Inhibition of mitogen-activated protein kinase kinase blocks T cell proliferation but does not induce or prevent anergy

Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes, the extracellular signal-regulated kinase (ERK), Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation, we used the inhibitor U0126, which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK), the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs, but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy, unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly, induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK, unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term, but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants.     

Hepatocyte growth factor/scatter factor and hepatocytes are potent downregulators of tyrosinase expression in B16 melanoma cells

A large-scale procedure was developed for the anaerobic purification of the human recombinant Ca2+- and Zn2+-binding protein S100A3 for spectroscopic studies. S100A3 eluted as a non-covalently bound dimer (20.8 kDa). It contained 7.5+/-0.1 free thiol groups/monomer, and bound Ca2+ with a Kd of approximately 4 mM, which corresponds to a tenfold increase in affinity compared to the aerobically purified protein. The transition metal ions Co2+, Zn2+ and Cd2+ were used as spectroscopic probes to investigate the role of the 10 cysteine residues per monomer S100A3 in metal binding. Spectrophotometric titrations suggest the formation of dinuclear thiolate-bridged clusters consisting of a Me2+(S(Cys))4 and a Me2+(S(Cys))3(N(His)) site as described for zinc finger proteins. A three-dimensional structural model of S100A3 was proposed on the basis of the NMR structure of the structurally related rabbit S100A6 protein, and taking into account the structural influence of cysteine residues.     

Inhibition of caspase activity prevents anti-IgM induced apoptosis but not ceramide generation in WEHI 231 B cells

In apoptosis induced by Reaper in Drosophila, as well as in a number of other systems, it has been suggested that the increased synthesis of ceramide might be a consequence of the activation of the caspase/ICE (Interleukin-1beta converting enzyme) protease pathway involved in cell death, implying that ceramide generation might often be the result rather than the cause of apoptosis. WEHI 231 B cells have previously been shown to undergo apoptosis following exposure to exogenous ceramide and to produce increased amounts of ceramide in response to anti-IgM crosslinking. We show here that in WEHI 231 cells a peptide inhibitor of caspase activity blocks cell death in response to both anti-IgM and exogenous ceramide. However, the induction of ceramide synthesis by WEHI 231 cells in response to anti-IgM crosslinking is not blocked by this peptide. These results indicate that antigen receptor induced ceramide generation in WEHI 231 cells does not require caspase activation, and support the view that ceramide generation in immature B cells may be the cause rather than the consequence of activation of the caspase dependent death pathway.     

Biological role of tyrosinase related protein and its biosynthesis and transport from TGN to stage I melanosome, late endosome, through gene transfection study

OBJECTIVES: To examine the use of tomography for dental implant planning. METHODS: A questionnaire was sent to oral radiology clinics in Sweden and to implantology clinics in different parts of the world with questions on selection criteria and techniques for, and frequency of, pre-implant tomography. Differences between mean values were assessed by t-test. A new method developed by the Swedish Radiation Protection Institute was used to assess radiation absorbed dose from CT. RESULTS: Tomography was used by 93.4% of the clinics, but there was marked variation both between and within different clinical situations. It was performed in all cases by 21% and the majority used it for the evaluation of the maxilla, the posterior mandible and in single implant cases. Small clinics (< 100 patients per year) used tomography frequently and clinics in Sweden significantly more often than those in other countries. The majority had changed their policy recently, using tomography more often. CT was used by 73% of respondents, mainly the small clinics. The majority of the large clinics (> 500 patients per year) used conventional tomography. The mean absorbed dose for CT scanning protocols was 65 mGy. The variation within and between different makes of CT was considerable. CONCLUSIONS: There are large variations in frequency of use of both conventional and computed tomography for dental implant planning by different clinics who also vary in the indications for their choice. A substantial factor influencing the technique chosen was its availability rather than clinical need.     

Ligation of CD5 on resting B cells, but not on resting T cells, results in apoptosis

The CD5 molecule is expressed by a B cell subset. We have demonstrated that resting B cells do not proliferate in response to CD5 ligation, whereas cells preactivated with anti-IgM and IL-2 do so. Here, we specifically studied the effects of anti-CD5 and anti-IgM on apoptosis of CD5+ B cells. Both ligation of CD5 or of surface IgM (sIgM) resulted in apoptosis. This started earlier following ligation of CD5 than with sIgM, and both responses were time dependent. CD5-induced apoptosis was independent of the epitope recognized or the way the antibody was presented to the B cells. CD5+ B cells were more sensitive to IgM-induced apoptosis than CD5 B cells. Engagement of CD5 or CD3 expressed by T cells failed to induce apoptosis. Our data indicate differences in the function of CD5 molecules on tonsillar B cells, compared with blood T cells and suggest that cross-linking CD5 on B cell activates specific pathways responsible for apoptosis.     

