Search

We established a canine NK-type cell line called CNK-89 derived from a dog with NK cell neoplasia. Immunophenotyping analysis showed positive staining for CD5, CD8, CD45, CD56, CD79a and NKp46, while negative for CD3, CD4, CD14, CD20, CD21, CD34, Thy1, IgG, IgM and MHCII. PCR analysis revealed the presence of CD56, NKG2D, NKp30, NKp44, NKp46 and perforin, but the absence of CD16, Ly49 and granzyme B mRNA. Treating CNK-89 cells with IL-2 did not change the expression of activating receptors, TNFα and IFNγ secretion and cytotoxic activity, however, treatment with IL-12 alone or in combinations with IL-15, IL-18 and IL-21 caused an increase in granzyme B and CD16 mRNA, IFNγ secretion and cytotoxic properties of the CNK-89 cell line. This article is protected by copyright. All rights reserved.

The aim of the work was to study the interaction between boron-rich boron carbide nanoparticles and selected tumor and immune phagocytic cells. Experiments were performed to investigate the feasibility of the application of boron carbide nanoparticles as a boron carrier in boron neutron capture therapy. Boron carbide powder was prepared by the direct reaction between boron and soot using the transport of reagents through the gas phase. The powder was ground, and a population of nanoparticles with an average particle size about 80 nm was selected by centrifugation. The aqueous suspension of the nanoparticles was functionalized with human immunoglobulins or FITC-labeled human immunoglobulins and was then added to the MC38 murine colon carcinoma and to the RAW 264.7 cell line of mouse macrophages. Flow cytometry analysis was used to determine interactions between the functionalized boron carbide nanoparticles and respective cells. It was shown that B4C–IgG nanoconjugates may bind to phagocytic cells to be internalized by them, at least partially, whereas such nanoconjugates can only slightly interact with molecules on the cancer cells’ surface.

Background Although fruit and vegetable-rich diets have beneficial effects on cardiovascular diseases, we have little knowledge of the impact of fruits and their constituents, iridoids and anthocyanins, on the l -arginine-ADMA-DDAH pathway. Our previous study demonstrated the modulation of those factors by the oral administration of the cornelian cherry fruit. Hypothesis/purpose We have assessed the effects of the oral administration of two main constituents isolated from the cornelian cherry fruit, iridoid loganic acid and anthocyanins, on l -arginine, its derivatives (ADMA, SDMA), metabolites (DMA, l -citrulline), and the hepatic DDAH activity and its isoform expression in rabbits fed a high-cholesterol diet. We have also analyzed eNOS expression in the thoracic aorta as well as the redox status in blood. Study Design In the present study, we used an animal model of diet induced atherosclerosis. For 60 days, white New Zealand rabbits were fed a standard diet, a 1% cholesterol enriched diet, or concomitantly with the investigated substances. l -arginine, ADMA, SDMA, DMA, and l -citrulline were assessed using the LC-MS/MS method. DDAH activity and redox parameters were analyzed spectrophotometrically. DDAH1 and DDAH2 isoform expressions were assessed by western blotting, mRNA expression of eNOS was quantified by real-time PCR. Results We demonstrated that the administration of loganic acid (20 mg/kg b.w.), and to a lesser extent of anthocyanins (10 mg/kg b.w.), caused an increase in the l -arginine level and the l -arginine/ADMA ratio. Also, both substances decreased ADMA, DMA, and l -citrulline, but not SDMA levels. Anthocyanins, but not loganic acid, enhanced the activity of DDAH in the liver. Anthocyanins also significantly enhanced both DDAH1 and DDAH2 expression, while loganic acid to a lesser extent enhanced DDAH1 but not DDAH2 expression. Both loganic acid and anthocyanins pronouncedly increased mRNA expression of eNOS in thoracic aortas. Both loganic acid and anthocyanins reversed the blood glutathione level depleted by dietary cholesterol. Cholesterol feeding decreased the blood GPx level, and the change was not reversed by anthocyanins or loganic acid. We did not observe any significant differences in the blood levels of MDA or SOD among the groups. Conclusion Iridoids and anthocyanins may modulate the l -arginine-ADMA pathway in subjects fed a high-cholesterol diet.

