MEK�CERK-dependent multiple caspase activation by mitochondrial proapoptotic Bcl-2 family proteins is essential for heavy ion irradiation-induced glioma cell death

Recently developed heavy ion irradiation therapy using a carbon beam (CB) against systemic malignancy has numerous advantages. However, the clinical results of CB therapy against glioblastoma still have room for improvement. Therefore, we tried to clarify the molecular mechanism of CB-induced glioma cell death. T98G and U251 human glioblastoma cell lines were irradiated by CB, and caspase-dependent apoptosis was induced in both cell lines in a dose-dependent manner. Knockdown of Bax (BCL-2-associated X protein) and Bak (BCL-2-associated killer) and overexpression of Bcl-2 or Bcl-xl (B-cell lymphoma-extra large) showed the involvement of Bcl-2 family proteins upstream of caspase activation, including caspase-8, in CB-induced glioma cell death. We also detected the activation of extracellular signal-regulated kinase (ERK) and the knockdown of ERK regulator mitogen-activated protein kinase kinase (MEK)1/2 or overexpression of a dominant-negative (DN) ERK inhibited CB-induced glioma cell death upstream of the mitochondria. In addition, application of MEK-specific inhibitors for defined periods showed that the recovery of activation of ERK between 2 and 36 h after irradiation is essential for CB-induced glioma cell death. Furthermore, MEK inhibitors or overexpression of a DN ERK failed to significantly inhibit X-ray-induced T98G and U251 cell death. These results suggested that the MEK–ERK cascade has a crucial role in CB-induced glioma cell death, which is known to have a limited contribution to X-ray-induced glioma cell death.     

Spatio-temporal expression profile of stem cell-associated gene LGR5 in the intestine during thyroid hormone-dependent metamorphosis in Xenopus laevis

Background

The intestinal epithelium undergoes constant self-renewal throughout adult life across vertebrates. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells. This self-renewal system is established in the so-called postembryonic developmental period in mammals when endogenous thyroid hormone (T3) levels are high.

Methodology/Principal Findings

The T3-dependent metamorphosis in anurans like Xenopus laevis resembles the mammalian postembryonic development and offers a unique opportunity to study how the adult stem cells are developed. The tadpole intestine is predominantly a monolayer of larval epithelial cells. During metamorphosis, the larval epithelial cells undergo apoptosis and, concurrently, adult epithelial stem/progenitor cells develop de novo, rapidly proliferate, and then differentiate to establish a trough-crest axis of the epithelial fold, resembling the crypt-villus axis in the adult mammalian intestine. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a well-established stem cell marker in the adult mouse intestinal crypt. Here we have cloned and analyzed the spatiotemporal expression profile of LGR5 gene during frog metamorphosis. We show that the two duplicated LGR5 genes in Xenopus laevis and the LGR5 gene in Xenopus tropicalis are highly homologous to the LGR5 in other vertebrates. The expression of LGR5 is induced in the limb, tail, and intestine by T3 during metamorphosis. More importantly, LGR5 mRNA is localized to the developing adult epithelial stem cells of the intestine.

Conclusions/Significance

These results suggest that LGR5-expressing cells are the stem/progenitor cells of the adult intestine and that LGR5 plays a role in the development and/or maintenance of the adult intestinal stem cells during postembryonic development in vertebrates.     

Ionotropic glutamate receptor AMPA 1 is associated with ovulation rate

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1(Asn) has a weaker affinity to glutamate than GRIA1(Ser), both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1(Asn) have a slower luteinizing hormone (LH) surge than cows with GRIA1(Ser). In addition, cows with GRIA1(Asn) possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1(Ser). Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy.     

Effects of escins Ia, Ib, IIa, and IIb from horse chestnuts on gastrointestinal transit and ileus in mice.

The effects of saponin fraction and its principal constituents escins Ia (1), Ib (2), IIa (3), and IIb (4) from horse chestnuts on gastrointestinal transit (GIT) and ileus were investigated in mice. Ileus was induced by acetic acid peritoneal irritation or by laparotomy with manipulation. One hour after the oral administration, the saponin fraction (12.5-100 mg/kg) and 14 (12.5-50 mg/ kg, except for 3 at 12.5 mg/kg) dose-dependently accelerated GIT. The optimal effects of the saponin fraction (25 mg/kg) occurred 5-240 min (applied intervals between the fraction and the charcoal meal) after the oral administration. The fraction (12.5-100 mg/ kg) and 1-4 (12.5-50 mg/kg, except for 1 and 2 at 12.5 mg/kg) dose-dependently prevented the inhibition of GIT induced by the acetic acid peritoneal irritation. They (12.5-100mg/kg) also dose-dependently prevented the inhibition of GIT induced by the laparotomy with manipulation. Desacylescins I (5) and II (6) (50 mg/kg) showed no such effects. These results demonstrated that the saponin fraction and 1-4 accelerated GIT and prevented the experimental ileus, and indicate that the 21, 22-acyl groups are essential for the accelerative effects of 1-4. The accelerations of GIT by 1-4 were completely abolished by the pretreatment with streptozotocin (100 mg/kg, iv), but not by the pretreatment with capsaicin (75 mg/kg in total, sc) or atropine (10 mg/kg, sc). These results imply that the sympathetic nervous system may be, but neither capsaicin-sensitive sensory nerves nor the cholinergic mechanism, involved in the accelerations of GIT by escins 1-4.     

