مجال التميز
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تميز دراسي و بحثي
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البحوث المنشورة
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البحث (1):
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عنوان البحث:
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Mass
spectrometric determination of early and advanced glycation in biology
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رابط إلى البحث:
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Click here
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تاريخ النشر:
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07/06/2016
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موجز عن البحث:
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Protein glycation in
biological systems occurs predominantly on lysine, arginine and Nterminal
residues of proteins. Major quantitative glycation adducts are found at mean
extents of modification of 1 – 5 mol percent of proteins. These are
glucose-derived fructosamine on lysine and N-terminal residues of proteins,
methylglyoxal-derived hydroimidazolone on arginine residues and
Nε-carboxymethyl-lysine residues mainly formed by the oxidative degradation
of fructosamine. Total glycation adducts of different types are quantified by
stable isotopic dilution analysis liquid chromatography-tandem mass
spectrometry (LCMS/ MS) in multiple reaction monitoring mode. Metabolism of
glycated proteins is followed by LC-MS/MS of glycation free adducts as minor
components of the amino acid metabolome. Glycated proteins and sites of
modification within them – amino acid residues modified by the glycating
agent moiety – are identified and quantified by label-free and stable isotope
labelling with amino acids in cell culture (SILAC) high resolution mass
spectrometry. Sites of glycation by glucose and methylglyoxal in selected
proteins are listed. Key issues in applying proteomics techniques to analysis
of glycated proteins are: (i) avoiding compromise of analysis by formation,
loss and relocation of glycation adducts in pre-analytic processing; (ii)
specificity of immunoaffinity enrichment procedures, (iii) maximizing protein
sequence coverage in mass spectrometric analysis for detection of glycation
sites, and (iv) development of bioinformatics tools for prediction of protein
glycation sites. Protein glycation studies have important applications in
biology, ageing and translational medicine – particularly on studies of
obesity, diabetes, cardiovascular disease, renal failure, neurological
disorders and cancer. Mass spectrometric analysis of glycated proteins has yet
to find widespread use clinically. Future use in health screening, disease
diagnosis and therapeutic monitoring, and drug and functional food
development is expected. A protocol for high resolution mass spectrometry
proteomics of glycated proteins is given.
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المؤتمرات العلمية:
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المؤتمر (1):
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عنوان المؤتمر:
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7th Saudi
Students’ Conference UK
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تاريخ الإنعقاد:
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01/02/2014
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مكان الإنعقاد:
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Edinburgh,
UK
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طبيعة المشاركة:
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Poster
presentation
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عنوان المشاركة:
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Dicarbonyl
Stress and the Glyoxalase System in Periodontal Ligament Fibroblasts in vitro
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ملخص المشاركة:
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Periodontal
ligament (PDL) inflammation or periodontitis is a common disease
characterized by gradual destruction of connective tissue fibres that attach
a tooth to the alveolar bone. Diabetes and inflammation enhances periodontal
bone loss through enhanced resorption and diminished bone formation. PDL
fibroblast attachment and function to type 1 collagen is impaired by
methylglyoxal (MG) modification in vitro. Glyoxalase 1 (Glo1) is the main
enzyme catalysing the metabolism of MG in PDL fibroblasts. It is hypothesised
that increased PDL detachment and dysfunction by MG may occur in biochemical
dysfunction in hyperglycaemia and by increased exposure to exogenous MG by
ingestion of high MG content food and beverages. The aim of this study was to
evaluate the effects of low and high glucose concentrations and exogenous MG
on the glyoxalase system in human periodontal ligament fibroblasts (hPDLFs)
in vitro. Primary hPDLFs were cultured for three days with high glucose (25
mM) and low (8 mM) glucose to mimic hyperglycaemic conditions. Glo1 activity
was determined by measuring the initial rate of formation of
S-D-lactoyl-glutathione from the hemithioacetal substrate formed
non-enzymatically from methylglyoxal and reduced glutathione (GSH).The
reaction is followed spectrophotometrically at 240 nm; Δε240 = 2.86 mM−1
cm−1. D-glucose consumption and D-lactate formation were determined using
end-point enzymatic assay spectrophotometrically at 340 nm and (λexcitation
340 and λemission 460 nm) respectively.
Dicarbonyl
content and protein damage markers of medium and cell protein were determined
by stable isotopic dilution analysis using LC-MS/MS. There was a 49% decrease
of glyoxalase 1 activity .
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المؤتمر (2):
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عنوان المؤتمر:
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8th Saudi
Students’ Conference UK
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تاريخ الإنعقاد:
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31/01/2015
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مكان الإنعقاد:
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London,
UK
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طبيعة المشاركة:
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Poster
presentation
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عنوان المشاركة:
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Dicarbonyls
stress in periodontal ligament fibroblasts is corrected by Resveratrol in
model hyperglycaemia in vitro
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ملخص المشاركة:
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Background
and aim Diabetes and inflammation enhances periodontal bone loss through
enhanced resorption and diminished bone formation. It has been shown that
human PDL fibroblast (hPDLF) attachment and function to type1 collagen was
impaired by methylglyoxal (MG) modification in vitro. We found that increased
hPDLFsdetachment and dysfunction was partly mediated by increased exposure to
high glucose concentration with increased formation of MG, decreased
metabolism of MG by glyoxalase1(Glo1) and increased formation of advanced
glycation endproducts(AGEs). The aim of this study was to evaluate the effect
of resveratrol (RES) on MG metabolism in hPDLFs in vitro in high and low
glucose concentration. Materials and methods hPDLFs were cultured for three
days in medium containing low glucose concentration(LG, 8mM) and also high
glucose concentration (HG, 25 mM) to model hyperglycaemic conditions. The
effect of addition of RES (10 μM) was studied. The activity of Glo1,
D-glucose consumption and D-lactate formation were measured. The
concentrations of MG and MG-derived AGE, MG-H1, were quantified by stable
isotopic dilution analysis LC-MS/MS. Results There was a significant decrease
in Glo1 activity in HG (527±97 versus 930 ± 85 mU/mg protein.
