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PubMed2001

Desiccation tolerance in human cells.

The ability to desiccate mammalian cells while maintaining a high degree of viability would have implications for many areas of biological science, including tissue engineering. Previously, we reported that introduction of the genes for trehalose biosynthesis allowed human cells in culture to be reversibly desiccated for up to 5 days. Here, we have further investigated the factors that allow human cells to survive in the desiccated state. The most important finding is that vacuum greatly enhances the ability of human cells in culture to withstand desiccation. In fact, cells dried slowly and stored under vacuum are able to withstand desiccation even in the absence of added carbohydrates or polyols. In addition to vacuum, the rate of desiccation, the temperature at which cells are maintained, the degree of confluence when dried, and the presence or absence of light have a large effect on the ability to retain viability in the desiccated state. Our data are consistent with a model in which cells can retain viability if they are desiccated in such a way that cellular structures are maintained. However, gradual loss of viability may be due to damage that occurs over time in the desiccated state, perhaps due to free radicals. Further optimization of the process for desiccating and maintaining cells is required before long-term storage of desiccated cells can be achieved.

Carbohydrates↗
PubMed1996

Effects of intermediate filament disruption on the early development of the peripheral nervous system of Xenopus laevis.

The principal function of intermediate filaments is to strengthen cells. Their developmentally regulated, tissue-specific patterns of expression further suggest that they modulate cellular structural properties during development. To explore the role of intermediate filaments in development, we injected RNA encoding a truncated form of the Xenopus laevis middle-molecular-weight neurofilament protein (NF-M) into embryonic frog blastomeres at the 2-cell stage. A similar truncated form of mammalian NF-M disrupts neurofilaments (Type IV) and vimentin (Type III) intermediate filaments in transfected fibroblasts. In cultures made from dissociated neural tubes and their adjacent myotomes, the resultant protein disrupted both desmin filaments in muscle cells and neurofilaments in neurons during the first day of culture, which corresponds to stage 35/36 in the intact embryo. We next examined the effects of this truncated neurofilament protein on development of the nervous system. The greatest effects were seen on development of cranial and primary motor nerves, which were severely stunted as late as stage 37/38. In addition to these effects, ectopic neurons also appeared immediately beneath the epidermis along the flank of tadpoles expressing the truncated neurofilament protein. Whereas the former effects on peripheral nerve development were nearly identical to effects obtained with injected neurofilament antibodies, the ectopic neurons were novel, suggesting they resulted from the disruption of intermediate filaments other than the neurofilaments. These experiments thus implicate intermediate filaments in several functions important for normal neural development.

Animals↗
PubMed1999

Measurements of mechanical properties of the blastula wall reveal which hypothesized mechanisms of primary invagination are physically plausible in the sea urchin Strongylocentrotus purpuratus.

Computer simulations showed that the elastic modulus of the cell layer relative to the elastic modulus of the extracellular layers predicted the effectiveness of different force-generating mechanisms for sea urchin primary invagination [L. A. Davidson, M. A. R. Koehl, R. Keller, and G. F. Oster (1995) Development 121, 2005-2018]. Here, we measured the composite elastic modulus of the cellular and extracellular matrix layers in the blastula wall of Strongylocentrotus purpuratus embryos at the mesenchyme blastula stage. Combined, these two layers exhibit a viscoelastic response with an initial stiffness ranging from 600 to 2300 Pa. To identify the cellular structures responsible for this stiffness we disrupted these structures and correlated the resulting lesions to changes in the elastic modulus. We treated embryos with cytochalasin D to disrupt the actin-based cytoskeleton, nocodazole to disrupt the microtubule-based cytoskeleton, and a gentle glycine extraction to disrupt the apical extracellular matrix (ECM). Embryos treated less than 60 min in cytochalasin D showed no change in their time-dependent elastic modulus even though F-actin was severely disrupted. Similarly, nocodazole had no effect on the elastic modulus even as the microtubules were severely disrupted. However, glycine extraction resulted in a 40 to 50% decrease in the elastic modulus along with a dramatic reduction in the hyalin protein at the apical ECM, thus implicating the apical ECM as a major mechanical component of the blastula wall. This finding bears on the mechanical plausibility of several models for primary invagination.

Actins↗
PubMed2000

Inhibition of the establishment of zygotic polarity by protein tyrosine kinase inhibitors leads to an alteration of embryo pattern in Fucus.

Fucoid algae, including the genus Fucus and Pelvetia, are recognized as model systems to study early embryogenesis in plants. In particular the zygotes of these fucoid algae are highly suitable experimental systems for investigating the establishment of polarity and its requirement for later embryogenesis. However, the transduction pathways involved in the initiation of polarization are still poorly understood, and the link between the early polarization processes and embryo long-term patterning has never been experimentally demonstrated. We, therefore, have investigated the putative role of protein phosphorylation in the regulation of early embryogenesis, using a combined pharmacological and biochemical approach. Among the various protein kinase inhibitors tested, a subset of well-known PTK inhibitors, including genistein, prevented germination but had no effect on growth of germinated zygotes and embryos. Inhibition of germination appeared to be a direct consequence of prevention of polarization since genistein and other PTK inhibitors specifically inhibited axis formation in a light-independent manner. Genistein inhibited cellular events associated with polarization such as polarized secretion of cell wall sulfated compounds. Anchorage of F-actin at the rhizoid pole was also inhibited and F-actin redistributed in response to a new light vector. Zygotes inhibited in the polarization process over the period of axis formation recovered from the treatment and displayed differentiated cellular structures after a few days. However, they exhibited a deeply disorganized pattern, suggesting that the early polarization process is essential for normal patterning of the embryo. Western blot analysis of protein phosphorylation showed that the patterns of protein phosphorylation changed during development and were disturbed by treatments with genistein. This drug also inhibited in vitro autophosphorylation. The nature of the genistein-sensitive kinases required for polarization and long-term patterning is discussed in light of these data.

Actins↗
PubMed1998

Expression of centrin isoforms in the mammalian retina.

Centrin is a calcium-binding phosphoprotein of centrosomes, mitotic spindle poles, and flagellar basal apparatus. Indirect immunofluorescence studies in human and rat retinas reveal centrin localization in two distinct cellular structures: at centrosomes of nonciliated neuronal cells as well as in basal bodies, and in larger amounts in the highly modified cilium--the connecting cilium--of photoreceptor cells. Western blot analyses of mammalian retinal proteins show two closely migrating centrin bands at about 20 kDa, the previously described molecular weight of centrins. Using isoform specific primers in PCR, the expression of two related but distinct forms of centrin (centrin 1 and centrin 2), can be identified in the retina of human and rat as well as in the mammalian testis, tissues where cilia are present. However, only one isoform (centrin 2) is expressed in nondifferentiated, nonciliated retinal cells (retinoblastoma cells), as well as in rat liver, skeletal muscle, and cardiac muscle. These observations suggest centrin 2 message may be universally expressed while centrin 1 message may be restricted to retina and testis which contain cells that have differentiated cilia or flagella, or their modifications.

Animals↗
PubMed2001

Flow cytometric assessment of haemocyte sub-populations in the European flat oyster, Ostrea edulis, haemolymph.

The morphology and functions of haemocytes from the haemolymph of the European oyster, Ostrea edulis, were analysed by flow cytometry on the basis of cellular structures and incorporation of fluorescent markers. O. edulis circulating haemocytes appear to be composed of one to three cell populations based on cell size and granularity, with many individual variations. Analysis of haemocytes after propidium iodide staining indicated that the majority of oyster haemocytes are alive after sampling. The phagocytic activity level of haemocytes was analysed using fluorescent beads and this cell activity varied greatly depending on the oysters. The use of 3,3'dihexyloxacarbocyanine iodide (DIOC6) allowed the demonstration of several cell populations on the basis of labelled intensity. One to three cell sub-populations can be observed depending on the oysters. The haemocytes characterised by high granularity showed a strong fluorescence intensity related to high mitochondrial activity.

