Wyniki wyszukiwania

1

Beta-actin in human colon adenocarcinoma cell lines with different metastatic potential

100%

Nowak D., Skwarek-Maruszewska A., Zemanek-Zboch M., Malicka-Blaszkiewicz M.

Acta Biochimica Polonica

|

2005

|

tom 52

|

nr 2

Human colon adenocarcinoma LS180 parental cell line and selected variants, characterized by different metastatic capacity were used to examine, whether a correlation exists between β-actin expression, its subcellular distribution and metastatic potential of these cells. Cytosolic fraction (supernatant 105 000 × g), isolated from the tumor cells was used as a source for actin quantification. The higher level of β-actin was observed in the cytosol of three selected sublines to compare with LS180 parental line. Statistically significant increase of β-actin level in highly motile EB3 cells variant should be underlined to compare with the other sublines. Distinct differences in the phenotype of adenocarcinoma cell variants were found, such as the changes in cells shape, cells spreading and ability to attach to the surface of culture dish. Actin cytoskeleton was visualized with fluorescence microscopy application and microfilaments rhodamine-conjugated phalloidin staining. β-actin subcellular localization was done by immunofluorescence staining with monoclonal anti-β actin antibodies. In the elongated cells (LS180, 3LNLN), this isoactin is dispersed in the whole cell body and concentrates in pseudopods and at the leading edges, when in the rounded variant (EB3) β-actin dominates mainly in cortical ring under cellular membrane and it is also seen in the subtle protrusions. Summary of our former (Nowak et al., 2002, Acta Biochim. Polon., 49: 823) and current data lead to the conclusion that there is a distinct correlation between metastatic capacity of examined human colon adenocarcinoma cells, the state of actin polymerization, actin cytoskeleton organization and β-actin expression.

2

Short, 12 mer fluorescently labeled methylphosphonated oligonucleotides to visualize beta-actin mRNA in vivo

100%

Lazowski K. W., Kaczmarek L.

Journal of Physiology and Pharmacology

|

2003

|

tom 54

|

nr 4

A pair of fluorescently labeled antisense ( complementary to ß-actin mRNA) or control methylphosphonated DNA 12-mers were introduced into live cells. After fixation their distribution throughout the cell was compared to the localization pattern for the pair of control oligos.The distribution of the two sets of oligos differed in that there was a distinct pool of antisense probes that were detected at elevated levels in the leading edge of fibroblast and cortical underlining. The resulting fluorescence patterns of antisense probes colocalized and were analogous to labeling pattern already described and produced by in situ hybridization . The length of each of the probe destabilized binding to mismatched sequences at physiological temperature, while the overall length of the pair gave a unique, highly sequence specific recognition of a target sequence. Simultaneous, in vivo application of multiple probes let include internal controls into the experimental setup, in order to distinguish different distributions of antisense and control probes in the same specimen.

3

Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged beta-actin [pEYFP-actin] and the neurokinin type 1 receptor [NK1-R]

84%

Hrovat A., Zavec A. B., Pogacnik A., Frangez R., Vrecl M.

Cellular and Molecular Biology Letters

|

2010

|

tom 15

|

nr 1

55-69

This study focused on establishing and making a comprehensive functional characterization of an HEK-293-transfected cell line that would coexpress the enhanced yellow fluorescent protein-actin (pEYFP-actin) construct and the neurokinin type 1 receptor (NK1-R), which is a member of the seven transmembrane (7TM) receptor family. In the initial selection procedure, the cloning ring technique was used alone, but failed to yield clones with homogenous pEYFP-actin expression. Flow cytometry sorting (FCS) was subsequently used to enrich the pEYFP-actin-expressing subpopulation of cells. The enzyme-linked immunosorbent assay (ELISA), FCS and quantitative real-time reverse transcription/polymerase chain reaction (RT-PCR) were then employed to monitor the passage-dependent effects on transgene expression and to estimate the total β-actin/pEYFP-actin ratio. NK1-R was characterized via radioactive ligand binding and the second messenger assay. The suitability of the pEYFP-actin as a marker of endogenous actin was assessed by colocalizing pEYFP-actin with rhodamine-phalloidine-stained F-actin and by comparing receptor- and jasplakinolide-induced changes in the actin cytoskeleton organization. These experiments demonstrated that: i) both constructs expressed in the generated transfected cell line are functional; ii) the estimated pEYFP-actin: endogenous β-actin ratio is within the limits required for the functional integrity of the actin filaments; and iii) pEYFP-actin and rhodamine-phalloidine-stained F-actin structures colocalize and display comparable reorganization patterns in pharmacologically challenged cells.

