Cytochrome b of fish mitochondria is strongly resistant to funiculosin, a powerful inhibitor of respiration.

We report here some unusual properties of ubiquinol: cytochrome c reductase of eel and other fish mitochondria. The turnover rate of the reductase is clearly higher than in mammalian mitochondria and the binding constant for ubiquinone seems to be larger than in other vertebrates. Additionally, the reductase activity of fish mitochondria is resistant to some powerful inhibitors that bind to cytochrome b, in particular to funiculosin. After sequencing most of the gene of eel cytochrome b and comparing the deduced amino acid sequence with that of other fish and animals, we hypothesize that the decreased binding of funiculosin could be due to a few amino acid replacements in the third and fourth transmembrane helix of the protein. In particular, the presence of methionine instead of alanine at position 125 seems to be largely responsible for the strong resistance to funiculosin and also to the partial resistance to myxothiazol in all fish mitochondria. Correlations between some residue substitutions in cytochrome b and the different effects of funiculosin in different species are also considered.     

A regulatory region from the mouse Hox-2.2 promoter directs gene expression into developing limbs

To characterize cis-acting regulatory elements of the murine homeobox gene, Hox-2.2, transgenic mouse lines were generated that contained the LacZ reporter gene under the control of different fragments from the presumptive Hox-2.2 promoter. A promoter region of 3600 base pairs (bp) was identified, which reproducibly directed reporter gene expression into specific regions of developing mouse embryos. At 8.5 days postcoitum (p.c.) reporter gene activity was detected in posterior regions of the lateral mesoderm and, in subsequent developmental stages, expression of the LacZ gene was restricted to specific regions of the developing limb buds and the mesenchyme of the ventrolateral body region. This pattern of Hox-2.2-LacZ expression was found in all transgenic embryos that have been generated with the 3.6 kb promoter fragment (two founder embryos and embryos from five transgenic lines). In addition, embryos from two transgenic mouse lines expressed the reporter gene at low levels in the developing central nervous system (CNS). Our results are consistent with the idea that in addition to their presumptive role in CNS and vertebrae development, Hox-2.2 gene products are involved in controlling pattern formation in developing limbs.     

Skin abnormalities of the back in diastematomyelia.

The presence of congenital skin abnormalities on the back may be associated with a serious underlying spinal anomaly, diastematomyelia. The significance of the progressive development of neurological deficits, due to the presence of a bony or cartilaginous spur in the spinal cord, is stressed. An awareness of this condition should lead to an early diagnosis with a neurological evaluation and, if necessary, prophylactic surgery before irreversible nerve damage develops.     

Disruption in the autophagic process underlies the sensory neuropathy in dystonia musculorum mice

A homozygous mutation in the DST (dystonin) gene causes a newly identified lethal form of hereditary sensory and autonomic neuropathy in humans (HSAN-VI). DST loss of function similarly leads to sensory neuron degeneration and severe ataxia in dystonia musculorum (Dst^dt) mice. DST is involved in maintaining cytoskeletal integrity and intracellular transport. As autophagy is highly reliant upon stable microtubules and motor proteins, we assessed the influence of DST loss of function on autophagy using the Dst^dt-Tg4 mouse model. Electron microscopy (EM) revealed an accumulation of autophagosomes in sensory neurons from these mice. Furthermore, we demonstrated that the autophagic flux was impaired. Levels of LC3-II, a marker of autophagosomes, were elevated. Consequently, Dst^dt-Tg4 sensory neurons displayed impaired protein turnover of autophagosome substrate SQTSM1/p62 and of polyubiquitinated proteins. Interestingly, in a previously described Dst^dt-Tg4 mouse model that is partially rescued by neuronal specific expression of the DST-A2 isoform, autophagosomes, autolysosomes, and damaged organelles were reduced when compared to Dst^dt-Tg4 mutant mice. LC3-II, SQTSM1, polyubiquitinated proteins and autophagic flux were also restored to wild-type levels in the rescued mice. Finally, a significant decrease in DNAIC1 (dynein, axonemal, intermediate chain 1; the mouse ortholog of human DNAI1), a member of the DMC (dynein/dynactin motor complex), was noted in Dst^dt-Tg4 dorsal root ganglia and sensory neurons. Thus, DST-A2 loss of function perturbs late stages of autophagy, and dysfunctional autophagy at least partially underlies Dst^dt pathogenesis. We therefore conclude that the DST-A2 isoform normally facilitates autophagy within sensory neurons to maintain cellular homeostasis.     

Structural and Functional Studies of the Biotin Protein Ligase from Aquifex aeolicus Reveal a Critical Role for a Conserved Residue in Target Specificity

Biotin protein ligase (BPL; EC 6.3.4.15) catalyses the formation of biotinyl-5′-AMP from biotin and ATP, and the succeeding biotinylation of the biotin carboxyl carrier protein. We describe the crystal structures, at 2.4 Å resolution, of the class I BPL from the hyperthermophilic bacteria Aquifex aeolicus (AaBPL) in its ligand-free form and in complex with biotin and ATP. The solvent-exposed β- and γ-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal domains. The Arg40 residue from the conserved GXGRXG motif is shown to interact with the carboxyl group of biotin and to stabilise the α- and β-phosphates of the nucleotide. The structure of the mutant AaBPL R40G in both the ligand-free and biotin-bound forms reveals that the mutated loop has collapsed, thus hindering ATP binding. Isothermal titration calorimetry indicated that the presence of biotin is not required for ATP binding to wild-type AaBPL in the absence of Mg²⁺, and the binding of biotin and ATP has been determined to occur via a random but cooperative process. The affinity for biotin is relatively unaffected by the R40G mutation. In contrast, the thermodynamic data indicate that binding of ATP to AaBPL R40G is very weak in the absence or in the presence of biotin. The AaBPL R40G mutant remains catalytically active but shows poor substrate specificity; mass spectrometry and Western blot studies revealed that the mutant biotinylates both the target A. aeolicus BCCPΔ67 fragment and BSA, and is subject to self-biotinylation.     

Biochemical studies on native and cross-linked aggregates of Aspergillus awamori feruloyl esterase

Thermal stabilities of a native freeze dried Aspergillus awamori feruloyl esterase (FAE-II) enzyme and a cross-linked feruloyl esterase aggregate (CLEAs) at 25–85 °C were evaluated and discussed. Effects of some metal ions and some chemicals on the activity of both native freeze dried FAE-II enzyme and CLEAs were examined and explained. Differential scanning calorimetry, thermogravimetry, and derived thermogravimetry, were used to observe and explain the thermal denaturation processes. Structural analyses were made for native FAE-II and CLEAs using FT-IR and SEM techniques to investigate whether the cross-linking had any effect on the powder structure of native FAE-II enzyme.     

RNA Silencing of Mcl-1 Enhances ABT-737-Mediated Apoptosis in Melanoma: Role for a Caspase-8-Dependent Pathway

Background

Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma''s striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance.

Methodology/Principal Findings

Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity.

Conclusions/Significance

Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.