Mammalian oocytes are arrested at the diplotene stage of the first meiotic division. Release of oocytes from their follicles induces meiotic resumption characterized by germinal vesicle breakdown (GVBD), followed by the chromosome formation and metaphase I spindle organization and finally the extrusion the first polar body. Recently it was shown that cellpermeant antioxidants significantly inhibit spontaneous resumption of meiosis in mouse oocytes, which may indicate a role of oxygen radicals in oocyte maturation. The regulation of mouse oocyte meiosis resumption is different from that of large domestic animals in that GVBD is independent of Ca2+ and protein synthesis. The present study investigated the influence of two cell-permeant antioxidants, 2(3)-ter-butyl-4-hydroxyanisole (BHA) and nordihydroguaiaretic acid (NDGA), on porcine oocyte meiosis resumption, chromatin behavior and spindle assembly. Our findings revealed a different role of antioxidants in porcine oocyte meiosis resumption than in mouse oocyte maturation.

Cortical granules are specialized Golgi-derived membrane-bound secretory granules that are located beneath the plasma membrane in unfertilized sea urchin eggs. Upon fertilization cortical granules discharge in a reaction induced by calcium and release their contents between the plasma membrane and a thin vitelline layer that lines the plasma membrane. Microvilli at the plasma membrane elongate incorporting cortical granule membranes during elongation. The vitelline layer elevates and becomes the egg's fertilization coat that hardens and serves as physical block to polyspermy. While we do not understand the precise mechanisms that participate in cortical granule discharge it is believed that actin plays a role in this process. Because actin and calcium metabolism is affected in aging cells we investigated if cortical granule secretion is affected in aging sea urchin eggs.

Lytechinus pictus eggs were obtained by intracoelomic injection of 0.5M KCI to release the eggs into sea water at 23°C.

Head-to-head sperm association was described in some species as a normal phenomenon during epididyma transit (Phillips and Bedford, 1987). Recently, a new sperm head association (SHA) was characterized in the rat (Fornés & Burgos 1990; 1994).

At this moment, the study of SHA was extended to mouse sperm. SHA is a type of cell-cell interaction clearly observed when an a drop of epididymal fluid, obtained by a puncture of epididymal duct, is placed in a culture medium (HMB media: Modified Ringers medium).

This preparation can be observed under contrast phase microscope (optiphot-2, Nikkon) with or without fixative (2% paraformaldehyde and 2% glutaraldehyde in 0.01 M PBS buffer, pH 7,2). The mass of spermatozoa begin to move but not as a single sperm. They move as a clump of sperm and without fixative the sperm motility alone disaggregate the SHA.

The interaction of surfaces with their surroundings can be described as arising from force fields generated by molecules at the surface of interest. This statement is true for electrostatic interactions of proteins on cell membranes as well as steric interactions of macromolecules on polymer surfaces. To better understand these systems and ultimately to design superior materials, it is necessary to make direct measurements of these near-surface molecular scale force fields. Scanning force microscopy (SFM) holds some promise in the endeavor to make surface force measurements in a full three dimensional submolecularly resolved space. In-vitro measurements present a problem in that the force fields tend to be very short ranged. This necessitates placing the SFM probe very near the surface in order to measure the fields. The ubiquitous van der Waals attraction tends to induce an instability in the SFM instrument under these conditions leading to a non-equilibrium state. We have carefully examined this state of affairs and found that it is essential to use a substantially more sophisticated method to analyze SFM data than is commonly performed.

The atomic force microscope (AFM) can be used both to manipulate molecules and to make measurements on individual molecules. Here, we describe manipulation of chromatin constructs as an example of the first type of measurement, and electronic measurements on single molecules as an example of the second type.

An AFM was used to both image and stretch single synthetic chromatin fibers consisting of twelve core nucleosomes with no linker histones. Peaks in the force-curves are attributed to sequential detachment of nucleosomes from the glass support (Figure 1). The short distances between peaks and reversibility of the pulling process show that the nucleosomes remain intact even at tensions on the order of 350 picoNewtons (pN). This is more than an order of magnitude larger than the force required to de-spool histone octamers from the nucleosomal DNA in laser optical tweezer measurements made with longer molecules, suggesting that loading rates and sample size are important factors in determining the force required to break inter-molecular bonds.

