Electron paramagnetic resonance of single crystal deoxycobaltohemoglobin

Single crystals of horse ^CoHb were obtained by reduction of ^CoHb⁺ crystals with dithionite. Epr measurements showed that the g? and ^Coà tensors are both axial and share the same principal axis systems. Of the four subunits, the “heme” normals of C? and d? subunits ãb?plane 29 ± 1° from b?; they have the same orientation as the hemes in methemoglobin. The normals of “hemes” à and B? are 47 above the ãb? plane as compared to 16° in methemoglobin.     

Conditionally activated E7 proteins of high-risk and low-risk human papillomaviruses induce S phase in postmitotic, differentiated human keratinocytes

The productive program of human papillomaviruses (HPVs) in epithelia is tightly linked to squamous differentiation. The E7 proteins of high-risk HPV genotypes efficiently inactivate the pRB family of proteins that control the cell cycle, triggering S phase in suprabasal keratinocytes. This ability has until now not been demonstrated for the low-risk HPV-6 or HPV-11 E7 proteins. An inducible system in which HPV-16 E7 is fused to the ligand binding domain of the human estrogen receptor (ER) was described by Smith-McCune et al. (K. Smith-McCune, D. Kalman, C. Robbins, S. Shivakumar, L. Yuschenkoff, and J. M. Bishop, Proc. Natl. Acad. Sci. USA 96:6999-7004, 1999). In the absence of hormone, E7ER is cytoplasmic, and upon addition of 17beta-estradiol, it translocates to the nucleus. Using organotypic epithelial raft cultures developed from primary human keratinocytes, we show that 16E7ER promotes either S-phase reentry or p21cip1 accumulation in differentiated keratinocytes in a stochastic manner as early as 6 h postinduction with 17beta-estradiol. A vector expressing the ER moiety alone had no effect. These observations prove unequivocally that the E7 protein drives S-phase reentry in postmitotic, differentiated keratinocytes rather than preventing S-phase exit while the cells ascend through the epithelium. HPV-11 E7ER and, much less efficiently, HPV-6 E7ER also promoted S-phase reentry by differentiated cells upon exposure to 17beta-estradiol. S-phase induction required the consensus pRB binding motif. We propose that the elevated nuclear levels of the low-risk HPV E7 protein afforded by the inducible system account for the positive results. These observations are entirely consistent with the fact that low-risk HPV genotypes replicate in the differentiated strata in patient specimens, as do the high-risk HPVs.     

Insights into interactions between the alpha-helical region of the salmon calcitonin antagonists and the human calcitonin receptor using photoaffinity labeling

Fish-like calcitonins (CTs), such as salmon CT (sCT), are widely used clinically in the treatment of bone-related disorders; however, the molecular basis for CT binding to its receptor, a class II G protein-coupled receptor, is not well defined. In this study we have used photoaffinity labeling to identify proximity sites between CT and its receptor. Two analogues of the antagonist sCT(8-32) containing a single photolabile p-benzoyl-l-phenylalanine (Bpa) residue in position 8 or 19 were used. Both analogues retained high affinity for the CT receptor and potently inhibited agonist-induced cAMP production. The [Bpa(19)]sCT(8-32) analogue cross-linked to the receptor at or near the equivalent cross-linking site of the full-length peptide, within the fragment Cys(134)-Lys(141) (within the amino terminus of the receptor, adjacent to transmembrane 1) (Pham, V., Wade, J. D., Purdue, B. W., and Sexton, P. M. (2004) J. Biol. Chem. 279, 6720-6729). In contrast, proteolytic mapping and mutational analysis identified Met(49) as the cross-linking site for [Bpa(8)]sCT(8-32). This site differed from the previously identified cross-linking site of the agonist [Bpa(8)]human CT (Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 31177-31182) and may provide evidence for conformational differences between interaction with active and inactive state receptors. Molecular modeling suggests that the difference in cross-linking between the two Bpa(8) analogues can be accounted for by a relatively small change in peptide orientation. The model was also consistent with cooperative interaction between the receptor amino terminus and the receptor core.     

Downregulation of p57kip2 promotes cell invasion via LIMK/cofilin pathway in human nasopharyngeal carcinoma cells

