Investigating apoptosis: Characterization and analysis of Trichoplusia ni-caspase-1 through overexpression and RNAi mediated silencing

In both mammals and invertebrates, caspases play a critical role in apoptosis. Although Lepidopteron caspases have been widely studied in Spodoptera frugiperda cells, this is not the case for Trichoplusia ni cells, despite their widespread use for the production of recombinant protein and differences in baculovirus infectivity between the two species. We have cloned, expressed, purified and characterized Tn-caspase-1 in several situations: in its overexpression, in silencing via RNA interference (RNAi), during baculovirus infection, and in interactions with baculovirus protein p35. Overexpression can transiently increase caspase activity in T. ni (High Five?) cells, while silencing results in a greater than 6-fold decrease. The reduction in caspase activity resulted in a reduction in the level of apoptosis, demonstrating the ability to affect apoptosis by modulating Tn-caspase-1. During baculovirus infection, caspase activity remains low until approximately 5 days post infection, at which point it increases dramatically, though not in those cells treated with dsRNA. Our results demonstrate that Tn-caspase-1 is presumably the principal effector caspase present in High Five cells, and that it is inhibited by baculovirus protein p35. Finally, our results indicate differences between RNAi and p35 as effector molecules for modulating caspase activity and apoptosis during cell growth and baculovirus infection.     

A twin study of mitochondrial DNA polymorphisms shows that heteroplasmy at multiple sites is associated with mtDNA variant 16093 but not with zygosity

The mitochondrial theory of ageing proposes that damage to mitochondria and diminished mitochondrial DNA (mtDNA) repair are major contributors to cellular dysfunction and age-related diseases. We investigate the prevalence of heteroplasmy in the mtDNA control region in buccal swab and blood derived samples for 178 women from the TwinsUK cohort (41 DZ pair 39 MZ pairs, 18 singletons, mean age 57.5 range 28–82) and its relationship to age, BMI and fasting insulin and glucose serum levels. The overall estimated prevalence of heteroplasmy for both tissues in the control region measured for 37 sites was 17%. The prevalence of heteroplasmy was higher among the older half of the study subjects than in the younger half (23% vs 10% p<0.03), primarily reflecting the increase in the prevalence of a heteroplasmic dinucleotide CA repeat in variable region II (VRII) with age. The VRII 523–524 heteroplasmic site (heteroplasmic in 25 subjects) was also associated with a decrease in BMI. In addition, concordance rates for common heteroplasmy were observed to be near complete for both dizygotic (DZ = 94%) and monozygotic twin pairs (MZ = 100%), consistent with previous reports that suggest variation in heteroplasmy rates between generations are determined by bottlenecks in maternal transmission of mitochondria. Differences in the prevalence of heteroplasmy were observed overall between samples derived from buccal swabs (19%) and blood (15%, p<0.04). These were particularly marked at position 16093 of hypervariable region I (HVI, 7% vs 0%, respectively, p<4×10⁻¹¹). The presence of the C allele at position 16093 in blood was associated with the presence of heteroplasmy in buccal swabs at this position (p = 3.5×10⁻¹⁴) and also at VRII (p = 2×10⁻⁴) suggesting a possible predisposing role for this site in the accumulation of heteroplasmy. Our data indicate that BMI is potentially associated with control region heteroplasmy.     

Draft Genome Sequence of the Extremely Acidophilic Bacterium Acidithiobacillus caldus ATCC 51756 Reveals Metabolic Versatility in the Genus Acidithiobacillus

