The Shortest Isoform of Dystrophin (Dp40) Interacts with a Group of Presynaptic Proteins to Form a Presumptive Novel Complex in the Mouse Brain

Duchenne muscular dystrophy (DMD) causes cognitive impairment in one third of the patients, although the underlying mechanisms remain to be elucidated. Recent studies showed that mutations in the distal part of the dystrophin gene correlate well with the cognitive impairment in DMD patients, which is attributed to Dp71. The study on the expression of the shortest isoform, Dp40, has not been possible due to the lack of an isoform specific antibody. Dp40 has the same promoter as that found in Dp71 and lacks the normal C-terminal end of Dp427. In the present study, we have raised polyclonal antibody against the N-terminal sequence common to short isoforms of dystrophin, including Dp40, and investigated the expression pattern of Dp40 in the mouse brain. Affinity chromatography with this antibody and the consecutive LC-MS/MS analysis on the interacting proteins revealed that Dp40 was abundantly expressed in synaptic vesicles and interacted with a group of presynaptic proteins, including syntaxin1A and SNAP25, which are involved in exocytosis of synaptic vesicles in neurons. We thus suggest that Dp40 may form a novel protein complex and play a crucial role in presynaptic function. Further studies on these aspects of Dp40 function might provide more insight into the molecular mechanisms of cognitive impairment found in patients with DMD

Extracellular Production of an RNA Aptamer by Ribonuclease-Free Marine Bacteria Harboring Engineered Plasmids: a Proposal for Industrial RNA Drug Production▿

Natural noncoding small RNAs have been shown to be involved in a number of cellular processes as regulators. Using the mechanisms thus elucidated, artificial small interfering RNAs (siRNAs), ribozymes, and RNA aptamers are also expected to be potential candidates for RNA therapeutic agents. However, current techniques are too costly for industrial production of these RNAs for use as drugs. Here, we propose a new method for in vivo production of artificial RNAs using the marine phototrophic bacterium Rhodovulum sulfidophilum. Using engineered plasmids and this bacterium, which produces extracellular nucleic acids in nature, we developed a method for extracellular production of a streptavidin RNA aptamer. As the bacterium does not produce any RNases in the culture medium, at least within the cultivation period tested, the designed RNA itself is produced and retained in the culture medium of the bacterium without any specific mechanism for protection against degradation by nucleases. Here, we report that the streptavidin RNA aptamer is produced in the culture medium and retains its specific function. This is the first demonstration of extracellular production of a functional artificial RNA in vivo, which will pave the way for inexpensive production of RNA drugs

Identification of novel long chain <i>N</i>-acylhomoserine lactones of chain length C₂₀ from the marine phototrophic bacterium <i>Rhodovulum sulfidophilum</i>

<p>Gram-negative bacterial quorum sensing is mainly regulated by an extracellularly produced <i>N</i>-acylhomoserine lactone (AHL). AHL consists of a lactone ring and an acyl chain, which generally varies from C₄ to C₁₈ in length and affords species-specific variety. In this study, we developed an ultra-high performance liquid chromatography tandem mass spectrometry system and detected two kinds of long chain AHLs with chain length C₂₀ from the reverse-phase thin layer chromatography-fractionated cultured supernatant of the marine photosynthetic bacterium <i>Rhodovulum sulfidophilum</i>. By fragmentation search analysis to detect compounds with a homoserine lactone ring moiety for data dependent acquisition, a minor AHL, presumed to be 3-OH-C₁₈-homoserine lactone (HSL), was also found. Among the detected C₂₀-HSLs, 3-OH-C₂₀-HSL was structurally identified and 3-OH-C_20:1-HSL was strongly suggested. To our knowledge, this is the first report to show a novel AHL with the longest C₂₀ acyl side chain found to date.</p> <p><b>Abbreviations:</b> AGC: automatic gain control; AHL: <i>N</i>-acylhomoserine lactone; CD: cyclodextrin; CID: collision induced dissociation; DDA: data dependent acquisition; EPI: enhanced product ion; FISh: fragment ion search; HCD: high energy collisional dissociation; HSL: homoserine lactone; IT: injection time; LC: liquid chromatography; MS: mass spectrometry; PRM: parallel reaction monitoring; RP: reverse phase; SRM: selected reaction monitoring; TLC: thin layer chromatography; UHPLC: ultra high performance liquid chromatography</p> <p>Novel longest acyl chained HSL produced by <i>R. sulfidophilum</i>.</p