Structure-related inhibitory activity of oleanolic acid glycosides on gastric emptying in mice

We examined the effects of various oleanolic acid oligoglycosides obtained from traditional herbs on gastric emptying in non-nutrient meal- or nutrient meal-loaded mice. Test samples were given orally to fasted mice 0.5 h before loading of test meals. Oleanolic acid 3-O-monodesmosides [oleanolic acid 3-O-glucuronide (3, 12.5-50 mg/kg), momordin Ic (4, 25 and 50 mg/kg), momordin I (6, 12.5-50 mg/kg), and 28-O-deglucosyl-chikusetsusaponins IV (8, 12.5-50 mg/kg) and V (10, 50 mg/kg)] were found to show inhibitory effects on gastric emptying in 1.5% CMC-Na test meal-loaded mice. 4, 6, and 8 also inhibited gastric emptying in mice given 40% glucose test meal, milk test meal, and 60% ethanol test meal. 3 inhibited gastric emptying in mice given milk test meal or 60% ethanol test meal, but lacked significant inhibition in 40% glucose test meal-loaded mice. 10 (50 mg/kg) also slightly inhibited gastric emptying in milk test meal-loaded mice, but lacked the significant inhibition in mice given 40% glucose or 60% ethanol test meal. Whereas oleanolic acid 3,28-0-bisdesmosides [momordin IIc (5), chikusetsusaponins IV (7) and V (9)], oleanolic acid 28-O-monodesmoside [compound O (2)], and their common aglycon [oleanolic acid (1)] showed no such effects at dose of 50 mg/kg. 28-O-Deglucosyl-chikusetsusaponin V (10) showed a little inhibition in these experiments. These results indicate that both the 3-O-monodesmoside structure and 28-carboxyl group were confirmed to be essential for such activity, and the 28-ester glucoside moiety and 2'-O-beta-D-glucopyranoside moiety reduce the activity.     

<Emphasis Type="Italic">Legionella</Emphasis> pneumonia due to non-<Emphasis Type="Italic">Legionella pneumophila</Emphasis> serogroup 1: usefulness of the six-point scoring system

Background

Because of a limited number of reports, we aimed to investigate the clinical characteristics of patients with Legionella pneumonia due to non-Legionella pneumophila serogroup 1 and the diagnostic usefulness of the six-point scoring system for such patients compared with patients with pneumonia caused by L. pneumophila serogroup 1.

Methods

We retrospectively analysed patients diagnosed with Legionella pneumonia due to non-L. pneumophila serogroup 1 between March 2001 and June 2016. We examined the clinical characteristics, including symptoms, laboratory findings, radiologic findings, pneumonia severity, initial treatment and prognosis. We also calculated scores using the six-point scoring system in these patients. Furthermore, we compared the clinical characteristics and six-point scores between non-L. pneumophila serogroup 1 patients and L. pneumophila serogroup 1 patients among hospitalized community-acquired pneumonia patients enrolled prospectively between October 2010 and July 2016.

Results

Eleven patients had pneumonia due to non-L. pneumophila serogroup 1; their median age was 66 years and 8 patients (72.7%) were male. The most common pathogen was L. pneumophila serogroup 3 (6/11), followed by L. pneumophila serogroup 9 (3/11), L. pneumophila serogroup 6 (1/11) and L. longbeachae (1/11). Non-specific symptoms, such as fever and cough, were common. Six patients (54.5%) had liver enzyme elevation, but no patient developed hyponatraemia at <130 mEq/L. Nine patients (81.8%) showed lobar pneumonia and 7 patients (63.6%) manifested with consolidation and ground-glass opacity. Patients with mild to moderate severity comprised 10 (90.9%) by CURB-65 and 5 (45.5%) by the Pneumonia Severity Index. Of all patients, 4 were admitted to the intensive care unit and 3 died despite appropriate empiric therapy. The clinical characteristics were not significantly different between non-L. pneumophila serogroup 1 patients and L. pneumophila serogroup 1 patients (n?=?23). At a cut-off value of ≥?2 points, the sensitivity of the six-point scoring system was 54.5% (6/11) for non-L. pneumophila serogroup 1 patients and 95.7% (22/23) for L. pneumophila serogroup 1 patients.

