Survey of chemical speciation of trace elements using synchrotron radiation

Information concerning the chemical state of trace elements in biological systems generally has not been available. Such information for toxic elements and metals in metalloproteins could prove extremely valuable in the elucidation of their metabolism and other biological processes. The shielding of core electrons by binding electrons affect the energy required for creating inner-shell holes. Furthermore, the molecular binding and symmetry of the local environment of an atom affect the absorption spectrum in the neighborhood of the absorption edge. X-ray absorption near-edge structure (XANES) using synchrotron radiation excitation can be used to provide chemical speciation information for trace elements at concentrations as low as 10 ppm. The structure and position of the absorption curve in the region of an edge can yield vital data about the local structure and oxidation state of the trace element in question. Data are most easily interpreted by comparing the observed edge structure and position with those of model compounds of the element covering the entire range of possible oxidation states. Examples of such analyses will be reviewed.     

Responses of the photosynthetic flagellate,Euglena gracilis,to hypergravity

Motility and orientation has been studied in the unicellular photosynthetic flagellate, Euglena gracilis, using real time image analysis capable of tracking up to 200 cells simultaneously in the slow rotating centrifuge microscope (NIZEMI) which allows one to observe the cells' swimming behavior during centrifugation accelerations between 1 g and 5 g. At 1 g the cells show a weak negative gravitaxis, which increases significantly at higher accelerations up to about 3 g. Though most cells were capable of swimming even against an acceleration of 4.5 g, the degree of gravitaxis decreased and some of the cells were passively moved downward by the acceleration force; this is true for most cells at 5 g. The velocity of cells swimming against 1 g is about 10% lower than that of cells swimming in other directions. The velocity decreases even more drastically in cells swimming against higher acceleration forces than those at 1 g. The degree of gravitactic orientation drastically decreases after short exposure to artificial UV radiation which indicates that gravitaxis may be due to an active physiological perception rather than a physical effect such as an asymmetry of the center of gravity within the cell. Offprint requests to: D.-P. Häder     

Interactions between intrinsic regulation and neural modulation of acetylcholinesterase in fast and slow skeletal muscles

1. Initiation of subsynaptic sarcolemmal specialization and expression of different molecular forms of AChE were studied in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle of the rat under different experimental conditions in order to understand better the interplay of neural influences with intrinsic regulatory mechanisms of muscle cells. 2. Former junctional sarcolemma still accumulated AChE and continued to differentiate morphologically for at least 3 weeks after early postnatal denervation of EDL and SOL muscles. In noninnervated regenerating muscles, postsynaptic-like sarcolemmal specializations with AChE appeared (a) in the former junctional region, possibly induced by a substance in the former junctional basal lamina, and (b) in circumscribed areas along the whole length of myotubes. Therefore, the muscle cells seem to be able to produce a postsynaptic organization guiding substance, located in the basal lamina. The nerve may enhance the production or accumulation of this substance at the site of the future motor end plate. 3. Significant differences in the patterns of AChE molecular forms in EDL and SOL muscles arise between day 4 and day 10 after birth. The developmental process of downregulation of the asymmetric AChE forms, eliminating them extrajunctionally in the EDL, is less efficient in the SOL. The presence of these AChE forms in the extrajunctional regions of the SOL correlates with the ability to accumulate AChE in myotendinous junctions. The typical distribution of the asymmetric AChE forms in the EDL and SOL is maintained for at least 3 weeks after muscle denervation. 4. Different patterns of AChE molecular forms were observed in noninnervated EDL and SOL muscles regenerating in situ. In innervated regenerates, patterns of AChE molecular forms typical for mature muscles were instituted during the first week after reinnervation. 5. These results are consistent with the hypothesis that intrinsic differences between slow and fast muscle fibers, concerning the response of their AChE regulating mechanism to neural influences, may contribute to different AChE expression in fast and slow muscles, in addition to the influence of different stimulation patterns.     

A continuum model to study the internal fluctuations in globular proteins

The salient features of the differential equation model to study protein dynamics are presented with results for 19 proteins.     

Slow potential changes due to transport number effects in cells with unstirred membrane invaginations or dendrites

Summary Many neurones are extremely invaginated and possess branching processes, axons and dendrites. In general, they are surrounded by a restricted diffusion space. Many of these cells exhibit large, slow potential changes during the passage of current across their membranes. Whenever currents cross membranes separating aqueous solutions, differences in transport numbers of the major permeant ions give rise to local concentration changes of these ions adjacent to the membranes, which will result in various electrical and osmotic effects. These transport number effects are expected to be enhanced by the presence of membrane invaginations. Dendrites are equivalent to reversed invaginations and there should be significant changes in concentrations of permeant ions within them. In general, the effects of such changes on the electrical response of a cell will be greater when the concentration of a major permeant ion is low. The effects have been modelled in terms of two nondimensional parameters: the invagination transport number parameter and the relative area occupied by the invaginations A. If these two parameters are known, the magnitudes and time course of the slow potential changes can immediately be estimated and the time course converted to real time, if the length of the invaginations (l) and ionic diffusion coefficient (D) within them are also known. Both analytical and numerical solutions have been given and predictions compared. It is shown that in the case of large currents and potentials the analytical solution predictions will underestimate the magnitudes and rates of onset of the voltage responses. The relative magnitude of the transport number effect within the invaginations (or dendrites) and other transport number contributions to slow potential changes have also been assessed and order-of-magnitude values of these are estimated for some biological data.     