ABSTRACT

Binding formation between a peptide sequence (GSSSGRGDSPA) which contains the cell adhesion sequence –RGD-found in fibrinogen, vWF, fibrinonectin, and vitronectin and human platelet intergrin GP Ilb/IIIa plays an important role in thrombus formation. Using atomic force microscopy (AFM), we visualized the detailed structures of membrane and submembrane of the cell, and measured the interaction forces between a peptide modified cantilever probe tip and platelet surface from pN to nN levels under physiological buffer. Direct measurements of the debonding force for the RGD ligand - GP Ilb/IIIa system are presented. To eliminate the possible measurement of the hgand-receptor pair, or of the ligand and AFM tip, following desorption of the ligand; the cantilever tip surface was modified with covalent coupling chemistry rather than physical absorption. Our results showed that the single molecular rupturing force was 93.3 ± 10.41 pN with considerable chain extension in the receptor. The rupturing forces showed a logarithmic dependence of the rate of loading

An atomic force microscope (AFM) can image real-time intermolecular interactions between complementary macromolecules, such as receptor-ligand and antigen-antibody with nanometer resolution in hydrated state. Recent developments in AFM imaging allow mapping of such interaction forces over a large surface area such that local as well as global distribution of complementary biomolecules could be ascertained simultaneously. Using the force-volume maps and AFM probe conjugated with antibody, we have mapped the reorganization of specific receptors on the cell surface as well as the resultant changes in cellular micromechanical properties, such as elasticity and cytoskeletal reorganization of the cell (Figure 1).

In present study, we have mapped vascular endothelial growth factor receptor (VEGF-R) in the plasma membrane of cultured endothelial cells using anti-VEGFR - antibody conjugated to AFM probe. VEGF induced changes in cytoskeleton reorganization in endothelial cells were observed by real-time AFM imaging.

Many pathological states are related to aberrant functioning of ion channels. Cures for these conditions involve the design of chemicals that will interact with the ion channels or a channel regulator to return their function to the normal level. A notable example of a pathological state associated with ion channel malfunction is the abnormal conductance of inward rectifying potassium channels associated with irregular heart beat. The development of drugs targeting ion channels requires efficient screening of thousands of potential channel ligands. Classically the function of ion channels are assayed by patch clamping which is a fairly elaborate technique and not efficient from the point of screening thousands of ligands. Here is described the development of a compact sensor device where an ion channel is made to function in a lipid bilayer in 50nm holes in silicon or polymer hardware.

Nature produces exquisite structures under ambient conditions using raw materials found in its immediate environment. Examples of naturally resilient materials seem nearly infinite and include (but not limited to) strong composites like seashells and tough fibers like spider dragline silk, which has a breakage energy per unit weight two orders of magnitude greater than high tensile steel.

These natural processes and the resulting materials are ultimately biodegradable and Eco-friendly. Scientists have been interested in nature's designs and processes for hundreds of years. The result of these studies has led to many unique synthetic materials such as KEVLAR™, styrofoam, PLEXIGLAS™, etc. Unfortunately, our “man-made” materials are usually produced under harsh conditions such as high temperatures or under vacuum. Rarely are these processes and the resulting products environmentally friendly.

In order to mimic nature's designs, researchers have attempted to understand its basic mechanisms for making these resilient materials by studying the starting materials that nature produces.

In this talk, the development of key elements for BioPOEMS (Biomedical Polymer-based Opto Electro Mechanical Systems) will be discussed as a new generation of microscopic and microanalysis biochips. Current BioMEMS (Biomedical Microelectromechanical Systems) technology is in its infancy stage. Just as silicon-based microelectronics technology went through many stages since the late ‘60's, BioMEMS technology will have to overcome many obstacles before becoming a mature industry. The IC industry was made possible by the development of fabrication processes, integration technologies, and design tools. Similarly, the field of BioMEMS must prepare new components with biomaterials and tools for the development of new microsystems, enabling the combination of biological sensors and actuators with the rapidly growing capabilities of bioinformatics.

Polymers are not only used in macroscopic systems, but are now increasingly finding use in the microscopic realm such as microfluidic devices. Among the many different classes of polymers, it is the fluoropolymers that provide the most unique material characteristics.

