Biophysical Alterations Research Articles

Molecular details of membrane fluidity changes during apoptosis and relationship to phospholipase A2 activity

Secretory phospholipase A(2) exhibits much greater activity toward apoptotic versus healthy cells. Various plasma membrane changes responsible for this phenomenon have been proposed, including biophysical alterations described as "membrane fluidity" and "order." Understanding of these membrane perturbations was refined by applying studies with model membranes to fluorescence measurements during thapsigargin-induced apoptosis of S49 cells using probes specific for the plasma membrane: Patman and trimethylammonium-diphenylhexatriene. Alterations in emission properties of these probes corresponded with enhanced susceptibility of the cells to hydrolysis by secretory phospholipase A(2). By applying a quantitative model, additional information was extracted from the kinetics of Patman equilibration with the membrane. Taken together, these data suggested that the phospholipids of apoptotic membranes display greater spacing between adjacent headgroups, reduced interactions between neighboring lipid tails, and increased penetration of water among the heads. The phase transition of artificial bilayers was used to calibrate quantitatively the relationship between probe fluorescence and the energy of interlipid interactions. This analysis was applied to results from apoptotic cells to estimate the frequency with which phospholipids protrude sufficiently at the membrane surface to enter the enzyme's active site. The data suggested that this frequency increases 50-100-fold as membranes become susceptible to hydrolysis during apoptosis.

Spatial Anisotropies and Temporal Fluctuations in Extracellular Matrix Network Texture during Early Embryogenesis

Early stages of vertebrate embryogenesis are characterized by a remarkable series of shape changes. The resulting morphological complexity is driven by molecular, cellular, and tissue-scale biophysical alterations. Operating at the cellular level, extracellular matrix (ECM) networks facilitate cell motility. At the tissue level, ECM networks provide material properties required to accommodate the large-scale deformations and forces that shape amniote embryos. In other words, the primordial biomaterial from which reptilian, avian, and mammalian embryos are molded is a dynamic composite comprised of cells and ECM. Despite its central importance during early morphogenesis we know little about the intrinsic micrometer-scale surface properties of primordial ECM networks. Here we computed, using avian embryos, five textural properties of fluorescently tagged ECM networks — (a) inertia, (b) correlation, (c) uniformity, (d) homogeneity, and (e) entropy. We analyzed fibronectin and fibrillin-2 as examples of fibrous ECM constituents. Our quantitative data demonstrated differences in the surface texture between the fibronectin and fibrillin-2 network in Day 1 (gastrulating) embryos, with the fibronectin network being relatively coarse compared to the fibrillin-2 network. Stage-specific regional anisotropy in fibronectin texture was also discovered. Relatively smooth fibronectin texture was exhibited in medial regions adjoining the primitive streak (PS) compared with the fibronectin network investing the lateral plate mesoderm (LPM), at embryonic stage 5. However, the texture differences had changed by embryonic stage 6, with the LPM fibronectin network exhibiting a relatively smooth texture compared with the medial PS-oriented network. Our data identify, and partially characterize, stage-specific regional anisotropy of fibronectin texture within tissues of a warm-blooded embryo. The data suggest that changes in ECM textural properties reflect orderly time-dependent rearrangements of a primordial biomaterial. We conclude that the ECM microenvironment changes markedly in time and space during the most important period of amniote morphogenesis—as determined by fluctuating textural properties.

Nanotechnology in stem cells research: advances and applications

Human beings suffer from a myriad of disorders caused by biochemical or biophysical alteration of physiological systems leading to organ failure. For a number of these conditions, stem cells and their enormous reparative potential may be the last hope for restoring function to these failing organ or tissue systems. To harness the potential of stem cells for biotherapeutic applications, we need to work at the size scale of molecules and processes that govern stem cells fate. Nanotechnology provides us with such capacity. Therefore, effective amalgamation of nanotechnology and stem cells - medical nanoscience or nanomedicine - offers immense benefits to the human race. The aim of this paper is to discuss the role and importance of nanotechnology in stem cell research by focusing on several important areas such as stem cell visualization and imaging, genetic modifications and reprogramming by gene delivery systems, creating stem cell niche, and similar therapeutic applications.

