Conditions of formation of the heparin-fibronectin-collagen complex and the effect of plasmin

The formation and composition of the insoluble heparin-fibronectin-collagen complex and its degradation by proteolysis was investigated. At fixed concentrations of the other molecular components of the complex, the maximal rate of complex formation, measured turbidimetrically, was reached at a concentration of 4 microM heparin and 0.9 microM collagen, while the rate of complex formation was linearly related to concentrations of fibronectin as high as 3 microM. Heparin was incorporated into the complex in a saturable manner, and was released in active anticoagulant form by plasmin but not by urokinase. The complex formation was inhibited by 5 mM calcium or 250 mM NaCl as well as by polybrene or spermin. It is suggested that fibronectin binds both heparin and collagen cooperatively to form an insoluble ternary complex of the extracellular matrix.     

Somatic expansion of the (CAG) n repeat in Huntington disease brains

The mutation causing Huntington disease (HD) has been identified as an expansion of a polymorphic (CAG) _n repeat in the 5 part of the huntingtin gene. The specific neuropathology of HD, viz. selective neuronal loss in the caudate nucleus and putamen, cannot be explained by the widespread expression of the gene. Since somatic expansion is observed in affected tissue in myotonic dystrophy, we have studied the length of the (CAG) _n repeat in various regions of the brain. Although we have not found clear differences when comparing severely and mildly affected regions, we have observed a minor increase in repeat length upon comparison of affected brain samples with cerebellum or peripheral blood. Hence, although further somatic amplification seems to occur in affected areas of the brain, the differences between affected and unaffected regions are too small to make this mechanism an obvious candidate for the cause of differential neuronal degeneration in HD.     

Bone tissue engineering using adipose‐derived stem cells and endothelial cells: Effects of the cell ratio

The microvascular endothelial network is essential for bone formation and regeneration. In this context, endothelial cells not only support vascularization but also influence bone physiology via cell contact‐dependent mechanisms. In order to improve vascularization and osteogenesis in tissue engineering applications, several strategies have been developed. One promising approach is the coapplication of endothelial and adipose derived stem cells (ADSCs). In this study, we aimed at investigating the best ratio of human umbilical vein endothelial cells (HUVECs) and osteogenic differentiated ADSCs with regard to proliferation, apoptosis, osteogenesis and angiogenesis. For this purpose, cocultures of ADSCs and HUVECs with ratios of 25%:75%, 50%:50% and 75%:25% were performed. We were able to prove that cocultivation supports proliferation whereas apoptosis was unidirectional decreased in cocultured HUVECs mediated by a p‐BAD‐dependent mechanism. Moreover, coculturing ADSCs and HUVECs stimulated matrix mineralization and the activity of alkaline phosphatase (ALP). Increased gene expression of the proangiogenic markers eNOS, Flt, Ang2 and MMP3 as well as sprouting phenomena in matrigel assays proved the angiogenic potential of the coculture. In summary, coculturing ADSCs and HUVECs stimulates proliferation, cell survival, osteogenesis and angiogenesis particularly in the 50%:50% coculture.     

An Olfactory Stimulus Modifies Nighttime Sleep in Young Men and Women

Aromatherapy is an anecdotal method for modifying sleep and mood. However, whether olfactory exposure to essential oils affects night‐time objective sleep remains untested. Previous studies also demonstrate superior olfactory abilities in women. Therefore, this study investigated the effects of an olfactory stimulus on subsequent sleep and assessed gender differences in such effects. Thirty‐one young healthy sleepers (16 men and 15 women, aged 18 to 30 yr, mean±SD, 20.5±2.4 yr) completed 3 consecutive overnight sessions in a sleep laboratory: one adaptation, one stimulus, and one control night (the latter 2 nights in counterbalanced order). Subjects received an intermittent presentation (first 2 min of each 10 min interval) of an olfactory (lavender oil) or a control (distilled water) stimulus between 23:10 and 23:40 h. Standard polysomnographic sleep and self‐rated sleepiness and mood data were collected. Lavender increased the percentage of deep or slow‐wave sleep (SWS) in men and women. All subjects reported higher vigor the morning after lavender exposure, corroborating the restorative SWS increase. Lavender also increased stage 2 (light) sleep, and decreased rapid‐eye movement (REM) sleep and the amount of time to reach wake after first falling asleep (wake after sleep onset latency) in women, with opposite effects in men. Thus, lavender serves as a mild sedative and has practical applications as a novel, nonphotic method for promoting deep sleep in young men and women and for producing gender‐dependent sleep effects.     

