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Whole cell patch clamp experiments show that hydraphiles integrate into the membranes of human embryonic kidney cells and increase membrane conductance to a large extent. The studies indicate that the combination of structural features designed into the hydraphiles afford true channel function in live cells.

A multilevel finite element approach is applied to predict local cell deformations in engineered tissue constructs. Cell deformations are predicted from detailed nonlinear FE analysis of the microstructure, consisting of an arrangement of cells embedded in matrix material. Effective macroscopic tissue behavior is derived by a computational homogenization procedure. To illustrate this approach, we simulated the compression of a skeletal muscle tissue construct and studied the influence of microstructural heterogeneity on local cell deformations. Results show that heterogeneity has a profound impact on local cell deformations, which highly exceed macroscopic deformations. Moreover, microstructural heterogeneity and the presence of neighboring cells leads to complex cell shapes and causes non-uniform deformations within a cell. [DOI: 10.1115/1.1449492]

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

Research has been conducted on the yeast Saccharomyces cerevisiae IRE1 encoding a bifunctional protein with transmembrane kinase and HAC1 encoding a transcription factor. Results indicate that both genes play a crucial role in the unfolded protein response.

Research reveals that Cryptococcus neoformans populations in tissue undergo dynamic morphological changes during the course of murine pulmonary infection. Data indicate that cells show increased cell wall thickness, melanization, and black color. The heterogeneous cell populations assume giant forms.

Unicellular organisms from South Australia's Ediacara Hills are described, along with evolutionary theories, interpretations and implications. The organisms, dating from the Precambrian era, are predominately radiate rather than bilaterally symmetrical, and evince little evidence of specialized organs.

The intestinal S-cells show few ultrastructural differences among the five species, whereas the vacuolar volume of the A-cells is higher in the mesoplanktonic species of Sagitta. Data further reveal differences in the degree of intestinal vacuolation among the species, whose ecophysiological implications influence their vertical distribution pattern.

Aspergillus nidulans has been characterized morphologically with consideration of growth, septation and fragmentation. The parental strain A28 and the sepA null mutant were compared. It is known that the sepA gene does affect the septation process in A. nidulans.

Research has been conducted on Myxococcus xanthus. Results indicate that this bacteria can sporulate in two ways and these sporulation pathways differ in certain respects.

A novel Drosophila gene, act up (acu), controls actin polymerization to alter cell shape and restrict hedgehog signaling in the eye disc of the insect. It appears that dynamic changes in actin filaments may change cell shape, controlling signal movement to coordinate a differentiation wave.

Results indicate that there are 12 known penicillin binding proteins in Escherichia coli composed of high and low-molecular weight proteins. Data show that the low molecular-weight protein 5 has a role in cell diameter determination, on surface uniformity, and topology of the peptidoglycan sacculus.

Results demonstrate that cell shape has indirect effects on positioning of the septum by spindle extension. Data reveal that in spherical cells, the cell division scars and septa lie in an angular position with respect to each other.

Luminal (LE) and glandular epithelial (GE) cells from d-13 pregnant pigs were cultured on plastic, matrix secreted from endometrial stroma, and EHS matrix (Matrigel) in culture medium (RPMI-1640) supplemented with 20 and 10% fetal bovine serum, respectively. After culture for 7 and 14 d, GE and LE cells were prepared for transmission and GE cells for scanning electron microscopy. The two types of endometrial epithelial cells displayed different morphological characteristics when grown on different culture substrates. On plastic, the GE and LE cells formed flattened monolayers. However, stroma-secreted matrix directed the polarization of endometrial epithelia. The GE and LE cells reacted differently to thick Matrigel coatings; LE cells formed a colony after 7 d of culture and then proliferated further to form a colony with a cavity, but GE cells organized to form a colony with a shallow depression in the center at 7 d and developed duct-like structures after 14 d in vitro. Luminal epithelial cells grown on either diluted or thin-coated Matrigel and grown on stroma-secreted matrix formed a monolayer but no three-dimensional structures. Key Words: Endometrium, Cell Culture, Pigs

