Spermiotoxicity of commercial condoms made from polyurethane, polyisoprene and latex, using domestic ruminants as an experimental animal model.

The use of condoms could provide a means of collecting high-quality spermatozoa from different species under physiological ejaculation conditions. However, certain condom materials may affect sperm functionality. This study examined the spermiotoxicity of different commercial condom materials towards ram and goat spermatozoa. Sperm samples were diluted in Tyrode's medium and placed in contact with a piece of condom material (polyurethane, polyisoprene or latex) and incubated for 30 or 90 min. Contact time in the polyisoprene and latex treatments affected some sperm variables; no such effects were seen, however, in the polyurethane treatments. For ram spermatozoa in contact with polyisoprene, the percentage of dead spermatozoa with a damaged acrosome increased at 90 min, while for spermatozoa in contact with latex, the percentage of live spermatozoa with an intact acrosome decreased. For goat spermatozoa in contact with both polyisoprene and latex, the percentage of dead spermatozoa with a damaged acrosome increased at 90 min, while for spermatozoa in contact with polyisoprene, the percentage of live spermatozoa with an intact acrosome decreased. In conclusion, latex and polyisoprene contain components that affect sperm motility, plasma membrane integrity and acrosome function. Polyurethane does not seem to reduce the quality of semen.

Ultrathin self-assembled fibrin sheets.

Fibrin polymerizes into the fibrous network that is the major structural component of blood clots and thrombi. We demonstrate that fibrin from three different species can also spontaneously polymerize into extensive, molecularly thin, 2D sheets. Sheet assembly occurs in physiologic buffers on both hydrophobic and hydrophilic surfaces, but is routinely observed only when polymerized using very low concentrations of fibrinogen and thrombin. Sheets may have been missed in previous studies because they may be very short-lived at higher concentrations of fibrinogen and thrombin, and their thinness makes them very difficult to detect. We were able to distinguish fluorescently labeled fibrin sheets by polymerizing fibrin onto micro-patterned structured surfaces that suspended polymers 10 microm above and parallel to the cover-glass surface. We used a combined fluorescence/atomic force microscope system to determine that sheets were approximately 5 nm thick, flat, elastic and mechanically continuous. Video microscopy of assembling sheets showed that they could polymerize across 25-microm channels at hundreds of microm(2)/sec (approximately 10(13) subunits/s x M), an apparent rate constant many times greater than those of other protein polymers. Structural transitions from sheets to fibers were observed by fluorescence, transmission, and scanning electron microscopy. Sheets appeared to fold and roll up into larger fibers, and also to develop oval holes to form fiber networks that were "pre-attached" to the substrate and other fibers. We propose a model of fiber formation from sheets and compare it with current models of end-wise polymerization from protofibrils. Sheets could be an unanticipated factor in clot formation and adhesion in vivo, and are a unique material in their own right.

Evidence that αC region is origin of low modulus, high extensibility, and strain stiffening in fibrin fibers.

Fibrin fibers form the structural scaffold of blood clots and perform the mechanical task of stemming blood flow. Several decades of investigation of fibrin fiber networks using macroscopic techniques have revealed remarkable mechanical properties. More recently, the microscopic origins of fibrin's mechanics have been probed through direct measurements on single fibrin fibers and individual fibrinogen molecules. Using a nanomanipulation system, we investigated the mechanical properties of individual fibrin fibers. The fibers were stretched with the atomic force microscope, and stress-versus-strain data was collected for fibers formed with and without ligation by the activated transglutaminase factor XIII (FXIIIa). We observed that ligation with FXIIIa nearly doubled the stiffness of the fibers. The stress-versus-strain behavior indicates that fibrin fibers exhibit properties similar to other elastomeric biopolymers. We propose a mechanical model that fits our observed force extension data, is consistent with the results of the ligation data, and suggests that the large observed extensibility in fibrin fibers is mediated by the natively unfolded regions of the molecule. Although some models attribute fibrin's force-versus-extension behavior to unfolding of structured regions within the monomer, our analysis argues that these models are inconsistent with the measured extensibility and elastic modulus.

A high throughput array microscope for the mechanical characterization of biomaterials.

In the last decade, the emergence of high throughput screening has enabled the development of novel drug therapies and elucidated many complex cellular processes. Concurrently, the mechanobiology community has developed tools and methods to show that the dysregulation of biophysical properties and the biochemical mechanisms controlling those properties contribute significantly to many human diseases. Despite these advances, a complete understanding of the connection between biomechanics and disease will require advances in instrumentation that enable parallelized, high throughput assays capable of probing complex signaling pathways, studying biology in physiologically relevant conditions, and capturing specimen and mechanical heterogeneity. Traditional biophysical instruments are unable to meet this need. To address the challenge of large-scale, parallelized biophysical measurements, we have developed an automated array high-throughput microscope system that utilizes passive microbead diffusion to characterize mechanical properties of biomaterials. The instrument is capable of acquiring data on twelve-channels simultaneously, where each channel in the system can independently drive two-channel fluorescence imaging at up to 50 frames per second. We employ this system to measure the concentration-dependent apparent viscosity of hyaluronan, an essential polymer found in connective tissue and whose expression has been implicated in cancer progression.

High throughput system for magnetic manipulation of cells, polymers, and biomaterials.

