The Group VIA Phospholipase A 2 (iPLA 2 ␤) is the first recognized cytosolic Ca 2؉ -independent PLA 2 and has been proposed to participate in arachidonic acid (20:4) incorporation into glycerophosphocholine lipids, cell proliferation, exocytosis, apoptosis, and other processes. To study iPLA 2 ␤ functions, we disrupted its gene by homologous recombination to generate mice that do not express iPLA 2 ␤. Heterozygous iPLA 2 ␤ ؉/؊ breeding pairs yield a Mendelian 1:2:1 ratio of iPLA 2 ␤ ؉/؉ , iPLA 2 ␤ ؉/؊ , and iPLA 2 ␤ ؊/؊ pups and a 1:1 male:female gender distribution of iPLA 2 ␤ ؊/؊ pups. Several tissues of wild-type mice express iPLA 2 ␤ mRNA, immunoreactive protein, and activity, and testes express the highest levels. Testes or other tissues of iPLA 2 ␤ ؊/؊ mice express no iPLA 2 ␤ mRNA or protein, but iPLA 2 ␤ ؊/؊ testes are not deficient in 20:4-containing glycerophosphocholine lipids, indicating that iPLA 2 ␤ does not play an obligatory role in formation of such lipids in that tissue. Spermatozoa from iPLA 2 ␤ ؊/؊ mice have reduced motility and impaired ability to fertilize mouse oocytes in vitro and in vivo, and inhibiting iPLA 2 ␤ with a bromoenol lactone suicide substrate reduces motility of wild-type spermatozoa in a time-and concentration-dependent manner. Mating iPLA 2 ␤ ؊/؊ male mice with iPLA 2 ␤ ؉/؉ , iPLA 2 ␤ ؉/؊ , or iPLA 2 ␤ ؊/؊ female mice yields only about 7% of the number of pups produced by mating pairs with an iPLA 2 ␤ ؉/؉ or iPLA 2 ␤ ؉/؊ male, but iPLA 2 ␤ ؊/؊ female mice have nearly normal fertility. These findings indicate that iPLA 2 ␤ plays an important functional role in spermatozoa, suggest a target for developing male contraceptive drugs, and complement reports that disruption of the Group IVA PLA 2 (cPLA 2 ␣) gene impairs female reproductive ability.

Leptin is a protein encoded by the ob gene that is expressed in adipocytes and regulates eating behavior via central neuroendocrine mechanisms. Serum leptin levels have been shown to correlate with weight and percent body fat in normal and obese individuals; however, it is not known whether the regulation of leptin is normal below a critical threshold of body fat in chronic undernutrition. We investigated serum leptin levels in 22 women, aged 23 +/- 4 yr, with anorexia nervosa. Duration of disease, weight, BMI, percent body fat, and serum leptin levels were determined for each patient. Nutritional status was assessed further by caloric intake and measurement of insulin and insulin-like growth factor I (IGF-I) levels. Twenty-three healthy women, aged 23 +/- 4 yr, taking no medications, with normal menstrual function and body mass index (BMI) between 20-26 kg/m2 (mean, 23.7 +/- 1.7 kg/m2), served as a control population for comparison of leptin levels. Subjects with anorexia nervosa were low weight (BMI, 16.3 +/- 1.6 kg/m2; normal, 20-26 kg/m2) and exhibited a striking reduction in percent body fat (7 +/- 2%; normal, 20-30%). The mean serum leptin level was significantly decreased in subjects with anorexia nervosa compared with that in age- and sex-matched controls of normal body weight (5.6 +/- 3.7 vs. 19.1 +/- 8.1 ng/mL; P < 0.0001). Serum leptin levels were correlated highly with weight, as expressed either BMI (r = 0.66; P = 0.002) or percent ideal body weight (r = 0.68; P = 0.0005), body fat (r = 0.70; P = 0.0003), and IGF-I (r = 0.64; P = 0.001), but not with caloric intake or serum levels of estradiol or insulin in subjects with anorexia nervosa. The correlation between leptin and body fat was linear, with progressively lower, but detectable, leptin levels measured even in patients with less than 5% body fat, but was not significant when the effects of weight were taken into account. In contrast, the correlation between leptin and IGF-I remained significant when the effects of weight, body fat, and caloric intake were taken into account. In normal controls, leptin correlated with BMI (r = 0.55; P = 0.007) and IGF-I (r = 0.44; P < 0.05), but not with fat mass. These data demonstrate that serum leptin levels are reduced in association with low weight and percent body fat in subjects with anorexia nervosa compared to normal controls. Leptin levels correlate highly with weight, percent body fat, and IGF-I in subjects with anorexia nervosa, suggesting that the physiological regulation of leptin is maintained in relation to nutritional status even at an extreme of low weight and body fat.

