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1.
Clin Case Rep ; 7(11): 2156-2164, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31788270

ABSTRACT

A 60-year-old woman with stage IV rectal cancer received adoptive cell therapy with autologous cancer antigen (AC-ACT) causing induction of anti-oncogenic and anti-PD-L1 miRNAs as assessed by miRNA microarray. More than 1 year after AC-ACT, metastases have been arrested, and the patient reports good quality of life.

2.
J AIDS Clin Res ; 5(11)2014 Nov 08.
Article in English | MEDLINE | ID: mdl-25750760

ABSTRACT

BACKGROUND: Clinical characteristics of HIV-1 infection in people inhabiting Western, Sub-Saharan African, and South-East Asian countries are well recognized. However, very little information is available with regard to HIV-1 infection and treatment outcome in MENA countries including the Gulf Cooperation Council (GCC) states. METHODS: Clinical, demographic and epidemiologic characteristics of 602 HIV-1 infected patients followed in the adult Infectious Diseases Clinic of King Faisal Specialist Hospital and Research Centre, in Riyadh, Kingdom of Saudi Arabia a tertiary referral center were longitudinally collected from 1989 to 2010. RESULTS: Of the 602 HIV-1 infected patients in this observation period, 70% were male. The major mode of HIV-1 transmission was heterosexual contact (55%). At diagnosis, opportunistic infections were found in 49% of patients, most commonly being pneumocysitis. AIDS associated neoplasia was also noted in 6% of patients. A hundred and forty-seven patients (24%) died from the cohort by the end of the observation period. The mortality rate peaked in 1992 at 90 deaths per 1000 person-year, whereas the mortality rate gradually decreased to <1% from 1993-2010. In 2010, 71% of the patients were receiving highly active retroviral therapy. CONCLUSIONS: These data describe the clinical characteristic of HIV-1-infected patients at a major tertiary referral hospital in KSA over a 20-year period. Initiation of antiretroviral therapy resulted in a significant reduction in both morbidity and mortality. Future studies are needed in the design and implementation of targeted treatment and prevention strategies for HIV-1 infection in KSA.

3.
BMC Cancer ; 13: 289, 2013 Jun 14.
Article in English | MEDLINE | ID: mdl-23768049

ABSTRACT

BACKGROUND: Accumulating evidence supports cancer to initiate and develop from a small population of stem-like cells termed as cancer stem cells (CSC). The exact phenotype of CSC and their counterparts in normal mammary gland is not well characterized. In this study our aim was to evaluate the phenotype and function of stem/progenitor cells in normal mammary epithelial cell populations and their malignant counterparts. METHODS: Freshly isolated cells from both normal and malignant human breasts were sorted using 13 widely used stem/progenitor cell markers individually or in combination by multi-parametric (up to 9 colors) cell sorting. The sorted populations were functionally evaluated by their ability to form colonies and mammospheres, in vitro. RESULTS: We have compared, for the first time, the stem/progenitor markers of normal and malignant breasts side-by-side. Amongst all markers tested, we found CD44high/CD24low cell surface marker combination to be the most efficient at selecting normal epithelial progenitors. Further fractionation of CD44high/CD24low positive cells showed that this phenotype selects for luminal progenitors within Ep-CAMhigh/CD49f + cells, and enriches for basal progenitors within Ep-CAM-/low/CD49f + cells. On the other hand, primary breast cancer samples, which were mainly luminal Ep-CAMhigh, had CD44high/CD24low cells among both CD49fneg and CD49f + cancer cell fractions. However, functionally, CSC were predominantly CD49f + proposing the use of CD44high/CD24low in combination with Ep-CAM/CD49f cell surface markers to further enrich for CSC. CONCLUSION: Our study clearly demonstrates that both normal and malignant breast cells with the CD44high/CD24low phenotype have the highest stem/progenitor cell ability when used in combination with Ep-CAM/CD49f reference markers. We believe that this extensive characterization study will help in understanding breast cancer carcinogenesis, heterogeneity and drug resistance.


Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/metabolism , Neoplastic Stem Cells/metabolism , Animals , Antigens, Neoplasm/analysis , Antigens, Neoplasm/biosynthesis , Breast Neoplasms/pathology , CD24 Antigen/analysis , CD24 Antigen/biosynthesis , Cell Adhesion Molecules/analysis , Cell Adhesion Molecules/biosynthesis , Epithelial Cell Adhesion Molecule , Female , Flow Cytometry , Humans , Hyaluronan Receptors/analysis , Hyaluronan Receptors/biosynthesis , Immunohistochemistry , Integrin alpha6/analysis , Integrin alpha6/biosynthesis , Mice , Neoplastic Stem Cells/pathology , Phenotype , Transcriptome , Transplantation, Heterologous
4.
Am J Kidney Dis ; 58(2): 186-95, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21658830

ABSTRACT

BACKGROUND: Accurate diagnosis of the primary cause of an individual's kidney disease can be essential for proper management. Some kidney diseases have overlapping histopathologic features despite being caused by defects in different genes. In this report, we describe 2 consanguineous Saudi Arabian families in which individuals presented with kidney failure and mixed clinical and histologic features initially believed to be consistent with focal segmental glomerulosclerosis. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: We studied members of 2 apparently unrelated families from Saudi Arabia with kidney disease. MEASUREMENTS: Whole-genome single-nucleotide polymorphism analysis followed by targeted isolation and sequencing of exons using genomic DNA samples from affected members of these families, followed by additional focused genotyping and sequence analysis. RESULTS: The 2 apparently unrelated families shared a region of homozygosity on chromosome 2q13. Exome sequence from affected individuals lacked sequence reads from the NPHP1 gene, which is located within this homozygous region. Additional polymerase chain reaction-based genotyping confirmed that affected individuals had NPHP1 deletions, rather than defects in a known focal segmental glomerulosclerosis-associated gene. LIMITATIONS: The methods used here may not result in a clear genetic diagnosis in many cases of apparent familial kidney disease. CONCLUSIONS: This analysis shows the power of new high-throughput genotyping and sequencing technologies to aid in the rapid genetic diagnosis of individuals with an inherited form of kidney disease. We believe it is likely that such tools may become useful clinical genetic tools and alter the manner in which diagnoses are made in nephrology.


Subject(s)
Consanguinity , Homozygote , Kidney Diseases/diagnosis , Kidney Diseases/genetics , Adolescent , Adult , Child , Female , Humans , Male , Pedigree , Sequence Analysis, DNA
5.
J Cell Mol Med ; 15(1): 86-93, 2011 Jan.
Article in English | MEDLINE | ID: mdl-19818099

ABSTRACT

HTm4 (MS4A3) is a member of a family of four-transmembrane proteins designated MS4A. MS4A proteins fulfil diverse functions, acting as cell surface signalling molecules and intracellular adapter proteins. Early reports demonstrated that HTm4 is largely restricted to the haematopoietic lineage, and is involved in cell cycle control, via a regulatory interaction with the kinase-associated phosphatase, cyclin A and cyclin-dependent kinase 2 (CDK2). Here we describe the expression pattern of HTm4 in peripheral blood cells using gene expression microarray technology, and in normal foetal and adult human tissues, as well as adult human cancers, using tissue microarray technology. Using oligonucleotide microarrays to evaluate HTm4 mRNA, all peripheral blood cell types demonstrated very low levels of HTm4 expression; however, HTm4 expression was greatest in basophils compared to eosinophils, which showed lower levels of HTm4 expression. Very weak HTm4 expression is found in monocytes, granulocytes and B cells, but not in T cells, by lineage specific haematopoietic cell flow cytometry analysis. Interestingly, phytohaemagglutinin stimulation increases HTm4 protein expression in peripheral blood CD4-T-lymphocytes over nearly undetectable baseline levels. Western blotting and immunohistochemical studies show strong HTm4 expression in the developing haematopoietic cells of human foetal liver. Immunohistochemical studies on normal tissue microarrays confirmed HTm4 expression in a subset of leucocytes in nodal, splenic tissues and thymic tissue, and weak staining in small numbers of cell types in non-haematopoietic tissues. Human foetal brain specimens from 19 to 31 gestational weeks showed that the strongest-staining cells are ventricular zone cells and the earliest-born, earliest-differentiating 'pioneer' neurons in the cortical plate, Cajal-Retzius and, to a lesser extent, subplate-like neurons. Malignant tissue microarray analysis showed HTm4 expression in a wide variety of adenocarcinomas, including breast, prostate and ovarian. These findings warrant the further study of the role of HTm4 in the cell cycle of both haematopoietic and tumour cells.


