Distinctly localized lipid phosphate phosphatases mediate endoplasmic reticulum glycerolipid metabolism in Arabidopsis.

Inter-organelle communication is an integral subcellular process in cellular homeostasis. In plants, cellular membrane lipids are synthesized in the plastids and endoplasmic reticulum (ER). However, the crosstalk between these organelles in lipid biosynthesis remains largely unknown. Here, we show that a pair of lipid phosphate phosphatases (LPPs) with differential subcellular localizations is required for ER glycerolipid metabolism in Arabidopsis (Arabidopsis thaliana). LPPα2 and LPPε1, which function as phosphatidic acid phosphatases and thus catalyze the core reaction in glycerolipid metabolism, were differentially localized at ER and chloroplast outer envelopes despite their similar tissue expression pattern. No mutant phenotype was observed in single knockout mutants; however, genetic suppression of these LPPs affected pollen growth and ER phospholipid biosynthesis in mature siliques and seeds with compromised triacylglycerol biosynthesis. Although chloroplast-localized, LPPε1 was localized close to the ER and ER-localized LPPα2. This proximal localization is functionally relevant, because overexpression of chloroplastic LPPε1 enhanced ER phospholipid and triacylglycerol biosynthesis similar to the effect of LPPα2 overexpression in mature siliques and seeds. Thus, ER glycerolipid metabolism requires a chloroplast-localized enzyme in Arabidopsis, representing the importance of inter-organelle communication in membrane lipid homeostasis.

The role of lysophosphatidic acid acyltransferase 1 in reproductive growth of Arabidopsis thaliana.

Lysophosphatidic acid acyltransferase1 (LPAT1) catalyzes the second step of de novo glycerolipid biosynthesis in chloroplasts. However, the embryonic-lethal phenotype of the knockout mutant suggested an unknown role for LPAT1 in non-photosynthetic reproductive organs. Reciprocal genetic crossing of the lpat1-1 heterozygous line suggested a female gametophytic defect of the lpat1-1 knockout mutant. By suppressing LPAT1 specifically during seed development, we showed that LPAT1 suppression affected silique growth and seed production. Glycerolipid analysis of the LPAT1 knockdown lines revealed a pronounced decrease of phosphatidylcholine (PC) content in mature siliques along with an altered polyunsaturation level of the polar glycerolipids. In seeds, the acyl composition of triacylglycerol (TAG) was altered albeit not the content. These results indicate that plastidic LPAT1 plays an important role in reproductive growth and extraplastidic glycerolipid metabolism involving PC and TAG.

Plastid LPAT1 is an integral inner envelope membrane protein with the acyltransferase domain located in the stroma.

KEY MESSAGE: The N-terminal transmembrane domain of LPAT1 crosses the inner membrane placing the N terminus in the intermembrane space and the C-terminal enzymatic domain in the stroma. Galactolipids mono- and di-galactosyl diacylglycerol are the major and vital lipids of photosynthetic membranes. They are synthesized by five enzymes hosted at different sub-chloroplast locations. However, localization and topology of the second-acting enzyme, lysophosphatidic acid acyltransferase 1 (LPAT1), which acylates the sn-2 position of glycerol-3-phosphate (G3P) to produce phosphatidic acid (PA), remain unclear. It is not known whether LPAT1 is located at the outer or the inner envelope membrane and whether its enzymatic domain faces the cytosol, the intermembrane space, or the stroma. Even the size of mature LPAT1 in chloroplasts is not known. More information is essential for understanding the pathways of metabolite flow and for future engineering endeavors to modify glycerolipid biosynthesis. We used LPAT1 preproteins translated in vitro for import assays to determine the precise size of the mature protein and found that the LPAT1 transit peptide is at least 85 residues in length, substantially longer than previously predicted. A construct comprising LPAT1 fused to the Venus fluorescent protein and driven by the LPAT1 promoter was used to complement an Arabidopsis lpat1 knockout mutant. To determine the sub-chloroplast location and topology of LPAT1, we performed protease treatment and alkaline extraction using chloroplasts containing in vitro-imported LPAT1 and chloroplasts isolated from LPAT1-Venus-complemented transgenic plants. We show that LPAT1 traverses the inner membrane via an N-terminal transmembrane domain, with its N terminus protruding into the intermembrane space and the C-terminal enzymatic domain residing in the stroma, hence displaying a different membrane topology from its bacterial homolog, PlsC.

