Læknablaðið : fylgirit - 05.01.2015, Blaðsíða 69

X V I I V Í S I N D A R Á Ð S T E F N A H Í
F Y L G I R I T 8 2
LÆKNAblaðið/Fylgirit 82 2015/101 69
V 37 Determination of the kinetic parameters of human
gluconokinase using isothermal titration calorimetry
Neha Rohatgi1,2, Óttar Rolfsson1,2
1Center for Systems Biology, University of Iceland, 2University of Iceland Biomedical Center
ner1@hi.is
Introduction: Gluconate is a commonly encountered nutrient, yet the
molecular details concerning its metabolism in humans are unaccounted
for. A key enzyme in the degradation of gluconate (Glcn) is gluconok-
inase which acts to phosphorylate the hydroxyl group at C-6 of Glcn
generating 6-phosphogluconate. Here we report the kinetics of this
reaction catalyzed by recombinantly produced isoform I of human
gluconokinase (hGntK).
Methods and data: Isothermal titration calorimetry (ITC) was used to
study the characterisitics of the reaction catalyzed by hGntK. ITC works
by measuring the heat changes during a binding event or a reaction.
Results: The phosphorylation of Glcn by ATP is shown to be associated
with an enthalpy decrease corresponding to 8.27 ± 0.2 kcal/mol. At
saturating concentrations of Glcn (5 mM), the KM of ATP was 0.404 ±
0.096 mM and kcat of the reaction was 10.69 ± 1.32 s-1. At saturating ATP
concentration (5mM), the KM for Glcn was 0.173 ± 0.004 mM, the kcat of
the reaction was 16.6 ± 1.6 s-1 and Ki (inhibition constant for Glcn) was
2.36 ± 0.37 mM. It is proposed that the reaction follows Compulsory-
Ordered Ternary Complex mechanism, with ATP binding first. The
order of binding is also confirmed by doing binding studies with ITC,
which show ATP binds strongly to the enzyme while gluconate binds
with very low affinity to the enzyme in absence of ATP.
Conclusions: We were able to use ITC to biochemically characterize
human gluconokinase whose context specific role in metabolism rema-
ins to be defined.
V 38 Polysaccharide from Achillea millefolium has
immunomodulatory effects on THP-1 monocytes
Oddný T. Logadóttir1,2,3,4, Sesselja Ómarsdóttir5, Arnór Víkingsson3, Jóna
Freysdóttir2,3,4, Ingibjörg Harðardóttir1
1Biochemistry and Molecular Biology, Faculty of Medicine, Biomedical Center, University of
Iceland, 2Department of Immunology, Faculty of Medicine, Biomedical Center, University of
Iceland, 3Center for Rheumatology Research Landspítali University Hospital, 4Department of
Immunology, Landspítali University Hospital, 5Faculty of Pharmaceutical Sciences, University
of Iceland
othl1@hi.is
Introduction: Achillea millefolium has been used in traditional medicine
to heal inflamed cuts and alleviate rheumatism. Aqueous extract from
Achillea millefolium has been shown to have anti-inflammatory effects on
dendritic cells in vitro. The objective of this study was to use bioguided
fractionation to isolate a polysaccharide from Achillea millefolium that has
immunomodulatory effects on THP-1 monocytes and to determine how
it affects intracellular signalling in the cells.
Methods and data: Bioguided fractionation using ion-exchange chroma-
tography was used to isolate a polysaccharide from Achillea millefolium.
THP-1 monocytes were primed with IFN-γ and then stimulated with
LPS in the absence or presence of the polysaccharide. The cells were
cultured for 48 h and cytokine concentration in the supernatant determ-
ined by ELISA. Phosphorylation of several intracellular kinases was
determined by Western blotting after one hour stimulation of the cells in
the presence or absence of the polysaccharide.
Results: The water-soluble compound isolated from Achillea millefolium
is most likely a polysaccharide with a molecular weight around 270 kDa.
THP-1 cells incubated with the polysaccharide increased IL-12p40 secre-
tion by around 80% and IL-10 secretion by around three fold, thus dec-
reasing the IL-12p40/IL-10 ratio by around 40%. THP-1 cells incubated
with the polysaccharide also secreted more IL-1β, IL-8, IL-23 and TNF-α
than cells incubated without the polysaccharide. The polysaccharide
also led to less phosphorylation of the ERK1/2 and Akt kinases.
Conclusions: The polysaccharide from Achillea millefolium increased
secretion of both pro- and anti-inflammatory cytokines, possibly dec-
reasing phosphorylation of the Akt kinase.
V 39 Pneumococcal carriage in healthy children and children with
upper
Páll Guðjónsson1, Helga Erlendsdóttir1,2, Sigríður Júlía Quirk1,2, Martha Á.
Hjálmarsdóttir1,2, Ásgeir Haraldsson1,3, Karl G. Kristinsson1,2
1Faculty of Medicine, University of Iceland, 2Department of Clinical Microbiology, Landspítali
University Hospital, 3Landspítali University Hospital
pag5@hi.is
Introduction: S. pneumoniae is a common pathogen colonizing the
nasopharynx of humans. The difference in carriage between healthy
children and children with upper respiratory tract infections (URTI)
usually remains speculative. The aim of the study was to compare
serotypes and antibiotic susceptibilities of pneumococci in healthy
carriage and children with URTI.
Methods and data: A comparison was made between isolates collected
during carriage studies of children attending day care centres in
2009-2012 (DCC group), and nasopharyngeal clinical samples from
children with upper respiratory tract infections (CS group) submitted
to Landspitalinn during the same period. Serotypes were determined
using latex agglutination and/or multiplex PCR. Comparisons were
made between children of the same age.
Results: There were 1281 isolates from children in the DCC group and
770 in the CS group. The five most common serotypes in DCC were 23F,
6A, 19F, 6B and non-typable and in the CS group 19F, 6A, 23F, 6B and
15B/C. The difference was significant for 6B (p=0,044), 14 (p=0,036), 19F
(p<0.0001), 23F (p=0.01) and non-typable (p<0.0001) isolates. Penicillin
non-susceptible pneumococci were more common in CS (265 isolates,
34%) compared DCC (171 isolate, 14%, p<0.0001). Multiresistant S.
pneumoniae was also more common in CS (33% of the isolates) comp-
ared to DCC (14%, p<0.0001).
Conclusions: There was a significant difference in carriage of the main
serotypes between the two study groups and a significantly higher
resistance rate in the CS group, probably because certain serotypes are
more likely to cause respiratory tract infections and infected children are
more likely to have received antibiotics.
V 40 Samlegðaráhrif meðferðar með TNFα hemli og
metótrexat við sóragigt
Björn Guðbjörnsson1,2, Stefán P. Jónsson3, Pétur S. Gunnarsson3,4, Anna I.
Gunnarsdóttir3,4, Þorvarður J. Löve1,5
1Læknadeild Háskóla Íslands, 2rannsóknastofu í gigtarsjúkdómum við Landspítala,
3lyfjafræðideild Háskóla Íslands, 4sjúkrahúsapóteki, 5vísinda- og þróunarsviði Landspítala
thl@hi.is
Inngangur: Sjúkdómsdempandi áhrif metótrexats og TNFα hemla við
sóragigt er vel þekkt, en takmarkaðar upplýsingar eru til um samhliða-
meðferð með þessum lyfjum við sóragigt. Markmið þessarar rann-
sóknar var því að skoða samlegðaráhrif meðferðar með TNFα hemli og