scholarly journals Expression ofN-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Shuji Mizumoto ◽  
Moto Watanabe ◽  
Shuhei Yamada ◽  
Kazuyuki Sugahara
Keyword(s):  
Chondroitin Sulfate ◽  
Pulmonary Metastasis ◽  
Drug Targets ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Future Drug ◽  
Invasive Capacity ◽  
Anticancer Treatment

Chondroitin sulfate (CS) containing E-disaccharide units, glucuronic acid-N-acetylgalactosamine(4, 6-O-disulfate), at surfaces of tumor cells plays a key role in tumor metastasis. However, the molecular mechanism of the metastasis involving the CS chain-containing E-units is not fully understood. In this study, to clarify the role of E-units in the metastasis and to search for potential molecular targets for anticancer drugs, the isolation and characterization of Lewis lung carcinoma (LLC) cells stably downregulated by the knockdown for the gene encodingN-acetylgalactosamine 4-O-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which is responsible for the formation of E-units in CS chains, were performed. Knockdown ofGalNAc4S-6STin LLC cells resulted in a reduction in the proportion of E-units, in adhesiveness to extracellular matrix adhesion molecules and in proliferationin vitro. Furthermore, the stable downregulation ofGalNAc4S-6STexpression in LLC cells markedly inhibited the colonization of the lungs by inoculated LLC cells and invasive capacity of LLC cells. These results provide clear evidence that CS chain-containing E-units and/or GalNAc4S-6ST play a crucial role in pulmonary metastasis at least through the increased adhesion and the invasive capacity of LLC cells and also provides insights into future drug targets for anticancer treatment.

Isolation and characterization of calcifying bacteria from “living rocks”: a possible carbon sink

2021 ◽  
Author(s):  
Maddalena del Gallo ◽  
Amedeo Mignini ◽  
Giulio Moretti ◽  
Marika Pellegrini ◽  
Paola Cacchio
Keyword(s):  
Calcium Carbonate ◽  
Carbon Sink ◽  
In Vitro Regeneration ◽  
Scientific Basis ◽  
16S Rrna Analysis ◽  
Environmental Threats ◽  
Isolation And Characterization ◽  
Definition Of

<p>CO<sub>2</sub> emissions triggered by anthropogenic and natural activities contribute to climate change, one of the current environmental threats of public and scientific concern. At present, microbially-induced biomineralization of CO<sub>2</sub> by calcium carbonate (CaCO<sub>3</sub>) is one of the highly topical study subjects as carbon stabilization process. In the present study we focused our attention on the calcifying bacteria of “living rocks”. The origin of these concretions, composed by a silicate skeleton of quartz and feldspars, merged by massive carbonate concrete, has so far been recognized as abiotic. Within this study we investigated the role of calcifying bacteria in their formation of these concretions and we isolated and characterized the species with CaCO<sub>3</sub> precipitation abilities. Concretions were sampled in Romania (Trovant) and Italy (Sibari and Rome). Samples were first analyzed for their culturable microflora (i.e. isolation, CaCO<sub>3 </sub>precipitation capability and molecular characterization). Then, in vitro regeneration tests were carried out to confirm the contribution of bacteria in the formation of these erratic masses. Moreover, natural samples and bioliths regenerated in vitro were (i) observed and analyzed by scanning electron microscopy (SEM-EDS) and (ii) characterized at molecular level by DNA extraction and 16S rRNA analysis (V3-V4 regions). By isolating and characterizing the culturable microflora, we obtained 19 calcifying isolates, with different morphological, bacteriological and mineral precipitation properties. These evidences have given a first relevant contribution for the definition of the biotic role to the formation of these concretions. These evidences were confirmed by the efficient in vitro regeneration and SEM-EDS analysis. The molecular identification of the isolates and the comparison of the data obtained from the Illumina sequencing with those present in the literature, allowed us to hypothesize the genera that most likely contributed to the formation of these concretions. The results obtained provide a good scientific basis for further studies, which should be directed towards the use of isolates in studies of environmental and socio-economic relevance. Several studies demonstrate that microbially mediated biomineralization has the potential to capture and sequester carbon. Calcium carbonate, is a stable pool of carbon and is an effective sealant to prevent CO<sub>2</sub> release back into the atmosphere.</p>