Functional divergence of a pair of Arabidopsis phospho-base methyltransferases, PMT1 and PMT3, conferred by distinct N-terminal sequences.

In seed plants, phospho-base N-methyltransferase (PMT) catalyzes a key step in the biosynthesis pathway of phosphatidylcholine (PC), the most abundant phospholipid class. Arabidopsis thaliana possesses three copies of PMT, with PMT1 and PMT3 play a primary role because the pmt1 pmt3 double mutant shows considerably reduced PC content with a pale seedling phenotype. Although the function of PMT1 and PMT3 may be redundant because neither of the parental single mutants showed a similar mutant phenotype, major developmental defects and possible functional divergence of these PMTs underlying the pale pmt1 pmt3 seedling phenotype are unknown. Here, we show the major developmental defect of the pale seedlings in xylem of the hypocotyl with partial impairments in chloroplast development and photosynthetic activity in leaves. Although PMT1 and PMT3 are localized at the endoplasmic reticulum, their tissue-specific expression pattern was distinct in hypocotyls and roots. Intriguingly, the function of PMT3 but not PMT1 requires its characteristic N-terminal sequence in addition to the promoter because truncation of the N-terminal sequence of PMT3 or substitution with PMT1 driven by the PMT3 promoter failed to rescue the pale pmt1 pmt3 seedling phenotype. Thus, PMT3 function requires the N-terminal sequence in addition to its promoter, whereas the PMT1 function is defined by the promoter.

Non-specific phospholipases C2 and C6 redundantly function in pollen tube growth via triacylglycerol production in Arabidopsis.

Non-specific phospholipase Cs (NPCs) are responsible for membrane lipid remodeling that involves hydrolysis of the polar head group of membrane phospholipids. Arabidopsis NPC2 and NPC6 are essential in gametogenesis, but their underlying role in the lipid remodeling remains elusive. Here, we show that these NPCs are required for triacylglycerol (TAG) production in pollen tube growth. NPC2 and NPC6 are highly expressed in developing pollen tubes and are localized at the endoplasmic reticulum. Mutants of NPC2 and NPC6 showed reduced rate of pollen germination, length of pollen tube and amount of lipid droplets (LDs). Overexpression of NPC2 or NPC6 induced LD accumulation, which suggests that these NPCs are involved in LD production. Furthermore, mutants defective in the biosynthesis of TAG, a major component of LDs, showed defective pollen tube growth. These results suggest that NPC2 and NPC6 are essential in gametogenesis for a role in hydrolyzing phospholipids and producing TAG required for pollen tube growth. Thus, lipid remodeling from phospholipids to TAG during pollen tube growth represents an emerging role for the NPC family in plant developmental control.

Primary Breast Tuberculosis Mastitis Manifested as Nonhealing Abscess.

Primary breast tuberculosis (TB) is a rare extrapulmonary TB mainly affecting young women of childbearing age from endemic countries. Its incidence is increasing in immunocompromised and HIV-infected people and with the emergence of drug-resistant strains of Mycobacterium tuberculosis (MTB). There are no specific clinical signs suggestive of this disease, it often presents as a hard mass or breast abscess. There is an overlap of features with other inflammatory, infectious, benign lesions, fat necrosis and malignant neoplasms of the breast. The detection of MTB remains the gold standard for diagnosis. Several other diagnostic modalities are used, with varying lack of sensitivity and specificity, and with a range of false negatives. A quarter of cases were treated solely on the basis of clinical, imaging or histological suspicion, without confirmation of the diagnosis. Therefore, we report the case of a young Vietnamese woman, presented for a nonhealing breast abscess, and diagnosed with breast TB based on the patient's ethnicity, histological findings, lack of clinical response to conventional antibiotic therapy, and a good clinical response to anti-TB treatment.