Natural killer (NK) cells are lymphocytes of the innate immune system that survey the body for stressed and abnormal cells. The integration of signals that they receive through various inhibitory and activating cell surface receptors controls their activation and ability to kill target cells and produce cytokines. In this manner, phenotypically and functionally distinct subsets of NK cells help protect against microbial infections and cancer and shape the adaptive immune response. NK cells can use two different mechanisms to kill their targets, either by cytotoxic granule exocytosis or by induction of death receptor-mediated apoptosis. Death ligands belong to the tumor necrosis factor (TNF) family of ligands. Upon release in close proximity to a cell slated for killing, perforin forms pores in the cell membrane of the target cell through which granzymes and associated molecules can enter and induce apoptosis. NK cells are also involved in antibody-dependent cellular toxicity via the CD16 receptor. In addition to target recognition, NK cells can be also activated by treatment with multiple compounds with stimulatory properties. Apart from interleukins, which belong to the best characterized group of NK cell-stimulating compounds, vitamins and constituents extracted from plants also display the ability to activate NK cells. The current review characterizes several groups of NK cell-activating compounds: vitamins belonging to classes A, B, C, D, and E, polysaccharides, lectins, and a number of phytochemicals used in cancer research, exhibiting stimulatory properties when applied to NK cells. Although in most cases the exact mechanism of action is not known, constituents described in this review seem to be promising candidates for NK cell-stimulating drugs.

The aim of this study was to accurately determine the profile of polyphenols using the highly sensitive LC&ndash, DAD&ndash, ESI&ndash, QTOF&ndash, MS/MS technique and to determine in vitro antioxidant activity, the ability of inhibition of &alpha, amylase, &alpha, glucoamylase, and pancreatic lipase activity, and antiproliferative activity in leaves, flowers, roots, and stalks of medical plant Sanguisorba officinalis L. The results of the analysis of the morphological parts indicated the presence of 130 polyphenols, including 62 that were detected in S. officinalis L. for the first time. The prevailing group was tannins, with contents ranging from 66.4% of total polyphenols in the flowers to 43.3% in the stalks. The highest content of polyphenols was identified in the flowers and reached 14,444.97 mg/100 g d.b., while the lowest was noted in the stalks and reached 4606.33 mg/100 g d.b. In turn, the highest values of the antiradical and reducing capacities were determined in the leaves and reached 6.63 and 0.30 mmol TE/g d.b, respectively. In turn, a high ability to inhibit activities of &alpha, amylase and &alpha, glucoamylase was noted in the flowers, while a high ability to inhibit the activity of pancreatic lipase was demonstrated in the leaves of S. officinalis L. In addition, the leaves and the flowers showed the most effective antiproliferative properties in pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, bladder cancer, and T-cell leukemia cells, whereas the weakest activity was noted in the stalks. Thus, the best dietetic material to be used when composing functional foods were the leaves and the flowers of S. officinalis L., while the roots and the stalks were equally valuable plant materials.

Betulinic acid and betulin show promising activity against cancers cells, but the mechanism of their action is still unclear. In this study, non-small cell lung cancer (NSCLC) cell lines: A549, H358 and NCI-H1703 were treated with betulinic acid and betulin under normoxic and hypoxic conditions as hypoxia is critically involved in the response of solid tumors to chemotherapy. The treatment inhibits viability and proliferation of NSCLC cells. The anti-proliferative effect was induced by G1 cell cycle arrest with increased p21 expression and decreased cyclin D1 and cyclin B1 expression. Additionally, downregulation of p-GSK3β activity and the Wnt/β-catenin pathway were also observed under hypoxia. We found that hypoxia increased apoptosis in NSCLC cells. Cell death was associated with changes in the expression of proteins involved in the mitochondrial apoptosis pathway and induction of apoptotic death by caspase activation. Additionally, hypoxia exposure deregulated HIF-1α and p53 expression levels. Importantly, treatment with betulinic acid and betulin reduced colony-forming ability under normoxia, however, only betulinic acid reduced clonogenic activity under hypoxia. Our findings that betulinic acid increases apoptotic cell death and clonogenic activity under hypoxic conditions reveal new attractive strategies for treating hypoxic cancer tumors, such as NSCLC.

