Site-specific labeling of ‘second generation’ annexin V with 99mTc(CO)3 for improved imaging of apoptosis in vivo

In this study ‘second generation’ AnxV was specifically labeled with ^99mTc in three different ways outside the binding region of the protein to obtain an improved target-to-background activity ratio. The compounds were tested in vitro and in vivo in normal mice and in a model of hepatic apoptosis (anti-Fas mAb). The apoptosis binding was most prominent for the HIS-tagged ‘second generation’ AnxV labeled with ^99mTc(CO)₃ in comparison to ^99mTc-HYNIC-cys-AnxV and ^99mTc(CO)₃-DTPA-cys-AnxV.     

Improved automated monitoring and new analysis algorithm for circadian phototaxis rhythms in Chlamydomonas

Automated monitoring of circadian rhythms is an efficient way of gaining insight into oscillation parameters like period and phase for the underlying pacemaker of the circadian clock. Measurement of the circadian rhythm of phototaxis (swimming towards light) exhibited by the green alga Chlamydomonas reinhardtii has been automated by directing a narrow and dim light beam through a culture at regular intervals and determining the decrease in light transmittance due to the accumulation of cells in the beam. In this study, the monitoring process was optimized by constructing a new computer-controlled measuring machine that limits the test beam to wavelengths reported to be specific for phototaxis and by choosing an algal strain, which does not need background illumination between test light cycles for proper expression of the rhythm. As a result, period and phase of the rhythm are now unaffected by the time a culture is placed into the machine. Analysis of the rhythm data was also optimized through a new algorithm, whose robustness was demonstrated using virtual rhythms with various noises. The algorithm differs in particular from other reported algorithms by maximizing the fit of the data to a sinusoidal curve that dampens exponentially. The algorithm was also used to confirm the reproducibility of rhythm monitoring by the machine. Machine and algorithm can now be used for a multitude of circadian clock studies that require unambiguous period and phase determinations such as light pulse experiments to identify the photoreceptor(s) that reset the circadian clock in C. reinhardtii.     

A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic

The mammalian class III phosphatidylinositol 3-kinase (PI3K-III) complex regulates fundamental cellular functions, including growth factor receptor degradation, cytokinesis and autophagy. Recent studies suggest the existence of distinct PI3K-III sub-complexes that can potentially confer functional specificity. While a substantial body of work has focused on the roles of individual PI3K-III subunits in autophagy, functional studies on their contribution to endocytic receptor downregulation and cytokinesis are limited. We therefore sought to elucidate the specific nature of the PI3K-III complexes involved in these two processes. High-content microscopy-based assays combined with siRNA-mediated depletion of individual subunits indicated that a specific sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates both receptor degradation and cytokinesis, whereas ATG14L, a PI3K-III subunit involved in autophagy, is not required. The unanticipated role of UVRAG and BIF-1 in cytokinesis was supported by a strong localisation of these proteins to the midbody. Importantly, while the tumour suppressive functions of Beclin 1, UVRAG and BIF-1 have previously been ascribed to their roles in autophagy, these results open the possibility that they may also contribute to tumour suppression via downregulation of mitogenic signalling by growth factor receptors or preclusion of aneuploidy by ensuring faithful completion of cell division.     

Identical TCR beta-chain rearrangements in streptococcal angina and skin lesions of patients with psoriasis vulgaris

Tonsillar infection with Streptococcus pyogenes may induce several nonsuppurative autoimmune sequelae. The precise pathogenetic mechanisms behind this clinically well-established association are still unresolved. Using TCR analysis, we sought to identify a link between streptococcal tonsillitis and the T cell-mediated autoimmune response in psoriasis. Three patients with streptococcal-induced psoriasis underwent tonsillectomy. Using size spectratyping and sequencing of TCR beta-chain variable region gene (TCRBV) rearrangements, we compared the TCR usage of psoriatic skin lesions, blood, tonsils, and tonsillar T cells fractionated according to the expression of the skin address in "cutaneous lymphocyte-associated Ag" (CLA). TCRBV-size spectratype analysis of the blood lymphocytes, tonsils, and the CLA-negative tonsillar T cells revealed largely unselected T cell populations. Instead, TCRBV gene families of the psoriatic lesions and skin-homing CLA-positive tonsillar T cells displayed highly restricted spectratypes. Sequencing of TCRBV cDNA identified various clonal TCRBV rearrangements within the psoriatic lesions that indicated Ag-driven T cell expansion. Several of these clonotypes were also detected within the tonsils and, in one of the patients, within the small subset of CLA-positive tonsillar T cells, suggesting that T cells from the same T cell clones were simultaneously present within skin and tonsillar tissue. Because after tonsillectomy psoriasis cleared in all three patients our observations indicate that T cells may connect psoriatic inflammation to streptococcal angina. They suggest that the chronic streptococcal immune stimulus within the tonsils could act as a source for pathogenic T cells in poststreptococcal disorders, and they may help to explain why eliminating this source with tonsillectomy may improve streptococcal-induced sequelae.     

Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom

L-amino acid oxidase (LAAO) from the Malayan pit viper induces both necrosis and apoptosis in Jurkat cells. Cell death by necrosis is attributed to H₂O₂ produced by oxidation of α-amino acids. In the presence of catalase that effectively scavenges H₂O_2, a switch to apoptosis is observed. The major factors contributing to apoptosis are proposed to be: (i) generation of toxic intermediates from fetal calf serum (ii) binding and internalization of LAAO. The latter process appears to be mediated by the glycan moiety of the enzyme as desialylation reduces cytotoxicity. D-amino acid oxidase (DAAO), which catalyzes the same reaction as LAAO but lacks glycosylation, triggers necrosis as a consequence of H₂O₂ production but not apoptosis in the presence of catalase. Thus induction of cell death by LAAO appears to involve both the generation of H₂O₂ and the molecular interaction of the glycan moiety of the enzyme with structures at the cell surface. S. R. Ande, P. R. Kommoju contributed equally to this work.     

Caspase-containing complexes in the regulation of cell death and inflammation

Caspases are a family of cysteine proteases that are essential in the initiation and execution of apoptosis and the proteolytic maturation of inflammatory cytokines such as IL-1beta and IL-18. Caspases can be subdivided into those that have a large prodomain and those that have not. In general, apoptotic and inflammatory signalling pathways are initiated when large-prodomain caspases are recruited to large protein complexes via homotypic interactions involving death domain folds. The formation of these specialised multimeric platforms involves three major functions: (1) the sensing of cellular stress, damage, infection or inflammation; (2) multimerisation of the platform; and (3) recruitment and conformational activation of caspases. In this overview we discuss the complexes implicated in the regulation of cell death and inflammatory processes such as the death-inducing signalling complex (DISC), the apoptosome, the inflammasomes and the PIDDosome. We describe their sensing functions, compositions and functional outcomes. Inhibitory protein families such as FLIPs and CARD-only proteins prevent the recruitment of caspases in these sensing complexes, avoiding inappropriate initiation of cell death or inflammation.     

Molecular basis of RNA recognition by the human alternative splicing factor Fox-1

The Fox-1 protein regulates alternative splicing of tissue-specific exons by binding to GCAUG elements. Here, we report the solution structure of the Fox-1 RNA binding domain (RBD) in complex with UGCAUGU. The last three nucleotides, UGU, are recognized in a canonical way by the four-stranded beta-sheet of the RBD. In contrast, the first four nucleotides, UGCA, are bound by two loops of the protein in an unprecedented manner. Nucleotides U1, G2, and C3 are wrapped around a single phenylalanine, while G2 and A4 form a base-pair. This novel RNA binding site is independent from the beta-sheet binding interface. Surface plasmon resonance analyses were used to quantify the energetic contributions of electrostatic and hydrogen bond interactions to complex formation and support our structural findings. These results demonstrate the unusual molecular mechanism of sequence-specific RNA recognition by Fox-1, which is exceptional in its high affinity for a defined but short sequence element.     

Quantification of Campylobacter Species Cross-Contamination during Handling of Contaminated Fresh Chicken Parts in Kitchens

Numerous outbreak investigations and case-control studies for campylobacteriosis have provided evidence that handling Campylobacter-contaminated chicken products is a risk factor for infection and illness. There is currently extremely limited quantitative data on the levels of Campylobacter cross-contamination in the kitchen, hindering risk assessments for the pathogen commodity combination of Campylobacter and chicken meat. An exposure assessment needs to quantify the transfer of the bacteria from chicken to hands and the kitchen environment and from there onto ready-to-eat foods. We simulated some typical situations in kitchens and quantified the Campylobacter transfer from naturally contaminated chicken parts most commonly used in Germany. One scenario simulated the seasoning of five chicken legs and the reuse of the same plate for cooked meat. In another, five chicken breast filets were cut into small slices on a wooden board where, without intermediate cleaning, a cucumber was sliced. We also investigated the transfer of the pathogen from chicken via hands to a bread roll. The numbers of Campylobacter present on the surfaces of the chicken parts, hands, utensils, and ready-to-eat foods were detected by using Preston enrichment and colony counting after surface plating on Karmali agar. The mean transfer rates from legs and filets to hands were 2.9 and 3.8%. The transfer from legs to the plate (0.3%) was significantly smaller (P < 0.01) than the percentage transferred from filets to the cutting board and knife (1.1%). Average transfer rates from hands or kitchen utensils to ready-to-eat foods ranged from 2.9 to 27.5%.