Impaired Shc, Ras, and MAPK activation but normal Akt activation in FL5.12 cells expressing an insulin-like growth factor I receptor mutated at tyrosines 1250 and 1251

The Y1250F/Y1251F mutant of the insulin-like growth factor I receptor (IGF-IR) has tyrosines 1250 and 1251 mutated to phenylalanines and is deficient in IGF-I-mediated suppression of apoptosis in FL5.12 lymphocytic cells. To address the mechanism of loss of function in this mutant we investigated signaling responses in FL5.12 cells overexpressing either a wild-type (WT) or Y1250F/Y1251F (mutant) IGF-IR. Cells expressing the mutant receptor were deficient in IGF-I-induced phosphorylation of the JNK pathway and had decreased ERK and p38 phosphorylation. IGF-I induced phosphorylation of Akt was comparable in WT and mutant expressing cells. The decreased activation of the mitogen-activated protein kinase (MAPK) pathways was accompanied by greatly decreased Ras activation in response to IGF-I. Although phosphorylation of Gab2 was similar in WT and mutant cell lines, phosphorylation of Shc on Tyr(313) in response to IGF-I was decreased in cells expressing the mutant receptor, as was recruitment of Grb2 and Ship to Shc. However, phosphorylation of Shc on Tyr(239), the Src phosphorylation site, was normal. A role for JNK in the survival of FL5.12 cells was supported by the observation that the JNK inhibitor SP600125 suppressed IGF-I-mediated protection from apoptosis. Altogether these data demonstrate that phosphorylation of Shc, and assembly of the Shc complex necessary for activation of Ras and the MAPK pathways are deficient in cells expressing the Y1250F/Y1251F mutant IGF-IR. This would explain the loss of IGF-I-mediated survival in FL5.12 cells expressing this mutant and may also explain why this mutant IGF-IR is deficient in functions associated with cellular transformation and cell migration in fibroblasts and epithelial tumor cells.     

A-type cranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity

Clinical, epidemiological and mechanistic studies support the role of cranberry (Vaccinium macrocarpon Ait.) in maintaining urinary tract health. Cranberry proanthocyanidins contain A-type linkages and have been associated with preventing adhesion of P-fimbriated uropathogenic Escherichia coli to uroepithelial cells. It is not known if the presence of the A-type linkage is a prerequisite for anti-adhesion activity. Other commercial sources of proanthocyanidins with all B-type linkages have not previously been screened for this activity. The goals of this study were to compare the in vitro anti-adhesion activity of A-linked proanthocyanidins from cranberry juice cocktail with the anti-adhesion activities of B-linked proanthocyanidins from commercial grape and apple juices, green tea and dark chocolate, and determine if anti-adhesion activity is detectable in human urine following consumption of single servings of each commercial food product. Structural heterogeneity and presence of the A-type linkage in cranberry proanthocyanidins was confirmed utilizing MALDI-TOF/MS and DI/ESI MS, as was the presence of all B-type linkages in the proanthocyanidins from the other commercial products. The isolated A-type proanthocyanidins from cranberry juice cocktail elicited in vitro anti-adhesion activity at 60 microg/ml, the B-type proanthocyanidins from grape exhibited minor activity at 1200 microg/ml, while other B-type proanthocyanidins were not active. Anti-adhesion activity in human urine was detected following cranberry juice cocktail consumption, but not after consumption of the non-cranberry food products. Results suggest that presence of the A-type linkage in cranberry proanthocyanidins may enhance both in vitro and urinary bacterial anti-adhesion activities and aid in maintaining urinary tract health.     

Quantification of the virus-host interaction in human T lymphotropic virus I infection

Background

Cellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection.

Results

In this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 ± 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells.

Conclusion

These observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.     

Inactivation of Giardia muris cysts by free chlorine

The chlorine resistance of cysts of the flagellate protozoan Giardia muris was examined. This organism, which is pathogenic to mice, is being considered as a model for the inactivation of the human pathogen Giardia lamblia. Excystation was used as the criterion for cyst viability. Experiments were performed at pH 5, 7, and 9 at 25 degrees C and pH 7 at 5 degrees C. Survival curves were "stepladder"-shaped, but concentration-time data generally conformed to Watson's Law. Chlorine was most effective at neutral pH and was only slightly less so in acidic solutions. Comparison of inactivation data based on equivalent hypochlorous acid concentrations, which corrects for chlorine ionization, showed that the cysts have a pH-dependent resistance to inactivation. Concentration-time (C X t') products for free chlorine obtained at 25 degrees C ranged from a low of 50 mg min/liter at pH 5 to a high of 218 mg min/liter at pH 9 and were as high as 1,000 mg min/liter at 5 degrees C. It appears that G. muris cysts are somewhat more resistant to inactivation than G. lamblia cysts and rank among the microorganisms that are most resistant to inactivation by free chlorine.     

