303 research outputs found
3D Telomeric Fingerprint of Advanced Cutaneous T-Cell Lymphoma: A Pilot Study
No full textOncogenic events of cutaneous T-cell lymphomas (CTCLs) progression remain elusive. Telomere remodeling, a manifestation of genetic instability, is associated with progression of some malignancies. We aim to characterize the three-dimensional (3D) telomeric organization in CTCLs. We performed 3D telomeric quantitative FISH (3D Telo-q-FISH) of skin tissue of 9 patients with CTCL and control lymphocytes. Reported parameters included telomeres of low intensity (TLI), number and intensity of telomeric signals, telomere aggregates and nuclear volume. Stratification was based on CD30 expression and clinical stage. CTCLs had more TLIs than controls (27% vs 16%, p<0.0001). TLI proportion was higher in CD30-high cells than CD30-low cells (34% vs 22%, p<0.0001) and in the advanced group compared to the early group (30% vs 24%, p<0.0001). CD30 expression and advanced stage were associated with larger nuclei and more telomeres and advanced stage cells had significantly more aggregates. We show that 3D Telo-QFISH is feasible in small skin biopsies with limited tissue, and we report evidence that advanced CTCLs are associated with an increased proportion of TLIs, a hallmark feature in many tumor cells. Our analysis suggests that CTCL cells undergo in a first step telomere shortening and loss compared to healthy controls. In a second step further telomeric shortening associated with chromosomal rearrangements and Breakage-fusion-Bridge cycles may be involved in the progression of CTCL.THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV
The adenomatous polyposis coli protein unambiguously localizes to microtubule plus-ends and is involved in establishing parallel arrays of microtubule bundles in highly polarised epithelial cells
No full textLoss of full-length adenomatous polyposis coli (APC) protein correlates with the development of colon cancers in familial and sporadic cases. In addition to its role in regulating ß-catenin levels in the Wnt signaling pathway, the APC protein is implicated in regulating cytoskeletal organization. APC stabilizes microtubules in vivo and in vitro, and this may play a role in cell migration (Näthke, I.S., C.L. Adams, P. Polakis, J.H. Sellin, and W.J. Nelson. 1996. J. Cell Biol. 134:165–179; Mimori-Kiyosue, Y., N. Shiina, and S. Tsukita. 2000. J. Cell Biol. 148:505–517; Zumbrunn, J., K. Inoshita, A.A. Hyman, and I.S. Näthke. 2001. Curr. Biol. 11:44–49) and in the attachment of microtubules to kinetochores during mitosis (Fodde, R., J. Kuipers, C. Rosenberg, R. Smits, M. Kielman, C. Gaspar, J.H. van Es, C. Breukel, J. Wiegant, R.H. Giles, and H. Clevers. 2001. Nat. Cell Biol. 3:433–438; Kaplan, K.B., A. Burds, J.R. Swedlow, S.S. Bekir, P.K. Sorger, and I.S. Näthke. 2001. Nat. Cell Biol. 3:429–432). The localization of endogenous APC protein is complex: actin- and microtubule-dependent pools of APC have been identified in cultured cells (Näthke et al., 1996; Mimori-Kiyosue et al., 2000; Reinacher-Schick, A., and B.M. Gumbiner. 2001. J. Cell Biol. 152:491–502; Rosin-Arbesfeld, R., G. Ihrke, and M. Bienz. 2001. EMBO J. 20:5929–5939). However, the localization of APC in tissues has not been identified at high resolution. Here, we show that in fully polarized epithelial cells from the inner ear, endogenous APC protein associates with the plus ends of microtubules located at the basal plasma membrane. Consistent with a role for APC in supporting the cytoskeletal organization of epithelial cells in vivo, the number of microtubules is significantly reduced in apico-basal arrays of microtubule bundles isolated from mice heterozygous for APC. © 2002 Mogensen et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). </p
Molecular epidemiology of meningococcal disease in Northern Ghana
No full textMeningococcal disease remains a major public health concern, especially in the
African Meningitis Belt where large meningitis epidemics with attack rates of up to
500/100,000 recur every 8-12 years. The factors precipitating epidemics are largely
unknown. Epidemics are therefore unpredictable which often leads to control
measures being initiated too late to be effective. Following a major meningitis
epidemic that occurred in northern Ghana in 1997, a collaborative research project
was developed between the Swiss Tropical Institute and the Navrongo Health
Research Center, in order to address several research questions relevant to the
epidemiology of meningococcal disease in Ghana. This research partnership built the
framework of the present thesis, which concentrated on the molecular epidemiological
aspects of the project. During the dry season of 1998, there was a second meningitis outbreak in the
Kassena-Nankana district (KND) of northern Ghana. All suspected meningitis
patients were recruited at the local health facilities, lumbar punctures carried out
before treatment and the cerebrospinal fluid (CSF) specimen sent to the field
laboratory for analysis. In 50 of 92 CSF samples analyzed, serogroup A Neisseria
meningitidis were detected. All serogroup A N. meningitidis isolates recovered were
of the A:4:P1.9,20 phenotype. Analysis of representative isolates by multilocus
sequence typing (MLST) and by restriction fragment length polymorphism (RFLP) of
opa, iga and ingA genes showed that they belonged to subgroup III (sequence type 5)
of N. meningitidis and had RFLP patterns characteristic of serogroup A subgroup III
bacteria isolated in Africa after the 1987 Mecca epidemic. RFLP analysis of six
polymorphic loci in a global collection of 502 isolates of subgroup III, serogroup A N.