Animals↗
PubMed1998

Non-mono-exponential attenuation of water and N-acetyl aspartate signals due to diffusion in brain tissue.

Diffusion measurements were performed on water and N-acetyl aspartate (NAA) molecules in excised brain tissue using a wide range of b-values (up to 28.3 x 10(6) and 35.8 x 10(6) s cm-2 for water and NAA, respectively). The attenuation of the signals of water and NAA due to diffusion was measured at fixed diffusion times (tD). These measurements, in which the echo time (TE) was set to 70 ms, were repeated for several diffusion times ranging from 35 to 305 ms. Signal attenuations were fitted to mono-, bi-, and triexponential functions to obtain the apparent diffusion coefficients (ADCs) of these molecules at each diffusion time. From these experiments the following observations and conclusions were made: (1) Signal attenuation of water and NAA due to diffusion over the entire range of b values examined is not monoexponential and the extracted ADCs depend on the diffusion time; (2) In the case of water the experimental data are best fitted by a triexponential function, while for b values up to 1 x 10(6) s cm-2, a biexponential function seems to reproduce the experimental data as well as the triexponential function; (3) If only the low range of b values are fitted (up to 0.5 x 10(6) s cm-2) signal attenuation of water is monoexponential and insensitive to tD; (4) Water ADCs decreased with the increase in tD but the relative population of the fast diffusing component increases such that at a tD of 305 ms there is nearly a single population; (5) The major fast diffusion component of the water shows only very limited restriction; (6) NAA signal attenuation is biexponential and analysis of the low b-value range gives only monoexponential decay, but the obtained ADC is sensitive to the diffusion time; (7) The ADCs obtained from fitting the data with a biexponential function decrease as diffusion time increases; (8) The relative population of the slow-diffusing component decreases with increasing tD; (9) Both the fast and the slow diffusing components of NAA show a considerable restriction by what seems to be a nonpermeable barrier from which two compartments, one of 7-8 micron and one of approximately 1 micron, were calculated using the Einstein equation. It is suggested that the two compartments represent the NAA in cell bodies and in the intra-axonal space. The effect of the range of the b value used in the diffusion experiments on the results is discussed and used to reconcile some of the apparent discrepancies obtained in different experiments concerning water diffusion in brain tissue. The potential of NAA diffusion experiments to probe cellular structure is discussed.

Algorithms↗
PubMed1999

A novel conception for liver preservation at a temperature just above freezing point.

BACKGROUND: It is generally accepted that 0 to 4 degreesC is a suitable temperature for organ preservation. The reason for this is based on the premise that at temperatures below 0 degreesC, intracellular ice is likely to form, with subsequent damage to cellular structures. However, it cannot be assumed that subzero temperatures will freeze the cell. In this study, we attempted to confirm the specific freezing point of rat liver and to preserve it at a temperature just above that point. METHODS: Rat livers were stored for 24, 48, 72, and 96 h either at 4 degreesC (Group N) or at -0.8 degreesC (just above the temperature ascertained to be the specific freezing point of rat liver; Group H). After cold storage, the livers were perfused for 60 min using an isolated perfused liver model for assessment of liver function. RESULTS: ATP and TAN (total adenine nucleotides) in reperfused liver tissues were significantly higher in Group H than in Group N for all preservation periods. ADP was significantly higher in Group H than in Group N for 24-, 72-, and 96-h preservation periods. Energy charge was significantly higher in Group H than in Group N for 24-, 48-, and 96-h preservation periods. CONCLUSIONS: Regarding the content of ATP, ADP, and TAN and the adenylate energy charge, our results indicate that preservation at -0.8 degreesC is advantageous. This novel preservation technique seems to prolong the period that organs can be stored.

Adenine Nucleotides↗
PubMed1999

Cellular uptake and nuclear delivery of recombinant adenovirus penton base.

