Survey of the Distribution of a Newly Characterized Receptor for Advanced Glycation End Products in Tissues

Advanced glycation end products (AGEs), the final products of nonenzymatic glycation and oxidation of proteins, are found in the plasma and accumulate in the tissues during aging and at an accelerated rate in diabetes. A novel integral membrane protein, termed receptor for AGE (RAGE), forms a central part of the cell surface binding site for AGEs. Using monospecific, polyclonal antibody raised to human recombinant and bovine RAGE, immunostaining of bovine tissues showed RAGE in the vasculature, endothelium, and smooth muscle cells and in mononuclear cells in the tissues. Consistent with these data, RAGE antigen and mRNA were identified in cultured bovine endothelium, vascular smooth muscle, and monocyte-derived macrophages. RAGE antigen was also visualized in bovine cardiac myocytes as well as in cultures of neonatal rat cardiac myocytes and in neural tissue where motor neurons, peripheral nerves, and a population of cortical neurons were positive. In situ hybridization confirmed the presence of RAGE mRNA in the tissues, and studies with rat PC12 pheochromocytes indicated that they provide a neuronal-related cell culture model for examining RAGE expression. Pathological studies of human atherosclerotic plaques showed infiltration of RAGE-expressing cells in the expanded intima. These results indicate that RAGE is present in multiple tissues and suggest the potential relevance of AGE-RAGE interactions for modulating properties of the vasculature as well as neural and cardiac function, prominent areas of involvement in diabetes and in the normal aging process.     

Bicycle helmet ventilation and comfort angle dependence

Five modern bicycle helmets were studied to elucidate some of the variations in ventilation performance, using both a heated manikin headform and human subjects (n=7). Wind speed and head angle were varied to test their influence on the measured steady-state heat exchange (cooling power) in the skull section of the headform. The cooling power transmitted by the helmets varied from about 60% to over 90% of that of the nude headform, illustrating the range of present manufacturer designs. Angling the head forward by 30° was found to provide better cooling power to the skull (up to 25%) for three of the helmets and almost equal cooling power in the remaining two cases. Comparisons of skull ventilation at these angles with human subjects strongly supported the headform results.     

Use of an automated multiple-locus, variable-number tandem repeat-based method for rapid and high-throughput genotyping of Staphylococcus aureus isolates

Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant Staphylococcus aureus. We describe an automated variable-number tandem repeat-based method for the rapid genotyping of Staphylococcus aureus. Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of S. aureus isolates in real time.     

Raman spectroscopic study of glutaraldehyde-stabilized collagen and pericardium tissue

For the first time, Raman spectroscopy has been employed to investigate formation of cross-links in collagen and porcine pericardium tissue upon glutaraldehyde (GA) treatment. GA treatment causes a very high fluorescence background, which overlaps Raman bands. It has been found that short fixation time, i.e. 2 h, reduces background radiation significantly, providing new possibilities for studying changes in molecular structure of collagen upon GA modification. The observed changes in position and intensity of Raman bands allowed us to recognize different types of GA-collagen interactions. Strong spectral evidence has been found for the peptide contribution to the formation of the GA-collagen cross-links and for the formation of secondary amines via Schiff base intermediates, and pyridinium-type cross-links. The results also revealed that different hydration levels and a more complex structure of intact tissue in comparison to collagen preparation strongly influence the formation of a GA cross-linking network, e.g. ether-type bond is preferred to form in a less hydrated collagen preparation. Our results have shown that GA treatment causes an increase in water content of pericardium tissue and collagen.     

Incidence and identification of Klebsiella planticola in clinical isolates with emphasis on newborns

Studies conducted in France and Germany suggest that up to 19% of clinically identified Klebsiella sp. are actually Klebsiella planticola, an environmental species that has been attributed to two cases of septicemia, with a rare isolate of Klebsiella terrigena (0. 4%) being identified. A 1-year survey of newborns on a neonatal ward, also conducted in Germany, reported that 72% of Klebsiella sp. were Klebsiella oxytoca and 8.7% were K. planticola. The tests necessary to identify these species are not found in most clinical identification schemes or in the database matrices of most commercial identification products. To determine the incidence of unrecognized K. planticola among the Klebsiella sp. isolates in our collection, we used the battery of seven supplemental tests amended from the work of Monnet and Freney to test 352 stock isolates and 84 fresh clinical isolates from four local hospitals. After testing 436 strains of Klebsiella, only one strain was identified as a possible K. planticola and none was identified as K. terrigena. We tested an additional 43 stock strains of K. oxytoca isolated from newborns by using eight biochemical tests and found one additional strain of K. planticola. The occurrence of K. planticola in our collection is far less frequent than that observed in other countries.     

