Resistance to azithromycin of Neisseria gonorrhoeae isolates from 2 cities in China.

BACKGROUND: Antimicrobial resistance in Neisseria gonorrhoeae (NG) is a public health concern globally, and azithromycin-resistant NG isolates have been identified in many countries. This study aimed to investigate azithromycin susceptibility in NG and to genetically characterize the azithromycin-resistant isolates identified in 2 cities in China. METHODS: NG isolates (n = 318) were cultured from patients attending 2 sexually transmitted infection clinics in Nanjing and Chongqing, China, between 2008 and 2009. Minimum inhibitory concentration (MIC) of azithromycin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone was determined using the agar dilution method. NG strains ATCC 49226 and WHO G, J, L, and P were used for quality control. Azithromycin-resistant isolates were defined as having an MIC value equal to or greater than that of strain WHO P, which is internationally recognized as azithromycin-resistant (MIC = 2 mg/L), and genotyped using NG multiantigen sequence typing. RESULTS: The MIC values of strain WHO P for azithromycin were 2 to 4 mg/L in all runs, which showed that the method provided consistent and reliable MIC values. Seventeen isolates (5.3%) showed resistance to azithromycin. Among these isolates (n = 17), 11 sequence types (STs) were identified by NG multiantigen sequence typing, of which 5 were novel. The most common ST was ST3356, represented by 6 isolates. ST1866 was represented by 2 isolates, which were isolated from patients with an unknown relationship, and both isolates were highly resistant to azithromycin, i.e., displayed an MIC of >64 mg/L. CONCLUSIONS: A relatively high prevalence of azithromycin-resistant NG strains implies that azithromycin should not be recommended for the treatment of gonococcal urethritis or cervicitis in China.

Breast cancer-associated PIK3CA mutations are oncogenic in mammary epithelial cells.

Activation of the phosphoinositide 3-kinase (PI3K) pathway has been implicated in the pathogenesis of a variety of cancers. Recently, mutations in the gene encoding the p110alpha catalytic subunit of PI3K (PIK3CA) have been identified in several human cancers. The mutations primarily result in single amino acid substitutions, with >85% of the mutations in either exon 9 or 20. Multiple studies have shown that these mutations are observed in 18% to 40% of breast cancers. However, the phenotypic effects of these PIK3CA mutations have not been examined in breast epithelial cells. Herein, we examine the activity of the two most common variants, E545K and H1047R, in the MCF-10A immortalized breast epithelial cell line. Both variants display higher PI3K activity than wild-type p110alpha yet remain sensitive to pharmacologic PI3K inhibition. In addition, expression of p110alpha mutants in mammary epithelial cells induces multiple phenotypic alterations characteristic of breast tumor cells, including anchorage-independent proliferation in soft agar, growth factor-independent proliferation, and protection from anoikis. Expression of these mutant p110alpha isoforms also confers increased resistance to paclitaxel and induces abnormal mammary acinar morphogenesis in three-dimensional basement membrane cultures. Together, these data support the notion that the cancer-associated mutations in PIK3CA may significantly contribute to breast cancer pathogenesis and represent attractive targets for therapeutic inhibition.

Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition.

The Akt family of kinases are activated by growth factors and regulate pleiotropic cellular activities. In this study, we provide evidence for isoform-specific positive and negative roles for Akt1 and -2 in regulating growth factor-stimulated phenotypes in breast epithelial cells. Insulin-like growth factor-I receptor (IGF-IR) hyperstimulation induced hyperproliferation and antiapoptotic activities that were reversed by Akt2 down-regulation. In contrast, Akt1 down-regulation in IGF-IR-stimulated cells promoted dramatic neomorphic effects characteristic of an epithelial-mesenchymal transition (EMT) and enhanced cell migration induced by IGF-I or EGF stimulation. The phenotypic effects of Akt1 down-regulation were accompanied by enhanced extracellular signal-related kinase (ERK) activation, which contributed to the induction of migration and EMT. Interestingly, down-regulation of Akt2 suppressed the EMT-like morphological conversion induced by Akt1 down-regulation in IGF-IR-overexpressing cells and inhibited migration in EGF-stimulated cells. These results highlight the distinct functions of Akt isoforms in regulating growth factor-stimulated EMT and cell migration, as well as the importance of Akt1 in cross-regulating the ERK signaling pathway.

Gamma integrase complementation at the level of DNA binding and complex formation.

Site-specific recombinases of the gamma Int family carry out two single-strand exchanges by binding as head-to-head dimers on inverted core-type DNA sites. Each protomer may cleave its own site as a monomer in cis (as for Cre recombinase), or it may recruit the tyrosine from its partner in trans to form a composite active site (as for Flp recombinase). The crystal structure of the gamma Int catalytic domain is compatible with both cleavage mechanisms, but two previous biochemical studies on gamma integrase (Int) generated data that were not in agreement. Support for cis and trans cleavage came from assays with bispecific DNA substrates for gamma and HK022 Ints and from functional complementation between recombination-deficient mutants, respectively. The data presented here do not provide new evidence for cis cleavage, but they strongly suggest that the previously described complementation results cannot be used in support of a trans-cleavage mechanism. We show here that IntR212Q retains some residual catalytic function but is impaired in binding to core-type DNA on linear substrates and in forming higher-order attL intasome structures. The binding-proficient mutant IntY342F can stabilize IntR212Q binding to core-type DNA through protein-protein interactions. Similarly, the formation of higher-order Int complexes with arm- and core-type DNA is boosted with both mutants present. This complementation precedes cleavage and thus precludes any conclusions about the mechanism of catalysis. Cross-core stimulation of wild-type HK022-Int cleavage on its cognate site (in cis) by mutant gamma Ints on bispecific core DNA suicide substrates is shown to be independent of the catalytic tyrosine but appears to be proportional to the respective core-binding affinities of the gamma Int mutants.