Publications by authors named "Olga A Koval"

14 Publications

  • Page 1 of 1

The Recombinant Fragment of Human κ-Casein Induces Cell Death by Targeting the Proteins of Mitochondrial Import in Breast Cancer Cells.

Cancers (Basel) 2020 May 31;12(6). Epub 2020 May 31.

Translational Inflammation Research, Medical Faculty, Center of Dynamic Systems (CDS), Otto von Guericke University, 39120 Magdeburg, Germany.

Breast cancer is still one of the most common cancers for women. Specified therapeutics are indispensable for optimal treatment. In previous studies, it has been shown that RL2, the recombinant fragment of human κ-Casein, induces cell death in breast cancer cells. However, the molecular mechanisms of RL2-induced cell death remain largely unknown. In this study, mechanisms of RL2-induced cell death in breast cancer cells were systematically investigated. In particular, we demonstrate that RL2 induces loss of mitochondrial membrane potential and cellular ATP loss followed by cell death in breast cancer cells. The mass spectrometry-based screen for RL2 interaction partners identified mitochondrial import protein TOM70 as a target of RL2, which was subsequently validated. Further to this, we show that RL2 is targeted to mitochondria after internalization into the cells, where it can also be found in the dimeric form. The importance of TOM70 and RL2 interaction in RL2-induced reduction in ATP levels was validated by siRNA-induced downregulation of TOM70, resulting in the partial rescue of ATP production. Taken together, this study demonstrates that RL2-TOM70 interaction plays a key role in RL2-mediated cell death and targeting this pathway may provide new therapeutic options for treating breast cancer.
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http://dx.doi.org/10.3390/cancers12061427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352597PMC
May 2020

Cytotoxic and Antitumor Activity of Lactaptin in Combination with Autophagy Inducers and Inhibitors.

Biomed Res Int 2019 17;2019:4087160. Epub 2019 Jun 17.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Lavrentiev Ave. 8, 630090 Novosibirsk, Russia.

Autophagy is a degradative process in which cellular organelles and proteins are recycled to restore homeostasis and cellular metabolism. Autophagy can be either a prosurvival or a prodeath process and remains one of the most fundamental processes for cell vitality. Thus autophagy modulation is an important approach for reinforcement anticancer therapeutics. Earlier we have demonstrated that recombinant analog of human milk protein lactaptin (RL2) induced apoptosis of various cultured cancer cells and activated lipidation of microtubule-associated protein 1 light chain 3 (LC3). In this study we investigated whether autophagy inhibitors-chloroquine (CQ), Ku55933 (Ku), and 3-methyladenine (3MA)-or inducer-rapamycin (Rap)-can enhance cytotoxic activity of lactaptin analog in cancer cells and its anticancer activity in the mice model. Western Blot analysis revealed that RL2 induced short-term autophagy in MDA-MB-231 and MCF-7 cells at early stages of incubation and that these data were confirmed by the transmission electron microscopy of autophagosome/autophagolysosome formation. RL2 stimulates reactive oxygen species (ROS) production, autophagosomes accumulation, upregulation of ATG5 with processing of LC3I to LC3II, and downregulation of p62/sequestosome 1 (p62). We have shown that autophagy modulators, CQ, Ku, and Rap, synergistically increased cytotoxicity of RL2, and RL2 with CQ induced autophagic cell death. In addition, CQ, Ku, and Rap in combination with RL2 decreased activity of lysosomal protease Cathepsin D. More importantly, combining RL2 with CQ, we improved antitumor effect in mice. Detected synergistic cytotoxic effects of both types of autophagy regulators, inhibitors, and inducers with RL2 against cancer cells allow us to believe that these combinations can be a basis for the new anticancer approach. Finally, we suppose that CQ and Rap promoting of short-term RL2-induced autophagy interlinks with final autophagic cell death.
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http://dx.doi.org/10.1155/2019/4087160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601476PMC
December 2019

Establishment of primary human breast cancer cell lines using "pulsed hypoxia" method and development of metastatic tumor model in immunodeficient mice.

Cancer Cell Int 2019 28;19:46. Epub 2019 Feb 28.

1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Lavrentiev Avenue, 8, Novosibirsk, 630090 Russia.

