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PubMed1985

Induction of heat shock protein synthesis in murine tumors during the development of thermotolerance.

The function of one or more heat shock proteins (HSPs) may be to confer protection of cells against thermal damage. We examined the induction kinetics of thermotolerance and the synthesis of HSPs in murine tumor models. Squamous cell carcinomas (SCC VII/SF) or radiation-induced fibrosarcomas (RIF) were implanted in the flanks of C3H mice. These flank tumors were first exposed to an elevated temperature (41 degrees-45 degrees C) for a fixed duration, for example, 43 degrees C for 15 min. Some of the tumors were excised immediately, and tumor cell suspensions were made. The other mice with tumors were returned to the cages and left undisturbed for various times up to 72 h before being sacrificed. Again, tumors were then removed and tumor cell suspensions were prepared. These tumor cells were either challenged with a second heat treatment at 45 degrees C in vitro or labeled with [35S]methionine at 37 degrees C in vitro. The tumor cell survival after the combined heat treatments was measured using the in vitro cloning assay. The cellular proteins were analyzed by one- or two-dimensional gel electrophoresis. We found that mild heat shock induced thermotolerance in murine tumors, a result consistent with those of others. The kinetics of induction and decay of thermotolerance depended on the temperature and duration of the priming treatment. Mild heat shock also enhanced the rate of synthesis and accumulation of some HSPs during the development of thermotolerance. For example, after an initial treatment at 43 degrees C for 15 min, the rates of synthesis of HSPs with molecular weights 68,000, 70,000, and 88,000 were greatly enhanced in SCC VII/SF tumors when compared to unheated controls. Qualitatively similar results were seen with radiation-induced fibrosarcoma tumors. The rate of synthesis of Mr 68,000 to 70,000 HSPs reached maximum value (300% of control value) 2 to 4 h after heat shock and decreased to the control value 6 to 24 h later. On the other hand, the rate of synthesis of actin, a major structural cellular protein, remained relatively constant throughout the 72 h of experiments. We then determined the relationship between the synthesis and accumulation of these HSPs and the expression of thermotolerance in murine tumors after a priming heat treatment. The data indicate that the levels of Mr 68,000 to 70,000 HSPs correlate well with thermotolerance.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Physiological↗

PubMed1996

Isoform-specific monoclonal antibodies to Na,K-ATPase alpha subunits. Evidence for a tissue-specific post-translational modification of the alpha subunit.

Monoclonal antibodies to isoforms of the Na,K-ATPase have become important tools in the study of the enzyme's distribution, physiological roles, and gene regulation, and when their epitopes are defined, they are useful in the study of enzyme structure as well. Evidence is presented that the alpha3-specific antibody McBX3 recognizes an unusual epitope that is not present on alpha3 in the heart. The epitope, which is also found in kidney alpha1 from some species, was mapped to a site on the large intracellular loop near the ATP binding site. DNA sequencing of reverse transcribed-PCR products encompassing the corresponding regions from alpha3 from brain (where McBX3 recognizes alpha3) and heart demonstrated that the tissue difference in epitope is not due to alternative splicing of the mRNA. Instead, hydroxylamine sensitivity indicated that the antibody recognizes a post-translational modification. The epitope for a new antibody for alpha3, XVIF9-G10, was mapped to a site near the N terminus, a location analogous to the sites for the well-characterized antibodies McK1 (alpha1) and McB2 (alpha2). The antibody XVIF9-G10 reacted with the alpha3 of the heart as well as that of the brain; however, McBX3 and XVIF9-G10 both stained the same cellular structures in sections of the rat retina. A new alpha1-specific antibody, 6F, was characterized and mapped to another site near the N terminus; this antibody has broader species specificity than the other well-characterized alpha1 antibody, McK1.

Amino Acid Sequence↗

PubMed1984

Passive stiffness of isolated cardiac and skeletal myocytes in the hamster.

