Altered permeability and modulatory character of connexin channels during mammary gland development

Abrupt developmental changes occur in structural form and function of connexin (Cx) channels in the mouse mammary gland. Microarray study shows that the principal connexin isoform in epithelial cells during pregnancy is Cx26, up-regulated and persisting from the virgin. After parturition, there is rapid induction of Cx32. In epithelial plasma membranes, size exclusion chromatography reveals that Cx32 organizes initially with Cx26 as heteromeric (Cx26-Cx32) hemichannels and later in heteromeric and homomeric Cx32 channels. Dramatic alterations of connexin channel function following these developmental changes in channel composition are characterized using native channels reconstituted into liposomes. Changes to channel stoichiometry increase the allowable physical size limits of permeant after parturition; the new Cx32 channels are wider than channels containing Cx26. Most remarkably, heteromeric Cx26-Cx32 channels are selectively permeability to adenosine 3',5' cyclic phosphate (cAMP), guanosine 3',5' cyclic phosphate (cGMP), and inositol 1,4,5-triphosphate (IP(3)), whereas homomeric channels are not. Homomeric Cx26 and heteromeric channels with high Cx26/Cx32 stoichiometry are also inhibited by taurine, an osmolyte playing a key role in milk protein synthesis. Taurine effect is reduced where heteromeric channels contain Cx32 > Cx26 and eliminated when channels contain only Cx32. Connexin channel stoichiometry, permeability, and chemical gating character change in precisely the desired fashion after parturition to maximize molecular and electrical coupling to support coordinated milk secretion.     

Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions

N-terminal pyroglutamate (pGlu) formation from glutaminyl precursors is a posttranslational event in the processing of bioactive neuropeptides such as thyrotropin-releasing hormone and neurotensin during their maturation in the secretory pathway. The reaction is facilitated by glutaminyl cyclase (QC), an enzyme highly abundant in mammalian brain. Here, we describe for the first time that human and papaya QC also catalyze N-terminal glutamate cyclization. Surprisingly, the enzymatic Glu(1) conversion is favored at pH 6.0 while Gln(1) conversion occurs with an optimum at pH 8.0. This unexpected finding might be of importance for deciphering the events leading to deposition of highly toxic pyroglutamyl peptides in amyloidotic diseases.     

Ratjadone and leptomycin B block CRM1-dependent nuclear export by identical mechanisms

Research on the export of proteins and nucleic acids from the nucleus to the cytoplasm has greatly gained from the discovery that the actinobacterial toxin leptomycin B (LMB) specifically inactivates the export receptor chromosomal region maintenance 1 (CRM1). Recently, it was shown that myxobacterial cytotoxins, named ratjadones (RATs), also bind to CRM1 and inhibit nuclear export. However, the reaction mechanism of RATs was not resolved. Here, we show that LMB and RAT A employ the same molecular mechanism to inactivate CRM1. Alkylation of residue Cys528 of CRM1 determines both LMB and RAT sensitivity and prevents nuclear export of CRM1 cargo proteins.     

Inhibition of the yeast cytochrome bc1 complex by ilicicolin H, a novel inhibitor that acts at the Qn site of the bc1 complex

Ilicicolin H is an antibiotic isolated from the "imperfect" fungus Cylindrocladium iliciola strain MFC-870. Ilicicolin inhibits mitochondrial respiration by inhibiting the cytochrome bc(1) complex. In order to identify the site of ilicicolin action within the bc(1) complex we have characterized the effects of ilicicolin on the cytochrome bc(1) complex of Saccharomyces cerevisiae. Ilicicolin inhibits ubiquinol-cytochrome c reductase activity of the yeast bc(1) complex with an IC(50) of 3-5 nM, while 200-250 nM ilicicolin was required to obtain comparable inhibition of the bovine bc(1) complex. Ilicicolin blocks oxidation-reduction of cytochrome b through center N of the bc(1) complex and promotes oxidant-induced reduction of cytochrome b but has no effect on oxidation of ubiquinol through center P. These results indicate that ilicicolin binds to the Qn site of the bc(1) complex. Ilicicolin induces a blue shift in the absorption spectrum of ferro-cytochrome b, and titration of the spectral shift indicates binding of one inhibitor molecule per Qn site. The effects of ilicicolin on electron transfer reactions in the bc(1) complex are similar to those of antimycin, another inhibitor that binds to the Qn site of the bc(1) complex. However, because the two inhibitors have different effects on the absorption spectrum of cytochrome b, they differ in their mode of binding to the Qn site.     

Phylogenetic position and biogeography of Hillebrandia sandwicensis (Begoniaceae): a rare Hawaiian relict

The Begoniaceae consist of two genera, Begonia, with approximately 1400 species that are widely distributed in the tropics, and Hillebrandia, with one species that is endemic to the Hawaiian Islands and the only member of the family native to those islands. To help explain the history of Hillebrandia on the Hawaiian Archipelago, phylogenetic relationships of the Begoniaceae and the Cucurbitales were inferred using sequence data from 18S, rbcL, and ITS, and the minimal age of both Begonia and the Begoniaceae were indirectly estimated. The analyses strongly support the placement of Hillebrandia as the sister group to the rest of the Begoniaceae and indicate that the Hillebrandia lineage is at least 51-65 million years old, an age that predates the current Hawaiian Islands by about 20 million years. Evidence that Hillebrandia sandwicensis has survived on the Hawaiian Archipelago by island hopping from older, now denuded islands to younger, more mountainous islands is presented. Various scenarios for the origin of ancestor to Hillebrandia are considered. The geographic origin of source populations unfortunately remains obscure; however, we suggest a boreotropic or a Malesian-Pacific origin is most likely. Hillebrandia represents the first example in the well-studied Hawaiian flora of a relict genus.     

