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PubMed2006

Processing body autoantibodies reconsidered.

Processing bodies (P-bodies) are cellular structures that have critical roles in mRNA degradation and post-transcriptional gene silencing. Some patients with autoimmune disease have high titer antibodies directed against P-bodies, and certain sera have been used as markers for the GW182 component of these structures. This study shows that available reference sera are unreliable markers for GW182 because the sera contain antibodies directed against Ge-1, a second P-body autoantigen.

Argonaute Proteins↗
PubMed2005

Ge-1 is a central component of the mammalian cytoplasmic mRNA processing body.

The mRNA processing body (P-body) is a cellular structure that regulates gene expression by degrading cytoplasmic mRNA. The objective of this study was to identify and characterize novel components of the mammalian P-body. Approximately 5% of patients with the autoimmune disease primary biliary cirrhosis have antibodies directed against this structure. Serum from one of these patients was used to identify a cDNA encoding Ge-1, a 1,401-amino-acid protein. Ge-1 contains an N-terminal WD 40 motif and C-terminal domains characterized by a repeating psi(X(2-3)) motif. Ge-1 co-localized with previously identified P-body components, including proteins involved in mRNA decapping (DCP1a and DCP2) and the autoantigen GW 182. The Ge-1 C-terminal domain was necessary and sufficient to target the protein to P-bodies. Following exposure of cells to oxidative stress, Ge-1-containing P-bodies were found adjacent to TIA-containing stress granules. During the recovery period, TIA returned to the nucleus while Ge-1-containing P-bodies localized to the perinuclear region. siRNA-mediated knock-down of Ge-1 resulted in loss of P-bodies containing Ge-1, DCP1a, and DCP2. In contrast, Ge-1-containing P-bodies persisted despite knock-down of DCP2. Taken together, the results of this study show that Ge-1 is a central component of P-bodies and suggest that Ge-1 may act prior to the 5(')-decapping step in mRNA degradation.

Amino Acid Motifs↗
PubMed2006

RNA-associated protein 55 (RAP55) localizes to mRNA processing bodies and stress granules.

The mRNA processing body (P-body) is a cellular structure that has an important role in mRNA degradation. P-bodies have also been implicated in RNAi-mediated post-transcriptional gene silencing. The objective of this study was to identify and characterize novel components of the mammalian P-body. Approximately 5% of patients with the autoimmune disease primary biliary cirrhosis have antibodies directed against this structure. Serum from one of these patients was used to identify a cDNA encoding RAP55, a 463-amino acid protein. RAP55 colocalized with previously identified P-body components DCP1a and Ge-1. RAP55 contains an N-terminal Sm-like domain and two C-terminal RGG-rich domains separated by an FDF motif. The two RGG domains and the FDF domain were necessary and sufficient to target the protein to P-bodies. A fragment of RAP55 consisting of the FDF and the second RGG domains did not localize to P-bodies, but was able to displace other P-body components from this structure. After cells were subjected to arsenite-induced stress, RAP55 was detected in TIA-containing stress granules. The second RGG domain was necessary and sufficient for stress granule localization. siRNA-mediated knock-down of RAP55 resulted in loss of P-bodies, suggesting that RAP55 acts prior to the 5'-decapping step in mRNA degradation. The results of this study show that RAP55 is a component of P-bodies in cells at rest and localizes in stress granules in arsenite-treated cells. RAP55 may serve to shuttle mRNAs between P-bodies and stress granules.

Blotting, Western↗
PubMed1984

Desmoplastic fibroma of bone. A report of six cases.

The clinical and pathological features of six cases of desmoplastic fibroma of bone are presented. Desmoplastic fibroma is rarely seen as a primary tumour of bone; when it does occur the sites of predilection are the long bones, but other sites such as the scapula and os calcis can be involved. Radiographically the lesion tends to expand the bone from within; it is well-demarcated and lytic, often with a trabeculated soap-bubble appearance. The cellular structure and the morphological arrangement are similar to those of aggressive fibromatosis of soft tissues. Differential diagnosis from malignant spindle-cell lesions of bone is important because the treatment of choice for desmoplastic fibroma of bone is simply excision with a thin layer of healthy tissue.

Adolescent↗
PubMed2004

Simultaneous two-photon spectral and lifetime fluorescence microscopy.