Bronchial smooth muscle (SM) mesenchymal cell precursors change their shape from round to spread/elongated while undergoing differentiation. Here we show that this change in cell shape induces the expression of laminin (LM) (alpha)2 chain not present in round mesenchymal cells. LM (alpha)2 expression is reversible and switched on and off by altering the cell's shape in culture. In comparison, the expression of LM (beta)1 and (gamma)1 remains unchanged. Functional studies showed that mesenchymal cell spreading and further differentiation into SM are inhibited by an antibody against LM (alpha)2. Dy/dy mice express very low levels of LM (alpha)2 and exhibit congenital muscular dystrophy. Lung SM cells isolated from adult dy/dy mice spread defectively and synthesized less SM (alpha)-actin, desmin, and SM-myosin than controls. These deficiencies were completely corrected by exogenous LM-2. On histological examination, dy/dy mouse airways and gastrointestinal tract had shorter SM cells, and lungs from dy/dy mice contained less SM-specific protein. The intestine, however, showed compensatory hyperplasia, perhaps related to its higher contractile activity. This study therefore demonstrated a novel role for the LM (alpha)2 chain in SM myogenesis and showed that its decrease in dy/dy mice results in abnormal SM.

Plexin-neuropilin(NP)-1 complexes, which form functional semaphorin-3A receptors, are discussed. Physiologic Sema3A receptors are made up of NP-1/plexin complexes. To explore the relationship between two semaphorin-binding proteins, an effort was made to characterize any molecular interactions between NP and plexin.

Gradual disruption of the actin cytoskeleton induces a series of structural shape changes in cells leading to a transformation of cylindrical cell extensions into a periodic chain of 'pearls.' Quantitative measurements of the pearling instability give a square-root behavior for the wavelength as a function of drug concentration. We present a theory that explains these observations in terms of the interplay between rigidity of the submembranous actin shell and tension that is induced by boundary conditions set by adhesion points. The theory allows estimation of the rigidity and thickness of this supporting shell. The same theoretical considerations explain the shape of nonadherent edges in the general case of untreated cells.

Using two-photon excitation of fluorescent indicator dyes, we measured calcium concentration transients in retinal ganglion and amacrine cells without destroying the light sensitivity of the retina by maximally activating or bleaching the photoreceptors. This allowed an immediate assessment of the cellular morphology and study of the calcium signals evoked by visual stimuli. Calcium dynamics in individual dendritic processes could be examined for extensive periods without deterioration and with little apparent phototoxicity at excitation wavelengths of from 930 to 990 nm. Light-evoked increases in calcium were resolved in ganglionand amacrine-cell neurites, making it possible to use optical recording to study the relationship between calcium signaling and retinal function.

Fruiting body formation in Myxococcus xanthus depends on ordered changes in cell movements from swarming to aggregation in response to starvation. We show that appropriately starved individual cells change behavior during fruiting body formation. Specifically, from the time of initiation of aggregation, individual wild-type cells began to move with increased gliding speeds, the duration of the mean gliding interval increased, and the stop frequency decreased whereas the duration of the mean stop interval and the reversal frequency remained unchanged. Mutants lacking the cell surface-associated, intercellular C-signal (csgA mutants) failed to aggregate. Likewise, appropriately starved individual csgA cells did not change their behavior during development. In the absence of other cell-cell interactions, the motility defect of individual csgA cells was corrected in a time- and concentration-dependent manner after C-signaling was reestablished by exogenous MalE-CsgA protein. The C-signal-induced stimulation of motility depended on the cytoplasmic Frz signal transduction system. We propose that C-signal instructs cells to move with high speed and low stop and reversal frequencies into aggregation centers during development.

A study describes a new approach for the investigation of the structure of in vitro assembled mitotic chromosomes in Xenopus egg extract, based on their elastic properties. For the first time, the force-extension curve of whole chromosomes is measured using specially designed micropipettes and their bending rigidity determined through analysis of the curvature fluctuation spectrum. Data from these measurements provide new critical information on chromosome structure.

The Rho family of GTPases help regulate the actin cytoskeleton, cell adhesion, smooth muscle contraction, neurite retraction, and cytokinesis. However, the precise molecular mechanisms by which these proteins operate is not fully understood.

The ultrastructure of the protozoon Cryptosporidium parvum's sporozoite was investigated through cryotechnique and energy-filtering transmission electron microscopy. The cryotechnique generated image thick sections of the parasite that preserved cell integrity. In the longitudinal section studied, the cell exhibited the typical conical-shaped apical region which contained most of the organelles. The sporozoite had a lone rhoptry having an organized lamellar body, an absent mitochondria or conventional Golgi apparatus.

The hypothesis that activation of store-operated calcium channels (SOCs) is sufficient to promote cell shape change necessary for leukocyte migration and initiation of angiogenesis in conduit-vessel endothelial cells was tested. Data indicated that store-operated calcium entry induces cell shape change in rat pulmonary endothelial cells expressing transient receptor potential 1. These suggested that calcium entry through SOCs involves site-specific rearrangement of the microfilamentous cytoskeleton.