Computational Predictions of Tissue-Specific Consequences of Long QT Mutations: Role of the Purkinje Fiber

The long QT syndrome (LQTS) is a heritable cardiac disorder that leads to prolongation of ventricular repolarization, episodes of ventricular arrhythmia, and sudden cardiac death. Mounting evidence has implicated the Purkinje fiber (PF) conduction system in the genesis of ventricular arrhythmias. This study assesses for tissue-specific consequences of the biophysical alterations induced by LQTS-related mutations, using computational models of ventricular and PF cells. Mutations causing LQT1 and LQT2 (in KCNQ1 and HERG, respectively) are first simulated by reducing density of the respective components of the delayed rectifier current. These mutations prolong action potential duration (APD) in ventricular myocytes more than in their PF counterparts, due to differences in the conductance and activation of plateau potassium currents between the two cell types. Next, the canonical LQT3 mutation delKPQ (in the cardiac NaV 1.5 sodium channel, encoded by SCN5A) is modeled in both tissue types (as described previously, by increasing entry into a bursting mode of channel gating, producing substantial late non-inactivating inward current). Marked APD prolongation is confirmed in both tissue types, exacerbated by slow stimulation rates or pauses in pacing. Simulation of another SCN5A mutation, F1473C, which clinically produces severe QT prolongation and heavy arrhythmia burden, is shown to have a markedly larger effect on PF cells than ventricular myocytes (including a propensity for repetitive early afterdepolarizations), owing to a depolarizing shift in the mutant channel availability and lower plateau potential in PF. Finally, the interactions between PF and ventricular cells at the tissue level are investigated in the context of these mutations in a cable model representing a section of ventricular wall. In conclusion, the biophysical alterations induced by LQT mutations may have significant tissue-specific consequences, with important implications for arrhythmia and its therapy.

In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology

In hematologic diseases, such as sickle cell disease (SCD) and hemolytic uremic syndrome (HUS), pathological biophysical interactions among blood cells, endothelial cells, and soluble factors lead to microvascular occlusion and thrombosis. Here, we report an in vitro "endothelialized" microfluidic microvasculature model that recapitulates and integrates this ensemble of pathophysiological processes. Under controlled flow conditions, the model enabled quantitative investigation of how biophysical alterations in hematologic disease collectively lead to microvascular occlusion and thrombosis. Using blood samples from patients with SCD, we investigated how the drug hydroxyurea quantitatively affects microvascular obstruction in SCD, an unresolved issue pivotal to understanding its clinical efficacy in such patients. In addition, we demonstrated that our microsystem can function as an in vitro model of HUS and showed that shear stress influences microvascular thrombosis/obstruction and the efficacy of the drug eptifibatide, which decreases platelet aggregation, in the context of HUS. These experiments establish the versatility and clinical relevance of our microvasculature-on-a-chip model as a biophysical assay of hematologic pathophysiology as well as a drug discovery platform.

Omics of Root-to-Shoot Signaling Under Salt Stress and Water Deficit

Maximizing crop yield depends on the leaves receiving an optimal supply of water, mineral nutrients, small organic molecules, proteins, and hormones from the root system via the xylem. Soil drying and salinization alter these xylem fluxes, and modern omics techniques offer unparalleled opportunities to understand the complexity of these responses. Although absolute xylem concentrations of any constituent depend on the genotype and xylem sap sampling methodology, analysis of the relative changes in concentrations has revealed some conserved behavior. Typically, these stresses increase xylem concentrations of the plant hormone abscisic acid (ABA) that limits crop water loss, but decrease the concentrations of certain cytokinins that stimulate expansive growth and prevent premature leaf senescence. Further understanding of the ionic and biophysical alterations in the rhizosphere environment that cause increased xylem concentrations of the ethylene precursor (ACC) is needed. Interactions of these plant hormones with plant nutrient status and xylem nutrient delivery may be important in tuning plant responses to their environment. Xylem proteomics is an emerging area that will help understand mechanisms of plant stress adaptation. Using omics techniques to underpin rootstock-mediate plant improvement is likely to improve crop yields in dry or saline soil.

