Leukotrienes (LTs) and sphingolipids are critical lipid mediators participating in numerous cellular signal transduction events and developing various disorders, such as bronchial hyperactivity leading to asthma. Enzymatic reactions initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to LTs and serine palmitoyltransferase (SPT)-mediated de novo synthesis of sphingolipids. Previous studies have shown that endoplasmic reticulum membrane protein ORM1-like protein 3 (ORMDL3) inhibits the activity of SPT and subsequent sphingolipid synthesis. However, the role of ORMDL3 in the synthesis of LTs is not known. In this study, we used peritoneal-derived mast cells isolated from ORMDL3 KO or control mice and examined their calcium mobilization, degranulation, NF-κB inhibitor-α phosphorylation, and TNF-α production. We found that peritoneal-derived mast cells with ORMDL3 KO exhibited increased responsiveness to antigen. Detailed lipid analysis showed that compared with WT cells, ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids but also of LT signaling mediators LTB4, 6t-LTB4, LTC4, LTB5, and 6t-LTB5. The crosstalk between ORMDL3 and 5-LO metabolic pathways was supported by the finding that endogenous ORMDL3 and 5-LO are localized in similar endoplasmic reticulum domains in human mast cells and that ORMDL3 physically interacts with 5-LO. Further experiments showed that 5-LO also interacts with the long-chain 1 and long-chain 2 subunits of SPT. In agreement with these findings, 5-LO knockdown increased ceramide levels, and silencing of SPTLC1 decreased arachidonic acid metabolism to LTs to levels observed upon 5-LO knockdown. These results demonstrate functional crosstalk between the LT and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators.

• MeSH
• Arachidonate 5-Lipoxygenase metabolism   MeSH
• Eicosanoids analysis   metabolism   MeSH
• Membrane Proteins metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Serine C-Palmitoyltransferase metabolism   MeSH
• Sphingolipids analysis   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Patients with the neurological disorder HSAN-I suffer frequent infections, attributed to a lack of pain sensation and failure to seek care for minor injuries. Whether protective CD8+ T cells are affected in HSAN-I patients remains unknown. Here, we report that HSAN-I-associated mutations in serine palmitoyltransferase subunit SPTLC2 dampened human T cell responses. Antigen stimulation and inflammation induced SPTLC2 expression, and murine T-cell-specific ablation of Sptlc2 impaired antiviral-T-cell expansion and effector function. Sptlc2 deficiency reduced sphingolipid biosynthetic flux and led to prolonged activation of the mechanistic target of rapamycin complex 1 (mTORC1), endoplasmic reticulum (ER) stress, and CD8+ T cell death. Protective CD8+ T cell responses in HSAN-I patient PBMCs and Sptlc2-deficient mice were restored by supplementing with sphingolipids and pharmacologically inhibiting ER stress-induced cell death. Therefore, SPTLC2 underpins protective immunity by translating extracellular stimuli into intracellular anabolic signals and antagonizes ER stress to promote T cell metabolic fitness.

• MeSH
• CD8-Positive T-Lymphocytes immunology   MeSH
• Cytokines biosynthesis   MeSH
• Hereditary Sensory and Autonomic Neuropathies genetics   MeSH
• Cells, Cultured MeSH
• Middle Aged MeSH
• Humans MeSH
• Lymphocytic Choriomeningitis immunology   virology   MeSH
• Mechanistic Target of Rapamycin Complex 1 metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Cell Proliferation MeSH
• Serine C-Palmitoyltransferase genetics   MeSH
• Sphingolipids biosynthesis   MeSH
• Signal Transduction immunology   MeSH
• Endoplasmic Reticulum Stress genetics   immunology   MeSH
• Lymphocytic choriomeningitis virus immunology   MeSH
• Animals MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

