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1.
Health Qual Life Outcomes ; 10: 155, 2012 Dec 27.
Article in English | MEDLINE | ID: mdl-23270428

ABSTRACT

BACKGROUND: Natalizumab (Tysabri, Biogen Idec and Elan Pharmaceuticals) significantly reduces the relapse rate and disability progression, and improves health-related quality of life (HRQoL), in patients with relapsing-remitting multiple sclerosis. We investigated the impact of natalizumab on patient-reported outcomes (PROs) in a real-world setting. METHODS: PRO data were collected from patients enrolled in a longitudinal real-world study using validated measures administered as surveys before the patients initiated natalizumab treatment and after the 3rd, 6th, and 12th monthly infusion. HRQoL, ability to carry out daily activities, disability level, and impact on cognitive functioning and fatigue were assessed. RESULTS: A total of 333 patients completed 12 months of assessments. After 12 months of natalizumab treatment, 69% to 88% of patients reported a positive outcome (either an improvement or no further decline) in all PRO measures assessed. Significant improvements in general and disease-specific HRQoL were observed after three infusions, both with physical (p < .01) and psychological (p < .001) measures, and were sustained after 12 infusions (all p < .001). The impact of multiple sclerosis on cognitive functioning and fatigue was significantly reduced (both p < .001 after 3 and 12 infusions). CONCLUSIONS: PRO measures were improved with natalizumab in a real-world setting. The improvements were observed as early as after 3 months and sustained over a 12-month period. The improvements in PROs show that, in clinical practice, the clinical benefits of natalizumab are translated into patient-reported benefits.


Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Multiple Sclerosis/drug therapy , Outcome Assessment, Health Care , Self Report , Adult , Female , Humans , Longitudinal Studies , Male , Middle Aged , Natalizumab
2.
Mol Membr Biol ; 28(7-8): 473-86, 2011.
Article in English | MEDLINE | ID: mdl-22034844

ABSTRACT

Lck is a non-receptor tyrosine kinase of the Src family that is essential for T cell activation. Dual N-terminal acylation of Lck with myristate (N-acylation) and palmitate (S-acylation) is essential for its membrane association and function. Reversible S-acylation of Lck is observed in vivo and may function as a control mechanism. Here we identify the DHHC family protein S-acyltransferase DHHC2 as an enzyme capable of palmitoylating of Lck in T cells. Reducing the DHHC2 level in Jurkat T cells using siRNA causes decreased Lck S-acylation and partial dislocation from membranes, and conversely overexpression of DHHC2 increases S-acylation of an Lck surrogate, LckN10-GFP. DHHC2 localizes primarily to the endoplasmic reticulum and Golgi apparatus suggesting that it is involved in S-acylation of newly-synthesized or recycling Lck involved in T cell signalling.


Subject(s)
Acyltransferases/metabolism , Endoplasmic Reticulum/enzymology , Golgi Apparatus/enzymology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , T-Lymphocytes/enzymology , Tumor Suppressor Proteins/metabolism , Acylation , Acyltransferases/chemistry , Endoplasmic Reticulum/chemistry , Gene Expression , Golgi Apparatus/chemistry , HEK293 Cells , HeLa Cells , Humans , Jurkat Cells , Lipoylation , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/chemistry , Myristic Acid/chemistry , Myristic Acid/metabolism , Palmitates/chemistry , Palmitates/metabolism , RNA, Small Interfering/genetics , Tumor Suppressor Proteins/chemistry
3.
Curr Protoc Protein Sci ; Chapter 14: Unit 14.2, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19235135

ABSTRACT

Proteins can be acylated with a variety of fatty acids attached by different covalent bonds, influencing, among other things, their function and intracellular localization. This unit describes methods to analyze protein acylation, both levels of acylation and also the identification of the fatty acid and the type of bond present in the protein of interest. Protocols are provided for metabolic labeling of proteins with tritiated fatty acids, for exploitation of the differential sensitivity to cleavage of different types of bonds, in order to distinguish between them, and for thin-layer chromatography to separate and identify the fatty acids associated with proteins.