An activating mutation in the kit receptor abolishes the stroma requirement for growth of ELM erythroleukemia cells, but does not prevent their differentiation in response to erythropoietin

We have previously shown that murine ELM erythroleukemia cells can only be grown in vitro in the presence of a stromal feeder layer, or alternatively stem cell factor (SCF), without which they differentiate. When grown in the presence of SCF, ELM cells can still differentiate in response to erythropoietin (Epo), but growth on stroma prevents this. We previously isolated a stroma-independent ELM variant, ELM-I-1, that is also defective in Epo-induced differentiation. We show here that this variant has an activating mutation in the Kit receptor, converting aspartic acid 814 to histidine. Expression of the mutant receptor in stroma-dependent ELM-D cells causes growth factor-independent proliferation and also gives the cells a selective advantage, in terms of proliferation rate and clonegenicity, compared with ELM-D cells grown in optimal amounts of SCF. Expression of the mutant receptor in ELM-D cells also prevents spontaneous differentiation, but not differentiation induced by Epo. Analysis of mitogenic signaling pathways in these cells shows that the mutant receptor induces constitutive activation of p42/p44 mitogen-activated protein kinases. It also selectively inhibits the expression of p66Shc but not the p46/p52 Shc isoforms (as did treatment of ELM cells with SCF), which is of interest, because p66Shc is known to play an inhibitory role in growth factor signaling.     

Molecular interactions within the melanogenic complex: formation of heterodimers of tyrosinase and TRP1 from B16 mouse melanoma

Melanin synthesis in mammals is catalyzed by three structurally related, membrane-bound proteins, tyrosinase, and the tyrosinase-related proteins 1 and 2 (TRP1 and TRP2). Current evidence suggests that in vivo these proteins may form a multienzyme complex. However, neither the precise composition of the complex, nor the specific interactions between its components have been characterized. This study used purified preparations of tyrosinase and TRP1 to analyze their interactions in non ionic detergent solution. Purified tyrosinase and TRP1 behaved as homodimers as judged by gel filtration chromatography and electrophoresis. Upon mixing of the purified proteins, the preferential formation of heterodimers was detected by: i) coelution in gel filtration chromatography with a shift to a common partition coefficient for both proteins, and ii) the occurrence of fluorescent energy transfer between fluorescein-labeled tyrosinase and rhodamine-labeled TRP1. However, the formation of heterodimers did not cause changes in the tyrosine hydroxylase activity of the enzymes, at least under standard assay conditions. Thus, tyrosinase and TRP1 interact strongly and specifically in detergent solution to form an heterodimer that might contribute to the formation of the melanogenic complex.     

Overexpression of Bcl-2 does not rescue impaired B lymphopoiesis in IL-7 receptor-deficient mice but can enhance survival of mature B cells

IL-7 receptor-deficient (IL-7R(-/-)) mice are lymphopenic as a result of defective cell production at early steps in both B and T lymphopoiesis. In the bone marrow, there is an incomplete block in B cell development at the transition from the pro-B to the pre-B cell stage. As a consequence, peripheral lymphoid organs of IL-7R(-/-) mice contain abnormally low numbers of mature surface (s) Ig-expressing B cells and this is accompanied by a relative increase in immature sIg- B cells. Transgenic expression of the anti-apoptotic protein Bcl-2 in IL-7R(-/-) mice rescues the defect in T cell development and in mature T cell function. The present report shows that constitutive expression of Bcl-2 is incapable of rescuing B lymphopoiesis in IL-7R(-/-) mice but can enhance survival of those mature B cells which escape the developmental arrest. Thus the essential role of IL-7R signaling in B lymphoid cells cannot be replaced by Bcl-2, indicating that in B lymphopoiesis IL-7R signaling is necessary for promoting cell division and/or for inhibiting a Bcl-2-insensitive pathway to apoptosis.     