Safflower Yellow Prevents Pulmonary Metastasis of Breast Cancer by Inhibiting Tumor Cell Invadopodia

2016 ◽  
Vol 44 (07) ◽  
pp. 1491-1506 ◽  
Author(s):  
Huiying Fu ◽  
Renjie Wu ◽  
Yuanyuan Li ◽  
Lizong Zhang ◽  
Xiaofang Tang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Pulmonary Metastasis ◽  
Breast Cancer Cells ◽  
Dependent Cell ◽  
Response Profiles ◽  
Dose Dependent

Carthamus tinctorius L. is a traditional Chinese medicine that activates blood circulation and dissipates blood stasis, and has been extensively used as antitumor treatment in a clinical setting in single or in compound preparation form. However, empirical evidence and a better understanding of the possible mechanisms involved are still required. Here, we investigated the role of safflower yellow (SY), the active ingredient of C. tinctorius, in the pulmonary metastasis of breast cancer, and the underlying mechanism of action. EGF-meditated time- and dose-dependent cell response profiles were applied to screen for the activity of SY in vitro, while orthotopic lung metastasis and intravenous injection were used to evaluate the antimetastatic role of SY in vivo. SY could dose-dependently inhibit EGF-mediated time- and dose-dependent cell response profiles by inhibiting cytoskeletal rearrangement. We also found that SY significantly inhibited the migration of breast cancer cells in vitro and pulmonary metastasis of breast cancer cells in vivo. Consistent with these phenotypes, formation of invadopodia and the expression of MMP-9 and p-Src proteins were decreased after EGF stimulation in MBA-MD-231 cells treat with SY, as well as in lung metastatic foci. Additionally, circulating tumor cells retained in lung capillaries were also reduced. These results suggest that the antimetastatic effect of SY is due to its inhibition of invadopodia formation, which occurs mainly through Src-dependent cytoskeleton rearrangement. We suggest that SY should be considered as a potential novel therapeutic agent for the treatment of breast cancer.

scholarly journals Defects in Tendon, Ligament, and Enthesis in Response to Genetic Alterations in Key Proteoglycans and Glycoproteins: A Review

Arthritis ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-30 ◽  
Author(s):  
Subhash C. Juneja ◽  
Christian Veillette
Keyword(s):  
Drug Targets ◽  
Dermatan Sulfate ◽  
Genetic Diseases ◽  
Genetic Alterations ◽  
Ligament Injury ◽  
Structural Development ◽  
Matricellular Proteins ◽  
Secreted Phosphoprotein 1

This review summarizes the genetic alterations and knockdown approaches published in the literature to assess the role of key proteoglycans and glycoproteins in the structural development, function, and repair of tendon, ligament, and enthesis. The information was collected from (i) genetically altered mice, (ii) in vitro knockdown studies, (iii) genetic variants predisposition to injury, and (iv) human genetic diseases. The genes reviewed are for small leucine-rich proteoglycans (lumican, fibromodulin, biglycan, decorin, and asporin); dermatan sulfate epimerase (Dse) that alters structure of glycosaminoglycan and hence the function of small leucine-rich proteoglycans by converting glucuronic to iduronic acid; matricellular proteins (thrombospondin 2, secreted phosphoprotein 1 (Spp1), secreted protein acidic and rich in cysteine (Sparc), periostin, and tenascin X) including human tenascin C variants; and others, such as tenomodulin, leukocyte cell derived chemotaxin 1 (chondromodulin-I, ChM-I), CD44 antigen (Cd44), lubricin (Prg4), and aggrecan degrading gene, a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 5 (Adamts5). Understanding these genes represents drug targets for disrupting pathological mechanisms that lead to tendinopathy, ligamentopathy, enthesopathy, enthesitis and tendon/ligament injury, that is, osteoarthritis and ankylosing spondylitis.

scholarly journals Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Bruno Lorusso ◽  
Angela Falco ◽  
Denise Madeddu ◽  
Caterina Frati ◽  
Stefano Cavalli ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinases ◽  
Human Lung ◽  
Lymphatic Endothelial Cells ◽  
Isolation And Characterization ◽  
Microbial Agents ◽  
Diseased Lung

Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retainin vitroseveral receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy.

