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2.
J Cancer Educ ; 37(3): 857-871, 2022 06.
Article in English | MEDLINE | ID: mdl-35098479

ABSTRACT

Summer internships serve important roles in training the next generation of biomedical researchers and healthcare providers through laboratory and clinical experiences that excite trainees about these fields and help them make informed decisions about career paths. The SARS-CoV-2 (COVID) pandemic and associated physical distancing restrictions precluded implementation of traditional in-person summer curricula and led to the cancellation of many internships across the USA. COVID-related disruptions also created opportunities for trainees to engage in remote research, become proficient in online learning platforms, and explore multidisciplinary topics. These skills are highly relevant to trainees as virtual interfaces occupy an increasingly mainstream role in their professional paths. The response to the COVID pandemic required real-time adaptations at all levels for major biomedical institutions including the University of Maryland Baltimore (UMB). Pivoting summer programs to a virtual format as part of this response provided a "teachable moment" to expose trainees to the innovation and resilience that are essential components of the biomedical profession. UMB summer programs, which span diverse biomedical disciplines from cancer research to diabetes, consolidated resources and identified mentors with online research projects to develop a robust virtual curriculum. Herein, data from a cancer-focused internship illustrate the collaborative adaptations to established components and creation of new learning modules in the transition to, and implementation of, online training. Outcomes are presented in the context of the COVID pandemic and significant societal issues that arose in the summer of 2020. The utility of virtual components and their impact on future programs is discussed.


Subject(s)
COVID-19 , Education, Distance , Neoplasms , COVID-19/epidemiology , Curriculum , Humans , Neoplasms/epidemiology , Pandemics , SARS-CoV-2
3.
Oncogene ; 35(31): 4112-21, 2016 08 04.
Article in English | MEDLINE | ID: mdl-26751774

ABSTRACT

MUC5AC is a secretory mucin aberrantly expressed in various cancers. In lung cancer, MUC5AC is overexpressed in both primary and metastatic lesions; however, its functional role is not well understood. The present study was aimed at evaluating mechanistic role of MUC5AC on metastasis of lung cancer cells. Clinically, the overexpression of MUC5AC was observed in lung cancer patient tissues and was associated with poor survival. In addition, the overexpression of Muc5ac was also observed in genetically engineered mouse lung adenocarcinoma tissues (Kras(G12D); Trp53(R172H/+); AdCre) in comparison with normal lung tissues. Our functional studies showed that MUC5AC knockdown resulted in significantly decreased migration in two lung cancer cell lines (A549 and H1437) as compared with scramble cells. Expression of integrins (α5, ß1, ß3, ß4 and ß5) was decreased in MUC5AC knockdown cells. As both integrins and MUC5AC have a von Willebrand factor domain, we assessed for possible interaction of MUC5AC and integrins in lung cancer cells. MUC5AC strongly interacted only with integrin ß4. The co-localization of MUC5AC and integrin ß4 was observed both in A549 lung cancer cells as well as genetically engineered mouse adenocarcinoma tissues. Activated integrins recruit focal adhesion kinase (FAK) that mediates metastatic downstream signaling pathways. Phosphorylation of FAK (Y397) was decreased in MUC5AC knockdown cells. MUC5AC/integrin ß4/FAK-mediated lung cancer cell migration was confirmed through experiments utilizing a phosphorylation (Y397)-specific FAK inhibitor. In conclusion, overexpression of MUC5AC is a poor prognostic marker in lung cancer. MUC5AC interacts with integrin ß4 that mediates phosphorylation of FAK at Y397 leading to lung cancer cell migration.


Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/physiology , Integrin beta4/physiology , Lung Neoplasms/pathology , Mucin 5AC/physiology , Signal Transduction/physiology , Animals , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cisplatin/therapeutic use , Drug Resistance, Neoplasm , Humans , Integrin beta4/analysis , Male , Mice , Mucin 5AC/analysis , Phosphorylation
4.
Oncogene ; 31(30): 3561-8, 2012 Jul 26.
Article in English | MEDLINE | ID: mdl-22081072

ABSTRACT

Missing in metastasis (MIM) is a member of newly emerged inverse Bin-Amphiphysin-Rvs (BAR) domain protein family and a putative metastasis suppressor. Although reduced MIM expression has been associated with bladder, breast and gastric cancers, evidence for the role of MIM in tumor progression remains scarce and controversial. Herein we characterized a MIM knockout mouse strain and observed that MIM-deficient mice often developed enlarged spleens. Autopsy and histological analysis revealed that nearly 78% of MIM(-/-) mice developed tumors with features similar to diffuse large B lymphoma during a period from 1 to 2 years. MIM(-/-) mice also exhibited abnormal distribution of B cells in lymphoid organs with decrease in the spleen but increase in the bone marrow and the peripheral blood. Furthermore, the bone marrow of MIM(-/-) mice contained a higher percentage of pre-B2 cells but fewer immature B-cells than wild-type mice. In response to CXCL13, a B-cell chemokine released from splenic stromal cells, MIM-deficient B-cells did not undergo chemotaxis or morphological changes in response to the chemokine and also did not internalize CXCR5, the receptor of CXCL13. Microarray analyses demonstrated that MIM is the only member of the I-BAR domain family that was highly expressed in human B cells. However, low or absent MIM expression was common in either primary B-cell malignancies or established B-cell acute lymphocytic leukemia or lymphomas. Thus, our data demonstrate for the first time an important role for MIM in B-cell development and suggest that predisposition of MIM-null mice to lymphomagenesis may involve aberrant interactions between B lineage cells and the lymphoid microenvironment.


