Structural requirements for selective binding of ISC1 to anionic phospholipids

Yeast ISC1 (Yer019w) encodes inositolphosphosphingolipid-phospholipase C and is activated by phosphatidylserine (PS) and cardiolipin (CL) (Sawai, H., Okamoto, Y., Lubert, C., Mao, C., Bielawska, A., Domae, M., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 39793-39798). In this study, the structural requirements for anionic phospholipid-selective binding of ISC1 were determined using site-directed and deletion mutants. FLAG-tagged Isc1p was activated by PS, CL, and phosphatidylglycerol (PG) in a dose-dependent manner. Using lipid-protein overlay assays, Isc1p interacted specifically and directly with PS/CL/PG. Lipid-protein binding studies of a series of deletion mutants demonstrated that the second transmembrane domain (TMII) and the C terminus were required for PS binding. Moreover, the TMII and the C terminus domain were sufficient to impart PS binding to a heterologous protein, green fluorescence protein. In addition, mutations of positively charged amino acid residues at the C terminus of ISC1 reduced the activating effects of PS, suggesting involvement of these amino acids in interaction with PS/CL/PG and in the activation of the enzyme. Finally, when separate fragments containing the N terminus-TMI and TMII-C terminus were expressed heterologously, enzyme activity was reconstituted, demonstrating that the interaction of the N terminus and the C terminus is required for activity of Isc1p. These results raise the hypothesis that in the presence of PS/CL/PG, the catalytic domain in the N terminus of Isc1p is "pulled" to the membrane to interact with substrate. These studies provide unique insights into the properties of ISC1 and define a novel mechanism for activation of enzymes by lipids cofactors.     

Trace elements and some extracellular antioxidant protein levels in serum of patients with laryngeal cancer

In this study, we investigated the levels of serum Zn, Cu, Mg, Mn, Fe, ceruloplasmin (Cp), transferrin (Trf), and albumin in laryngeal carcinoma and correlated their levels with the cancer stage. The sera from 35 patients with laryngeal cancer (10 at stage II, 12 at stage III, and 13 at stage IV) were extracted before treatment and compared with those from the healthy control group (n=15). Although serum Fe and Mn concentrations were lower in the laryngeal cancer groups (for all stages) than in the control group, the difference was not statistically significant (p>0.05). The higher Cu (p<0.001) and Cp (p<0.01) and lower Zn (p<0.01), Mg (p<0.001), and Trf (p<0.01) concentrations were found in laryngeal cancer groups (for each stage) when compared to the control group. In the comparison of stages II, III, and IV (with each other), all parameters were found to be statistically not significant (p>0.05). On the other hand, no meaningful difference was found in terms of the serum albumin level. In our opinion, alterations in the level of trace elements and antioxidant proteins, important for many metabolic processes, in laryngeal cancer may not be a reason for but is, in fact, a consequence of the disease itself.     

Activation of p38 Mitogen-Activated Protein Kinase in Gaucher’s Disease

Gaucher’s disease is caused by defects in acid β-glucosidase 1 (GBA1) and has been also proposed as an inflammatory disease. GBA1 cleaves glucosylceramide to form ceramide, an established bioactive lipid, and defects in GBA1 lead to aberrant accumulation in glucosylceramide and insufficient formation of ceramide. We investigated if the pro-inflammatory kinase p38 is activated in Gaucher’s disease, since ceramide has been proposed to suppress p38 activation. Three Gaucher’s disease mouse models were employed, and p38 was found to be activated in lung and liver tissues of all Gaucher’s disease mice. Most interestingly, neuronopathic Gaucher’s disease type mice, but not non-neuronopathic ones, displayed significant activation of p38 and up-regulation of p38-inducible proinflammatory cytokines in brain tissues. In addition, all type of Gaucher’s disease mice also showed increases in serum IL-6. As cellular signalling is believed to represent an in vivo inflammatory phenotype in Gaucher’s disease, activation of p38 and possibly its-associated formation of proinflammatory cytokines were assessed in fibroblasts established from neuronopathic Gaucher’s disease mice. In mouse Gaucher’s disease cells, p38 activation and IL-6 formation by TNF-α treatment were enhanced as compared to those of wild type. Furthermore, human fibroblasts from Gaucher’s disease patients also displayed increases in p38 activation and IL-6 formation as comparison to healthy counterpart. These results raise the potential that proinflammatory responses such as p38 activation and IL-6 formation are augmented in Gaucher’s disease.     

Overexpression of γ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: Physiological and chlorophyll a fluorescence measurements

Tocopherols (vitamin E) are lipid soluble antioxidants synthesized by plants and some cyanobacteria. We have earlier reported that overexpression of the γ-tocopherol methyl transferase (γ-TMT) gene from Arabidopsis thaliana in transgenic Brassica juncea plants resulted in an over six-fold increase in the level of α-tocopherol, the most active form of all the tocopherols. Tocopherol levels have been shown to increase in response to a variety of abiotic stresses. In the present study on Brassica juncea, we found that salt, heavy metal and osmotic stress induced an increase in the total tocopherol levels. Measurements of seed germination, shoot growth and leaf disc senescence showed that transgenic Brassica juncea plants overexpressing the γ-TMT gene had enhanced tolerance to the induced stresses. Analysis of the chlorophyll a fluorescence rise kinetics, from the initial “O” level to the “P” (the peak) level, showed that there were differential effects of the applied stresses on different sites of the photosynthetic machinery; further, these effects were alleviated in the transgenic (line 16.1) Brassica juncea plants. We show that α-tocopherol plays an important role in the alleviation of stress induced by salt, heavy metal and osmoticum in Brassica juncea.     

