High level of GUS gene expression driven by pollen-specific promoters in electroporated lily pollen protoplasts

Gene constructs that contained the -glucuronidase (GUS) gene under the control of a pollen-specific Zm13 promoter from maize and a LAT52 promoter from tomato were introduced by electroporation into pollen protoplasts isolated from bicellular pollen grains of Lilium longiflorum. After 20 h in culture, the pollen protoplasts exhibited the apparent expression of GUS in a fluorometric assay. The GUS activity induced under the control of the Zm13 promoter was over 10 000 times higher than activity in the control (with no DNA or without electroporation). By contrast, the GUS gene was nearly silent in the lily microspore protoplasts and generative cell protoplasts. The GUS activity driven by the Zm13 and LAT52 promoters was also detected by a cytochemical assay. The frequency of blue-staining pollen protoplasts was about 70% in the case of the Zm13 promoter. The efficiency of gene transfer by electroporation was much higher than by particle bombardment. This protoplast-specific electroporation system is suitable for rapid and reliable examination of pollen-specific promoters, being as good as the particle bombardment system.     

Role of side-edge site of sphingomyelinase from Bacillus cereus

Bacillus cereus sphingomyelinase (Bc-SMase) belongs to the Mg(2+)-dependent neutral sphingomyelinase (nSMase) which hydrolyzes sphingomyelin (SM) to produce phosphocholine and ceramide, and acts as an extracellular hemolysin. Bc-SMase has two metal ion-binding sites in a long horizontal cleft across the molecule, with one Mg(2+) in the central region of the cleft and one divalent metal ion at the side-edge of the cleft. The role of the Mg(2+) at the side-edge of the long horizontal cleft in Bc-SMase remains unresolved. The replacement of Asn-57, Glu-99, and Asp-100 located in close proximity to Mg(2+) at the side-edge with alanine resulted in a striking reduction in binding to and hydrolysis of sphingomyelin in membranes of sheep erythrocytes or SM-liposomes but that of Phe-55 did not. However, the replacement of these residues had little effect on the enzymatic activity. N57A, E99A, and D100A contained 2 mol of Mg(2+) per mol of protein, and the wild type and F55A contained 3 mol. A crystal analysis showed that N57A with Mg(2+) had no metal ion at the side-edge. These results indicate that the Mg(2+) at the side-edge of Bc-SMase plays an important role in the binding to membranes.     

Intake of dissolved organic matter from deep seawater inhibits atherosclerosis progression

Dissolved organic matter (DOM) in seawater can be defined as the fraction of organic matter that passes through a filter of sub micron pore size. In this study, we have examined the effect of DOM of deep seawater (DSW) from Pacific Ocean on platelet aggregation and atherosclerosis progression. DSW was passed through a series of filters and then through an Octadecyl C18 filter; the retained substance in ethanol was designated as C18 extractable DOM (C18-DOM). Our studies showed that C18-DOM treatment inhibited platelet aggregation, P-selectin expression and activity of COX-1 significantly. C18-DOM increased the expression of anti-atherogenic molecule namely heme oxygenase-1 in endothelial cells and all these data showed that C18-DOM is exhibiting aspirin-like effects. Moreover our in vivo studies showed that C18-DOM feeding slowed remarkably the progression of atherosclerosis. Our study demonstrated a novel biological effect of oceanic DOM, which has several important implications, including a possible therapeutic strategy for atherosclerosis.     

Adipose triglyceride lipase regulates basal lipolysis and lipid droplet size in adipocytes

In adipocytes, lipid droplet (LD) size reflects a balance of triglyceride synthesis (lipogenesis) and hydrolysis (lipolysis). Perilipin A (Peri A) is the most abundant phosphoprotein on the surface of adipocyte LDs and has a crucial role in lipid storage and lipolysis. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major rate-determining enzymes for lipolysis in adipocytes. Each of these proteins (Peri A, ATGL, and HSL) has been demonstrated to regulate lipid storage and release in the adipocyte. However, in the absence of protein kinase A (PKA) stimulation (basal state), the lipases (ATGL and HSL) are located mainly in the cytoplasm, and their contribution to basal rates of lipolysis and influence on LD size are poorly understood. In this study, we utilize an adenoviral system to knockdown or overexpress ATGL and HSL in an engineered model system of adipocytes in the presence or absence of Peri A. We are able to demonstrate in our experimental model system that in the basal state, LD size, triglyceride storage, and fatty acid release are mainly influenced by the expression of ATGL. These results demonstrate for the first time the relative contributions of ATGL, HSL, and Peri A on determination of LD size in the absence of PKA stimulation.     

Immature Pacific bluefin tuna, <Emphasis Type="Italic">Thunnus orientalis</Emphasis>, utilizes cold waters in the Subarctic Frontal Zone for trans-Pacific migration

The habitat and movements of a Pacific bluefin tuna were investigated by reanalyzing archival tag data with sea surface temperature data. During its trans-Pacific migration to the eastern Pacific, the fish took a direct path and primarily utilized waters, in the Subarctic Frontal Zone (SFZ). Mean ambient temperature during the trans-Pacific migration was 14.5 ± 2.9 (°C ± SD), which is significantly colder than the waters typically inhabited by bluefin tuna in their primary feeding grounds in the western and eastern Pacific (17.6 ± 2.1). The fish moved rapidly through the colder water, and the heat produced during swimming and the thermoconservation ability of bluefin tuna likely enabled it to migrate through the cold waters of the SFZ.     

The Role of Individual Domains and the Significance of Shedding of ATP6AP2/(pro)renin Receptor in Vacuolar H+-ATPase Biogenesis

The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H⁺-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.     

Nicotine Dependence and Cost-Effectiveness of Individualized Support for Smoking Cessation: Evidence from Practice at a Worksite in Japan

Given the lack of economic studies evaluating the outcomes of smoking cessation programs from the viewpoint of program sponsors, we conducted a case study to provide relevant information for worksites. The present study was carried out between 2006 and 2008 at a manufacturing factory in the Toyama Prefecture of Japan and included subjects who voluntarily entered a smoking cessation program. The program included face-to-face counselling followed by weekly contact to provide encouragement over six months using e-mail or inter-office mail. Nicotine patches were available if required. All 151 participants stopped smoking immediately. Over the 24-month study period, self-report showed 49.7% abstained continuously from smoking. The rate of 24-month consecutive abstinence was higher in participants with lower Fagerström Test scores for Nicotine Dependence at baseline than in those with higher scores (63.6% for 0–2 points vs. 46.5% for 3–6 points vs. 43.8% for 7–10 points; chi-square test p = 0.19). A logistic regression model showed a significant linear trend for the association between the score and abstinence status after adjustment for possible confounding factors (p = 0.03). The crude incremental cost for one individual to successfully quit smoking due to the support program was ¥46,379 (i.e., ¥100 = $1.28, £0.83, or €1.03 at foreign exchange rates). The corresponding costs for the three categories of the Fagerström Test score for Nicotine Dependence were ¥31,953, ¥47,450 and ¥64,956, respectively. When a sensitivity analysis was conducted based on the 95% confidence interval of the success rate, the variance in the corresponding costs was ¥25,514–45,034 for 0–2 points, ¥38,344–61,824 for 3–6 points, and ¥45,698–108,260 for 7–10 points. The degree of nicotine dependence may therefore be an important determinant of the cost-effectiveness of smoking cessation programs.