Subject(s)
Cell Transformation, Neoplastic/genetics , Genetic Predisposition to Disease , Lymphoma, Large B-Cell, Diffuse/genetics , Microfilament Proteins/genetics , Neoplasm Proteins/genetics , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Bone Marrow/metabolism , Cells, Cultured , Chemokine CXCL13/pharmacology , Chemotaxis/drug effects , Female , Granulocytes/metabolism , Humans , Leukocytes, Mononuclear/metabolism , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Male , Mice , Mice, Knockout , Microfilament Proteins/metabolism , Neoplasm Proteins/metabolism , Receptors, CXCR5/metabolism , Spleen/metabolism , Spleen/pathology , T-Lymphocytes/metabolism
5.
J Anim Sci ; 80(5): 1294-8, 2002 May.
Article in English | MEDLINE | ID: mdl-12019618

ABSTRACT

The purpose of this study was to examine the 133xenon washout technique as a viable method for measuring adipose tissue blood flow (ATBF) in swine. Using a total of 32 female Yucatan miniature swine (Sus scrofa), the partition coefficient for 133xenon in swine subcutaneous adipose tissue was determined and ATBF was measured at rest and under various physiological conditions. These conditions included feeding, anesthesia, epinephrine infusion, and acute exercise. The effects of epinephrine and acute exercise were examined in both sedentary and exercise-trained swine. The partition coefficient value for 133xenon in swine subcutaneous adipose tissue was 9.23+/-0.26 mL/g (mean +/- SD, n = 10). The average value for resting ATBF in swine was 3.98+/-2.72 mL/(100 g tissue-min) (n = 19). Feeding increased ATBF by approximately fivefold over fasting values, and isoflurane anesthesia significantly decreased ATBF compared to rest (1.64+/-1.12 vs 3.92+/-4.22 mL/[100 g x min], n = 10). A 30-min epinephrine infusion (1 microg/[kg BW x min]) significantly increased ATBF from a resting value of 3.13+/-2.61 to 10.35+/-5.31 mL/(100 g x min) (n = 12). Epinephrine infusion into exercise-trained swine increased ATBF to the same extent as when infused into sedentary swine. An acute, 20-min bout of exercise significantly increased ATBF in swine, and the sedentary swine showed a larger increase in ATBF than their exercise-trained littermates relative to rest: 7.83 vs 2.98 mL/(100 g x min). In conclusion, the 133xenon washout technique appears to be a viable method for measuring ATBF in swine; our findings are comparable to swine ATBF values reported using the microsphere method and are consistent with values reported in animal and human studies.


Subject(s)
Adipose Tissue/blood supply , Swine, Miniature/physiology , Xenon Radioisotopes , Adrenergic Agonists/administration & dosage , Adrenergic Agonists/pharmacology , Anesthesia/veterinary , Animals , Epinephrine/administration & dosage , Epinephrine/pharmacology , Female , Infusions, Parenteral/veterinary , Physical Conditioning, Animal/physiology , Regional Blood Flow/drug effects , Swine
6.
J Immunol ; 166(3): 1618-26, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11160203

ABSTRACT

Cross-linking of surface Ig receptors with anti IgM (anti-mu heavy chain, anti-mu), but not anti-IgD (anti-delta heavy chain, anti-delta), Abs leads to growth arrest and apoptosis in several extensively characterized B cell lymphomas. By poorly understood mechanisms, both Igs transiently stimulate c-Myc protein expression. However, ultimately, only anti-mu causes a severe loss in c-Myc and a large induction of p27(Kip1) protein expression. Because phosphatidylinositol 3-kinase (PI3K) has been established as a major modulator of cellular growth and survival, we investigated its role in mediating anti-Ig-stimulated outcomes. Herein, we show that PI3K pathways regulate cell cycle progression and apoptosis in the ECH408 B cell lymphoma. Anti-mu and anti-delta driven c-Myc protein changes precisely follow their effects on the PI3K effector, p70(S6K). Upstream of p70(S6K), signaling through both Ig receptors depresses PI3K pathway phospholipids below control with time, which is followed by p27(Kip1) induction. Conversely, anti-delta, but not anti-mu stimulated PI3K-dependent phospholipid return to control levels by 4-8 h. Abrogation of the PI3K pathway with specific inhibitors mimics anti-mu action, potentiates anti-mu-induced cell death and, importantly, converts anti-delta to a death signal. Transfection with active PI3K kinase construct induces anti-mu resistance, whereas transfection with dominant negative PI3K augments anti-mu sensitivity. Our results show that prolonged disengagement of PI3K or down-regulation of its products by anti-mu (and not anti-delta) determines B cell fate.


