Polysaccharide-protein surface modification of titanium via a layer-by-layer technique: characterization and cell behaviour aspects

To improve the surface biocompatibility of titanium films, a layer-by-layer (LBL) self-assembly technique, based on the polyelectrolyte-mediated electrostatic adsorption of chitosan (Chi) and gelatin (Gel), was used leading to the formation of multilayers on the titanium thin film surfaces. The film growth was initialized by deposition of one layer of positively charged poly(ethylene imine) (PEI). Then the thin film was formed by the alternate deposition of negatively charged Gel and positively charged Chi utilizing electrostatic interactions. The LBL film growth was monitored by several techniques. The chemical composition, surface topography as well as wettability were investigated by using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM) and water contact angle measurement, respectively. Quantitative XPS analysis showed the alternative change of C/N ratio after four sequential cycles coating of Ti/PEI/Gel/Chi/Gel, which indicated the discrete layer structure of coatings. Uncoated titanium (control sample) displayed a smooth surface morphology (root mean square (RMS) roughness was around 2.5 nm). A full coverage of coating with Gel/Chi layers was achieved on the titanium surface only after the deposition layers of PEI/(Gel/Chi)2. The PEI/Gel/(Chi/Gel)3 layer displayed a rough surface morphology with a tree-like structure (RMS roughness is around 82 nm). These results showed that titanium films could be modified with Chi/Gel which may affect the biocompatibility of the modified titanium films. To confirm this hypothesis, cell proliferation and cell viability of osteoblasts on LBL-modified titanium films as well as control samples were investigated in vitro. The proliferation of osteoblasts on modified titanium films was found to be greater than that on control (p<0.05) after 1 and 7 days culture, respectively. Cell viability measurement showed that the Chi/Gel-modified films have higher cell viability (p<0.05) than the control. These data suggest that Chi/Gel were successfully employed to surface engineer titanium via LBL technique, and enhanced its cell biocompatibility. The approach presented here may be exploited for fabrication of titanium-based implant surfaces.     

Human papillomavirus type 16 DNA sequence

The complete nucleotide sequence of HPV16 DNA (7904 bp) cloned from an invasive cervical carcinoma was determined. Homology comparisons allowed us to align the major open reading frames with the other published papilloma virus DNA sequences. The general organization of the open reading frames is similar to that of the other four papillomavirus (BPV1, HPV1a, HPV6b, CRPV) already sequenced. The sequence reveals an interruption of the reading frame coding for a suspected E1 protein.     

In vivo monitoring of age-related changes in rat brain using quantitative diffusion magnetic resonance imaging and magnetic resonance relaxometry

Ghrelin is a novel peptide that stimulates the release of growth hormone from the pituitary and is involved in hypothalamic feeding regulation. A pre-embedding immunostaining technique was used to study the ultrastructure and synaptic relationships of ghrelin-containing neurons in the rat arcuate nucleus (ARC). Ghrelin-like immunoreactive (ghrelin-LI) neurons were found in the ARC, and were especially abundant in its ventral part. At the electron microscopic level, ghrelin-LI neurons received afferent synapses from many unknown axon terminals. Ghrelin-LI products in the immunoreactive cell bodies, processes, and axon terminals were detected mainly in dense granular vesicles about 110 nm in diameter. Ghrelin-LI presynaptic axon terminals often made synapses with unknown immunonegative neurons. These results suggest that ghrelin acts to regulate food intake through synaptic connections in hypothalamic neuronal networks.     

Selective targeting of antibody-conjugated nanoparticles to leukemic cells and primary T-lymphocytes

In the present study, surface-modified nanoparticles based on biodegradable material were used for antibody coupling in order to get a selective drug carrier systems. Gelatin nanoparticles were prepared by a desolvation process. Sulfhydryl groups were introduced which enabled the linkage of NeutrAvidin (NAv). Antibodies specific for the CD3 antigen on lymphocytic cells were conjugated to the nanoparticles surface. The binding of biotinylated anti-CD3 antibody was achieved by NAv-biotin-complex formation. Cellular binding and uptake were determined by flow cytometry and confocal laser scanning microscopy (CLSM). Cell-type-specific targeting of anti-CD3-conjugated nanoparticles into CD3-positive human T-cell leukemia cells and primary T-lymphocytes could be shown. Celluar uptake and effective internalization of antibody-conjugated nanoparticles into CD3 expressing cells were demonstrated. Uptake rates of about 84% into T-cell leukemia cells were observed. To confirm selectivity of T-cell targeting, competition experiments were carried out adding excessive free anti-CD3 prior to nanoparticle incubation leading to significantly reduced cellular uptake of antibody-conjugated nanoparticles. Further analysis on the mechanism of uptake confirmed a receptor-mediated endocytotic process. Protein-based nanoparticles conjugated with an antibody against a specific cellular antigen hold promise as selective drug delivery systems for specific cell types.     