Background: Among breast cancer (BC) patients the outcomes of anticancer therapy vary dramatically due to the highly heterogeneous molecular characteristics of BC. Therefore, an extended panel of BC cell lines are required for in vitro and in vivo studies to find out new characteristic of carcinogenesis and metastasis. The purpose of this study was to develop patient-derived BC cell cultures and metastatic tumor models representing a tool for personal therapy and translational research.

Methods: Breast cancer cells were prepared by optimizing technique from tumor samples. We used real-time RT-PCR, flow cytometry, western blotting, cytotoxicity assay, karyotyping and fluorescent and electron microscopy analyses to characterize the established cell lines. BC xenografts in mice were used for in vivo tumorigenicity studies.

Results: The technique of preparing primary cells was optimized and this resulted in a high output of viable and active proliferated cells of nine patient-derived breast cancer cell lines and one breast non-malignant cell line. High E-cadherine and EpCAM expression correlated positively with epithelial phenotype while high expression of N-cadherine and Vimentin were shown in cells with mesenchymal phenotype. All mesenchymal-like cell lines were high HER3-positive-up to 90%. More interesting than that, is that two cell lines under specific culturing conditions (pulsed hypoxia and conditioned media) progressively transformed from mesenchymal to epithelial phenotypes displaying the expression of respective molecular markers proving that the mesenchymal-to-epithelial transition occurred. Becoming epithelial, these cells have lost HER3 and decreased HER2 membrane receptors. Three of the established epithelial cancer cell lines were tumorigenic in SCID mice and the generated tumors exhibited lobules-like structures. Ultrastructure analysis revealed low-differentiate phenotype of tumorigenic cell lines. These cells were in near-triploid range with multiple chromosome rearrangements. Tumorigenic BrCCh4e cells, originated from the patient of four-course chemotherapy, initiated metastasis when they were grafted subcutaneous with colonization of mediastinum lymph nodes.

Conclusions: The developed BC cells metastasizing to mediastinum lymph nodes are a relevant model for downstream applications. Moreover, our findings demonstrate that pulsed hypoxia induces transformation of primary fibroblastoid breast cancer cells to epithelial-like cells and both of these cultures-induced and original-don't show tumor initiating capacity.
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http://dx.doi.org/10.1186/s12935-019-0766-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6394017PMC
February 2019

Characterization of primary normal and malignant breast cancer cell and their response to chemotherapy and immunostimulatory agents.

BMC Cancer 2018 Jul 9;18(1):728. Epub 2018 Jul 9.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Lavrentiev Avenue, 8, 630090, Novosibirsk, Russia.

Background: The phenomenon of chemotherapy-resistant cancers has necessitated the development of new therapeutics as well as the identification of specific prognostic markers to predict the response to novel drugs. Primary cancer cells provide a model to study the multiplicity of tumourigenic transformation, to investigate alterations of the cellular response to various molecular stimuli, and to test therapeutics for cancer treatment.

Methods: Here, we developed primary cultures of human breast tissue - normal cells (BN1), cancer cells (BC5), and cells from a chemotherapy-treated tumour (BrCCh1) to compare their response to conventional chemotherapeutics and to innate immunity stimulators with that of the immortalized breast cells MCF7, MDA-MB-231, and MCF10A. Expression of the progesterone receptor (PGR), oestrogen receptor (ER) α and β, human epidermal growth factor receptor (HER) 2 and 3 and aromatase CYP19, as well as expression of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) mRNA in human breast cells were characterized.

Results: We revealed that BC5 carcinoma cells were PGR/ERb/ERa/Cyp19, the BrCCh1 cells that originated from the recurrent tumour were PGR/ERb/ERa/Cyp19, and normal BN cells were PGR/ERb/ERa/Cyp19. The treatment of primary culture cells with antitumour therapeutics revealed that BrCCh1 cells were doxorubicine-resistant and sensitive to cisplatin. BC5 cells exhibited low sensitivity to tamoxifen and cisplatin. The innate immunity activators interferon-α and an artificial small nucleolar RNA analogue increased expression of IFIT3 at different levels in primary cells and in the immortalized breast cells MCF7, MDA-MB-231, and MCF10A. The relative level of activation of IFIT3 expression was inversely correlated with the baseline level of IFIT3 mRNA expression in breast cell lines.

