Changes in S-(2-succinyl)cysteine and advanced glycation end-products levels in mouse tissues associated with aging

Cysteine is non-enzymatically modified by fumarate, which is an intermediate of the tricarboxylic acid cycle, leading to the formation of S-(2-succinyl)cysteine (2SC). Post-translational modification of physiological proteins by fumarate causes enzyme dysfunction. The aim of the study was to evaluate the changes in 2SC accumulation in physiological tissues associated with aging. Brain, liver, kidney, and serum samples were collected from 4-, 12-, and 96-week-old male C57BL/6J mice, and the level of 2SC was determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS) after pretreatment, including delipidation, protein precipitation, and hydrolysis using hydrochloric acid. The 2SC level in the brain was higher than that in other tissues, and its accumulation significantly increased with age. Similarly, N^ε-(carboxymethyl)lysine levels, an advanced glycation end-products (AGEs) that accumulates in tissues in an age-dependent manner, was found to be increased in the brain and kidneys of elderly mice. Accumulation of N^δ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine increased significantly with age, but only in the kidneys. The fumarate content in the brain was similar to that in the liver and kidney at 4 and 12 weeks of age. Furthermore, fumarate contents increased in the liver and kidney at 96 weeks of age, whereas its level did not change in the brain. Our results demonstrated that the changes in 2SC and AGEs levels in tissues reflected differing metabolism and enhanced oxidative stress in each organ; in particular, the metabolism in the brain and kidneys is highly affected by aging.

     

Closed embryo culture system improved embryological and clinical outcome for single vitrified-warmed blastocyst transfer: A single-center large cohort study

While some studies have shown that the closed embryo culture system (CCS) is a possible improvement over standard embryo culture systems (STS) in terms of early embryonic development, information on clinical outcomes of culturing blastocysts following single vitrified-warmed blastocyst transfer (SVBT) in the CCS and STS remains limited. Therefore, the objective of this single-center, large-cohort, retrospective study was to compare embryonic development until the blastocyst stage and clinical outcomes following SVBT between CCS and STS. From May 2017 to October 2018, 2420 oocytes from 1402 patients who underwent in vitro fertilization and blastocyst culture after minimal stimulation were divided into two groups (CCS and STS). The main outcome measures in the two groups were embryological (blastocyst formation rates and utilized blastocyst rates) and clinical outcomes (ongoing pregnancy rates) after a single vitrified-warmed blastocyst transfer (SVBT). There were no significant differences in the blastocyst formation rates between the CCS and STS groups (59.6% versus 59.1%, p = 0.81). However, there were significant differences in utilized blastocyst rates (51.0% versus 46.6%, p < 0.05). Ongoing pregnancy rates per SVBT cycle were significantly higher in the CCS group than in the STS group (41.4% versus 34.4%, p < 0.05). Moreover, after applying multivariable logistic regression analysis, the type of embryo culture system (CCS to STS, adjusted odds ratios: 1.41, 95% CI: 1.04–1.91) was correlated with ongoing pregnancy. Our study suggests that compared to STS, CCS could improve utilized blastocyst rates and ongoing pregnancy rates to a greater extent, following SVBT.     

Predictors for Stroke and Death in Non-Anticoagulated Asian Patients with Atrial Fibrillation: The Fushimi AF Registry

Background

Atrial fibrillation (AF) increases the risk of stroke and death. Data on the predictors for stroke and death in ‘real-world’ AF patients are limited, especially from large prospective Asian cohorts.

Methods

The Fushimi AF Registry is a community-based prospective survey designed to enroll all AF patients who visited the participating medical institutions in Fushimi-ku, Kyoto, Japan. Follow-up data were available for 3,304 patients (median follow-up period 741 days). We explored the predictors for ‘death, stroke, and systemic embolism (SE)’ during follow-up in 1,541 patients not receiving oral anticoagulants (OAC) at baseline.

Results

The mean age was 73.1 ± 12.5 years, and 673 (44%) patients were female. The mean CHADS₂ and CHA₂DS₂-VASc scores were 1.76 and 3.08, respectively. Cumulative events were as follows: stroke/SE in 61 (4%) and death in 230 (15%), respectively. On multivariate analysis, advanced age (hazard ratio (HR): 1.68, 95% confidence interval (CI): 1.24–2.29), underweight (body mass index <18.5 kg/m²) (HR: 1.71, 95% CI: 1.25–2.32), previous stroke/SE/transient ischemic attack (HR: 1.70, 95% CI: 1.25–2.30), heart failure (HR: 1.59, 95% CI: 1.17–2.15), chronic kidney disease (HR: 1.53, 95% CI: 1.16–2.02), and anemia (HR: 2.41, 95% CI: 1.78–3.28) were independent predictors for death/stroke/SE. Cumulative numbers of these 6 risk predictors could stratify the incidence of death/stroke/SE in patients without OAC, as well as those with OAC in our registry.

Conclusions

Advanced age, underweight, previous stroke/SE/transient ischemic attack, heart failure, chronic kidney disease, and anemia were independently associated with the risk of death/stroke/SE in non-anticoagulated Japanese AF patients.     