Bone marrow-derived mesenchymal stem cells in repair of the injured lung

We sought to determine whether an intact bone marrow is essential to lung repair following bleomycin-induced lung injury in mice, and the mechanisms of any protective effects conferred by bone marrow-derived mesenchymal stem cell (BMDMSC) transfer. We found that myelosupression increased susceptibility to bleomycin injury and that BMDMSC transfer was protective. Protection was associated with the differentiation of engrafted BMDMSC into specific and distinct lung cell phenotypes, with an increase in circulating levels of G-CSF and GM-CSF (known for their ability to promote the mobilization of endogenous stem cells) and with a decrease in inflammatory cytokines. In vitro, cells from injured, but not from normal, mouse lung produced soluble factors that caused BMDMSC to proliferate and migrate toward the injured lung. We conclude that bone marrow stem cells are important in the repair of bleomycin-injured lung and that transfer of mesenchymal stem cells protects against the injury. BMDMSC localize to the injured lung and assume lung cell phenotypes, but protection from injury and fibrosis also involves suppression of inflammation and triggering production of reparative growth factors.     

Dependence of force and immediate stiffness on sarcomere length and Ca2+ activation in frog skinned muscle fibres

1. Skinned fibres prepared from semitendinosus muscle of the frog (Rana temporaria) by a modified Natori's method were suspended in ATP-salt solution (pCa 5, pH 6.7, 3°C). Isometric tension was studied as a function of sarcomere length (determined by laser diffraction) and stiffness was measured by recording tension changes in response to quick changes in length performed within 0.5 ms during Ca²⁺ activated contractions.
2. There was a sigmoidal relationship between contractile tension or stiffness and pCa. The threshold Ca ion concentration was 5×10^?7 M at a sarcomere length of 2.2 μm and a little lower at larger sarcomere lengths (as also described by Endo 1972). At all sarcomere lengths peak tension was reached at about 10^?5 M Ca²⁺.
3. The skinned fibres produced maximum tension at sarcomere lengths of 2.0–2.3 μm. With a further increase in sarcomere length, contractile tension decreased. The relation between tension and sarcomere length was linear up to 3.2 μm above which value the relationship ‘tailed’.
4. Quick releases in the range of 0.1–0.5%L ₀ applied during Ca²⁺ activation produced an immediate elastic fall in tension in phase with the length change followed by a quick recovery phase completed within about 10 ms. Conversely, a quick stretch produced an elastic increase followed by a rapid tension decay completed within about 8–10 ms. When the extreme tensions obtained during the length step were plotted versus the size of the length step, a force-extension diagram was obtained corresponding to the T₁-curve of Huxley and Simmons (1973) which intercepted the length axis at about ?8 nm/half sarcomere at all sarcomere lengths investigated. The slope of the linear portion of the T₁-curve was taken to determine immediate stiffness which was proportional to tension when either sarcomere length or Ca²+ ion concentration were varied.
5. In conclusion tension and immediated stiffness are proportional to the extent of actin myosin filament overlap and hence to the number of possible crossbridges between thick and thin filaments.
6. At very low calcium ion concentrations (10^?7 M) skinned fibres develop tension and become stiff when the Mg-ATP concentration is lowered (at constant [ATP] total) to values below 10^?5 M. Under these conditions a quick release causes a drop in tension which is—as in the case of rigor—not followed by a fast recovery of tension. Again stiffness was independent of the direction and amplitude of quick length changes; but — as in the case of rigor — the stiffness to tension ratio was much higher than in Ca²⁺ activated contraction.

     

Increased expression and release of functional tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by T cells from lupus patients with active disease

Increased expression of TRAIL in membrane-bound and soluble form in patients with systemic lupus erythematosus (SLE) has been previously reported. In this study, we characterized the upregulation of T-cell-associated and soluble TRAIL (sTRAIL) in vivo and the modulation of TRAIL expression and soluble protein release in vitro following T cell activation and IFNalpha exposure. The expression of membrane-bound TRAIL as determined by flow cytometry was higher on CD4(+) and CD8(+) T cells from lupus patients compared to controls, particularly on activated CD69(+)CD8(+) T cells. Similarly, sTRAIL levels determined by ELISA were significantly elevated in serum from patients with active SLE and correlated with levels of IFNalpha. In vitro, both T-cell-associated and sTRAIL were maximally induced by T cell activation plus IFNalpha in patients and controls. By Western blot analysis, sTRAIL was detected in sera in both the monomeric and multimeric, functional form. Both forms of TRAIL were functional in vitro as determined by Annexin V staining and (51)Cr release assay but the apoptotic activity of membrane TRAIL was 2.5-fold higher compared to that of sTRAIL. These results indicate that IFNalpha-induced enhancement of TRAIL expression and of TRAIL-mediated apoptosis may amplify the abnormal apoptotic process in SLE.