Amino acid sequence of rabbit ventricular myosin light chain-2: Identity with the slow skeletal muscle isoform

Many studies have established a correlation of differences in the activities of various muscle types with differences in the expression of myosin isoforms. In this paper we report the sequence determination of myosin light chain-2 from rabbit slow skeletal (LC2s) and ventricular (LC2v) nmscles. We sequenced tryptic peptides from LC2v which account for all except a few terminal amino acid residues. The major part (87 residues) of the rabbit LC2s sequence, obtained from tryptic and cyanogen bromide (CNBr) peptides, was found to be identical to rabbit LC2v. Our results provide the first sequence information on LC2s from any species, and lend strong support to the hypothesis that LC2s and LC2v are identical. Comparisons of rabbit LC2v and LC2s with rabbit LC2f (from fast skeletal muscle), and also with chicken LC2f and LC2v, show clearly that LC2s and LC2v from mammalian and avian species are more closely related to each other than they are to LC2f isoforms from the same species.     

Flavonoids: potent inhibitors of arachidonate 5-lipoxygenase

Various flavonoids were found to be relatively selective inhibitors of arachidonate 5-lipoxygenase which initiates the biosynthesis of leukotrienes with the activity of slow reacting substance of anaphylaxis. Cirsiliol (3',4',5-trihydroxy-6,7-dimethoxyflavone) was most potent, and the enzyme partially purified from rat basophilic leukemia cells was inhibited by 97% at a concentration of 10 microM (IC50, about 0.1 microM). 12-Lipoxygenases from bovine platelets and porcine leukocytes were also inhibited but at higher concentrations (IC50, about 1 microM), and fatty acid cyclooxygenase purified from bovine vesicular gland was scarcely affected. The compound at 10 microM suppressed by 99% the immunological release of slow reacting substance of anaphylaxis from passively sensitized guinea pig lung (IC50, about 0.4 microM).     

轴浆转运在神经再生中的作用

夹伤坐骨神经阻断标记蛋白在轴浆中的快、慢转运。3天后转运再现。第14天的转运距离与对照相似,说明再生神经的转运动能基本恢复。用快、慢转运测出的14天平均再生速度分别为1.77±0.14与1.96±0.07mm/d,比对照神经的正常生长速度快6.3—7倍,提示再生需要更多的转运物质。进一步发现再生神经中某些标记蛋白(慢转运波W1)的转运速度为10.25±0.66mm/d,约比对照快1倍,因此这些标记蛋白可能包含适应再生需要而加速转运的结构和功能物质。     

Do salt bridges stabilize proteins? A continuum electrostatic analysis

The electrostatic contribution to the free energy of folding was calculated for 21 salt bridges in 9 protein X-ray crystal structures using a continuum electrostatic approach with the DELPHI computer-program package. The majority (17) were found to be electrostatically destabilizing; the average free energy change, which is analogous to mutation of salt bridging side chains to hydrophobic isosteres, was calculated to be 3.5 kcal/mol. This is fundamentally different from stability measurements using pKa shifts, which effectively measure the strength of a salt bridge relative to 1 or more charged hydrogen bonds. The calculated effect was due to a large, unfavorable desolvation contribution that was not fully compensated by favorable interactions within the salt bridge and between salt-bridge partners and other polar and charged groups in the folded protein. Some of the salt bridges were studied in further detail to determine the effect of the choice of values for atomic radii, internal protein dielectric constant, and ionic strength used in the calculations. Increased ionic strength resulted in little or no change in calculated stability for 3 of 4 salt bridges over a range of 0.1-0.9 M. The results suggest that mutation of salt bridges, particularly those that are buried, to "hydrophobic bridges" (that pack at least as well as wild type) can result in proteins with increased stability. Due to the large penalty for burying uncompensated ionizable groups, salt bridges could help to limit the number of low free energy conformations of a molecule or complex and thus play a role in determining specificity (i.e., the uniqueness of a protein fold or protein-ligand binding geometry).     

Slow cortical potential biofeedback and the startle reflex

The negativity of slow cortical potentials (SCP) of the surface EEG is a measure of brain excitability, correlating with motor and cognitive preparation. Selfcontrol of SCP positivity has been shown to reduce seizure activity. Following SCP biofeedback from a central EEG electrode position, subjects gained bidirectional control over their SCP. The current study used a modified feedback methodology, and found a positive relationship between negativity and magnitude of EMG startle response (a measure of cortical and subcortical arousal, particularly aversive response disposition). Greater success in SCP differentiation was associated with self-report of less relaxation during negativity training.This research was supported by the Deutsche Forschungsgemeinschaft under grant No. SFB 307.