Conclusion: Our data demonstrated that primary cancer cells are a useful model for the development of novel cancer treatments. Our findings suggest that expression of IFIT3 mRNA can be used as a prognostic marker of breast cancer cell sensitivity to immunostimulating therapeutics.
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http://dx.doi.org/10.1186/s12885-018-4635-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038312PMC
July 2018

Artificial Analogues of Circulating Box C/D RNAs Induce Strong Innate Immune Response and MicroRNA Activation in Human Adenocarcinoma Cells.

Adv Exp Med Biol 2016 ;924:121-125

Institute of Chemical Biology and Fundamental Medicine SB of RAS, Lavrentiev ave. 8, Novosibirsk, 630090, Russia.

Fragments of small nucleolar RNAs (snoRNAs) were found among various non-coding RNAs (ncRNAs) circulating in human blood. Currently, the function of such cell-free sno-derived-RNAs is not clearly defined. This work is aimed at identifying regulatory pathways controlled by extracellular snoRNAs. In order to determine the molecular targets and pathways affected by artificial snoRNAs, we performed Illumina array analysis of MCF-7 human adenocarcinoma cells transfected with box C/D RNAs. The genes related to the innate immune response and apoptotic cascades were found to be activated in transfected cells compared with control cells. Intriguingly, the transfection of MCF-7 cells with artificial box C/D snoRNAs also increased the transcription of several microRNAs, such as mir-574, mir-599 and mir-21. Our data demonstrated that extracellular snoRNAs introduced into human cells may function as gene expression modulators, with activation of microRNA genes being one of the regulatory mechanisms.
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http://dx.doi.org/10.1007/978-3-319-42044-8_24DOI Listing
September 2017

Tumor-Specific Peptide, Selected from a Phage Peptide Library, Enhances Antitumor Activity of Lactaptin.

PLoS One 2016 11;11(8):e0160980. Epub 2016 Aug 11.

Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia.

A recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, induces apoptosis in cultured tumor cells. The tumor suppression efficacy of RL2 was shown against mouse hepatoma-1 cells and MDA-MB-231 human breast adenocarcinoma cells. The RL2-based therapeutic drug lactaptin is distributed evenly throughout the organism, which reduces its antitumor efficacy. In the current study, we obtained a genetic construct that allows production of the recombinant fusion protein T3-RL2, consisting of RL2 and T3 peptide (YTYDPWLIFPAN), in E. coli cells. T3 peptide was selected from a phage peptide library as a result of two screenings: in vitro using MDA-MB-231 cell culture and in vivo using a mouse xenograft model of breast cancer MDA-MB-231. It was shown that the displayed peptide T3 provides binding and internalization of phage particles by MDA-MB-231 cells and their specific accumulation in MDA-MB-231 tumor tissue. In addition, based on the nucleotide sequences coding RL2 and the known tumor-targeting peptide iRGD, we obtained genetic constructs that provide synthesis of fusion proteins RL2-iRGD and RL-iRGD-His. We studied the cytotoxic activity of fusion proteins T3-RL2, RL2-iRGD and RL-iRGD-His in vitro using MDA-MB-231 and MCF-7 human adenocarcinoma cells. The in vitro results showed that the fusion proteins inhibit proliferation of both cell cultures, and their cytotoxic activity is higher than that of RL2. In vivo experiments on the study of the antitumor efficacy of the obtained fusion proteins demonstrated that T3-RL2 protein significantly inhibits MDA-MB-231 tumor growth in a xenograft model compared with RL2, while the antitumor effect of RL2-iRGD and RL-iRGD-His proteins is comparable to the effect of RL2.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160980PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981335PMC
August 2017

Design of protein homocystamides with enhanced tumor uptake properties for (19)F magnetic resonance imaging.

Bioorg Med Chem 2015 Nov 30;23(21):6943-54. Epub 2015 Sep 30.