Subject(s)
Antibodies, Anti-Idiotypic/pharmacology , Apoptosis/immunology , Cell Cycle Proteins , Immunoglobulin D/immunology , Immunoglobulin M/immunology , Lymphoma, B-Cell/enzymology , Lymphoma, B-Cell/immunology , Phosphatidylinositol 3-Kinases/physiology , Tumor Suppressor Proteins , Animals , Chromones/pharmacology , Cyclin-Dependent Kinase Inhibitor p27 , Enzyme Activation/drug effects , Enzyme Activation/immunology , Enzyme Inhibitors/pharmacology , Growth Inhibitors/pharmacology , Immunoglobulin delta-Chains/immunology , Immunoglobulin mu-Chains/immunology , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/pathology , Mice , Microtubule-Associated Proteins/biosynthesis , Microtubule-Associated Proteins/metabolism , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-myc/biosynthesis , Proto-Oncogene Proteins c-myc/metabolism , Ribosomal Protein S6 Kinases/antagonists & inhibitors , Ribosomal Protein S6 Kinases/metabolism , Signal Transduction/immunology , Transfection , Tumor Cells, Cultured
7.
Int Immunol ; 13(2): 211-22, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11157854

ABSTRACT

The T cell receptor (TCR) beta chain transmembrane domain contains two evolutionarily conserved tyrosines (Y). In this study, the functional basis for the evolutionary conservation is addressed by mutation of the residues, expression of the mutants in hybridoma and primary T cells, and examination of TCR signaling function. We find that the phenotype of the mutants, both surface expression and ability to signal for IL-2 production, is highly variable in different mouse T hybridoma lines. Although we have not been able to determine the basis for these differences in the hybridomas, expression of the mutants in primary T cells provides a definitive assessment of mutant phenotype. We show that mutation of the N-terminal Y to either leucine (L) or alanine (A) results in low surface expression in primary T cells, while mutation of both N- and C-terminal Y to A or L abrogates surface expression. However, the more conservative mutation of both transmembrane Y to phenylalanine maintained receptor surface expression and assembly while severely disrupting signaling in primary T cells. Our data demonstrate that TCR beta chain transmembrane Y are essential for TCR signal transduction as well as complex assembly. These findings suggest that protein-protein interactions involving membrane-spanning domains are likely relevant for TCR signal transduction mechanisms.


Subject(s)
Conserved Sequence/immunology , Hybridomas/metabolism , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , T-Lymphocyte Subsets/metabolism , Tyrosine/physiology , Animals , Base Sequence , Cell Line , Cell Membrane/genetics , Cell Membrane/immunology , Cell Membrane/metabolism , Cells, Cultured , Gene Expression Regulation/immunology , Hybridomas/immunology , Immunophenotyping , Lymphocyte Activation/genetics , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Transgenic , Mutagenesis, Site-Directed , Protein Structure, Tertiary/genetics , Receptors, Antigen, T-Cell/deficiency , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell, alpha-beta/deficiency , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/physiology , Signal Transduction/genetics , Signal Transduction/immunology , Spleen/immunology , Spleen/metabolism , T-Lymphocyte Subsets/immunology , Tumor Cells, Cultured , Tyrosine/genetics
8.
J Biol Chem ; 276(5): 3361-70, 2001 Feb 02.
Article in English | MEDLINE | ID: mdl-11058590

ABSTRACT

To determine how hyaluronidase increases certain cancer cell sensitivity to tumor necrosis factor (TNF) cytotoxicity, we report here the isolation and characterization of a hyaluronidase-induced murine WW domain-containing oxidoreductase (WOX1). WOX1 is composed of two N-terminal WW domains, a nuclear localization sequence, and a C-terminal alcohol dehydrogenase (ADH) domain. WOX1 is mainly located in the mitochondria, and the mitochondrial targeting sequence was mapped within the ADH domain. Induction of mitochondrial permeability transition by TNF, staurosporine, and atractyloside resulted in WOX1 release from mitochondria and subsequent nuclear translocation. TNF-mediated WOX1 nuclear translocation occurred shortly after that of nuclear factor-kappaB nuclear translocation, whereas both were independent events. WOX1 enhanced TNF cytotoxicity in L929 cells via its WW and ADH domains as determined using stable cell transfectants. In parallel with this observation, WOX1 also enhanced TRADD (TNF receptor-associated death domain protein)-mediated cell death in transient expression experiments. Antisense expression of WOX1 raised TNF resistance in L929 cells. Enhancement of TNF cytotoxicity by WOX1 is due, in part, to its significant down-regulation of the apoptosis inhibitors Bcl-2 and Bcl-x(L) (>85%), but up-regulation of pro-apoptotic p53 ( approximately 200%) by the ADH domain. When overexpressed, the ADH domain mediated apoptosis, probably due to modulation of expression of these proteins. The WW domains failed to modulate the expression of these proteins, but sensitized COS-7 cells to TNF killing and mediated apoptosis in various cancer cells independently of caspases. Transient cotransfection of cells with both p53 and WOX1 induced apoptosis in a synergistic manner. WOX1 colocalizes with p53 in the cytosol and binds to the proline-rich region of p53 via its WW domains. Blocking of WOX1 expression by antisense mRNA abolished p53 apoptosis. Thus, WOX1 is a mitochondrial apoptogenic protein and an essential partner of p53 in cell death.