4

The role of the membrane skeleton in formation of the irreversibly sickled cell: a review

84%

Goodman S. R.

Cellular and Molecular Biology Letters

|

1996

|

tom 01

|

nr 1

In this review we discuss the evidence in support of the concept that a posttranslational modification in β-actin, in which a disulfide bridge is formed between cysteine284 and cysteine373, is the major cause of the formation of the irreversibly sickled cell (ISC). This ISC β-actin modification caused a decreased ability of the ISC membrane skeletal proteins to disassemble, as compared to the control and reversible sickled cell (RSC) membrane skeleton, because of altered actin filament formation. The slow disassembly of the ISC membrane skeleton proteins gives a reasonable explanation for the inability of the ISC to remodel its shape. An understanding of the molecular basis of the irreversibly sickled cells formation has helped initiate a rationale for development of drugs to block ISC formation in vivo.

5

New therapeutic approaches to sickle cell disease: targeting RBC membrane oxidative damage

67%

Goodman S. R., Pace B. S., Shartava A.

Cellular and Molecular Biology Letters

|

1998

|

tom 03

|

nr 4

In this brief review, we discuss evidence leading to the conclusion that diminished levels of reduced glutathione combined with increased oxygen radical production leads to oxidative damage to membrane proteins in sickle cell disease erythrocytes. Among these oxidatively damaged proteins are K+-channels (Gardos channel and K+-Cl--channel) and β-actin. Oxidative damage to the K+-channels leads to K+ leakage and H2O loss from light density reversibly sickled cells (RSCs). The resulting dense RSCs are primarily sickled in shape due to increased [HbS] and increased polymerization. The oxidation of β-actin converts the dense RSCs to dense irreversibly sickled cells (ISCs) as explained in our two step model. Furthermore, we discuss recent in vitro evidence that N-acetylcysteine (NAC) can block the formation of dense cells and ISCs by protecting the K+-channels and β-actin respectively from oxidative damage. Finally we describe an ongoing Phase II human trial to determine whether NAC can also lower dense cells and ISCs in vivo and, if so, result in fewer painful vasooclusive episodes.

6

Influence of 4-hydroxynonenal and spleen cells on primary hepatocyte culture and a novel liver-derived cell line resembling hepatocyte stem cells

51%

Cipak A., Borovic S., Jaganjac M., Bresgen N., Kirac I., Grbesa I., Mrakovcic L., Cindric M., Scukanec-Spoljar M., Gall-Troselj K., Coric M., Eckl P., Zarkovic N.

Acta Biochimica Polonica

|

2010

|

tom 57

|

nr 2

Liver is a unique mammalian organ with a great capacity of regeneration related to its function. After surgical resection or injury, hepatic cells, especially hepatocytes, can proliferate rapidly to repair the damage and to regenerate the structure without affecting the function of the liver. Loss of catalase activity during regeneration indicates that oxidative stress is present in the liver not only in pathological conditions but also as a "physiological" factor during regeneration. As we have shown in our previous work, liver stem cell-like cells treated with 4-hydroxynonenal (HNE), a cytotoxic and growth regulating lipid peroxidation product, recover in the presence of spleen cells. In the current study we characterized this novel cell line as liver-derived progenitor/oval-like cells, (LDP/OCs), i.e. functional liver stem-like cells. We showed that LDP/OC were OV6 positive, with abundant glycogen content in the cytoplasm and expressed α-fetoprotein, albumin, biliverdin reductase and γ-glutamyl transferase. Also, we compared their growth in vitro with the growth of cultured primary hepatocytes stressed with HNE and co-cultured with autologous spleen cells. The influence of spleen cells on HNE-treated primary hepatocytes and on LDP/OCs showed that spleen cells support in a similar manner the recovery of both types of liver cells indicating their important role in regeneration. Hence, LDP/OC cells may provide a valuable tool to study cell interactions and the role on HNE in liver regeneration.

Ograniczanie wyników

Beta-actin in human colon adenocarcinoma cell lines with different metastatic potential

Short, 12 mer fluorescently labeled methylphosphonated oligonucleotides to visualize beta-actin mRNA in vivo

Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged beta-actin [pEYFP-actin] and the neurokinin type 1 receptor [NK1-R]

The role of the membrane skeleton in formation of the irreversibly sickled cell: a review

New therapeutic approaches to sickle cell disease: targeting RBC membrane oxidative damage

Influence of 4-hydroxynonenal and spleen cells on primary hepatocyte culture and a novel liver-derived cell line resembling hepatocyte stem cells