Single cardiac myocytes and skeletal myocyte fragments, devoid of interstitial collagen but with intact glycocalyx, were prepared by mechanical disaggregation of hamster ventricular myocardium and caudal gracilis muscle, respectively. Passive stiffness was studied by examining the sarcomere length-tension relationship over the approximate Eulerian stress range of 0-20 mN/mm2 for cardiac myocytes and 0-120 mN/mm2 for skeletal myocytes. Creep and stress-relaxation became apparent only when cells were stretched to sarcomere lengths close to, or exceeding, 2.2 micron for the cardiac myocytes, and 2.7 micron for the skeletal myocytes. Stress-relaxation and creep occurred simultaneously, suggesting that the sarcomere is at least one of the structural components responsible for viscoelasticity. The differential strain stiffness constant was calculated from the regression of natural stress [Ln(mN/mm2)] against differential strain [(L-Lo)/Lo] and found to be 7.48 +/- 1.73 for the ventricular myocytes and 5.77 +/- 0.87 for the skeletal myocyte fragments. The natural strain stiffness constant was obtained from the regression of natural stress against natural strain [Ln(L/Lo)]. The natural strain stiffness constant was 30-50% higher than the differential strain constant. The high correlation coefficients obtained for both regressions indicate that the length-tension relationships for these isolated cardiac and skeletal myocytes can be very closely fitted to the single exponential function, sigma = C X exp[K(epsilon)]. The length-tension curves obtained for the skeletal myocyte fragments are qualitatively and quantitatively similar to those obtained by others with intact skeletal muscle. The cardiac myocyte length-tension curves are qualitatively, but not quantitatively, similar to those obtained with cardiac muscle. Isolated ventricular myocytes are stiffer than similarly isolated skeletal myocytes. These findings suggest that cellular structures contribute to myocardial stiffness in the hamster.

Animals↗

PubMed2001

Distribution of transcript and protein isoforms of the synaptic glycoprotein neuroplastin in rat retina.

PURPOSE: To examine the expression and localization of the neuroplastins (np), two synapse-enriched members of the immunoglobulin (Ig) superfamily of cell-adhesion molecules, in the developing and adult retina and optic nerve. METHODS: Expressions of the two isoforms np55 and np65 and carboxyl-terminal splice variants were investigated by immunocytochemistry, Western blot analysis, RT-PCR, and in situ hybridization. RESULTS: Immunoreactivity for both neuroplastins was confined to the two synaptic layers of the retina: the inner (IPL) and outer plexiform layer (OPL). Significant overlap was found in staining at synaptic structures with synaptophysin. A large proportion of immunoreactivity for both isoforms, however, was of perisynaptic origin. In situ hybridization studies were suggestive of a pre- and postsynaptic localization of np65 in the OPL. Transcripts for np55 were already present at birth in the inner retina, but the hybridization signals increased during postnatal development. Np65 transcripts and immunosignals appeared at later developmental ages, concomitant with synapse formation in the OPL. Several C-terminal neuroplastin cDNA clones harbor an insert of 12 bp, coding for four amino acids (DDEP) in the intracellular domain of neuroplastins. Splice isoforms containing the insert exhibited a developmental expression pattern similar to that of np55; however, both neuroplastins could harbor the C-terminal insert. Neuroplastins were also detected in optic nerve homogenates. RT-PCR and blockade of axonal transport by nerve crush confirmed transcript and protein expression in optic nerve tissue. CONCLUSIONS: The findings suggest a role for neuroplastins in cell adhesion in the plexiform layers during histogenesis, as well as in maintenance of connections between specific cellular structures.

Animals↗

PubMed1995

Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms.

In the last few years there has been a veritable explosion of knowledge about cyclic nucleotide phosphodiesterases. In particular, the accumulating data showing that there are a large number of different phosphodiesterase isozymes have triggered an equally large increase in interest about these enzymes. At least seven different gene families of cyclic nucleotide phosphodiesterase are currently known to exist in mammalian tissues. Most families contain several distinct genes, and many of these genes are expressed in different tissues as functionally unique alternative splice variants. This article reviews many of the more important aspects about the structure, cellular localization, and regulation of each family of phosphodiesterases. Particular emphasis is placed on new information obtained in the last few years about how differential expression and regulation of individual phosphodiesterase isozymes relate to their function(s) in the body. A substantial discussion of the currently accepted nomenclature is also included. Finally, a brief discussion is included about how the differences among distinct phosphodiesterase isozymes are beginning to be used as the basis for developing therapeutic agents.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2000

Calbindin28kDa is specifically associated with extranuclear constituents of the dense particulate fraction.