Applied tissue engineering in the closure of severe burns and chronic wounds using cultured human autologous keratinocytes in a natural fibrin matrix

Whereas in severe burns cultured human epithelial cells may well serve as a life saving method, the true value of tissue-engineered skin products in chronic wound care has yet to be clearly defined. Among other well-known clinical problems, the engraftment rate of commercially available multilayered "sheet grafts" has been shown to vary extremely. Adherence of transplanted cells to the wound bed--especially in the presence of potential wound contamination-- is one of the crucial aspects of this technique. Keratinocyte suspensions in a natural fibrin sealant matrix can potentially treat a variety of skin defects. In acute burn wounds, as well as in chronic wounds the clinical application of this type of tissue-engineered skin substitute demonstrates the capacity of cultured human autologous keratinocytes in a fibrin sealant matrix to adhere to wound beds, attach and spread over the wound resulting in reepithelialization of both acute and chronic wounds. In full thickness burns the combination of this new tool with allogenic dermis is a promising option to achieve complete dermal-epidermal reconstitution by means of tissue engineering and guided tissue repair. When transferring this technique into the treatment of chronic wounds we found an optimal preparation of such recipient wound beds to be crucial to the success. The additional application of continuous negative pressure (vacuum therapy) and preliminary chip skin grafting to optimally prepare the recipient site may be helpful tools to achieve such well-prepared and graftable surfaces. Prospective controlled comparative studies should be designed to further assess the clinical efficacy of this technique.     

A phylogenetically based secondary structure for the yeast telomerase RNA

BACKGROUND: Telomerase is a ribonucleoprotein complex whose RNA moiety dictates the addition of specific simple sequences onto chromosomes ends. While relevant for certain human genetic diseases, the contribution of the essential telomerase RNA to RNP assembly still remains unclear. Phylogenetic analyses of vertebrate and ciliate telomerase RNAs revealed conserved elements that potentially organize protein subunits for RNP function. In contrast, the yeast telomerase RNA could not be fitted to any known structural model, and the limited number of known sequences from Saccharomyces species did not permit the prediction of a yeast specific conserved structure. RESULTS: We cloned and analyzed the complete telomerase RNA loci (TLC1) from all known Saccharomyces species belonging to the "sensu stricto" group. Complementation analyses in S. cerevisiae and end mappings of mature RNAs ensured the relevance of the cloned sequences. By using phylogenetic comparative analysis coupled with in vitro enzymatic probing, we derived a secondary structure prediction of the Saccharomyces cerevisiae TLC1 RNA. This conserved secondary structure prediction includes a central domain that is likely to orchestrate DNA synthesis and at least two accessory domains important for RNA stability and telomerase recruitment. The structure also reveals a potential tertiary interaction between two loops in the central core. CONCLUSIONS: The predicted secondary structure of the TLC1 RNA of S. cerevisiae reveals a distinct folding pattern featuring well-separated but conserved functional elements. The predicted structure now allows for a detailed and rationally designed study to the structure-function relationships within the telomerase RNP-complex in a genetically tractable system.     

Edaphic races and phylogenetic taxa in the Lasthenia californica complex (Asteraceae: Heliantheae): an hypothesis of parallel evolution

Lasthenia californica sensu Ornduff consists of two races that differ in their flavonoid pigments and edaphic tolerances. Recent phylogenetic studies of Lasthenia have revealed that members of L. californica sensu Ornduff belong to two phylogenetic species. The relationship of the edaphic races to these new species and to each other is the focus of this study. Characterization of flavonoid profiles and phylogenetic placement of 33 populations demonstrates that races and phylogenetic taxa are not concordant, suggesting that one or both edaphic races evolved in parallel in the two clades. We hypothesize an edaphically linked ecological role for flavonoid differences that first revealed the existence of two races.     

Myoglobin as a model system for designing heme protein based blood substitutes

The ligand binding properties and resistances to denaturation of >300 different site-directed mutants of sperm whale, pig, and human myoglobin have been examined over the past 15 years. This library of recombinant proteins has been used to derive chemical mechanisms for ligand binding and to examine the factors governing holo- and apoglobin stability. We have also examined the effects of mutagenesis on the dioxygenation of NO by MbO(2) to form NO(3)(-) and metMb. This reaction rapidly detoxifies NO and is a key physiological function of both myoglobins and hemoglobins. The mechanisms derived for O(2) binding and NO dioxygenation have been used to design safer, more efficient, and more stable heme protein-prototypes for use as O(2) delivery pharmaceuticals in transfusion therapy (i.e. blood substitutes). An interactive database is being developed (http://olsonnt1.bioc.rice.edu/web/myoglobinhome.asp) to allow rapid access to the ligand binding parameters, stability properties, and crystal structures of the entire set of recombinant myoglobins. The long-range goal is to use this library for developing general protein engineering principles and for designing individual heme proteins for specific pharmacological and industrial uses.