An Ad2 capsid component, the penton base, expressed as recombinant protein, was found to be capable of affecting the entire entry pathway of adenovirion in HeLa cells, i.e., cell attachment, endocytosis, vesicular escape, intracytoplasmic movement, and translocation through the nuclear pore complex. Data with pentamerization-defective mutants suggested that none of these successive steps depended upon penton base pentamer status, indicating that the peptide domains responsible for these functions were carried by the monomer. Observations performed with wild-type (WT) and an integrin-binding-site double-mutant (K288E340) suggested that the penton base could enter the cell via an alternative, RGD- and LDV-independent, pathway. Of three mutants that were found to be defective in nuclear addressing in insect cells, only one, W165H, was also altered in nuclear transport in HeLa cells. The other two, W119H and RRR547EQQ, showed a WT pattern of nuclear localization in HeLa cells, suggesting that the region including tryptophan-119 and the basic signal at position 547 did not act as a nuclear localization signal in the human cell context. The integrity of cellular structures and the cytoskeleton seemed to be required for the vectorial movement and nuclear import of WT penton base, as suggested by experiments using permeabilized HeLa cells, isolated nuclear membranes, and cytoskeleton-targeted drugs.

Adenoviruses, Human↗
PubMed1999

Entry of rotaviruses is a multistep process.

The infection of epithelial cells by some animal rotavirus strains requires the presence of sialic acid (SA) on the cell surface. Recently, we isolated rhesus rotavirus variants, named nar, whose infectivity, like that of human rotaviruses, is not dependent on SA. In this work, we have determined the binding properties of these SA-dependent and -independent rotavirus strains to MA104 cells. The half-time of attachment of the SA-dependent porcine rotavirus YM and reassortant virus DS1xRRV was found to be about 10 times longer in neuraminidase-treated cells than in untreated cells. On the other hand, human rotaviruses Wa and DS1, and the variant nar3, bound to cells two to three times more rapidly in the absence of SA. To investigate whether the SA-independent cellular structure recognized by the variant and human rotaviruses was the same, we used an infection assay designed to detect competition for cell surface molecules at both attachment and post-attachment steps. In this assay, human rotavirus Wa efficiently competed the infectivity of YM in untreated cells and that of the variant nar3 in untreated, as well as neuraminidase-treated, cells. This competition was nonreciprocal, since YM and nar3 did not compete, but rather increased three- to fivefold the infectivity of Wa. In contrast, a two-direction competition between the variant nar3 and DS1xRRV was found. Similar results were obtained when psoralen-inactivated viruses were used as competitors, indicating that the competition observed was during the early stages of infection. Altogether, these results suggest the existence of multiple interactions between rotaviruses and the cell surface and revealed the existence of common steps during the entry of human and animal rotavirus strains.

Adsorption↗
PubMed1981

Metabolism of benzo(a)pyrene and benzo(a)pyrene 4,5-oxide in rabbit lung.

For several years our laboratory has been investigating the biotransformation of various environmental pollutants by lung. Studies have been performed with pulmonary subcellular fractions, purified monooxygenase and glutathione transferase enzymes, and preparations having intact cellular structure including the isolated perfused lung and cell fractions enriched in alveolar macrophages, Clara cells and alveolar type II cells. Collectively, these investigations have identified several metabolic factors which may contribute to the pulmonary toxicity mediated by certain polycyclic aromatic hydrocarbons (PAH). First, although lung has low overall cytochrome P-450-dependent monooxygenase activity for many substrates, relative to liver, this activity is localized in only a few cell types and specific activity in certain cell types, such as the non-ciliated bronchiolar epithelial (Clara) cell, can be high. Second, oxidative metabolites of benzo(a)pyrene tend to accumulate in pulmonary tissue due, at least in part, to the low ability of lung (relative to liver) to conjugate and detoxify phenolic, dihydrodiol and epoxide metabolites. Thus, products such as benzo(a)pyrene 7,8-dihydrodiol are available for further cytochrome P-450-dependent oxidation to ultimate carcinogens and cytotoxins. Moreover, the lung is efficient in removing benzo(a)pyrene 4,5-oxide and presumably other oxidized PAH metabolites, from the bloodstream. Consequently, the uptake of relatively stable electrophilic metabolites released by the liver may also contribute to pulmonary toxicity.