Institute of Chemical Biology and Fundamental Medicine, SB RAS, 630090 Novosibirsk, Russia; Novosibirsk State University, 630090 Novosibirsk, Russia. Electronic address:

Straightforward and reliable tools for in vivo imaging of tumors can benefit the studies of cancer development, as well as contribute to successful diagnosis and treatment of cancer. (19)F NMR offers an exceptional quantitative way of in vivo imaging of the infused agents because of the lack of (19)F signals from the endogenous molecules in the body. The purpose of this study is to develop molecular probes with appropriate NMR characteristics and the biocompatibility for in vivo applications using (19)F MRI. We have studied the reaction between perfluorotoluene and homocysteine thiolactone resulting in the formation of N-substituted homocysteine thiolactone derivative. It has been shown that the reaction occurs selectively at the para position. This fluorine-labeled homocysteine thiolactone has been employed for the introduction of a perfluorotoluene group as a (19)F-containing tag into human serum albumin. The modified protein has been studied in terms of its ability to aggregate and promote the formation of free radicals. By comparing the properties of N-perfluorotoluene-homocystamide of albumin with N-homocysteinylated albumin, it has been revealed that blocking of the alpha-amino group of the homocysteine residue in the fluorinated albumin conjugate inhibits the dangerous aggregation process, as well as free radical formation. A dual-labeled albumin-based molecular probe for (19)F MRI and fluorescence microscopy has been obtained by functionalizing the protein with both maleimide of a fluorescent dye and a fluorinated thiolactone derivative. The incubation of cells with this conjugate did not reveal any significant reduction in cell viability with respect to the parent albumin. The perfluorotoluene-labeled albumin has been demonstrated to act as a promising agent for in vivo (19)F MRI.
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http://dx.doi.org/10.1016/j.bmc.2015.09.043DOI Listing
November 2015

Sensitivity of endometrial cancer cells from primary human tumor samples to new potential anticancer peptide lactaptin.

J Cancer Res Ther 2015 Apr-Jun;11(2):345-51

Institute of Chemical Biology and Fundamental Medicine SB RAS; Novosibirsk State University, Novosibirsk, Russia.

Purpose: Endometrial carcinoma is the most common gynecologic malignancy which is associated with a poor prognosis when diagnosed at an advanced stage; therefore, the discovery of efficacious new drugs is required to reinforce conventional chemotherapy. Short-term cultures of primary cells from endometrial tumors could be used for testing new anticancer therapeutics as well as for the development of personalized cancer therapy strategy. Here, the antitumor effect of a recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, was examined against primary human endometrial cancer cells.

Materials And Methods: Primary cell cultures of malignant and normal human endometrium were performed by enzymatic digestion of endometrial tissue from biopsy material. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the messenger ribonucleic acid (mRNA) state of estrogen (ERs) and progesterone (PRs) hormone receptors and aromatase (Cyp 19) in cell cultures. Dynamic monitoring of cell adhesion and proliferation was made using the iCELLigence system (ASEA Biosciences). The sensitivity of cell cultures to conventional anticancer drugs and the lactaptin analog was estimated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, flow cytometry, and the iCELLligence system.

Results: Established short-term primary cultures of endometrial cancer cells were ERα/ERβ/PR-positive and sensitive for RL2. The IC 50 values of doxorubicin and cisplatin were determined for all of the primary cultures designed. KE normal cells displaying low Cyp19 mRNA levels and high ERβ and PR mRNA levels were more resistant to RL2 treatment as well as to cisplatin and doxorubicin.

Conclusions: Our results indicate that the recombinant analog of lactaptin, RL2, exerts cytotoxic effects against primary hormone-dependent endometrial tumor cells in vitro with features of apoptosis.
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http://dx.doi.org/10.4103/0973-1482.157301DOI Listing
March 2016

Lactaptin induces p53-independent cell death associated with features of apoptosis and autophagy and delays growth of breast cancer cells in mouse xenografts.

PLoS One 2014 7;9(4):e93921. Epub 2014 Apr 7.

Institute of Chemical Biology and Fundamental Medicine SB RUS, Novosibirsk, Russia.