We demonstrated an ultra-broadband, polarization-insensitive, and wide-angle metamaterial absorber for terahertz (THz) frequencies using arrays of truncated pyramid unit structure made of metal-dielectric multilayer composite. In our design, each sub-layer behaving as an effective waveguide is gradually modified in their lateral width to realize a wideband response by effectively stitching together the resonance bands of different waveguide modes. Experimentally, our five layer sample with a total thickness 21 μm is capable of producing a large absorptivity above 80% from 0.7 to 2.3 THz up to the maximum measurement angle 40°. The full absorption width at half maximum of our device is around 127%, greater than those previously reported for THz frequencies. Our absorber design has high practical feasibility and can be easily integrated with the semiconductor technology to make high efficient THz-oriented devices.

The death of insulin-secreting β-cells that causes type I diabetes mellitus (DM) occurs in part by apoptosis, and apoptosis also contributes to progressive β-cell dysfunction in type II DM. Recent reports indicate that ER stress-induced apoptosis contributes to β-cell loss in diabetes. Agents that deplete ER calcium levels induce β-cell apoptosis by a process that is independent of increases in [Ca 2+ ] i . Here we report that the SERCA inhibitor thapsigargin induces apoptosis in INS-1 insulinoma cells and that this is inhibited by a bromoenol lactone (BEL) inhibitor of group VIA calcium-independent phospholipase A 2 (iPLA 2 β). Overexpression of iPLA 2 β amplifies thapsigargin-induced apoptosis of INS-1 cells, and this is also suppressed by BEL. The magnitude of thapsigargin-induced INS-1 cell apoptosis correlates with the level of iPLA 2 β expression in various cell lines, and apoptosis is associated with stimulation of iPLA 2 β activity, perinuclear accumulation of iPLA 2 β protein and activity, and caspase-3-catalyzed cleavage of full-length 84 kDa iPLA 2 β to a 62 kDa product that associates with nuclei. Thapsigargin also induces ceramide accumulation in INS-1 cells, and this response is amplified in cells that overexpress iPLA 2 β. These findings indicate that iPLA 2 β participates in ER stress-induced apoptosis, a pathway that promotes β-cell death in diabetes.Diabetes mellitus (DM) 1 is the most prevalent human endocrine disease, and it results from loss and/or dysfunction of insulin-secreting β-cells in pancreatic islets. Type I DM is caused † This research was supported in part by grants from the National Institutes of Health (R37-DK34388, P41-RR00954, P01-HL57278, P60-DK20579, and P30-DK56341) and by an Award (to S.R.) from the American Diabetes Association.© 2004 American Chemical Society * To whom correspondence should be addressed: Department of Medicine, Washington University School of Medicine, Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110. Phone: (314) 362-8194. Fax: (314) 362-8188. sramanad@im.wustl.edu. 1 Abbreviations: AA, arachidonic acid; BEL, bromoenol lactone suicide inhibitor of iPLA 2 β; BME, β-mercaptoethanol; BSA, bovine serum albumin; CAD, collisionally activated dissociation; CM, ceramide; CNL, constant neutral loss; C3-I, caspase-3 inhibitor; cPLA 2 , group IV cytosolic phospholipase A2; ECL, enhanced chemiluminescence; EGFP, enhanced green fluorescence protein; ER, endoplasmic reticulum; ESI, electrospray ionization; FBS, fetal bovine serum; IF, immunocytofluorescence; iPLA 2 β, β-isoform of group VIA calcium-independent phospholipase A 2 ; IS, internal standard; MS, mass spectrometry; OE, iPLA 2 β-overexpressing cells; O/N, overnight; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PIC, protease inhibitor cocktail; PLA 2 , phospholipase A 2 ; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; SERCA, sarcoen-doplasmic reticulum Ca 2+ -ATPase; TBS-T, Tris-buffered saline-tween; TIC, total ion current; TLC, thin-layer chromatography;...