Subject(s)
Blood Cells/metabolism , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinase 2/metabolism , Membrane Proteins/metabolism , Neoplasms/metabolism , Blotting, Western , Cell Cycle Proteins/genetics , Cell Differentiation/drug effects , Cells, Cultured , Fetus/cytology , Fetus/drug effects , Fetus/metabolism , Flow Cytometry , Humans , Lung/cytology , Lung/metabolism , Mast Cells/metabolism , Membrane Proteins/genetics , Mitogens/pharmacology , Neoplasms/genetics , Neoplasms/pathology , Phytohemagglutinins/pharmacology , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
6.
Transplantation ; 90(2): 184-8, 2010 Jul 27.
Article in English | MEDLINE | ID: mdl-20463642

ABSTRACT

BACKGROUND: Transplant tourism is the term used for patients who travel abroad for transplantation. Transplant tourism has always been surrounded with controversy regarding how these organs were obtained, the donor's care after transplantation, and the recipient outcome. Many authors have found that the outcome of the recipients in transplant tourism is inferior to those transplanted in their own countries. However, most these studies were small, with the latest one including only 33 patients. Here, we describe the outcome of 93 patients who were transplanted abroad compared with local transplantation. MATERIAL AND METHODS: All transplant patients who were followed up at our Nephrology Clinic from 1998 until 2008 were identified using our data base system. We selected patients transplanted from 2003 and forward because the computerized system for laboratory and electronic records began operation that year. RESULTS: A total of 165 patients were identified (93 in the tourist group and 72 in the local one). Transplant tourists had a higher rate of acute rejection in the first year compared with local transplantation (27.9% vs. 9.9, P=0.005), higher mean creatinine at 6 months and 1 year (120 vs. 101 micromol/L, P=0.0007, 113 vs. 98 micromol/L, P=0.008). There was no statistical difference in graft or patient survival in 1 or 2 years after transplantation. However, transplant tourist had a higher rate of cytomegalovirus infection (15.1% vs. 5.6%, P=0.05) and hepatitis C seroconversion (7.5% vs. 0%, P=0.02). CONCLUSION: Transplant tourists had a more complex posttransplantation course with higher incidence of acute rejection and infectious complications.


Subject(s)
Kidney Transplantation/statistics & numerical data , Medical Tourism/statistics & numerical data , Adult , Creatinine/blood , Cytomegalovirus Infections/epidemiology , Egypt , Female , Follow-Up Studies , Graft Rejection/epidemiology , Graft Survival , Hepatitis C/epidemiology , Humans , Immunosuppression Therapy/methods , Kidney Transplantation/adverse effects , Kidney Transplantation/mortality , Male , Middle Aged , Pakistan , Philippines , Renal Replacement Therapy/statistics & numerical data , Reoperation/statistics & numerical data , Survival Analysis , Tissue Donors/statistics & numerical data
7.
Cancer Genomics Proteomics ; 7(2): 93-103, 2010.
Article in English | MEDLINE | ID: mdl-20335524

ABSTRACT

BACKGROUND: The incidence of prostate cancer (PCa) has increased in recent years due to the aging of the population and increased testing; however, mortality rates have remained largely unchanged. Studies have shown deficiencies in predicting patient outcome for both of the major PCa diagnostic tools, namely prostate specific antigen (PSA) and transrectal ultrasound-guided biopsy. Therefore, serum biomarkers are needed that accurately predict prognosis of PCa (indolent vs. aggressive) and can thus inform clinical management. AIM: This study uses surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) mass spectrometry analysis to identify differential serum protein expression between PCa patients with indolent vs. aggressive disease categorised by Gleason grade and biochemical recurrence. MATERIALS AND METHODS: A total of 99 serum samples were selected for analysis. According to Gleason score, indolent (45 samples) and aggressive (54) forms of PCa were compared using univariate analysis. The same samples were then separated into groups of different recurrence status (10 metastatic, 15 biochemical recurrences and 70 non-recurrences) and subjected to univariate analysis in the same way. The data from Gleason score and recurrence groups were then analysed using multivariate statistical analysis to improve PCa biomarker classification. RESULTS: The comparison between serum protein spectra from indolent and aggressive samples resulted in the identification of twenty-six differentially expressed protein peaks (p<0.05), of which twenty proteins were found with 99% confidence. A total of 18 differentially expressed proteins (p<0.05) were found to distinguish between recurrence groups; three of these were robust with p<0.01. Sensitivity and specificity within the Gleason score group was 73.3% and 60% respectively and for the recurrence group 70% and 62.5%. CONCLUSION: SELDI-TOF-MS technology has facilitated the discovery of prognostic biomarkers in serum that can successfully discriminate aggressive from indolent PCa and also differentiate between recurrence groups.