Recent attempts to understand the function of calbindin28kDa, a widely expressed calcium-binding protein, are confounded by uncertainties over its subcellular location. Using immunoblot analysis of rat brain subregions, we found that the proportion of particulate calbindin28kDa (24-43% of total) was independent of expression level and location. The association of calbindin28kDa with particulate structures appeared to be specific, since it persisted when soluble calbindin28kDa was sequestered by antibodies added before tissue disruption. Moreover, when exogenous calbindin28kDa was added during homogenisation of brain from calbindin28kDa-nullmutant mice, only 10% partitioned to the particulate fraction compared with 33% of endogenous calbindin28kDa in wildtype controls. Confocal microscopy showed that calbindin28kDa was predominantly extranuclear in all tissues analysed (i.e. various brain regions, isolated neurons, and dental enamel epithelium). Dual-label microscopy of neural dense particulate fractions confirmed the extranuclear location of calbindin28kDa and also showed that it partly colocalised with synaptosome and microtubule markers. Using sucrose step gradients, calbindin28kDa was separated from nuclei in parallel with synaptosome and endoplasmic reticulum markers. However, no association with the marker proteins (synaptophysin, ERp29, alpha/beta-tubulin) was detected by calbindin28kDa-immunoprecipitation analysis. Together these findings provide the first consistent picture that calbindin28kDa is located predominantly outside of the nucleus, irrespective of tissue type (neuronal vs. non-neuronal) and experimental approach (biochemical vs morphological). The evidence of a substantial, strong and specific association with insoluble cellular structures challenges the widely held view of calbindin28kDa as a mobile calcium buffer, and supports the existence of important alternative roles that involve target proteins.

Animals↗

PubMed1978

Mast cells of the Mongolian gerbil (Meriones unguiculatus). Morphology, histamine content and role of calcium in the histamine release process.

The fine structure, histamine conten;, and role of calcium in the histamine release process were studied in peritoneal mast cells of the Mongolian gerbil (Meriones unguiculatus). Stereological methods were applied to obtain quantitative data on their structure. The findings were compared with results obtained from the same type of cells in the rat. The gerbil mast cells were smaller in size (mean volume 242 micrometer3 vs 684 micrometer3 in the rat), and the nuclei were also smaller (55 micrometer3 vs 102 micrometer3). There were fewer granules in the gerbil mast cells and their diameter averaged 0.54 micrometer as compared with 0.78 micrometer in the rat). Only 20% of the cytoplasm of the gerbil mast cell was occupied by granules. This figure is approximately one third of that obtained in rat mast cells. The mean total histamine content per cell was 9 pg as compared to an estimated 30 pg/cell in rats. Calculated molar concentration of histamine in the mast granules, however, was higher in the gerbil than in the rat (2.3 M vs. 0.9 M). The mast cells of the gerbil were much more sensitive to the histamine-releasing agent compound 48/80 and in contrast to rat mast cells they were entirely dependent on calcium for their amine release. The fine cellular structure of both species showed multitudinous plasma membrane folds on their surfaces. In addition gerbil mast cells showed extensive surface invaginations. Apart from this were no major differences at the ultrastructural level between unstimulated cells of the two species. During histamine release, however, the mast cells of the gerbil showed a much greater tendency to form large, intracytoplasmic vacuoles and a decreased propensity for fusion of perigranular and plasma membranes (exocytosis) as compared with the corresponding cells in the rat.

Animals↗

PubMed1996

Molecular dissection of a protein SopB essential for Escherichia coli F plasmid partition.