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المؤتمر (3):
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عنوان المؤتمر:
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9th Saudi Students’ Conference UK
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تاريخ الإنعقاد:
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19/02/2016
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مكان الإنعقاد:
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Birmingham,
UK
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طبيعة المشاركة:
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Poster
presentation
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عنوان المشاركة:
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The
effect of resveratrol on dicarbonyl stress in periodontal ligament
fibroblasts in model hyperglycaemia. An in vitro study
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ملخص المشاركة:
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Periodontal
ligament inflammation (periodontitis) is a common disease characterized by
gradual destruction of connective tissue fibres that attach a tooth to the
alveolar bone within which it sits. Diabetes and inflammation enhances
periodontal bone loss through enhanced resorption and diminished bone
formation. PDL fibroblast attachment to type 1 collagen and function was
impaired by methylglyoxal (MG) modification in vitro. We hypothesise that
increased PDL detachment and dysfunction may be exacerbated by increased
endogenous MG in hyperglycemia associated with diabetes. The aim of this
study was to evaluate the effects of high and low glucose concentrations on
MG metabolism in human periodontal ligament fibroblasts (hPDLFs) with and
without resveratrol (RSV) treatment Primary hPDLFs were cultured for three
days in medium containing low glucose (LG, 8 mM) and high glucose
concentration (HG, 25 mM) to model hyperglycaemic conditions with and without
RSV (10 μM). The activity of Glo1, D-glucose consumption and D-lactate
formation were measured. The concentrations of MG and MG-derived AGE, MG-H1,
were quantified by stable isotopic dilution analysis using LC-MS/MS. The
efficiency of hPDLFs adhesion to collagen type 1 was tested by colorimetric
cell adhesion assay. In hPDLFs incubated with HG there was a significant
decrease in Glo1 activity, increase in D-lactate flux, increase in cellular
concentration of MG and increase in MG-H1 residue content of cell protein,
compared to LG control. Decrease of Glo1 activity and increase in MG and
MG-G1 residue content of cell protein was corrected with RSV treatment. The
results are summarised in the table below. Analyte Low glucose (8 mM) High
glucose (25 mM) High glucose +RSV (10μM) Glo1 activity (mU/mg protein) 928 ±
84 529 ± 109** 964 ± 87ooo D-Lactate (nmol per million cells / day) 7.00 ±
0.36 9.90 ± 1.08** 9.78 ± 0.78 *** MG (pmol / million cells) 3.98 ± 1.00 10.6
± 1.07 6.98 ± 0.89oo MG-H1 (mmol/mol arg) 0.431 ± 0.061 0.806 ± 0.184 * 0.560
± 0.088oo Data are mean ± SD, n = 4. Significance: *, ** and ***.
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المؤتمر (4):
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عنوان المؤتمر:
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Midlands
Academy of Medical Sciences Festival
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تاريخ الإنعقاد:
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15/04/2016
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مكان الإنعقاد:
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Birmingham, UK
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طبيعة المشاركة:
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Poster
presentation
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عنوان المشاركة:
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Resveratrol
corrects dicarbonyl stress in periodontal ligament fibroblasts in model
hyperglycaemia in vitro
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ملخص المشاركة:
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Periodontal ligament
inflammation (periodontitis) is a common disease characterized by gradual
destruction of connective tissue fibres that attach a tooth to the alveolar
bone within which it sits. Diabetes and inflammation enhances periodontal
bone loss through enhanced resorption and diminished bone formation. PDL
fibroblast attachment to type 1 collagen and function was impaired by methylglyoxal
(MG) modification in vitro. We hypothesise that increased PDL detachment and
dysfunction may be exacerbated by increased endogenous MG in hyperglycemia
associated with diabetes. The aim of this study was to evaluate the effects
of high and low glucose concentrations on MG metabolism in human periodontal
ligament fibroblasts (hPDLFs) with and without resveratrol (RSV) treatment.
Primary hPDLFs were
cultured for three days in medium containing low glucose (LG, 8 mM) and high
glucose concentration (HG, 25 mM) to model hyperglycaemic conditions with and
without RSV (10 μM). The activity of Glo1, D-glucose consumption and
D-lactate formation were measured. The concentrations of MG and MG-derived
AGE, MG-H1, were quantified by stable isotopic dilution analysis using
LC-MS/MS. The efficiency of hPDLFs adhesion to collagen type 1 was tested by
colorimetric cell adhesion assay.
In hPDLFs incubated
with HG there was a significant decrease in Glo1 activity, increase in D-lactate
flux, increase in cellular concentration of MG and increase in MG-H1 residue
content of cell protein, compared to LG control. Decrease of Glo1 activity
and increase in MG and MG-G1 residue content of cell protein was corrected
with RSV treatment. The results are summarised in the table below.
Analyte Low glucose
(8 mM) High glucose (25 mM) High glucose +RSV (10μM) Glo1 activity (mU/mg
protein) 928 ± 84 529 ± 109** 964 ± 87ooo D-Lactate (nmol per million cells /
day) 7.00 ± 0.36 9.90 ± 1.08** 9.78 ± 0.78 *** MG (pmol / million cells) 3.98
± 1.00 10.6 ± 1.07 6.98 ± 0.89oo MG-H1 (mmol/mol arg) 0.431 ± 0.061 0.806 ±
0.184 * 0.560 ± 0.088oo Data are mean ± SD, n = 4. Significance: *.
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