Measuring the Direct Effects of Sickle Cell Vaso-Occlusion on Endothelial Cells Using Microfluidic Technology

Abstract 897Sickle cell disease is a complex process involving biophysical and biological phenomenon such as microvascular occlusion due to rigid sickle erythrocytes, hemolysis, and aberrant cellular interactions involving endothelial cells and sickle erythrocytes and leukocytes. Indeed, a key aspect of sickle cell pathophysiology is endothelial cell dysfunction. Cardiovascular research in recent years has shown that endothelial cells biologically respond to the local mechanical environment, particularly to the changes in the applied shear stresses (Chiu and Chien, Physiological Reviews, 2011). Interestingly, no studies investigating how the biophysical alterations in sickle cell disease may directly affect endothelial function have been published. The classic view has been that vaso-occlusion is simply due to sickled erythrocytes becoming stuck in microvasculature at low oxygen tensions leading to decreased blood flow and tissue ischemia. However, the mechanical aspects of sickle cell vaso-occlusion themselves, that is, the physical phenomenon of sickling erythrocytes tightly packed in an occluded blood vessel, may directly affect endothelial biology and lead to dysfunction. We hypothesize that these pathologic forces induced by sickling erythrocytes directly lead to dysfunction of endothelial cells, which are mechanosensitive, and contribute to sickle cell pathophysiology. However, these sickling-induced forces and their effects on endothelial cells have been difficult to measure, in part due to a lack of available tools.To that end, we have developed two microfluidic tools to assess the role of sickle-cell vaso-occlusion on endothelial cells. The first device is an in vitro microfluidic platform featuring microchannels the size of post-capillary venules (30 μm) with human endothelial cells cultured within and completely lining the entire inner surface of those microchannels (Figure 1A). This “microvasculature-on-a-chip” enables the visualization of blood cell-endothelial cell interactions during vaso-occlusion under a controlled hemodynamic environment and provides a platform to study the effect of vaso-occlusion on endothelial cells. To date we have characterized this “endothelialized” microfluidic device, showing that endothelial cells are confluent using anti-VE-cadherin immunostaining and adequately generate nitric oxide. Furthermore, we have flowed blood samples from patients with sickle cell disease and found that hydroxyurea treatment both reduces the number of occlusions and increases the mean velocity of the blood traveling through the device, as expected (Figure 1B–E).To decouple whether it is a biochemical or biophysical phenomenon that causes endothelial cell dysfunction during vaso-occlusion, a second micromechanical device was created to quantitatively measure the forces generated by sickling events. The device captures whole blood and will deform outward when forces are applied by the sickle erythrocytes as shown in Figure 2. The membrane above the sickle cells has been coated with 2 μm fluorescent beads which will change focus during deflection. Deflections of one or two beads indicates that a single sickle cell is locally applying force, whereas deflections of large numbers of beads indicates that the cells are collectively applying a pressure to the membrane. The device has been fully fabricated and loaded with blood cells. An accompanying experimental setup enabling the deoxygenation of the device coupled with microscopy has also been created and preliminary tests show successful deoxygenation of sickle erythrocytes from patients with hemoglobin SS disease and the Berkeley sickle cell mouse model.By combining insights gained from each device, future work will determine how the mechanical process of sickling and vaso-occlusion directly affect endothelial function and will lead to a new understanding of sickle cell pathophysiology. Sickle cell vaso-occlusion will be induced in the “endothelialized” microfluidic device while monitoring nitric oxide production and the upregulation of inflammatory markers, such as adhesion molecules and free radicals. The second device will provide quantitative numbers of forces produced by sickling erythrocytes, leading to experiments in which these forces are applied to endothelial cells while monitoring the same metrics. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Assessment of Alterations in the Electrical Impedance of Muscle After Experimental Nerve Injury via Finite-Element Analysis

The surface measurement of electrical impedance of muscle, incorporated as the technique of electrical impedance myography (EIM), provides a noninvasive approach for evaluating neuromuscular diseases, including amyotrophic lateral sclerosis. However, the relationship between alterations in surface impedance and the electrical properties of muscle remains uncertain. In order to investigate this further, a group of healthy adult rats, a group of rats two weeks postsciatic crush, and a group of animals six months postcrush underwent EIM of the gastrocnemius-soleus complex. The animals were then killed and the conductivity and permittivity of the extracted muscle measured. Finite-element models based on MRI data were then constructed for each group. The characteristic EIM parameter, 50 kHz phase (±standard error), obtained with surface impedance measurements was 17.3° ± 0.3° for normal animals, 13.8° ± 0.7° for acutely injured animals, and 16.1° ± 0.5° for chronically injured animals. The models predicted parallel changes with phase values of 24.3°, 18.8°, and 21.2° for the normal, acute, and chronic groups, respectively. Other multifrequency impedance parameters showed similar alterations. These results confirm that surface impedance measurements taken in conjunction with anatomical data and finite-element models may offer a noninvasive approach for assessing biophysical alterations in muscle in neuromuscular disease states.