Acidithiobacillus caldus is an extremely acidophilic, moderately thermophilic, chemolithoautotrophic gammaproteobacterium that derives energy from the oxidation of sulfur and reduced inorganic sulfur compounds. Here we present the draft genome sequence of Acidithiobacillus caldus ATCC 51756 (the type strain of the species), which has permitted the prediction of genes for survival in extremely acidic environments, including genes for sulfur oxidation and nutrient assimilation.Acidothiobacillus caldus is one of the three recognized species of the genus Acidithiobacillus, which also circumscribes A. thiooxidans and A. ferrooxidans. These bacteria live in extremely acidic environments (pH 1 to 3) typically associated with copper mining operations (bioleaching) (15, 17) and natural acid drainage systems (7). All of these bacteria have the capacity to gain energy by the oxidation of sulfur and reduced inorganic sulfur compounds and to thrive in extremely high concentrations of heavy metals (16). Of the three species, A. ferrooxidans is unique in also being able to obtain energy through the oxidation of ferrous iron, as well as being a facultative anaerobe capable of using ferric iron as an alternative electron acceptor. Acidithiobacilli have been shown to be able to fix atmospheric carbon via the Calvin-Benson-Bassham cycle (1, 11, 21) and to synthesize extracellular polymeric substances that are postulated to promote adhesion to mineral surfaces (3).As opposed to A. ferrooxidans, for which substantial bioinformatic and experimental evidence exists for these and other properties (4, 14, 19, 20), A. caldus is poorly characterized, although it is known that it cannot carry out iron oxidation or nitrogen fixation (13). In contrast to the other two species of the genus, A. caldus thrives at temperatures up to 45 to 50°C. In order to unravel strategies for energy and nutrient assimilation used by A. caldus to survive and proliferate in extremely acidic environments, we have generated and annotated a draft genome sequence of A. caldus and performed a genome-based metabolic reconstruction to address these questions.The draft genome sequence of the type strain of A. caldus, ATCC 51756, was determined by a whole-genome shotgun strategy. Genomic libraries of 4 kb and 40 kb were constructed and sequenced, assembled using the Phred/Phrap programs (5), leading to the generation of a draft assembly based on 41,813 high-quality reads. Using Consed (8), assemblies that contained only contig segments with at least twofold coverage were edited and curated. Gene modeling was performed using CRITICA (2) and Glimmer (6). Predicted coding sequences were annotated based on comparisons with public databases (COG, KEGG, Pfam, TIGRFAMs, Unipprot, and NR-NCBI). Automatic metabolic reconstruction was carried out using the PRIAM and Pathways tools for prediction and curation.The A. caldus ATCC 51756 draft genome sequence has a total of 2,946,159 bp distributed in 139 contigs with an average GC content of 61.4%. Two 5S-16S-23S operons and 47 tRNAs on the draft assembly were identified, as were complete sets of genes for the synthesis of amino acids, nucleotides, and prosthetic groups. As in the case of A. ferrooxidans ATCC 23270 and other chemolithoautotrophic representatives, an incomplete tricarboxylic acid (TCA) cycle was detected, in which genes for the 2-oxogluatarate dehydrogenase enzyme complex were absent. The incomplete TCA cycle has been hypothesized to be an ancient biosynthetic pathway, instead of an energy generation cycle characteristic of the complete TCA cycle (22).A detailed inspection of the genome sequence revealed the presence of a complete set of genes encoding flagellum formation and chemotaxis. All of the genes of the classical Calvin-Benson-Bassham pathway were predicted, including genes for two RuBisCO (ribulose-1,5-bisphosphate carboxylase oxygenase) enzymes (type I and type II) and carboxysome formation genes. In addition, genes for sulfur and reduced inorganic sulfur compound oxidation were identified, including two gene clusters harboring the genes encoding the sulfur oxidation complex SOX (soxYZB-hyp-resB-soxAX-resC and soxYZA-hyp-SoxB), previously characterized in neutrophilic, chemoautotrophic bacteria (9); genes for the sulfur quinone oxidoreductase enzyme (sqr); a sulfur oxygenase:reductase gene (sor); and genes for a tetrathionate hydrolase and a thiosulfate quinone oxidoreductase (doxD) previously characterized in this strain (18). Several terminal oxidases were identified, including two copies of the genes encoding a bo₃-type quinol oxidase (cyoBACD), six copies of the genes encoding a bd-type quinol oxidase (cydAB), and one putative aa₃-type quinol oxidase gene cluster termed “quoxBACD.” No gene was detected that could encode rusticyanin, which has been shown to be involved in electron flow during iron oxidation in A. ferrooxidans ATCC 23270 (10).Genes for ammonia uptake were predicted, while those for atmospheric nitrogen fixation were not found in the draft genome as expected. In addition, an alternative nitrogen assimilatory pathway in A. caldus can be proposed based on the presence of a gene cluster encoding a membrane-associated nitrate reductase (narGHJI). A similar complex has been shown to carry out nitrogen assimilation in Mycobacterium tuberculosis (12).The information provided in the draft genome sequence of A. caldus ATCC 51756 reported here will facilitate additional bioinformatic and experimental investigations to elucidate the role of this microorganism in bioleaching and in natural and man-made acidic environments. This information also provides a first overview of the comparative metabolic diversity of the genus Acidithiobacillus and generates a more comprehensive picture of the metabolic diversity and adaptability of organisms that dwell in extreme acidic environments.     

