Titration properties and thermodynamics of the transition state for folding: comparison of two-state and multi-state folding pathways

CI2 folds and unfolds as a single cooperative unit by simple two-state kinetics, which enables the properties of the transition state to be measured from both the forward and backward rate constants. We have examined how the free energy of the transition state for the folding of chymotrypsin inhibitor 2 (CI2) changes with pH and temperature. In addition to the standard thermodynamic quantities, we have measured the overall acid-titration properties of the transition state and its heat capacity relative to both the denatured and native states. We were able to determine the latter by a method analogous to a well-established procedure for measuring the change in heat capacity for equilibrium unfolding: the enthalpy of activation of unfolding at different values of acid pH were plotted against the average temperature of each determination. Our results show that the transition state of CI2 has lost most of the electrostatic and van der Waals' interactions that are found in the native state, but it remains compact and this prevents water molecules from entering some parts of the hydrophobic core. The properties of the transition state of CI2 are then compared with the major folding transition state of the larger protein barnase, which folds by a multi-state mechanism, with the accumulation of a partly structured intermediate (Dphys or I). CI2 folds from a largely unstructured denatured state under physiological conditions via a transition state which is compact but relatively uniformly unstructured, with tertiary and secondary structure being formed in parallel. We term this an expanded pathway. Conversely, barnase folds from a largely structured denatured state in which elements of structure are well formed through a transition state that has islands of folded elements of structure. We term this a compact pathway. These two pathways may correspond to the two extreme ends of a continuous spectrum of protein folding mechanisms. Although the properties of the two transition states are very different, the activation barrier for folding (Dphys-->++) is very similar for both proteins.     

Is a mutagenic event involved in radiation induced malignant transformation?

A method is described using counterflow centrifugation elutriation to isolate macrophages from murine liver and spleen. In this study three, size fractionated, macrophage populations were collected. Isolation resulted in a high yield of pure Kupffer cells (total of 10 x 10(6) /g liver) and enrichment of splenic macrophages to 20%. In addition to standard methods such as non-specific esterase staining, the isolated macrophages were also characterized by flow cytometry using specific monoclonal antibodies. In addition, a rapid flow cytometry method was introduced to determine the percentage of macrophages based on autofluorescence. A strong correlation was found between the percentages of macrophages found by non-specific esterase staining and autofluorescence. Functional tests revealed differences between the isolated macrophages in terms of tumor necrosis factor-alpha (TNF-alpha) production, oxygen metabolism and the production of nitric oxide. However, no significant differences in phagocytic activity was observed between the fractions. After two weeks of culture without the addition of antibiotics the cells still exhibited the above mentioned functions.     

The Jupiter-2 slow-rotation system

The experience of space missions shows that functional disorders in crewmembers on the type of space motion sickness (SMS) may develop on the initial stage of flight. Longer exposure in micro-g causes a wide range of debilitative changes in the vital body systems. Artificial gravity produced by spacecraft rotation might be a universal tool to counteract the impacts of prolonged microgravity on the human body. However, the significance of SMS does not become less high because of a new factor, i.e. the rotating environment. Research system Jupiter 2 is a stand-alone slow-rotating ground facility for simulating motion sickness equivalent to its space form. The merits of this facility are the possibilities to control the intensity of exposure, perform long-term investigations of two active subjects simultaneously, and study the stages of body adaptation to this agent, and assess physical and operator's performance. The facility carries large expectations to occupational selection.     

Screening for cervical cancer in Brazil

The vibration-induced finger flexion reflex (VFR) and the inhibitory effect of acupuncture on this reflex were studied in five cervical spinal cord injury patients (C-SCIs). VFR, which is a tonic finger flexion reflex induced by vibratory stimulation on the finger tip, was induced before and after acupuncture was carried out on the same hand. A stainless steel needle was inserted to the Hoku point. As in healthy subjects, VFR was performed and it was significantly inhibited by acupuncture in the C-SCIs; mean maximum VFR was 204.2 +/- S.E. 68.6 g before and 119.8 +/- S.E. 42.2 g after acupuncture. The present results suggest that at least part of the reflex center for VFR is located in the spinal cord and that part of VFR inhibition by acupuncture may be mediated via the spinal cord.