Cornelian cherry (Cornus mas L.) fruits possess potential cardiovascular, lipid-lowering and hypoglycemic bioactivities. The aim of this study is to evaluate the influence of resin-purified cornelian cherry extract rich in iridoids and anthocyanins on several transcription factors, intima/media ratio in aorta and serum parameters, which determine or are valuable indicators of the adverse changes observed in the course of atherosclerosis, cardiovascular disease, and metabolic syndrome. For this purpose, male New Zealand rabbits were fed a diet enriched in 1% cholesterol for 60 days. Additionally, one group received 10 mg/kg b.w. of cornelian cherry extract and the second group 50 mg/kg b.w. of cornelian cherry extract. PPAR-α and PPAR-γ expression in the aorta, LXR-α expression in the liver, cholesterol, triglycerides, adipokines, apolipoproteins, glucose and insulin levels in serum, the intima and media diameter in the thoracic and abdominal aorta were determined. Administration of cornelian cherry extract resulted in an enhancement in the expression of all tested transcription factors, a decrease in triglycerides, leptin and resistin, and an increase in adiponectin levels. In addition, a significant reduction in the I/M ratio was observed for both the thoracic and abdominal aorta. The results we have obtained confirm the potential contribution of cornelian cherry extract to mitigation of the risk of developing and the intensity of symptoms of obesity-related cardiovascular diseases and metabolic disorders such as atherosclerosis or metabolic syndrome.

The highly selective multi-targeted agent sorafenib is an inhibitor of a number of intracellular signaling kinases with anti-proliferative, anti-angiogenic and pro-apoptotic effects in various types of tumors, including human non-small cell lung cancer (NSCLC). Betulin displays a broad spectrum of biological and pharmacological properties, including anticancer and chemopreventive activity. Combination of drugs with different targets is a logical approach to overcome multilevel cross-stimulation among key signaling pathways in NSCLC progression. NSCLC cell lines, A549, H358 and A427, with different KRAS mutations, and normal human peripheral blood lymphocyte cells, were treated with sorafenib and betulinic acid alone and in combination. We examined the effect of different combined treatments on viability (MTS test), proliferation and apoptotic susceptibility based on flow cytometry, alterations in signaling pathways by western blotting and colony-forming ability. The combination of sorafenib with betulinic acid had a strong effect on the induction of apoptosis of different NSCLC cell lines. In addition, this combination was not toxic for human peripheral blood lymphocytes. Combination treatment changed the expression of proteins involved in the mitochondrial apoptosis pathway and induced apoptotic death by caspase activation. Importantly, combination treatment with low drug concentrations tremendously reduced the colony-forming ability of A549, H358 and A427 cells, as compared to both compounds alone. In this study, we showed that combination therapy with low concentrations of sorafenib and betulinic acid had the capacity to induce high levels of cell death and abolish clonogenic activity in some NSCLC cell lines regardless of KRAS mutations.

We established a new B-cell leukaemia cell line CLB70 from a dog with chronic lymphocytic leukaemia. This cell line is positive for CD20, CD45, CD79a, MHC class II, IgG, IgM; weakly positive for CD21; and negative for CD3, CD4, CD5, CD8, CD14, CD34, CD117. PCR for antigen receptor gene rearrangement (PARR) analysis revealed a biclonal immunoglobulin heavy chain (IgH) gene rearrangement and negative result for TCRγ. Western blot analysis of anti- and pro-apoptotic proteins showed increased expression of Bcl-2, Mcl-1, NF-kB, and Ras, and decreased expression of p53. CLB70 cells grow rapidly in vitro and are tumourigenic in nude mice. The CLB70 line is highly sensitive to doxorubicin, less sensitive to etoposide and imatinib, and resistant to piroxicam, celecoxib and dexamethasone. Our results indicate that CLB70 cells are derived from mature B-cells and they may be a useful tool for the development of new therapeutic strategies for both dogs and humans.

