Induction and rescue from apoptosis by glucocorticoids Whereas glucocorticoids (GCs) induce activation-induced cell
death (AICD) in activated human T cells, pretreatment of activated T cells
is protective of AICD when these cells are reactivated. Our results demonstrate
that anti-proliferative concentrations of GCs accelerated AICD in mitogen-stimulated
T cells. The specificity of GCs effects was demonstrated by the failure
of non-GCs steroids to affect AICD and by the capacity of GCs receptor
antagonist, RU-486, to abrogate GCs effects. GC-induced enhancement of
AICD was not due to modulation of bcl-2 expression, since GCs failed to
affect bcl-2 mRNA expression. We are currently addressing the mechanism
of action of GCs in modulating AICD, specifically with regard to GC effects
on anti-apoptotic (bcl-xL) and pro-apoptotic (bcl-xS and p 53) gene products.
This is of significance in relation to a better understanding of the mechanism
of action of GCs with regard to their regulation of cellular viability
and entry into the cell cycle. W. Y. Almawi. (Supported by AUB Medical
Practice Plan; LNSCR.)
Association of altered T cell immunity with insulin-dependent diabetes mellitus (IDDM) Aberrant T cell immunity is the hallmark of several autoimmune
diseases, including IDDM. The purpose of this study was to investigate
the status of T cell immunity in IDDM patients at distinct stages of the
disease. PBMC were isolated from acute and chronic IDDM patients and sex
and age-matched healthy subjects. PBMC proliferation was stimulated with
mitogens, receptor cross-linking antibodies, and cytokines in conjunction
with anti-CD3 mAb or PMA, and assessed by measuring the cellular uptake
of tritiated thymidine; cytokine mRNA expression was determined by RT-PCR.
Whereas the proliferation of lymphocytes from acute IDDM patients were
significantly lower than those of controls and chronic IDDM patients to
all stimulation regimen, the proliferative responses of lymphocytes from
chronic IDDM patients were either significantly higher than those of healthy
controls, or unchanged, depending on the nature of the stimulation regimen
tested. In essence, the obtained results underscore the differential regulation
of T cell activation in IDDM, in relation to the time of post-disease onset.
W.Y. Almawi, H. N. Beyhum and S.T. Azar.
(Supported by LNCSR.)
Upregulation of cytokine receptor expression by glucocorticoids Insofar as glucocorticoids (GCs) inhibit cytokine expression
and upregulate the expression of cytokine receptors on target cells, we
tested the effect of GCs pretreatment on cytokine/cytokine receptor expression
and T cell proliferation. Whereas GCs inhibited cytokine mRNA expression
and T cell proliferation, pretreatment of activated T cell with GCs resulted
in enhanced accumulation of cytokine mRNA upon reactivation. Changes in
mRNA expression paralleled a concentration-dependent increase in IL-1R,
IL-2R, and IL-6R expression, and augmentation in T cell proliferation.
This was specific for GCs, and was blocked by the GCs receptor antagonist
RU-486. Furthermore, GCs-induced rebound was not stimulus-specific, and
was not associated with alteration in apoptosis. Taken together, our results
underline the dual effects of GCs in regulating T cell activation and cytokine
expression. In essence, GCs directly inhibit T cell proliferation by inhibiting
cytokine production and, via enhancement of cytokine receptor expression,
pretreatment by GCs augments T cell proliferation. W. Y. Almawi, N. A.
Jaffal and M. J. Rieder*. (Supported by AUB
Medical Practice Plan; URB.)
Regulations of cytokine gene expression and T cell proliferation by the Na-K-2Cl cotransporter The Na-K-2Cl cotransporter regulates several aspects of T
cell activation, and is inhibited by the loop diuretics bumetanide and
furosemide. In this study, we examined the effect of bumetanide and furosemide
on cytokine gene expression and proliferation of human T cells. Bumetanide
and furosemide, at concentrations that inhibited Na-K-2Cl activity (86Rb
uptake), blocked IL-2, IL-4, IFN-gamma, and TNF-alpha steady state mRNA
expression and protein secretion, and profoundly inhibited T cell proliferation
stimulated by mitogens and receptor cross-linking antibodies. Bumetanide-mediated
anti-proliferative effects were not the result of induction of and/or acceleration
of activation-induced cell death (apoptosis) in activated T cell cultures.
Whereas bumetanide blocked cytokine steady-state mRNA expression, it failed
to suppress T cell activation stimulated by IL-2, thereby indicating that
bumetanide affects proximal events of T cell activation. Our data underline
the importance of the Na-K-2Cl cotransporter in regulating early events
of T cell activation. W. Almawi, M. J. Reider*, R. D. Feldman* and A. Karnoub.
(Supported by the Department of Biochemistry.)
In vivo stimulation of crypt cells protein kinase C by ergocalciferol and cycloheximide The effect of ergocalciferol (vit D2)
on the activity of intestinal protein Kinase C (PKC) and the possible role
of this enzyme on the differentiation of crypt cells was examined. Crypt
cells were isolated from rat intestine following intramuscular injection
of vit D2 PKC specific activity
was determined from the rate of incorporation of 32P
- ATP into protamine. Vit D2
(60 m
g/200 g of body Wt) resulted in 30% activation of PKC specific activity.
Optimal effect was observed within 72 hours of injection. Cycloheximide
blocked the activation but when injected alone stimulated endogenous PKC
by 34%, 24 hours after injection. Our students tentatively rule out a PKC
dependent differentiation of Crypt to Villi regardless of Vit D2
effect. I. F Durr and J. Usta. (Supported by LNCSR.)
Molecular characterization of X chromosome derived markers associated with Turner syndrome Turner
syndrome is characterized by a wide spectrum of physical features including
short stature, gonadal dysgenesis, lymphedema, webbed neck, broad chest,
cubitus valgus, nail dysplasia, renal structural anomalies, and cardiac
malformations. Nearly 50% of patients with Turner syndrome have a mosaic
condition with one 45,X cell line and a second cell line with a normal
or a structurally rearranged X or Y chromosome. However, individuals with
a small X derived chromosome may have additional abnormalities uncharacteristic
of Turner syndrome. These additional abnormalities are due to the loss
of the X Inactivation Center, resulting in two active copies of pericentromeric
genes. We were presented with several cases of Turner mosaics with structurally
abnormal X chromosomes. We intend to map the X derived abnormal chromosomes
in these patients using the FISH technique and attempt to assess their
X-inactivation status. This should aid in genetic counseling and may contribute
to igenes responsible for mental retardation and dysmorphic features.
N. Mogharbel, L. Zahed, S. Najjar, M. Shoucair, M. Saleh and
R. Slim. (Supported by AUB Medical Practice Plan.)
Evidence for the presence of new genes responsible for Usher syndrome type I and II in the Lebanese population The
Usher syndrome (MIM 276900, 276901) is an autosomal recessive disorder
causing congenital hearing loss and progressive retinitis pigmentosa resulting
in a definitive deaf-blind condition. This severe disease affects approximately
3.6/100,000 in the general population. The Usher syndrome is clinically
and genetically heterogeneous. So far genes responsible for the Usher syndrome
have been mapped to seven loci; among them only the gene responsible for
the Usher syndrome type IB is cloned. In the last two years we have collected,
from Lebanon and from other Middle Eastern countries, 10 families having
Usher patients (5 Usher type I families and 5 Usher type II families).
Segregation analysis of alleles at loci surrounding each of the known Usher
genes demonstrated no linkage of 4 families (1 type I and 3 type II) to
any of the known loci. These results demonstrated the presence of at least
2 additional new genes responsible for Usher syndrome. M. Saoud,
A. Mansour, M. Moustapha*, A. Megarbane*, E. El Zir*and
R. Slim. (Supported by LNCSR; URB; AUB Medical Practice Plan.)
Refinement of the Pendred syndrome candidate region by homozygosity analysis Pendred
syndrome is an autosomal recessive disease characterized by congenital
sensorineural deafness and goiter. The gene responsible for the Pendred
syndrome has been mapped to chromosome 7q31, in a 5.5-centiMorgan interval
flanked by D7S501 and D7S523. This interval was recently refined to 1.7-cM
interval located between D7S501 and D7S692. A large Lebanese family was
genotyped with 8 microsatellite markers located between D7S501 and D7S523.
Our data showed a complete co-segregation with the disease locus for all
analyzed loci, which further supports the locus homogeneity of the Pendred
syndrome and its close linkage to this region. Haplotype analysis allowed
us to narrow down the Pendred syndrome candidate region facilitating, therefore,
the construction of a physical map of this region and the identification
of candidate genes. R. Slim, M. Moustapha*, S. Azar, Y. Bou Moglabey,
M. Saouda, G. Zeitoun and J. Loiselet*. (Supported by LNCSR
and AUB Medical Practice Plan.)
Purification of a copper-stimulated adenosine triphosphatase Purification
of the copper-stimulated ATPase enzyme from rat liver is now in progress
using: detergent solubilization; gel filtration; isoelectric focusing and
polyacrylamide gel electrophoresis. The purified protein will then be sequenced
and compared to the sequence deduced from the cloned gene. (Ongoing project.)
J. Usta, H. Barakeh and N. Cortas. (Supported
by AUB Medical Practice Plan; URB.)
