Overexpression of Aurora-C interferes with the spindle checkpoint by promoting the degradation of Aurora-B.

The chromosomal passenger complex (CPC) plays a pivotal role in controlling accurate chromosome segregation and cytokinesis during cell division. Aurora-B, one of the chromosomal passenger proteins, is important for the mitotic spindle assembly checkpoint (SAC). Previous reports noted that Aurora-C is predominantly expressed in male germ cells and has the same subcellular localization as Aurora-B. Increasing evidence indicates that Aurora-C is overexpressed in many somatic cancers, although its function is uncertain. Our previous study showed that the aberrant expression of Aurora-C increases the tumorigenicity of cancer cells. Here, we demonstrate that overexpressed Aurora-C displaces the centromeric localization of CPCs, including INCENP, survivin, and Aurora-B. When cells were treated with nocodazole to turn on SAC, both the Aurora-B protein stability and kinase activity were affected by overexpressed Aurora-C. As a result, the activation of spindle checkpoint protein, BubR1, and phosphorylation of histone H3 and MCAK were also eliminated in Aurora-C-overexpressing cells. Thus, our results suggest that aberrantly expressed Aurora-C in somatic cancer cells may impair SAC by displacing the centromeric localization of CPCs.

Kinome siRNA-phosphoproteomic screen identifies networks regulating AKT signaling.

To identify regulators of intracellular signaling, we targeted 541 kinases and kinase-related molecules with small interfering RNAs (siRNAs), and determined their effects on signaling with a functional proteomics reverse-phase protein array (RPPA) platform assessing 42 phospho and total proteins. The kinome-wide screen demonstrated a strong inverse correlation between phosphorylation of AKT and mitogen-activated protein kinase (MAPK) with 115 genes that, when targeted by siRNAs, demonstrated opposite effects on MAPK and AKT phosphorylation. Network-based analysis identified the MAPK subnetwork of genes along with p70S6K and FRAP1 as the most prominent targets that increased phosphorylation of AKT, a key regulator of cell survival. The regulatory loops induced by the MAPK pathway are dependent on tuberous sclerosis complex 2 but demonstrate a lesser dependence on p70S6K than the previously identified FRAP1 feedback loop. The siRNA screen also revealed novel bi-directionality in the AKT and GSK3 (Glycogen synthase kinase 3) interaction, whereby genetic ablation of GSK3 significantly blocks AKT phosphorylation, an unexpected observation as GSK3 has only been predicted to be downstream of AKT. This method uncovered novel modulators of AKT phosphorylation and facilitated the mapping of regulatory loops.

A novel RING finger protein, Znf179, modulates cell cycle exit and neuronal differentiation of P19 embryonal carcinoma cells.

Znf179 is a member of the RING finger protein family. During embryogenesis, Znf179 is expressed in a restricted manner in the brain, suggesting a potential role in nervous system development. In this report, we show that the expression of Znf179 is upregulated during P19 cell neuronal differentiation. Inhibition of Znf179 expression by RNA interference significantly attenuated neuronal differentiation of P19 cells and a primary culture of cerebellar granule cells. Using a microarray approach and subsequent functional annotation analysis, we identified differentially expressed genes in Znf179-knockdown cells and found that several genes are involved in development, cellular growth, and cell cycle control. Flow cytometric analyses revealed that the population of G0/G1 cells decreased in Znf179-knockdown cells. In agreement with the flow cytometric data, the number of BrdU-incorporated cells significantly increased in Znf179-knockdown cells. Moreover, in Znf179-knockdown cells, p35, a neuronal-specific Cdk5 activator that is known to activate Cdk5 and may affect the cell cycle, and p27, a cell cycle inhibitor, also decreased. Collectively, these results show that induction of the Znf179 gene may be associated with p35 expression and p27 protein accumulation, which lead to cell cycle arrest in the G0/G1 phase, and is critical for neuronal differentiation of P19 cells.

Plasma-cell cheilitis: successful treatment with intralesional injections of corticosteroids.