The anticancer activity of novel platinum derivative, a complex of platinum with tris(2-carboxyethyl)phosphine (Pt-TCEP), has been evaluated in canine (D-17) and human osteosarcoma (U2-OS) cell lines. Viability of cells after incubation for 24 or 72 hours with increasing concentrations (0.625, 1.25, 2.50, 5, 10 and 20 μM) of Pt-TCEP was tested in an MTT assay and compared to effect of cisplatin. Longer-term effect of Pt-TCEP was evaluated in the colony-forming unit assay after 24 hours exposure to the Pt-TCEP (2 and 3 μM) and subsequent incubation for 2 weeks. The influence of the compound on the cell cycle was measured after 24 hours treatment with Pt-TCEP (3 μM). Its pro-apoptotic activity was examined after 24 hours treatment with Pt-TCEP (1.25, 2.50, 5, 10 and 20 μM) using flow cytometry. Expression of main proteins involved in apoptosis was measured after exposure for 24 hours to 3 or 5 μM Pt-TCEP in Western Blot. The compound much more effectively decreased cell viability than cisplatin in case of both cell lines. IC50 of Pt-TCEP was 5.93 ± 0.12 in D-17 and 3.45 ± 0.14 in U2-OS cell lines after 24 hours, and 1.77 ± 0.14 in D-17 and 1.53 ± 0.11 in U2-OS after 72 hours (P < .05). The compound arrested cells in the G2/M phase and inhibited the ability of cells to form colonies. Pt-TCEP induced caspase-dependent apoptosis. The expression of the anti-apoptotic Bcl-XL protein was decreased after Pt-TCEP treatment in both cell lines. The results confirmed anti-cancer activity of Pt-TCEP against canine and human osteosarcoma cell lines.

For many years, studies focused on developing new natural or synthetic compounds with antineoplastic activity have attracted the attention of researchers. An interesting group of such compounds seem to be those with both lactone moiety and an aromatic ring which, in addition to antimicrobial or antiviral activity, also exhibit antitumor properties. The study shows antitumor activity of two enantiomeric trans isomers of 5-(1-iodoethyl)-4-(2′,5′-dimethylphenyl)dihydrofuran-2-one. Our aim was to determine their antitumor activity manifested as an ability to induce apoptosis in selected canine cancer cell lines as well as to evaluate differences in their strength depending on the configuration of their stereogenic centers. The enantiomers (+)-(4R,5S,6R)-1 and (−)-(4S,5R,6S)-2 were found to induce classical caspase-dependent apoptosis through downregulation of the expression of anti-apoptotic proteins Bcl-xL and Bcl-2. Although the mechanism of apoptosis induction was the same for both enantiomers, they differed in their strength, as stronger antineoplastic activity in vitro was exhibited by isomer (+)-(4R,5S,6R)-1.

Four stereoisomers of δ-iodo-γ-lactones with p-isopropylphenyl substituent at β-position: cis-(4R,5R,6S)-1, cis-(4S,5S,6R)-2, trans-(4R,5S,6R)-3, trans-(4S,5R,6S)-4 with proved antiproliferative activity were subjected to in vitro tests for a better understanding of their anticancer activity. The subject of our interest was a possible relationship between a configuration of chiral centers of the studied lactones and their anticancer potency against a panel of canine cell lines representing hematopoietic (CLBL-1, GL-1, CL-1, CLB70) and mammary gland cancers (P114, CMT-U27, CMT-U309). To determine the anticancer activity of the tested compounds, cell viability and cell metabolic activity were checked using propidium iodide staining and the MTT test. To determine whether the studied compounds cause necrotic or apoptotic cell death, two assays for apoptosis evaluation were performed, annexin V staining and detection of caspase 3/7 activation. Simultaneously, the effects of the compounds on the cell cycle were also examined. The conducted research confirmed the anticancer potential of the tested lactones against canine cancers. The investigated isomers exerted higher activity against canine lymphoma/leukemia cell lines than against mammary tumors, whereas the configuration of stereogenic centers of the examined compounds affected their activity. It has been shown that stereoisomers with 4S configuration (2,4) were more active, and the most potent one was the cis-(4S,5S,6R)-2 isomer. The investigated lactones seemed to initiate the process of apoptosis rather than acting as typical cytostatic agents, as cell death via apoptosis, and no increase in G2-M population in the cell cycle analysis were observed. The presented study demonstrated that all four stereoisomers of δ-iodo-γ-lactones with p-isopropylphenyl substituent at β-position induced apoptosis via a mitochondrial-mediated, caspase-dependent pathway.