Subject(s)
Biomarkers, Tumor/blood , Blood Proteins/analysis , Prostatic Neoplasms/blood , Prostatic Neoplasms/pathology , Proteomics/methods , Algorithms , Blood Proteins/chemistry , Disease Progression , Humans , Male , Molecular Weight , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Prostatic Neoplasms/classification , Recurrence , Reproducibility of Results , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
8.
Eur J Med Chem ; 45(6): 2689-94, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20226574

ABSTRACT

3-aminothiazolo[3,2-a]benzimidazole-2-carbonitrile (2) was prepared and upon hydrolysis using concentrated sulfuric acid or phosphoric acid resulted in the corresponding 3-aminothiazolo[3,2-a]benzimidazole-2-carboxamide derivative (3). Cyclization of the 2 using acetic anhydride or formic acid gave the corresponding pyrimido[4',5':4,5]thiazolo[3,2-a]benzimidazol-4(3H)-one (5) in good yields. Acetylation of 2 with acetic anhydride in pyridine afforded N-acetylaminothiazolo[3,2-a]benzimidazole-2-carbonitrile (6). In vitro antiproliferative activities of synthesized compounds were investigated at The National Cancer Institute (NCI), USA, according to their applied protocol. Compound 6 revealed significant antiproliferative activity, however, weak activity was shown by the other derivatives. Cell cycle disruption and apoptotic activity of 6 were studied, interestingly, 6 has the ability to arrest G2/M phase and it can induce apoptosis in time dependent manner.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Cell Cycle/drug effects , Nitriles/chemistry , Nitriles/pharmacology , Antineoplastic Agents/chemical synthesis , Benzimidazoles/chemical synthesis , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Nitriles/chemical synthesis
9.
J Immunol ; 183(2): 993-1004, 2009 Jul 15.
Article in English | MEDLINE | ID: mdl-19561093

ABSTRACT

Human clinical trials in type 1 diabetes (T1D) patients using mesenchymal stem cells (MSC) are presently underway without prior validation in a mouse model for the disease. In response to this void, we characterized bone marrow-derived murine MSC for their ability to modulate immune responses in the context of T1D, as represented in NOD mice. In comparison to NOD mice, BALB/c-MSC mice were found to express higher levels of the negative costimulatory molecule PD-L1 and to promote a shift toward Th2-like responses in treated NOD mice. In addition, transfer of MSC from resistant strains (i.e., nonobese resistant mice or BALB/c), but not from NOD mice, delayed the onset of diabetes when administered to prediabetic NOD mice. The number of BALB/c-MSC trafficking to the pancreatic lymph nodes of NOD mice was higher than in NOD mice provided autologous NOD-MSC. Administration of BALB/c-MSC temporarily resulted in reversal of hyperglycemia in 90% of NOD mice (p = 0.002). Transfer of autologous NOD-MSC imparted no such therapeutic benefit. We also noted soft tissue and visceral tumors in NOD-MSC-treated mice, which were uniquely observed in this setting (i.e., no tumors were present with BALB/c- or nonobese resistant mice-MSC transfer). The importance of this observation remains to be explored in humans, as inbred mice such as NOD may be more susceptible to tumor formation. These data provide important preclinical data supporting the basis for further development of allogeneic MSC-based therapies for T1D and, potentially, for other autoimmune disorders.