In situ X-ray absorption spectroscopy at the K-edge of red phosphorus in polyamide 6,6 during a thermo-oxidative degradation

We present a new method for studying the influence of flame retardants as a function of time and temperature by measuring the X-ray absorption spectra of the corresponding additive. Here, red phosphorus in polyamide 6,6 was investigated at the phosphorus K-edge using synchrotron radiation. The thermo-oxidative degradation of the polymer was simulated by heating the sample up to 300°C. XANES

1 XANES, EXAFS: X-ray absorption spectroscopy investigating the near edge structure or the fine structure of the extended region, respectively.

spectra were monitored during the degradation process at different temperatures and at a constant reaction temperature as a function of time. The degradation reaction was analyzed by comparing the XANES spectra of red phosphorus and orthophosphoric acid, and the reaction was identified as an oxidation of red phosphorus to H₃PO₄. The results so obtained are confirmed by the EXAFS spectra of the additive in the polymer sample recorded before and after the thermo-oxidative degradation process, and by the EXAFS spectra of suitable reference compounds.     

The psbA-gene from a red alga resembles those from Cyanobacteria and Cyanelles

Summary Plastid DNA (ptDNA) from the unicellular red alga Cyanidium caldarium was isolated. A 5.8 kb Eco RI, fragment containing the entire psbA-gene was cloned and the nucleotide sequence of the psbA-gene determined. At the carboxyl terminus the encoded protein (D1) contains the seven amino acid-insertion which was found to be typical of the cyanobacteria and the cyanelles of Cyanophora paradoxa. However, the overall sequence homology does not support a direct relationship between the plastids of Cyanidium, cyanelles and the cyanobacteria. As in other photosynthetic organisms the psbA-gene is transcribed as a monocistronic mRNA. The ribosomal RNA operon was located 4 kb upstream of the psbA-gene.     

Effect of increased distal sodium delivery on organic osmolytes and cell electrolytes in the renal outer medulla

Sodium absorption in distal tubule segments was stimulated by increasing the distal delivery via infusion of hypertonic saline. In these animals, and in control rats, electrolyte concentrations in thick ascending limb cells, light and dark cells of the collecting duct in the outer and inner stripe of the outer medulla and in cells of the proximal straight tubule (outer stripe only) were studied. The measurements were performed by electron microprobe analysis of freeze-dried cryosections of the outer medulla. In addition, organic osmolytes (glycerophosphorylcholine, betaine and myo-inositol) were measured by high performance liquid chromatography in cortex and outer medulla. Augmented delivery of sodium chloride to the distal tubule was associated with increased sodium concentrations of thick ascending limb cells both in the outer and inner stripe and of medullary collecting duct light and dark cells in the outer stripe. While the sum of organic osmolyte concentrations was 28% higher in the outer medulla of the salt-loaded animals compared with controls, this value was unchanged in the renal cortex. These findings indicate that the primary event underlying stimulation of sodium absorption along the thick ascending limb during increased distal sodium delivery is enhanced entry of sodium across the apical cell membrane. This would be expected to lead to higher cell sodium concentrations and stimulation of basolateral active Na-K-exchange. The enhanced transport activity of outer medullary tubules may be associated with increased interstitial tonicities and intracellular retention of organic osmolytes.     

High diversity of ankA sequences of Anaplasma phagocytophilum among Ixodes ricinus ticks in Germany

In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes of Anaplasma phagocytophilum strains infecting German Ixodes ricinus ticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022 I. ricinus ticks were investigated for infection with A. phagocytophilum by nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA and groESL genes were sequenced. The complete coding sequence of the ankA gene could be determined in 24 samples. The 16S rRNA and groESL gene sequences were as much as 100% identical to known sequences. Fifteen ankA sequences were >/=99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, German I. ricinus ticks most likely harbor A. phagocytophilum strains that can cause disease in humans. Nine additional sequences were clearly different from known ankA sequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use the ankA gene for further phylogenetic analyses of A. phagocytophilum and to investigate the biology and pathogenicity of strains that differ in the ankA gene.     

The early phase of experimental acute renal failure

Summary Experiments were designed to determine whether leakage of substances across the tubular epithelium, which are impermeant in the normal kidney, falsifies the measurement of glomerular filtration rate in acute renal failure. Permeability to those substances most commonly used for filtration rate determination, polyfructosan, inulin and ferrocyanide, was estimated by measuring their recoveries following perfusion through various nephron segments in haeme pigment, ischaemic and nephrotoxic models of actue renal failure. Late proximal recovery of¹⁴C ferrocyanide was only marginally decreased compared to controls, by a maximum of 6%. Distal recovery of polyfructosan,¹⁴C and³H inulin were depressed somewhat more, by a maximum of 11%. Urinary recovery of¹⁴C inulin was reduced by only 15% in kidneys showing severely restricted renal function. It is concluded that tubular leakage is not a feature of significance in the early phase of moderate acute renal failure, that ferrocyanide and inulin are reliable markers for the determination of nephron filtration rate and water reabsorption, and that the reduction in whole kidney inulin or polyfructosan clearance reflects primarily a reduction in glomerular filtration rate.