The aliphatic nitroalkene (E)-1-nitropentadec-1-ene (NPD), reported in early seventies in soldiers of the termite genus Prorhinotermes, was the first documented nitro compound produced by insects. Yet, its biosynthetic origin has long remained unknown. Here, we investigated in detail the biosynthesis of NPD in P. simplex soldiers. First, we track the dynamics in major metabolic pathways during soldier ontogeny, with emphasis on likely NPD precursors and intermediates. Second, we propose a hypothesis of NPD formation and verify its individual steps using in vivo incubations of putative precursors and intermediates. Third, we use a de novo assembled RNA-Seq profiles of workers and soldiers to identify putative enzymes underlying NPD formation. And fourth, we describe the caste- and age-specific expression dynamics of candidate initial genes of the proposed biosynthetic pathway. Our observations provide a strong support to the following biosynthetic scenario of NPD formation, representing an analogy of the sphingolipid pathway starting with the condensation of tetradecanoic acid with l-serine and leading to the formation of a C16 sphinganine. The C16 sphinganine is then oxidized at the terminal carbon to give rise to 2-amino-3-hydroxyhexadecanoic acid, further oxidized to 2-amino-3-oxohexadecanoic acid. Subsequent decarboxylation yields 1-aminopentadecan-2-one, which then proceeds through six-electron oxidation of the amino moiety to give rise to 1-nitropentadecan-2-one. Keto group reduction and hydroxyl moiety elimination lead to NPD. The proposed biosynthetic sequence has been constructed from age-related quantitative dynamics of individual intermediates and confirmed by the detection of labeled products downstream of the administered labeled intermediates. Comparative RNA-Seq analyses followed by qRT-PCR validation identified orthologs of serine palmitoyltransferase and 3-ketodihydrosphingosine reductase genes as highly expressed in the NPD production site, i.e. the frontal gland of soldiers. A dramatic onset of expression of the two genes in the first days of soldier's life coincides with the start of NPD biosynthesis, giving further support to the proposed biosynthetic hypothesis.

• MeSH
• Alcohol Oxidoreductases metabolism   MeSH
• Amino Acids metabolism   MeSH
• Insect Proteins metabolism   MeSH
• Isoptera metabolism   MeSH
• Ketones metabolism   MeSH
• Fatty Acids metabolism   MeSH
• Metabolome MeSH
• Nitroparaffins metabolism   MeSH
• Serine C-Palmitoyltransferase metabolism   MeSH
• Sphingolipids metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

Cíl: Cílem práce je charakterizovat klinické a elektrofyziologické nálezy dvou typů ulceromutilující dědičné neuropatie CMT2B a HSN1 u tří českých rodin s molekulárně geneticky objasněnou příčinou. Soubor: Popisujeme tři rodiny s dědičnou senzitivní neuropatií s celkem 16 postiženými. Metodika: Na základě neurologického a elektromyografického vyšetření, výskytu podobných obtíží u příbuzných byla stanovena dia­gnóza senzitivní, převážně axonální neuropatie. Následně bylo provedeno sekvenování genů SPTLC1 a RAB7 v rodinách A, B, C a u dalších 24 nepříbuzných pacientů s klinickým podezřením na dědičnou senzitivní neuropatii. Výsledky: V rodině B byla nálezem mutace p.C133Y v SPTLC1 genu prokázána hereditární senzitivní neuropatie typ 1 (HSN1). V rodinách A a C byla nálezem mutací p.L129F a p.V162M v genu RAB7 prokázána choroba Charcot‑Marie‑Tooth typ 2B (CMT2B). Všechny tři mutace již byly dříve popsány a fenotyp odpovídá popisu pacientů z jiných zemí. DNA vyšetření dalších 24 nepříbuzných pacientů však příčinu onemocnění neobjasnilo. Závěr: Ulceromutilující dědičné neuropatie CMT2B a HSN1 jsou v české populaci, podobně jako v jiných zemích vzácné, ale klinické projevy jsou jasně poznatelné, pokud jsou zohledněny a správně získány genealogické údaje – rodinná anamnéza. Tyto tři popsané rodiny jsou dosud jediné známé v ČR s objasněnou příčinou dědičné senzitivní neuropatie v důsledku mutací v RAB7 a SPTLC1. Objasnění příčiny ulceromutilující senzitivní neuropatie má význam nejen pro upřesnění genetické a klinické prognózy a pro cílenou genetickou prevenci, ale u pacientů s mutacemi v SPTLC1 genu možná i pro cílenou terapii se substitucí L‑serinem, která bude testována v klinické studii (Boston, USA)