Subject(s)
Hyaluronoglucosaminidase/metabolism , Oxidoreductases/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , 3T3 Cells , Amino Acid Sequence , Animals , Apoptosis , Caspases/metabolism , Cloning, Molecular , Down-Regulation/drug effects , Gene Expression/drug effects , HeLa Cells , Humans , Mice , Molecular Sequence Data , Oxidoreductases/genetics , Oxidoreductases/physiology , Proline/chemistry , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcl-2/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Sequence Homology, Amino Acid , Tumor Suppressor Protein p53/drug effects , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins , Up-Regulation/drug effects , WW Domain-Containing Oxidoreductase , bcl-X Protein
9.
Immunol Rev ; 176: 105-15, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11043771

ABSTRACT

A series of B-cell lymphoma lines with an immature phenotype has been used as a model system to study molecular events associated with receptor ligation induced death. B-cell receptor (BCR) cross-linking with antibodies to membrane IgM (but not with anti IgD) induces c-Myc downregulation via nuclear factor kappaB inactivation and p27(Kip1) accumulation in these B lymphomas. Anti-mu-treated cells then undergo G1 arrest and die by apoptosis independent of Fas. Steroids and retinoids similarly downregulate c-Myc and induce apoptosis in these B cells and synergize with anti-mu. Rescue from apoptosis induced by anti-mu or steroids occurs with T-cell signals, like CD40L, or a broad-range caspase inhibitor, but only CD40L prevents the loss of c-Myc, p27 accumulation and growth arrest. Both IgM and IgD signaling lead to modulation of phosphatidylinositol 3-kinase (PI3K) signals, including the activation of p70(S6K), but this pathway recovers under anti-IgD treatment. Blockade of the PI3K pathway augments anti-mu-induced death and converts anti-delta to an apoptotic signal. Resistance to Fas-mediated death may be an important factor in B-cell transformation in vivo. Many of our panel of lymphomas are insensitive to Fas-mediated death signals, although all can form a death-inducing signaling complex (DISC). Additional studies suggest that some lymphomas can be blocked at the DISC complex by anti-apoptotic proteins, whereas others are inhibited downstream of caspase 8 activation. Anti-Ig treatment of a Fas-sensitive line, A20.2J, activated a number of genes whose products may block apoptosis proximally (like FLICE-inhibitory protein (FLIP1)) or at late points, such as bcl-2-family members. Our data suggest that B lymphomas develop multiple pathways of resistance to Fas-mediated signals during lymphomagenesis, in part via signaling through the BCR.


Subject(s)
B-Lymphocytes/cytology , B-Lymphocytes/immunology , Receptors, Antigen, B-Cell/metabolism , fas Receptor/metabolism , Animals , Apoptosis , B-Lymphocytes/metabolism , Cell Division , Humans , Immunoglobulin D/metabolism , Immunoglobulin M/metabolism , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/pathology , Signal Transduction , Tumor Cells, Cultured
10.
J Appl Physiol (1985) ; 88(3): 881-7, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10710382

ABSTRACT

This study examined the influence of energy expenditure and energy intake on cellular mechanisms regulating adipose tissue metabolism. Twenty-four swine were assigned to restricted-fed sedentary, restricted-fed exercise-trained, full-fed sedentary, or full-fed exercise-trained groups. After 3 mo of treatment, adipocytes were isolated and adipocyte size, adenosine A(1) receptor characteristics, and lipolytic sensitivity were measured. Swine were infused with epinephrine during which adipose tissue extracellular adenosine, plasma fatty acids, and plasma glycerol were measured. Results revealed that adipocytes isolated from restricted-fed exercised swine had a smaller diameter, a lower number of A(1) receptors, and a greater sensitivity to lipolytic stimulation, compared with adipocytes from full-fed exercised swine. Extracellular adenosine levels were transiently increased on infusion of epinephrine in adipose tissue of restricted-fed exercised but not full-fed exercised swine. These results suggest a role for adenosine in explaining the discrepancy between in vitro and in vivo lipolysis findings and underscore the notion that excess energy intake dampens the lipolytic sensitivity of adipocytes to beta-agonists and adenosine, even if accompanied by exercise training.


Subject(s)
Adaptation, Physiological , Adipocytes/metabolism , Energy Intake/physiology , Adenosine/metabolism , Adipocytes/cytology , Adipocytes/drug effects , Adrenergic beta-Agonists/pharmacology , Animals , Cell Size , Dipyridamole/pharmacology , Energy Metabolism , Epinephrine/pharmacology , Female , In Vitro Techniques , Lipolysis/drug effects , Physical Conditioning, Animal , Receptors, Purinergic P1/metabolism , Swine , Swine, Miniature , Theophylline/pharmacology
11.
Curr Top Microbiol Immunol ; 252: 151-9, 2000.
Article in English | MEDLINE | ID: mdl-11187083

ABSTRACT

Crosslinking of surface immunoglobulin (Ig) receptors with anti-IgM (anti-mu) but not anti-IgD (anti-delta) antibodies causes growth arrest and apoptosis in several extensively characterized B1-like lymphoma cell lines. While anti-mu stimulates a transient increase in c-myc mRNA and protein expression, followed by a rapid decline below the baseline level, anti-delta only causes a moderate increase in the expression of this oncogene, which returns to baseline levels within 24-48 hours. However, signals downstream from anti-delta can be converted into an apoptotic pathway by modulating PI3K activity, suggesting that PI3K is a critical rheostat controlling survival signals in B1 cell lines. Anti-mu-induced down-regulation of c-Myc is followed in time with an increase in the cyclin dependent kinase inhibitor, p27Kip1, in all anti-mu sensitive lymphoma lines. This increase correlates with growth arrest and apoptosis. The anti-mu-mediated decrease in c-Myc, increase in p27Kip1, growth arrest and apoptosis, can all be prevented via CD40/CD40L signaling. Inhibition of caspase activation, on the other hand, prevents anti-mu-induced apoptosis, but has no effect on c-Myc, p27Kip1, and G1 arrest. Interestingly, we also found that steroids and retinoids can mimic anti-mu-mediated signaling and lead to a loss of c-Myc, an increase in p27Kip1, G1 arrest, and apoptosis. Together, these data suggest that modulation of c-Myc and p27Kip1 protein levels is crucial for the life versus death decisions in murine immature B1-like lymphoma cells lines.


Subject(s)
B-Lymphocyte Subsets/cytology , Cell Cycle Proteins , Ecdysterone/analogs & derivatives , Lymphoma, B-Cell/pathology , Microtubule-Associated Proteins/physiology , Neoplasm Proteins/physiology , Neoplastic Stem Cells/cytology , Proto-Oncogene Proteins c-myc/physiology , Tumor Suppressor Proteins , Antibodies, Anti-Idiotypic/immunology , Apoptosis/drug effects , B-Lymphocyte Subsets/drug effects , CD40 Antigens/physiology , CD40 Ligand/physiology , Caspases/physiology , Cell Cycle/drug effects , Cell Survival/drug effects , Cyclin-Dependent Kinase Inhibitor p27 , Dexamethasone/pharmacology , Ecdysterone/pharmacology , Enzyme Activation/drug effects , Enzyme Induction/drug effects , Enzyme Precursors/physiology , G1 Phase/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Genes, myc , Humans , Intracellular Signaling Peptides and Proteins , Microtubule-Associated Proteins/biosynthesis , Microtubule-Associated Proteins/genetics , Neoplastic Stem Cells/drug effects , Phosphatidylinositol 3-Kinases/physiology , Protein-Tyrosine Kinases/physiology , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Receptors, Retinoic Acid/drug effects , Receptors, Retinoic Acid/genetics , Recombinant Fusion Proteins/physiology , Retinoid X Receptors , Signal Transduction/drug effects , Syk Kinase , Transcription Factors/drug effects , Transcription Factors/genetics , Transfection , Tretinoin/pharmacology
12.
Article in English | MEDLINE | ID: mdl-10582321

ABSTRACT

Extracellular cyclic AMP is source of extracellular adenosine in brain and kidney. Whether this occurs in adipose tissue is unknown. The present study evaluated the capacity of swine adipocyte plasma membranes to metabolize cyclic AMP to AMP and adenosine, via phosphodiesterase (PDE) and 5'-nucleotidase (5'-NT), respectively. Plasma membranes (PM) and microsomal membranes (MM) were isolated from over-the-shoulder subcutaneous adipose tissue of 3 month-old male miniature swine. The purity of the membrane fractions was determined and PDE and 5'-NT activities in PM and MM fractions were corrected for cross-contamination. The maximal activity of MM-PDE was 7-fold greater than that of PM-PDE. MM-PDE was 100% inhibited by 5 microM cilostamide, while PM-PDE was unaffected by this PDE3B inhibitor. Inhibitors of PDE1, PDE2, PDE4 and PDE5 also failed to inhibit PM-PDE. However, 1 mM DPSPX inhibited PM-PDE activity by 72%. When PM were incubated with 0.8 microM cyclic AMP for 20 min, AMP accumulation was four times that of adenosine. These data demonstrate that cyclic AMP can be converted to AMP and adenosine by the PM-bound enzymes 5'-NT and PDE, and suggest that the PM-PDE responsible for extracellular cyclic AMP metabolism to AMP is distinct from the intracellular MM-PDE.