Isolation and characterization of a copper stimulated adenosine triphosphatase activity. A novel P-type ATPase. Cloning
of Wilson’s disease gene showed that it encodes a putative copper activated
ATPase not yet identified. We have isolated a liver membrane fraction rich
with a Mg++ requiring
copper activated ATPase with an apparent half maximal activation of 20
m
M and an apparent maximal velocity of 63.44 ±
16.10 m
mole Pi/mg Protein/hr. Copper activation curve best fits Hill kinetics
with a coefficient n =
1.6 suggesting that at least two Cu binding sites exhibiting positive cooperativity
exist. K+ had no effect on Cu-ATPase activity. Substituting
chloride with nitrate or sulfate salts of copper resulted in no change
in Cu-ATPase activity. Substituting Cu++ with Zn++,
Mn++ or Co++ did not stimulate ATPase activity above
basal activity. On the other hand cadmium resulted in an ATPase activity
which is about 50% of that produced by copper. We therefore have identified
a specific Cu++ ATPase in rat liver, established its assay and
substrate kinetics. J. Usta, H. Barakeh, H. Mahfouz and
N. Cortas. (Supported by Department of Biochemistry.)
Cu-ATPase in rat kidney: effect of thiols Wilson’s
disease is characterized by excessive copper accumulation in liver, kidney
and brain. We have recently isolated and characterized Cu-ATPase activity
in liver. The activity of Cu-ATPase in rat kidney is now being investigated.
Kinetic parameters Km and Vmax of kidney Cu-ATPase will be determined and
compared to those of liver to establish whether isozymes exit. The role
of reduced and oxidized thiols on the activity of the enzyme will be tested.
This helps in elucidating the mechanistic role of the cysteine-rich metal
binding domains on the activity. (Ongoing project). J. Usta,
I. Chakroun and N. Cortas. (Supported by URB, AUB Medical
Practice Plan.)
Electro spray ionization-mass spectrometry (ESI-MS) of mitochondrial subunit C The
recent development of ESI-MS and its applicability to protein analysis
made it possible to determine the stoichiometry of substrate binding, metabolic
intermediates and post-translational modifications of proteins. The properties
of subunit C (a mitochondrial component of ATP Synthetase of major role
in energy conservation) was investigated by developing a rapid micro-extraction
procedure, yielding a chloroform / methanol (C/M) extract suitable for
ESI-MS analysis. Results show: (1) an occasional component of relative
molecular mass (RMM) = 7608 Da; (2) a component of RMM = 7650 Da arising
from an extra 42.1 Da adduct due to oxidation or acetylation of methionine
residue in subunit C. The addition of Na+/K+ to C/M
extract led to formation of Na+/K+ adduct of subunit
C. The role of this on the mechanism of ATP synthetase and Fo proton translocation
remains to be established. J. Usta, D. E. Griffiths* and
K. Jennings*. (Supported by Department of Chemistry, University
of Warwick, UK.)
Subcellular localization of copper-stimulated adenosine triphosphatase activity in rat liver Copper
is an essential trace element in eukaryotic cells, functioning as a cofactor
for a variety of enzymes. The lethal effects of hepatic copper accumulation
and hence toxicity characterizes Wilson’s disease. The predicted amino
acid sequence of the cDNA clone of Wilson’s gene showed several transmembrane
spanning regions, Cu and ATP binding sites. Though transport studies using
radio active copper have shown that Cu transport is ATP dependent, there
is no conclusion yet regarding the site of cellular defect in copper transport
or metabolism. Hence, the objective is to determine the intracellular localization
of Cu-ATPase using the enzymatic assay developed in our laboratory. Systematic
isolation of plasma (Canalicular, Basolateral), mitochondrial and lysosomal
membranes followed by checking Cu-ATPase activity is now being investigated.
(Ongoing project.) J. Usta, A. Rahme and N. Cortas.
(Supported by URB; AUB Medical Practice Plan.)
Almawi, W., Beyhum, H.N., Rahme, A.A., and Reider, M.J.*, Regulation of cytokine and cytokine receptor expression by glucocorticoids. Journal of Leukocyte Biology, 60 (3), 563-572, 1996. Almawi, W.Y., Saouda, M.S., Stevens, A.C.*, Lipman, M.L.*, Barth, C.M.*, and Strom, T.B.*, Partial mediation of glucocorticoid anti-proliferative effects by lipocortins. Journal of Immunology, 157 (2), 5231-5239, 1996. Griffiths, D.E.*, Millar, A.*, Usta, J. and Jennings, K.R.*, ESI-MS studies of heart mitochondrial subunit C. Biochemical Society Transactions, 25, 387S ,1997. Hess D.A.*, Bird I.A.*, Almawi W.Y., and Rieder M.J.*, The hydroxylamine of sulfamethoxazole synergizes with FK506 and cyclosporin A in inhibiting T cell proliferation. Journal of Pharmacology and Experimental Therapeutics, 281 (1), 540-548, 1997. Li, X.-C.*, Almawi, W., Jevnikar, A.*, Tucker, J.*, Zhong, R.* and Grant, D.*, Allogeneic lymphocyte proliferation stimulated by small intestine-derived epithelial cells. Transplantation, 60 (1), 82-89, 1995. Stevens, C.*, Lipman, M.*, Fabry, S.*, Moscovitch-Lopatin, M.*, Almawi W., Keresztes, S.*, Peppercorn, M.A.* and Strom, T.B.*, 5-Aminosalicylic acid abrogates T cell proliferation by blocking interleukin-2 production in peripheral bmononuclear cells. Journal of Pharmacology and Experimental Therapeutics, 272 (1), 399-406, 1995. Usta,
J. and Durr, I.F., Ergocalciferol and cycloheximide in vivo stimulate
protein kinase C of intestinal crypt cells. International Journal of
Biochemistry and Cell Biology, 28, 91-95, 1996.
ABSTRACT, PRESENTATIONS AND PROCEEDINGS Almawi, W.Y., Paradoxical regulation of cytokine and cytokine receptor expression by glucocorticoids. Proceedings of the Ninth International Congress of Immunology, San Francisco, USA, 1995. ———, Effect of FK506 on the production of and signaling by IL-7 in human T cells. Proceedings of the American Society of Transplant Physicians Meeting, Chicago, USA, 1995. Almawi, W. Y., Beyhum, H. N., Kaidbey, H. H., Nuwayri-Salti, N. and Azar, S. Dysregulation of cytokine expression in diabetes. Proceedings of the American Society for Biochemistry and Molecular Biology/American Society for Ivestigative Pathology/American Association of Immunologists, Joint Meeting, New Orleans, USA, 1996. Almawi, W.Y. and Jaffal, N.A. Pretreatment with glucocorticoids enhance T cell activation and cytokine expression. Journal of Allergy and Clinical Immunology, 99, S399 (1623), 1997. Beyhum, H.N., Azar, S.T. and Almawi, W.Y. Alterations of T cell activation and cytokine expression in IDDM. Proceedings of the American Academy of Allergy, Asthma and Immunology/American Association of Immunologists/Clinical Immunology Society, Joint Meeting, San Francisco, USA. Journal of Allergy and Clinical Immunology, 99 (1922), 1997. Beyhum, H.N., Azar, S., Nuwayri-Salti, N. and Almawi, W.Y. Association of altered T cell immunity with the onset and progress of type I diabetes. Proceedings of the Tenth Annual Clinical Immunology Society Meeting, New Orleans, USA, 1996. Karnoub, A.E., Kaidbey, H. and Almawi, W.Y. Inhibition of T cell proliferation and cytokine gene expression by bumetanide, and inhibitor of the Na-K-Cl contransporter. Proceedings of the ASBMB/ASIP/AAI Joint Meeting, New Orleans, USA, 1996. ———, Regulation of T cell activation by the Na-K-2Cl contransporter. Proceedings of the Tenth Annual Clinical Immunology Society Meeting, New Orleans, USA, 1996. Rahme, A.A., Kaidbey, H.H. and Almawi, W.Y. Induction of and rescue from activation induced cell death in activated T cell by glucocorticoids. Proceedings of the ASBMB/ASIP/AAI Joint Meeting, New Orleans, USA, 1996. Usta, J., Barakeh, H. and Cortas, N., A biochemical evidence for a copper stimulated ATPase activity in rat liver plasma membrane. Comparative Aspects of translocating ATPases, Seventh Federation of Asian and Oceanic Biochemists and Molecular Biologists, Congress Leura, Blue Mountains, NSW, Australia. 29 Sep. - 2 Oct. 1995. Usta,
J., Barakeh, H., Mahfouz, H., and Cortas, N., Isolation and kinetic characterization
of copper stimulated adenosine Triphosphatase from rat liver. Eighth
International Conference on the Na/K-ATPase (and related transport
Na/K-ATPases), Mar del Plata, Argentina. August 26-30, 1996.
Barakeh, H., Isolation and characterization of a rat liver copper activated ATPase: A putative candidate of Wilson’s Disease gene product (1996). Advisor: J. Usta. Beyhum, H., Association of altered T cell immunity with insulin-dependent diabetes mellitus (1997). Advisor: W. Almawi. Karnoub, A., Inhibition of a cytokine gene expression and T cell proliferation by bumetanide, a select inhibitor of the Na-K-2CI cotransporter (1996). Advisor: W. Almawi.
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