Aim: The goal was to clinically and electrophysiologically characterize two types of ulceromutilating hereditary neuropathy CMT2B and HSN1 in three Czech families where molecular genetic cause was confirmed. Patients: We describe three families, overall 16 affected patients, with hereditary sensory neuropathy. Methods: The diagnosis of sensory, predominantly axonal neuropathy was done on the basis of neurological and electrophysiological examination. Sequencing of the SPTLC1 and RAB7 genes was done in families A, B, C and 24 unrelated patients with clinical suspicion for HSN. Results: Hereditary sensory neuropathy type 1 (HSN1) caused by the p.C133Y mutation in the SPTLC1 gene was confirmed in family B and the Charcot-Marie-Tooth type 2B (CMT2B) caused by p.L129F and p.V162M mutations in the RAB7 gene was confirmed in families A and C. All three mutations have been previously described. DNA examination of 24 unrelated patients did not reveal the cause of their disease. Conclusion: As in other countries, ulceromutilating hereditary neuropathies CMT2B and HSN1 are rare in the Czech population. However, clinical manifestations are clearly recognizable if correctly obtained genealogical data – family history – is properly taken into account. The three families described here are the only known families with hereditary neuropathies caused by mutations in RAB7 and SPTLC1 in the Czech Republic. Clarification of the cause of ulceromutilating sensory neuropathy is crucial for genetic and clinical prognosis, including targeted genetic prevention, but possibly also for an L-serin therapy in SPTLC1 mutation patients to be tested in a clinical study (Boston, USA).

• MeSH
• Amputation, Surgical MeSH
• Sensation * genetics   MeSH
• Hereditary Sensory and Autonomic Neuropathies * diagnosis   genetics   physiopathology   MeSH
• Genetic Testing MeSH
• Haplotypes MeSH
• Extremities MeSH
• Humans MeSH
• Mutation genetics   MeSH
• Peripheral Nervous System Diseases genetics   MeSH
• Protein Subunits * genetics   MeSH
• Foot Injuries genetics   MeSH
• Sensation Disorders * genetics   MeSH
• rab GTP-Binding Proteins genetics   MeSH
• Family MeSH
• Pedigree MeSH
• Serine C-Palmitoyltransferase * genetics   MeSH
• Serine MeSH
• Sphingolipids biosynthesis   genetics   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

The highly repetitive nature of the Trichomonas vaginalis genome and massive expansion of various gene families has caused difficulties in genome assembly and has hampered genome mapping. Here, we adapted fluorescence in situ hybridization (FISH) for T. vaginalis, which is sensitive enough to detect single copy genes on metaphase chromosomes. Sensitivity of conventional FISH, which did not allow single copy gene detection in T. vaginalis, was increased by means of tyramide signal amplification. Two selected single copy genes, coding for serine palmitoyltransferase and tryptophanase, were mapped to chromosome I and II, respectively, and thus could be used as chromosome markers. This established protocol provides an amenable tool for the physical mapping of the T. vaginalis genome and other essential applications, such as development of genetic markers for T. vaginalis genotyping.

• MeSH
• Antigens, Protozoan genetics   MeSH
• Chromosomes chemistry   genetics   ultrastructure   MeSH
• DNA Fingerprinting methods   MeSH
• Fluorescence MeSH
• Genetic Markers MeSH
• Genome MeSH
• Genotype MeSH
• Gene Dosage MeSH
• In Situ Hybridization, Fluorescence methods   MeSH
• Humans MeSH
• Chromosome Mapping methods   MeSH
• Sensitivity and Specificity MeSH
• Serine C-Palmitoyltransferase genetics   MeSH
• Trichomonas vaginalis chemistry   cytology   genetics   MeSH
• Trichomonas Infections genetics   parasitology   MeSH
• Tryptophanase genetics   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH