Modulation by centrifugation of cell susceptibility to chlamydial infection.

Enhancement of chlamydial infection of cell monolayers by centrifugation was shown to depend on induced cell surface changes. Evidence for this came from analysis of two forms of organism attachment which take place during centrifugation. In 'productive binding', organisms attached to cells and then entered and infected them. In 'unproductive binding', organisms became attached to cells but were not ingested. These organisms could be stripped from the cells by treatment with trypsin and could then infect fresh monolayers. Measurement of attachment kinetics during centrifugation showed that cells passed through three different susceptibility states. Only productive binding occurred in the first 20 min; cells then entered a refractory state during which no attachment took place At about 45 min, attachment recommenced but this allowed only unproductive binding. Induced movement of cell surface structures may enhance infection by promoting specific or non-specific interactions. Failure of ingestion may result from insufficient cell 'receptors' for circumferential binding of the whole chlamydial surface so that engulfment cannot take place.     

The surface membrane as a regulator of animal cell division

Summary The surface membrane of an animal cell is proposed to be the target for regulation of cell division. It undergoes regular periodic changes during the cell division cycle. Interference with these changes by cell-cell surface contacts is proposed to prevent the normal progression of events, and thereby can change the metabolic pattern so as to put the cells into a resting state. Through external influences, cells can escape from this resting state; when this occurs surface changes are the earliest ones observed. Cells that have become malignant, particularly after virus infection, show marked changes in their surface properties. Infection is proposed to prevent the surface changes that lead to the resting state. Recent evidence from in vitro experiments is summarized, and some speculations are made on the connection between the surface and processes of division such as nuclear replication. Presented in the Symposium on Regulation in Tumor Cells at the 22nd Annual Meeting of the Tissue Culture Association. Lake Placid, New York. This work was supported by Public Health Service Grant CA-A1-1195 and Grant E-555 from the American Cancer Society.     

Determination of cell form in cultured fibroblasts : Role of surface components and cytokinetic elements

Functions involved in the determination of the morphology of mouse embryo fibroblasts have been studied using as a system the cell rounding and reflattening related to the presence or absence of a protein component of the cell surface [18]. Cells that had undergone rounding following the removal of the surface protein gradually reflattened if their protein synthesis was not impaired but could rapidly revert to the flattened state if a fraction containing the surface protein was returned to the cells. Studies with sodium azide and potassium cyanide showed that both the rounding and reflattening were energy-dependent. When the rounded state was induced the organised appearance of the microfilaments present in the cortical layer adjacent to the plasma membrane was lost. However, microfilament structures reappeared when rounded cells respread over the glass substrate. On the other hand no reflattening occurred if microfilament function was impaired by treatment with cytochalasin B (CB), showing a requirement of microfilament integrity for the flattened form to be manifest. Microtubules also were shown to be involved in shape determination as their integrity was necessary for cells to progress from the flattened form to the rounded state, but they did not appear to be primarily involved in the process of reflattening. The distribution of macromolecular components of the plasma membrane was also shown to affect changes of cell shape as cross-linking of surface receptors by concanavalin A (ConA) or specific anti-cell immunoglobulins prevented cells from acquiring the rounded form and treatment of cells with media of low pH, which causes aggregation of intra-membrane particles, also prevented rounding. A comparison between quiescent cells and cells in S phase showed the changes of morphology to be similar in nature, but more prompt and marked in the S population. The indication of the studies presented is that determination of cell form requires the cooperative interaction between the protein component of the cell surface, integral constituents of the plasma membrane and cytokinetic organelles.     

Positional reorganization in compound janus cells of Tetrahymena thermophila

The janus mutations of Tetrahymena thermophila convert the large-scale organization of the dorsal surface of the cell into a mirror-image of the ventral surface, which is characterized by a second, abnormal, oral apparatus and by contractile vacuole pores to the left of the second oral area rather than the usual right. This conversion could be due either to a local change in the response to an unaltered positional system or to a more global reorganization of the system itself. janus homopolar doublets were used to distinguish between these two alternatives. Homopolar doublets can be made by fusing two similarly oriented cells in side-by-side parabiosis. Non-janus homopolar doublets typically possess two sets of normal oral structures with contractile vacuole pores to the right of each of them. In janus doublets, there are up to four sets of oral structures, with the abnormal oral structures located between the two sets of normal oral structures; contractile vacuole pores are situated to the right of the normal oral areas and to the left of the abnormal oral structures. Non-janus homopolar doublets are known to propagate their compound condition for a number of cell divisions, but also to regulate toward the singlet state through a progressive reduction in number of ciliary rows followed by loss of one of the two sets of major cell surface structures. janus homopolar doublets go through a corresponding regulation. As a consequence, the location of the abnormal oral structures relative to the normal ones is more variable in janus doublets than in janus singlets. Sometimes the abnormal oral structures shift to a position close to their normal counterparts and then the intervening CVP sets disappear. There is evidence for occasional fusion of an abnormal oral area with an adjacent normal oral apparatus, a condition that may be transitional to the singlet state. These observations are inconsistent with the idea of a fixed positional system and strongly suggest a global reorganization of the surface pattern in a manner consistent with predictions of an intercalation model that was first proposed to explain the regulation of non-janus doublets to singlets.     

Cristae formation-linking ultrastructure and function of mitochondria

Mitochondria are double-membrane enclosed eukaryotic organelles with a central role in numerous cellular functions. The ultrastructure of mitochondria varies considerably between tissues, organisms, and the physiological state of cells. Alterations and remodeling of inner membrane structures are evident in numerous human disorders and during apoptosis. The inner membrane is composed of two subcompartments, the cristae membrane and the inner boundary membrane. Recent advances in electron tomography led to the current view that these membrane domains are connected by rather small tubular structures, termed crista junctions. They have been proposed to regulate the dynamic distribution of proteins and lipids as well as of soluble metabolites between individual mitochondrial subcompartments. One example is the release of cytochrome c upon induction of apoptosis. However, only little is known on the molecular mechanisms mediating the formation and maintenance of cristae and crista junctions. Here we review the current knowledge of the factors that determine cristae morphology and how the latter is linked to mitochondrial function. Further, we formulate several theoretical models which could account for the de novo formation of cristae as well as their propagation from existing cristae.     

Redox control and the evolution of multicellularity

Redox chemistry, involving the transfer of electrons and hydrogen atoms, is central to energy conversion in respiration; in addition, control of gene expression by redox state commonly occurs in bacteria, allowing a rapid response to environmental changes, such as altered food supply. Colonial metazoans often encrust surfaces over which the food supply varies in time or space; hence, in these organisms redox control of the development of feeding structures and gastrovascular connections could be similarly adaptive, allowing colonies to adjust the timing of development and spacing of structures in response to a variable food supply and other environmental factors. Experimental perturbations of redox state in colonial hydroids support this notion of adaptive redox control, and redox signaling in metazoans may have evolved in this ecological context. At the same time, redox signaling has important consequences for the evolutionary transition from unicellular to multicellular organisms. Unlike protein or peptide signaling, redox signaling acting in concert with programmed cell death may automatically inflict a cost on those cells that "defect," that is, selfishly favor their own replication rate over that of the multicellular group. In this way, redox signaling may have allowed multicellular individuality to evolve and more easily be maintained.     

Improved method for visualizing cells revealed dynamic morphological changes of ventral neuroblasts during ventral cleft closure of Caenorhabditis elegans

The formation of intricate and functional biological structures depends on the dynamic changes of cellular morphology. Confocal laser scanning microscopy (CLSM) is a widely used method to reveal the three-dimensional (3-D) structure of cells during the development of Caenorhabditis elegans (C. elegans) and other model organisms. Improving the efficiency and image quality of CLSM would benefit studies using this method. We found that CED-10::GFP::CED-10, a green fluorescent protein (GFP) marker, is intensely expressed beneath the cell surface, facilitating visualization of cellular morphology in C. elegans embryos. By combining the unique properties of this marker, and with the help of direct 3-D rendering of images obtained by CLSM, we developed a simple but powerful method for investigating cellular morphology in developing embryos. Using this method we, for the first time, document the dynamic changes in the morphology of ventral neuroblasts in vivo during ventral cleft closure.     

Periplasmic Structure of Frozen-Etched and Negatively Stained Cells of Bacillus licheniformis as Correlated with Penicillinase Formation

Bacillus licheniformis strain 749/C (constitutive for penicillinase formation) and uninduced cells of strain 749 (penicillinase-inducible) were examined after freezeetching. In the early stationary phase, strain 749/C organisms had clusters of vesicles (30 to 40 nm in diameter) on the outer surface of the plasma membrane. These are randomly distributed on the membrane, including the region of septum formation. The vesicles are not intimately associated with the plasma membrane, and their inner and outer surfaces are devoid of particles. Periplasmic vesicles were not detected by freeze-etching in strain 749 (uninduced) or in young cells of 749/C; however, the membrane of mid-logarithmic phase 749/C cells had a corrugated appearance. Negatively stained 749/C cells (logarithmic phase) also showed many vesicular and tubular bodies in the periplasm as well as septal and cytoplasmic mesosomes of typical morphology. The periplasmic structures appear to be formed either by evagination of plasma membrane or by migration of vesicular bodies from the membranous pockets of the cytoplasm. Stationary phase cells of 749/C still have many periplasmic vesicular bodies; however, the mesosomes are greatly reduced both in number and size. In sharp contrast, strain 749 organisms have very few structures similar to the periplasmic bodies of strain 749/C. These findings support our previous view that penicillinase-producing cells of 749/C have periplasmic membranous structures that are rare in the uninduced strain 749, though there is some lack of correspondence between freeze-etching, negative staining, and thin section data. These structures may be important for the retention or storage of penicillinase in the cell.     

The effects of temperature on the composition and physical properties of the lipids of Pseudomonas fluorescens

1. Pseudomonas fluorescens was grown at various temperatures between 5 degrees C and 33 degrees C. The extractable lipids from organisms at various stages of growth and grown at different temperatures were examined. 2. The extractable lipids contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, and an ornithine-containing lipid. The relative amounts of these lipids did not vary significantly during growth or with the changes in growth temperature. 3. The major fatty acids were hexadecanoic, hexadecenoic and octadecenoic acids and the cyclopropane acids methylene-hexadecanoic and methylene-octadecanoic acids. The relative amount of unsaturated acids (including cyclopropane acids) did not change significantly during growth, but increased with decreasing temperature. 4. Phosphatidylethanolamines with different degrees of unsaturation and containing different amounts of cyclopropane acids were isolated from organisms grown at 5 degrees C and 22 degrees C and their surface and phase behaviour in water was investigated. Thermodynamic parameters for fusion and monolayer results for cyclopropane and other fatty acids were examined. 5. The surface pressure-area isotherms of phosphatidylethanolamines containing different amounts of unsaturated fatty acids show small differences but the individual isotherms remain essentially unchanged over the temperature range 5-22 degrees C. X-ray-diffraction methods show that the structures (lamellar+hexagonal) formed in water by phosphatidylethanolamine, isolated from organisms grown at 5 degrees C and 22 degrees C, are identical when compared at the respective growth temperatures. This points to a control mechanism of the physical state of the lipids that is sensitive to the operating temperature of the organism. 6. The molecular packing of cyclopropane acids is intermediate between that of the corresponding cis- and trans-monoenoic acids. However, substitution of a cyclopropane acid for a cis-unsaturated acid has insignificant effects on the molecular packing of phospholipids containing these acids.     

Formation of High-Order Oligomers by a Hyperthemostable Fe-Superoxide Dismutase (tcSOD)

Hyperthermostable proteins are highly resistant to various extreme conditions. Many factors have been proposed to contribute to their ultrahigh structural stability. Some thermostable proteins have larger oligomeric size when compared to their mesophilic homologues. The formation of compact oligomers can minimize the solvent accessible surface area and increase the changes of Gibbs free energy for unfolding. Similar to mesophilic proteins, hyperthermostable proteins also face the problem of unproductive aggregation. In this research, we investigated the role of high-order oligomerization in the fight against aggregation by a hyperthermostable superoxide dismutase identified from Tengchong, China (tcSOD). Besides the predominant tetramers, tcSOD could also form active high-order oligomers containing at least eight subunits. The dynamic equilibrium between tetramers and high-order oligomers was not significantly affected by pH, salt concentration or moderate temperature. The secondary and tertiary structures of tcSOD remained unchanged during heating, while cross-linking experiments showed that there were conformational changes or structural fluctuations at high temperatures. Mutational analysis indicated that the last helix at the C-terminus was involved in the formation of high-order oligomers, probably via domain swapping. Based on these results, we proposed that the reversible conversion between the active tetramers and high-order oligomers might provide a buffering system for tcSOD to fight against the irreversible protein aggregation pathway. The formation of active high-order oligomers not only increases the energy barrier between the native state and unfolded/aggregated state, but also provides the enzyme the ability to reproduce the predominant oligomers from the active high-order oligomers.     

Hydrophobic interactions of group A streptococci with hexadecane droplets.

The adherence of Streptococcus pyogenes cells to hexadecane droplets was measured by vortexing water suspensions of streptococci with hexadecane. It was found that adherence of the organisms to hexadecane droplets was abolished by pretreating the organisms with trypsin, pepsin at pH 4.5, or HCl solutions at 95 degrees C. Streptococcal adherence was best expressed in organisms harvested during the stationary phase of growth and was inhibited by fatty acid-free albumin because of the interaction of the protein with the streptococcal surfaces. The data suggest that adherence to hexadecane droplets measures the availability on the surface of S. pyogenes cells of lipophilic residues that are either hydrophobic regions of surface protein structures or, more likely, glycolipids complexed with and oriented by surface proteins.     

Method of live microscopy of the neurons of the cat brain]

Method of microscopic study of living nervous elements of the brain stained with vital dyes in the falling light was developed. Investigation of the morphology of neurons, glial cells and vasculo-nervous relations in the cat's cerebral cortex in living state in situ became possible thanks to the proposed method. Conditions were made to observe the patterns of dynamic changes in these structures under effects of different stimuli.     

Physiology of synapse: from molecular modules to retrograde modulation

Synapses are highly organized, specific structures assuring rapid and highly selective interactions between cells. Synaptic transmission involves the release of neurotransmitter from presynaptic neurons and its detection by specific ligand-gated ion channels at the surface membrane of postsynaptic neurons. The protenomic analysis shows that for self-formation and functioning of synapses nearly 2000 proteins are involved in mammalian brain. The core complex in excitatory synapses includes glutamate receptors, potassium channels, CaMKII, scaffolding protein and actin. These proteins exist as part of a highly organized protein complex known as the postsynaptic density (PSD). The coordinated functioning of the different PSD components determines the strength of signalling between the pre- and postsynaptic neurons. Synaptic plasticity is regulated by changes in the amount of receptors on the postsynaptic membrane, changes in the shape and size of dendritic spines, posttranslational modification of PSD components, modulation kinetics of synthesis and degradation of proteins. Integration of these processes leads to long-lasting changes in synaptic function and neuronal networks underlying learning-related plasticity, memory and information treatment in nervous system of multicellular organisms.     

'Sloughing-off' of heterochromatin in Werner's syndrome cells during high-temperature phosphate incubation

Previous investigations of cells undergoing rapid division revealed the presence of heterochromatic 'dots' in chromosomes as well as numerous chromocentres in interphase nuclei. Such structures were seen in human embryonic cells, as well as cells from organisms capable of regeneration, and cells from various malignancies. Cells with a reduced capacity for reproduction were found to be virtually devoid of nuclear chromocentres and chromosome dots after incubation in phosphate buffer at high temperature. The lack of heterochromatin in such cells (Werner's syndrome) thereby explained their reduced capacity for cell division and the resultant rapid rate of aging in individuals afflicted. Re-examination of such slides containing these cells revealed that chromocentres and chromosome dots were present initially, but the incubation process resulted in a 'sloughing-off' of such structures. The incubation process left these heterochromatic structures intact in malignant and control cells, inferring a link between cell proliferation and stable intact heterochromatin. These findings implicate heterochromatin as the object of the purported chromosomal instability factor characteristic of Werner's syndrome. The loss of heterochromatin did not result in chromosome breakage, suggesting that heterochromatin may not be an integral part of chromosome structure, but rather a surface feature or covering.     

