Core and periphery functionalized dendrimers for transition metal catalysis; a covalent and a non-covalent approach

Dendrimers are well-defined hyperbranched macromolecules with characteristic globular structures for the larger systems. The recent impressive strides in synthetic procedures increased the accessibility of functionalized dendrimers at a practicable scale, resulting in a rapid development of dendrimer chemistry. Dendrimers have inspired many chemists to develop new materials and several applications have been explored, catalysis being one of them. The position of the catalytic site(s) as well as the spatial separation of the catalysts within the dendritic framework is of crucial importance. Dendrimers that are functionalized with transition metals in the core can potentially mimic properties of enzymes, their efficient natural counterparts, whereas the surface-functionalized systems have been proposed to fill the gap between homogeneous and heterogeneous catalysis. We prepared both core- and periphery-functionalized dendritic catalysts that are sufficiently large to enable separation by modern nanofiltration techniques. Here we review our recent findings using these promising novel transition metal-functionalized dendrimers as catalysts in several reactions. We will discuss some of the consequences of the architecturally different systems that have been studied and will elaborate on a novel non-covalent strategy of dendrimer functionalization.     

Spectrally resolved microscopy of GFP trafficking.

Folding and chromophore cyclization-oxidation processes of green and cyan fluorescent fusion proteins (GFP and CFP) in subcellular microenvironments of transfected C6 glioma cells were studied by multipixel spectrally resolved microscopy (SRM). Discrete time-dependent spectral transitions were characterized during protein folding and chromophore maturation in the cytosol, nucleus, mitochondria, endoplasmic reticulum (ER), and Golgi. Spectral similarity mapping of fluorophore transition phases demarcated spatio-temporal fluorescence correlation at a subcellular level. Folding stages were characterized by a transition from red-shifted spectral populations in the time interval of 7-10 hr after transfection to a fully matured fluorophore emitting typical GFP or CFP fluorescence after 10-15 hr. The nascent protein revealed an initial focal accumulation in cytosol emitting in the range of 580-680 nm. After 10 hr, mixed pixel population spectra were measured and at 15 hr GFP was visualized in the cytoplasm by its specific spectral fingerprints with maxima at 545 nm. For nucleus- and mitochondrion-targeted CFPs, the mature conformer was discovered only in its final destination, whereas intermediate steps of fluorophore synthesis (at 10 hr) were found in the cytoplasm. Enhanced fluorescence maturation was manifested only by the ER-Golgi-targeted CFP after 10 hr post transfection by spectral imaging. Moreover, only remnants of initial intermediate fluorescent pixels were localized externally to the Golgi framework at 15 hr. SRM assessed the competence of ER-Golgi to maintain efficient CFP folding in comparison to the rest of the cellular compartments.     

Isolation and function of a human endothelial cell C1q receptor

It has been shown previously that cultured human venous and arterial endothelial cells (EC) bind C1q in a time- and dose-dependent manner. Cultured human endothelial cells express an average number of 5.2 x 10(5) binding sites/cell. In the present study the putative receptor for C1q (C1qR) was isolated from the membranes of 1-5 x 10(9) human umbilical cord EC by affinity chromatography on C1q-Sepharose. During isolation, C1qR was detected by its capacity to inhibit the lysis of EAC1q in C1q-deficient serum. The eluate from C1q-Sepharose was concentrated, dialysed and subjected to QAE-A50 chromatography and subsequently to gel filtration on HPLC-TSK 3000. C1qR filtered at an apparent molecular weight of 60 kDa. Purified C1qR exhibited an apparent molecular weight of 55-62 kDa in the unreduced state and a molecular weight of 64-68 kDa in reduced form. Two IgM monoclonal antibodies (mAb) D3 and D5 were raised following immunization of mice with purified receptor preparations. Both monoclonal antibodies increased the binding of (125)I-C1q to endothelial cells but F(ab')(2) anti-C1qR mAb inhibited the binding of a(125)I-C1q to EC in a dosedependent manner. The D3 mAb recognized a band of 54-60 kDa in Western blots of membranes of human EC and polymorphonuclear leukocytes. Previously, the authors showed that C1q induces the binding of IgM-containing immune complexes to EC. Therefore, it was hypothesized that during a primary immune response generation of IgM-IC may occur, resulting in binding and activation of C1, dissociation of activated C1 by C1 inhibitor and subsequent interaction of IgM-IC bearing C1q with EC-C1qR.     

A 13C NMR method for determination of the transbilayer distribution of phosphatidylcholine in large, unilamellar, protein-free and protein-containing vesicles.

(1) Large unilamellar vesicles have been prepared from N-[Ne3-13C]-18 : 1c/18 : 1c-phosphatidylcholine, both with and without the major intrinsic proteins from the human erythrocyte membrane incorporated in the bilayer. (2) It is shown that the inside-outside distribution of the lipid molecules in these large unilamellar structures can be determined using 13C NMR. (3) Large vesicles of 18 : 1c/18 : 1c-phosphatidylcholine containing glycophorin show an enhanced permeability to Dy3+. It is shown that the permeability barrier of these vesicles can be restored by addition of 10 mol% 18 : 1c/18 : 1c-phosphatidylethanolamine or 1-18 : 1c-lysophosphatidylcholine.     

Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy.