Lactaptin, the proteolytic fragment of human milk kappa-casein, induces the death of various cultured cancer cells. The mechanisms leading to cell death after lactaptin treatment have not been well characterized. In this study the in vivo and in vitro effects of a recombinant analogue of lactaptin (RL2) were examined. Following treatment with the recombinant analogue of lactaptin strong caspase -3, -7 activation was detected. As a consequence of caspase activation we observed the appearance of a sub-G1 population of cells with subdiploid DNA content. Dynamic changes in the mRNA and protein levels of apoptosis-related genes were estimated. No statistically reliable differences in p53 mRNA level or protein level were found between control and RL2-treated cells. We observed that RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2 protein expression in MDA-MB-231 cells. We demonstrated that RL2 penetrates cancer and non-transformed cells. Identification of the cellular targets of the lactaptin analogue revealed that α/β-tubulin and α-actinin-1 were RL2-bound proteins. As the alteration in cellular viability in response to protein stimulus can be realized not only by way of apoptosis but also by autophagy, we examined the implications of autophagy in RL2-dependent cell death. We also found that RL2 treatment induces LC3-processing, which is a hallmark of autophagy. The autophagy inhibitor chloroquine enhanced RL2 cytotoxicity to MDA-MB-231 cells, indicating the pro-survival effect of RL2-dependent autophagy. The antitumour potential of RL2 was investigated in vivo in mouse xenografts bearing MDA-MB-231 cells. We demonstrated that the recombinant analogue of lactaptin significantly suppressed the growth of solid tumours. Our results indicate that lactaptin could be a new molecule for the development of anticancer drugs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093921PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3978064PMC
January 2015

Artificial box C/D RNAs affect pre-mRNA maturation in human cells.

Biomed Res Int 2013 31;2013:656158. Epub 2013 Mar 31.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Lavrentiev Avenue 8, Novosibirsk 630090, Russia.

Box C/D small nucleolar RNAs (snoRNAs) are known to guide the 2'-O-ribose methylation of nucleotides in eukaryotic ribosomal RNAs and small nuclear RNAs. Recently snoRNAs are predicted to regulate posttranscriptional modifications of pre-mRNA. To expand understanding of the role of snoRNAs in control of gene expression, in this study we tested the ability of artificial box C/D RNAs to affect the maturation of target pre-mRNA. We found that transfection of artificial box C/D snoRNA analogues directed to HSPA8 pre-mRNAs into human cells induced suppression of the target mRNA expression in a time- and dose-dependent manner. The artificial box C/D RNA directed to the branch point adenosine of the second intron, as well as the analogue directed to the last nucleotide of the second exon of the HSPA8 pre-mRNA caused the most prominent influence on the level of HSPA8 mRNAs. Neither box D nor the ability to direct 2'-O-methylation of nucleotides in target RNA was essential for the knockdown activity of artificial snoRNAs. Inasmuch as artificial box C/D RNAs decreased viability of transfected human cells, we propose that natural snoRNAs as well as their artificial analogues can influence the maturation of complementary pre-mRNA and can be effective regulators of vital cellular processes.
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http://dx.doi.org/10.1155/2013/656158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626359PMC
September 2013

A novel pro-apoptotic effector lactaptin inhibits tumor growth in mice models.

Biochimie 2012 Dec 30;94(12):2467-74. Epub 2012 Aug 30.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentiev av. 8, Novosibirsk 630090, Russia.

Lactaptin, a human milk-derived protein, induces apoptosis in cultured tumor cells. We designed a recombinant analog of lactaptin (RL2) and tested its antitumor activity. The sensitivity of hepatocarcinoma A-1 (HA-1), Lewis lung carcinoma, and Ehrlich carcinoma to RL2 were tested to determine the most reliable in vitro animal model. HA-1 cells, which had the highest sensitivity to RL2, were transplanted into A/Sn mice to investigate RL2 antitumor activity in vivo. Investigation of the molecular effects of RL2 shows that RL2 induces apoptotic transformation of HA-1 cells in vitro: phosphatidylserine translocation from inner side of the lipid bilayer to the outer one and dissipation of the mitochondrial membrane potential. Repetitive injections of RL2 (5-50 mg/kg) for 3-5 days effectively inhibited ascites and solid tumor transplant growth when administered intravenously or intraperitoneally, without obvious side effects. The solid tumor inhibitory effect of RL2 (5 i.v. injections, cumulative dose 125 mg/kg) was comparable with that of cyclophosphamide at a therapeutic dose (5 i.v. injections, cumulative dose 150 mg/kg). In combination therapy with cyclophosphamide, RL2 had an additive antitumor effect for ascites-producing tumors. Histomorphometric analysis indicated a three-fold reduction of spontaneous metastases in the liver of RL2-treated mice with solid tumor transplants in comparison with control animals. Repeated RL2 treatment substantially prolonged the lifespan of mice with intravenously injected tumor cells. Recombinant analog of lactaptin effectively induced apoptosis of tumor cells in vitro and suppressed the growth of sensitive tumors and metastases in vivo.
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http://dx.doi.org/10.1016/j.biochi.2012.08.017DOI Listing
December 2012

Recombinant analogs of a novel milk pro-apoptotic peptide, lactaptin, and their effect on cultured human cells.