Expression Profiles of 2 Phosphate Starvation-Inducible Phosphocholine/Phosphoethanolamine Phosphatases, PECP1 and PS2, in Arabidopsis.

Phosphorus is essential for plant viability. Phosphate-starved plants trigger membrane lipid remodeling to replace membrane phospholipids by non-phosphorus galactolipids presumably to acquire scarce phosphate source. Phosphoethanolamine/phosphocholine phosphatase 1 (PECP1) and phosphate starvation-induced gene 2 (PS2) belong to an emerging class of phosphatase induced by phosphate starvation and dephosphorylates phosphocholine and phosphoethanolamine (PEtn) in vivo. However, detailed spatiotemporal expression pattern as well as subcellular localization has not been investigated yet. Here, by constructing transgenic plants harboring functional translational promoter-reporter fusion system, we showed the expression pattern of PECP1 and PS2 in different tissues and in response to phosphate starvation. Besides, the Venus fluorescent reporter revealed that both are localized at the ER. Characterization of transgenic plants that overexpress PECP1 or PS2 showed that their activity toward PEtn may be different in vivo. We suggest that PECP1 and PS2 are ER-localized phosphatases that show similar expression pattern yet have a distinct substrate specificity in vivo.

Adult neural stem cell fate is determined by thyroid hormone activation of mitochondrial metabolism.

OBJECTIVE: In the adult brain, neural stem cells (NSCs) located in the subventricular zone (SVZ) produce both neuronal and glial cells. Thyroid hormones (THs) regulate adult NSC differentiation towards a neuronal phenotype, but also have major roles in mitochondrial metabolism. As NSC metabolism relies mainly on glycolysis, whereas mature cells preferentially use oxidative phosphorylation, we studied how THs and mitochondrial metabolism interact on NSC fate determination. METHODS: We used a mitochondrial membrane potential marker in vivo to analyze mitochondrial activity in the different cell types in the SVZ of euthyroid and hypothyroid mice. Using primary adult NSC cultures, we analyzed ROS production, SIRT1 expression, and phosphorylation of DRP1 (a mitochondrial fission mediator) as a function of TH availability. RESULTS: We observed significantly higher mitochondrial activity in cells adopting a neuronal phenotype in vivo in euthyroid mice. However, prolonged hypothyroidism reduced not only neuroblast numbers but also their mitochondrial activity. In vitro studies showed that TH availability favored a neuronal phenotype and that blocking mitochondrial respiration abrogated TH-induced neuronal fate determination. DRP1 phosphorylation was preferentially activated in cells within the neuronal lineage and was stimulated by TH availability. CONCLUSIONS: These results indicate that THs favor NSC fate choice towards a neuronal phenotype in the adult mouse SVZ through effects on mitochondrial metabolism.

High prevalence of anti-Trichinella IgG in domestic pigs of the Son La province, Vietnam.

Although several outbreaks of Trichinella infection among humans have occurred in northwestern Vietnam, no information is available on the circulation of Trichinella among domestic pigs. The objective of the present study was to estimate the seroprevalence of anti-Trichinella IgG in free-roaming pigs (Sus scrofa) in the Son La province of northwestern Vietnam, where a human outbreak of trichinellosis occurred in June 2008. Serum samples were collected from free-roaming pigs of four communes of the Bac Yen district (Son La province) and tested for Trichinella antibodies with a commercial ELISA kit using excretory/secretory antigens. Of 1035 pigs from which serum samples were collected, 206 were positive (19.9%). There was a significant difference in the prevalence among communes (chi(2)=22.87, 3 d.f., p<0.0001). Muscle samples from 76 serologically positive pigs were tested by artificial digestion. Trichinella larvae were detected in 11 (14.5%) of them. The larvae were identified by multiplex PCR as Trichinella spiralis. This study provides the first data on the circulation of T. spiralis in domestic pigs reared in Vietnam, and the results are useful for evaluating the risk of infection for humans. The results indicate that pigs act as a reservoir and play an important role in the maintenance of the domestic cycle of T. spiralis in northwestern Vietnam.