Splicing of unnatural amino acids into proteins: a peptide model study

S-Ethyl 2-azidohexanethioate (N3-Hex-SEt), an unnatural amino acid analog of leucine, is coupled with L-cysteine ethyl ester (NH2-Cys-OEt) to obtain N3-Hex-Cys-OEt by native chemical ligation. Coupling of this dipeptide with N-t-butoxycarbonyl-2-diphenylphosphinoethanethioglycinate produces the tripeptide, t-Boc-Gly-Hex-Cys-OEt, in high yield. These reactions suggest an approach for the incorporation of unnatural amino acids into proteins by successive native chemical ligation and Staudinger ligation.     

Shear-dependent 'stick-and-roll' adhesion of type 1 fimbriated Escherichia coli

It is generally assumed that bacteria are washed off surfaces as fluid flow increases because they adhere through 'slip-bonds' that weaken under mechanical force. However, we show here that the opposite is true for Escherichia coli attachment to monomannose-coated surfaces via the type 1 fimbrial adhesive subunit, FimH. Raising the shear stress (within the physiologically relevant range) increased accumulation of type 1 fimbriated bacteria on monomannose surfaces by up to two orders of magnitude, and reducing the shear stress caused them to detach. In contrast, bacterial binding to anti-FimH antibody-coated surfaces showed essentially the opposite behaviour, detaching when the shear stress was increased. These results can be explained if FimH is force-activated; that is, that FimH mediates 'catch-bonds' with mannose that are strengthened by tensile mechanical force. As a result, on monomannose-coated surfaces, bacteria displayed a complex 'stick-and-roll' adhesion in which they tended to roll over the surface at low shear but increasingly halted to stick firmly as the shear was increased. Mutations in FimH that were predicted earlier to increase or decrease force-induced conformational changes in FimH were furthermore shown here to increase or decrease the probability that bacteria exhibited the stationary versus the rolling mode of adhesion. This 'stick-and-roll' adhesion could allow type 1 fimbriated bacteria to move along mannosylated surfaces under relatively low flow conditions and to accumulate preferentially in high shear regions.     

Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence sensing of high- curvature membranes and 3-phosphoinositides

BACKGROUND: Sorting nexins (SNXs) are phox homology (PX) domain-containing proteins thought to regulate endosomal sorting of internalized receptors. The prototypical SNX is sorting nexin-1 (SNX1), a protein that through its PX domain binds phosphatidylinositol 3-monophosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. SNX1 is associated with early endosomes, from where it has been proposed to regulate the degradation of internalized epidermal growth factor (EGF) receptors through modulating endosomal-to-lysosomal sorting. RESULTS: We show here that SNX1 contains a BAR (Bin/Amphiphysin/Rvs) domain, a membrane binding domain that endows SNX1 with the ability to form dimers and to sense membrane curvature. We present evidence that through coincidence detection, the BAR and PX domains efficiently target SNX1 to a microdomain of the early endosome defined by high curvature and the presence of 3-phosphoinositides. In addition, we show that the BAR domain endows SNX1 with an ability to tubulate membranes in-vitro and drive the tubulation of the endosomal compartment in-vivo. Using RNA interference (RNAi), we establish that SNX1 does not play a role in EGF or transferrin receptor sorting; rather it specifically perturbs endosome-to-trans Golgi network (TGN) transport of the cation-independent mannose-6-phosphate receptor (CI-MPR). Our data support an evolutionarily conserved function for SNX1 from yeast to mammals and provide functional insight into the molecular mechanisms underlying lipid-mediated protein targeting and tubular-based protein sorting. CONCLUSIONS: We conclude that through coincidence detection SNX1 associates with a microdomain of the early endosome-characterized by high membrane curvature and the presence of 3-phosphoinositides-from where it regulates tubular-based endosome-to-TGN retrieval of the CI-MPR.     

Essential role for mitochondrial thioredoxin reductase in hematopoiesis, heart development, and heart function

Oxygen radicals regulate many physiological processes, such as signaling, proliferation, and apoptosis, and thus play a pivotal role in pathophysiology and disease development. There are at least two thioredoxin reductase/thioredoxin/peroxiredoxin systems participating in the cellular defense against oxygen radicals. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. To begin to address this question, we generated and characterized mice lacking functional mitochondrial thioredoxin reductase (TrxR2). Ubiquitous Cre-mediated inactivation of TrxR2 is associated with embryonic death at embryonic day 13. TrxR2(TrxR2(-/-)minus;/TrxR2(-/-)minus;) embryos are smaller and severely anemic and show increased apoptosis in the liver. The size of hematopoietic colonies cultured ex vivo is dramatically reduced. TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous oxygen radicals when glutathione synthesis is inhibited. Besides the defect in hematopoiesis, the ventricular heart wall of TrxR2(TrxR2(-/-)minus;/TrxR2(-/-)minus;) embryos is thinned and proliferation of cardiomyocytes is decreased. Cardiac tissue-restricted ablation of TrxR2 results in fatal dilated cardiomyopathy, a condition reminiscent of that in Keshan disease and Friedreich's ataxia. We conclude that TrxR2 plays a pivotal role in both hematopoiesis and heart function.     