scholarly journals Pharmacological Antagonism of Kainate Receptor Rescues Dysfunction and Loss of Dopamine Neurons in a Mouse Model of Human Parkin-induced Toxicity

2020 ◽  
Author(s):  
Maria Regoni ◽  
Stefano Cattaneo ◽  
Daniela Mercatelli ◽  
Salvatore Novello ◽  
Alice Passoni ◽  
...  
Keyword(s):  
Mouse Model ◽  
Neuroprotective Effect ◽  
Chronic Administration ◽  
Dopamine Neurons ◽  
Kainate Receptors ◽  
Substantia Nigra Pars Compacta ◽  
Gene Encoding ◽  
Neuroprotective Therapy

Abstract BackgroundMutations in the PARK2 gene encoding the protein parkin cause autosomal recessive juvenile parkinsonism (ARJP), a neurodegenerative disease characterized by dysfunction and death of dopamine (DA) neurons in the substantia nigra pars compacta. Since a neuroprotective therapy for ARJP does not exist, research efforts aimed at discovering potential targets for neuroprotection are critically needed.A previous study demonstrated that loss of parkin function or expression of parkin mutants associated with ARJP causesan accumulation of glutamate kainate receptors (KARs) in human brain tissues and an increase of KAR-mediated currents in neuronsin vitro. MethodsBased on the hypothesisthat such KAR hyper-activation may contribute to the death of nigralDA neurons, we investigated the effect of KAR antagonism on the DA neuron dysfunction and death that occur in the parkinQ311X mouse, a model of human parkin-induced toxicity. ResultsWe found that early accumulation of KARs occurs in the DA neurons of the parkinQ311X mouse model and that chronic administration of the KAR antagonist UBP310 prevents DA neuron loss. This neuroprotective effect was associated with rescue of the abnormal firing rate of nigral DA neurons and downregulation of GluK2, the key KAR subunit. ConclusionsThis study provides novel evidence ofa causal role of glutamate KARs in the DA neuron dysfunction and loss occurring in a mouse model of human parkin-induced toxicity. Our results support KAR as a potential target in the development of a neuroprotective therapy for ARJP.

scholarly journals CENPF Promotes Proliferation of Renal Cell Carcinoma In Vitro

2021 ◽  
Author(s):  
Ji Zhang ◽  
Shushu Yuan ◽  
Hua Zhu ◽  
Zhan Chen ◽  
Zhenmin Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cancer Progression ◽  
Renal Cell ◽  
Drug Targets ◽  
Tissue Samples ◽  
Clinical Biomarkers

Abstract Background: Metastasis and drug resistance are the main causes of renal cell carcinoma (RCC) mortality. Currently, there are still limited number of targeted therapies against advanced RCC. It is critical to develop new effective clinical biomarkers and drug targets in RCC. Several studies have shown that Centromere protein F (CENPF), a microtubule binding protein, promotes cancer progression in various types of cancer. The purpose of this study is to explore the role of CENPF in RCC.Materials and methods: Peripheral blood and corresponding tissue samples of 23 RCC patients and 23 normal physical examination patients who were treated in our hospital from 2018 to 2020 were collected, and the CENPF expression was detected by qRT-PCR, Western-blot and immunohistochemical methods. Down-regulate the expression of CENPF by siRNA transfection, and detect the proliferation of the corresponding RCC cells and the corresponding cell cycle.Results: According to TCGA data analysis, CENPF is highly expressed in RCC, and its expression level is significantly related to the overall survival and recurrence-free survival of RCC. In addition, high expression of CENPF was found in the tissues of RCC patients in our hospital. Knockdown of CENPF can significantly reduce the proliferation of RCC cells in in vitro experiments, and knockdown of CENPF can regulate the cell cycle by inhibiting the expression of cyclins such as CDK4, CDK6 and CyclinD1. Conclusion: CENPF can be used as an independent prognostic factor of RCC and regulate the proliferation ability and cell cycle of RCC cells. CENPF is a potential oncogene and prognostic marker in RCC.

