Reduction in egg hatch after a sublethal dose of chlorfluazuron to larvae of the common cutworm, Spodoptera litura

Abstract.  The effects of a sublethal dose of chlorfluazuron on egg hatching in Spodoptera litura are examined under laboratory conditions. When LD₁₀ of chlorfluazuron is topically applied to newly moulted fifth-instar larvae of either sex, a significant reduction in both the number of eggs laid and subsequent hatching is observed after mating but no significant differences in daily of oviposition are observed when compared with the controls. In addition, examination of the unhatched eggs reveals that the number of unfertilized eggs is greater than those that were fertilized but there are significantly more unfertilized eggs laid by treated insects. Interference by chlorfluazuron, transferred by copulation through sperm fluids or ova, appears improbable. All the fertilized unhatched eggs in the treated crosses die at an earlier stage than those of the controls. In the female-treated crosses, the egg size is significantly reduced compared with the control or male-treated crosses. During mating, the treated-males transferred significantly lower-weight spermatophores into the females. The weight of spermatophores transferred by untreated males is the same to both treated and untreated females. The duration of mating is not affected by chlorfluazuron treatment.     

The kv4.2 potassium channel subunit is required for pain plasticity

A-type potassium currents are important determinants of neuronal excitability. In spinal cord dorsal horn neurons, A-type currents are modulated by extracellular signal-regulated kinases (ERKs), which mediate central sensitization during inflammatory pain. Here, we report that Kv4.2 mediates the majority of A-type current in dorsal horn neurons and is a critical site for modulation of neuronal excitability and nociceptive behaviors. Genetic elimination of Kv4.2 reduces A-type currents and increases excitability of dorsal horn neurons, resulting in enhanced sensitivity to tactile and thermal stimuli. Furthermore, ERK-mediated modulation of excitability in dorsal horn neurons and ERK-dependent forms of pain hypersensitivity are absent in Kv4.2(-/-) mice compared to wild-type littermates. Finally, mutational analysis of Kv4.2 indicates that S616 is the functionally relevant ERK phosphorylation site for modulation of Kv4.2-mediated currents in neurons. These results show that Kv4.2 is a downstream target of ERK in spinal cord and plays a crucial role in pain plasticity.     

Serum antibody responses to Wolbachia surface protein in patients with human lymphatic filariasis

Wolbachia surface protein (WSP), which is the most abundantly expressed protein of Wolbachia from the human filarial parasite Brugia malayi, was chosen for the present study. B‐cell epitope prediction of the WSP protein sequence indicates a high antigenicity, surface probability and hydrophilicity by DNA STAR software analysis. ProPred analysis suggests the presence of HLA class II binding regions in the WSP protein that contribute to T‐cell responses and isotype reactivity. In order to validate these findings, the gene coding for endosymbiont WSP was PCR‐amplified from the genomic DNA of the human filarial parasite Brugia malayi and cloned in T‐7 expression vector pRSET‐A. Western blot and ELISA at the total IgG level with recombiant WSP indicated a significantly elevated reactivity in CP compared to MF, EN and NEN individuals. Isotype ELISA also suggested an elevated reactivity in CP patients at the IgG1 level. In contrast, WSP‐specific IgG4 levels were found to be elevated in MF patients compared to CP and EN. Besides this, WSP‐specific IgE levels indicated an elevated reactivity in CP and MF patients compared to normals. Observations from ELISA supported the in silico predictions that indicate the presence of B‐ and T‐cell epitopes. Hence, a combinatorial approach of in silico predictions and wet‐lab studies provides interesting insights into the role of Wolbachia proteins in filarial pathogenesis.     

On-chip detection of myoglobin based on fluorescence

A disposable immunosensor cartridge was developed that allows antibodies to be immobilized on the surface for the detection of myoglobin, a marker for the early assessment of acute myocardial infarction (AMI) using fluorescence techniques. The anti-myoglobin antibody was immobilized on a polystyrene substrate based on covalent bonding via silanization. The immunosensor chip layers were fabricated from sheets by CO(2)-laser ablation. The functionalized polystyrene surfaces were characterized by contact angle measurement, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). After the antigen-antibody reaction as a sandwich enzyme-linked immunosorbent assay (ELISA) with a horseradish peroxidase-conjugated secondary antibody (HRP-anti-myoglobin), addition of fluorogenic substrate produced a fluorescent dye which was quantified on-chip using fluorescent technique. The immunosensor response was linear for myoglobin concentrations between 20 and 230 ng/ml (r=0.991, n=3). The detection limit was found to be 16 ng/ml, which is lower than the clinical cut-off value for myoglobin in healthy patients. This protocol could be extended to the detection of other important cardiac markers simultaneously in microchannels.     

<Emphasis Type="Italic">In silico</Emphasis> modeling of the specific inhibitory potential of thiophene-2,3-dihydro-1,5-benzothiazepine against BChE in the formation of β-amyloid plaques associated with Alzheimer's disease

Background  

Alzheimer's disease, known to be associated with the gradual loss of memory, is characterized by low concentration of acetylcholine in the hippocampus and cortex part of the brain. Inhibition of acetylcholinesterase has successfully been used as a drug target to treat Alzheimer's disease but drug resistance shown by butyrylcholinesterase remains a matter of concern in treating Alzheimer's disease. Apart from the many other reasons for Alzheimer's disease, its association with the genesis of fibrils by β-amyloid plaques is closely related to the increased activity of butyrylcholinesterase. Although few data are available on the inhibition of butyrylcholinesterase, studies have shown that that butyrylcholinesterase is a genetically validated drug target and its selective inhibition reduces the formation of β-amyloid plaques.     

