Læknaneminn - 01.10.1995, Page 133

ABSTRAKTAR
several cell types. This happens through a cannabinoid receptor, by an
unknown pathway, causing an increase in internal Ca2+. Aortic rings
were used to investigate if cannabinoid and/or AA receptors are
expressed on the aorta and whether AA might cause Ca2+ entry and
therefore tissue contraction when given exogenously by binding to the
cannabinoid receptor.
Materials and Methods: The aorta of guinea pigs was cut into 2-
3 mm rings and contraction measured. After precontracting the rings
in a buffer containing 40 mM KCI, the drugs were given to the rings in
accumulative doses, from 10-10 M to 10-4 M, and the contractile
response recorded, calculated as a percentage of the response with KCl
and expressed as a mean curve + SEM. The experiment was also
carried out in a Ca2+ free buffer, in order to investigate whether the
drugs had any effect on Ca2+ mobilization from intracellular stores.
Finally, the interaction of the compounds was studied by subsequently
applying the drugs to the aortic rings.
Results: Arachidonic acid, d9-THC and anandamide all caused a
biphasic response, the first part of the response reaching equilibrium at
10-7 M, where the mean response of d9-THC was 53% + 18,
anandamide 41% + 9, and for AA 19% + 8% of the KCl response. The
half maximal values (EC50) were estimated to be 3 x 10-9 M, 5 x 10-9
M, and 3 x 10-9 M respectively. These results suggest that they are all
binding to a receptor, saturated at 10-7 M concentration. Concentrations
higher than 10-6 M caused an almost linear increase in concentration,
most likely due to an aspecific effect of the drugs at higher
concentrations. In Ca2+ free buffer, the response with 10-8 M d9-THC
was 15% + 14, anandamide 11% + 3, AA 0 at 10-8 M but 9% + 2% at
10-6 M, suggesting that the effect of the drugs is mainly on Ca2+ entry.
Because of thc aspecific effect it was not possible to conclude whether
the compounds are competing on the same receptor at nanomolar
concentrations.
Discussion: In conclusion, according to these results cannabinoid
receptors and AA receptors are expressed on the aorta of guinea pigs
and can be stimulated in the nanomolar range to cause contraction
mainly via Ca2+ influx mechanism.
IN SITU HYBRIDIZATION WITH
DIGOXIGENIN-LABELLED RNA PROBES.
Optimizing the Method for Application in Testicular Tissue Sections
Elisabet S. Guömundsdóttir* 1.
Karsten Kristiansen2 * 4.
'LHÍ, 2Department ofMolecular Biology.University ofOdense
Introduction: Testicular germ cell tumors (TGCTs) represent
about 1-2% of all male human malignant tumors. The peak incidence is
between the ages 20 and 34 years and it is the most common
malignancy in men in that age group. The nature of genetic lesions and
molecular mechanisms underlying the pathogenesis of TGCTs is largely
unknown. Mutations in the p53 tumor suppressor gene have been
shown not to occur in testicular tumors, but it appears to be expressed at
abnormally high levels. The MDM2 gene is a cellular oncogene and
the MDM2 protein can bind to and inactivate p53. The WAFl gene
encodes a protein that can suppress the growth of human tumor cells.
Wild-type p53 can induce the transcription of MDM2 and WAFl. This
study was undertaken to investigate the expression patterns of p53,
MDM2 and WAFl genes in human TGCTs. For this analysis I was
going to use in situ hybridization (ISH) with digoxigenin-labelled RNA
probes.
Materials and Methods: For optimization of the method, tissue
sections from normal rat testis were used. ACBP and RXR RNA
probes which are known to be expressed in normal rat testis were
prepared. The plasmid cDNA templates were purified and after
linearization with restriction enzymes, in vitro transcription witli
digoxigenin-labelled dUTP was performed. After various
pretreatments, hybridization was carried out overnight. Detection of thc
digoxigenin-labelled probes was achieved by incubation with peroxidase
conjugated anti-digoxigenin antibodies followed by addition of a
mixture of H202 and 3,3-diaminobenzidinehydrochloride.