Validating targets for antiparasite chemotherapy

Parasitology ◽  
1997 ◽  
Vol 114 (7) ◽  
pp. 31-44 ◽  
Author(s):  
C. C. WANG
Keyword(s):  
Drug Targets ◽  
Drug Transport ◽  
Gene Knockout ◽  
Specific Inhibitor ◽  
Selective Inhibition ◽  
Conclusive Evidence ◽  
Gene Encoding ◽  
Multiple Copies ◽  
Antiparasitic Agents

The enzymes and receptors in parasites that can be qualified as targets for antiparasite chemotherapy should perform essential functions in the parasites and demonstrate some feasibility for selective inhibition. They can be tentatively identified through detailed analysis of various aspects of metabolisms in the parasites or elucidation of the mechanisms of action among proven antiparasitic agents. Preliminary verifications of these putative targets can be indicated by in vitro antiparasite activity of an inhibitor of the target. However, before a major long-term effort to pursue in-depth structure-activity analysis of the target is to be committed for specific inhibitor design, further validations of the target are essential to insure that future studies are not misguided. One old-fashioned approach to validate a target in the pharmaceutical industry is by correlating target inhibitions with antiparasitic activities among large numbers of drug derivatives. The results are often indicative but hardly ever conclusive. Another method is by comparing the putative drug targets between the drug-sensitive and the drug-resistant parasites for potential discrepancies. Unfortunately, the latter often result from indirect causes, such as reduced drug transport, instead of an alteration of the drug target itself. The third experimental approach is by disrupting the gene encoding the putative target in parasite, which can provide the most conclusive evidence on whether the target plays an indispensible role in the parasite. But special conditions are needed for the gene knockout mutants to survive to exhibit their phenotypes and to allow genetic complementation studies for further verifications. Furthermore, gene knockout experiments are often difficult to perform on cells of multiple ploidy or genes of multiple copies, and are currently applicable only to a limited number of protozoan parasites. In the current article I have tried to take a cursory look at some eleven putative drug targets among various parasites, each supported by well-established antiparasitic agents identified as its inhibitors. I have also considered the evidence for validity of each of them and the potential means of further verifying their validity.

Atypical Protein Kinase Cs Promote CSF-1-Dependent Erk Activation and Proliferation in Myeloid Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4227-4227 ◽  
Author(s):  
Angel W. Lee
Keyword(s):  
Drug Targets ◽  
Phorbol Esters ◽  
Myeloid Cells ◽  
Dominant Negative ◽  
Erk Pathway ◽  
Erk Activation ◽  
Cell Biol ◽  
Pkc Inhibitors

Abstract Macrophages are integral components of the innate immune system and essential players in inflammation. Enhanced macrophage numbers underlie these pathological states. Colony stimulating factor-1 (CSF-1) is the major physiological regulator of proliferation and survival of cells of the monocyte/macrophage lineage. CSF-1 binds to a receptor tyrosine kinase, the CSF-1 receptor (CSF-1R). CSF-1 and CSF-1R have emerged as drug targets in several diseases where inflammation is a critical component, e.g. breast cancer and rheumatoid arthritis. Multiple pathways are activated downstream of the CSF-1R; however, it is not clear which of these pathways regulate proliferation and survival. Atypical PKCs (aPKCs) are implicated in cell proliferation and survival. They include the isoforms PKCζ and PKCλ/ι. Unlike the classical and novel PKCs, aPKCs are insensitive to Ca2+ and phorbol esters. In this study, we investigated the role of aPKCs in CSF-1-mediated proliferation in myeloid cells. CSF-1 is a proliferation and survival factor for 32D.R cells, a myeloid progenitor cell line transfected with the CSF-1R. Western blotting shows that PKCα, PKCδ, PKCε and PKCζ/λ/I are expressed in 32D.R. Based on studies with PKC inhibitors that have different specificities towards aPKCs (GF109203X, Ro-31-8220, Go6983 and a Myr-PKCζ peptide), maximal CSF-1-dependent proliferation in 32D.R cells appears to depend on the activity of either aPKCs or PKCε. Using phospho-specific antibodies that detect the activation state of PKCζ as well as in vitro kinase assays, we showed that CSF-1 activates aPKCs in 32D.R and bone marrow derived macrophages. In contrast, CSF-1-induced activation of PKCε was not observed. We next asked how aPKC affects CSF-1 signaling. PKCζ promotes activation of the MEK-Erk pathway in different cell types (Corbit, K.C. et al. Mol. Cell. Biol. 20, 5392). In 32D.R cells, treatment with the MEK inhibitor, U0126, reduced CSF-1-provoked proliferation by 60–70%, consistent with the inhibition observed with PKC inhibitors. Previous work from our lab showed that CSF-1 activates the Erk pathway through A-Raf and not Raf-1 (Lee and States, Mol. Cell. Biol. 18, 6779). We found that aPKC inhibitors do not affect CSF-1 induced Ras and A-Raf activity but markedly reduce MEK and Erk activity, implying that aPKC inputs into the CSF-1 Erk pathway at the level of MEK. Transient transfections with dominant-negative and constitutively active (CA) PKCζ confirmed that aPKC promotes CSF-1-induced Erk activation. aPKC inhibition does not affect CSF-1-stimulated Akt activation. To investigate the role of PKCζ in CSF-1-dependent proliferation, we established stable 32D.R mass populations overexpressing wildtype (WT) or CA PKCζ at levels 2-fold above endogenous. Comparing cells expressing CA-PKCζ to WT-PKCζ, the EC50 for CSF-1-dependent proliferation and the cell doubling time at maximal CSF-1 concentration were both reduced, consistent with a role for PKCζ in CSF-1 dependent proliferation. We will use our stable cell lines to elucidate the pathways modulated by PKCζ. Altogether, our results identify atypical PKCs as new targets of CSF-1 signaling.