When a fluorescence photon is emitted from a molecule within a living cell it carries a signature that can potentially identify the molecule and provide information on the microenvironment in which it resides, thereby providing insights into the physiology of the cell. To unambiguously identify fluorescent probes and monitor their physiological environment within living specimens by their fluorescent signatures, one must exploit as much of this information as possible. We describe the development and implementation of a combined two-photon spectral and lifetime microscope. Fluorescence lifetime images from 16 individual wavelength components of the emission spectrum can be acquired with 10-nm resolution on a pixel-by-pixel basis. The instrument provides a unique visualization of cellular structures and processes through spectrally and temporally resolved information and may ultimately find applications in live cell and tissue imaging.

Animals↗
PubMed2025

Tissue tropism, pathology, and pathogenesis of West Nile virus infection in saltwater crocodile (Crocodylus porosus).

West Nile virus (WNV) is one of the leading causes of economic losses to the saltwater crocodile farming industry due to skin lesions, known as "pix", induced by the infection. Our previous study suggested a possible immunopathological pathway causing these lesions. We therefore resolved to investigate the kinetics of WNV-infection and the elicited immune responses in experimentally challenged saltwater crocodile hatchlings. Employing virus isolation, quantitation of viral genome loads in tissues by RT-qPCR and immunohistochemistry, we demonstrated that upon infection, the virus replicates in the spleen, liver and later in the pancreas. Transcriptomic analysis, based on RNA sequencing and RT-qPCR of kidney and liver tissues, revealed that the early host response is primarily via alteration of cellular structure and metabolism. As the infection progresses, the response becomes predominantly inflammatory and antiviral. The results suggest that the kidney and gastrointestinal tract are primary nidi of viral replication leading to cloacal shedding, but a link to skin lesion development remains to be fully clarified.

Animals↗
PubMed2006

Mast cell apoptosis.

Apoptosis is a physiological form of cell death. Cells undergoing apoptosis execute a genetically controlled program that leads to organized breakdown of cellular structures and ends in phagocytosis of their remains. In mast cells, several mechanisms regulating apoptosis have been identified including growth factors, tumor necrosis factor-alpha receptors, monomeric IgE, Toll-like receptors, and proteins of the bcl-2 family. Methods used to characterize apoptosis of mast cells are reviewed, with special attention to flow cytometric analysis of annexin V staining, analysis of deoxyribonucleic acid fragmentation by gel electrophoresis and end-labeling techniques, measurement of caspase activity by enzymatic assays, and characterization of pro- and anti-apoptotic proteins by immunoblotting.

Animals↗
PubMed2004

Effects of halothane, enflurane, and isoflurane on plasma and erythrocyte antioxidant enzymes and trace elements.

Alterations of the normal redox balance in mammals might be attributed to increases of plasma free-radical concentrations and/or a disruption of the protective mechanisms. These conditions lead to damage to cellular structure by the mechanism of lipoperoxidation, particularly in the liver, kidney, and central nervous system. In this study, the effect of general anesthesia on the oxidative metabolism of human plasma and erythrocytes was investigated. Forty-five patients undergoing anesthesia by using halothane, enflurane, or isoflurane were included in this study. Blood samples were taken preoperatively, the first hour, the first day, and the third day after the operation. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzyme activities and trace elements such as cofactor copper (Cu), zinc (Zn) and selenium (Se) levels were measured in plasma and red blood cells. Our results showed that halothane and enflurane administration increased the plasma GSH-Px activity and reduced zinc levels. In addition, they lowered SOD and GSH-Px activities and trace element levels on erythrocytes. Isoflurane had no effect on plasma antioxidant enzymes, but, similar to the others, isoflurane decreased the plasma zinc levels, erythrocyte SOD and GSH-Px activities and trace element levels.

Adult↗
PubMed2000

Isolation of peroxisome-defective CHO mutant cells using green fluorescent protein.