Linear Mixed Model Selection for False Discovery Rate Control in Microarray Data Analysis

Summary In a microarray experiment, one experimental design is used to obtain expression measures for all genes. One popular analysis method involves fitting the same linear mixed model for each gene, obtaining gene‐specific p‐values for tests of interest involving fixed effects, and then choosing a threshold for significance that is intended to control false discovery rate (FDR) at a desired level. When one or more random factors have zero variance components for some genes, the standard practice of fitting the same full linear mixed model for all genes can result in failure to control FDR. We propose a new method that combines results from the fit of full and selected linear mixed models to identify differentially expressed genes and provide FDR control at target levels when the true underlying random effects structure varies across genes.     

Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium

Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in Escherichia coli (E. coli). The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-beta-thiogalactopyranoside (IPTG), temperature, and time of induction, which were identified to be critical for the optimal production of recombinant pCDase. The recombinant pCDase was purified using nickel-nitrilotriacetic acid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg/l purified protein of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with optimum activity in the neutral pH range. Both the hydrolytic and the reverse activities of CDase were stimulated by calcium with an affinity constant (K(a)) of 1.5 microM. Kinetics studies showed that calcium caused a decrease of K(m) and an increase in V(max) of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near ultraviolet circular dichroism (CD) spectra and the changes were inhibited in the presence of EGTA. These results identify important interactions between calcium and pCDase, which may play an essential role in the interaction of pCDase and its substrate.     

Patterns of contraceptive use in Australia: analysis of the 2001 National Health Survey

The purpose of this paper is to review the patterns of contraceptive use in Australia, using data from a nationally representative sample of 5872 women aged 18 to 49. This survey was conducted by the Australian Bureau of Statistics in 2001 as part of the National Health Survey. Results of the analysis indicate that the oral contraceptive pill and condom were the two most frequently used methods. More than 76% of the respondents reported having ever used the pill. Over 23% of women were currently using condoms; of these 80% of the condom users used them for contraception - this included 36% who used condoms for both protection against infection and for contraception - and the remainder used them only for protection. Withdrawal was the third most popular non-surgical method up to age 40. Few women used IUDs, injections or diaphragms. Just over 3% of the respondents were using natural methods with the highest rate reported among those in their 30s. The 'morning-after pill' was reported mostly by women aged 18-24; however, there was no evidence to suggest that it was being used as a primary method of birth control. Contraceptive use declined in older women who turned to sterilization for themselves and/or their partners. Use of the contraceptive pill was somewhat higher among better-educated women, but lower among less-educated women and those from non-English-speaking backgrounds.     

Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis

Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death （PCD）. However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B1 r_esistant11-1 （/br11-1）, which fails to generate reactive oxygen intermediates （ROIs）, is incapable of initiating PCD when the mutant is challenged by fumonisin B l （FB0, a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain base 1 （LCB 1） subunit of serine palmitoyltransferase （SPT）, which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr11-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FBl. By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine- 1-phosphate in a dosedependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.     

Investigation of asymptomatic visceral leishmaniasis cases using western blot in an endemic area in Turkey

In Turkey, Leishmania infantum is responsible for human visceral leishmaniasis (VL), which is seen mainly in the Aegean, Mediterranean, and Central Anatolia Regions. This study aimed to determine asymptomatic infections in an endemic area of VL in Turkey using the western blot technique. A total of 82 persons including children and adults were chosen randomly in Denizli province which is one of the endemic sites for VL. Serum samples were collected and screened using indirect immunofluorescent test (IFAT), enzyme-linked immunosorbent assay (ELISA) and western blot (WB). One year later, 35 of the 82 persons were sampled and screened serologically for the second time. Seven out of 82 samples were found to be positive by western blot analysis with the presence of 14 and/or 18 kDa bands. Two of these seven sera were also positive by IFAT, but only one of these two was positive by ELISA. Only one person showing seropositivity with all three tests had clinical symptoms and was diagnosed as VL with the presence of amastigotes in bone marrow aspirate. Because six people, including the one found to be seropositive in all two tests, had no clinical symptoms, they were accepted as asymptomatic carriers. The ratio of asymptomatic infection was calculated as 7.41% (6/81) in the region. In the second sampling, the western blot revealed antibodies against the same antigens in all seven subjects. Our findings showed that the presence of antibodies against 14 and 18 kDa antigens are important for the diagnosis of symptomatic and asymptomatic infections. Western blot was found to be effective in the detection of asymptomatic persons in the epidemiological studies in endemic areas.     

RNAi-mediated male sterility of tobacco by silencing TA29

The superior performance of F₁ hybrids has a significant impact on agricultural productivity. For commercial application, the availability of an efficient system for obtaining male-sterile lines of crops is an essential prerequisite. Here we have investigated the use of RNA interference (RNAi) technology to silence a male-specific gene in the model host tobacco. TA29 is expressed exclusively in anthers at the time of microspore development. About 10 out of 13 tobacco lines transformed with a hairpin RNAi construct containing TA29 sequences were male sterile. Transgenic plants were phenotypically indistinguishable from non-transgenic plants. At the anthesis stage, pollen grains from transgenic, male-sterile plants were aborted and lysed in comparison to the round and fully developed pollen in non-transgenic plants. Microscopic analysis of anthers showed selective degradation of tapetum in transgenic plants with no microspore development. One week after self-pollination, the ovules of non-transgenic plants were double the size of those in transgenic plants, due to successful self-fertilization. Male sterile transgenic plants set seed normally, when cross-pollinated with pollen from non-transgenic plants, confirming no adverse effect on the female parts of the flower. These results show that silencing of male-specific genes by RNAi is potentially a useful tool for generating male-sterile lines for producing hybrid seed.