A novel biosensor for mercuric ions based on motor proteins.

We explored the potential of contractile proteins, actin and myosin, as biosensors of solutions containing mercuric ions. We demonstrate that the reaction of HgCl2 with myosin rapidly inhibits actin-activated myosin ATPase activity. Mercuric ions inhibit the in vitro analog of contraction, namely the ATP-initiated superprecipitation of the reconstituted actomyosin complex. Hg reduces both the rate and extent of this reaction. Direct observation of the propulsive movement of actin filaments (10 nm in diameter and 1 microm long) in a motility assay driven by a proteolytic fragment of myosin (heavy meromyosin or HMM) is also inhibited by mercuric ions. Thus, we have demonstrated the biochemical, biophysical and nanotechnological basis of what may prove to be a useful nano-device.

Actin binding proteins: regulation of cytoskeletal microfilaments.

The actin cytoskeleton is a complex structure that performs a wide range of cellular functions. In 2001, significant advances were made to our understanding of the structure and function of actin monomers. Many of these are likely to help us understand and distinguish between the structural models of actin microfilaments. In particular, 1) the structure of actin was resolved from crystals in the absence of cocrystallized actin binding proteins (ABPs), 2) the prokaryotic ancestral gene of actin was crystallized and its function as a bacterial cytoskeleton was revealed, and 3) the structure of the Arp2/3 complex was described for the first time. In this review we selected several ABPs (ADF/cofilin, profilin, gelsolin, thymosin beta4, DNase I, CapZ, tropomodulin, and Arp2/3) that regulate actin-driven assembly, i.e., movement that is independent of motor proteins. They were chosen because 1) they represent a family of related proteins, 2) they are widely distributed in nature, 3) an atomic structure (or at least a plausible model) is available for each of them, and 4) each is expressed in significant quantities in cells. These ABPs perform the following cellular functions: 1) they maintain the population of unassembled but assembly-ready actin monomers (profilin), 2) they regulate the state of polymerization of filaments (ADF/cofilin, profilin), 3) they bind to and block the growing ends of actin filaments (gelsolin), 4) they nucleate actin assembly (gelsolin, Arp2/3, cofilin), 5) they sever actin filaments (gelsolin, ADF/cofilin), 6) they bind to the sides of actin filaments (gelsolin, Arp2/3), and 7) they cross-link actin filaments (Arp2/3). Some of these ABPs are essential, whereas others may form regulatory ternary complexes. Some play crucial roles in human disorders, and for all of them, there are good reasons why investigations into their structures and functions should continue.

The affinity of chick cofilin for actin increases when actin is complexed with DNase I: formation of a cofilin-actin-DNase I ternary complex.

Cofilin, an actin-binding protein, regulates the rate, nature and extent of assembly of the actin cytoskeleton. Native Phast gels show that the addition of cofilin to an actin-DNase I complex (74 kDa) results in the formation of a ternary complex of 94 kDa indicating an equimolar stoichiometry in the ternary complex. Furthermore, native gels show that the addition of cofilin to a solution containing free actin and actin-DNase I and run at pH 8.3 results in cofilin complexing preferentially to the actin-DNase I complex. Conversely, the addition of DNase I to a solution containing an actin-cofilin complex and free actin results in the preferential binding of DNase I to the actin-cofilin complex. These results show that the affinity of cofilin for actin can be increased when actin forms binary complexes. When native gels were run at pH 6.8 the affinity of cofilin for monomeric actin was greater than for the actin-DNase I complex indicating that the cofilin-actin interaction can be regulated by changes in pH. The addition of cofilin to actin resulted in the polymerisation of actin at pH 6.8 whereas at alkaline pH a stable cofilin-actin binary complex could be formed. The biological implications are discussed.

Electrophoretic monitoring of pollutants: effect of cations and organic compounds on protein interactions monitored by native gel electrophoresis.

We describe how the interaction between actin and its protein ligands can be used to evaluate the presence of certain metal (Cd, Cu, Hg, Zn) ions and organic compounds (2,4-dioxin or Picloram) which are common components of environmental pollution. The assay detects the high-affinity binding of actin to actin-binding proteins (ABPs), cofilin or DNase I. The actin-ABP complex was analyzed using native polyacrylamide gel electrophoresis and quantified by scanning densitometry. These proteins are widely distributed in animals and plant cells. The assay involves allowing the proteins to form an actin-ABP complex into which increasing amounts of pollutants are titrated. Thus, the assay directly tests for inhibition of protein-protein interaction. It is sensitive to common pollutants using concentration ranges over which they are known to exert a biological toxicity. A convenient feature of the assay is the fact that all the proteins can be stored in freeze-dried form, and can be purchased commercially. We suggest that if this molecular assay is sensitive to a wide range of environmental pollutants, it could be used as a rapid and convenient assay of the environment in combination with currently available tests.

Learning relational anatomy by correlating thin plastinated sections and magnetic resonance images: preparation of specimens.

Plastination is a process whereby the tissue water and part of the tissue fat of anatomical specimens is replaced with a curable polymer. Several variations of this technique are available, depending on both the type of specimen and polymer being used. In this study, the efficiency of BIODUR PEM 11-prepared cross sections as a teaching aid for radiographic anatomy is investigated. A human cadaveric head was scanned on a magnetic resonance (MR) imager, recording images in a coronal plane of approximately 6 mm in thickness. Corresponding slices were then taken of this head and plastinated by the BIODUR PEM 11 method. In general, a good correlation existed between the plastinated sections and the MR scans. The plastinated slices displayed superior differentiation between musculature compared to the scans. In conclusion, the plastinated sections, used in conjunction with the MR scans, are a valuable tool in learning radiographic anatomy. Additionally, BIODUR PEM 11 is a viable alternative to plastination by other methods.

Distance measurements near the myosin head-rod junction using fluorescence spectroscopy.

We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) bound to the myosin "head". We used LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of chicken skeletal muscle myosin (Cys154). The atomic coordinates of these LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominantly alpha-helical structure. We have constructed a model based on our FRET distance data combined with the known structure of chicken skeletal muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic changes in this region.

Reduced active thyroid hormone levels after delivery.

The effect of delivery on the serum concentration of thyroid hormones was studied in 25 euthyroid women. After delivery serum free and total T3 and T4 fell transiently with a simultaneous increase in reverse T3 while serum TSH and thyroxine binding globulin (TBG) concentrations showed no significant variation. These data suggest that i) similar to what happens in other stressful situations, delivery influences peripheral T4 metabolism, and ii) an elevation of TBG in serum in the early puerperium does not prevent these changes.

[Evaluation of diagnostic methods in anatomic urinary incontinence in gynecology]. / Valorizacija dijagnostickih metoda kod anatomske inkontinencije urina u ginekologiji

The present study was undertaken with the aim of verifying the value of diagnostic methods in female stress incontinence. In their diagnostic work the authors used urethrocystography, measuring of the urethral length and resistance. Displacement of the internal urinary meatus related to the interior border of the symphysis was also observed. In demonstrable incontinence the average posterior urethrovesical angle was 140 degrees, and in the absence of incontinence 120 degrees. It was also found that the difference between the pressure of the internal urinary meatus in straining and non-straining women with incontinence was +0,7 mm Hg and in those without incontinence +4,5 mm Hg. It has been observed, furthermore, that the relation between the vertical displacement of the internal urinary meatus in rest and stress was less than 5 in women with incontinence. In women without incontinence it was more than 5. There was no difference in the range of the urethral length in women either with or without stress incontinence.