Maternal-fetal T4 transfer does not suffice to prevent the effects of in utero hypothyroidism

It has been suggested recently that in congenitally hypothyroid infants with organification defect there is a maternal-fetal transfer of thyroxine (T4). The present study was conducted to evaluate how effective the maternal-fetal transfer is and whether the maternal T4 can prevent intrauterine hypothyroidism. The clinical, laboratory and radiological data on 271 full-term infants with persistent primary congenital hypothyroidism, detected by the national screening program, were used to assess the degree of in utero hypothyroidism. For 6 out of 50 athyroid infants, two pretreatment blood samples spotted on filter paper were available for calculating the T4 disappearance rate. Most infants with agenesis of the thyroid had very low T4 and very high levels of thyroid-stimulating hormone compared to infants with ectopic thyroid. In the athyroid infants the initial T4 declined to low and undetectable levels. Bone maturation was significantly delayed while the clinical symptomatology was more prominent in the athyroid congenital hypothyroid infants, as compared with the ectopic thyroid infants. In conclusion, there is some maternal-fetal transfer of T4. However, this transfer is insufficient to suppress the fetal levels of thyroid-stimulating hormone and prevent intrauterine hypothyroidism.     

Regulation of melanogenesis in B16 mouse melanoma cells by protein kinase C

Serotonergic neuronal networks are important for food intake and body weight regulation. Dexfenfluramine (dF), a serotonin releaser and reuptake inhibitor, was used to investigate changes in food intake, body weight development, energy expenditure, respiratory quotient, and substrate oxidation rates for 12 days. Rats, which had been made obese by early postnatal overfeeding, received an energy-controlled mash diet and water ad lib and were intraperitoneally injected daily with either saline, 5 or 10 mg dF/kg. Compared to controls, food intake, body weight development, and energy expenditure were decreased in a dose-dependent manner, especially during the first 6 days. Lipid oxidation was increased while oxidation of carbohydrates was decreased. Pair-feeding experiments over 2 days revealed that this was not solely a result of diminished food intake but also an additional metabolic effect of dF, different from its anorectic effect. At the end of these experiments, plasma glucose and liver glycogen were unchanged after dF, but plasma free fatty acids were significantly decreased. Insulin-sensitivity was probably improved, indicated by decreased insulin levels and increases in muscle glycogen contents and activities of muscle pyruvate kinase. Liver-glutamine and contents of valine, leucine, and isoleucine in the muscle were significantly decreased after dF-treatment, the latter indicating a diminished proteolysis. The plasma tryptophan/large neutral amino acids ratio of the dF-rats was unchanged but that of the paired-fed rats was changed, despite similar changes in food intake. It is concluded that both increased oxidation of endogenous fat and reduced food intake could mediate the body weight reducing effect of dF.     

Transforming growth factor-beta1 inhibits basal melanogenesis in B16/F10 mouse melanoma cells by increasing the rate of degradation of tyrosinase and tyrosinase-related protein-1

Current evidence suggests that melanogenesis is controlled by epidermal paracrine modulators. We have analyzed the effects of transforming growth factor-beta1 (TGF-beta1) on the basal melanogenic activities of B16/F10 mouse melanoma cells. TGF-beta1 treatment (48 h) elicited a concentration-dependent decrease in basal tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (Dopa) oxidase activities, to less than 30% of the control values but had no effect on dopachrome tautomerase activity (TRP-2). The inhibition affected to similar extents the Dopa oxidase activity associated to tyrosinase-related protein-1 (TRP-1) and tyrosinase. This inhibition was noticeable between 1 and 3 h after the addition of the cytokine, and maximal after 6 h of treatment. The decrease in the enzymatic activity was paralleled by a decrease in the abundance of the TRP-1 and tyrosinase proteins. TGF-beta1 mediated this effect by increasing the rate of degradation of tyrosinase and TRP-1. Conversely, after 48 h of treatment, the expression of the tyrosinase gene decreased only slightly, while TRP-1 and TRP-2 gene expression was not affected. An increased rate of proteolytic degradation of TRP-1 and tyrosinase seems the main mechanism accounting for the inhibitory effect of TGF-beta1 on the melanogenic activity of B16/F10 cells.     

