Acid ceramidase controls apoptosis and increases autophagy in human melanoma cells treated with doxorubicin

Acid ceramidase (AC) is a lysosomal hydrolase encoded by the ASAH1 gene, which cleaves ceramides into sphingosine and fatty acid. AC is expressed at high levels in most human melanoma cell lines and may confer resistance against chemotherapeutic agents. One such agent, doxorubicin, was shown to increase ceramide levels in melanoma cells. Ceramides contribute to the regulation of autophagy and apoptosis. Here we investigated the impact of AC ablation via CRISPR-Cas9 gene editing on the response of A375 melanoma cells to doxorubicin. We found that doxorubicin activates the autophagic response in wild-type A375 cells, which effectively resist apoptotic cell death. In striking contrast, doxorubicin fails to stimulate autophagy in A375 AC-null cells, which rapidly undergo apoptosis when exposed to the drug. The present work highlights changes that affect melanoma cells during incubation with doxorubicin, in A375 melanoma cells lacking AC. We found that the remarkable reduction in recovery rate after doxorubicin treatment is strictly associated with the impairment of autophagy, that forces the AC-inhibited cells into apoptotic path

Hepatoprotective effects of B-1,3-(D)-Glucan on bortezomib-induced liver damage in rats

The aim of this study was to evaluate the effects of beta -1,3-(D)-glucan as an antioxidant and tissue protective agent and study the biochemical, histopathologic, and immunohistochemical effects of first therapeutic proteasome inhibitor bortezomib on the liver for treating relapsed multiple myeloma. The experiment included 36 adult male rats, which were divided into four treatment groups: control (healthy); bortezomib-treated; beta-1,3-D-glucan-treated; and bortezomib + beta-1,3-(D)-glucan-treated. Each group was subdivided into two subgroups based on time of sacrifice (48 or 72 h). After the experiments, superoxide dismutase (SOD) activity and lipid peroxidation (LPO) amounts were determined, and immunohistochemical and histopathological changes were examined in all rat liver tissues. beta -1,3-(D)-Glucan treatment normalized changes of LPO and stimulated an over activity of endogenous SOD. The results of the histopathologic parameters showed that treatment with beta -1,3-(D)-Glucan in the bortezomib group ameliorated the development of non-specific reactive hepatitis (NSRH) and Kupffer cell activation via NF-kB. Administration of beta -1,3-(D)-Glucan is effective in reversing tissue damage induced by bortezomib in rat livers.Bu çalışmanın amacı, relaps multiple miyelom tedavi etmek için kullanılan ilk terapötik proteazom inhibitörü olan bortezomibin karaciğer üzerine immunohistokimyasal, histopatolojik ve biyokimyasal etkilerini araştırmak ve bir antioksidant ve doku koruyucu ajan olarak B-1,3-(D)-glukanın etkilerini değerlendirmekdi. Deney; kontrol (sağlıklı), bortezomib ile tedavi, B-1,3-(D)-glukan ile tedavi ve bortezomib + B-1,3-(D)-glukan ile tedavi olmak üzere dört tedavi grubuna bölünen 36 yetişkin erkek sıçan içerdi. Her bir grup sakrifikasyon zamanına (48 veya 72 saat) göre iki alt gruba ayrıldı. Deneylerin bitiminden sonra, süperoksit dismutaz (SOD) aktivitesi ve lipid peroksidasyon (LPO) miktarları ölçüldü ve tüm sıçan karaciğer dokularında immünohistokimyasal ve histopatolojik değişiklikler incelendi. B-1,3-(D)-glukan ile tedavi LPO değişikliğini normalize etti ve endojen SOD aktivitesi aşırı uyardı. Histopatolojik parametrelerin sonuçları, bortezomib grubunda B-1,3-(D)-glukan ile tedavi NF-kB yoluyla Kupffer hücre aktivasyonunu ve non-spesifik reaktif hepatit (NSRH) gelişimini regüle ettiğini gösterdi. B-1,3-(D)-glukan uygulaması, sıçan karaciğerinde bortezomibin neden olduğu geri döndürülebilir doku hasarında efektifdir

Gem1 and ERMES Do Not Directly Affect Phosphatidylserine Transport from ER to Mitochondria or Mitochondrial Inheritance

In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology

Gastrointestinal quality of life in patients with asymptomatic cholelithiasis after laparoscopic cholecystectomy