Vascular endothelial cells experience a wide range of mechanical stimuli in vivo, including dynamic stretching and time-varying shear stress due to pulsatile blood flow. Under physiologic conditions, these hemodynamic forces constitute an important component of the cells' environment and are necessary for the normal maintenance of the structure and function of the vessel wall. Implantation of artificial vascular graft materials can significantly alter the mechanical environment of the cell. Furthermore, an important design criterion for engineered tissue constructs is the production of a mechanical environment that, ideally, stimulates normal growth and structural adaptation and. At least, avoids extremes in mechanical loading that might cause cellular trauma. In this paper, we investigate the mechanical loading conditions associated with normal and pathological mechanotransduction and cellular injury with an emphasis on the loading rate dependence of the cellular responses.

A custom designed cone-and-plate device] allows arbitrary cone velocity waveforms to be generated.

Type I collagen comprises between 75-95% of the stationary extracellular matrix of most tissues, forming a continuum in which most of the static cells are anchored. Collagen serves as the track for haptotactic cell migration. The junction between collagen, its receptor integrins, and the cells’ cytoskeleton plays a crucial role in cell differentiation and morphogenesis by serving as the agent for transducing mechanical forces into chemical and biochemical work. The physiological, functional organization of collagen is the solid state in the form of a network of fibers. The only molecules available for engaging the receptors are those located on the fiber surface. Cryogenic scanning force microscopy (SFM) of single molecules of collagen allowed us to correlate surface features with known sequence and stereochemical markers within collagen. Theoretical conformational studies to locate markers for intermolecular recognition and allosteric binding showed that collagen a 1(1) chain residues 766GTPGPQGIAGQRGVV780 generate a distinctive conformation, characterized by a relatively stable (3-bend at the core (Fig. 1) within the triple helical polyproline II conformation extant in the rest of the molecule (1).

Introduction: There is considerable interest in the use of three-dimensional porous scaffolds for tissue regeneration. The presence of an interconnected framework of pores with large surface area facilitates the formation of extracellular matrix and permits cellular ingrowth into implanted structures. For scaffolds to be useful for tissue regeneration, they must maintain good dimensional stability during the lifetime of the implant. While the initial scaffold architecture is often well characterized, a systematic study of the influence of incubation on the scaffold architecture is critical to ensure that the scaffolds retain their interconnected network of pores during their useful lifetime. Herein, we report on the evaluation of the architecture of polyarylate scaffolds and their stability under in vitro conditions using scanning electron microscopy (SEM).

The polymers used in this study were selected from a library of degradable polyarylates. This library is the first reported combinatorial library of biodegradable condensation polymers.

Central to the successful advance of tissue engineering (e.g. 1, 2) is the development of highly engineered materials which trigger particular body responses to regenerate specific tissue or sets of tissue. Such properties as hydrophilicity, hydrolytic susceptibility, and protein-recognition behavior are critical to the success of tissue scaffolds. Poly(ethylene glycol) [PEG] is often introduced into scaffold polymers to tune hydrolytically controlled properties. Relatively little is known about the local morphology and nature of phase separation in these systems, however. This research studies the development of phase-separated morphology in blends and randommultiblock copolymers of a tyrosine-based poly(psuedo amino acid) [poly(DTE carbonate)] and PEG 1000 (fig. 1). This system exhibits attractive biocompatability, strength and modulus, and resorption behavior which can be controlled by main-chain and pendant-chain chemistries (3, 4).

The nature of phase separation was studied by bright-field TEM (Philips CM30 ST; CM20 FEG TEM/STEM) using solvent-cast thin films.

Microscopy and microanalysis techniques have played an important role in our understanding of how biomaterials interact with their environment. In first part of this study, we will focus on the behavior of bioactive glass, whereas in the second a model surface will be investigated. Upon implantation bioactive glass undergoes a series of reactions that leads to the formation of a calcium phosphate-rich layer. Most in vitro studies of the changes that occur on the surface of bioactive glass have employed the use of buffer solutions with compositions reflecting the ionic composition of interstitial fluid. Although these studies have documented the physical and chemical changes associated with bioactive glass immersed in aqueous media, they do not reveal the effect of serum proteins and cells which are present at the implantation site. In the present study, we document, using atomic force microscopy (AFM) and Rutherford backscattering spectrometry (RBS), significant differences in reaction layer composition, thickness, morphology and kinetics of formation arising from the presence of serum proteins.