Biophysical alteration of the secretory track in β-cells due to molecular overcrowding: the relevance for diabetes

Recent data demonstrate that accumulation of misfolded proteins within the early part of the secretory track of β-cells causes impaired insulin synthesis and development of diabetes. The molecular mechanism of this cellular dysfunction remains largely unknown. Using basic molecular principles and computer simulations, we suggested recently that hyperglycemic conditions can generate substantial molecular crowding effects in the secretory track of β-cells leading to significant alterations of the insulin biosynthesis capabilities. Here, we review the major molecular mechanisms that may be implicated in the alteration of insulin synthesis in susceptible β-cells. Steric repulsions and volume exclusion in the endoplasmic reticulum (ER) increase the propensity of misfolding of proinsulin (the precursor molecule of insulin). In addition, similar forces might act in the next secretory compartments (Golgi and vesicles) leading to (i) altered packaging of proinsulin in vesicles (ii) entrapment of proinsulin convertases and/or restricted accessibility for these convertases to the cleavage sites on the surface of the proinsulin and (iii) depressed kinetic rate of the transformation of the native proinsulin in active insulin and C-peptide. These concepts are expressed in simple mathematical terms relating the kinetic coefficient of proinsulin to insulin conversion to the levels of proinsulin misfolding and hyperglycemic stress. The present approach is useful for understanding molecular phenomena associated with the pathogenesis of diabetes. It also offers practical means for predicting the state of pancreatic β-cells from measurements of the insulin to proinsulin ratio in the blood. This is of immediate clinical relevance and may improve the diagnosis of diabetes.

β-Cell Dysfunction under Hyperglycemic Stress: A Molecular Model

Pancreatic β cells respond to chronic hyperglycemia by increasing the synthesis of proinsulin (the precursor molecule of insulin). Prolonged stimulations lead to accumulation of misfolded proinsulin in the secretory track, delayed insulin secretion, and release of unprocessed proinsulin in the blood. The molecular mechanisms connecting the state of endoplasmic reticulum overloading with the efficiency of proinsulin to insulin conversion remain largely unknown. Computer simulations can help us to understand mechanistic features of the β-cell secretory defect and to design experiments that may reveal the molecular basis of this dysfunction. We used molecular crowding concepts and statistical thermodynamics to dissect possible biophysical mechanisms underlying the alteration of the secretory track of β cells and to elucidate the chemistry aspects of the secretory dysfunction. We then used numerical algorithms to relate the degree of biophysical alteration of these secretory compartments with the change of proinsulin to insulin conversion rate. Our computer simulations suggest that overloading the endoplasmic reticulum initiates downstream molecular crowding effects that affect protein translational mechanisms, including proinsulin misfolding, delayed packing of proinsulin in secretory vesicles, and low kinetic coefficient of proinsulin to insulin conversion. Together with previous experimental data, the present study can help us to better understand chemistry aspects related to the secondary translational mechanisms in β cells and how hyperglycemic stress can alter secretory function. This can give a further impetus to the development of novel software to be used in a clinical setup for prediction and assessment of diabetic states in susceptible patients.

Do Sodium Channel α - α Interactions Contribute to Loss-of-Function Observed in Brugada Syndrome?

The pathogenesis of Brugada Syndrome (BrS) has been associated with mutations in the cardiac sodium channel gene SCN5A, resulting in loss-of-function. Recently, the L325R mutation has been proposed to cause BrS through a dominant-negative effect. Dominant-negative effects are usually the consequence of mutant subunits assembling with wild-type (WT) into non-functional channel multimers. In contrast, sodium channel α-subunits are not believed to oligomerize. However, there is increasing evidence suggesting the existence of α-α interactions between sodium channels. Therefore, we tested whether the dominant-negative effect seen in some BrS mutations is due to interactions between sodium channel α-subunits. We co-expressed a dominant-negative BrS mutation, L325R, with WT channels at different molar ratios. Channels containing the mutation alone did not elicit current. When WT and L325R channels were co-transfected in a 1:1 and 4:1 WT:L325R ratios, the normalized peak INa densities were reduced respectively to 29.8±6.2% and 57.3±5.8% of the control WT confirming the dominant-negative effect of this mutation. When using a binomial distribution, our results were fitted best by a configuration suggesting the interaction of two channel monomers. We also investigated the existence of channel-channel interactions using the BrS mutation L567Q. This mutation displays biophysical alterations possibly too small to explain the clinical phenotype. Interestingly, co-expression of L567Q with WT channels produced a significant reduction in INa density which could possibly also be caused by channel-channel interactions and therefore explain the clinical manifestation of the disease. In conclusion, our experiments using BrS mutations, now suggest the idea of a dimerization of sodium channel α-subunits.