Animals↗
PubMed1983

Shape, motility and locomotor responses of neutrophil granulocytes.

Relationships among neutrophil shape, motility and locomotion were analysed. Spherical cells were always non-motile. Two types of motile cells could be distinguished 1) motile polarized neutrophils that were capable of locomotion under conditions of limited adhesion and 2) motile non-polar cells that did not locomote under the same conditions. Polarity appears to be a secondary phenomenon related to locomotion or motility. It can also be reversed, indicating that it is not a characteristic based on fixed cellular structures. The fact that a neutrophil is polarized on the substratum does not provide conclusive evidence of cell motility or locomotion. Neutrophils were found to exhibit orthokinetic but not klinokinetic responses. The speed was mainly or exclusively determined by changes in the proportion of migrating cells. Changes in the speed of the migrating subset play a less important role. Locomotion depends on the activity of the motile apparatus and on adhesion to the substratum. By measuring motility in suspension and adhesion to the substratum the proportion of migrating cells can be predicted. The results show that adhesion and changes in motility play a variable role in chemokinesis. Chemokinetic factors may change only adhesion (example: HSA) or adhesion and the activity of the motile apparatus. The effects of chemokinetic factors on motility and adhesion may be synergistic or antagonistic. The separate assessment of adhesion to the substratum and of motility of floating cells may also be useful for the analysis of biological activities of chemokinetic factors, mechanisms regulating chemokinesis and defects of locomotion.

Cell Adhesion↗
PubMed2006

The engrailed transcription factors and the mesencephalic dopaminergic neurons.

The engrailed genes belong to a large family of homeobox transcription factors. They are found throughout the animal kingdom, are highly conserved in the DNA binding domain and have been investigated for more than half a century. In the murine genome, two engrailed genes exist, called Engrailed-1 and Engrailed-2. Here, we summarize the properties of the engrailed genes and their functions, such as conserved structures, cellular localisation, secretion and internalisation, transcription factor activity, potential target genes and review their role in the development of mesencephalic dopaminergic neurons. During early development, they take part in the regionalization event, which specifies the neuroepithelium that provides the precursor cells of the mesencephalic dopaminergic neurons with the necessary signals for their induction. Later in the post-mitotic neurons, the two transcription factors participate in their specification and are cell-autonomously required for their survival.

Animals↗
PubMed1989

Strategies for ultrastructural visualization of biotinated probes hybridized to messenger RNA in situ.

A progressive development of the application of in situ methodology to ultrastructural procedures has resulted in the ability to detect individual molecules of mRNA with high probability. Beginning with whole-mount cells and then developing myotubes, both in culture and detergent extracted before fixation, we were able to progress to methods which allow detection of mRNA in tissue sections. Initial results confirm that the detection of mRNA in thin-sectioned tissue is very similar to observations on the extracted, cultured cells, and that the same methods of data analysis apply. Current work is devoted to the application of the methodology to other cellular structures, such as the nucleus, and to other tissue-probe systems, such as brain. Acknowledgements. The authors appreciate the skilled help from John McNeil and Shirwin Pockwinse in the laborious and time-consuming preparations of material and photography. FS was on sabbatical leave from the Department of Pathology at Southwestern Medical Center.

Animals↗
PubMed1983

Ultrastructural changes in the dog liver cell after isolated liver perfusion with various cytotoxins.

Following isolated liver perfusion with different cytotoxins - 5-fluorouracil (5-FU), methotrexate (MTX), dacarbazine (DTIC) cis-platinum (cis-PT) - liver tissue of dogs was examined with the electron microscope (a) directly after the perfusion and (b) after a survival time of 4 weeks (5-FU and DTIC). Acute disintegration of the granular endoplasmic reticulum and depletion of the glycogen stores occurred after perfusion with 5-FU, MTX, and DTIC, while cis-PT induced disintegration of the granular endoplasmic reticulum accompanied by an accumulation of glycogen. Four weeks after perfusion with DTIC signs of remarkably increased cellular activity were observed, while 4 weeks after perfusion with 5-FU the parenchymal liver cells revealed a well-balanced and moderate recovery of the cellular structures.