Two mutant alpha subunit genes of the position-specific 2 (PS2) integrins were developed to determine whether the function PS2 is regulated during embryonic development. The entire cytoplasmic domain was removed in one mutants, while the alpha(sub PS2) variant form was deleted from the other. Results showed that the removal of the alpha(sub PS2) cytoplasmic domain promotes formation of PS2 integrin that mediates development of an abnormal size and number of muscle attachments. This indicates that modulation of integrin function is essential for embryonic morphogenesis.

A century ago, Camillo Golgi discovered in neurons an intracellular network of anastomosing threads, impregnated by the chromoargentic reaction he had devised to stain the nervous tissue. This structure, designated by Golgi as 'internal reticular apparatus', was soon detected in a wide variety of eukaryotic cells. However, skepticism arose on the existence of the Golgi apparatus in the first decades of this century, when it was fiercely debated whether this structure represented a genuine new cell constituent or an artifact due to the deposit of metallic impregnation on diverse cytoplasmic structures. The reality of the Golgi apparatus became established unequivocally only with the application of electron microscopy;with the visualization of its fine structure, the apparatus finally achieved the status of cytoplasmic organelle, and thus, linked with Golgi's name, entered the modern era of investigation on its components, chemistry and function.

Jagged2 gene and Notch signaling, which is a intercellular signaling mechanism, play an important part in the limb, thymic and craniofacial development of mice. Mutant mice without a domain of the Jagged2 protein had fatal defects in craniofacial morphogenesis. The mice also had fusion in the limbs and modified thymic morphology.

The centrosome plays an important role in maintenance of cell polarity and in progression through the cell cycle by determining the number, polarity, and organization of interphase and mitotic microtubules. By examining a set of 35 high grade human breast tumors, we show that centrosomes of adenocarcinoma cells generally display abnormal structure, aberrant protein phosphorylation, and increased microtubule nucleating capacity in comparison to centrosomes of normal breast epithelial and stromal tissues. These structural and functional centrosome defects have important implications for understanding the mechanisms by which genomic instability and loss of cell polarity develop in solid tumors.

Molecular mechanisms that orient left-right (LR) axis in embryos have been unknown. It has been shown recently that the inverted viscerum mutation in mice may come from a mutation in a gene that encodes an axonemal dynein heavy chain. Along with answers, this has brought new questions: what the role of axonemal dynein is as opposed to that of cytoplasmic dynein and how dynein action is moved across fields of cells. Timing of initial LR actions in embryogenesis and evolutionary functions of asymmetry are of interest.

We have addressed the question of how keratin intermediate filaments are associated with the cell envelope at the periphery of cornified epidermal cells. Many peptides from human epidermal cell envelopes containing isopeptide crosslinks inserted by transglutaminases in vivo have been characterized. A major subset involves the type II keratin chains keratin 1, 2e, 5, or 6 crosslinked to several protein partners through a lysine residue located in a conserved region of the V1 subdomain of their head domains. This sequence specificity was confirmed in in vitro crosslinking experiments. Previously the causative mutation in a family with diffuse nonepidermolytic palmar-plantar keratoderma was shown to be the loss in one allele of the same lysine residue of the keratin 1 chain. Ultrastructural studies of affected palm epidermis have revealed abnormalities in the organization of keratin filaments subjacent to the cell envelope and in the shape of the cornified cells. Together, these data suggest a mechanism for the coordination of cornified cell structure by permanent covalent attachment of the keratin intermediate filament cytoskeleton to the cell envelope by transglutaminase crosslinking. Furthermore, these studies identify the essential role of a conserved lysine residue on the head domains of type II keratins in the supramolecular organization of keratin filaments in cells.

A study was conducted on the cloning and characterization of PRA1, a gene encoding a pH-regulated antigen of opportunistic fungal pathogen, Candida albicans. The results indicate the homology of the PRA1 protein to surface antigens of Aspergillus spp which react with serum from aspergillosis patients. This implies that the PRA1 protein may play a role in the host-parasite interaction during candidal infection.

A study was conducted on the process of DNA replication, cell morphology and viability under conditions of Mycoplasma capricolum. The results indicate that transfer of a temperature-sensitive mutant deficient in DNA replication to the restrictive temperature induces a rise in the proportion of branched cells. Moreover, viable cells stopped dividing upon cell type conversion. However, the reproductive capacity of branched cells was maintained. A model of cellular reproduction for M capricolum is proposed.

