The Mitochondrial Acyl-carrier Protein Interaction Network Highlights Important Roles for LYRM Family Members in Complex I and Mitoribosome Assembly

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Highlights

• •The interaction network of NDUFAB1 reveal associations with 9 known LYRM proteins as well as more than 20 other proteins involved in mitochondrial respiratory chain complex and mitochondrial ribosome assembly.
• •The LYRM protein AltMiD51 is required for the stability of assembly factor MALSU1 and optimal assembly of the mitoribosome.
• •LYRM2 is important for integration of the N-module into respiratory chain complex I.

     

In vitro migration of Dictyocaulus viviparus larvae: migration of the infective stage inagar gel.

The infective stage of the cattle lungworm, Dictyocaulus viviparus, was able to migrate in agar gel when activated by bile. The number of larvae which penetrated the upper surface of a 2 mm thick agar layer was counted and found to be independent from the agar concentration. Larvae which had migrated out of the agar remained on the surface and did not re-enter. The agar migration process was temperature dependent. Influence of time as well as dependency of agar thickness was investigated. The significance of these findings is discussed.     

The Specific Elongation Factor to Selenocysteine Incorporation in Escherichia coli: Unique tRNASec Recognition and its Interactions

Several molecular mechanisms are involved in the genetic code interpretation during translation, as codon degeneration for the incorporation of rare amino acids. One mechanism that stands out is selenocysteine (Sec), which requires a specific biosynthesis and incorporation pathway. In Bacteria, the Sec biosynthesis pathway has unique features compared with the eukaryote pathway as Ser to Sec conversion mechanism is accomplished by a homodecameric enzyme (selenocysteine synthase, SelA) followed by the action of an elongation factor (SelB) responsible for delivering the mature Sec-tRNA^Sec into the ribosome by the interaction with the Selenocysteine Insertion Sequence (SECIS). Besides this mechanism being already described, the sequential events for Sec-tRNA^Sec and SECIS specific recognition remain unclear. In this study, we determined the order of events of the interactions between the proteins and RNAs involved in Sec incorporation. Dissociation constants between SelB and the native as well as unacylated-tRNA^Sec variants demonstrated that the acceptor stem and variable arm are essential for SelB recognition. Moreover, our data support the sequence of molecular events where GTP-activated SelB strongly interacts with SelA.tRNA^Sec. Subsequently, SelB.GTP.tRNA^Sec recognizes the mRNA SECIS to deliver the tRNA^Sec to the ribosome. SelB in complex with its specific RNAs were examined using Hydrogen/Deuterium exchange mapping that allowed the determination of the molecular envelopes and its secondary structural variations during the complex assembly. Our results demonstrate the ordering of events in Sec incorporation and contribute to the full comprehension of the tRNA^Sec role in the Sec amino acid biosynthesis, as well as extending the knowledge of synthetic biology and the expansion of the genetic code.     

Identification of Osteoclastic Factors in the Nuclear Envelope of Mature,Multinucleated Osteoclasts

Multinucleation is indispensable to the bone-resorbing activity of mature osteoclasts. Nevertheless, little is known about the regulatory networks among multi-nuclei in a single mature osteoclast. For this reason, we purified osteoclastic factors from the nuclear envelope by two-dimensional gel electrophoresis. Two annexin family proteins and ferritin light chain 1 protein were identified as osteoclastic candidates.     

Virus elimination during the purification of monoclonal antibodies by column chromatography and additional steps