Preferential vitrification of water in small alginate microcapsules significantly augments cell cryopreservation by vitrification

The morphological changes of small (approximately 100 microm) alginate microcapsules and the biophysical alterations of water in the microcapsules during cryopreservation were studied using cryomicroscopy and scanning calorimetry. It was found that water in the small microcapsules can be preferentially vitrified over water in the bulk solution in the presence of 10% (v/v) or more dimethylsulfoxide (DMSO, a cryoprotectant), which resulted in an intact morphology of the microcapsules post cryopreservation with a cooling rate of 100 degrees Celsius/min. A small amount of Ca(2+) (up to 0.15 M) was also found to help maintain the microcapsule integrity during cryopreservation, which is attributed to the enhancement of the alginate matrix strength by Ca(2+) rather than promoting vitrification of water in the microcapsules. The preferential vitrification of water in small microcapsules was further found to significantly augment cell cryopreservation by vitrification at a low concentration of cryoprotectants (i.e., 10% (v/v)) using a small quartz microcapillary (400 microm in diameter). Therefore, the small alginate microcapsule could be a great system for protecting living cells that are highly sensitive to stresses due to freezing (i.e., ice formation) and high concentration of cryoprotectants from injury during cryopreservation.

Analyses of a novel SCN5A mutation (C1850S): conduction vs. repolarization disorder hypotheses in the Brugada syndrome

Brugada syndrome (BrS) is characterized by arrhythmias leading to sudden cardiac death. BrS is caused, in part, by mutations in the SCN5A gene, which encodes the sodium channel alpha-subunit Na(v)1.5. Here, we aimed to characterize the biophysical properties and consequences of a novel BrS SCN5A mutation. SCN5A was screened for mutations in a male patient with type-1 BrS pattern ECG. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in HEK293 cells. Sodium currents (I(Na)) were analysed using the whole-cell patch-clamp technique at 37 degrees C. The electrophysiological effects of the mutation were simulated using the Luo-Rudy model, into which the transient outward current (I(to)) was incorporated. A new mutation (C1850S) was identified in the Na(v)1.5 C-terminal domain. In HEK293 cells, mutant I(Na) density was decreased by 62% at -20 mV. Inactivation of mutant I(Na) was accelerated in a voltage-dependent manner and the steady-state inactivation curve was shifted by 11.6 mV towards negative potentials. No change was observed regarding activation characteristics. Altogether, these biophysical alterations decreased the availability of I(Na). In the simulations, the I(to) density necessary to precipitate repolarization differed minimally between the two genotypes. In contrast, the mutation greatly affected conduction across a structural heterogeneity and precipitated conduction block. Our data confirm that mutations of the C-terminal domain of Na(v)1.5 alter the inactivation of the channel and support the notion that conduction alterations may play a significant role in the pathogenesis of BrS.

Seminal hyperviscosity is associated with poor outcome of in vitro fertilization and embryo transfer: a prospective study

To investigate the effect of increased seminal viscosity on fertilization, implantation, and pregnancy outcome in patients undergoing in vitro fertilization/ intracytoplasmic sperm injection (ICSI). Prospective study. University-affiliated infertility center. One hundred fifty-eight infertile couples (168 cycles) with increased seminal viscosity and 129 infertile couples (138 cycles) with normal seminal viscosity as controls. In vitro fertilization using conventional insemination or ICSI. Fertilization, embryo quality, implantation, and pregnancy rates (PR) were evaluated. Patient age and the cause of infertility were similar between the two groups. Sperm count (36.9 +/- 34.0 vs. 28.1 +/- 25.6) and motility (41.5 +/- 19.2 vs. 37.0 +/- 20.5) was higher in the control group compared to the study group. Fertilization rate after IVF (50.4% vs. 64.6%), clinical PR (28% vs. 39.9%), and the implantation rates (10.5% vs. 16.5%) were significantly lower in patients with hyperviscosity compared to the control group. The lower implantation rate and clinical PR in couples with seminal hyperviscosity may be attributed to biophysical alterations or chemical changes of the ejaculate that could impact sperm DNA despite the presence of apparently normal motile sperm for the IVF/ICSI procedure.