Deep microbial biofilms are a major problem in many industrial, environmental, and medical settings. Novel approaches are needed to understand the structure and metabolism of these biofilms. Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM) were compared quantitatively for the ability to visualize bacteria within deep in vitro biofilms. pH gradients within these biofilms were determined by fluorescence lifetime imaging, together with TPE. A constant-depth film fermentor (CDFF) was inoculated for 8 h at 50 ml. h(-1) with a defined mixed culture of 10 species of bacteria grown in continuous culture. Biofilms of fixed depths were developed in the CDFF for 10 or 11 days. The microbial compositions of the biofilms were determined by using viable counts on selective and nonselective agar media; diverse mixed-culture biofilms developed, including aerobic, facultative, and anaerobic species. TPE was able to record images four times deeper than CLSM. Importantly, in contrast to CLSM images, TPE images recorded deep within the biofilm showed no loss of contrast. The pH within the biofilms was measured directly by means of fluorescence lifetime imaging; the fluorescence decay of carboxyfluorescein was correlated with biofilm pH and was used to construct a calibration curve. pH gradients were detectable, in both the lateral and axial directions, in steady-state biofilms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gradients developed. Microcolonies with pH values of below pH 3.0 were visible, in some cases adjacent to areas with a much higher pH (>5.0). TPE allowed resolution of images at significantly greater depths (as deep as 140 microm) than were possible with CLSM. Fluorescence lifetime imaging allowed the in situ, real-time imaging of pH and the detection of sharp gradients of pH within microbial biofilms.     

Three new observational scales for use in Dutch nursing homes: scales from the Resident Assessment Instrument for Activities of Daily Living, cognition and depression

The reliability and validity of three MDS scales for ADL, cognition and depression are described. The scales consist of items of the Minimum Data Set of the Resident Assessment Instrument and are available just after an MDS assessment. Data collection took place in nine Dutch nursing homes (N = 227) and consisted of three MDS assessments within one month to determine reliability. Several criterion measures were assessed in order to determine convergent validity. Intra- and inter-rater reliability and internal consistency were determined as well as correlation coefficients of the criterion measures and the MDS scales. All three MDS scales appear reliable, especially the ADL-Hierarchy has very good psychometric properties (intra- and inter-rater Intra Class Correlation were 0.81 and 0.83, respectively). Convergent validity of the ADL-Hierarchy and the Cognitive Performance Scale is good, the Depression Rating Scale appears valid in residents with moderate cognitive disorders at the most, but the results are more difficult to interpret in residents with severe cognitive disorders. The MDS scales appear useful in clinical practice and for research purposes in the Dutch nursing homes.     

Effects of (artificial) boar stimuli on uterine activity in estrous sows

This study aims to examine influences of specific boar stimuli on uterine activity in estrous sows, by comparing uterine activity in presence of a mature teaser boar and a robot boar with variable stimuli. Nineteen multiparous, cyclic, commercial crossbred sows were used. Intra-luminal uterine pressure was measured using a non-surgical method for 45 min before applying one of four treatments in combination with a back-pressure-test (BPT): (1) robot with olfactory and auditory stimuli (R+O+A) (n=16), (2) robot with auditory stimuli (R+A) (n=16), (3) robot without additional stimuli (R) (n=16), (4) a mature boar (boar) (n=15). After treatment, measurements continued for 30 min. For each measurement, frequency, mean amplitude and mean duration of uterine contractions were determined. Spontaneous frequency of uterine contractions was 18.6+/-0.7 h(-1) on average and did not differ between treatments. Frequency of contractions increased significantly for the boar (+5.6+/-1.3 h(-1); P<0.01), R+O+A (+3.9+/-1.3; P<0.01) and R+A (+2.6+/-1.3; P<0.05). The effect of boar presence on frequency of contractions was greater than the effect of R (P<0.05). Amplitude and duration of contractions were not affected by treatment. The change in frequency was dependent on spontaneous frequency (P<0.01). In conclusion, the higher the level of boar stimuli, the greater the increase in frequency of uterine contractions. The results indicate that the used combinations of artificial boar stimuli do not mimic a 'whole' boar. It is unclear which boar stimuli stimulate maximal uterine activity during estrus.     

Proinflammatory bacterial peptidoglycan as a cofactor for the development of central nervous system autoimmune disease

Upon stimulation by microbial products through TLR, dendritic cells (DC) acquire the capacity to prime naive T cells and to initiate a proinflammatory immune response. Recently, we have shown that APC within the CNS of multiple sclerosis (MS) patients contain peptidoglycan (PGN), a major cell wall component of Gram-positive bacteria, which signals through TLR and NOD. In this study, we report that Staphylococcus aureus PGN as a single component can support the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model for MS. Mice immunized with an encephalitogenic myelin oligodendrocyte glycoprotein peptide in IFA did not develop EAE. In contrast, addition of PGN to the emulsion was sufficient for priming of autoreactive Th1 cells and development of EAE. In vitro studies demonstrate that PGN stimulates DC-mediated processes, reflected by increased Ag uptake, DC maturation, Th1 cell expansion, activation, and proinflammatory cytokine production. These data indicate that PGN-mediated interactions result in proinflammatory stimulation of Ag-specific effector functions, which are important in the development of EAE. These PGN-mediated processes may occur both within the peripheral lymph nodes as well as in the CNS and likely involve recognition by TLR on DC. Thus, PGN may provide a physiological trigger of DC maturation, and in this way disrupt the normal tolerance to self Ag. As such, PGN signaling pathways may serve as novel targets for the treatment of MS.