The heat shock protein HSP70 promotes mouse NK cell activity against tumors that express inducible NKG2D ligands

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease.     

A fluorescence cell biology approach to map the second integrin-binding site of talin to a 130-amino acid sequence within the rod domain

The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin beta subunits. Here, we report the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site. Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing alpha(IIb)beta(3), linked to green fluorescent protein (GFP). Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments. The rod domain fragment G (residues 1984-2344), devoid of any known actin- or vinculin-binding sites, colocalized with beta(3)-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin alpha(IIb)beta(3) or with the recombinant wild type, but not the Y747A mutant beta(3) cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively. Here, we demonstrate for the first time the in vivo relevance of this interaction by fluorescence resonance energy transfer between beta(3)-GFP and DsRed-talin fragment G. Further in vitro pull-down studies allowed us to map out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984-2113) that also localized to focal adhesions. Finally, we show by a cell biology approach that this integrin-binding site within the talin rod domain is important for beta(3)-cytoskeletal interactions but does not participate in alpha(IIb)beta(3) activation.     

Clinical studies on the appearance of natural anterior teeth in young and old adults

Objectives: To evaluate the typical appearance of natural anterior teeth in young and elderly people and to develop guidelines for the natural appearance of dentures. Design: Clinical examination and photography. Inclusion criteria were a dental state with natural anterior teeth and no more than two crowns. Subjects: A study group (SG) consisting of 64 patients ranging in age from 60 to 86 years (67.3 ± 5.8) and a control group (CG) composed of 64 dental students aged 21–33 years (25.8 ± 2.4). Setting: Dental schools of the Johannes‐Gutenberg‐University Mainz and the University of Leipzig. Results: Most participants were content with the appearance of their own natural teeth. With relaxed lips a mean of 1.5 (±0.19) mm of upper incisors and 1.6 (±0.19) mm of lower incisors were visible in the SG compared with 2.5 (±0.16) and 0.4 (±0.07) mm in the CG, respectively. The subjects in the SG showed a greater diversity in position of teeth in the anterior arch and this was more often with incisors than with canines. Features such as stains, cracks and defects were much more noticeable in the SG. An inter‐dental ‘black triangle’ seemed to be a typical feature of an aged dental appearance. Conclusion: The majority of people older than 60 years who still have their own natural anterior teeth would not wish for a change in appearance if they needed a prosthodontic reconstruction. Thus techniques for customisation should be promoted to obtain a natural appearance in dentures.     

Cobalt(II) and cadmium(II) chelates with nitrogen donors and O2 bonding to Co(II) derivatives

The formation of Cd(II) and Co(II) complexes with N-methylethylenediamine (men) has been studied at 298 K in dimethylsulfoxide (dmso) in an ionic medium set to 0.1 mol dm⁻³ with Et₄NClO₄ in anaerobic conditions by means of potentiometric, UV-Vis, calorimetric and FT-IR technique. Mononuclear ML_j (M=Cd, Co; j=1-3) complexes are formed in exothermic reactions, whereas the entropy changes oppose the complexes formation. The results are discussed in terms of different basicities and steric requirements and the whole of the thermodynamic data reported till now for the two ions with a number of diamines are summarized to visualize the selectivity of the ligands. The dioxygen uptake of Co(men)₂ species has also been studied by means of UV-Vis and EPR techniques. The kinetic parameters and stability constants obtained for the formation of the superoxo and μ-peroxo species are discussed in terms of solvent effect and steric hindrance due to methyl group.Cyclic voltammetry was used to confirm the stability constant for the Co(dmen)₂ (dmen=N,N^′-dimethylethylenediamine) superoxo adduct formation but was not successful to investigate this Co(men)₂-O₂ system.     

Aspergillus fumigatus AR04 obeys Arrhenius' rule in cultivation temperature shifts from 30 to 40°C

To set a benchmark in fungal growth rate, a differential analysis of prototrophic Aspergillus fumigatus AR04 with three ascomycetes applied in > 10³ t year^-1 scale was performed, i.e. Ashbya gosspyii (riboflavin), Aspergillus niger (citric acid) and Aspergillus oryzae (food-processing). While radial colony growth decreased 0.5-fold when A. gossypii was cultivated at 40°C instead of 28°C, A. fumigatus AR04 responded with 1.7-fold faster hyphal growth. A. niger and A. oryzae formed colonies at 40°C, but not at 43°C. Moreover, all A. fumigatus strains tested grew even at 49°C. In chemostat experiments, A. fumigatus AR04 reached steady state at a dilution rate of 0.7 h^-1 at 40°C, 120% more than reported for A. gossypii at 28°C. To study mycelial growth rates under unlimited conditions, carbon dioxide increase rates were calculated from concentrations detected online in the exhaust of batch fermentations for 3 h only. All rates calculated suggest that A. fumigatus AR04 approximates Arrhenius’ rule when comparing short cultivations at 30°C with those at 40°C. Linearization of the exponential phase and comparison of the slopes revealed an increase to 192% by the 10°C up-shift.     