Background and aims Cardiovascular benefits of fruits are attributed mainly to their (poly)phenolic constituents, especially anthocyanins. The main aim of our study is to compare effects of iridoids and anthocyanins from one fruit on diet-induced atherosclerosis. The cornelian cherry is a native or cultivated plant that grows in many European countries, used in cuisine and folk medicine. In our previous study, we showed its constituents and proved that oral administration of lyophilized fruits to hypercholesterolemic rabbits had preventive effects on atherosclerosis through the activation of PPARα expression. In this study, we have compared the effects of the main constituents of the cornelian cherry:iridoid loganic acid and anthocyanins. Methods Our experiment followed the model used in our previous study, in which rabbits were fed 1% cholesterol. Results We showed that both loganic acid (20 mg/kg b.w.) and a mixture of anthocyanins (10 mg/kg b.w.) administered orally for 60 days had a positive impact on dyslipidemia caused by cholesterol-rich diet, although the effects of anthocyanins were more pronounced. Anthocyanins decreased total and LDL-cholesterol and triglycerides and increased HDL-cholesterol. Loganic acid showed similar effects, but only the triglycerides and HDL-cholesterol changes achieved statistical significance. Anthocyanins, and to a lesser extent loganic acid, significantly decreased intima thickness and intima/media ratio in the thoracic aorta. Both substances decrease ox-LDL in the plasma. Anthocyanins significantly increased expression of PPARγ and α in the liver. Loganic acid also increased their expression, but to a lesser extent. Conversely, loganic acid showed pronounced anti-inflammatory effects, decreasing TNF-α and IL-6 activity. Conclusions Our results imply that both substances have a positive effect on factors contributing to the development of diet-induced atherosclerosis. Our results also indicate the potential health benefits of fruits containing anthocyanins and iridoids, and support the idea of creating composed phytopharmaceuticals containing both groups of substances.

Receptory aktywowane proliferatorami peroksysomów, oznaczane skrótem PPAR (peroxisome proliferator-activated receptors), to czynniki transkrypcyjne, należące do rodziny jądrowych receptorów hormonów. Ich główną rolą jest kontrola metabolizmu kwasów tłuszczowych i utrzymanie homeostazy glukozowej. Izotyp γ PPAR może również wpływać na proliferację, przeżywalność i różnicowanie się komórek zarówno prawidłowych, jak i nowotworowych. Związki, które są ligandami PPARγ negatywnie wpływają na komórki nowotworowe, indukując apoptozę, hamując ich proliferację i promując prawidłowe różnicowanie. W pracy przedstawiono ogólne wiadomości na temat receptorów PPAR oraz skupiono się na właściwościach antyproliferacyjnych ligandów PPARγ i ich wykorzystaniu w terapii skojarzonej. Terapia skojarzona z użyciem ligandów PPARγ i innych związków, zwłaszcza retinoidów i swoistych inhibitorów kinaz, może być efektywną strategią w chemioprewencji i leczeniu niektórych typów nowotworów.

Thymic lymphoma cells restore their sensitivity to ionomycin-induced apoptosis when treated with FK506 or HA1004. In apoptosis-resistant cells, ionomycin-induced Nur77 strongly binds DNA during the first 2 h of response, in contrast to lymphoma cells treated with ionomycin together with FK506 or HA1004, which undergo massive apoptosis. We show that Nur77 could discriminate between calcium signals sensitive to FK506 and those sensitive to HA1004, as the inhibitors differentially regulate the kinetics of Nur77 nuclear import, and FK506, unlike HA1004, inhibits Nur77 DNA-binding activity. In the presence of HA1004, NBRE binding by Nur77 protein increases with time (6 h vs 2 h), whereas the final outcome of both inhibitors is apoptosis of thymic lymphoma cells.

The orphan nuclear receptors Nur77 and Nurr1 are the members of the Nur77 family of transcription factors. We demonstrate that transcription of the Nur77 family genes was upregulated in PC12 cells following incubation with Ca2+ ionophore as well as cyclic AMP (cAMP) analog. On the other hand, cAMP analog induced strong increase, while Ca2+ ionophore induced weak increase in the transactivation activity of Nur77. We found that Nur77 and Nurr1 proteins were expressed in the nucleus following stimulation with cAMP analog but not after stimulation with Ca2+ ionophore. However, expression of Nur77 protein was increased in the cytoplasm of cells treated with Ca2+ ionophore. In conclusion, our results suggest that cAMP-induced and Ca2+-induced processes may differentially regulate activity of Nur77 at the level of translocation of Nur77 protein from the cytoplasm into the nucleus.