Results: The result is a method that can be used as originally
intended. In the pretreatment procedure incubation with proteinase K
was found to be optimal with a concentration of 0. lg/ml. Optimal probe
concentration was 20ng/l. It was necessary to treat sections with 20g/ml
RNase A after hybridization. Vigorous high stringency washings were
found to be unnecessary. Finally, specific hybridization signals were
obtained for RXR and ACBP where control sections showed no
staining.
Discussion: ISH with digoxigenin-labelled RNA probes is a
sensitive technique for detection of mRNA in cells. The method has
many variables and requires optimization before satisfactory results are
obtained. My work allows the following conclusions to be made: (a)
proteinase K treatment in order to increase accessibility of target RNA is
the most important pretreatment step and must be optimized for each
new tissue block to be used; (b) higher probe concentrations arc required
for dig-labelled probes than what is advised for radioactive probes; (c)
RNase A treatment is necessary to reduce background and; (d) vigorous
high stringency posthybridization washings are unnecessary and lead to
diminution of the signal.
HIV-1 BÓLUEFNI; ÁHRIF MISMUNANDI
BÓLUSETNINGARAÐFERÐA Á MÓTEFNASVÖRUN
Elisabet Revkdal Jóhannesdóttir1.
Dr. Sveinbjörn Gizurarson2.
1LHÍ, 2Lyfjafrœöi lyfsala LHÍ.
Inngangur: Var aó kanna áhrif mismunandi bólusetningaraðferða
á tegund og styrk mótefna í mismunandi líffærum.
Brýn þörf er á virku og öruggu bóluefni gegn útbreiðslu HIV
veirunnar sem í árslok 1994 hafði m.a. lagt alls 91 íslending að velli.
Einstaklingsbreytileiki(genetic variability) HIV veirunnarer mjög mikill
og af þeim sökum hefur þróun bóluefna einna helst beinst gegn
yfirborössameindum hennar, gpl20 og gp41, sem eru nauðsynlegar
fyrir viðloöun og samruna viö hýsilfrumuna. HIV veiran getur sýkt
frumur og orðið þögul án þess aö ónæmiskerfió verði þess vart og þegar
hún virkjast, skert starfsemi þess. Vegna þessa og fjölda annarra
eiginleika veirunnar þykir nokkuð víst að cf bóluefni ætti að koma í veg
fyrir sjúkdóm yrði þaö aö stööva veiruna strax viö slímhimnur, en þar
er aðal inngönguleiö veirunnar í líkamann.
Rannsóknir hafa sýnt fram á að líkaminn búi yfir sérstöku
slímhúðarónæmiskerfi. Þetta kerfi hefur nýlega fengiö aukna athygli í
tengslum við smitsjúkdóma og framleiðslu nýrra bóluefna.
Slímhúöarónæmiskerfinu má skipta anatómískt og starfrænt í tvö svæði,
mucosal inductive og mucosal effector svæói. Inductive svæðin
samanstanda af bronchial-associated lymphoreticular tissue, nasal-
associated lymphoreticular tissue og gut-associated lymphoreticular
tissue (BALT, NALT og GALT). Þar verða fyrstu kynnin við
mótefnavakann, sem leióa til virkjunar á T-hjálparfrumum og IgA
sérhæfðum B-frumum, sem rata sérhæft til effector svæða, sem mynduð
eru m.a. af lamina propria meltingarvegs, efri öndunarvega og þvag-og
kynfæra og útkirtlum. Þar verður svo vækis-sérhæft S-IgA svar sem er
einn mikilvægasti þátturinn í vernd slímhúða gegn innrás sýkla.
Efniviður og aðferðir: Rannsóknarhópurinn samanstóð af 30
karlkyns BALB/c músum, sem var svo skipt jafnt í sex hópa, (1-6). I
upphafi tilraunarinnar fengu hópar 1 og 2 bóluefni undir húð, hópar 3 og
4 fengu sama skammt af bóluefni í nef, hópur 5 fékk helmingin af
skammtinum undir húð en restina í nef og hópur 6 fékk bóluefnið um
munn(buccalt). Mýsnar voru endurbólusettar á degi 35, þá fengu hópar
LÆKNANEMINN 2. tbl. 1995 48. árg.
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