Assay of zofenopril and its active metabolite zofenoprilat by liquid chromatography coupled with tandem mass spectrometry

Zofenopril is a pro-drug designed to undergo metabolic hydrolysis yielding the active free sulfhydryl compound zofenoprilat, which is an angiotensin converting enzyme (ACE) inhibitor, endowed also with a marked cardioprotective activity. A simple, highly sensitive specific LC–MS–MS method was developed for the determination of zofenopril and zofenoprilat in human plasma. In order to prevent oxidative degradation of zofenoprilat and its internal standard, their free sulfhydryl groups were protected by treatment with N-ethylmaleimide (NEM), which produced the succinimide derivatives. The compounds and their corresponding fluorine derivatives, used as internal standards, were extracted from plasma with toluene. The reconstituted dried extracts were chromatographed and then monitored by a triple-stage-quadrupole instrument operating in the negative ion spray ionization mode. The method was validated over the concentration range of 1–300 ng/ml for zofenopril and 2–600 ng/ml for zofenoprilat. Inter- and intra-assay precision and accuracy of both zofenopril and zofenoprilat were better than 10%. The limit of quantitation was 1 ng/ml with zofenopril and 2 ng/ml with zofenoprilat. Extraction recovery proved to be on average 84.8% with zofenopril and 70.1% with zofenoprilat. Similar recoveries were shown by the above two internal standards. The method was applied to measure plasma concentrations of zofenopril and zofenoprilat in 18 healthy volunteers treated orally with zofenopril calcium salt at the dose of 60 mg.     

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.     

Cardiac Fibroblasts Contribute to Myocardial Dysfunction in Mice with Sepsis: The Role of NLRP3 Inflammasome Activation

Myocardial contractile dysfunction in sepsis is associated with the increased morbidity and mortality. Although the underlying mechanisms of the cardiac depression have not been fully elucidated, an exaggerated inflammatory response is believed to be responsible. Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome is an intracellular platform that is involved in the maturation and release of interleukin (IL)-1β. The aim of the present study is to evaluate whether sepsis activates NLRP3 inflammasome/caspase-1/IL-1β pathway in cardiac fibroblasts (CFs) and whether this cytokine can subsequently impact the function of cardiomyocytes (cardiac fibroblast-myocyte cross-talk). We show that treatment of CFs with lipopolysaccharide (LPS) induces upregulation of NLRP3, activation of caspase-1, as well as the maturation (activation) and release of IL-1β. In addition, the genetic (small interfering ribonucleic acid [siRNA]) and pharmacological (glyburide) inhibition of the NLRP3 inflammasome in CFs can block this signaling pathway. Furthermore, the inhibition of the NLRP3 inflammasome in cardiac fibroblasts ameliorated the ability of LPS-chalenged CFs to impact cardiomyocyte function as assessed by intracellular cyclic adenosine monophosphate (cAMP) responses in cardiomyocytes. Salient features of this the NLP3 inflammasome/ caspase-1 pathway were confirmed in in vivo models of endotoxemia/sepsis. We found that inhibition of the NLRP3 inflammasome attenuated myocardial dysfunction in mice with LPS and increased the survival rate in mice with feces-induced peritonitis. Our results indicate that the activation of the NLRP3 inflammasome in cardiac fibroblasts is pivotal in the induction of myocardial dysfunction in sepsis.     

Modulatory effect of Lactobacillus acidophilus KLDS 1.0738 on intestinal short‐chain fatty acids metabolism and GPR41/43 expression in β‐lactoglobulin–sensitized mice

We investigated the correlation between the beneficial effect of Lactobacillus acidophilus on gut microbiota composition, metabolic activities, and reducing cow's milk protein allergy. Mice sensitized with β‐lactoglobulin (β‐Lg) were treated with different doses of L. acidophilus KLDS 1.0738 for 4 weeks, starting 1 week before allergen induction. The results showed that intake of L. acidophilus significantly suppressed the hypersensitivity responses, together with increased fecal microbiota diversity and short‐chain fatty acids (SCFAs) concentration (including propionate, butyrate, isobutyrate, and isovalerate) when compared with the allergic group. Moreover, treatment with L. acidophilus induced the expression of SCFAs receptors, G‐protein–coupled receptors 41 (GPR41) and 43 (GPR43), in the spleen and colon of the allergic mice. Further analysis revealed that the GPR41 and GPR43 messenger RNA expression both positively correlated with the serum concentrations of transforming growth factor‐β and IFN‐γ (p < .05), but negatively with the serum concentrations of IL‐17, IL‐4, and IL‐6 in the L. acidophilus–treated group compared with the allergic group (p < .05). These results suggested that L. acidophilus protected against the development of allergic inflammation by improving the intestinal flora, as well as upregulating SCFAs and their receptors GPR41/43.