Adhesion of Plasmodium gallinaceum Ookinetes to the Aedes aegypti Midgut: Sites of Parasite Attachment and Morphological Changes in the Ookinete

Plasmodium gallinaceum ookinetes adhered to Aedes aegypti midgut epithelia when purified ookinetes and isolated midguts were combined in vitro. Ookinetes preferentially bound to the microvillated luminal surface of the midgut, and they seemed to interact with three types of structures on the midgut surface. First, they adhered lo and migrated through a network-like matrix, which we have termed microvilli-associated network, that covers the surface of the microvilli. This network forms on the luminal midgut surface in response to blood or protein meals. Second, the ookinetes bound directly to the microvilli on the surface of the midgut and were occasionally found immersed in the thick microvillar layer. Third, the ookinetes associated with accumulations of vesicular structures found interspersed between the microvillated cells of the midgut. The origin of these vesicular structures is unknown, but they correlated with the surface of midgut cells invaded by ookinetes as observed by TEM. After binding to the midgut. ookinetes underwent extensive morphological changes: they frequently developed one or more annular constrictions, and their surface roughened considerably, suggesting that midgut components remain bound to the parasite surface. Our observations suggest that, in a natural infection, the ookinete interacts in a sequential manner with specific components of the midgut surface. Initial binding to the midgut surface may activate the ookinete and cause morphological changes in preparation for invasion of the midgut cells.     

Analysis of intracellular state based on controlled 3D nanostructures mediated surface enhanced Raman scattering

Near-infrared surface-enhanced Raman spectroscopy (SERS) is a powerful technique for analyzing the chemical composition within a single living cell at unprecedented resolution. However, current SERS methods employing uncontrollable colloidal metal particles or non-uniformly distributed metal particles on a substrate as SERS-active sites show relatively low reliability and reproducibility. Here, we report a highly-ordered SERS-active surface that is provided by a gold nano-dots array based on thermal evaporation of gold onto an ITO surface through a nanoporous alumina mask. This new combined technique showed a broader distribution of hot spots and a higher signal-to-noise ratio than current SERS techniques due to the highly reproducible and uniform geometrical structures over a large area. This SERS-active surface was applied as cell culture system to study living cells in situ within their culture environment without any external preparation processes. We applied this newly developed method to cell-based research to differentiate cell lines, cells at different cell cycle stages, and live/dead cells. The enhanced Raman signals achieved from each cell, which represent the changes in biochemical compositions, enabled differentiation of each state and the conditions of the cells. This SERS technique employing a tightly controlled nanostructure array can potentially be applied to single cell analysis, early cancer diagnosis and cell physiology research.     

血清白蛋白在多聚阳离子－壳聚糖共混材料表面的构象变化及其与细胞生长的关系

用L-多聚赖氨酸、聚乙烯亚胺及L-多聚鸟氨酸三种多聚阳离子对壳聚糖进行共混修饰,制备了三种共混材料.在这些材料表面吸附了血清白蛋白,并利用圆二色(CD)光谱研究了白蛋白吸附到材料表面后的构象变化.结果显示,与天然状态相比,白蛋白吸附到共混材料表面后,其α-螺旋、β-折叠及无规则卷曲的含量均发生了明显改变.通过研究MC3T3-E1细胞在这些材料表面的生长情况,发现细胞的增殖与血清白蛋白的构象变化有一定关系,在吸附的白蛋白构象与天然构象最接近的共混材料表面,MC3T3-E1细胞增殖水平最高.     