Isolation of four new pterocarpans from Zygophyllum eurypterum (Syn. Z. atriplicoides) with enzyme-inhibition properties

Four new pterocarpans, atricarpan A (=(-)-1,2-dihydroxy-4-(hydroxymethyl)-3,9-dimethoxypterocarpan; 1), atricarpan B (=(-)-2,3-ethylenedioxy)-1,4-dihydroxy-9-methoxypterocarpan; 2), atricarpan C (=(-)-1,9-dimethoxypterocarpan-3-carboxylic acid; 3), and atricarpan D (=(-)-2,9-dimethoxy-4-(5-oxohexyl)pterocarpan; 4) were isolated from the BuOH extract of the whole plant of Zygophyllum eurypterum. The structure elucidations of those compounds were based primarily on 1D- and 2D-NMR analysis, including COSY, HMBC, and HMQC correlations. Compounds 1-4 also inhibited butyrylcholinesterase (BChE; EC 3.1.1.8) enzyme in a concentration-dependent manner with IC(50) values between 12.5-65.0 microM. Similarly, compounds 1 and 4 inhibited lipoxygenase (LOX; EC 1.13.11.12) and acetylcholinesterase (AChE; EC 3.1.1.7) enzymes with IC50 values of 13.5 and 20.5 muM, respectively.     

Cyclin-dependent kinase TPK2 is a critical cell cycle regulator in Toxoplasma gondii

The Apicomplexan parasite Toxoplasma gondii replicates by endodyogeny, an unusual form of binary fission. We tested the role of TPK2, a homologue of the CDC2 cyclin-dependent kinases, in cell cycle regulation. TPK2 tagged with HA epitope (TPK2-HA-wt) was expressed in mammalian cells as confirmed by Western blot analysis using HA tag and PSTAIRE antibodies. TPK2-HA-wt phosphorylated a peptide from Histone H1, proving that TPK2 is a functional kinase. TPK2-HA-wt coimmunoprecipitated with mammalian cyclins A, B1, D3 and E. Despite being a functional kinase, TPK2 did not rescue Schizosaccharomyces pombe cdc2 and Saccharomyces cerevisiae cdc28 mutant strains. Overexpression of a dominant-negative mutant of TPK2 (TPK2-HA-dn) in T. gondii tachyzoites arrested replication. FACS analysis of tachyzoites expressing TPK2-HA-dn revealed an increase in the fraction of cells in S-phase when compared with TPK2-HA-wt transfected parasites. Expression of TPK2-HA-wt did not arrest tachyzoite replication. No discernable G2 cell cycle block was evident suggesting that cell cycle checkpoints differ in T. gondii from most other eukaryotic cells. These data suggest that TPK2 executes an essential function in T. gondii cell cycle and is likely to be the T. gondii CDC2 orthologue.     

Differential SLP-76 expression and TCR-mediated signaling in effector and memory CD4 T cells

We present in this study novel findings on TCR-mediated signaling in naive, effector, and memory CD4 T cells that identify critical biochemical markers to distinguish these subsets. We demonstrate that relative to naive CD4 T cells, memory CD4 T cells exhibit a profound decrease in expression of the linker/adapter molecule SLP-76, while effector T cells express normal to elevated levels of SLP-76. The reduced level of SLP-76 is memory CD4 T cells is coincident with reduced phosphorylation overall, yet the residual SLP-76 couples to a subset of TCR-associated linker molecules, leading to downstream mitogen-activated protein (MAP) kinase activation. By contrast, effector CD4 T cells strongly phosphorylate SLP-76, linker for activation of T cells, and additional Grb2-coupled proteins, exhibit increased associations of SLP-76 to phosphorylated linkers, and hyperphosphorylate downstream Erk1/2 MAP kinases. Our results suggest distinct coupling of signaling intermediates to the TCR in naive, effector, and memory CD4 T cells. Whereas effector CD4 T cells amplify existing TCR signaling events accounting for rapid effector responses, memory T cells engage fewer signaling intermediates to efficiently link TCR triggering directly to downstream MAP kinase activation.     

Variations in green plant regeneration response from anthers of indica rice and their hybrids with japonica cv. Taipei 309

The green plant regeneration ability from anthers of BR-7, a high yielding indica cultivar, Binnatoa (BA), a salt tolerant indica land race and IR-43 was tested in N6, M8, He2 and R2 media. The response was calculated on the basis of number of anthers producing green plants. The number of green plants per responding anther was also recorded. The response of BR-7 and BA was poor compared to the indica cultivar IR-43 in three of the media that were tested. In N6 medium, green plant regeneration of BA and BR-7 was respectively 10-fold and 100-fold less than the japonica cultivar Taipei 309 (T-309). No anther-derived green regenerant was obtained from another salt tolerant indica land race, Rajashail (RAJ). The N6 medium was selected to test green plant regeneration frequency from anthers obtained from the F1 crosses of T-309 × BR-7, T-309 × BA, T-309 × RAJ and T-309 × BR-7 AC regenerants backcrossed with BA. Our objective was to combine the salt tolerant trait of BA and the high yield of BR-7 in a single line. The intermediate crossing step with T-309 was performed to increase the green plant regenerability of the anthers. All F1 progeny from the crosses with T-309 showed significantly increased callus induction compared to the indica parent although the values were lower than the midparent means. Green plant regeneration compared to their respective indica parents either increased or decreased but never approached the level of T-309. This revised version was published online in June 2006 with corrections to the Cover Date.