Characterization of two classes of small molecule inhibitors of Arp2/3 complex.

Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movements. However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones; this is because of the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-0993548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying the Arp2/3 complex in living cells.

Germ line transformation of the yellow fever mosquito, Aedes aegypti, mediated by transpositional insertion of a piggyBac vector.

Mosquito-vectored diseases such as yellow fever and dengue fever continue to have a substantial impact on human populations world-wide. Novel strategies for control of these mosquito vectored diseases can arise through the development of reliable systems for genetic manipulation of the insect vector. A piggyBac vector marked with the Drosophila melanogaster cinnabar (cn) gene was used to transform the white-eyed khw strain of Aedes aegypti. Microinjection of preblastoderm embryos resulted in four families of cinnabar transformed insects. An overall transformation frequency of 4%, with a range of 0% to as high as 13% for individual experiments, was achieved when using a heat-shock induced transposase providing helper plasmid. Southern hybridizations indicated multiple insertion events in three of four transgenic lines, while the presence of duplicated target TTAA sites at either ends of individual insertions confirmed characteristic piggyBac transposition events in these three transgenic lines. The transgenic phenotype has remained stable for more than twenty generations. The transformations effected using the piggyBac element establish the potential of this element as a germ-line transformation vector for Aedine mosquitoes.

The structure of Sky1p reveals a novel mechanism for constitutive activity.

Sky1p is the only member of the SR protein kinase (SRPK) family in Saccharomyces cerevisiae. SRPKs are constitutively active kinases that display remarkable substrate specificity and have been implicated in RNA processing. Here we present the three-dimensional structure of a fully active truncated Sky1p. Analysis of the structure and structure-based functional studies reveal that the C-terminal tail, an unusual Glu residue located in the P+1 loop, and a unique mechanism for the positioning of helix alpha C act together to render Sky1p constitutively active. We have modeled a substrate peptide bound to Sky1p. The modeled complex combined with mutagenesis studies illustrate the molecular basis for substrate recognition by this kinase and suggest a mechanism by which SRPKs catalyze a sequential phosphorylation reaction of the consecutive RS dipeptide repeats characteristic of mammalian SRPK substrates.

Stimulus repetition rate effect on the auditory brainstem response in systemic lupus erythematosus.

Neuropsychiatric involvement is common in systemic lupus erythematosus (SLE), but, in early half of cases, indications are not present on examination. Auditory brainstem response (ABR) measures using differential stimulus repetition rates have been reported as sensitive indicators of subclinical central nervous system (CNS) disorders associated with SLE. In the present study, ABRs were measured in a group of normal-hearing subjects with SLE, as well as in a group of subject controls. Differences in interpeak latency (IPL) measures obtained using low- and high-stimulus repetition rates did not reach statistical significance (P greater than .05). Clinical utility of ABRs using high- and low-stimulus repetition rates for the identification of occult CNS disorder in patients with SLE was not demonstrated.

Insert earphones for audiometry: applications, advantages, and limitations.

The measurement of hearing sensitivity during audiometry requires precise presentation of auditory stimuli. In most cases, these stimuli are introduced to the listener via standard earphones. Technical problems associated with these earphones have troubled clinicians for years. An alternative earphone system for audiometry was introduced that uses shoulder-worn transducers with inserts for connection with the external auditory canal. Although insert earphones are not intended to supplant standard earphones, experimental evidence and clinical observations support the advantageousness of these earphones under a variety of clinical conditions.

Clinical applicability of insert earphones for audiometry.

Insert transducers for audiometry are available which may offer significant advantages over older 'standard' headphones. Clinicians have remained cautious in using such devices due to the paucity of experimental data demonstrating their comparability with more widely used devices. The purpose of this investigation was to compare and contrast pure-tone audiometric thresholds obtained using insert earphones with those measured utilizing conventional supra-aural transducers in normal and hearing-impaired subjects. A second purpose was to examine the differential effects on the hearing threshold level of two coupling systems for the insert device. There were no clinically significant differences among pure-tone thresholds measured with the three earphone/coupler arrangements for subjects with normal and impaired hearing. Advantages and limitations of insert earphones are discussed.