Subject(s)
Diabetes Mellitus, Type 1/therapy , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/immunology , Animals , Bone Marrow Cells , Cell Movement , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/immunology , Diabetes Mellitus, Experimental/therapy , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/immunology , Hyperglycemia/therapy , Immunologic Factors/immunology , Mesenchymal Stem Cell Transplantation/adverse effects , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Neoplasms/etiology , Treatment Outcome
10.
Diabetes ; 57(7): 1759-67, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18586907

ABSTRACT

Mesenchymal stem cells (MSCs) are pluripotent stromal cells that have the potential to give rise to cells of diverse lineages. Interestingly, MSCs can be found in virtually all postnatal tissues. The main criteria currently used to characterize and identify these cells are the capacity for self-renewal and differentiation into tissues of mesodermal origin, combined with a lack in expression of certain hematopoietic molecules. Because of their developmental plasticity, the notion of MSC-based therapeutic intervention has become an emerging strategy for the replacement of injured tissues. MSCs have also been noted to possess the ability to impart profound immunomodulatory effects in vivo. Indeed, some of the initial observations regarding MSC protection from tissue injury once thought mediated by tissue regeneration may, in reality, result from immunomodulation. Whereas the exact mechanisms underlying the immunomodulatory functions of MSC remain largely unknown, these cells have been exploited in a variety of clinical trials aimed at reducing the burden of immune-mediated disease. This article focuses on recent advances that have broadened our understanding of the immunomodulatory properties of MSC and provides insight as to their potential for clinical use as a cell-based therapy for immune-mediated disorders and, in particular, type 1 diabetes.


Subject(s)
Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/surgery , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/immunology , Animals , B-Lymphocytes/immunology , Biomarkers , Dendritic Cells/immunology , HLA-D Antigens/immunology , Histocompatibility Antigens Class II/immunology , Humans , Immunologic Factors , Killer Cells, Natural/immunology , Mice , T-Lymphocytes/immunology
11.
Cell Signal ; 18(10): 1584-94, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16500080

ABSTRACT

Certain TRP cation channels confer the ability to sense environmental stimuli (heat, cold, pressure, osmolarity) across physiological and pathophysiological ranges. TRPA1 is a TRP-related channel that responds to cold temperatures, and pungent compounds that include the cold-mimetic icilin and cannabinoids. The initial report of TRPA1 as a transformation-associated gene product in lung epithelia is at odds with subsequent descriptions of a tissue distribution for TRPA1 that is restricted to sensory neurons. Here, we report that the human TRPA1 protein is widely expressed outside the CNS, and is indeed dys-regulated during oncogenic transformation. We describe that TRPA1 associates with the tumor-suppressor protein CYLD. TRPA1 is a novel substrate for the de-ubiquitinating activity of CYLD, and this de-ubiquitination has the net effect of increasing the cellular pool of TRPA1 proteins. Oncogenic mutations in the CYLD gene may therefore be predicted to alter cellular levels of TRPA1.


Subject(s)
Calcium Channels/metabolism , Nerve Tissue Proteins/metabolism , Transient Receptor Potential Channels/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin/metabolism , Cells, Cultured , Central Nervous System/metabolism , Deubiquitinating Enzyme CYLD , Gene Expression , Gene Expression Profiling , Humans , Protein Binding , Substrate Specificity , TRPA1 Cation Channel
12.
Hum Genet ; 117(1): 16-26, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15806396

ABSTRACT

Asthma is a phenotypically heterogeneous disorder with many etiologic factors and clinical characteristics. T-bet, a Th1-specific transcription factor of T-box family, has been found to control interferon-gamma (IFN-gamma) expression in T cells. Mice lacking the T-bet gene (tbx21) demonstrate multiple physiological and inflammatory features reminiscent of human asthma. In order to examine whether polymorphisms in the candidate gene, TBX21, located on chromosome 17q21.32, are related to the risk of human asthma phenotypes, we have searched for genetic variations in the human TBX21 gene and identified 24 single nucleotide polymorphisms (SNPs), including five novel SNPs, by direct sequencing in Japanese subjects. Among asthma phenotypes, a promoter -1993T-->C SNP, which is in linkage disequilibrium with a synonymous coding 390A-->G SNP in exon 1, is significantly associated with a risk of aspirin-induced asthma (AIA; P = 0.004, P(c) = 0.016). This association has also been confirmed in additional independent samples of asthma with nasal polyposis (P = 0.008), regardless of aspirin hypersensitivity. Furthermore, our data indicate that the -1993T-->C substitution increases the affinity of a particular nuclear protein to the binding site of TBX21 covering the -1993 position, resulting in increased transcriptional activity of the TBX21 gene. Thus, in addition to the antigen-driven excess Th2 response, increased T-bet (and subsequent IFN-gamma) production in human airways of individuals with the -1993T-->C polymorphism could contribute to the development of certain asthma-related phenotypes, such as AIA.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Aspirin/adverse effects , Asthma/chemically induced , Asthma/genetics , Polymorphism, Single Nucleotide , T-Box Domain Proteins/genetics , Adolescent , Anti-Inflammatory Agents, Non-Steroidal/immunology , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Aspirin/immunology , Aspirin/therapeutic use , Binding Sites , Child , Child, Preschool , Chromosomes, Human, Pair 17 , Drug Hypersensitivity/genetics , Female , Genetic Predisposition to Disease , Humans , Interferon-gamma/immunology , Japan , Male , Phenotype , Promoter Regions, Genetic/genetics , Risk Factors , T-Box Domain Proteins/immunology , Th2 Cells/immunology , Transcription Factors/immunology , Transcription, Genetic
13.
Biochem J ; 388(Pt 2): 465-73, 2005 Jun 01.
Article in English | MEDLINE | ID: mdl-15669919