Characterization of a novel SCN5A mutation associated with Brugada syndrome reveals involvement of DIIIS4–S5 linker in slow inactivation

Mutations in SCN5A, the gene encoding the alpha-subunit of the cardiac sodium channel (Na(v)1.5), have been associated with various inherited arrhythmia syndromes, including Brugada syndrome (BrS). Here, we report the functional consequences of a novel missense SCN5A mutation, G1319V, identified in a BrS patient. The G1319V mutation is located in the loop connecting transmembrane segments 4 and 5 in domain III (DIIIS4-S5), a region so far considered to be exclusively involved in fast inactivation. Whole-cell mutant (G1319V) and wild-type (WT) sodium currents (I(Na)) were studied in the Human Embryonic Kidney cell line (HEK-293) transfected with Na(v)1.5 alpha-subunit cDNA (WT or mutant) together with h beta(1)-subunit cDNA, using the patch-clamp technique. Maximal peak I(Na) and persistent sodium current were similar in WT and channel G1319V channels. The G1319V mutation shifted the potential of half-maximal (V(1/2)) activation towards more positive potentials (+3.7 mV), thereby increasing the degree of depolarization required for activation. The V(1/2) of inactivation of G1319V channels was shifted by -6.0 mV compared to WT, resulting in a reduced channel availability. The change in the steady-state inactivation was completely due to a negative shift (-6.8 mV) of the voltage-dependence of slow inactivation, while the voltage-dependence of fast inactivation was unaffected. The fast component of recovery from inactivation of G1319V channels was slowed down. Finally, the G1319V mutation caused a two-fold increase in the propensity of the channels to enter the slow inactivated state. Reduction in I(Na) peak amplitude on repetitive depolarizations at short interpulse intervals (40 ms) was significantly more pronounced in G1319V compared to WT. Accordingly, carriers of the G1319V mutation showed marked QRS widening upon increases in heart rate during exercise testing, pointing to enhancement of slow inactivation. We identified the DIIIS4-S5 linker as a new region involved in slow inactivation of Na(v)1.5. The biophysical alterations of the G1319V mutation all contribute to a reduction in I(Na), in line with the proposed mechanism underlying BrS.

ANDROLOGY LAB CORNER*: Filling Time of a Lamellar Capillary‐Filling Semen Analysis Chamber Is a Rapid, Precise, and Accurate Method to Assess Viscosity of Seminal Plasma

SANNE RIJNDERS,* JAN G. M. BOLSCHER,{JOSEPH MCDONNELL,* ANDJAN P. W. VERMEIDEN*{From the *IVF Center, Vrije Universiteit MedicalCenter, Amsterdam, The Netherlands; gametes (Gonzales et al, 1992). Deviant viscosity canDepartment ofOral Biochemistry, Academic Centre Dentistry,Amsterdam, The Netherlands; and`Leja Products BV, Nieuw-Vennep, The Netherlands.Among men from infertile couples, increased viscosityof the ejaculate has been reported to occur morefrequently than in fertile men (Moon and Bunge, 1968;Bunge, 1970). During standard semen analysis, severalsemen variables are evaluated (Oehninger, 2000), butviscosity is rarely quantified. Since the current seminalvariables have limited prognostic value (Castilla et al,2006), extra variables are needed to improve malefertility diagnostics.Viscosity is a semen variable that has received relativelylittle attention in the literature. Hyperviscosity might bedue to dysfunction of accessory sex glands. Associationshave been made between hypofunction of seminal vesiclesand hyperviscosity (Gonzales et al, 1993), and alteredprostate function is associated with low zinc content insperm chromatin and low chromatin stability (Bjo¨rndahland Kvist, 1990). When this integrity is altered, spermquality declines. Seminal viscosity abnormality has beenshown tobe associatedwithmaleinfertility and was foundto accompany poor semen quality (Elzanaty et al, 2004)and altered sperm motility (Eliasson, 1973, Gonzales et al,1993). It also has been shown that the epididymis andaccessory sex glands play a role in the function of maleaffect the quality of sperm cells. Hyperviscosity canattribute to biophysical alterations or chemical changes ofthe ejaculate thatcouldimpact sperm quality despiteothernormal sperm variables seen during semen analysis(Gonzales and Sanchez, 1994).To make viscosity a diagnostic variable, the relation-ship between viscosity and fertility needs to be de-termined. First the viscosity of semen must be quantifiedproperly. Currently this remains a time-consuming jobbecause specialized machinery is required. The methodfor viscosity assessment suggested by the World HealthOrganization (1999) is semiquantitative and in ouropinion, inadequate for good quantitative assessments.A method that can be widely used and that expresses itsresults in centipoise (cP), the international unit ofviscosity, is needed.It is known that the flow velocity in a capillarydepends on the diameter of the capillary, the angle ofcontact, and the viscosity of the fluid. For this study, weused capillary-loaded semen analysis chambers fromLeja. The properties of these chambers are well defined.According to theoretic assumptions, a linear relation-ship exists between filling time of the capillary andviscosity of the sample (Douglas-Hamilton et al, 2005).The aims of this study were to assess the relationshipbetween the filling time of a capillary-loaded chamberand the viscosity of seminal plasma, to express theviscosity in cP, and to assess the accuracy and precision ofthe proposed method. Such a method will make itpossible to describe the relationship between fertility andthe semen variable viscosity. This will extend the numberof variables available for the assessment of male fertilityand will contribute to better models of male fertility.