Subject(s)
Adipocytes/metabolism , Cell Membrane/metabolism , Cyclic AMP/metabolism , Microsomes/metabolism , 5'-Nucleotidase/antagonists & inhibitors , 5'-Nucleotidase/metabolism , Adenosine/metabolism , Adenosine Monophosphate , Adipocytes/enzymology , Adipocytes/ultrastructure , Animals , Biomarkers , Cell Membrane/enzymology , Dose-Response Relationship, Drug , Intracellular Membranes/enzymology , Intracellular Membranes/metabolism , Kinetics , Membrane Proteins/chemistry , Microsomes/enzymology , Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/metabolism , Protein Isoforms/analysis , Purinergic P1 Receptor Antagonists , Quinolones/pharmacology , Second Messenger Systems , Swine , Xanthines/pharmacology
13.
Med Sci Sports Exerc ; 31(1): 105-10, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9927017

ABSTRACT

PURPOSE: This study examined the relationships among diet, exercise intensity, and breast milk composition in lactating women. METHODS: Twelve lactating women were randomly assigned to either a high (N = 6; 5.03 g carbohydrate (CHO) x kg body mass (BM)(-1)) or moderate (N = 6; 3.89 g CHO x kg BM(-1)) carbohydrate diet. Milk and blood samples were collected before and after a nonexercise session (control) and maximal, lactic acid-threshold (LAT), and 20% below the LAT (LAT-20) intensities. RESULTS: The 30-min exercise LAT bout was more stressful than the 30-min LAT-20 bout (rating of perceived exertion (RPE) = 15 vs 12, respectively, P < 0.05). Milk LA was significantly higher at 0 min following maximal exercise in the high and moderate CHO groups (1.27+/-0.56 and 1.52+/-0.49 mM, respectively) and following LAT exercise (0.19+/-0.16 and 0.25+/-0.12 mM, respectively), when compared with the control session (0.08+/-0.03 and 0.09+/-0.05 mM, respectively). This was not observed following the LAT-20 exercise in the high and moderate CHO groups (0.11+/-0.04 and 0.12+/-0.08 mM, respectively). Elevated milk LA persisted in the 30-min collection point after maximal exercise only. There was no significant effect of dietary treatment on milk or blood LA at any of the collection points. CONCLUSIONS: In lactating women whose caloric needs are being met: 1) dietary CHO intake, within a practical range, does not influence LA levels in breast milk at rest or after exercise; 2) LA appearance in the milk is a function of exercise intensity; and 3) moderate intensity exercise (RPE = 12) will not increase breast milk LA levels.


Subject(s)
Dietary Carbohydrates , Exercise/physiology , Lactic Acid/analysis , Milk, Human/chemistry , Adult , Female , Humans , Lactation/physiology , Nutritional Requirements , Physical Endurance
15.
Adv Exp Med Biol ; 441: 157-70, 1998.
Article in English | MEDLINE | ID: mdl-9781323

ABSTRACT

Mechanisms regulating adipocyte lipolysis are reviewed in three stages. The first stage examines plasma membrane hormone receptors and G-proteins. The primary regulators of adipose tissue lipolysis, the catecholamines, bind to the alpha 2, beta 1, beta 2, and beta 3 adrenergic receptors. The alpha 2 receptor couples with Gi-proteins to inhibit cyclic AMP formation and lipolysis, while the beta receptors couple with Gs-proteins to stimulate cyclic AMP formation and lipolysis. The beta 1 receptor may mediate low level catecholamine stimulation, while the beta 3 receptor, which is activated by higher levels of catecholamines, may deliver a more sustained signal. The second stage examines the regulation of cyclic AMP, the intracellular messenger that activates protein kinase A. Adenylyl cyclase synthesizes cyclic AMP from ATP and is regulated by the G-proteins. Phosphodiesterase 3B hydrolyzes cyclic AMP to AMP and is activated and phosphorylated by both insulin and the catecholamines norepinephrine and epinephrine. The third stage focuses on the rate-limiting enzyme of lipolysis, hormone-sensitive lipase (HSL). This 82 to 88 kDa protein is regulated by reversible phosphorylation. Protein kinase A activates and phosphorylates the enzyme at 2 sites, and 3 phosphatases have been implicated in HSL dephosphorylation. The translocation of HSL from the cytosol to the lipid droplet in response to lipolytic stimulation may be facilitated by a family of lipid-associated droplets called perilipins that are heavily phosphorylated by protein kinase A and dephosphorylated by insulin. As the mechanisms regulating adipocyte lipolysis continue to be uncovered, we look forward to the challenges of integrating these findings with research at the in situ and in vivo levels.