Inhibition of VLA-4 and up-regulation of TIMP-1 expression in B16BL6 melanoma cells transfected with MHC class I genes

The effect of MHC class I gene transfection on the metastatic properties of B16BL6 melanoma cells was investigated. BL6-8 melanoma cells transfected with H-2Kb or H-2Kd, but not H-2Dd or H-2Ld, genes showed a dramatic reduction in their ability to generate experimental metastases in immunosuppressed CB6F1 mice. This observation suggested that some changes in the metastatic phenotype may have been induced in the H-2K- transfected melanoma cells. Analyses of adhesive and invasive properties of BL6-8 melanoma cells transfected with H-2 class I genes have been performed. We found that the loss of metastatic properties in the H-2Kb or H-2Kd gene-transfected melanoma cells was associated with reduced adherence to endothelial cells, laminin and collagen IV, decreased ability to form homotypic cell aggregates and with a complete loss of VLA-4 integrin expression. In addition, BL6-8 melanoma cells transfected with H-2K genes demonstrated reduced ability to invade Matrigel that paralleled up-regulation of TIMP-1 expression. Incubation of untransfected BL6-8 clone or B16F1 cells with 5-azacytidine similarly resulted in up-regulation of TIMP-1, suggesting that the changes in methylation of TIMP-1 gene could be responsible for TIMP-1 expression in the H-2K-transfected BL6-8 melanoma cells. Transfection of BL6-8 cells with the H-2Dd/Ld genes did not affect their adhesive and invasive properties. Previously we reported that reduction in the metastatic properties of the H-2Kb transfected cells was associated with alterations in cell surface carbohydrates with appearance of alpha-galactosyl epitopes and reduction in cell surface sialylation. The present data indicate that, in addition to changes in cell surface carbohydrates, reduction in adhesive properties and up-regulation of TIMP-1 may be responsible for the observed loss of metastatic potential of BL6-8 cells transfected with the H-2K genes.     

Exposure of macrophage-like cells to titanium particles does not affect bone resorption, but inhibits bone formation

Endocrine tumours of the pancreas, even in case of liver involvement, are generally characterized by a slower evolution and a better prognosis, if compared with ductal carcinoma. This fact gives reason to a radical surgical approach, whenever possible, and to the research of any effective adjuvant treatment. For this purpose, hepatic transarterial chemoembolization (TACE) has been proposed in recent years for the treatment of metastatic endocrine tumours. Out of 80 patients suffering from endocrine tumours of the pancreas, observed between January 1985 and December 1996, 28 (35%) presented liver metastases at the time of diagnosis. Twelve of these patients were submitted to palliative resection of pancreatic tumour and one or more cycles of TACE. Overall survival was 50% (6/12); median survival was 35.4 months (range 4-75). These results suggest that chemoembolization, combined with surgical resection of primary malignancy, appears to be able to control the disease for a certain time and to increase the survival rate.     

Dendritic cells but not B cells present antigenic complexes to class II-restricted T cells after administration of protein in adjuvant

We have analyzed the relative contribution of dendritic cells (DC) and B cells in the presentation of peptide-class II complexes in an inflammatory situation in vivo. Draining lymph node cells from mice immunized subcutaneously with hen egg-white lysozyme (HEL) in adjuvant display HEL peptide-major histocompatibility complex class II complexes able to stimulate, in the absence of any further antigen addition, specific T hybridoma cells. The antigen-presenting capacity of three different antigen-presenting cell (APC) populations recruited in lymph nodes, DC (N418+, class II+, B220-, low buoyant density), large B cells (B220+, low buoyant density), and small B cells (B220+, high buoyant density), was analyzed. After immunization with HEL in adjuvant, DC are the only lymph node APC population expressing detectable HEL peptide-class II complexes. These results indicate that lymph node DC and not B cells are the APC initiating the immune response in vivo after administration of antigen in adjuvant.     

Spatial disorientation blocks reliable goal location on a plus maze but does not prevent goal location in the Morris maze.

Cue control in spatial learning was investigated in a plus maze and a Morris maze. Rats transported in opaque containers with prior rotation to a plus maze, but not a Morris maze, could not find a goal defined by external cues. Rats transported in clear containers without rotation found the goal in both mazes. In the Morris maze, goal location was readily relearned following cue removal by rats in clear containers but not by rats in the opaque/rotation group. B. L. McNaughton et al's (1996) theory that during spatial learning sensory information is bound to preconfigured internal maps in the hippocampus, whose metric is self-notion and whose orientation depends on input from an inertial based head direction system, may explain this study's findings. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Stimulation of the protein tyrosine kinase c-Yes but not c-Src by neurotrophins in human brain-metastatic melanoma cells