ABSTRACT

Cannabinoids are broadly immunosuppressive, and anti-inflammatory properties have been reported for certain marijuana constituents and endogenously produced cannabinoids. The CB2 cannabinoid receptor is an established constituent of immune system cells, and we have recently established that the CB1 cannabinoid receptor is expressed in mast cells. In the present study, we sought to define a role for CB1 in mast cells and to identify the signalling pathways that may mediate the suppressive effects of CB1 ligation on mast cell activation. Our results show that CB1 and CB2 mediate diametrically opposed effects on cAMP levels in mast cells. The observed long-term stimulation of cAMP levels by the Galpha(i/o)-coupled CB1 is paradoxical, and our results indicate that it may be attributed to CB1-mediated transcriptional regulation of specific adenylate cyclase isoenzymes that exhibit superactivatable kinetics. Taken together, these results reveal the complexity in signalling of natively co-expressed cannabinoid receptors and suggest that some anti-inflammatory effects of CB1 ligands may be attributable to sustained cAMP elevation that, in turn, causes suppression of mast cell degranulation.


Subject(s)
Cyclic AMP/biosynthesis , Mast Cells/metabolism , Receptor, Cannabinoid, CB1/physiology , Receptor, Cannabinoid, CB2/physiology , Adenylyl Cyclases/metabolism , Animals , Arachidonic Acids/pharmacology , Camphanes/pharmacology , Cell Line , Colforsin/pharmacology , Cytosol/drug effects , Cytosol/metabolism , Down-Regulation , GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Humans , Indoles/pharmacology , Ligands , Mast Cells/drug effects , Morpholines/pharmacology , Pertussis Toxin/pharmacology , Pyrazoles/pharmacology , Receptor, Cannabinoid, CB1/agonists , Receptor, Cannabinoid, CB2/agonists , Signal Transduction , Time Factors , Transcription, Genetic , Up-Regulation
14.
J Biol Chem ; 280(17): 17235-42, 2005 Apr 29.
Article in English | MEDLINE | ID: mdl-15671017

ABSTRACT

Cyclin-dependent kinase 2 (cdk2) activation requires phosphorylation of Thr160 and dissociation from cyclin A. The T-loop of cdk2 contains a regulatory phosphorylation site at Thr160. An interaction between cdc-associated phosphatase (KAP) and cdk2 compromises the interaction between cdk2 and cyclin A, which permits access of KAP, a Thr160-directed phosphatase, to its substrate, cdk2. We have reported that KAP is bound and activated by a nuclear membrane protein, HTm4. Here, we present in vitro data showing the direct interaction between the HTm4 C terminus and KAP Tyr141. We show that this interaction not only facilitates access of KAP to Thr160 and accelerates KAP kinetics, but also forces exclusion of cyclin A from the KAP.cdk2 complex.