Conclusions

Cases of non-L. pneumophila serogroup 1 pneumonia varied in severity from mild to severe and the clinical characteristics were often non-specific. The six-point scoring system was not useful in predicting such Legionella pneumonia cases.

     

Lipophagy maintains energy homeostasis in the kidney proximal tubule during prolonged starvation

Macroautophagy/autophagy is a self-degradation process that combats starvation. Lipids are the main energy source in kidney proximal tubular cells (PTCs). During starvation, PTCs increase fatty acid (FA) uptake, form intracellular lipid droplets (LDs), and hydrolyze them for use. The involvement of autophagy in lipid metabolism in the kidney remains largely unknown. Here, we investigated the autophagy-mediated regulation of renal lipid metabolism during prolonged starvation using PTC-specific Atg5-deficient (atg5-TSKO) mice and an in vitro serum starvation model. Twenty-four h of starvation comparably induced LD formation in the PTCs of control and atg5-TSKO mice; however, additional 24 h of starvation reduced the number of LDs in control mice, whereas increases were observed in atg5-TSKO mice. Autophagic degradation of LDs (lipophagy) in PTCs was demonstrated by electron microscopic observation and biochemical analysis. In vitro pulse-chase assays demonstrated that lipophagy mobilizes FAs from LDs to mitochondria during starvation, whereas impaired LD degradation in autophagy-deficient PTCs led to decreased ATP production and subsequent cell death. In contrast to the in vitro assay, despite impaired LD degradation, kidney ATP content was preserved in 48-h starved atg5-TSKO mice, probably due to increased utilization of ketone bodies. This compensatory mechanism was accompanied by a higher plasma FGF21 (fibroblast growth factor 21) level and its expression in the PTCs; however, this was not essential for the production of ketone bodies in the liver during prolonged starvation. In conclusion, lipophagy combats prolonged starvation in PTCs to avoid cellular energy depletion.     

Cryptic diversity and diversification processes in three cis-Andean Rhamdia species (Siluriformes: Heptapteridae) revealed by DNA barcoding

The wide distribution of the Neotropical freshwater catfish Rhamdia offers an excellent opportunity to investigate the historical processes responsible for modeling South America’s hydrogeological structure. We used sequences from cis-Andean and Mesoamerican Rhamdia species to reconstruct and estimate divergence times among cis-Andean lineages, correlating the results with known geological events. Species delimitation methods based on distance (DNA barcoding and BIN) and coalescence (GMYC) approaches identified nine well-supported lineages from the cis-Andean region from sequences available in the BOLD dataset. The cis-Andean Rhamdia lineages diversification process began in Eocene and represented the split between cis-Andean and Mesoamerican clades. The cis-Andean clade contains two principal groups: Northwest clade (MOTUs from Amazon, Essequibo, Paraguay, and Itapecuru basins) and Southeast clade (Eastern Brazilian shield basins (Paraná, Uruguay, Iguaçu, and São Francisco) plus eastern coastal basins). The diversification of the cis-Andean Rhamdia lineages results from vicariance and geodispersion events, which played a key role in the current intricate distribution pattern of the Rhamdia lineages. The wide geographical distribution and large size of the specimens make it attractive to cultivate in different countries of the Neotropical region. The lineages delimitation minimizes identification mistakes, unintentional crossings by aquaculture, and reduces natural stocks contamination.     

Sphingolipid synthesis is involved in autophagy in Saccharomyces cerevisiae

In eukaryotes, autophagy is a conserved protein degradation system that degrades cytoplasmic components by encompassing them with double-membrane structures, called autophagosomes, and delivering them to the lytic compartments of vacuoles/lysosomes. Certain Atg proteins are known to be involved in autophagy, yet the identity and function of lipid molecules involved remain largely unknown. We investigated the involvement of sphingolipids in autophagy using Saccharomyces cerevisiae. Inhibiting synthesis of the simplest complex sphingolipid, inositol phosphorylceramide (IPC), resulted in reduced autophagic activities. Similar results were obtained using myriocin, an inhibitor of the first step in sphingolipid synthesis. Our results indicate that sphingolipids, especially IPC, are required for autophagy. Inhibition of sphingolipid synthesis had no effect on formation of Atg12-Atg5 or Atg8-phosphatidylethanolamine conjugates, on maturation of vacuolar proteases, or on formation of the pre-autophagosomal structure (PAS). These results suggest that sphingolipids are not involved in the cellular signaling that leads to formation of the PAS, but may be involved in the process of autophagosome formation.