Subject(s)
Fatty Acids/metabolism , Proteins/metabolism , Acylation , Animals , Biochemistry/methods , Cells, Cultured , Chromatography, Thin Layer , Fatty Acids/analysis , Humans , Isotope Labeling , Mammals/metabolism , Protein Binding , Proteins/chemistry
4.
J Neurooncol ; 91(1): 19-26, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18759130

ABSTRACT

PURPOSE: To interrogate grade II, III, and IV gliomas and characterize the critical effectors within the PI3-kinase pathway upstream and downstream of mTOR. Experimental design Tissues from 87 patients who were treated at UCSF between 1990 and 2004 were analyzed. Twenty-eight grade II, 17 grade III glioma, 26 grade IV gliomas, and 16 non-tumor brain specimens were analyzed. Protein levels were assessed by immunoblots; RNA levels were determined by polymerase chain reaction amplification. To address the multiple comparisons, first an overall analysis was done comparing the four groups using Spearman's Correlation Coefficient. Only if this analysis was statistically significant were individual pairwise comparisons done. RESULTS: Multiple comparison analyses revealed a significant correlation with grade for all variables examined, except phosphorylated-S6. Expression of phosphorylated-4E-BP1, phosphorylated-PKB/Akt, PTEN, TSC1, and TSC2 correlated with grade (P < 0.01 for all). We extended our analyses to ask whether decreases in TSC proteins levels were due to changes in mRNA levels, or due to changes in post-transcriptional alterations. We found significantly lower levels of TSC1 and TSC2 mRNA in GBMs than in grade II gliomas or non-tumor brain (P < 0.01). CONCLUSIONS: Expression levels of critical signaling molecules upstream and downstream of mTOR differ between non-tumor brain and gliomas of any grade. The single variable whose expression did not differ between non-tumor brain and gliomas was phosphorylated-S6, suggesting that other protein kinases, in addition to mTOR, contribute significantly to S6 phosphorylation. mTOR provides a rational therapeutic target in gliomas of all grades, and clinical benefit may emerge as mTOR inhibitors are combined with additional agents.


Subject(s)
Brain Neoplasms/genetics , Gene Expression Regulation, Neoplastic/genetics , Glioma/genetics , Phosphatidylinositol 3-Kinases/genetics , Protein Kinases/metabolism , Signal Transduction/genetics , Brain Neoplasms/diagnosis , Brain Neoplasms/metabolism , Glioma/diagnosis , Glioma/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinases/genetics , Retrospective Studies , Statistics, Nonparametric , TOR Serine-Threonine Kinases
5.
Mol Membr Biol ; 26(1): 32-41, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19115143

ABSTRACT

Many proteins are S-acylated, affecting their localization and function. Dynamic S-acylation in response to various stimuli has been seen for several proteins in vivo. The regulation of S-acylation is beginning to be elucidated. Proteins can autoacylate or be S-acylated by protein acyl transferases (PATs). Deacylation, on the other hand, is an enzymatic process catalyzed by protein thioesterases (APT1 and PPT1) but only APT1 appears to be involved in the regulation of the reversible S-acylation of cytoplasmic proteins seen in vivo. PPT1, on the other hand, is involved in the lysosomal degradation of S-acylated proteins and PPT1 deficiency causes the disease infant neuronal ceroid lipofuscinosis.


Subject(s)
Acyltransferases/metabolism , Proteins/metabolism , Thiolester Hydrolases/metabolism , Acylation , Humans
6.
J Biol Chem ; 280(18): 17910-9, 2005 May 06.
Article in English | MEDLINE | ID: mdl-15718244

ABSTRACT

We have shown previously that protein kinase C (PKC) epsilon can induce neurite outgrowth independently of its catalytic activity via a region encompassing its C1 domains. In this study we aimed at identifying specific amino acids in this region crucial for induction of neurite outgrowth. Deletion studies demonstrated that only 4 amino acids N-terminal and 20 residues C-terminal of the C1 domains are necessary for neurite induction. The corresponding regions from all other novel isoforms but not from PKCalpha were also neuritogenic. Further mutation studies indicated that amino acids immediately N-terminal of the C1a domain are important for plasma membrane localization and thereby for neurite induction. Addition of phorbol ester made this construct neurite-inducing. However, mutation of amino acids flanking the C1b domain reduced the neurite-inducing capacity even in the presence of phorbol esters. Sequence alignment highlighted an 8-amino acid-long sequence N-terminal of the C1b domain that is conserved in all novel PKC isoforms. Specifically, we found that mutations of either Phe-237, Val-239, or Met-241 in PKCepsilon completely abolished the neurite-inducing capacity of PKCepsilon C1 domains. Phorbol ester treatment could not restore neurite induction but led to a plasma membrane translocation. Furthermore, if 12 amino acids were included N-terminal of the C1b domain, the C1a domain was dispensable for neurite induction. In conclusion, we have identified a highly conserved sequence N-terminal of the C1b domain that is crucial for neurite induction by PKCepsilon, indicating that this motif may be critical for some morphological effects of PKC.