No difference in pubertal growth and final height between treated hypogonadal and non-hypogonadal thalassemic patients

BACKGROUND: Many factors can negatively affect growth in thalassemic patients, and hypogonadism has been considered as the main factor responsible for their pubertal growth failure. OBJECTIVE: To evaluate the influence of hypogonadism and its treatment on pubertal growth and final height in thalassemic patients. METHODS: We compared the growth of 28 hypogonadal thalassemic patients in whom puberty was induced to that of 25 patients in whom puberty occurred spontaneously. RESULTS: In both groups of patients we observed reduced peak height velocity (induced puberty: females 4.9 +/- 2.1, males 6.0 +/- 1.8 cm/year; spontaneous puberty: females 6.1 +/- 1.5, males 7.3 +/- 2.1 cm/year) and pubertal height gain (induced puberty: females 11.3 +/- 4.0, males 18.0 +/- 4.5 cm/year; spontaneous puberty: females 15.8 +/- 2.7, males 18.1 +/- 5.3 cm/year) and a short final height (induced puberty: females -1.8 +/- 0.7, males -2.1 +/- 1.0 SDS; spontaneous puberty: females -2.3 +/- 1.0, males -1.9 +/- 1.0 SDS). CONCLUSIONS: Poor pubertal growth is present in thalassemic patients regardless of hypogonadism. Other factors are responsible for the reduced growth spurt and the final short stature observed in these patients.     

Gene expression profiling of mouse Sertoli cell lines

The proliferation and differentiation of Sertoli cells is regulated by follicle-stimulating hormone (FSH). The molecular events following FSH stimulation are only partially known. To investigate FSH action in Sertoli cells, we established two novel FSH-responsive mouse Sertoli-cell-derived lines expressing human wild-type (WT) FSH receptor (FSHR) or overexpressing mutated (Asp567Gly) constitutively active FSHR (MUT). Gene expression profiling with commercially available cDNA arrays, including 588 mouse genes, revealed 146 genes expressed in both cell lines. Compared with the expression pattern of WT cells, 20 genes were identified as being either up- or down-regulated (>two-fold) in the MUT cells. We observed a strong differential expression of factors involved in cellular proliferation, e.g. cyclin D2 (repressed to nearly undetectable levels), proliferating cell nuclear antigen (2.5-fold repression) and Eps-8 (six-fold repression), and in genes involved in cellular differentiation, e.g. cytokeratin-18 (13-fold induction). The cDNA array results for six representative genes were confirmed by Northern blotting, which also included the parental SK-11 cell line devoid of FSHR expression. We found no further acute FSH- or forskolin-induced change in expression levels after 3-h stimulations, suggesting that the observed differences between the two cell lines is a consequence of mild, chronically increased, cAMP production in MUT cells. These results provide a platform for the further investigation of selected candidate genes in primary cultures and/or in vivo.Electronic Supplementary Material Supplementary material is available in the online version of this article at This work was supported by grants from the Deutsche Forschungsgemeinschaft (Confocal Research Group The Male Gamete: Production, Maturation, Function, grant FOR 197-3) and from the German Academic Exchange Service (DAAD) to P. Mathur     

New clues on the origin of the Friedreich ataxia expanded alleles from the analysis of new polymorphisms closely linked to the mutation

Friedreichs ataxia (FRDA) is an autosomal recessive neurodegenerative disorder commonly caused by large expansions of a GAA repeat in the first intron of the frataxin gene, FRDA. The expansion of the triplet repeat is localized within an Alu sequence. FRDA GAA-repeat alleles can be divided into three classes depending on their lengths: short normal alleles (SN), long normal alleles (LN) and expanded pathological alleles (E). We made an accurate analysis of the Alu sequence containing the GAA repeat. We found a new single-nucleotide polymorphism (SNP) that is the closest one to the GAA repeat. We studied this new SNP and the polymorphic polyA region contiguous to the GAA triplets in two populations with different frequencies of FRDA. We found that, while both E and LN alleles seem to be genetically homogeneous and likely related, SN represents a more heterogeneous class of alleles. Indeed, one SNP variation (T) was more frequently associated with (GAA)₈ alleles, whereas the other one (C) with (GAA)₉ repeat(s). The long normal and expanded alleles presented the C haplotype. The same correlation was described for polyA-tract polymorphisms. Thus, 14A was commonly associated with (GAA)₈ alleles and 17A with (GAA)₉ alleles. The long normal alleles more frequently showed the 17A haplotype. Our data seem to suggest that all the E alleles come from LN alleles, while LN alleles come from a defined subclass of SN alleles.     

Immunoprevention of mammary carcinoma in HER-2/neu transgenic mice is IFN-gamma and B cell dependent

A vaccine combining IL-12 and allogeneic mammary carcinoma cells expressing p185(neu) completely prevents tumor onset in HER-2/neu transgenic BALB/c mice (NeuT mice). The immune protection elicited was independent from CTL activity. We now formally prove that tumor prevention is mainly based on the production of anti-p185(neu) Abs. In the present studies, NeuT mice were crossed with knockout mice lacking IFN-gamma production (IFN-gamma(-/-)) or with B cell-deficient mice (microMT). Vaccination did not protect NeuT-IFN-gamma(-/-) mice, thus confirming a central role of IFN-gamma. The block of Ab production in NeuT-microMT mice was incomplete. About one third of NeuT-microMT mice failed to produce Abs and displayed a rapid tumor onset. By contrast, those NeuT-microMT mice that responded to the vaccine with a robust production of anti-p185(neu) Ab displayed a markedly delayed tumor onset. In these NeuT-microMT mice, the vaccine induced a lower level of IgG2a and IgG3 and a higher level of IgG2b than in NeuT mice. Moreover, NeuT-microMT mice failed to produce anti-MHC class I Abs in response to allogeneic H-2(q) molecules present in the cell vaccine. These findings show that inhibition of HER-2/neu carcinogenesis depends on cytokines and specific Abs, and that a highly effective vaccine can rescue Ab production even in B cell-deficient mice.