Ricin acts by translocating to the cytosol the enzymatically active toxin A-chain, which inactivates ribosomes. Retrograde intracellular transport and translocation of ricin was studied under conditions that alter the sensitivity of cells to the toxin. For this purpose tyrosine sulfation of mutant A-chain in the Golgi apparatus, glycosylation in the endoplasmic reticulum (ER) and appearance of A-chain in the cytosolic fraction was monitored. Introduction of an ER retrieval signal, a C-terminal KDEL sequence, into the A-chain increased the toxicity and resulted in more efficient glycosylation, indicating enhanced transport from Golgi to ER. Calcium depletion inhibited neither sulfation nor glycosylation but inhibited translocation and toxicity, suggesting that the toxin is translocated to the cytosol by the pathway used by misfolded proteins that are targeted to the proteasomes for degradation. Slightly acidified medium had a similar effect. The proteasome inhibitor, lactacystin, sensitized cells to ricin and increased the amount of ricin A-chain in the cytosol. Anti-Sec61alpha precipitated sulfated and glycosylated ricin A-chain, suggesting that retrograde toxin translocation involves Sec61p. The data indicate that retrograde translocation across the ER membrane is required for intoxication.

Producción Científica, Acidic fibroblast growth factor (aFGF) is a potent mitogen. It acts through activation of specific cell surface receptors leading to intracellular tyrosine phosphorylation cascades, but several reports also indicate that aFGF enters cells and that it has an intracellular function as well. The aFGF(K132E) mutant binds to and activates fibroblast growth factor receptors equally strongly as the wild-type, but it is a poor mitogen. We demonstrate that aFGF(K132E) enters NIH 3T3 cells and is transported to the nuclear fraction like wild-type aFGF. A fusion protein of aFGF(K132E) and diphtheria toxin A-fragment (aFGF(K132E)-DT-A) and a similar fusion protein containing wild-type aFGF (aFGF-DT-A) were reconstituted with diphtheria toxin B-fragment. Both fusion proteins were translocated to the cytosol by the diphtheria toxin pathway and subsequently recovered from the nuclear fraction. Whereas translocation of aFGF-DT-A stimulated DNA synthesis in U2OSDR1 cells lacking functional fibroblast growth factor receptors, aFGF(K132E)-DT-A did not. The mutation disrupts a protein kinase C phosphorylation site in the growth factor making it unable to be phosphorylated. The data indicate that a defect in the intracellular action of aFGF(K132E) is the reason for its strongly reduced mitogenicity, possibly due to inability to be phosphorylated.

Endocytosis and intracellular transport of ricin were studied in stable transfected HeLa cells where overexpression of wild-type (WT) or mutant dynamin is regulated by tetracycline. Overexpression of the temperature-sensitive mutant dynG273D at the nonpermissive temperature or the dynK44A mutant inhibits clathrin-dependent endocytosis (Damke, H., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131: 69–80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915–934). Under these conditions, ricin was endocytosed at a normal level. Surprisingly, overexpression of both mutants made the cells less sensitive to ricin. Butyric acid and trichostatin A treatment enhanced dynamin overexpression and increased the difference in toxin sensitivity between cells with normal and mutant dynamin. Intoxication with ricin seems to require toxin transport to the Golgi apparatus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949–966), and this process was monitored by measuring the incorporation of radioactive sulfate into a modified ricin molecule containing a tyrosine sulfation site. The sulfation of ricin was much greater in cells expressing dynWT than in cells expressing dynK44A. Ultrastructural analysis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dynK44A. In contrast, ricin transport to lysosomes as measured by degradation of 125I-ricin was essentially unchanged in cells expressing dynK44A. These data demonstrate that although ricin is internalized by clathrin-independent endocytosis in cells expressing mutant dynamin, there is a strong and apparently selective inhibition of ricin transport to the Golgi apparatus. Also, in cells with mutant dynamin, there is a redistribution of the mannose-6-phosphate receptor.