Moesin, ezrin, and radixin (MER) are components of the cortical actin cytoskeleton and membrane processes such as filopodia and microvilli. Their C-terminal tails contain an extended region that is predicted to be helical, an actin binding domain, and a region(s) that participates in self-association. We engineered an in vivo fluorescent actin binding protein (GFP-moe) by joining sequences that encode the jellyfish green fluorescent protein (GFP) to sequences that encode the C-terminal end of the sole Drosophila MER homolog, moesin [Moesin-like gene product, referred to previously as the D17 MER-like protein; Edwards et al., 1994, Proc. Natl. Acad. Sci. USA 91, 4589], and Dmoesin [McCartney and Fehon, 1996, J. Cell Biol. 133, 843]. Transgenic flies expressing this fusion protein under control of the hsp70 promoter were generated and used for analysis of cell shape changes during morphogenesis of various developmental stages and tissues. Following heat shock, high levels of stable fusion protein are produced by all somatic tissues. GFP-moe localizes to the cortical actin cytoskeleton, providing a strong in vivo marker for cell shape and pattern during epithelial morphogenesis. The protein also becomes highly enriched in pseudopods, microvilli, axons, denticles, the border cell process, and other membrane projections, potentially by binding to endogenous moesin as well as actin. We show that GFP-moe can be used to examine the development and behavior of these dynamic structures in live specimens. We observe a bright green fluorescent, presumably actin-rich, polar cell proboscis that inserts itself into the forming micropyle and appears to maintain an opening for sperm passage around which the chorion is formed. We also confirm the existence of an actin-rich purse string at the leading edge of the lateral epidermis and provide a dynamic analysis of its behavior as it migrates during dorsal closure. Observations of embryos, larvae, and pupae show that GFP-moe is also useful for labeling the developing nervous system and will be a good general marker of dynamic cell behavior during morphogenesis in live tissues and demonstrate that fusion of a subcellular localization signal to GFP greatly increases its utility as a cell marker.

Researchers constructed a simple system of microtubules and molecular motors to investigate the self-organization process in cellular structure. They observed the production of asters both within a system comprising only stabilized microtubules and multi-headed constructs of the motor protein kinesin, and also from a homogenous solution of motors and tubulin. They also obtained a range of self-organized structures by changing the relative concentrations of the components, and when they studied the process in a micro-fabricated glass chamber.

Fourier Transform methods were used to quantify mean elongation, mean orientation, and standard deviation of orientations of cultured vascular endothelial cells. Images of cell populations, which had been subjected to 11 and 20 hours of shear stress at 30 dynes/[cm.sup.2] and 20 hours of no shear, were analyzed by Fourier Transform methods. Measurements of cell morphology and orientation characteristics were also obtained using a manual method for comparison purposes. The results of the study showed that mean cell orientation can be determined accurately with the Fourier Transform methods. Attempts to determine the standard deviation of cell orientations, however, resulted in poorer estimates of mean elongation and standard deviation of orientations except in the case of exposure of endothelial cells to 20 hours of shear, where the actual standard deviation of orientations was low. When the value for standard deviation of orientations was constrained to zero, a minimum possible mean elongation was determined reliably using the Fourier Transform methods. Use of the Fourier Transform methods in determining morphological and orientation characteristics of cell monolayers is fast and objective and may provide a basis for identifying other characteristics of cell shape.

The apical distribution of amiloride-sensitive sodium channels in A6 renal epithelial cell cultures was analyzed by high-resolution atomic force microscopy (AFM). NanoScope III and BioScope AFM imaging of antibody-labeled Na+ channels on the apical surface of the A6 epithelium indicated the localized distribution of amiloride-sensitive sodium channels in the apical microvilli. Furthermore, AFM techniques provided high-resolution imaging of cell surface topography and distribution of ion channels in Na+ transporting renal epithelial cells.

The biological realm has inherited symmetries from the physicochemical realm, but with the increasing complexity at higher phenomenological levels of life, some inherited symmetries are broken while novel symmetries appear. These symmetries are of two types, structural and operational. Biological novelties result from breaking operational symmetries. They are followed by acquisition of regularity and stability, in a recurrent process throughout complexity levels.

The cold domain (0 to 17 degree celsius) and the suboptimal domain of growth temperature (17 to 30 degree celsius) have variant effects on growth and cellular protein production in psychotropic Pseudomonas fluorescens MF0. The specific growth rate decreases with the increase in the temperature beyond the optimal growth temperature of 30 degree celsius. The RNA and cellular protein levels are maximum, and the average cell size is minimum at 8 degree celsius. Increase in the growth rate leads to a decrease in protein level and increase in RNA level at 28 degree celsius.