The c-Yes proto-oncogene (pp62c-Yes) encodes a non-receptor-type protein tyrosine kinase (NRPTK) of the Src family. c-Yes activities and protein levels are elevated in human melanoma and melanocyte cell lines. Because the neurotrophins (NT) are important in the progression of melanoma to the brain-metastatic phenotype, we determined whether NT stimulate c-Yes activity in human MeWo melanoma cells and two variant sublines with opposite metastatic capabilities, 3 S 5 and 70W. The highly brain-metastatic 70W subline had an intrinsically higher c-Yes activity than parental MeWo or poorly metastatic 3 S 5 cells. c-Yes kinase was further induced by the prototypic human NT, nerve growth factor (NGF) in a dose and time-dependent manner. In contrast, c-Src activity (pp60-Src) was similar in all these cells and unaffected by NGF exposure. Additionally, human NGF and neurotrophin-3 stimulated c-Yes in brain-metastatic 70W cells. The magnitude of c-Yes activation correlated with the degree of invasion of 70 W cells following incubation of these neurotrophins. To further examine NT stimulation of c-Yes in melanoma cells, three additional cell lines were examined. Metastatic TXM-13 and TXM-18 increased c-Yes activity in response to NGF. In contrast, no increase was observed in low-metastatic TXM-40 cells. Together, these data suggest that altered c-Yes expression may play a role in the malignant progression of the human melanocyte towards the brain-metastatic phenotype and that NT enhance the activity of c-Yes in signaling penetration into the matrix of NT-rich stromal microenvironments such as the brain.     

Effect induced by interleukin-1 on the behaviour of B16F10 melanoma cells

Ethanol caused a concentration-dependent loss of PC12 cells over a 24 h interval, accompanied by an increase in intracellular calcium. The specific alpha7 nicotinic receptor partial agonist DMXB attenuated both of these ethanol-induced actions at a concentration (3 microM) found previously to protect against apoptotic and necrotic cell loss. The alpha7 nicotinic receptor antagonist methylylaconitine blocked the neuroprotective action of DMXB when applied with but not 30 min after the agonist. These results indicate that activation of alpha7 nicotinic receptors may be therapeutically useful in preventing ethanol-neurotoxicity.     

Local anesthetic treatment significantly attenuates acute pain responding but does not prevent the neonatal injury-induced reduction in adult spinal behavioral plasticity.

Recent findings indicate that neonatal injury results in decreased spinal plasticity in adult subjects (E. E. Young, K. M. Baumbauer, A. E. Elliot, & R. L. Joynes, 2007). Previous research has shown that acute manipulations of pain processing (i.e., administration of formalin, carrageenan, capsaicin) result in a loss of spinal behavioral plasticity (A. R. Ferguson, E. D. Crown, & J. W. Grau, 2006). Moreover, neonatal injury results in a lasting reduction in adult spinally mediated plasticity resembling the deficit seen following acute manipulations in adults (E. E. Young et al., 2007). The present study was designed to determine whether the effects of neonatal injury could be prevented by lidocaine administration during the initial healing period. Subjects (injured or uninjured) received lidocaine or saline on 1 of 4 administration schedules (preinjury only, postinjury only, for 24 hr postsurgery, or for 72 hr postsurgery). Results demonstrated that lidocaine administration did not prevent the hypersensitivity and reduced spinal plasticity associated with neonatal injury. This suggests that (a) the mechanisms underlying neonatal injury are independent of peripheral input in the initial healing period and (b) lidocaine is ineffective at preventing long-term spinal plasticity changes following neonatal injury. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Transforming growth factor-alpha expands progenitor cells of the basal forebrain, but does not promote cholinergic differentiation

Specific transport activities package classical neurotransmitters into secretory vesicles for release by regulated exocytosis, but the proteins responsible for the vesicular transport of neurotransmitters are still being identified. One family of proteins includes vesicular transporters for monoamines and acetylcholine. Genetic manipulation in cells and in mice now shows that changes in the expression of these proteins can alter the amount of neurotransmitter stored per synaptic vesicle, the amount released and behavior. Although the mechanisms responsible for regulating these transporters in vivo remains unknown, recent work has demonstrated the potential for regulation by changes in intrinsic activity and in location. In addition, a recently identified vesicular transporter for GABA defines a novel family of proteins that mediates the packaging of amino acid neurotransmitters.