Cell-trafficking impairment in disease-associated LPA6 missense mutants and a potential pharmacoperone therapy for autosomal recessive woolly hair/hypotrichosis.

In human autosomal recessive woolly hair/hypotrichosis (ARWH/HT), many mutations have been identified in a gene encoding LPA6, a G protein-coupled receptor (GPCR) for lysophosphatidic acid (LPA). However, information regarding the effects of such mutations on receptor function is limited. In this study, we examined functional impacts of selected amino acid changes in LPA6 identified in ARWH/HT patients. In our exogenous expression experiments, all mutants except S3T failed to respond to LPA, indicating that they are loss-of-function mutants. Among the nine mutants, five (D63V, G146R, N246D, L277P and C278Y) displayed impaired expression at the cell surface because of endoplasmic reticulum (ER) retention, indicating that these mutants are trafficking-defective, as reported in other disease-associated GPCRs. Notably, alkyl-OMPT, a potent synthetic agonist for LPA6 restored the defective cell surface expression of two of the ER-retained mutants, D63V and N246D, possibly by its chaperoning function that allows them to escape intracellular retention as well as proteasomal degradation. Furthermore, the alkyl-OMPT-rescued N246D mutant was shown be functional. Our findings encourage future application of pharmacoperone therapy for ARWH/HT patients with specific LPA6 mutations.

Lysophosphatidic acid receptor, LPA6, regulates endothelial blood-brain barrier function: Implication for hepatic encephalopathy.

Cerebral edema is a life-threatening neurological condition characterized by brain swelling due to the accumulation of excess fluid both intracellularly and extracellularly. Fulminant hepatic failure (FHF) develops cerebral edema by disrupting blood-brain barrier (BBB). However, the mechanisms by which mediator induces brain edema in FHF remain to be elucidated. Here, we assessed a linkage between brain edema and lysophosphatidic acid (LPA) signaling by utilizing an animal model of FHF and in vitro BBB model. Azoxymethane-treated mice developed FHF and hepatic encephalopathy, associated with higher autotaxin (ATX) activities in serum than controls. Using in vitro BBB model, LPA disrupted the structural integrity of tight junction proteins including claudin-5, occludin, and ZO-1. Furthermore, LPA decreased transendothelial electrical resistances in in vitro BBB model, and induced cell contraction in brain endothelial monolayer cultures, both being inhibited by a Rho-associated protein kinase inhibitor, Y-27632. The brain capillary endothelial cells predominantly expressed LPA6 mRNA, whose knockdown blocked the LPA-induced endothelial cell contraction. Taken together, the up-regulation of serum ATX in hepatic encephalopathy may activate the LPA-LPA6-G12/13-Rho pathway in brain capillary endothelial cells, leading to enhancement of BBB permeability and brain edema.

Platelet-activating factor production in the spinal cord of experimental allergic encephalomyelitis mice via the group IVA cytosolic phospholipase A2-lyso-PAFAT axis.

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) plays a critical role in inflammatory disorders including experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Although PAF accumulation in the spinal cord (SC) of EAE mice and cerebrospinal fluid of MS patients has been reported, little is known about the metabolic processing of PAF in these diseases. In this study, we demonstrate that the activities of phospholipase A(2) (PLA(2)) and acetyl-CoA:lyso-PAF acetyltransferase (LysoPAFAT) are elevated in the SC of EAE mice on a C57BL/6 genetic background compared with those of naive mice and correlate with disease severity. Correspondingly, levels of groups IVA, IVB, and IVF cytosolic PLA(2)s, group V secretory PLA(2), and LysoPAFAT transcripts are up-regulated in the SC of EAE mice. PAF acetylhydrolase activity is unchanged during the disease course. In addition, we show that LysoPAFAT mRNA and protein are predominantly expressed in microglia. Considering the substrate specificity and involvement of PAF production, group IVA cytosolic PLA(2) is likely to be responsible for the increased PLA(2) activity. These data suggest that PAF accumulation in the SC of EAE mice is profoundly dependent on the group IVA cytosolic PLA(2)/LysoPAFAT axis present in the infiltrating macrophages and activated microglia.

Inhibitors of bacterial multidrug efflux pumps potentiate antimicrobial photoinactivation.

Antimicrobial photodynamic inactivation (APDI) combines a nontoxic photoactivatable dye or photosensitizer (PS) with harmless visible light to generate singlet oxygen and reactive oxygen species that kill microbial cells. Cationic phenothiazinium dyes, such as toluidine blue O (TBO), are the only PS used clinically for APDI, and we recently reported that this class of PS are substrates of multidrug efflux pumps in both gram-positive and gram-negative bacteria. We now report that APDI can be significantly potentiated by combining the PS with an efflux pump inhibitor (EPI). Killing of Staphylococcus aureus mediated by TBO and red light is greatly increased by coincubation with known inhibitors of the major facilitator pump (NorA): the diphenyl urea INF271, reserpine, 5'-methoxyhydnocarpin, and the polyacylated neohesperidoside, ADH7. The potentiation effect is greatest in the case of S. aureus mutants that overexpress NorA and least in NorA null cells. Addition of the EPI before TBO has a bigger effect than addition of the EPI after TBO. Cellular uptake of TBO is increased by EPI. EPI increased photodynamic inactivation killing mediated by other phenothiazinium dyes, such as methylene blue and dimethylmethylene blue, but not that mediated by nonphenothiazinium PS, such as Rose Bengal and benzoporphyrin derivative. Killing of Pseudomonas aeruginosa mediated by TBO and light was also potentiated by the resistance nodulation division pump (MexAB-OprM) inhibitor phenylalanine-arginine beta-naphthylamide but to a lesser extent than for S. aureus. These data suggest that EPI could be used in combination with phenothiazinium salts and light to enhance their antimicrobial effect against localized infections.

