Chemical modification of the hydroxylase of soluble methane monooxygenase gives one form of the protein with significantly increased thermostability and another that functions well in organic solvents

Proteolysis of the hydroxylase component of soluble methane monooxygenase (MMO) with trypsin yielded a protein which retained 50% activity in a standard MMO assay. In an H₂O₂-driven assay, in which H₂O₂ replaced two of the protein components, NADH and O₂ used in the standard assay, the proteolysed hydroxylase retained full activity for ethane, propane and propene, but had a 2–3 fold increase with methane as substrate. Several crosslinking reagents have been tested for their ability to stabilise the proteolysed form of the hydroxylase. Using polyoxyethylene bis(imidazolyl carbonyl) (M_r 3350) as the crosslinking agent, increased thermostability of the hydroxylase was observed. Activated methoxypolyethylene glycol (M_r 5000) was used to modify the hydroxylase which was now soluble in organic solvents as well as water and could be activated by H₂O₂. The glycol-modified hydroxylase functioned well in organic solvents in the catalysis of propene oxidation.     

Identification two novel nacrein-like proteins involved in the shell formation of the Pacific oyster Crassostrea gigas

Nacrein-like proteins have carbonic anhydrase (CA)-like domains, but their coding regions are flanked by inserted repeat sequence, such as Gly-X-Asn. Reportedly, nacrein-like proteins show the highest similarity to human carbonic anhydrase 1(α-CA1), possess CA catalytic functions, and play a key role in shell biomineralization. In the present study, two novel nacrein-like proteins were firstly identified from the shell-forming mantle of the Pacific oyster Crassostrea gigas. With numerous analyses, it was identified and characterized that both the nacrein-like proteins F1 and F2 were secreted and most closely related to the nacrein-like protein of California mussel Mytilus californianus via phylogenetic analysis. RT-PCR analysis showed that the nacrein-like proteins F1 and F2 were expressed in multiple tissues and the expression levels remarkably rose after entering the spat stage, which were basically consistent with the increase of calcite fractions in the total shell volume. Surprisingly, the Gly-X-Asn repeat domain, which is distinctive in most nacrein-like proteins, was absent in the two newly identified nacrein-like proteins in C. gigas and replaced with a series of acidic amino acids (D/E). Regardless, nacrein-like proteins in mollusks seem to be vital to the deposition of calcium carbonate and likely perform diverse functions.     

Comparative study on the surface ultrastructures of fertilized eggs of three Sebastes species

Fertilized eggs of Sebastes schlegelii, Sebastes pachycephalus and Sebastes hubbsi are morphologically similar under stereomicroscope. However, under the scanning electron microscope, significant differences in the ultrastructures of the egg surface among the three species were observed, and herein, a taxonomic key was proposed for future applications. A clustering analysis based on the ultrastructures of the egg surface and the diameter of the oil globule suggested that among the three species, S. hubbsi and S. pachycephalus were more genetically similar, while S. schlegelii had diverged earlier. The results agree with the conclusions drawn from morphological and molecular analyses on adult samples of the same species.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Electrically-Evoked Frequency-Following Response (EFFR) in the Auditory Brainstem of Guinea Pigs

It is still a difficult clinical issue to decide whether a patient is a suitable candidate for a cochlear implant and to plan postoperative rehabilitation, especially for some special cases, such as auditory neuropathy. A partial solution to these problems is to preoperatively evaluate the functional integrity of the auditory neural pathways. For evaluating the strength of phase-locking of auditory neurons, which was not reflected in previous methods using electrically evoked auditory brainstem response (EABR), a new method for recording phase-locking related auditory responses to electrical stimulation, called the electrically evoked frequency-following response (EFFR), was developed and evaluated using guinea pigs. The main objective was to assess feasibility of the method by testing whether the recorded signals reflected auditory neural responses or artifacts. The results showed the following: 1) the recorded signals were evoked by neuron responses rather than by artifact; 2) responses evoked by periodic signals were significantly higher than those evoked by the white noise; 3) the latency of the responses fell in the expected range; 4) the responses decreased significantly after death of the guinea pigs; and 5) the responses decreased significantly when the animal was replaced by an electrical resistance. All of these results suggest the method was valid. Recording obtained using complex tones with a missing fundamental component and using pure tones with various frequencies were consistent with those obtained using acoustic stimulation in previous studies.     

Effects of SARA on Oxygen–Glucose Deprivation in PC12 Cell Line

Ischemic stroke is a major composition of cerebrovascular disease, seriously threatening to human health in the world. Activin A (ActA), belonging to transforming growth factor-beta (TGF-β) super family, plays an important role in the hypoxic-ischemic brain injury through ActA/Smads pathway. While as an essential phosphorylation assistor in TGF-β signaling, the functions and mechanisms of smad anchor for receptor activation (SARA) in ischemic brain injury remain poorly understood. To solve this problem and explore the pathological processes of ischemic stroke, we used an Oxygen–Glucose deprivation (OGD) model in nerve growth factor-induced differentiated rattus PC12 pheochromocytoma cells and down regulated the expressions of SARA by RNA interference technology. Our results showed that the repression of SARA before OGD exposure reduced the expressions of Smad2, 3, 4 mRNA and the phosphorylation rate of Smad2 protein, but it did not affect the mRNA expressions of Smad7. After OGD treatment, ActA/Smads pathway was activated and the expression of SARA in the SARA pre-repression group was significantly up-regulated. The pre-repression of SARA increased the sensitivities of nerve-like cells to OGD damage. Moreover, the mRNA expression of Smad7 which was supposed to participate in the negative feedback of ActA/Smads pathway was also elevated due to OGD injury. Taken together, these results suggest a positive role of SARA in assisting the phosphorylation of Smad2 and maintaining the neuron protective effect of ActA/Smads pathway.