Subject(s)
Amino Acids/chemistry , Conserved Sequence , Neurites/enzymology , Peptide Fragments/chemistry , Protein Kinase C/chemistry , Protein Kinase C/physiology , Amino Acid Sequence , Amino Acids/genetics , Amino Acids/physiology , Animals , Cell Line, Tumor , Humans , Isoenzymes/chemistry , Isoenzymes/physiology , Molecular Sequence Data , Neurites/physiology , Peptide Fragments/genetics , Peptide Fragments/physiology , Point Mutation , Protein Kinase C/genetics , Protein Kinase C-epsilon , Protein Structure, Tertiary/genetics
7.
Exp Cell Res ; 292(1): 135-50, 2004 Jan 01.
Article in English | MEDLINE | ID: mdl-14720513

ABSTRACT

We have shown that protein kinase C (PKC) epsilon, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKCepsilon resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKCepsilon-overexpressing cells and led to neurite induction in cells overexpressing PKCdelta. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCdelta induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCdelta-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCdelta can be counteracted by PKCdelta kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth.


Subject(s)
Isoenzymes/chemistry , Neurites/physiology , Neurons/physiology , Protein Kinase C/chemistry , rhoA GTP-Binding Protein/metabolism , Actins/metabolism , Amides/pharmacology , Catalysis , Cell Line , Enzyme Inhibitors/pharmacology , Green Fluorescent Proteins , Humans , Indoles/pharmacology , Isoenzymes/genetics , Isoenzymes/metabolism , Luminescent Proteins/metabolism , Maleimides/pharmacology , Models, Biological , Neurites/drug effects , Neuroblastoma/pathology , Neurons/drug effects , Phorbol Esters/pharmacology , Protein Kinase C/genetics , Protein Kinase C/metabolism , Protein Kinase C/pharmacology , Protein Structure, Tertiary , Pyridines/pharmacology , Recombinant Fusion Proteins/metabolism , Stress Fibers/drug effects , Stress Fibers/metabolism , Time Factors , Transfection , Tumor Cells, Cultured
8.
Mol Biol Cell ; 13(1): 12-24, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11809819

ABSTRACT

We have previously shown that protein kinase Cepsilon (PKCepsilon) induces neurite outgrowth via its regulatory domain and independently of its kinase activity. This study aimed at identifying mechanisms regulating PKCepsilon-mediated neurite induction. We show an increased association of PKCepsilon to the cytoskeleton during neuronal differentiation. Furthermore, neurite induction by overexpression of full-length PKCepsilon is suppressed if serum is removed from the cultures or if an actin-binding site is deleted from the protein. A peptide corresponding to the PKCepsilon actin-binding site suppresses neurite outgrowth during neuronal differentiation and outgrowth elicited by PKCepsilon overexpression. Neither serum removal, deletion of the actin-binding site, nor introduction of the peptide affects neurite induction by the isolated regulatory domain. Membrane targeting by myristoylation renders full-length PKCepsilon independent of both serum and the actin-binding site, and PKCepsilon colocalized with F-actin at the cortical cytoskeleton during neurite outgrowth. These results demonstrate that the actin-binding site is of importance for signals acting on PKCepsilon in a pathway leading to neurite outgrowth. Localization of PKCepsilon to the plasma membrane and/or the cortical cytoskeleton is conceivably important for its effect on neurite outgrowth.


Subject(s)
Actins/metabolism , Isoenzymes/chemistry , Isoenzymes/metabolism , Neurites/physiology , Protein Kinase C/chemistry , Protein Kinase C/metabolism , Binding Sites , Cell Differentiation/physiology , Cytoskeleton/metabolism , Humans , Image Cytometry , Isoenzymes/genetics , Microscopy, Confocal , Microscopy, Fluorescence , Neuroblastoma , Protein Conformation , Protein Kinase C/genetics , Protein Kinase C-epsilon , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Transfection , Tumor Cells, Cultured
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