Plasma-cell cheilitis is a rare inflammatory disorder of the lip characterized histologically by a band-like infiltrate of plasma cells in the upper dermis. It is considered an oral counterpart of plasma-cell balanitis. Clinically, it presents as a circumscribed, flat to slightly raised, eroded area of the lip. The cause of plasma-cell cheilitis is unknown, and the treatment is often disappointing. We describe a 55-year-old woman who had a long-lasting painful, swollen, and eroded area on her lips, which responded poorly to various topical treatments. Biopsy showed a band-like infiltrate composed mainly of mature plasma cells in the dermis. A diagnosis of plasma-cell cheilitis was made after excluding contact dermatitis, lichen planus, bacterial, fungal and spirochaete infections, and an extramedullary plasmacytoma. Dramatic improvements were observed after intralesional injections of corticosteroids. The lesion cleared up after two treatments, and there has been no recurrence in 1 year of follow-up.

Mechanisms of R factor R931 and chromosomal tetracycline resistance in Pseudomonas aeruginosa.

The mechanism of tetracycline resistance mediated by R931 (a Pseudomonas aeruginosa R factor not yet successfully transferred to Escherichia coli recipients) was examined. In strain 931 (R931) (minimal inhibitory concentration [MIC] 200 mug/ml) significant tetracycline uptake did not occur until 100 mug of tetracycline per ml was included in uptake studies. The introduction of R931 into strain 280 resulted in a significant decline in (3)H-tetracycline uptake. In both strains 931 (R931) and 280 (R931), a further reduction in tetracycline uptake resulted from pre-incubation with 1 mug of tetracycline per ml. Tetracycline resistance in R(-)P. aeruginosa strains 1731, 1885, and 494, considered to be of chromosomal origin, was associated with a lack of tetracycline uptake until the MIC of the strain was obtained. No evidence of tetracycline inactivation or ribosomal resistance was detected in R(-) or R(+) strains. The MIC for R(-) strains was generally about 25 mug/ml and that for R(+) strains was 75 to 200 mug/ml.

Transferable drug resistance in Pseudomonas aeruginosa.

Three strains of Pseudomonas aeruginosa were demonstrated to transfer double-drug resistance by conjugation to a P. aeruginosa recipient at frequencies of 10(-4) to 10(-2) per recipient cell. Two of the three strains also transferred to Escherichia coli at frequencies which were 10(3)- to 10(5)-fold lower, but the third strain could not be demonstrated to do so. The latter strain, however, conferred maleness on the Pseudomonas recipient. The transfer of streptomycin resistance was associated with the acquisition of streptomycin phosphorylase by both P. aeruginosa and E. coli recipients. Maximal broth mating frequencies were obtained with nonagitated cultures less than 1 mm in depth. A pyocine selection system based on donor sensitivity and recipient resistance is described and appears to have future value as a generalized selective device for use after matings.

Mechanisms and spectrum of streptomycin resistance in a natural population of Pseudomonas aeruginosa.

A survey of 200 strains of Pseudomonas aeruginosa isolated from clinical specimens was made in an attempt to correlate the spectrum of their streptomycin resistance and the mechanism of resistance. The strains can be classified into three groups, according to their level of resistance to streptomycin: susceptible, low-level resistant, and high-level resistant strains. The mechanism of resistance of high-level resistant strains is either an R factor-mediated inactivation of streptomycin by phosphorylation or streptomycin-resistant ribosomes. However, such high-level resistant strains comprised less than 10% of the total strains isolated; the majority of the strains resistant to streptomycin were of low-level resistance. The latter are associated with a diminished uptake of streptomycin, and no evidence of streptomycin inactivation, resistant ribosomes, or R factors could be detected. The most probable explanation of low-level resistance is reduced permeability to streptomycin. Modification of the growth medium used in uptake studies simultaneously affected strongly both streptomycin incorporation and the minimal inhibitory concentration of streptomycin.