Subject(s)
Adipocytes/metabolism , Lipolysis , Animals , Catecholamines/metabolism , Catecholamines/pharmacology , Humans , Lipolysis/drug effects
16.
Proc Soc Exp Biol Med ; 219(1): 57-63, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9751223

ABSTRACT

We previously reported that prolactin-mediated macromolecular synthesis and mitogenesis are coupled to the activation of mitogen-activated protein kinase (MAPK) and p70 S6-kinase (p70S6K). Full activation of MAPK requires tyrosine and threonine phosphorylation whereas that of p70S6K requires serine phosphorylation. In the present study, okadaic acid, which inhibits serine/threonine protein phosphatase activity, was used to explore the linkage of MAPK and p70S6K activation to down-stream effects of prolactin in Nb2 cells. The results show that 1 nM okadaic acid augmented prolactin-stimulated mitogenesis and synthesis of protein and DNA 250%, 42%, and 70%, respectively. Addition of okadaic acid alone a) stimulated and sustained p70S6K activity (5- to 8-fold) and MAPK (3.5- to 5-fold); and b) increased protein synthesis with the maximum effect being about equal to that of prolactin (2.1-fold with 1 nMokadaic acid versus 2.3-fold with 0.2 nMprolactin). However, okadaic acid did not affect DNA synthesis or mitogenesis. These results indicate that the activation of MAPK and p70S6K is necessary for stimulation of protein synthesis but not sufficient for prolactin-driven mitogenesis.


Subject(s)
Enzyme Inhibitors/pharmacology , Okadaic Acid/pharmacology , Prolactin/pharmacology , Signal Transduction/drug effects , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA, Neoplasm/biosynthesis , Lymphoma/enzymology , Mitosis/drug effects , Mitosis/physiology , Molecular Mimicry , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/biosynthesis , Protein Biosynthesis , Ribosomal Protein S6 Kinases/metabolism , Tumor Cells, Cultured/drug effects
17.
Biochem Biophys Res Commun ; 249(1): 283-6, 1998 Aug 10.
Article in English | MEDLINE | ID: mdl-9705873

ABSTRACT

Transforming growth factor-beta (TGF-beta) antagonizes the cytotoxic function of tumor necrosis factor (TNF). By differential display and library screening, we isolated a murine TIF2 (TGF-beta-induced factor 2) cDNA, encoding a putative 15-kDa membrane adhesion protein, which possesses an RGD sequence at the extracellular region. When TNF-sensitive murine L929 fibroblasts were stably transfected with TIF2 cDNA, these cells significantly resisted TNF killing. In contrast, L929 cells, which stably expressed the TIF2 antisense mRNA, acquired enhanced TNF susceptibility. Calculated EC50 values, i.e., the amount of TNF needed for killing 50% cells, are 10, 55, and 1.5 ng/ml, respectively, for vector control, sense transfectant, and antisense transfectant. TGF-beta 1 rapidly induces TIF2 gene expression (approximately 1 hr), which correlates with time-related acquisition of TNF-resistance in TGF-beta 1-treated L929 cells. Notably, TIF2 gene expression is markedly increased in human breast cancer and lymphoid leukemia cells, compared to normal human cells, suggesting its potential role in cancer development. Together, the anti-apoptotic function of TIF2 is responsible in part for TGF-beta-mediated protection of L929 cells against TNF cytotoxicity.


Subject(s)
Apoptosis/genetics , Cell Adhesion Molecules/genetics , Gene Expression Regulation/drug effects , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/pharmacology , Amino Acid Sequence , Animals , Humans , Mice , Molecular Sequence Data , Sequence Analysis
18.
Obes Res ; 6(4): 292-8, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9688106

ABSTRACT

OBJECTIVE: A variety of cell types transport cyclic AMP (cAMP) to the extracellular fluid; the purpose of this study was to determine if and how this process occurs in adipocytes. RESEARCH METHODS AND PROCEDURES: Adipocytes were isolated from 3-month-old swine and incubated with stimulators of adenylate cyclase for 2 to 120 minutes to promote cAMP synthesis and efflux. Efflux was characterized in the presence of agents that inhibit ATP production, anion transport, intracellular cAMP metabolism, and extracellular cAMP metabolism. Extracellular cAMP was measured by enzyme immunoassay, then corrected for cell lysis by measuring lactate dehydrogenase release. RESULTS: cAMP efflux averaged 24.7 fmol/min/cm2 adipocyte surface area, was linear for 2 hours, and was proportional to adipocyte surface area (r=0.94, p<0.05). Efflux was reduced by approximately 35% in cells incubated with 1 microM antimycin, an inhibitor of ATP synthesis (p<0.05), and by approximately 55% in cells incubated with 2 mM probenecid, an anion-specific transport blocker (p<0.05). Extracellular cAMP levels more than doubled by the addition of 1 microM 1,3-dipropyl-8-p-sulfophenylxanthine, a purported inhibitor of extracellular phosphodiesterase. DISCUSSION: Our data demonstrate that cAMP is transported from swine adipocytes by an energy-dependent anion transporter and can be metabolized extracellularly. Future studies will evaluate extracellular cAMP as a potential source of extracellular adenosine, a potent inhibitor of adipocyte lipolysis.


Subject(s)
Adipocytes/metabolism , Cyclic AMP/metabolism , Adipocytes/drug effects , Adrenergic beta-Agonists/pharmacology , Animals , Antimycin A/analogs & derivatives , Antimycin A/pharmacology , Cells, Cultured , Colforsin/pharmacology , Isoproterenol/pharmacology , Probenecid/pharmacology , Quinolones/pharmacology , Swine , Swine, Miniature , Xanthines/pharmacology
19.
Med Sci Sports Exerc ; 29(11): 1437-43, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9372479

ABSTRACT

The swine has many similarities to humans, making it an excellent research model in which to study the role of exercise on lipid metabolism. Swine adapt to exercise-training by increasing muscle oxidative enzymes, maximal stroke volume, cardiac output, VO2max, and high density lipoprotein cholesterol levels, while decreasing total cholesterol levels and resting heart rate. The lipoprotein profile of swine and humans is also similar, and low density lipoprotein is the major cholesterol transporting lipoprotein in both species. Several studies in swine report conflicting results on the effect of exercise-training on lipoprotein profile and atherosclerotic lesion appearance. This may result from differences in total exercise time between the studies. With sufficient total exercise, atherosclerosis was reduced and high density lipoprotein cholesterol levels were increased. Exercise may also play a role in reducing obesity, a risk factor for cardiovascular disease, by enhancing lipid mobilization from adipocytes. Recent research suggests that swine adipocyte sensitivity to adenosine, a locally-produced antilipolytic agent, is reduced after exercise treatment. Cellular mechanisms responsible for this metabolic change include a reduction in adenosine A1 receptor number. Current studies are examining the transport of extracellular cyclic AMP from adipocytes and its role as a potential adenosine precursor.


Subject(s)
Arteriosclerosis/etiology , Cardiovascular Diseases/etiology , Disease Models, Animal , Lipid Metabolism , Physical Conditioning, Animal/physiology , Swine/physiology , Animals , Arteriosclerosis/prevention & control , Cardiovascular Diseases/prevention & control , Cholesterol, HDL/blood , Humans , Obesity/physiopathology , Receptors, Purinergic P1/physiology , Risk Factors
20.
J Anim Sci ; 75(9): 2368-77, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9303455

ABSTRACT

Obese and crossbred (X-Bred) pigs were removed from sows at 14 d of age and given ad libitum access to a high-fat, milk-based diet. A subset of pigs fed this diet were killed at 28 and 49 d of age. At 28 d, another subset of pigs was given ad libitum access to a low-fat, grain-based diet and were killed at 31, 35, and 49 d of age (nutritionally weaned for 3, 7, and 21 d, respectively). Dorsal subcutaneous adipose tissue was obtained at death and adipocytes were prepared by incubation with collagenase. A portion of the cells was fixed with osmium to determine size and number; remaining cells were lysed in hypotonic media and centrifuged to yield a crude membrane fraction. The beta-adrenergic receptor (beta-AR) and A1 adenosine receptor (A1R) affinity and number were measured in the membranes by equilibrium saturation ligand binding. Obese pigs had a lower body weight than X-Bred pigs at all ages (P < .05). Obese pigs tended (P for volume > .1 but < .2) to have larger adipocytes than X-Bred pigs. The beta-AR affinity did not differ between obese and X-Bred pigs. There were fewer beta-AR per milligram of membrane protein in obese than in X-Bred pigs at 28 and 49 d of age when fed the high-fat, milk-based diet (P < .01). However, beta-AR number expressed per cell or unit cell surface area did not differ between genetic groups. As pigs of either genetic group continued to be fed the low-fat, grain-based diet, the beta-AR decreased when expressed per milligram of protein or unit cell surface area (P < .05) and tended to decrease when expressed per cell. Obese and X-Bred pigs fed the high-fat, milk-based diet had more beta-AR than respective pigs fed the low-fat, grain-based diet when data were expressed per milligram of protein (P < .01) but not when expressed per cell or unit surface area. The A1R were only detectable in 2 of 16 X-Bred litters but were more developed in adipocytes of obese pigs, being measurable in 8 of 14 litters. The A1R number, expressed per milligram of protein, was lower in obese pigs fed the milk-based diet than in those fed the grain-based diet (P < .05). These findings suggest the decreased beta-AR number after nutritional weaning, or the transition from a high-fat to low-fat diet, may contribute to fat accretion in pigs. Furthermore, the lower number of beta-AR in obese than in X-Bred pigs may contribute to the obesity.


Subject(s)
Adipocytes/chemistry , Animal Nutritional Physiological Phenomena , Receptors, Adrenergic, beta/analysis , Receptors, Purinergic P1/analysis , Swine/physiology , Weaning , Adipocytes/cytology , Adipocytes/metabolism , Aging/metabolism , Aging/physiology , Analysis of Variance , Animals , Body Weight/physiology , Breeding , Diet/veterinary , Dietary Fats/pharmacology , Female , Lipid Metabolism , Male , Obesity/metabolism , Obesity/physiopathology , Obesity/veterinary , Rabbits , Receptors, Adrenergic, beta/metabolism , Receptors, Purinergic P1/metabolism , Swine/genetics , Swine/metabolism , Swine Diseases/metabolism , Swine Diseases/physiopathology , Swine, Miniature
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