NMDA receptor antagonists: tools in neuroscience with promise for treating CNS pathologies

NMDA receptors (NMDARs) are essential for normal physiological processes in the central nervous system, e.g. development, induction of synaptic plasticity, learning and memory. Excessive activation of NMDARs can lead to neuronal damage in many acute (hypoxic–ischaemic injury) and chronic neurodegenerative diseases (Alzheimer, Parkinson, Huntington). This dual role of NMDARs in normal and abnormal brain functioning imposes constraints on possible therapeutic strategies involving NMDAR antagonists. Blockade of excessive NMDAR activity must therefore be achieved without interference with physiological activity.

P-844: Relationship between seminal viscosity and oxidative stress in infertile men

P-844: Relationship between seminal viscosity and oxidative stress in infertile men

Blastocyst implantation failure in mice due to “nonreceptive endometrium”: endometrial alterations by Hibiscus rosa-sinensis leaf extract

Many plants are known to possess antifertility activity. However, limited attempts have been made to scientifically evaluate these claims. Hibiscus rosa-sinensis flowers have been shown to possess antifertility and abortifacient activity. In this report, antiimplantation activity of water extract of leaves of H. rosa-sinensis was investigated. Pregnant female mice were dosed with extract (100 mg/kg body weight) from days 1 to 6 of pregnancy. No implantation sites were observed in treated animals when they were surgically opened on day 15 of pregnancy. Biochemical and biophysical alterations were observed in the endometrium in treated animals, especially on day 5, at 4:40 a.m., the day of implantation. A sharp increase in superoxide anion radical and a sharp fall in superoxide dismutase (SOD) activity, as seen in the endometrium from control animals, were altered in treated animals. The extract also exhibited antiestrogenic activity, as judged by increase in uterine weight. The physiological alterations induced by water extract of H. rosa-sinensis are discussed.

Biophysical alterations of hippocampal pyramidal neurons in learning, ageing and Alzheimer's disease

A series of behavioral, electrophysiological, and molecular biochemical experiments are reviewed indicating that when animals learn hippocampus-dependent tasks, output neurons in the CA1 and CA3 hippocampal subfields show reductions in the slow, post-burst afterhyperpolarization (AHP). The slow AHP is mediated by an apamin-insensitive calcium-activated potassium current. A reduction in the slow AHP makes hippocampal neurons more excitable and facilitates NMDA receptor-mediated response and temporal summation. During normal aging and in a mouse model of Alzheimer's disease (AD), the slow AHP is increased, making neurons less excitable and making learning more difficult. The subgroup of aging animals that are able to learn demonstrates the capacity to increase neuronal excitability by reducing the size of the slow AHP. Similarly, in a mouse model of AD, mice that are able to learn normally after a genetic alteration have a normal capacity for increasing hippocampal neuron excitability by reducing their slow AHP. We suggest that reduction in the slow AHP is basic to learning in young and aging animals. Inability to modulate the slow AHP contributes to learning deficits that occur during aging and early stages of AD.