A two-component Cole-Cole model was used to obtain statistically significant fits to 100-Hz-10-MHz impedance data for EMT-6 mouse tumors during the progressive histological changes induced by hyperthermia. The resulting fitting parameters were used to deconvolute and reconstruct the two dispersions which confer the predominant impedance features to this tissue. The time-dependent changes of these two dispersions were correlated with the concurrent, heat-induced morphological changes of the tumors' cells. The higher frequency dispersion ([f.sub.c] [approximately equal to] 1 MHz) was identified with a Maxwell - Wagner relaxation process linked to the overall volume response of the cells. The lower frequency dispersion ([f.sub.c] [approximately equal to] 10 kHz) represented an alpha-relaxation associated with the surface morphology and integrity of the plasma membranes. Thus, two aspects of the characteristic cellular damage sequence in these tumors were found to be separately discernable and trackable in real-time using the impedance data.

A study was conducted to determine the impacts of salmon protamine on cell morphology, cell wall and cytoplasmic constituents of Listeria monocytogenes and Shewanella putrefaciens. TRansmission immunoelectron microscopy and atomic force microscopy were used to evaluate the effects. Protamine was found to disrupt the outer surface structure and condensed the cytoplasm of sensitive cells. It was also observed to change membrane structures in sublethal concentrations, consequently eliminating respiration.

The Holliday junction is a well-known intermediate of homologous recombination. Recently, the proteins involved in the correct processing of the Holliday structure into mature recombinant molecules, namely RuvA, RuvB, RuvC and RecG have been isolated and characterized. This has culminated in a model for their synergistic mechanism of action and the solving of the RuvC crystal structure.

Proteasomes are large multi-subunit protease complexes that selectively degrade intracellular proteins. Most of the proteins removed by these proteases are tagged for destruction by ubiquitination. Proteasomes have a role to play in controlling cellular processes, such as metabolism and the cell cycle, through signal-mediated proteolysis of key enzymes and regulatory proteins. They also operate in the stress response, by removing abnormal proteins, and in the immune response, by generating antigenic peptides.

Rhodopsin is the G protein-coupled receptor that upon light activation triggers the visual transduction cascade. Rod cell outer segment disc membranes were isolated from dark-adapted frog retinas and were extracted with Tween detergents to obtain two-dimensional rhodopsin crystals for electron crystallography. When Tween 80 was used, tubular structures with p2 lattice (a = 32 [Amstrong], b = 83 [Amstrong], [gamma] = 91 [degrees]) were formed. The use of a Tween 80/Tween 20 mixture favored the formation of larger [p22.sub.1][2.sub.1] lattices (a = 40 [Amstrong], b = 146 [Amstrong], [gamma] = 90 [degrees]). Micrographs from frozen hydrated frog rhodopsin crystals were processed, and projection structures to 7-[Amstrong] resolution for the [p22.sub.1][2.sub.1] form and to 6-[Amstrong] resolution for the p2 form were calculated. The maps of frog rhodopsin in both crystal forms are very similar to the 9-[Amstrong] map obtained previously for bovine rhodopsin and show that the arrangement of the helices is the same. In a tentative topographic model, helices 4, 6, and 7 are nearly perpendicular to the plane of the membrane. In the higher-resolution projection maps of frog rhodopsin, helix 5 looks more tilted than it appeared previously. The quality of the two frog rhodopsin crystals suggests that they would be suitable to obtain a three-dimensional structure in which all helices would be resolved.

Study examines how M-1 mouse cortical collecting duct (CCD) cells cope with the osmotic challenge related to the formation of hypertonic urine. The addition of 100 mM sucrose to an isotonic NaCl bath solution induces cell shrinkage and increases whole-cell currents within 5-10 min by 12 fold. The entire process reverses upon return to isotonic solution. During the early phase of hypertonic stimulation single-channel transitions is detected in whole-cell current recordings. The results indicate that the nonselective cation channel may play a role in volume regulation.

The in vivo inhibition of the mevalonate pathway leads to the alteration of cytoarchitecture in the adult rat retina due to the inhibition of protein prenylation. The incorporation of (3H) acetate into retinal lipids was studied in the presence and absence of metabolic inhibitors to investigate the biochemical specificity of lovastatin-induced alterations and the branches of the mevalonate pathway.

Endothelial cells grown on Matrigel matrix form three-dimensional, capillary-like structures and these structures affect the morphology, biochemistry, and proliferation of human umbilical vein endothelial (HUVE) cells in vitro. HUVE cells make a 'cobblestone' monolayer on culture dishes, and these cells proliferate faster compared with human microvascular endothelial cells.