To assess the outcome of laparoscopic cholecystectomy for asymptomatic cholelithiasis before and after laparoscopic cholecystectomy using a specific quality of life instrument for gastrointestinal disorders in adults : The Gastrointestinal Quality of Life Index (GIQLI) was used to study the quality of life in patients before and after laparoscopic cholecystectomy : Seventy one patients completed the GIQLI questionnaire both preoperatively and after a minimum postoperative follow-up of three months. Mean preoperative score was 126.8±14.07 out of a theoretical maximum score of 144.After three months, the score had significantly improved to 136.6±9.31, close to the range for the normal population. Not only items assessing gastrointestinal symptoms but also the domains of physical, social, and emotional function improved significantly. The most marked improvements were achieved in patients with the lowest preoperative scores. Laparoscopic cholecystectomy significantly improves the quality of life in patients with cholelithiasis who are asymptomatic or have nonspecific gastrointestinal symptoms that cannot be explained by another gastrointestinal pathology.Bu çalısmada asemptomatik kolelitiyazis olgularının ameliyat öncesi ve ameliyat sonrası hayat kalitesi degerlendirilmistir. Hastaların ameliyat öncesi ve sonrası hayat kalitelerinin ölçümü gastrointestinal hayat kalitesi indeksi parametreleri kullanılarak belirlenmistir. Çalısmaya alınan 71 hasta ameliyat öncesi ve ameliyattan en az 3 ay sonra gastrointestinal hayat kalitesi indeksine göre sorgulanmıstır. Preoperatif dönemde ortalama skor 126.8±14.07, 3 ay sonra yapılan sorgulamada ise ortalama 136.6±9.31 olup normal populasyona yakın bir oranda saptanmıstır. (toplam skor 144). Gastrointestinal semptomlarının yanı sıra fiziksel, sosyal ve duygusal durumlarında da anlamlı düzelme gözlenmistir.Düsük skorlu hastalarda bu iyilesmedaha belirgindir. Asemptomatik kolelitiyazisli hastalarda baska gastrointestinal patolojilerle açıklanamayan nonspesifik semptomların laparoskopik kolesistektomiyle iyilesme gösterdigi, hastaların hayat kalitelerinde anlamlı bir artıs oldugu görülmektedir

PCSK9 deficiency alters brain lipid composition without affecting brain development and function

PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor (LDLR) in the liver, hereby preventing removal of LDL cholesterol from the circulation. Accordingly, PCSK9 inhibitory antibodies and siRNA potently reduce LDL cholesterol to unprecedented low levels and are approved for treatment of hypercholesterolemia. In addition, PCSK9 inactivation alters the levels of several other circulating lipid classes and species. Brain function is critically influenced by cholesterol and lipid composition. However, it remains unclear how the brain is affected long-term by the reduction in circulating lipids as achieved with potent lipid lowering therapeutics such as PCSK9 inhibitors. Furthermore, it is unknown if locally expressed PCSK9 affects neuronal circuits through regulation of receptor levels. We have studied the effect of lifelong low peripheral cholesterol levels on brain lipid composition and behavior in adult PCSK9 KO mice. In addition, we studied the effect of PCSK9 on neurons in culture and in vivo in the developing cerebral cortex. We found that PCSK9 reduced LDLR and neurite complexity in cultured neurons, but neither PCSK9 KO nor overexpression affected cortical development in vivo. Interestingly, PCSK9 deficiency resulted in changes of several lipid classes in the adult cortex and cerebellum. Despite the observed changes, PCSK9 KO mice had unchanged behavior compared to WT controls. In conclusion, our findings demonstrate that altered PCSK9 levels do not compromise brain development or function in mice, and are in line with clinical trials showing that PCSK9 inhibitors have no adverse effects on cognitive function

Quantitative Profiling of Long-Chain Bases by Mass Tagging and Parallel Reaction Monitoring

Long-chain bases (LCBs) are both intermediates in sphingolipid metabolism and potent signaling molecules that control cellular processes. To understand how regulation of sphingolipid metabolism and levels of individual LCB species impinge upon physiological and pathophysiological processes requires sensitive and specific assays for monitoring these molecules. Here we describe a shotgun lipidomics method for quantitative profiling of LCB molecules. The method employs a "mass-tag" strategy where LCBs are chemically derivatized with deuterated methyliodide (CD3I) to produce trimethylated derivatives having a positively charged quaternary amine group. This chemical derivatization minimizes unwanted in-source fragmentation of LCB analytes and prompts a characteristic trimethylaminium fragment ion that enables sensitive and quantitative profiling of LCB molecules by parallel reaction monitoring on a hybrid quadrupole time-of-flight mass spectrometer. Notably, the strategy provides, for the first time, a routine for monitoring endogenous 3-ketosphinganine molecules and distinguishing them from more abundant isomeric sphingosine molecules. To demonstrate the efficacy of the methodology we report an in-depth characterization of the LCB composition of yeast mutants with defective sphingolipid metabolism and the absolute levels of LCBs in mammalian cells. The strategy is generic, applicable to other types of mass spectrometers and can readily be applied as an additional routine in workflows for global lipidome quantification and for functional studies of sphingolipid metabolism