Molecular dynamics simulations of transitions for ECD epidermal growth factor receptors show key differences between human and drosophila forms of the receptors

Recent X‐ray structural work on the Drosophila epidermal growth factor receptor (EFGR) has suggested an asymmetric dimer that rationalizes binding affinity measurements that go back decades (Alvarado et al., Cell 2010;142:568–579; Dawson et al., Structure 2007;15:942–954; Lemmon et al., Embo J 1997;16:281–294; Mattoon et al., Proc Natl Acad Sci USA 2004;101:923–928; Mayawala et al., Febs Lett 2005;579:3043–3047; Ozcan et al., Proc Natl Acad Sci USA 2006;103:5735–5740). This type of asymmetric structure has not been seen for the human EGF receptor family and it may or may not be important for function in that realm. We hypothesize that conformational changes in the Drosophila system have been optimized for the transition, whereas the barrier for the same transition is much higher in the human forms. To address our hypothesis we perform dynamic importance sampling (DIMS) (Perilla et al., J Comput Chem 2010;32:196–209) for barrier crossing transitions in both Drosophila and human EFGRs. For each set of transitions, we work from the hypothesis, based on results from the AdK system, that salt‐bridge pairs making and breaking connections are central to the conformational change. To evaluate the effectiveness of the salt‐bridges as drivers for the conformational change, we use the effective transfer entropy based on stable state MD calculations (Kamberaj and Der Vaart, Biophys J 2009;97:1747–1755) to define a reduced subset of degrees of freedom that seem to be important for driving the transition (Perilla and Woolf, J Chem Phys 2012;136:164101). Our results suggest that salt‐bridge making and breaking is not the dominant factor in driving the symmetric to asymmetric transition, but that instead it is a result of more concerted and correlated functional motions within a subset of the dimer structures. Furthermore, the analysis suggests that the set of residues involved in the transitions from the Drosophila relative to the human forms differs and that this difference in substate distributions relates to why the asymmetric form may be more common to Drosophila than to the human forms. We close with a discussion about the residues that may be changed in the human and the Drosophila forms to potentially shift the kinetics of the symmetric to asymmetric transition. Proteins 2013; 81:1113–1126. © 2013 Wiley Periodicals, Inc.     

Topography of the interaction of recA protein with single-stranded deoxyoligonucleotides

recA protein, in the presence of adenosine 5'-(gamma-thio)triphosphate, formed stable complexes with single-stranded deoxyoligonucleotides between 9 and 20 residues in length but not with those 8-residues long. The binding of recA protein to a 15-mer and 20-mer completely protected the sugar-phosphate backbone of the nucleic acid from digestion by pancreatic deoxyribonuclease I and protected the 5'-terminal phosphate from cleavage by calf intestinal alkaline phosphatase. Ethylation of the phosphate backbone at any position by ethylnitrosourea blocked the binding of recA protein to the 15-mer but not to the 20-mer. Ethylation of phosphates near the ends of the 15-mer interfered less, suggesting a minimum binding site requirement. In contrast to the protection of the nucleic acid backbone, recA protein did not protect the N-7 position of guanine or the N-3 position of adenine from methylation by dimethyl sulfate, but rather enhanced the methylation of guanine. These results indicate that recA protein binds primarily to the phosphate backbone of single-stranded DNA, leaving the bases free for homologous pairing. We present a model for the organization of the presynaptic filament.     

Cylindrocladium angustatum sp. nov., a new leaf spot pathogen ofTillandsia capitata from Florida, U.S.A.

Cylindrocladium angustatum is described as a new species fromTillandsia introduced with plant material into the U.S.A. from Central America. Koch's postulates are established to prove its pathogenicity to this host. The species is compared with and distinguished fromC. heptaseptatum andC. rumohrae based on morphology, cultural characteristics and phylogenetic analysis of sequence data of the beta-tubulin gene.