Analysis of cell surface interactions by measurements of lateral mobility

Interactions of cell surface components with one another and with structures inside and outside the cell may have important physiological functions in the transmission of signals and the assembly of specialized structures. These interactions may be detected and analyzed through their effects on the lateral mobility of cell surface molecules. Measurements by a fluorescence photobleaching method have shown that in general lipid-like molecules diffuse rapidly and freely through the plasma membrane, whereas proteins move much more slowly or appear to be immobile. This dichotomy has been supposed to result from forces beyond the viscosity of the lipid bilayer, which specifically retard the diffusion of membrane proteins. This general picture should be qualified, however, by noting that the lateral mobility of lipid-like molecules can be influenced in detail by changes in the state of the plasma membrane such as result from mitosis or fertilization. The interactions of cell surface proteins that limit their lateral mobility are unknown. The effects of binding concanavalin A to localized regions of cell surface show that these interactions can vary in subtle and complex ways. It may soon be useful to interpret mobility experiments in terms of simple reaction models that attempt to describe surface interactions in physicochemical terms. More experimental data are needed to carry out this program and to relate interactions that affect mobility to the structural connections between cell surface components and the cytoskeleton, which have been detected by biochemical methods and electron and immunofluorescence microscopy.     

Effects of cell surface damage on surface properties and adhesion of Pseudomonas aeruginosa

Bacterial cell surfaces play a crucial role in their adhesion to surfaces. In the present study, physico-chemical cell surface properties of Pseudomonas aeruginosa, isolated from a case of contact lens associated keratitis, are determined for mid-exponential and early stationary phase cells and for cells after exposure to a lens care solution or after mechanical damage by sonication. Exposure to a lens care solution and mechanical cell surface damage reduced the cell surface hydrophobicity and water contact angles decreased from 129 degrees to 96 degrees and 83 degrees, respectively. Zeta potentials in saline (-9 mV) were hardly affected after mechanical damage, but tri-modal zeta potential distributions, with subpopulation zeta potentials at -11, -28 and -41 mV, were observed after exposure of bacteria to a lens care solution. X-ray photoelectron spectroscopy indicated changes in the amounts of oxygen-, nitrogen- and phosphorus-rich cell surface components. Mid-exponential phase cells had more nitrogen-rich cell surface components than early stationary phase cells, but water contact angles and zeta potentials were not very different. In addition, mid-exponential phase cells adhered better than early stationary phase cells to hydrophobic and hydrophilic substrata in a parallel plate flow chamber. The capacity of P. aeruginosa to adhere was decreased after inflicting cell surface damage. Exposure to a lens care solution yielded a larger reduction in adhesion capacity than sonication, likely because sonication left most of the cells in a viable state, in contrast to exposure to a lens care solution. It is argued that for clinically relevant experiments, it may be preferable to work with surface damaged cells rather than with gently harvested organisms.     

Correlation of codon biases and potential secondary structures with mRNA translation efficiency in unicellular organisms

Gene expression is known to correlate with degree of codon bias in many unicellular organisms. However, such correlation is absent in some organisms. Recently we demonstrated that inverted complementary repeats within coding DNA sequence must be considered for proper estimation of translation efficiency, since they may form secondary structures that obstruct ribosome movement. We have developed a program for estimation of potential coding DNA sequence expression in defined unicellular organism using its genome sequence. The program computes elongation efficiency index. Computation is based on estimation of coding DNA sequence elongation efficiency, taking into account three key factors: codon bias, average number of inverted complementary repeats, and free energy of potential stem-loop structures formed by the repeats. The influence of these factors on translation is numerically estimated. An optimal proportion of these factors is computed for each organism individually. Quantitative translational characteristics of 384 unicellular organisms (351 bacteria, 28 archaea, 5 eukaryota) have been computed using their annotated genomes from NCBI GenBank. Five potential evolutionary strategies of translational optimization have been determined among studied organisms. A considerable difference of preferred translational strategies between Bacteria and Archaea has been revealed. Significant correlations between elongation efficiency index and gene expression levels have been shown for two organisms (S. cerevisiae and H. pylori) using available microarray data. The proposed method allows to estimate numerically the coding DNA sequence translation efficiency and to optimize nucleotide composition of heterologous genes in unicellular organisms. Availability: http://www.mgs.bionet.nsc.ru/mgs/programs/eei-calculator/.