Translocation of ricin A chain to the cytosol has been proposed to take place from the endoplasmic reticulum (ER), but attempts to visualize ricin in this organelle have failed. Here we modified ricin A chain to contain a tyrosine sulfation site alone or in combination with N-glycosylation sites. When reconstituted with ricin B chain and incubated with cells in the presence of Na 2 35 SO 4 , the modified A chains were labeled. The labeling was prevented by brefeldin A and ilimaquinone, and it appears to take place in the Golgi apparatus. This method allows selective labeling of ricin molecules that have already been transported retrograde to this organelle. A chain containing C-terminal N-glycosylation sites became core glycosylated, indicating retrograde transport to the ER. In part of the toxin molecules, the A chain was released from the B chain and translocated to the cytosol. The finding that glycosylated A chain was present in the cytosol indicates that translocation takes place after transport of the toxin to the ER.

Diphtheria toxin binds to receptor-positive cells through its B-fragment, the toxin is then endocytosed, and the low pH in endosomes triggers the translocation of the enzymatically active A-fragment to the cytosol. A synchronous release of A-fragments into the cytosol can be induced by exposing cells with surface-bound toxin to low pH. We have used this protein translocation system to develop a novel method to study whether or not a protein is exposed to the cytosol. Protein farnesylation is a cytosolic modification signaled by a C-terminal CaaX motif, and to visualize the translocation process, we added a farnesylation signal to the toxin A-fragment. The A-fragment with an added CaaX motif was farnesylated within 1 h after exposure of cells with surface-bound toxin to low pH, and also A-fragment translocated from endosomes was quantitatively farnesylated. The results indicate that all cell-mediated reduction of the toxin implicates translocation of the A-fragment to the cytosol. The farnesylation was inhibited by lovastatin, the alkylating agent NEM, and the peptidomimetic farnesylation inhibitor B581. Farnesylated A-fragment partitioned preferentially into the detergent phase upon extraction with Triton X-114. Our data suggest that farnesylation of a CaaX tag is generally applicable as a cytosolic marker, and this strategy for monitoring protein transfer to the cytosol may have considerable potential for studying the transport to the cytosol of proteins added externally to cells.

Acidic fibroblast growth factor (aFGF) added externally to cells has been proposed to enter the nucleus and stimulate DNA synthesis, but it has remained controversial whether or not exogenous aFGF has the capability of crossing cellular membranes. To test this, a novel principle to study translocation of proteins to the cytosol was developed by fusing a C-terminal farnesylation signal, a CAAX tag (C = Cys, A = an aliphatic amino acid, and X = any amino acid), onto aFGF. Farnesylation is only known to occur in the cytosol and possibly in the nucleus. When incubated with NIH3T3 cells overnight, about one-third of the cell-associated, CAAX-tagged growth factor was farnesylated, indicating that efficient translocation had taken place. Binding to specific FGF receptors was required for translocation to occur. Part of the farnesylated growth factor was found in the nuclear fraction. The data indicate that CAAX-tagged aFGF added externally to cells is able to cross cellular membranes and enter the cytosol and the nucleus.

The reaction of chloromethylated poly(styrene-co-divinylbenzene) (1 wt.-% of divinylbenzene) with potassium superoxide in the presence of phase-transfer catalysts was studied. It was found that in dimethyl sulfoxide and in the presence of 18-crown-6, the hydroxymethylated polymer is the main product with a yield of 45%. In dimethylformamide the transformation of chloromethyl groups was found to be the highest, but only 60% of alcohol groups are present in the resulting product. In benzene the transformation reaches only 25%. With Bu4NI as catalyst in a mixture of solvents, the transformation of chloromethyl groups proceeds with 85% yield and the product contains 80% hydroxyl groups.

The induction of thymocyte apoptosis through the Nur77-mediated intrinsic pathway can be of physiological importance in the clonal deletion of autoreactive thymocytes during negative selection in the thymus and/or in thymocytes undergoing oncogenic transformation. Ionomycin treatment induces endogenous Nur77 expression as well as apoptosis and cytochrome c release in thymocytes. Here it is shown for the first time that in normal thymocytes undergoing apoptosis, ionomycin induces translocation of endogenous Nur77 not only to the nucleus, but also to mitochondria. Immunosuppressant FK506 inhibits Nur77 NBRE and NurRE binding activity but has no effect on thymocytes apoptosis, the subcellular localization of Nur77, or cytochrome c release. This indicates that thymocytes can undergo apoptosis through the intrinsic Nur77-mediated mitochondrial pathway and that the transactivation activity of Nur77 monomers or dimers is not necessary for thymocyte apoptosis.