scholarly journals Effect of Farnesol on a Mouse Model of Systemic Candidiasis, Determined by Use of a DPP3 Knockout Mutant of Candida albicans

2007 ◽  
Vol 75 (4) ◽  
pp. 1609-1618 ◽  
Author(s):  
Dhammika H. M. L. P. Navarathna ◽  
Jacob M. Hornby ◽  
Navasona Krishnan ◽  
Anne Parkhurst ◽  
Gerald E. Duhamel ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Mouse Model ◽  
Tween 80 ◽  
Sublethal Dose ◽  
Systemic Candidiasis ◽  
Knockout Mutant ◽  
Gene Encoding

ABSTRACTThis work extends our previous observation that the fungusCandida albicanssecretes micromolar levels of farnesol and that accumulation of farnesol in vitro prevents the yeast-to-mycelium conversion in a quorum-sensing manner. What does farnesol do in vivo? The purpose of this study was to determine the role of farnesol during infection with a well-established mouse model of systemic candidiasis withC. albicansA72 administered by tail vein injection. This question was addressed by altering both endogenous and exogenous farnesol. For endogenous farnesol, we created a knockout mutation inDPP3, the gene encoding a phosphatase which converts farnesyl pyrophosphate to farnesol. This mutant (KWN2) produced six times less farnesol and was ca. 4.2 times less pathogenic than its SN152 parent. The strain withDPP3reconstituted (KWN4) regained both its farnesol production levels and pathogenicity. These mutants (KWN1 to KWN4) retained their full dimorphic capability. With regard to exogenous farnesol, farnesol was administered either intraperitoneally (i.p.) or orally in the drinking water. Mice receivingC. albicansintravenously and farnesol (20 mM) orally had enhanced mortality (P< 0.03). Similarly, mice (n= 40) injected with 1.0 ml of 20 mM farnesol i.p. had enhanced mortality (P< 0.03), and the onset of mortality was 30 h sooner than for mice which received a control injection without farnesol. The effect of i.p. farnesol was more pronounced (P< 0.04) when mice were inoculated with a sublethal dose ofC. albicans. These mice started to die 4 days earlier, and the percent survival on day 6 postinoculation (p.i.) was five times lower than for mice receivingC. albicanswith control i.p. injections. In all experiments, mice administered farnesol alone or Tween 80 alone remained normal throughout a 14-day observation period. Finally, beginning at 12 h p.i., higher numbers ofC. albicanscells were detected in kidneys from mice receiving i.p. farnesol than in those from mice receiving control i.p. injections. Thus, reduced endogenous farnesol decreased virulence, while providing exogenous farnesol increased virulence. Taken together, these data suggest that farnesol may play a role in disease pathogenesis, either directly or indirectly, and thus may represent a newly identified virulence factor.

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