The theoretical potential for virus transmission by monoclonal antibody based therapeutic products has led to the inclusion of appropriate virus reduction steps. In this study, virus elimination by the chromatographic steps used during the purification process for two (IgG‐1 & ?3) monoclonal antibodies (MAbs) have been investigated. Both the Protein G (>7log) and ion‐exchange (5 log) chromatography steps were very effective for eliminating both enveloped and non‐enveloped viruses over the life‐time of the chromatographic gel. However, the contribution made by the final gel filtration step was more limited, i.e., 3 log. Because these chromatographic columns were recycled between uses, the effectiveness of the column sanitization procedures (guanidinium chloride for protein G or NaOH for ion‐exchange) were tested. By evaluating standard column runs immediately after each virus spiked run, it was possible to directly confirm that there was no cross contamination with virus between column runs (guanidinium chloride or NaOH). To further ensure the virus safety of the product, two specific virus elimination steps have also been included in the process. A solvent/detergent step based on 1% triton X‐100 rapidly inactivating a range of enveloped viruses by >6 log inactivation within 1 min of a 60 min treatment time. Virus removal by virus filtration step was also confirmed to be effective for those viruses of about 50 nm or greater. In conclusion, the combination of these multiple steps ensures a high margin of virus safety for this purification process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1341–1347, 2014     

The dominant bacteria shifted from the order “ Lactobacillales ” to Bacillales and Actinomycetales during a start-up period of large-scale, completely-mixed composting reactor using plastic bottle flakes as bulking agent

Bacterial community succession in the start-up of a large-scale, completely-mixed composting reactor was analyzed by 16S rRNA gene (16S rDNA) clone analysis and denaturing gradient gel electrophoresis (DGGE) combined with measurements of temperature, pH, moisture contents, and decomposing rate. DGGE analysis and physicochemical parameters showed that bacterial community succession occurred in four phases; (1) at the start of operation and pH decreasing period (day 0–3), (2) pH decreased and increased period (day 4–11), (3) middle term, moisture content decreasing and maximum temperature increased period (day 12–16) and (4) latter term, temperature decreasing period (day 17–24). Lactobacillus spp. and Bacillus coagulans were detected from the initial phase and middle term, respectively. 16S rDNA clone analysis showed that the dominant bacteria shifted from the order “Lactobacillales” to Bacillales and Actinomycetales. The order “Lactobacillales” was unique which may be caused by using the plastic bottle flakes (polyethylene terephthalate, PET) as bulking agent.     

Phenotypic and proteomic analysis of positively regulated gellan biosynthesis pathway in Sphingomonas elodea

Sphingomonas elodea is a Gram-negative bacterium capable of producing ‘gellan gum’ exopolysaccharide that is the most extensively studied expolysaccharides of microbial origin. In this study, we investigated the phenotypic and proteomic alterations in S. elodea by homogeneously expressing both gelA and gelN involved in positive regulation and extracellular secretion of metabolites in gellan biosynthesis, respectively. Expression of six histidine-tagged GelA and GelN was determined by Western blot analysis. Successful expression of GelA and GelN resulted in both morphological changes of colonies and enhanced secretion of gellan into the growth medium (GelA, 21.2% more and GelN, 48.3% more) overexpressed compared to the wile-type. Comparative two-dimensional gel electrophoresis analysis revealed a differential proteome expression in S. elodea overexpressing GelA and GelN. Proteins up- or down-regulated by GelA and GelN overexpression were found to be mainly sugar transportation proteins, two-component regulatory proteins, and proteins involved in secretion pathways. The results suggest that the effect of GelA and GelN overexpression on gellan biosynthesis might be mainly caused by increased transportation of sugar units or enhanced exportation of gellan.     

3-[18F]Acetylcyclofoxy: a useful probe for the visualization of opiate receptors in living animals

A fluoro-analogue of the potent narcotic antagonist, naltrexone, was synthesized and shown to bind with high affinity to opiate receptors in vitro. 3-[18F]acetylcyclofoxy was prepared via a one-step triflate displacement reaction with the positron emitting 18F ion from tetraethylammonium [18F] fluoride. 3-[18F]acetylcyclofoxy accumulation in opiate receptor rich brain regions of both rat and baboon is shown to be completely displaced by the active enantiomer of naloxone [-)-naloxone) while the identical dose of the pharmacologically inert (+)-naloxone has no detectable effect. Moreover, both rat and baboon brain showed the well documented, typical opiate receptor distribution so that basal ganglia and thalamus are clearly visible in the living baboon brain up to 95 min after intravenous injection of 3-[18F] acetylcyclofoxy. We expect that 3-[18F )acetylcyclofoxy will be a useful probe for visualizing opiate receptors in living humans.