Toxicity, repellency, and horizontal transmission of fipronil in the formosan subterranean termite (Isoptera: Rhinotermitidae)

Toxicity of fipronil was evaluated against field-collected Coptoteres formosanus Shiraki. In topical application assays, fipronil was highly effective against both workers and soldiers at very low doses. Acute toxicity after 24 h was significantly greater in workers than in soldiers. The LD50s were 2.59- and 2.91-fold greater with soldiers than with workers from the two tested colonies. The LD50s of fipronil at 72 h after treatment were <2.0 ng/insect, with no significant differences regarding the tested workers/soldiers or colonies. Treated soldiers placed with untreated workers significantly increased worker mortality. However, there was no significant horizontal transmission of fipronil from treated workers to untreated soldiers. Fipronil at rates of 0.063% or less showed no repellency, whereas sand treatments of 0.125% fipronil were repellent to termite workers.     

A fluorescence-based,high-performance liquid chromatographic assay to determine acid sphingomyelinase activity and diagnose types A and B Niemann-Pick disease

Acid sphingomyelinase (ASM; sphingomyelin phosphodiesterase, EC 3.1.4.12) is the lysosomal enzyme that hydrolyzes sphingomyelin (SPM) to phosphorylcholine and ceramide. An inherited deficiency of ASM activity results in Types A and B Niemann-Pick disease (NPD). In this study we report a new assay method to detect ASM activity and diagnose NPD using the fluorescent substrate BODIPY C12-SPM and reverse-phase high-performance liquid chromatography (HPLC). The reaction product, BODIPY C12-ceramide (B12Cer), could be clearly and efficiently separated from the substrate within 4 min using a reverse-phase column (Aquasil C18, Keystone Scientific). Femtomole quantities of B12Cer could be detected in as little as 1.0 micro l of human plasma, providing a sensitive measure of ASM activity. The mean ASM activity in human plasma from NPD patients (36 pmol/ml/h) was only 2.7% of that in normal plasma (1334 pmol/ml/h), confirming the specificity and diagnostic value of this new assay method. Importantly, the mean ASM activity in human plasma from NPD carriers (258.3 pmol/ml/h) also was significantly reduced (19.5% of normal). The ranges of ASM plasma activities in NPD patients (N=19), NPD carriers (N=11), and normal subjects (N=15) were 2.5-97.3, 108-551, and 1030-2124 pmol/ml/h, respectively. Based on these results, we suggest that this fluorescence-based HPLC assay method is a reliable, rapid, and highly sensitive technique to determine ASM activity and that plasma is a very reliable and simple source for the accurate diagnosis of NPD patients and carriers based on ASM activity.     

Regulation of Fas (CD95)-induced apoptosis by nuclear factor-kappaB and tumor necrosis factor-alpha in macrophages

The APO-1/Fasligand (FasL) and tumor necrosis factor- (TNF-) are twofunctionally related molecules that induce apoptosis ofsusceptible cells. Although the two molecules have been reported toinduce apoptosis via distinct signaling pathways, we have shown that FasL can also upregulate the expression of TNF-, raising thepossibility that TNF- may be involved in FasL-inducedapoptosis. Because TNF- gene expression is under the controlof nuclear factor-B (NF-B), we investigated whether FasL caninduce NF-B activation and whether such activation plays a role inFasL-mediated cell death in macrophages. Gene transfection studiesusing NF-B-dependent reporter plasmid showed that FasL did activateNF-B promoter activity. Gel shift studies also revealed that FasLmobilized the p50/p65 heterodimeric form of NF-B. Inhibition ofNF-B by a specific NF-B inhibitor, caffeic acid phenylethylester, or by dominant expression of the NF-B inhibitory subunitIB caused an increase in FasL-induced apoptosis and areduction in TNF- expression. However, neutralization of TNF- byspecific anti-TNF- antibody had no effect on FasL-inducedapoptosis. These results indicate that FasL-mediated cell deathin macrophages is regulated through NF-B and is independent ofTNF- activation, suggesting the antiapoptotic role of NF-Band a separate death signaling pathway mediated by FasL.

     

Cysteine string protein interacts with and modulates the maturation of the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel whose phosphorylation regulates both channel gating and its trafficking at the plasma membrane. Cysteine string proteins (Csps) are J-domain-containing, membrane-associated proteins that have been functionally implicated in regulated exocytosis. Therefore, we evaluated the possibility that Csp is involved in regulated CFTR trafficking. We found Csp expressed in mammalian epithelial cell lines, several of which express CFTR. In Calu-3 airway cells, immunofluorescence colocalized Csp with calnexin in the endoplasmic reticulum and with CFTR at the apical membrane domain. CFTR coprecipitated with Csp from Calu-3 cell lysates. Csp associated with both core-glycosylated immature and fully glycosylated mature CFTRs (bands B and C); however, in relation to the endogenous levels of the B and C bands expressed in Calu-3 cells, the Csp interaction with band B predominated. In vitro protein binding assays detected physical interactions of both mammalian Csp isoforms with the CFTR R-domain and the N terminus, having submicromolar affinities. In Xenopus oocytes expressing CFTR, Csp overexpression decreased the chloride current and membrane capacitance increases evoked by cAMP stimulation and decreased the levels of CFTR protein detected by immunoblot. In mammalian cells, the steady-state expression of CFTR band C was eliminated, and pulse-chase studies showed that Csp coexpression blocked the conversion of immature to mature CFTR and stabilized band B. These results demonstrate a primary role for Csp in CFTR protein maturation. The physical interaction of this Hsc70-binding protein with immature CFTR, its localization in the endoplasmic reticulum, and the decrease in production of mature CFTR observed during Csp overexpression reflect a role for Csp in CFTR biogenesis. The documented role of Csp in regulated exocytosis, its interaction with mature CFTR, and its coexpression with CFTR at the apical membrane domain of epithelial cells may reflect also a role for Csp in regulated CFTR trafficking at the plasma membrane.     