Biochemical and genetic experiments were carried out to deduce the structural and functional domains of SopB protein involved in the equipartition of F plasmid. The protein is dimeric. Proteolytic and chemical footprinting studies support earlier genetic analyses that the binding of SopB to specific sites within the F plasmid sopC locus involves mainly the C-terminal region. In vivo, the expression of a high level of SopB protein is known to repress sopC-linked genes. This silencing activity is shown to be unaffected by the deletion of 35 N-terminal residues, but abolished when 71 or more were removed from the N terminus. An excess of SopB protein does not extend its in vitro binding outside sopC, implicating participation of a host factor(s) in SopB-mediated gene silencing. A data base search identified a number of SopB homologues, including both chromosomally encoded bacterial proteins and phage- and plasmid-encoded proteins known to be involved in partition. Sequence homology is limited to the N-terminal half, suggesting that the N-terminal regions of these proteins are conserved to interact with a conserved cellular structure(s), whereas the C-terminal regions have diverged to bind different nucleotide sequences.

Amino Acid Sequence↗

PubMed1996

Tau-like proteins associated with centrosomes in cultured cells.

The subcellular association of tau-like proteins with centrosomes in cultured cell lines and its effects in nucleating microtubule assembly were analyzed using biochemical and immunocytochemical approaches. Tau proteins, major components of microtubules, appear to be tightly associated with actin filaments in a variety of cell lines, while in pathological conditions of neurons, they are part of paired helical filaments found in Alzheimer's disease. Different studies suggest that, in addition to tau interactions with the components of the cytoskeletal network, tau polypeptides appear to be associated with highly structured cellular elements, in both interphase and mitotic cells. An in-depth analysis of tau subcellular distribution us- ing different polyclonal and monoclonal antibodies showed colocalization of tau-like components with centrosomes in interphase cells of the human Huh-7 hepatoma, in SW-13 adenocarcinoma, and in normal human fibroblasts. Tau associated with centrosomes in mitotic Huh-7 cells was also identified. However, antibodies against the tau binding repeats did not stain centrosomes. A set of different tau isoforms was also identified by Western blot analysis on isolated centrosomal preparations from Huh-7 cells, obtained by differential centrifugation through sucrose gradients. Microtubule nucleation in vitro over isolated centrosomes was inhibited by both the polyclonal antibody against native tau and an antibody to the N-terminal tau sequence, as revealed by immunofluorescence analysis and assembly kinetics experiments. The antibody TRS1.2 against the fragment containing the first binding repeat on tau did not affect nucleation. These studies allowed us to characterize tau association with the isolated centrosomal preparation and its involvement in microtubule assembly nucleated over centrosomes, thus suggesting possible structural and functional roles for these interactions.

Cells, Cultured↗

PubMed1965

FINE STRUCTURAL CHANGES IN RESPONSE TO HORMONAL STIMULATION OF THE PERFUSED CANINE PANCREAS.

The dog pancreas isolated in situ was perfused with oxygenated dog blood and stimulated with pancreozymin, secretin, or both. There were no significant changes in the fine structure of the acinar, centroacinar, or duct cells attributable to the perfusion. Combined glutaraldehyde and osmium fixation gave good preservation of the secretory products of the acinar cell. Before stimulation, the lumen of the acini is filled with material similar in texture to the content of the zymogen granules, but of somewhat lower density. Release of secretion commonly takes place by coalescence of the limiting membrane of zymogen granules with the plasmalemma, but one granule opening at the surface may frequently be joined by others coalescing with its membrane and forming an interconnected series all with contents having the same texture as the released zymogen. Such a mechanism seems to permit a more rapid release of secretory product than discharge of individual granules. Pancreozymin stimulation caused marked depletion of zymogen granules, but no obvious changes in the Golgi apparatus. It is clear, therefore, that this hormone exerts its effect upon release of granules rather than upon their formation. Secretin stimulation of water and bicarbonate secretion caused a marked washing out of the luminal contents, but had little detectable effect on cellular structure.

Animals↗

PubMed1999

Comparative anatomy of the claustrum in selected species: A morphometric analysis.