Gene delivery with biocompatible cationic polymer: pharmacogenomic analysis on cell bioactivity.

The availability of non-viral gene delivery systems is determined by their capacity and safety during gene introduction. In this study, the safety issues of polyplex were analyzed from the standpoint of the biomolecular mechanisms. P[Asp(DET)], a newly developed polymer, polyasparagine carrying the N-(2-aminoethyl)aminoethyl group as the side chain which was recently revealed to show good transfection efficiency to primary cells, was compared to conventional linear poly(ethylenimine) (LPEI). After transfection toward a bioluminescent cell line, P[Asp(DET)] maintained the expression level of stably expressing luciferase. In contrast, LPEI showed a decrease in the luciferase expression, while the similar expression of exogenous reporter gene was obtained. Evaluation of the housekeeping genes expression as well as the profiles of pDNA uptake after transfection suggested the time-dependent toxicity of LPEI that perturbs cellular homeostasis. Consistently, the induction of osteogenic differentiation by functional gene introduction was achieved only by P[Asp(DET)], even though appreciable expression of the gene was achieved by LPEI. It is crucial that this aspect of safety be taken into account, especially when the gene introduction is applied to primary cells to regulate such cell function as differentiation. This biomolecular analysis focusing on cellular homeostasis is beneficial for assessing the practicability of the gene delivery systems for clinical application.

Lobar emphysema due to ductus arteriosus compressing right upper bronchus in an infant with congenital heart disease.

A 1-month-old boy with tetralogy of Fallot, pulmonary atresia, right aortic arch, and right ductus arteriosus, exhibited progressive right upper lobar emphysema since his birth. The emphysema was caused by the right ductus arteriosus compressing the right upper bronchus. After division of the ductus arteriosus the emphysema completely regressed. We should explore the cause of lobar emphysema thoroughly before lobectomy especially when it is extrinsic. The emphysema may regress by eliminating the extrinsic factor.

[Successful management of a patient for cardiac surgery with difficulty in weaning from cardiopulmonary bypass by using both isosorbide dinitrate and olprinone hydrochloride].

A 57-year-old man with mitral stenosis underwent mitral valve plasty under general anesthesia. He had a history of cerebral infarction. Although he was with atrial fibrillation, his left ventricular function was good. Preoperative coronary angiography revealed no significant coronary stenosis. Induction of anesthesia and the surgical procedure had been uneventful, but the patient had difficulty to wean the patient from cardiopulmonary bypass because of unexpected low cardiac output syndrome. O1-prinone hydrochloride, a newly developed phosphodiesterase III inhibitor, was initiated in addition to high doses of dopamine and dobutamine. This increased the amplitude of the electrocardiogram and caused ST elevation of the lead II. A full dose of isosorbide dinitrate was administered intravenously to differentiate coronary artery spasm from coronary air embolism. This drastically improved the ventricular function and mixed venous oxygen saturation, and weaning from CPB was finally accomplished. The heart showed hypercontraction and inotropes were tapered gradually without further cardiac events. Although there are various etiologies for low cardiac output syndrome after CPB, the possibility of myocardial ischemia must be the first consideration. Full pharmacological support must be tried before initiating a mechanical assist modality. Coronary dilators, nitrates in particular, and phosphodiesterase III inhibitors are promising agents in such cases.

[Spinal anesthesia in a parturient complicated with idiopathic thrombocytopenic purpura].

A 27-yr-old parturient with idiopathic thrombocytopenic purpura was scheduled to undergo resection of a left ovarian cyst at 15 weeks gestation. Platelet counts were between 46,000 and 64,000.microliter-1, bleeding time was 2 min, and she denied having unusual bleeding diathesis. As the patient was reluctant to receive general anesthesia for fear of latent adverse effects of the drugs on the fetus, we selected spinal anesthesia and the perioperative course was uneventful. However, it is questionable to perform regional anesthesia in patients with coagulation disorders, for spinal hematomas leading to paraplegia can be a rare but devastating complication of regional anesthesia. According to our extensive literature review, it was revealed that platelet insufficiency, both in terms of function and count, did not represent a major risk factor for spinal hematomas associated with regional anesthesia, especially for spinal anesthesia. We suggest that spinal anesthesia may be safely performed in patients if their platelet counts exceed around 50,000.microliter-1.

Identification and characterization of a novel lysophosphatidic acid receptor, p2y5/LPA6.

p2y5 is an orphan G protein-coupled receptor that is closely related to the fourth lysophosphatidic acid (LPA) receptor, LPA4. Here we report that p2y5 is a novel LPA receptor coupling to the G13-Rho signaling pathway. "LPA receptor-null" RH7777 and B103 cells exogenously expressing p2y5 showed [3H]LPA binding, LPA-induced [35S]guanosine 5'-3-O-(thio)triphosphate binding, Rho-dependent alternation of cellular morphology, and Gs/13 chimeric protein-mediated cAMP accumulation. LPA-induced contraction of human umbilical vein endothelial cells was suppressed by small interfering RNA knockdown of endogenously expressed p2y5. We also found that 2-acyl-LPA had higher activity to p2y5 than 1-acyl-LPA. A recent study has suggested that p2y5 is an LPA receptor essential for human hair growth. We confirmed that p2y5 is a functional LPA receptor and propose to designate this receptor LPA6.