The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

Invasive aspergillosis promotes tumor growth and severity in a tumor-bearing mouse model

Invasive aspergillosis increases in chronic immunosuppressive diseases such as cancer. There is little information about the mechanisms by which Aspergillus infection affects the immune regulation and microenvironment of cancer cells. Hence, this study was aimed at investigating the effect of invasive aspergillosis on immunosurveillance, metastasis, and prognosis of cancer in tumor-bearing mice. After implantation of mouse mammary tumor in BALB/c mice, they were infected with Aspergillus conidia intravenously. For comparison, groups of mice were experimentally infected with Aspergillus conidia or implanted with tumor cells separately. Seven days after Aspergillus infection, the serum levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured by ELISA, and subsequently regulatory T lymphocytes were analyzed by flow cytometry. The survival of animals and mean tumor size were then determined. Our results indicated that tumor sizes in mice increased significantly after infection with Aspergillus conidia. Moreover, invasive aspergillosis enhanced the population of regulatory lymphocytes and level of TIMP-1. This study supports the idea that massive Aspergillus infection could stimulate tumor growth and increases the possibility of a bad prognosis. As a result, treatment of Aspergillus infection could be considered an important issue for efficient cancer therapy.     

Structure–toxicity relationship of phenolic analogs as anti-melanoma agents: An enzyme directed prodrug approach

The aim of this study was to identify a phenolic prodrug compound that is minimally metabolized by rat liver microsomes, but yet could form quinone reactive intermediates in melanoma cells as a result of its bioactivation by tyrosinase. In current work, we investigated 24 phenolic compounds for their metabolism by tyrosinase, rat liver microsomes and their toxicity towards murine B16-F0 and human SK-MEL-28 melanoma cells. A linear correlation was found between toxicities of phenolic analogs towards SK-MEL-28 and B16-F0 melanoma cells, suggesting similar mechanisms of toxicity in both cell lines. 4-HEB was identified as the lead compound. 4-HEB (IC₅₀ 48 h, 75 μM) showed selective toxicity towards five melanocytic melanoma cell lines SK-MEL-28, SK-MEL-5, MeWo, B16-F0 and B16-F10, which express functional tyrosinase, compared to four non-melanoma cells lines SW-620, Saos-2, PC3 and BJ cells and two amelanotic SK-MEL-24, C32 cells, which do not express functional tyrosinase. 4-HEB caused significant intracellular GSH depletion, ROS formation, and showed significantly less toxicity to tyrosinase specific shRNA transfected SK-MEL-28 cells. Our findings suggest that presence of a phenolic group in 4-HEB is critical for its selective toxicity towards melanoma cells.     

Role of disulfide bonds in modulating internal motions of proteins to tune their function: molecular dynamics simulation of scorpion toxin Lqh III

A series of 1-ns MD simulations were performed on the scorpion toxin Lqh III in native and disulfide bond broken states. The removal of disulfide bonds has caused hydrogen bond network alteration in the five-residue turn, the long loop, the alpha-helix, the loop connecting strands II and III, and the C-terminal region. In addition and more importantly, it has influenced the amplitude of the fluctuations of five-residue turn, loops, and C-terminal region with a minor effect on the fluctuations of the cysteines in the broken bond sites. These findings suggest that disulfide bonds are not the most important factors in rigidifying their own locations, while they have more important effects at a global scale. Furthermore, our results reveal that disulfide bonds have considerable influence on the functionally important essential modes of motions and the correlations between the motions of the binding site residues. Therefore, we can conclude that disulfide bonds have a crucial role in modulating the function via adjusting the dynamics of scorpion toxin molecules. Although this conclusion cannot be generalized to all peptides and proteins, it demonstrates the importance of more investigations on this aspect of disulfide bond efficacy.     