There are several established strategies for visualizing proteins by transmission electron microscopy (TEM). These include negative staining, cryo-TEM, glycerol-spray/rotary-replication, mica-flake/freeze-fracture. Of these, low-angle, rotary-replication of proteins dried out of glycerol has proven to be a reliable and accurate method for studying surface topologies of macromolecular assemblies and particles. We have characterized many proteins, both chimeras and wild type; methods, caveats and the surface structure of a couple examples are described here.

The technique of using heavy metals to cast replicas of proteins and particles has been around for decades (metal replication of proteins was first described in 1956) and continues to provide an excellent method for evaluating protein topology. The success of this method is based on the physical properties of protein in contact with freshly cleaved mica, glycerol and atomic platinum. Mica is easily split and atomically flat making it an excellent substrate.

Crystallization is fundamental to much drug manufacture. Consequently a great deal of attention is focused on trying to attain a reproducible, robust process with predictable outcomes.

Crystallization may be interrogated by various means including laser light scattering, infra-red spectroscopy, calorimetery, x-ray diffraction, solid state NMR analysis and optical and electron microscopy. Although laser backscattering is applicable to real time, in-situ studies it detects chord length and hence it is most accurate when all particles are spherical. This is clearly is not so with drug crystals. Crystal analysis by microscopy is usually conducted on samples removed from the reactor. While this approach has value it may lead to post-sampling changes and is not capable of fully characterising the real time dynamics of particle morphology. In-situ epifluorescence microscopy of cells within a bioreactor has been reported. However, this required insertion of a quartz window into the reactor wall which limited the observation position to one fixed location, which would be less appropriate for batch crystallization reactors.

Microscopy plays a vital role in assessing the safety of New Chemical Entities (NCE) in the pre-clinical phase of drug development. Light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are used at the Schering-Plough Research Institute (SPRI) for evaluation of NCE. To support regulatory submissions, NCE are routinely tested in rodents in short-term studies such as one-month toxicity studies, and in longterm studies such as oncogenicity studies that may last 24 months. At the completion of a study, the animals are necropsied and the required tissues collected and stored in fixative. The tissues for LM are processed to slides and stained with Hematoxylin and Eosin (H&E). The information derived from the examination of these tissues by LM becomes an essential part of the pathology report. The LM examination of these tissues usually yields the information needed to either progress a NCE or otherwise deter or halt development.

Chemical polymorphism refers to the ability of compounds to exist in different solid state forms. In general, these differ solely in their arrangement in space (i.e., their crystal form). Polymorphs typically have different solid state properties such as melting point, solubility, density, etc. They also commonly exhibit distinctive optical properties such as refractive index, extinction position, optic sign and optic angle. The choice of polymorph can be an important consideration in the drug development process if the solid state properties have wide differences. Further, it is generally desirable to develop the polymorph which is stable at room temperature and pressure, since metastable forms convert spontaneously to the stable ones, albeit often at a very slow rate. Optical, thermal and scanning electron microscopy each play important roles in the study of chemical polymorphism.

Optical microscopy, and more specifically polarized light microscopy, contributes both to the discovery of new polymorphs and to the elucidation of the relationship among polymorphs.

Development of a potential drug candidate was discontinued when rats treated with the compound developed severe renal toxicity after one week of daily dosing. Tubular degeneration in the kidney was accompanied by deposition of large amounts of crystalline material, which was also present in spleen and pancreas (Fig. 1). It was presumed that the crystals were precipitated parent drug or one of its metabolites. Using microscopy and mass spectrometry, it was confirmed that the crystals were indeed derived from the drug and not endogenously formed; in addition, the precise metabolite that had precipitated in the tissues was identified, providing clues to the metabolic pathways involved.

Frozen sections of phosphate-buffered formaldehyde-fixed kidney, pancreas, and spleen were evaluated by polarized light microscopy (PLM), scanning electron microscopy (SEM) with energy dispersive x-ray spectroscopy (EDXS), and matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS). For reference, crystals of the trifluoroacetate salt of the parent drug were also analyzed using these methods.