The morphology of the claustrum was studied by stereological methods in representatives of five mammalian orders (Insectivora, Rodentia, Lagomorpha, Carnivora and Primates). In each species under study, a dorsal and a ventral part of the nucleus can be distinguished. Based on differences in shape and separation from surrounding structures, five morphological types of the claustrum occur. The claustrum of Insectivora and some rodents represents the least complicated morphological type. The nucleus is very poorly separated from the surrounding structures. The human claustrum is morphologically the most complicated, although the two above-mentioned principal divisions are apparent. The ventrally situated paraamygdalar part of the human claustrum may correspond to the endopiriform nucleus or ventral part of the claustrum of other mammals, because of its morphological characteristics and connections with the limbic system. In guinea pigs, traditionally classified as members of the Rodentia, a characteristic morphological type of the claustrum is present. This observation may support arguments questioning the current position of this species in mammalian classification. Based on stereological studies, the increase of the claustral volume that occurs with increase of the hemispheric volume is significantly smaller than the increase of the isocortical volume and larger than the increase of the allocortical volume. The increase of the volume of the dorsal and ventral parts of the claustrum does not differ significantly in the species under study. Neurons of the claustrum represent differentiated morphology. The numerical density of neurons in the dorsal part of the claustrum is significantly higher than in the ventral one. Differences in the morphology and cellular structure of the two parts of the claustrum may suggest differences in function of the two parts of the nucleus, most probably concerned with transfer of information among various cortical regions. Changes in the claustrum, a cortico-related structure, that occur with increased brain volume, may suggest that its development is less dynamic than that of the isocortex.

Animals↗

PubMed2002

Proteomics characterization of novel spore proteins of Bacillus subtilis.

The spores of Bacillus subtilis have characteristic properties and consist of complex structures including various types of proteins. To perform comprehensive analysis of the protein composition of the spores, the proteins extracted from the spore were analysed by a combination of one-dimensional PAGE and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using Turboquest SEQUEST software interfaced with the DNA sequence database of B. subtilis. A total of 154 proteins were identified, and 69 of them were novel. The remaining 85 proteins have been previously reported as sporulation-specific proteins or as proteins that are synthesized in vegetative cells. The expression pattern of each gene deduced to encode novel spore proteins was analysed using a series of strains carrying a lacZ reporter gene. The results revealed that the expression of 26 genes was dependent on sporulation-specific sigma factors, namely sigma(F), sigma(E), sigma(G) and sigma(K). In this study, it is demonstrated that the combination of the techniques of SDS-PAGE and LC-MS/MS, with the mutant library of B. subtilis, is an effective tool for the analysis of complicated cellular structures.

Bacillus subtilis↗

PubMed1985

Immunocytochemical study of mammalian progesterone receptor using monoclonal antibodies.

Five monoclonal antibodies were used for the immunocytochemical study of mammalian progesterone receptor (PR). Initial studies were aimed at defining the optimal experimental conditions for the detection of the receptor, with special emphasis on techniques likely to be used in clinical determinations and in immunoelectron microscopic localization. Specific immunoperoxidase staining was observed either in fixed, frozen sections or in sections of paraffin-embedded tissue. The latter method allowed a better preservation of cellular structures. Among the eight fixatives tested, glutaraldehyde, picric-acid formaldehyde, and paraformaldehyde proved satisfactory. Both indirect immunoperoxidase and the indirect antibody peroxidase-antiperoxidase methods could be used. In immature rabbits or castrated guinea pigs primed by estrogen, i.e. in conditions where its ligand was absent (or present in very low concentration), the PR was confined to the nucleus of immunoreactive cells. This was the case for all the cell types of the endometrium and the myometrium, for the immunostained cells of the oviduct, cervix, vagina, pituitary gland, and for the very weakly stained cells in the liver. No staining was observed in nontarget tissues for progesterone, such as diaphragm, spleen, and small intestine. Nuclear staining was also absent when various control antibodies replaced anti-PR antibodies. This result thus generalizes the observations made on the estrogen receptor, showing that there is no translocation of the receptor from cytoplasm to nucleus under the influence of the hormone. Moreover, a marked heterogeneity in immunostaining was observed among cells of the same type in several tissues, suggesting that there could be large differences in the hormonal sensitivity of individual cells. Cellular distribution of PR immunoreactivity was studied in the uterus, cervix, oviduct, and pituitary gland of rabbits and in the uterus and vagina of guinea pigs. A labeling was observed in all the cell types of the uterus (luminal and glandular epithelium, stroma, and muscularis). In the cervix, nuclear immunostaining was observed in the connective tissue of the lamina propria and in some epithelial and muscle cells. In the vagina, PR immunoreactivity was seen in the basal layers of the stratified squamous epithelium, in the connective tissue of the lamina propria, and in the smooth muscle. In the oviduct, the luminal epithelium, the connective tissue, and the muscularis were stained. In the pituitary gland, selective nuclear labeling was observed in a few scattered cells.