Nucleocytoplasmic shuttling by nucleoporins Nup153 and Nup214 and CRM1-dependent nuclear export control the subcellular distribution of latent Stat1

Interferon stimulation of cells leads to the tyrosine phosphorylation of latent Stat1 and subsequent transient accumulation in the nucleus that requires canonical transport factors. However, the mechanisms that control the predominantly cytoplasmic localization in unstimulated cells have not been resolved. We uncovered that constitutive energy- and transport factor-independent nucleocytoplasmic shuttling is a property of unphosphorylated Stat1, Stat3, and Stat5. The NH(2)- and COOH-terminal Stat domains are generally dispensable, whereas alkylation of a single cysteine residue blocked cytokine-independent nuclear translocation and thus implicated the linker domain into the cycling of Stat1. It is revealed that constitutive nucleocytoplasmic shuttling of Stat1 is mediated by direct interactions with the FG repeat regions of nucleoporin 153 and nucleoporin 214 of the nuclear pore. Concurrent active nuclear export by CRM1 created a nucleocytoplasmic Stat1 concentration gradient that is significantly reduced by the blocking of energy-requiring translocation mechanisms or the specific inactivation of CRM1. Thus, we propose that two independent translocation pathways cooperate to determine the steady-state distribution of Stat1.     

Human intestinal microvascular endothelial cells express Toll-like receptor 5: a binding partner for bacterial flagellin

Bacterial flagellin has recently been identified as a ligand for Toll-like receptor 5 (TLR5). Human sites known to specifically express TLR5 include macrophages and gastric and intestinal epithelium. Because infection of intestinal epithelial cells with Salmonella leads to an active transport of flagellin to the subepithelial compartment in proximity to microvessels, we hypothesized that human intestinal endothelial cells functionally express TLR5, thus enabling an active inflammatory response upon binding of translocated flagellin. Endothelial expression of TLR5 in human macro- and microvascular endothelial cells was examined by RT-PCR, immunoblot analysis, and immunofluorescence. Endothelial expression of TLR5 in vivo was verified by immunohistochemistry. Endothelial modulation of ICAM-1 expression was quantitated using flow cytometry, and leukocyte transmigration in vitro was assessed by an endothelial transmigration assay. Epithelial-endothelial cellular interactions upon infection with viable Salmonella were investigated using a coculture system in vitro. We found that Salmonella-infected intestinal epithelial cells induce endothelial ICAM-1 expression in cocultured human endothelial cells. Both macro- (HUVEC) and microvascular endothelial cells derived from human skin (human dermal microvascular endothelial cell 1) and human colon (human intestinal microvascular endothelial cells) were found to express high constitutive amounts of TLR5 mRNA and protein. These findings were paralleled by strong immunoreactivity for TLR5 of normal human colonic microvessels in vivo. Furthermore, incubation of human dermal microvascular endothelial cells with flagellin from clinical isolates of Escherichia and Salmonella strains led to a marked up-regulation of ICAM-1, as well as to an enhanced leukocyte transendothelial cell migration. These results suggest that endothelially expressed TLR5 might play a previously unrecognized role in the innate immune response toward bacterial Ags.     

Theoretical 49Ti NMR chemical shifts

⁴⁹Ti chemical shifts for a total of 20 titanium complexes are reported, and several levels of theory are evaluated in order to identify a reliable approach for the calculation of titanium NMR data. The popular B3LYP/6–31G(d)//B3LYP/6–31G(d) proves to give very good agreement with experimental data over a range from 1,400 to −1,300 ppm. The MP2/6–31G(d)//MP2/6–31G(d) level computes even smaller average deviations but fails for TiI₄. This behavior together with its huge demand for computational resources requires careful handling of this theoretical level. In addition, NMR data for five titanium fulvene (or related) complexes are given. Dedicated to Professor Dr. Paul von Ragué Schleyer on the occasion of his 75th birthday     

Multiplex amplification of ancient DNA

This method is designed to assemble long, continuous DNA sequences using minimal amounts of fragmented ancient DNA as template. This is achieved by a two-step approach. In the first step, multiple fragments are simultaneously amplified in a single multiplex reaction. Subsequently, each of the generated fragments is amplified individually using a single primer pair, in a standard simplex (monoplex) PCR. The ability to amplify multiple fragments simultaneously in the first step allows the generation of large amounts of sequence from rare template DNA, whereas the second nested step increases specificity and decreases amplification of contaminating DNA. In contrast to current protocols using many template-consuming simplex PCRs, the method described allows amplification of several kilobases of sequence in just one reaction. It thus combines optimal template usage with a high specificity and can be performed within a day.