Modified Cetyltrimethylammonium bromide method improves robustness and versatility: The benchmark for plant RNA extraction

A wide range of plant RNA extraction methods are available; however, many of these are limited in their application for a diverse range of plant species. With special emphasis on robustness and versatility, we have improved the cetyltrimethylammonium bromide (CTAB) method and isolated high-quality RNA from 16 different plant species. The major modifications made to the protocol described here were a reduction of sample treatment steps and an increase in β-mercaptoethanol concentration (to 3%) resulting in a robust, rapid and reproducible plant RNA extraction protocol that can be used for a broad range of plant species and tissue types.     

Pharmacokinetic drug interactions of azoles

Drug-drug interactions are a major concern with triazoles. Azoles are potent metabolic inhibitors and interactions commonly occur via metabolizing enzymes (ie, cytochrome P450 isoenzyme superfamily) or drug transporters (ie, P-glycoprotein). However, the clinical relevance of these interactions may vary upon the azole involved and upon the “target” drug. Azoles may also be under influence of and become targets of metabolic drug-drug interactions. Potential interactions between antifungal agents and over-the-counter or alternative medicines and herbs should not be underestimated. Here, we provide a comprehensive overview of different types of pharmacokinetic drug interactions involving azoles with selected examples.     

Lateral packing of mineral crystals in bone collagen fibrils

Combined small-angle x-ray scattering and transmission electron microscopy studies of intramuscular fish bone (shad and herring) indicate that the lateral packing of nanoscale calcium-phosphate crystals in collagen fibrils can be represented by irregular stacks of platelet-shaped crystals, intercalated with organic layers of collagen molecules. The scattering intensity distribution in this system can be described by a modified Zernike-Prins model, taking preferred orientation effects into account. Using the model, the diffuse fan-shaped small-angle x-ray scattering intensity profile, dominating the equatorial region of the scattering pattern, could be quantitatively analyzed as a function of the degree of mineralization. The mineral platelets were found to be very thin (1.5 nm ∼ 2.0 nm), having a narrow thickness distribution. The thickness of the organic layers between adjacent mineral platelets within a stack is more broadly distributed with the average value varying from 6 nm to 10 nm, depending on the extent of mineralization. The two-dimensional analytical scheme also leads to quantitative information about the preferred orientation of mineral stacks and the average height of crystals along the crystallographic c axis.     

Basic fibroblast growth factor modulates the expression of glycophorin A and c-kit and inhibits erythroid differentiation in K562 cells.

Basic fibroblast growth factor (bFGF) is produced by bone marrow stromal cells as well as by normal and leukemic hematopoietic cells. In this study, we examine the direct effects of bFGF on erythroid differentiation in K562 cells in order to determine whether bFGF can promote the expression of a primitive phenotype. Low levels of bFGF inhibited erythroid differentiation as evidenced by decreased expression of glycophorin A and increased expression of c-kit. bFGF also increased both the numbers and the sizes of colonies of K562 cells in soft agar assays. The addition of TGF-beta to these cells induced erythroid differentiation which resulted in an increase in glycophorin A and a decrease in c-kit. The simultaneous addition of bFGF and TGF-beta to K562 cells prevented both the TGF-beta-mediated increase in glycophorin A expression and the decrease in c-kit expression associated with erythroid differentiation. bFGF antagonised the TGF-beta-mediated promotion of erythroid differentiation in K562 cells in a dose dependent manner and these two cytokines counteracted each other on an approximately molar basis. These results indicate that bFGF alone increases expression of c-kit and promotes a primitive phenotype in K562 cells. In addition, bFGF counteracts the effects of differentiation-inducing cytokines, such as TGF-beta, on hematopoietic cells. It is therefore possible that enhanced production of bFGF by leukemic cells could contribute to their neoplastic phenotype by opposing the effects of negative regulators or cytokines that induce differentiation.     

Point mutation causing constitutive signaling of CXCR2 leads to transforming activity similar to Kaposi's sarcoma herpesvirus-G protein-coupled receptor.

The chemokine receptor CXCR2 is the closest homologue to Kaposi's sarcoma herpesvirus-G protein-coupled receptor (KSHV-GPCR), which is known to be constitutively activated and able to cause oncogenic transformation. Among G protein-coupled receptors, a DRY sequence in the second intracellular loop is highly conserved. However, the KSHV-GPCR shows a VRY sequence instead. In this study, we exchanged Asp138 of the DRY sequence in the CXCR2 with a Val (D138V), the corresponding amino acid in KSHV-GPCR, or with a Gln (D138Q), and investigated the functional consequences of these mutations. In focus formation and soft agar growth assays in NIH 3T3 cells, the D138V mutant exhibited transforming potential similar to the KSHV-GPCR. Surprisingly, the CXCR2 wild type itself showed transforming activity, although not as potently, due to continuous autocrine stimulation, whereas the D138Q mutant formed no foci. In agreement with these results were high levels of inositol phosphate accumulation in the D138V mutant and the KSHV-GPCR, indicating constitutive activity. These data emphasize the importance of the DRY sequence for G protein-coupled signaling of the CXCR2. Either constitutive activation or persistent autocrine stimulation of the CXCR2 causes transformation similar to KSHV-GPCR-transfected cells, probably activating the same signal transduction cascade that can abrogate normal growth control mechanisms.     

Cell-mediated immunity in experimental allergic encephalomyelitis: cross reactivity between myelin basic protein and mycobacteria antigens.

Guinea pigs injected with Freund's incomplete adjuvant emulsified with guinea pig spinal cord, purified guinea pig myelin basic protein, or human myelin basic protein showed dermal reactivity to both of the basic proteins as well as to mycobacteria antigens. Animals receiving only mycobacteria antigens expressed dermal reactivity to the sensitizing antigen in addition to basic protein. This cross reactivity may help explain the role of mycobacteria in inducing and protecting against EAE, and may have important implications concerning human demyelinating diseases.     

All KLH preparations are not created equal

Keyhole limpet hemocyanin (KLH) preparations used for dermal testing in human volunteers may contain factors which elicit false positive reactions. These factors can be associated with the hemolymph collection procedure and possibly to a change in collection beds by the limpet supplier. Rapid collection of hemolymph from animals obtained from specified limpet beds resulted in KLH which produced negligible dermal reactivity in normal individuals.