Animals↗

PubMedDate not supplied

Neurosteroids, brain damage, and mental illness.

The steroidal environment of the brain has marked consequences for both its structure and function. Social or physical stress has deleterious results on hippocampal function. This can be replicated by raising corticoids, which are also highly responsive to stress. Corticosterone, the major glucocorticoid in the rat, induces neuronal death in primary hippocampal cultures. Elevated corticoids also induce mood changes, and these are well known to be associated with stress, particularly chronic stress such as social adversity accentuated by intercurrent aversive life events. DHEA, a second adrenal steroid, has a very different developmental history, increasing rapidly during childhood, reaching a peak in youth, and declining thereafter in both blood and CSF. DHEA, in contrast to corticoids, has brain protective actions. It reduces the neurotoxic actions of glutamate analogues (such as NMDA) as well as those of corticoids. Evidence from several sources suggests that DHEA can act as an antiglucocorticoid. DHEA levels are reduced in major depressive disorders in both adolescents and adults, and a raised cortisol/DHEA ratio (together with intercurrent life events) predicts delayed recovery. DHEA may have a role in the treatment of depression. Together, these findings suggest that altered steroidal environment, whether induced by stress or aging, can have appreciable results on the cellular structure of the brain as well as on its function, although links between the two sets of findings are still tentative.

Adrenal Cortex Hormones↗

PubMed2004

Anthocyanins induce cell cycle perturbations and apoptosis in different human cell lines.

To investigate the mechanistic basis for the biological properties of anthocyanins, two aglycone anthocyanins [delphinidin (DY) and cyanidin (CY)] were used to examine their effects on cell cycle progression and on induction of apoptosis in human cancer cells (uterine carcinoma and colon adenocarcinoma cells) and in normal human fibroblasts. These compounds differ in the number and position of hydroxyl groups on the beta ring in the molecular structure. Cellular uptake of anthocyanins was confirmed by HPLC analysis and no metabolites were detected. The clonogenic assay showed that CY induces a dose-dependent growth inhibitory effect only in fibroblasts. This effect was confirmed by flow cytometric analysis, showing a significant reduction of cells in S phase. In contrast, DP inhibited cell growth in normal and tumour cell lines. This event is accompanied in fibroblasts by an accumulation of cells in the S phase suggesting a block in the transition from S to G2 phase. On the other hand, in tumour cell lines we observed a reduction of cells in G1 phase, paralleled by the appearance of a fraction of cells with a hypodiploid DNA content, thus demonstrating an apoptotic effect by DP. The occurrence of apoptosis induced by DP was confirmed by morphological and biochemical features, including nuclear condensation and fragmentation, annexin V staining, DNA laddering and poly(ADP-ribose) polymerase-1-proteolysis. Furthermore, the mitochondrial membrane potential of apoptotic cells after treatment with DP was significantly lost. The different effects exerted by DP as compared with CY suggest that the presence of the three hydroxyl groups on the beta ring in the molecular structure of DP may be important for its greater biological activity.

Annexin A5↗

PubMed1996

Identification of a novel human Rho protein with unusual properties: GTPase deficiency and in vivo farnesylation.