Protein J 2010 Apr;29(3):174-80

Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev Ave., 8, 630090, Novosibirsk, Russia.

We recently isolated and characterized a human milk peptide, lactaptin, which induced apoptosis of cultured human MCF-7 cells. Lactaptin was identified as a proteolytic fragment of human kappa-casein. Here, we generated two recombinant analogs of the peptide, RL1 and RL2, containing truncated and complete amino acid sequences of lactaptin, respectively. Analogs were produced in E.coli, purified and assayed for biological activity on cultured human MCF-7 cells. RL1 was shown to induce only a small decrease in cell viability, whereas RL2 lowered the viability of MCF-7 cells by 60%. This reduction in MCF-7 cell viability was associated with apoptosis, which was indicated by phosphatidilserine externalization and caspase-7 activation. The viability of A549 and Hep-2 cells was also reduced by RL2, albeit to a lesser degree than seen with MCF-7 cells; this reduced viability was not accompanied by apoptosis. Non-malignant human mesenchymal stem cells (MSC) were completely resistant to RL2 action.
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http://dx.doi.org/10.1007/s10930-010-9237-5DOI Listing
April 2010

Preventative antiinflammatory effect of potamogetonan, a pectin from the common pondweed Potamogeton natans L.

Phytother Res 2007 Jul;21(7):609-14

Institute of Physiology, Komi Science Centre, The Urals Branch of the Russian Academy of Sciences, 50, Pervomaiskaya str., 167982 Syktyvkar, Republic Komi, Russia.

The pectic polysaccharide named potamogetonan (PN) was obtained using extraction of the leaves and stems of the common pondweed Potamogeton natans L. by an aqueous ammonium oxalate. The purified potamogetonan PN-300 was obtained using membrane ultrafiltration of PN and proved to be pectin with a molecular weight of 300 kDa. The capacity of potamogetonan PN-300 to prevent inflammation was assessed using a carrageenan paw edema test in mice. Oral administration of PN-300 24 h prior to induction of inflammation was found to reduce edema formation in a dose-related manner. The maximal effect of PN-300 was observed at 1 h after carrageenan injection (60% reduction of footpad swelling) and was comparable to that of indomethacin. The delayed edema (5 h) was less affected by pre-administration of PN-300 (33% reduction). PN-300 was found to improve the survival of mice subjected to a lethal dose of LPS. The anti-endotoxemic effect of PN-300 was shown to be mediated by decreased TNF-alpha and IL-1beta and increased IL-10 production.Thus, a pectin named potamogetonan PN-300 was isolated from P. natans and was shown to possess a preventive antiinflammatory effect following oral administration.
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http://dx.doi.org/10.1002/ptr.2125DOI Listing
July 2007

ROLL: a method of preparation of gene-specific oligonucleotide libraries.

Oligonucleotides 2004 ;14(3):210-20

SomaGenics, Inc., Santa Cruz, CA 95060, USA.

The selection of nucleic acid sequences capable of specifically and efficiently hybridizing to target sequences is crucial to the success of many applications, including microarrays, PCR and other amplification procedures, antisense inhibition, ribozyme-mediated cleavage, and RNA interference (RNAi). Methods of selection using nucleotide sequence libraries have several advantages over rational approaches using defined sequences. However, the high complexity of completely random (degenerate) libraries and their high toxicity in cell-based assays make their use in many applications impractical. Gene-specific oligonucleotide libraries, which contain all possible sequences of a certain length occurring within a given gene, have much lower complexity and, thus, can significantly simplify and accelerate sequence screening. Here, we describe a new method for the preparation of gene-specific libraries using the ligation of randomized oligonucleotide probes hybridized adjacently on target polynucleotide templates followed by PCR amplification. We call this method random oligonucleotide ligated libraries (ROLL).
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http://dx.doi.org/10.1089/oli.2004.14.210DOI Listing
August 2005