Subject(s)
CDC2-CDC28 Kinases/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cyclin A/metabolism , Membrane Proteins/chemistry , Protein Tyrosine Phosphatases/metabolism , Amino Acid Sequence , Binding Sites , Blotting, Western , CDC2-CDC28 Kinases/chemistry , Cell Nucleus/metabolism , Circular Dichroism , Cloning, Molecular , Cyclin A/chemistry , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase Inhibitor Proteins , Dose-Response Relationship, Drug , Dual-Specificity Phosphatases , Escherichia coli/metabolism , Humans , Ions , Kinetics , Membrane Proteins/metabolism , Models, Biological , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Protein Tyrosine Phosphatases/chemistry , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Threonine/chemistry , Time Factors , Tyrosine/chemistry
15.
J Exp Med ; 200(2): 137-47, 2004 Jul 19.
Article in English | MEDLINE | ID: mdl-15249591

ABSTRACT

Cutaneous mast cell responses to physical (thermal, mechanical, or osmotic) stimuli underlie the pathology of physical urticarias. In vitro experiments suggest that mast cells respond directly to these stimuli, implying that a signaling mechanism couples functional responses to physical inputs in mast cells. We asked whether transient receptor potential (vanilloid) (TRPV) cation channels were present and functionally coupled to signaling pathways in mast cells, since expression of this channel subfamily confers sensitivity to thermal, osmotic, and pressure inputs. Transcripts for a range of TRPVs were detected in mast cells, and we report the expression, surface localization, and oligomerization of TRPV2 protein subunits in these cells. We describe the functional coupling of TRPV2 protein to calcium fluxes and proinflammatory degranulation events in mast cells. In addition, we describe a novel protein kinase A (PKA)-dependent signaling module, containing PKA and a putative A kinase adapter protein, Acyl CoA binding domain protein (ACBD)3, that interacts with TRPV2 in mast cells. We propose that regulated phosphorylation by PKA may be a common pathway for TRPV modulation.


Subject(s)
Calcium Channels/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Mast Cells/metabolism , Signal Transduction , ATP-Binding Cassette Transporters/metabolism , Animals , Blotting, Northern , Blotting, Western , Calcium/metabolism , Cations , Cell Line , DNA, Complementary/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Membrane Potentials , Membrane Proteins/metabolism , Phosphorylation , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , RNA, Messenger/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Serotonin/metabolism , TRPV Cation Channels , Temperature , Time Factors
16.
J Allergy Clin Immunol ; 113(3): 528-35, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15007357

ABSTRACT

BACKGROUND: During inflammation, neutrophils, basophils, and eosinophils release cell type-specific mediators and proteases through signaling molecules, such as G protein-coupled receptors and ion channels. As such, ion channels and receptors, including G protein-coupled receptors, are common drug targets. OBJECTIVE: We sought to identify, for the first time, ion channels and receptors preferentially expressed by each granulocyte subtype. METHODS: Using GeneChip, we compared approximately 20,000 transcripts present in 7 leukocyte types, platelets, mast cells, and fibroblasts to identify granulocyte subtype-selective transcripts for receptors and ion channels. Granulocyte subtype-selective transcripts were chosen on the basis of several conditions, such as the transcript having a 5-fold or greater expression level compared with the maximum level of other leukocytes. RESULTS: Fifty-one transcripts were chosen to be preferentially expressed by each granulocyte subtype. Seventeen of the 51 transcripts have not been previously reported as granulocyte subtype selective. Among the 17 receptors and ion channels, 6 were basophil selective, eosinophil selective, or both and were not highly expressed by other organs, indicating that they might be potential targets for antiallergy drugs. CONCLUSION: Use of this database of potential cell type-selective drug targets should minimize the efforts required for pharmaceutical development.


Subject(s)
Granulocytes/classification , Granulocytes/metabolism , Ion Channels/genetics , Receptors, G-Protein-Coupled/genetics , Basophils/metabolism , Eosinophils/metabolism , Gene Expression Profiling , Humans , In Vitro Techniques , Neutrophils/metabolism , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism
17.
J Cell Biochem ; 91(4): 808-20, 2004 Mar 01.
Article in English | MEDLINE | ID: mdl-14991772

ABSTRACT

TRPV ion channels transduce a range of temperature stimuli. We proposed that analysis of the protein-protein interactions made by TRPV2 might give insight into the key issues surrounding this channel. These issues include the potential functional significance of TRPV2 in non-sensory tissues, the molecules involved in transducing its activation signal(s) and the mechanism by which its trafficking to the cell surface is regulated. Here we describe the interaction of TRPV2 channel with the RGA gene product. RGA is a four-transmembrane domain, intracellularly localized protein. RGA associates with TRPV2 in a rat mast cell line that is a native context for both proteins. The interaction between TRPV2 and RGA is transient and occurs intracellularly. RGA does not accompany TRPV2 to the cell surface. Formation of the TRPV2/RGA complex is dependent upon a cellular glycosylation event, suggesting that RGA may play a chaperone or targeting role for TRPV2 during the maturation of the ion channel protein. These data record a novel protein-protein interaction for TRPV2 and provide a foundation for future study of the potential regulatory contribution of RGA to TRPV2 function.