Pancreatic islets express a Ca 2؉-independent phospholipase A 2 (CaI-PLA 2 ) activity that is sensitive to inhibition by a haloenol lactone suicide substrate that also attenuates glucose-induced hydrolysis of arachidonic acid from islet phospholipids and insulin secretion. A cDNA has been cloned from a rat islet cDNA library that encodes a protein with a deduced amino acid sequence of 751 residues that is homologous to a CaI-PLA 2 enzyme recently cloned from Chinese hamster ovary cells. Transient transfection of both COS-7 cells and Chinese hamster ovary cells with the cloned islet CaI-PLA 2 cDNA resulted in an increase in cellular CaI-PLA 2 activity, and this activity was susceptible to inhibition by haloenol lactone suicide substrate. The domain of the islet CaI-PLA 2 from amino acid residues 150 -414 is composed of eight stretches of a repeating sequence motif of approximately 33-amino acid residues in length that is highly homologous to domains of ankyrin that bind both tubulin and integral membrane proteins, including several proteins that regulate ionic fluxes across membranes. These findings complement previous pharmacologic observations that suggest that CaI-PLA 2 may participate in regulating transmembrane ion flux in glucose-stimulated ␤-cells.Glucose-induced insulin secretion from pancreatic islet ␤-cells requires that glucose be transported into the ␤-cell and metabolized (1). Signals derived from glucose metabolism result in inactivation of plasma membrane ATP-sensitive K ϩ channels (K ATP ), 1 membrane depolarization, activation of voltage-operated Ca 2ϩ channels, influx of Ca 2ϩ, and a rise in cytosolic [Ca 2ϩ ], which triggers insulin exocytosis (2). Stimulation of islets with glucose also induces hydrolysis of arachidonic acid from islet membrane phospholipids (3), and the resultant accumulation of nonesterified arachidonic acid (4) may facilitate Ca 2ϩ entry into ␤-cells (5) and amplify depolarization-induced insulin secretion (6).Hydrolysis of arachidonic acid from membrane phospholipids in glucose-stimulated islets appears to be mediated in part by a phospholipase A 2 (PLA 2 ) enzyme that is catalytically active in the absence of Ca 2ϩ and that is inactivated by a haloenol lactone suicide substrate (HELSS) (7-10). Treatment of islets with HELSS results in attenuation of the glucose-induced rise in ␤-cell cytosolic [Ca 2ϩ ] and in inhibition of insulin secretion (8 -10). The structure of islet Ca 2ϩ -independent phospholipase A 2 (CaI-PLA 2 ) is not known, but a CaI-PLA 2 enzyme has recently been cloned from CHO cells (11) and its sequence determined (GenBank accession number 115470). We report here the cloning, expression, and sequence analysis of a homologous enzyme from a rat pancreatic islet cDNA library (12). IN); rodent Chow 5001 was from Ralston Purina (St. Louis, MO); ampicillin and kanamycin were from Sigma; and D-glucose was from the National Bureau of Standards. Media included KRB (Krebs-Ringer bicarbonate buffer; 25 mM HEPES, pH 7.4, 115 mM NaCl, 24 mM NaHCO 3 , 5 mM KCl...