Phenotypical evidence for a gender difference in cardiac norepinephrine transporter function

Norepinephrine transporter (NET) function has a central role in the regulation of synaptic norepinephrine concentrations. Clinical observations in orthostatic intolerance patients suggest a gender difference in NET function. We compared the cardiovascular response to selective NET inhibition with reboxetine between 12 healthy men and 12 age-matched women. Finger blood pressure, brachial blood pressure, and heart rate were measured. The subjects underwent cardiovascular autonomic reflex testing and a graded head-up tilt test. In a separate study, we applied incremental concentrations of tyramine and isoproterenol through subcutaneous microdialysis catheters in eight men and in eight women. NET inhibition elicited a threefold greater increase in supine blood pressure in men than women (P < 0.05). The pressor response was driven by an increased cardiac output. The orthostatic heart rate increase during NET inhibition was greater in men than women (56 +/- 5 beats/min in men, 42 +/- 4 beats/min in women, P < 0.001). In contrast, NET inhibition resulted in a similar suppression in the cold pressor and handgrip response, low-frequency blood pressure oscillations, and venous norepinephrine in the supine position. Men and women were similarly sensitive to the lipolytic effect of isoproterenol and tyramine. We conclude that NET inhibition results in more pronounced changes in cardiac regulation in men than women. Our observations suggest that the NET contribution to cardiac norepinephrine turnover may be decreased in women. The gender difference in NET function may not be expressed in tissues that are less NET dependent than the heart.     

Application of sterilised human amnion for reconstruction of the ocular surface

BACKGROUND: Despite improved surgical techniques and the use of new medication, healing of corneal and conjunctival defects cannot always be achieved. In this connection the clinical use of human amnion, produced by different techniques, has represented a successful alternative for the past ten years. The purpose of the present investigation was the development of a clinically secure, therapeutically efficient, and easy-to-handle (transport, storage, application) allogenic amnion transplant. PATIENTS AND METHODS: A new method for an amnion preparation, which contains a sterilisation process in peracetic acid and a drying process in a laminar flow cabinet, was developed as an alternative to previous techniques described in the literature. Amnion transplantation was used to treat 41 patients, 36 of them with corneal ulcer. Further indications for amnion transplantation were symblepharon, descemetocele, as well as dehiscence of conjunctiva after cerclage. RESULTS: Seventy-three percent of cases showed postoperative improvement evidenced by constant vision, while 15 percent showed decreased vision. CONCLUSIONS: The study confirms the observations of previous investigators who consider amnion transplantation an efficient therapeutic method for a multitude of eye diseases. The new method described in this report, guarantees patients' safety by using a validated new sterilisation process against infections that can be transmitted by human tissue. At present this method constitutes the only process available in Germany, and is approved by the Federal Institute for Drugs and Medical Products (BfArM) for the manufacture of human amnion transplants as a finished medical product.     

The redox protein construction kit: pre-last universal common ancestor evolution of energy-conserving enzymes

Genome analyses and the resolution of three-dimensional structures have provided evidence in recent years for hitherto unexpected family relationships between redox proteins of very diverse enzymes involved in bioenergetic electron transport. Many of these enzymes appear in fact to be constructed from only a limited set of building blocks. Phylogenetic analysis of selected units from this "redox enzyme construction kit" indicates an origin for several prominent bioenergetic enzymes that is very early, lying before the divergence of Bacteria and Archaea. Possible scenarios for the early evolution of selected complexes are proposed based on the obtained tree topologies.     

The naphthoquinol oxidizing cytochrome bc1 complex of the hyperthermophilic knallgasbacterium Aquifex aeolicus: properties and phylogenetic relationships

Phylogenetic analysis of constituent proteins of Rieske/cytochrome b complexes [Schütz et al. (2000) J. Mol. Biol. 300, 663-675] indicated that the respective enzyme from the hyperthermophile Aquifex (A.) aeolicus is closely related to proteobacterial counterparts, in disagreement with positioning of its parent species on small subunit rRNA trees. An assessment of the details and possible reasons for this discrepancy necessitates a thorough understanding of the biochemical and biophysical properties of the enzyme in addition to the bioinformatic data. The cytochrome bc(1) complex from A. aeolicus, which is part of the "Knallgasreaction" pathway, was therefore studied in membranes and in detergent-solubilized, isolated complex. Hemes b(L) (E(m,7) = -190 mV; g(z)= 3.7), b(H) (E(m,7) = -60 mV; g(z )= 3.45), and c(1) (E(m,7) = +160 mV; g(z )= 3.55) were identified by EPR and optical spectroscopy in combination with electrochemical methods. Two electrochemically distinct (E(m,7) = +95 mV; E(m,7) = +210 mV) Rieske centers were detected in membranes, and the +210 mV species was shown to correspond to the Rieske center of the cyt bc(1) complex. The gene coding for this latter Rieske protein was heterologously expressed in Escherichia coli, and the resulting protein was characterized in detail. The pool quinone of A. aeolicus was determined to be naphthoquinone. The redox poises of the individual electron-transfer steps are compared to those of other Rieske/cyt b complexes. The Aquifex enzyme was found to represent the only extant naphthoquinol oxidizing true cyt bc(1) complex described so far. An improved scenario for the phylogenetic positioning of the Aquifex cyt bc(1) complex is proposed.     

The disulphide bonds in the catalytic domain of BACE are critical but not essential for amyloid precursor protein processing activity

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartic protease responsible for the beta-secretase cleavage of APP producing a soluble form of the ectodomain (sAPPbeta) and the membrane-bound, carboxy-terminal intermediates C99 and C89. BACE maturation involves cysteine bridge formation, N -glycosylation and propeptide removal. We investigated variants of BACE in which the disulphide bonds of the catalytic domain spanning between Cys216/Cys420, Cys278/Cys443 and Cys330/Cys380 were removed by mutagenesis. When transfected in cultured cells, these mutants showed impaired maturation. Nevertheless, a fraction of mutated protein retained both the competence to mature as well as the activity to process APP. For the generation of a functional enzyme the conserved Cys330/Cys380 bond was the most critical, whereas the two bonds between Cys216/Cys420 and Cys278/Cys443, which are typical for the membrane-bound BACE, appeared to be less important.