The members of Rho family are well known for their regulation of actin cytoskeleton to control cell migration. The Cip/kip members of cyclin‐dependent (CDK) inhibitors have shown to implicate in cell migration and cytoskeletal dynamics. p57^kip2, a CDK inhibitor, is frequently down‐regulated in several malignancy tumors. However, its biological roles in human nasopharyngeal carcinoma (NPC) cells remained to be investigated. Here, we found p57^kip2 has nuclear and cytoplasm distributions and depletion of endogenous p57^kip2 did not change the cell‐cycle progression. Inhibition of cell proliferation by mitomycin C promoted FBS‐mediated cell migration and accompanied with the downregulation of ΔNp63α and p57^kip2, but did not change the level of p27^kip1, another CDK inhibitor. By using siRNA transfection and cell migration/invasion assays, we found that knockdown of p57^kip2, but not ΔNp63α, involved in promotion of NPC cell migration and invasion via decrease of phospho‐cofilin (p‐cofilin). Treatment with Y‐27632, a specific ROCK inhibitor, we found that dysregulation of ROCK/cofilin pathway decreased p‐cofilin expression and induced cell migration. This change of p‐cofilin induced actin remodeling and pronounced increase of membrane protrusions. Further, silence of p57^kip2 not only decreased the interaction between p57^kip2 and LIMK‐1 assayed by immunoprecipitation but also reduced the level of phospho‐LIMK1/2. Therefore, this study indicated that dysregulation of p57^kip2 promoted cell migration and invasion through modulation of LIMK/cofilin signaling and suggested this induction of inappropriate cell motility might contribute to promoting tumor cell for metastasis. J. Cell. Biochem. 112: 3459–3468, 2011. © 2011 Wiley Periodicals, Inc.     

Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure

Three trypsin inhibitors (TIs), from the seeds of the squash Momordica cochinchinensis (MCo), have been isolated and purified using gel filtration, ion exchange chromatography, and reverse-phase HPLC. Their sequences could be determined only after proteolytic cleavages. In the case of MCoTI-I and -II, it was shown that their polypeptide backbones are cyclic, a structure that has never been described in squash TIs. They contain 34 amino acid residues with 3 disulfide bridges and measured molecular masses of 3453.0 and 3480.7, respectively. They are the largest known macrocyclic peptides containing disulfide bridges. Their sequences show strong homology to other squash TIs, suggesting a similar three-dimensional structure and an analogous mechanism of action. A model of MCoTI-II was constructed by analogy to the crystal structure of the complex between bovine trypsin and CMTI-I, indicating that the linker connecting the two termini is flexible and does not impose significant geometrical constraints. This flexibility allows an Asp-Gly peptide bond rearrangement to occur in this region, giving rise to two isoforms of MCoTI-II. Although the importance of cyclization is not clear, it might confer increased stability and resistance to proteolysis. A minor species, MCoTI-III, was also characterized as containing 30 amino acid residues with a molecular mass of 3379.6. This component possesses a linear backbone with a blocked N-terminus. MCoTIs represent interesting candidates for drug design, either by changing their specificity of inhibition or by using their structure as natural scaffolds bearing new binding activities.     

Bactericidal Activity of Glycinecin A, a Bacteriocin Derived from Xanthomonas campestris pv. glycines, on Phytopathogenic Xanthomonas campestris pv. vesicatoria Cells

The ability of glycinecin A, a bacteriocin derived from Xanthomonas campestris pv. glycines 8ra, to kill closely related bacteria has been demonstrated previously by our group (S. G. Heu et al., Appl. Environ. Microbiol. 67:4105-4110, 2001). In the present study, we aimed at determining the glycinecin A-induced cause of death. Treatment with glycinecin A caused slow dissipation of membrane potential and rapid depletion of the pH gradient. Glycinecin A treatment also induced leakage of potassium ions from X. campestris pv. vesicatoria YK93-4 cells and killed sensitive bacterial cells in a dose-dependent manner. Sensitive cells were killed within 2 h of incubation, most likely due to the potassium ion efflux caused by glycinecin A. These results suggest that the bactericidal mechanism of action of glycinecin A is correlated with the permeability of membranes to hydroxyl and potassium ions, leading to the lethal activity of the bacteriocin on the target bacteria.     

Gene expression monitoring for gene discovery in models of peripheral and central nervous system differentiation, regeneration, and trauma

Gene expression monitoring using gene expression microarrays represents an extremely powerful technology for gene discovery in a variety of systems. We describe the results of seven experiments using Incyte GEM technology to compile a proprietary portfolio of data concerning differential gene expression in six different models of neuronal differentiation and regeneration, and recovery from injury or disease. Our first two experiments cataloged genes significantly up- or down-regulated during two phases of the retinoic acid-induced differentiation of the embryonal carcinoma line Ntera-2. To identify genes involved in neuronal regeneration we performed three GEM experiments, which included changes in gene expression in rat dorsal root ganglia during the healing of experimentally injured sciatic nerve, in regenerating neonatal opossum spinal cord, and during lipopolysaccharide stimulation of primary cultures of rat Schwann cells. Finally we have monitored genes involved in the recovery phase of the inflammatory disease of the rat spinal cord, experimental allergic encephalomyelitis, as well as those responsible for protection from oxidative stress in a glutamate-resistant rat hippocampal cell line. Analysis of the results of the approximately 70,000 data points collected is presented.