Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action

Fibroblast growth factor 1 (FGF-1) shows strong angiogenic, osteogenic and tissue-injury repair properties that might be relevant to medical applications. Since FGF-1 is partially unfolded at physiological temperature we decided to increase significantly its conformational stability and test how such an improvement will affect its biological function. Using an homology approach and rational strategy we designed two new single FGF-1 mutations: Q40P and S47I that appeared to be the most strongly stabilizing substitutions among those reported so far, increasing the denaturation temperature by 7.8 deg. C and 9.0 deg. C, respectively. As our goal was to produce highly stable variants of the growth factor, we combined these two mutations with five previously described stabilizing substitutions. The multiple mutants showed denaturation temperatures up to 27 deg. C higher than the wild-type and exhibited full additivity of the mutational effects. All those mutants were biologically competent in several cell culture assays, maintaining typical FGF-1 activities, such as binding to specific cell surface receptors and activation of downstream signaling pathways. Thus, we demonstrate that the low denaturation temperature of wild-type FGF-1 is not related to its fundamental cellular functions, and that FGF-1 action is not affected by its stability. A more detailed analysis of the biological behavior of stable FGF-1 mutants revealed that, compared with the wild-type, their mitogenic properties, as probed by the DNA synthesis assay, were significantly increased in the absence of heparin, and that their half-lives were extensively prolonged. We found that the biological action of the mutants was dictated by their susceptibility to proteases, which strongly correlated with the stability. Mutants which were much more resistant to proteolytic degradation always displayed a significant improvement in the half-life and mitogenesis. Our results show that engineered stable growth factor variants exhibit enhanced and prolonged activity, which can be advantageous in terms of the potential therapeutic applications of FGF-1.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

The development of patient information leaflets. Care of the mouth after radiotherapy

Objectives: Describe the methodology used to produce a patient leaflet on care of the mouth after radiotherapy. Design: Prospective study to design a patient information leaflet. Setting and Subjects : Patients undergoing radiotherapy for oral cancer. Intervention: After a review of the relevant literature, a patient questionnaire was produced. This was then completed by patients undergoing radiotherapy treatment and gave an indication of the problems being encountered. Healthcare professionals were surveyed for their views. A leaflet was then produced according to published guidelines. It was taken back to another group of patients and a further version produced after input from a health promotion group. Results: A leaflet was produced after wide consultation and survey. Conclusion: Considerable effort must to be put into the production of patient information leaflet. It must be discussed with patients in the target group if it is to be sensitive, understood and find acceptance among its users     

Sequence and pH effects of LNA-containing triple helix-forming oligonucleotides: physical chemistry, biochemistry, and modeling studies

Triple helix-forming oligonucleotides (TFOs) have been demonstrated to be capable of interfering with gene expression and modifying genomic DNA in a sequence-specific manner. Partial incorporation of 2'-O,4'-C-methylene linked locked nucleic acid (LNA) residues in TFOs has been shown to enhance significantly triple helix formation, whereas the full-length LNA TFO failed to form a stable triplex. This work is aimed at understanding the triple helix-forming properties of LNA-containing TFOs and at optimally designing their sequences. Both DNA thermal melting, gel retardation, and restriction enzyme experiments as well as modeling studies by molecular mechanics were carried out to investigate the base composition/sequence and pH-dependence effects of LNA-containing TFOs, as well as their structural features underlying triple helix formation. Alternating LNA substitution every 2-3 nucleotides in TFOs is mandatory, whereas the use of thymine LNA residues should be favored under neutral pH conditions. A rule for designing optimal LNA-containing TFOs is proposed. In addition, alternative LNA and 2'-O-methyl residues in TFOs do not significantly improve triple helix formation.     

The effects of long-term endurance training on the immune and endocrine systems of elderly men: the role of cytokines and anabolic hormones

Background  

a decline in immune and endocrine function occurs with aging. The main purpose of this study was to investigate the impact of long-term endurance training on the immune and endocrine system of elderly men. The possible interaction between these systems was also analysed.     

Magnitude and direction of DNA bending induced by screw-axis orientation: influence of sequence, mismatches and abasic sites

DNA-bending flexibility is central for its many biological functions. A new bending restraining method for use in molecular mechanics calculations and molecular dynamics simulations was developed. It is based on an average screw rotation axis definition for DNA segments and allows inducing continuous and smooth bending deformations of a DNA oligonucleotide. In addition to controlling the magnitude of induced bending it is also possible to control the bending direction so that the calculation of a complete (2-dimensional) directional DNA-bending map is now possible. The method was applied to several DNA oligonucleotides including A(adenine)-tract containing sequences known to form stable bent structures and to DNA containing mismatches or an abasic site. In case of G:A and C:C mismatches a greater variety of conformations bent in various directions compared to regular B-DNA was found. For comparison, a molecular dynamics implementation of the approach was also applied to calculate the free energy change associated with bending of A-tract containing DNA, including deformations significantly beyond the optimal curvature. Good agreement with available experimental data was obtained offering an atomic level explanation for stable bending of A-tract containing DNA molecules. The DNA-bending persistence length estimated from the explicit solvent simulations is also in good agreement with experiment whereas the adiabatic mapping calculations with a GB solvent model predict a bending rigidity roughly two times larger.