Processing of McCoy cell cultures infected with Chlamydia trachomatis: sequential isolation of chlamydial elementary bodies and lipopolysaccharide

Abstract Triton X-100 (TX100) enhances the liberation of chlamydial elementary bodies (EB) from host cells and dissolves the host cell membrane. In the presence of TX100 only differential centrifugation is needed to isolate reasonably pure EBs. The remaining high-speed supernatant still contains a large part of the chlamydial lipopolysaccharide (LPS), which can be isolated with the standard phenol-chloroform-petroleum ether extraction.     

Binding of bifunctional ethidium intercalators to transfer RNA

Two bifunctional intercalating dimers, an ethidium homodimer and an acridine ethidium heterodimer, bind to yeast tRNA^phe through two classes of sites, I and II (K_I ≥ 10⁹ M^?1, K_II ～ 10⁶ M^?1), as indicated by fluorescence titration, fluorescence lifetime, “contact” energy transfer and equilibrium dialysis measurements. Binding appears to involve mono-intercalation of the phenanthridinium moiety of these dimers and it is sensitive to, or possibly coupled with, conformational changes within the tRNA macromolecule. These observations raise the possibility that tRNA may represent a pharmacological target of the bifunctional intercalators.     

Aggregation of human lymphoblastoid cells by tumor-promoting phorbol esters and dihydroteleocidin B

Human lymphoblastoid cells transformed by Epstein-Barr virus aggregated rapidly in the presence of tumor-promoting phorbol esters and dihydroteleocidin B. Cell aggregation was almost complete after incubation for 6 hours. In amounts of a few ng, they induced significant aggregation. Their abilities to aggregate cells could be measured quantitatively and correlated well with their effects in promoting skin tumors.     

MA 160 and EB 33 cell lines: HeLa cell contaminants,hybrids or prostatic epithelial cells?

Summary Studies of acid phosphates produced by cell lines MA 160 and EB 33 demonstrated immunochemically their prostatic origin. MA 160 and EB 33, rather than being HeLa contaminants, may be hybrids of prostatic epithelial and HeLa cells or true prostatic cell lines with chromosomal changes common to all long-term cultivated cell lines. This research was supported by NIH (Cancer) Research Grants Nos. 18748 and 16426; and Detroit General Hospital Research Corporation.     

Microtubules cut loose at the cell cortex

The ability of the microtubule cytoskeleton to rapidly and locally reorganize itself in response to intra- and extracellular signals is essential to its wide range of functions. A site of tightly regulated microtubule dynamics—and the major interface between the microtubule cytoskeleton and the extracellular environment—is the cell cortex, where the selective stabilization and destabilization of microtubule plus-ends is required for normal cell division, morphogenesis and migration. In a recent study, we found that the cortex of Drosophila S2 and D17 cells is coated with the microtubule severing enzyme and plus-end depolymerase, Kat-60, which actively suppresses microtubule growth and stability along the cell edge. We have proposed that cortical Kat-60 functions by uncapping plus-ends, thereby activating another microtubule depolymerase, KLP10A, preloaded onto the end. The localized destruction of microtubule plus-ends at a specific cortical could feed into larger regulatory pathways, such as those in control of the actin cytoskeleton, to influence cell polarization and motility.     

Overcoming resistance to rapalogs in gliomas by combinatory therapies

Glioblastoma is the most common and aggressive brain tumor type, with a mean patient survival of approximately 1 year. Many previous analyses of the glioma kinome have identified key deregulated pathways that converge and activate mammalian target of rapamycin (mTOR). Following the identification and characterization of mTOR-promoting activity in gliomagenesis, data from preclinical studies suggested the targeting of mTOR by rapamycin or its analogs (rapalogs) as a promising therapeutic approach. However, clinical trials with rapalogs have shown very limited efficacy on glioma due to the development of resistance mechanisms. Analysis of rapalog-insensitive glioma cells has revealed increased activity of growth and survival pathways compensating for mTOR inhibition by rapalogs that are suitable for therapeutic intervention. In addition, recently developed mTOR inhibitors show high anti-glioma activity. In this review, we recapitulate the regulation of mTOR signaling and its involvement in gliomagenesis, discuss mechanisms resulting in resistance to rapalogs, and speculate on strategies to overcome resistance. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Analysis of genetic variability and relationships among Mentha L. using the limonene synthase gene, LS

The genus Mentha comprises a group of aromatic plants with worldwide distribution. Because of frequent interspecific hybridization, the genetic relationships within the genus are not clearly understood. Limonene synthase, which catalyses the first committed step in the essential oil monoterpene biosynthetic pathway, is considered to be a possible rate limiting enzyme. With the homology-based cloning method, primers were designed according to cDNA sequence to amplify full-length DNA sequences in 13 Mentha samples from five species, using Perilla as an outgroup. Analyses of gene structure, length variation, GC-content, Ts/Tv ratio and evolutionary diversity were carried out. Consensus phylogenetic trees were obtained using maximum likelihood, neighbor-joining, and maximum parsimony, respectively, based on the full-length genomic DNA sequences, complete ORF coding sequences and predicted amino acid sequences. The results presented here based on the sequence of MhLS provide the first credibly supported genetic relationships for Mentha, which enables a basis for further mint taxonomy, cultivation and breeding.