Cloning and characterization of microbial activated Aedes aegypti†MEK4 (AaMEK4): influences of noncatalytic domains on enzymatic activity.

Protein kinases are known to be involved in a number of signal transduction cascades. Both the stress-activated Jun N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) p38 pathways have been shown to correlate with the insect immune response to microbial infection. MAP kinase kinase 4 (MEK4) is an upstream kinase of JNK and p38 kinase. The cDNA of AaMEK4 was cloned and characterized. AaMEK4 was activated by microbial lysates of Gram-positive, Gram-negative bacteria and yeast. The conserved lysine (K112 ) and the putative phosphorylation sites (S238 and T242 ) were shown to be important for kinase activity by site-directed mutagenesis. A common MAPK docking site (MAPK_dsA) was found and in addition, a new nearby docking site, MAPK_dsB, was identified in the N-terminal noncatalytic domain of AaMEK4. MAPK_dsB was shown to be a unique element in the MEK4 family. In this study, both MAPK_dsA and _dsB were demonstrated to be important to AaMEK4 enzymatic activity for the downstream protein kinase, Aap38.

Comparison of one-tube multiplex PCR, automated ribotyping and intergenic spacer (ITS) sequencing for rapid identification of Acinetobacter baumannii.

Acinetobacter baumannii has emerged as a serious cause of nosocomial infections. Rapid identification of this pathogen is required so that appropriate therapy can be given and outbreaks controlled. This study evaluated a multiplex PCR and an automated ribotyping system for the rapid identification of Acinetobacter baumannii. In total, 22 different reference strains and 138 clinical isolates of Acinetobacter spp., identified by 16S-23S rRNA intergenic spacer (ITS) sequence analysis, were evaluated. All A. baumannii isolates (82 clinical isolates and one reference strain) were identified by the multiplex PCR method (specificity 100%). The sensitivity and specificity of the ribotyping system for identification of A. baumannii were 85.5% (71/83) and 93.5% (72/77), respectively. An additional 100 clinical isolates belonging to the Acinetobacter calcoaceticus-A. baumannii complex were used to compare these two methods for identification of A. baumannii, and this comparison revealed a level of disagreement of 14% (14 isolates). The accuracy of the multiplex PCR was 100%, which was confirmed by sequence analysis of the ITS and recA gene of these isolates. Thus, the multiplex PCR method dramatically increased the efficiency and speed of A. baumannii identification.

An immune signalling kinase AaMEK3 from mosquitoes: cDNA cloning and characterization.

In mammals, the mitogen-activated protein (MAP) kinase pathway is one of the four major signalling systems that respond to stress and inflammatory stimuli. A full-length cDNA corresponding to Aedes aegypti MAP kinase kinase 3 (AaMEK3) was cloned and sequenced. It is 1.7 kb and contains an open reading frame of 334 amino acids and eleven conserved kinase domains, including signatures of a putative serine/threonine kinase active site and an ATP binding site. The messenger (mRNA) and protein expression levels of AaMEK3 are enhanced post bacterial inoculation. The in vitro kinase activity assay reveals that (1) AaMEK3 is not autophosphorylated but can phosphorylate myelin basic protein successfully, and (2) it is slightly enhanced by lipopolysaccharide stimulation. This suggests that AaMEK3 may be involved in mosquito immune signalling.