CUP-1 is a novel protein involved in dietary cholesterol uptake in Caenorhabditis elegans

Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (CUP-1), is involved in dietary cholesterol uptake in C. elegans. Animals lacking CUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A CUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane "cholesterol recognition/interaction amino acid consensus" (CRAC) motif present in C. elegans CUP-1. In-silico analysis identified two mammalian homologues of CUP-1. Most interestingly, CRAC motifs are conserved in mammalian CUP-1 homologous. Our results suggest a role of CUP-1 in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals.     

G Protein–Coupled Receptor-Type G Proteins Are Required for Light-Dependent Seedling Growth and Fertility in Arabidopsis

G protein–coupled receptor-type G proteins (GTGs) are highly conserved membrane proteins in plants, animals, and fungi that have eight to nine predicted transmembrane domains. They have been classified as G protein–coupled receptor-type G proteins that function as abscisic acid (ABA) receptors in Arabidopsis thaliana. We cloned Arabidopsis GTG1 and GTG2 and isolated new T-DNA insertion alleles of GTG1 and GTG2 in both Wassilewskija and Columbia backgrounds. These gtg1 gtg2 double mutants show defects in fertility, hypocotyl and root growth, and responses to light and sugars. Histological studies of shoot tissue reveal cellular distortions that are particularly evident in the epidermal layer. Stable expression of GTG1_pro:GTG1-GFP (for green fluorescent protein) in Arabidopsis and transient expression in tobacco (Nicotiana tabacum) indicate that GTG1 is localized primarily to Golgi bodies and to the endoplasmic reticulum. Microarray analysis comparing gene expression profiles in the wild type and double mutant revealed differences in expression of genes important for cell wall function, hormone response, and amino acid metabolism. The double mutants isolated here respond normally to ABA in seed germination assays, root growth inhibition, and gene expression analysis. These results are inconsistent with their proposed role as ABA receptors but demonstrate that GTGs are fundamentally important for plant growth and development.     

Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related Gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.     

Methane oxidation and its coupled electron-sink reactions in ruminal fluid

AIMS: This study was conducted to investigate the occurrence of methane oxidation in the rumen, and to identify the electron-sink reaction coupled to the oxidation if it occurred. METHODS AND RESULTS: Mixed ruminal microbes taken from sheep were incubated with 13CH4. Oxidation of methane, estimated from the flux of 13C to CO2 and microbial cells, occurred, but represented only 0.2-0.5% of the methane produced. Methane oxidation was suppressed by the presence of oxygen, and was also inhibited by 2-bromoethane-sulphonate, and molybdate, but not by tungstate. CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: Methane could be oxidized anaerobically in the rumen by reverse methanogenesis in consort with sulphate reduction.     

Enhancement of organophosphorus hydrolase yield in Escherichia coli using multiple gene fusions

It was previously shown that organophosphorus hydrolase (OPH) expression and purification could be tracked by fluorescence of green fluorescent protein (GFP) when synthesized as an N-terminal fusion with GFP (Cha et al., 2000; Wu et al., 2000). In order to enhance OPH productivity while utilizing the advantage of the reporter protein (GFP), two copies of OPH were cloned in tandem following the gfp(uv) gene (e.g., GFP-OPH(n=2)). Both anti-GFP and anti-OPH Western blots demonstrated that a higher yield was achieved in comparison to the one copy fusion (GFP-OPH). Importantly, the fusion protein was still fluorescent as determined via microscopy. In contrast, a fusion containing two copies of OPH without GFP, and an operon fusion of two OPHs with two independent ribosomal binding sites, did not result in a higher yield than one OPH expressed alone.