Depot-specific gene expression profiles during differentiation and transdifferentiation of bovine muscle satellite cells, and differentiation of preadipocytes

We report a systematic study of gene expression during myogenesis and transdifferentiation in four bovine muscle tissues and of adipogenesis in three bovine fat tissues using DNA microarray analysis. One hundred hybridizations were performed and 7245 genes of known and unknown function were identified as being differentially expressed. Supervised hierarchical cluster analysis of gene expression patterns revealed the tissue specificity of genes. A close relationship in global gene expression observed for adipocyte-like cells derived from muscle and adipocytes derived from intramuscular fat suggests a common origin for these cells. The role of transthyretin in myogenesis is a novel finding. Different genes were highly induced during the transdifferentiation of myogenic satellite cells and in the adipogenesis of preadipocytes, indicating the involvement of different molecular mechanisms in these processes. Induction of CD36 and FABP4 expression in adipocyte-like cells and adipocytes may share a common pathway.     

Cytochrome b mutation Y268S conferring atovaquone resistance phenotype in malaria parasite results in reduced parasite bc1 catalytic turnover and protein expression

Atovaquone is an anti-malarial drug used in combination with proguanil (e.g. Malarone(TM)) for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Q(o)) site of the mitochondrial cytochrome bc(1) complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential, disruption of pyrimidine biosynthesis, and subsequent parasite death. Resistance to atovaquone in the field is associated with point mutations in the Q(o) pocket of cytochrome b, most notably near the conserved Pro(260)-Glu(261)-Trp(262)-Tyr(263) (PEWY) region in the ef loop). The effect of this mutation has been extensively studied in model organisms but hitherto not in the parasite itself. Here, we have performed a molecular and biochemical characterization of an atovaquone-resistant field isolate, TM902CB. Molecular analysis of this strain reveals the presence of the Y268S mutation in cytochrome b. The Y268S mutation is shown to confer a 270-fold shift of the inhibitory constant (K(i)) for atovaquone with a concomitant reduction in the V(max) of the bc(1) complex of ～40% and a 3-fold increase in the observed K(m) for decylubiquinol. Western blotting analyses reveal a reduced iron-sulfur protein content in Y268S bc(1) suggestive of a weakened interaction between this subunit and cytochrome b. Gene expression analysis of the TM902CB strain reveals higher levels of expression, compared with the 3D7 (atovaquone-sensitive) control strain in bc(1) and cytochrome c oxidase genes. It is hypothesized that the observed differential expression of these and other key genes offsets the fitness cost resulting from reduced bc(1) activity.     

Demographic History of Shorea curtisii (Dipterocarpaceae) Inferred from Chloroplast DNA Sequence Variations

We assessed the variability of chloroplast DNA sequences in populations of the dipterocarp forest tree, Shorea curtisii. This species is widely distributed in hill and coastal hill dipterocarp forests of the Malay Peninsula, whereas isolated populations are found in the coastal hills of north Borneo. Two chloroplast DNA regions (1555 bp of trnH‐psbA‐trnK and 925 bp of trnL‐trnF) were sequenced from 123 individuals collected from six Malay Peninsula and two Bornean populations. There were 15 chloroplast haplotypes derived from 16 polymorphic sites. A haplotype network revealed two distinct haplogroups that correlate with two geographic regions, the Malay Peninsula and Borneo. These two haplogroups differed by a number of mutations, and no haplotypes were shared between populations from the different geographic regions. This suggests an ancient diversification of these haplogroups, and that long‐distance seed dispersal was unlikely to have occurred during the Pleistocene when the Sunda Shelf was a contiguous landmass. Phylogenetic analysis of the haplotypes together with those found in other Shorea species showed that two haplogroups in S. curtisii appear in different positions of the phylogenetic tree. This could be explained by the persistence of ancestral polymorphisms or by ancient chloroplast capture. Low levels of genetic differentiation were found between populations within each geographic region. Signature of a bottleneck followed by demographic expansion was detected in the Malay Peninsula haplogroup. The presence of two distinct evolutionary lineages in the different regions suggests that they should be managed independently to conserve the major sources of genetic diversity in S. curtisii.