Animals↗
PubMed2000

The role of proteolytic enzymes in focal ischaemic brain damage.

Although various neuroprotective and fibrinolytic drugs are currently under evaluation in the acute stages of ischaemic stroke, their therapeutic potential is likely to be limited by unwanted side effects and a narrow time window of opportunity for intervention. Proteolytic enzymes are involved in the catabolism of peptide neurotransmitters and structural cellular proteins in normal brain and have been implicated in the pathogenesis of neurodegenerative disorders. We hypothesised that activation of these enzymes might also play a crucial role in effecting ischaemic neuronal injury, thereby providing a potential site for therapeutic intervention in human stroke. Focal cerebral ischaemia was induced by thermocoagulation of the left middle cerebral artery in aged (30 month) male Wistar rats who were pre-treated with saline or the competitive N-methyl-D-Aspartate antagonist D-CPP-ene, which has been shown to be neuroprotective in young animal models of stroke. Major protease activities were analysed in the left (ischaemic) and right (non-ischaemic) hemispheres, following tissue homogenisation. Data have been analysed using Mann-Whitney tests and are presented as means +/- standard errors. Enzyme activity decreased in ischaemic brain; for example, the mean activity of dipeptidyl aminopeptidase I was 23 +/- 3 and 43 +/- 6 nmol substrate/hour/ml brain extract in the left and right hemispheres respectively (n = 10, p < 0.05). Ischaemic neuronal injury is not effected by the early activation of proteolytic enzymes and protease inhibitors are therefore unlikely to be of benefit in human stroke.

Animals↗
PubMed2005

Deconvolution microscopy.

Since its introduction in 1983, deconvolution microscopy has become a key image-processing tool for visualizing the cellular structures of fixed and living specimens in three dimensions and at subresolution scale. The last 20 years have seen the development of many different applications based on deconvolution microscopy, including a wide variety of optical setup and deconvolution algorithms. This chapter aims to summarize and to describe in detail the major features of this technology, from theoretical aspects to practical solutions. It will begin by explaining the principle of image formation in three-dimensional optical sectioning microscopy. As deconvolution microscopy provides, in essence, a means of overcoming the limits of optical microscopy, the second part of this chapter is dedicated to the theoretical and experimental description of image generation through a microscope. Methods will be detailed for the determination of point spread function, as a crucial step for the characterization of any optical system and a key preliminary step for image deconvolution. The challenges faced and the various possibilities for determining this function precisely will be discussed. All possible sources of aberrations and image degradation processes will be discussed. In the third part of this chapter, we will introduce the acquisition setup and requirements for compliance between acquisition and deconvolution processes. Typical setups for fixed and living cell observation will be detailed, with key features for optimizing speed and reducing artifacts. In the fourth and last part of this chapter, we will describe, in theoretical terms, the various restoration algorithms commonly used in the field of optical microscopy and will provide results obtained with some of the commercially available packages. We shall conclude by considering the prospects for future solutions (currently under development) aiming to handle more easily the huge amounts of data generated by rapid multi-dimensional living cell microscopy. Designed for use by standard cell biologists and hardware and software engineers and developers, this chapter has been written to provide a clear explanation of the wide-reaching and powerful domain of deconvolution microscopy.

Algorithms↗
PubMed1993

Indirect immunofluorescence modified to display two antigens with one light filter.

We present a modification of double indirect immunofluorescence in which we used four antibodies raised in three species to visualize two different antigens. The procedure, which relies on dual recognition of a secondary antibody, requires that one primary antibody and one of the secondary antibodies be raised in the same species. As the two secondary antibodies are conjugated to two different fluorochromes, both of the antigens studied are visualized with one light filter while only one antigen is displayed with another filter. This, in turn, allows more efficient comparison of the distribution of the two antigens in a single field or photograph than is possible by comparing two fields or photographs by conventional double staining. The method is especially useful for determining possible co-localization of two cellular structures. We illustrate the method in adrenal cells in which mitochondria and intermediate filaments are seen to be co-localized.

Adrenal Gland Neoplasms↗