The authors constructed a recombinant green fluorescent protein (GFP) (PTS-GFP), which carries peroxisome targeting signal (PTS1 or PTS2) as an additional sequence, by polymerase chain reaction. The gene encoding for the recombinant GFP was constructed into an eukaryotic expression vector, and stable transformant of CHO cell expressing PTS-GFP was isolated, following the transfection of the plasmid encoding for the GFP. Each expressed PTS-GFP appeared to be localized in peroxisomes, because the GFP was observed in cellular structures, as was catalase. The observation proposed a visual screening procedure for isolating peroxisome-defective mutant. Following an enrichment of mutant cells by use of 9-(1'-pyrene)nonanol/ultraviolet irradiation (P9OH/UV) method, five peroxisome-defective mutants were isolated by pursuing the fluorescent signals from GFP. Two mutants (SK24 and SK32) were isolated from CHO cells expressing PTS1-GFP, and three mutants (PT13, PT32, and PT54) were isolated from cells expressing PTS2-GFP. Four mutants, except for PT13, showed cytosolic distributions of both PTS-GFP and catalase. On the other hand, mutant PT13 showed a cytosolic distribution on PTS2-GFP, but a peroxisomal distribution on catalase. Cell fusion analysis between SK24 mutant and other mutants indicated that PT54 mutant is in the same complementation group (CG) as SK24, but that SK32, PT13, and PT32 mutants are in different complementation group(s) from SK24.

Animals↗
PubMed2005

The role of oxidative stress in diabetic complications.

The morbidity and mortality associated with diabetes is the result of the myriad complications related to the disease. One of the most explored hypotheses to explain the onset of complications is a hyperglycemia-induced increase in oxidative stress. Reactive oxygen species (ROS) are produced by oxidative phosphorylation, nicotinamide adenine dinucleotide phosphate oxidase (NADPH), xanthine oxidase, the uncoupling of lipoxygenases, cytochrome P450 monooxygenases, and glucose autoxidation. Once formed, ROS deplete antioxidant defenses, rendering the affected cells and tissues more susceptible to oxidative damage. Lipid, DNA, and protein are the cellular targets for oxidation, leading to changes in cellular structure and function. Recent evidence suggests ROS are also important as second messengers in the regulation of intracellular signaling pathways and, ultimately, gene expression. This review explores the production of ROS and the propagation and consequences of oxidative stress in diabetes.

Animals↗
PubMed2001

The cadherin-catenin superfamily in endocrine tumors.

It has been well-known that the cadherin-catenin complexes bind with intracellular skeleton actin, which result in stabilization of cellular structure and tissue organization. Therefore, the cadherin-catenin family has been considered prerequisite for normal cell function and the preservation of tissue integrity. In human malignancies especially colon cancers, dysfunction and/or decrease of expression of these proteins have been proposed to prevent differentiation of tumors and to increase invasiveness and poor prognosis. However, recent studies also revealed that a member of this superfamily, beta-catenin, may play an important role in Wnt/wingless intracellular signaling pathway. Decreased expression of this protein or somatic mutation of the beta-catenin gene has been also reported in human carcinomas including various endocrine tumors. Mutant beta-catenin is associated with abnormal nuclear accumulation in tumor cells and subsequently to activate other transcription factors such as Tcf/Lef. This activation eventually results in which upregulation of mRNA and protein levels of various cell growth mediators in these endocrine tumors. Therefore, dysfunction of the cadherin-catenin system is considered to be closely correlated with tumorigenesis and development in human endocrine tumors.

Animals↗
PubMed2006

Rafts are required for acetylcholine receptor clustering.

Cholesterol-sphingolipid microdomains, or lipid rafts, are major regulators of molecular interactions in membrane organization. Because lipid rafts can move laterally and cluster into larger patches, they have been proposed to play a role in the redistribution of specific molecules to specialized cellular structures. Rafts have been shown to favor formation and maintenance of synaptic receptor clusters in neurons of the central nervous system. However, little is known about their role in formation of the neuromuscular junction (NMJ). To determine whether lipid rafts are involved in acetylcholine receptor (AChR) cluster formation and stabilization in myogenic cells, two standard tools were employed: (1) Perturbation of lipid rafts by drugs that deplete membrane cholesterol was carried out to verify that cholesterol is required for AChR clustering in agrin-treated C2C12 myotubes; and (2) detergent resistance of lipid-ordered domains was also used to demonstrate that AChRs, as well as key components of the postsynaptic membrane of the NMJ, are associated with rafts.

Animals↗
PubMed2004

Gap junctions: their importance for the dynamics of neural circuits.