In the past decade, high throughput screening (HTS) has changed the way biochemical assays are performed, but manipulation and mechanical measurement of micro- and nanoscale systems have not benefited from this trend. Techniques using microbeads (particles approximately 0.1-10 mum) show promise for enabling high throughput mechanical measurements of microscopic systems. We demonstrate instrumentation to magnetically drive microbeads in a biocompatible, multiwell magnetic force system. It is based on commercial HTS standards and is scalable to 96 wells. Cells can be cultured in this magnetic high throughput system (MHTS). The MHTS can apply independently controlled forces to 16 specimen wells. Force calibrations demonstrate forces in excess of 1 nN, predicted force saturation as a function of pole material, and powerlaw dependence of F approximately r(-2.7+/-0.1). We employ this system to measure the stiffness of SR2+ Drosophila cells. MHTS technology is a key step toward a high throughput screening system for micro- and nanoscale biophysical experiments.

Inhibition of bovine brain microtubule assembly in vitro by stypoldione.

Stypolidione, an orthoquinone derived from the brown seaweed Stypopodium zonale, inhibited the polymerization of three-cycle-purified bovine brain microtubule protein in vitro in a concentration-dependent manner. Fifty per cent inhibition of the extent of polymerization beginning under initiating conditions occurred at a stypoldione concentration of approximately 25 microM, and 50% inhibition of tubulin addition to the assembly ends of microtubules at steady state occurred at a concentration of approximately 8 microM. Only slight structural abnormalities could be detected by negative stain electron microscopy in some of the microtubules that did assemble in the presence of the drug, and no aberrant structural forms of microtubule protein were detected. Stypoldione inhibited the binding of [3H]colchicine to tubulin, with 50% inhibition of colchicine binding activity occurring at a stypoldione concentration of 12-15 microM. Inhibition of colchicine binding activity appeared noncompetitive and was at least partially reversible, suggesting that stypoldione and colchicine bind at separate sites. By assuming that the inhibition constant for the ability of stypoldione to prevent the binding of colchicine to tubulin was equivalent to the dissociation constant for the binding of stypoldione to tubulin, we calculated that approximately 62% of the tubulin present free in solution under initiating conditions and 35-37% of the soluble tubulin under steady-state conditions was complexed with stypoldione when polymerization was inhibited by 50%. These data are consistent with a mechanism in which stypoldione interacts with soluble tubulin and inactivates the tubulin so that it is unable to add to microtubule ends, although a colchicine-like mechanism involving an action of stypoldione at microtubule ends has not been eliminated.

How calcium causes microtubule depolymerization.

The effects of calcium (Ca) were assessed using video-enhanced differential interference contrast light microscopy on individual microtubules in vitro. Phosphocellulose-purified (PC) and microtubule associated protein (MAP)-containing preparations of porcine brain tubulin were assembled in a flow chamber onto sperm axoneme fragments and the pattern of growth and shortening of the microtubules was observed. Tubulin plus Ca was then added to the chamber and observation continued. Ca promoted the disassembly of microtubules by specifically promoting the catastrophe reaction in both PC- and MAP-containing microtubules, without an appreciable change in elongation rate. The effect on catastrophe frequency increased very rapidly above 0.5 mM free Ca, implying a possible cooperative effect. The rescue rate remained very high after Ca addition in MAP-containing microtubules, and the shortening rate was unchanged, while in phosphocellulose-purified microtubules, rescue appeared to be decreased by Ca addition and shortening rates increased 4 to 6-fold. These results illustrate that Ca can directly destabilize growing microtubule ends without changing the effective concentration of free tubulin, and that this effect can be seen even against the background of the profound differences in dynamics conferred by the microtubule-associated proteins. Considered within models of the GTP cap, the results imply that high Ca may act to increase the rate of GTP hydrolysis within the cap.

Symptoms reported by elderly patients with isolated systolic hypertension: baseline data from the SYST-EUR trial. Systolic Hypertension in Europe.

OBJECTIVES: To determine the symptomatic well-being of elderly persons with isolated systolic hypertension. DESIGN AND SETTING: Well-being determined during the placebo run-in period prior to entry to the Systolic Hypertension in Europe (SYST-EUR) trial. SUBJECTS: 641 People, 60 years or older with an average sitting blood pressure of 173/86 mm Hg. OUTCOME MEASURES: 33 Symptomatic complaints determined by a standard interview. RESULTS: The 437 women complained of 25% of the symptoms and the 204 men 21% (P<0.001). A markedly higher prevalence was observed in women compared with men for: pain in the joints of the hands (35% of women complained of this against 22% of men); 'racing heart' (33% against 17%); dry eyes (16% against 6%); blurring of vision (35% against 23%); cramps in the legs (43% against 31%); and a sore throat (15% against 7%). Nocturia was the most frequent complaint (68% in both sexes). Eight symptoms increased with age and one (rash) tended to decline. With increasing systolic pressure women also reported more headaches, unsteadiness, blurring of vision, irregular heart beat and 'racing heart' but, of these, only headaches increased with diastolic pressure. These observations were made after adjusting for age, blood sugar and body mass index (BMI) and were not observed in men. Higher blood sugars were associated with mouth ulcers, 'racing heart', blurring of vision and cramps in the legs. A higher BMI was associated with six symptoms, and a lower age of leaving education with eight. In men, alcohol consumption was related to 'racing heart', and smoking to wheezing and having a dry cough. CONCLUSIONS: A high level of complaint was associated with female gender, increasing age, blood sugar and BMI and a low age of leaving education.