Receptors for calcitonin gene-related peptide on the rat liver plasma membranes

Specific binding sites for calcitonin gene-related peptide (CGRP) were identified in the rat liver plasma membrane. The binding of 125I-[TyrO]rat CGRP to rat liver plasma membrane was time dependent, saturable and reversible. Scatchard analysis of the data revealed a single class of binding sites with apparent dissociation constant of 260.8 pM and a maximal binding capacity of 26.6 fmol/mg of protein. Rat, chick, and human CGRP and their synthetic analogues inhibited label binding in a dose-dependent manner with relative potencies as follows; chick greater than rat greater than human greater than [TyrO]rat CGRP. Salmon, human and [Asu1'7]eel calcitonin also inhibited label binding but only at higher concentrations. These results clearly indicate the presence of specific binding sites for CGRP in rat liver plasma membrane and suggest that CGRP has possible biological actions on the rat liver.     

Lint‐O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain

Loss‐of‐function mutations in Drosophila lethal(3)malignant brain tumor [l(3)mbt] cause ectopic expression of germline genes and brain tumors. Loss of L(3)mbt function in ovarian somatic cells (OSCs) aberrantly activates germ‐specific piRNA amplification and leads to infertility. However, the underlying mechanism remains unclear. Here, ChIP‐seq for L(3)mbt in cultured OSCs and RNA‐seq before and after L(3)mbt depletion shows that L(3)mbt genomic binding is not necessarily linked to gene regulation and that L(3)mbt controls piRNA pathway genes in multiple ways. Lack of known L(3)mbt co‐repressors, such as Lint‐1, has little effect on the levels of piRNA amplifiers. Identification of L(3)mbt interactors in OSCs and subsequent analysis reveals CG2662 as a novel co‐regulator of L(3)mbt, termed “L(3)mbt interactor in OSCs” (Lint‐O). Most of the L(3)mbt‐bound piRNA amplifier genes are also bound by Lint‐O in a similar fashion. Loss of Lint‐O impacts the levels of piRNA amplifiers, similar to the lack of L(3)mbt. The lint‐O‐deficient flies exhibit female sterility and tumorous brains. Thus, L(3)mbt and its novel co‐suppressor Lint‐O cooperate in suppressing target genes to maintain homeostasis in the ovary and brain.     

Isolation and mass spectrometry characterization of molecular species of lactosylceramides using liquid chromatography-electrospray ion trap mass spectrometry

Reverse-phase liquid chromatography/electrospray ion trap mass spectrometry (LC-ESI-MSn) was established for identification of the molecular species of lactosylceramides. Lactosylceramides derived from porcine blood cells were separated on a CapcellPak C8 column using a mixture of methanol and 1 mM ammonium formate from the C16 to C26 fatty acyl chains based on the length of total carbon chains and the nature of sphingoid bases (w') and fatty acyl chains (Y0'-w') was identified by MS3 as their [M+H]+ ions. The same number of fatty acyl moieties appeared in the order of unsaturated, (2-)hydroxylated, and saturated components. The molecular species of lactosylceramides derived from porcine blood cells totaled more than 33 and included mainly C24:0-d18:1, Ch24:0-d18:1, Ch24:1-d18:1, C24:1-d18:1, and C22:0-d18:1 in addition to 28 minor species from C16:0 to C26:0 fatty acyl moieties. The molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells (70% were differentiated into macrophage-lineage cells) were identified as C24:0-d18:1, C24:1-d18:1, C22:0-d18:1, C16:0-d18:1, and more than 21 other minor species. Our results suggest that reverse-phase LC-ESI-MSn is a useful and simple method for identification of lactosylceramide molecular species.     

The transmembrane adaptor Cbp/PAG1 controls the malignant potential of human non-small cell lung cancers that have c-src upregulation

The tyrosine kinase c-Src is upregulated in various human cancers, although the precise regulatory mechanism underlying this upregulation is unclear. We previously reported that a transmembrane adaptor Csk-binding protein (Cbp; PAG1) plays an important role in controlling the cell transformation that is induced by the activation of c-Src. To elucidate the in vivo role of Cbp, we examined the function of Cbp in lung cancer cell lines and tissues. In this study, we found that Cbp was markedly downregulated in human non-small cell lung cancer (NSCLC) cells. The ectopic expression of Cbp suppressed the anchorage-independent growth of the NSCLC cell lines (A549 and Lu99) that had upregulated c-Src, whereas the Cbp expression had little effect on other NSCLC cell lines (PC9 and Lu65) that express normal levels of c-Src. The expression of Cbp suppressed the kinase activity of c-Src in A549 cells by recruiting c-Src and its negative regulator, C-terminal Src kinase (Csk), to lipid rafts. The treatment with Src inhibitors, such as PP2, dasatinib, and saracatinib, also suppressed the growth of A549 cells. Furthermore, Cbp expression attenuated the ability of A549 cells to form tumors in nude mice, invade in vitro, and metastasize in vivo. In addition, we found a significant inverse correlation between the level of Cbp expression and the extent of lymph node metastasis in human lung cancers. These results indicate that Cbp is required for the Csk-mediated inactivation of c-Src and may control the promotion of malignancy in NSCLC tumors that are characterized by c-Src upregulation.     

Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses

Background

Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.

Methods

BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.

Results

Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.

Conclusion

These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.