Electrical coupling through gap junctions constitutes a mode of signal transmission between neurons (electrical synaptic transmission). Originally discovered in invertebrates and in lower vertebrates, electrical synapses have recently been reported in immature and adult mammalian nervous systems. This has renewed the interest in understanding the role of electrical synapses in neural circuit function and signal processing. The present review focuses on the role of gap junctions in shaping the dynamics of neural networks by forming electrical synapses between neurons. Electrical synapses have been shown to be important elements in coincidence detection mechanisms and they can produce complex input-output functions when arranged in combination with chemical synapses. We postulate that these synapses may also be important in redefining neuronal compartments, associating anatomically distinct cellular structures into functional units. The original view of electrical synapses as static connecting elements in neural circuits has been revised and a considerable amount of evidence suggests that electrical synapses substantially affect the dynamics of neural circuits.

Animals↗
PubMedDate not supplied

Electromagnetically induced fluid streaming as a possible mechanism of the biomagnetic orientation of organisms.

The results show that both the direction and the intensity of the geomagnetic field can be sensed within the chamber of a Zeiss Cytopherometer. This suggests that electromagnetically induced fluid streaming might play a role in the perception of the geomagnetic field by organisms, although it is not clear at present in which organ the perception occurs. A basic requirement for such an organ would be an anisotropy of cells or cellular structures. The nervous system with its parallel axons, or specific cells associated with the nervous system, could thus be possible locations of the sensitivity towards magnetic fields.

Electromagnetic Fields↗
PubMed2004

Mechanical properties of Xenopus egg cytoplasmic extracts.

Cytoplasmic extracts prepared from Xenopus laevis eggs are used for the reconstitution of a wide range of processes in cell biology, and offer a unique environment in which to investigate the role of cytoplasmic mechanics without the complication of preorganized cellular structures. As a step toward understanding the mechanical properties of this system, we have characterized the rheology of crude interphase extracts. At macroscopic length scales, the extract forms a soft viscoelastic solid. Using a conventional mechanical rheometer, we measure the elastic modulus to be in the range of 2-10 Pa, and loss modulus in the range of 0.5-5 Pa. Using pharmacological and immunological disruption methods, we establish that actin filaments and microtubules cooperate to give mechanical strength, whereas the intermediate filament cytokeratin does not contribute to viscoelasticity. At microscopic length scales smaller than the average network mesh size, the response is predominantly viscous. We use multiple particle tracking methods to measure the thermal fluctuations of 1 microm embedded tracer particles, and measure the viscosity to be approximately 20 mPa-s. We explore the impact of rheology on actin-dependent cytoplasmic contraction, and find that although microtubules modulate contractile forces in vitro, their interactions are not purely mechanical.

Actins↗
PubMed2005

Second harmonic imaging of exocytosis at fertilization.

Second harmonic generation offers an important alternative and complement to fluorescence for the imaging of cellular structure and function. Staining the eggs of the sea urchin, Lytechinus pictus, with the styryl dye di-8-ANEPPS, we have observed large changes in both second harmonic generation and two-photon fluorescence after fertilization, consistent with the dynamics of exocytosis of cortical granules. With nonlinear imaging on a scanning microscope, we are able to visualize the wave of exocytosis in real time.

Animals↗
PubMed1975

Presence of "receptors" for growth hormone on membranes of rat thymocytes.

Growth hormone conjugated to Sepharose-4 B was found to mimic the action of the free hormone in the stimulation of the uptake and incorporation of radioactive uridine into RNA in thymocytes in vitro. The integrity of the cellular structure was essential for the stimulating effect of the hormone. The bonding of the immunologically reactive determinants of the hormone on membranes of thymocytes was demonstrated by electron micrographs of preparations incubated sequentially with rabbit antigrowth hormone globulins and monospecific sheep anti-rabbit globulins tagged with horse-radish peroxidase.

Animals↗
PubMed1977

Effect of alpha-MSH on the tyrosinase activity and the rate of melanin accumulation of melanoma cells in vitro.

A stimulation of the tyrosinase activity was observed when melanoma cells isolated from transplantable mouse melanomas were incubated at 37 degrees C for 6 h in the presence of 1-10 X 10(-6) M alpha-MSH. All strains of mouse melanomas studied (B-16, Cloudman S-91 and Harding-Passey), exhibited similar responses. It was also observed that the intact cellular structure of melanoma cells was not required for the ability to respond to alpha-MSH. The stimulation of the enzymic activity was accompanied by an increase of the rate of incorporation of radioactivity into melanin from L-[U-14C]tyrosine, indicating an enhanced melanogenesis of tumour cells under in vitro conditions.

Animals↗