We have identified a human Rho protein, RhoE, which has unusual structural and biochemical properties that suggest a novel mechanism of regulation. Within a region that is highly conserved among small GTPases, RhoE contains amino acid differences specifically at three positions that confer oncogenicity to Ras (12, 59, and 61). As predicted by these substitutions, which impair GTP hydrolysis in Ras, RhoE binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specific GTPase-activating proteins. Replacing all three positions in RhoE with conventional amino acids completely restores GTPase activity. In vivo, RhoE is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally maintained in an activated state. Thus, amino acid changes in Ras that are selected during tumorigenesis have evolved naturally in this Rho protein and have similar consequences for catalytic function. All previously described Rho family proteins are modified by geranylgeranylation, a lipid attachment required for proper membrane localization. In contrast, the carboxy-terminal sequence of RhoE predicts that, like Ras proteins, RhoE is normally farnesylated. Indeed, we have found that RhoE in farnesylated in vivo and that this modification is required for association with the plasma membrane and with an unidentified cellular structure that may play a role in adhesion. Thus, two unusual structural features of this novel Rho protein suggest a striking evolutionary divergence from the Rho family of GTPases.

Amino Acid Sequence↗

PubMed2004

Survival of retinal pigment epithelium after exposure to prolonged oxidative injury: a detailed gene expression and cellular analysis.

PURPOSE: To detail, by DNA microarrays and cellular structure labeling, the in vitro responses of retinal pigment epithelial (RPE) cells to a nonlethal dose of the oxidant agent hydroquinone (HQ). METHODS: The viability of growth-quiescent ARPE-19 cells after treatment with HQ was measured by XTT conversion, (3)H-leucine incorporation, trypan blue exclusion, and the presence of DNA laddering. The effect of a nonlethal dose of HQ on the localization of apoptosis-induced factor (AIF) and phosphorylation of stress-activated kinase-2/p38 (SAPK2/p38) was detected by immunocytochemistry. Actin structures were visualized by phalloidin staining. Cell membrane blebbing was detected using GFP-membrane-labeled RPE cells (ARPE-GFP-c'-rRas). Changes in gene expression patterns of RPE cells within 48 hours of prolonged treatment with a nonlethal dose of HQ were evaluated by microarray analysis and confirmed by Northern blotting. RESULTS: The viability of RPE after a prolonged sublethal injury dose of HQ was determined by multiple assays and confirmed by the absence of AIF translocation or DNA laddering. Prolonged exposure (16 hours) of RPE cells to a nonlethal dose of HQ resulted in actin rearrangement into globular aggregates and cell membrane blebbing. Kinetic microarray analysis at several time points over a 48-hour recovery period revealed significant upregulation of genes involved in ameliorating the oxidative stress, chaperone proteins, anti-apoptotic factors, and DNA repair factors, and downregulation of pro-apoptotic genes. Genes involved in extracellular matrix functions were also dysregulated. Recovery of RPE cells after the injury was confirmed by the normalization of gene expression dysregulation back to baseline levels within 48 hours. CONCLUSIONS: RPE cells avoided cell death and recovered from prolonged oxidative injury by activating a host of defense mechanisms while simultaneously triggering genes and cellular responses that may be involved in RPE disease development.

Actins↗

PubMed2002

Specific effects of fructo- and gluco-oligosaccharides in the preservation of liposomes during drying.

The fructan family of oligo- and polysaccharides is a group of molecules that have long been implicated as protective agents in the drought and freezing tolerance of many plant species. However, it has been unclear whether fructans have properties that make them better protectants for cellular structures than other sugars. We compared the effects of fructans and glucans on membrane stability during air-drying. Although glucans of increasing chain length were progressively less able to stabilize liposomes against leakage of aqueous content after rehydration, fructans showed increased protection. On the other hand, glucans became more effective in protecting liposomes against membrane fusion with increasing chain length, whereas fructans became less effective. Fourier transform infrared spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature (T(m)) of air-dried liposomes by approximately 25 degrees C in the presence of sucrose and maltose. For the respective pentasaccharides, the reduction of T(m) of the lipids was 9 degrees C lower for samples containing fructan than for those containing glucan, indicating increased sugar--membrane interactions for the fructan compared to the glucan. A reduced interaction of the longer-chain glucans and an increased interaction of the respective fructans with the phospholipid head groups in the dry state was also indicated by dramatic differences in the phosphate asymmetric stretch region of the infrared spectrum. Collectively, our data indicate that the fructo-oligosaccharides accumulated in many plant species under stress conditions could indeed play an important role in cellular dehydration tolerance.

Cichorium intybus↗