inhibits TNF-α production, tends to increase IL-10 levels. Priming with GM-CSF or TNF-α was more effective than IFN-γ in compensating for the inhibitory PGE(2) effect, since these cytokines induce cells to produce higher H(2)O(2) and TNF-α levels.     

At least two nuclear-encoded factors are involved together with a mitochondrial factor (MR1) in spontaneous mitochondrial frameshift-suppression of the yeast S. cerevisiae

Summary Earlier genetic analyses have identified a mitochondrial +1 frameshift suppressor (MF1) in the 15S rRNA region of a leaky mitochondrial frameshift mutant and the respective wild-type strain 777-3A (Weiss-Brummer et al. 1987). Further genetic analyses revealed that for the observed spontaneous frameshift suppression in M5631 the mitochondrial factor (MF1) must act together with at least two dominant nuclear-encoded factors.     

The ogd1 and kgd1 mutants lacking 2-oxoglutarate dehydrogenase activity in yeast are allelic and can be differentiated by the cloned amber suppressor

The activity of mitochondrial 2-oxoglutarate dehydrogenase in S. cerevisiae can be impaired either by the ogd1 or the kgd1 mutation. The OGD1 gene and two suppressor genes were isolated by complementation of the ogd1 mutant. The complementation of the kdg1 mutant by the OGD1 gene, an allelism test, and meiotic mapping, revealed that the ogd1 and kgd1 mutations are allelic. The two mutations were differentiated by the cloned suppressor gene which was able to partially complement ogd1, but not kgd1. The molecular analysis of the suppressor gene revealed its identity with the natural tRNA _CAG ^Gln gene found in the upstream region of URA10.     

The effect of lignocaine on sperm phagocytosis in the peritoneal fluid from women with or without endometriosis

The study was undertaken to investigate a possible mechanism for reducing the phagocytosis of spermatozoa by leukocytes in the peritoneal fluid from women suffering from endometriosis. Peritoneal fluids were collected during laparoscopy from women undergoing laparoscopic sterilization or from women under investigation for cause of infertility where the laparoscopic findings were endometriosis. Prepared spermatozoa from one healthy man were incubated in vitro with peritoneal fluid with or without lignocaine. Samples from the incubations were studied daily and the number of viable and dead spermatozoa were counted. The number of free spermatozoa, not adhered to leukocytes, was significantly increased when incubated in human peritoneal fluid supplemented with lignocaine. Thus lignocaine contributes to increasing the number of free spermatozoa and maintaining the possibility of fertilizing an oocyte. For patients with endometriosis, treatment with lignocaine might be a means of increasing the chances of conception. A clinical study is in progress to evaluate this effect in vivo and to search for alternative methods of assisting the fertilization process.      

The mtDNA T8993G (NARP) mutation results in an impairment of oxidative phosphorylation that can be improved by antioxidants

A T8993G point mutation in the mtDNA results in a Leu156Arg substitution in the MTATP6 subunit of the mitochondrial F1F0-ATPase. The T8993G mutation causes impaired oxidative phosphorylation (OXPHOS) in two mitochondrial disorders, NARP (neuropathy, ataxia and retinitis pigmentosa) and MILS (maternally inherited Leigh's syndrome). It has been reported, in some studies, that the T8993G mutation results in loss of assembled F1F0-ATPase. Others reported that the mutation causes impairment of proton flow through F0. In addition, it was shown that fibroblasts from NARP subjects have a tendency to undergo apoptotic cell death, perhaps as a result of increased free radical production. Here, we show that the T8993G mutation inhibits oxidative phosphorylation and results in enhanced free radical production. We suggest that free radical-mediated inhibition of OXPHOS contributes to the loss of ATP synthesis. Importantly, we show that antioxidants restore respiration and partially rescue ATP synthesis in cells harboring the T8993G mutation. Our results indicate that free radicals might play an important role in the pathogenesis of NARP/MILS and that this can be prevented by antioxidants. The effectiveness of antioxidant agents in cultured NARP/MILS cells suggests that they might have a potential beneficial role in the treatment of patients with NARP.