Subject(s)
Ion Channels/metabolism , Membrane Proteins/metabolism , Receptors, Drug/metabolism , Animals , Cell Line , Cell Membrane/metabolism , Cytoplasm/metabolism , Humans , Immunoprecipitation , Ion Channels/genetics , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Phylogeny , Protein Binding/drug effects , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Receptors, Drug/genetics , Sequence Analysis, Protein , TRPV Cation Channels , Tunicamycin/pharmacology , Two-Hybrid System Techniques
18.
J Allergy Clin Immunol ; 109(6): 980-7, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12063528

ABSTRACT

BACKGROUND: A substantial body of evidence exists to support the pivotal role of IL-13 in the pathogenesis of bronchial asthma. We recently found that a variant of the IL13 gene (Arg110Gln) is genetically associated with bronchial asthma, which is concordant with animal experiments using IL-13 in the development of asthma. OBJECTIVE: To address whether the Gln110 variant of IL13 influences IL-13 function, contributing to the pathogenesis of bronchial asthma, we studied the functional properties of the variant. METHODS: We generated 2 types of recombinant IL-13 proteins, the amino acids of which at 110 were arginine or glutamine, and analyzed the binding affinities with the IL-13 receptors, as well as the stability of the proteins. We further compared the relationship between the genotype and serum levels of IL-13. RESULTS: The variant showed a lower affinity with the IL-13 receptor alpha2 chain, a decoy receptor, causing less clearance. The variant also demonstrated an enhanced stability in both human and mouse plasma. We further identified that asthmatic patients homozygous for the Gln110 variant have higher serum levels of IL-13 than those without the variant. CONCLUSION: These results suggested that the variant might act as a functional genetic factor of bronchial asthma with a unique mechanism to upregulate local and systemic IL-13 concentration in vivo.


Subject(s)
Asthma/genetics , Genetic Variation , Interleukin-13/biosynthesis , Up-Regulation , Amino Acid Substitution , Asthma/metabolism , Cell Line , Genotype , Humans , Interleukin-13/blood , Interleukin-13/genetics , Interleukin-13/physiology , Interleukin-13 Receptor alpha1 Subunit , Receptors, Interleukin/metabolism , Receptors, Interleukin-13 , Recombinant Proteins/metabolism
19.
J Clin Invest ; 109(1): 51-8, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11781350

ABSTRACT

Proper control of cell cycle progression is critical for the constant self-renewal, differentiation, and homeostasis of the hematopoietic system. Cells of all types share the common cell cycle regulators. The different expression patterns of common regulators, in a broad sense, define cell-type or lineage specificity. However, there remains the possibility of hematopoietic cell cycle regulators tailored to the demands of the hematopoietic system. Here we describe a novel protein, HTm4, which serves as a hematopoietic cell cycle regulator. Our data indicate that HTm4 is expressed in hematopoietic tissues and is tightly regulated during the differentiation of hematopoietic stem cells. It binds to cyclin-dependent kinase-associated (CDK-associated) phosphatase-CDK2 (KAP-CDK2) complexes, and the three proteins demonstrate similar patterns of cellular expression in human lymphoid tissues. HTm4 stimulates the phosphatase activity of KAP, and its C-terminal region is required for binding to KAP-CDK2 complexes and the modulation of KAP activity. Overexpression of HTm4 can cause cell cycle arrest at the G(0)/G(1) phase. Thus, HTm4 is a novel hematopoietic modulator for the G(1)-S cell cycle transition.


Subject(s)
CDC2-CDC28 Kinases , Cell Cycle Proteins , Cell Cycle/physiology , Hematopoiesis/physiology , Membrane Proteins/physiology , Amino Acid Sequence , Cells, Cultured , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase Inhibitor Proteins , Cyclin-Dependent Kinases/metabolism , Dual-Specificity Phosphatases , Gene Expression Regulation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Humans , Immunohistochemistry , Membrane Proteins/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Tyrosine Phosphatases/metabolism , Transfection , Two-Hybrid System Techniques
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