Overexpression of a soybean cytosolic glutamine synthetase gene linked to organ-specific promoters in pea plants grown in different concentrations of nitrate

A glutamine synthetase gene ( GS15) coding for soybean cytosolic glutamine synthetase (GS1) fused to a constitutive promoter (CaMV 35S), a putative nodule-specific promoter (LBC(3)) and a putative root-specific promoter (rolD) was transformed into Pisum sativum L. cv. Greenfeast. Four lines with single copies of GS15 (one 35S-GS15 line, one LBC (3) -GS15 line, and two rolD-GS15 lines) were tested for the expression of GS15, levels of GS1, GS activity, N accumulation, N(2) fixation, and plant growth at different levels of nitrate. Enhanced levels of GS1 were detected in leaves of three transformed lines (the 35S-GS15 and rolD-GS15 transformants), in nodules of three lines (the LBC (3) -GS15 and rolD-GS15 transformants), and in roots of all four transformants. Despite increased levels of GS1 in leaves and nodules, there were no differences in GS activity in these tissues or in whole-plant N content, N(2) fixation, or biomass accumulation among all the transgenic lines and the wild-type control. However, the rolD-GS15 transformants, which displayed the highest levels of GS1 in the roots of all the transformants, had significantly higher GS activity in roots than the wild type. In one of the rolD-GS15 transformed lines (Line 8), increased root GS activity resulted in a lower N content and biomass accumulation, supporting the findings of earlier studies with Lotus japonicus (Limami et al. 1999 ). However, N content and biomass accumulation was not negatively affected in the other rolD-GS15 transformant (Line 9) and, in fact, these parameters were positively affected in the 0.1 mM treatment. These findings indicate that overexpression of GS15 in various tissues of pea does not consistently result in increases in GS activity. The current study also indicates that the increase in root GS activity is not always consistent with decreases in plant N and biomass accumulation and that further investigation of the relationship between root GS activity and growth responses is warranted.     

Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy

A novel method for the covalent attachment of erythrocytes to glass microscope coverslips that can be used to image intact cells and the cytoplasmic side of the cell membrane with either solid or liquid mode atomic force microscopy (AFM) is described. The strong binding of cells to the glass surface is achieved by the interaction of cell membrane carbohydrates to lectin, which is bound to N-5-azido-2-nitrobenzoyloxysuccinimide (ANBNOS)-coated coverslips (1). The effectiveness of this method is compared with the other commonly used methods of immobilizing intact erythrocytes on glass coverslips for AFM observations. Experimental conditions of AFM imaging of biologic tissue are discussed, and typical topographies of the extracellular and the cytoplasmic surfaces of the plasma membrane in the dry state and in the liquid state are presented. Comparison of the spectrin network of cell age-separated erythrocytes has demonstrated significant loss in the network order in older erythrocytes. The changes are quantitatively described using the pixel height histogram and window size grain analysis.     

Site-directed mutagenesis and preliminary x-ray crystallographic studies of the tabtoxin resistance protein

Tabtoxin resistance protein (TTR) is an enzyme that catalyzes the acetylation of tabtoxin rendering tabtoxin-producing pathogens tolerant to their own phytotoxins. According to the structure based detoxification mechanism of TTR, three site-directed mutants Y141F, D130N and Y141F-D130N were constructed and overexpressed in E. coli. The products were then purified and their properties were analyzed by CD and DLS. Crystallization trials of two mutants Y141F andY141F-D130N were preformed.     

Flexoelectric origin of nanomechanic deflection in DNA-microcantilever system

The membrane theory is used to study the recently observed nanomechanical bending of cantilevers, which have processed biomolecular adsorption or biochemical reactions. To be different from entropy-controlling bending mechanism discussed before, we propose that the flexoelectric effect induces cantilever bending. With the introduction of flexoelectric spontaneous curvature, the relation between the bending and biopolymer character is constructed by a simple analytical formula. The cantilever motion induced by adsorption of single-strand DNA and DNA hybridization reaction is quantified analytically and our results show good agreement with experiments.     

Alterations of H19 imprinting and IGF2 replication timing are infrequent in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.