meningitidis identified nine ‘genoclouds’, consisting of genotypes that were isolated
repeatedly, plus 48 less frequent descendent genotypes. Starting during the second outbreak, a series of five 6-monthly carriage surveys
of 37 randomly selected households were carried out in KND. As serogroup A N.
meningitidis carriage decreased, that of X meningococci increased dramatically to
reach 18% (53/298) of the people sampled during the dry season of 2000. This
coincided with a further outbreak of disease, this time caused by serogroup X. The
Ghanaian serogroup X strains were analyzed by MLST and pulsed-field gel
electrophoresis (PFGE) along with other serogroup X isolates from different countries of Africa, Europe and North America. The European and American isolates were
highly diverse. However, one clonal grouping was identified among sporadic disease
and carrier strains isolated over the last two decades in the UK, The Netherlands,
Germany and the USA. In contrast to the diversity among the European and American
isolates, most carrier and disease isolates recovered in Ghana and other countries of
the African Meningitis Belt over the last thirty years belong to a second clonal
grouping. Based on the PFGE results, two genoclouds were identified within the
second clonal grouping, one of which caused an outbreak in Niger in 1997 and the
other of which caused the outbreak in KND in 2000. Patterns of carriage of N. lactamica in KND were unrelated to those of N.
meningitidis. Non-serogroupable (NG) strains of N. meningitidis were infrequent.
This contrasts with industrialized countries where asymptomatic nasopharyngeal
carriage of N. meningitidis is common and up to 50% of the strains carried are NG. The nine genoclouds of subgroup III meningococci have caused three pandemic
waves of disease since the mid-1960’s, with the 1997-8 outbreaks in KND forming
part of the second wave. The third wave was imported from East Asia to Europe and
Africa in the mid-1990s, and may well lead to renewed epidemic serogroup A disease
in Europe and the Americas. The finding that a serogroup X meningococcal clonal
grouping has caused outbreaks in Africa, supports concerns that polysaccharide
vaccines, which have been in use for more than a decade might be selecting for nonvaccine
serogroups and argues for the development of a comprehensive conjugate
vaccine including serogroup X polysaccharide. The dynamics of meningococcal
carriage that were observed in KND suggest that in the African meningitis belt, the
populations become colonized in waves of different meningococcal strains, and the
occurrence of epidemics of disease depends on the virulence of these strains. Carriage
of NG meningococci may protect against meningococcal disease by eliciting crossreactive
immunity against pathogenic strains and the low levels of carriage of such
organisms in the African meningitis belt may thus increase susceptibility to
epidemics
Synthesis and rearrangement of silylated oxy-cope systems
No full textAddition of allyl or vinyl organometallic reagents to chiral a,b- or b,g-unsaturated acylsilanes afforded stereoselectively 3-silylated 3-hydroxy-1,5-dienes that are stereoselectively converted to d,e-unsaturated acylsilanes by the thermal oxy-Cope rearrangement. The rearrangement is restricted to compounds possessing an (alkoxy)methyl substituent at the silicon moiety; upon heating, analogous compounds with the t-BuMe2Si group in the 3-position led to decomposition only. The (alkoxy)methyl group at silicon is supposed to act as a weak internal base, which accelerates the rearrangment reaction
Synthesis of -pyrenolide B
No full textIn the synthesis of the title compound 12, the important intermediate 7 was obtained in good yield from the easily available ethyl 5,5-ethylenedioxy-2-oxocyclohexane1 --c arboxylate (1) via ring enlargement of the bicyclic enol ether 5 (Scheme). Its reduction (NaBH,, CeCI, in EtOH) and subsequent protection with (t-Bu)Me2Si resulted in the highly functionalized ten-membered lactone 9. Introduction of the (2)-configurated double bond, followed by deprotection and elimination of H20, gave (A)-pyrenolide B (12) in 16% overall yield
Wnt/beta-catenin/Tcf signaling: A critical pathway in gastrointestinal tumorigenesis
No full textCancers of the gastrointestinal tract, including the liver, bile ducts, and pancreas, constitute the largest group of malignant tumors. Colorectal cancer is one of the most common neoplastic diseases in Western countries and one of the leading causes of cancer-related deaths. Inactivation of the adenomatous polyposis coli (APC) tumor-suppressor gene during early adenoma formation is thought to be the first genetic event in the process of colorectal carcinogenesis followed by mutations in oncogenes like K-Ras and tumor-suppressor genes like p53. Identification of the interaction of APC with the proto-oncogene beta-catenin has linked colorectal carcinogenesis to the Wnt-signal transduction pathway. The main function of APC is thought to be the regulation of free beta-catenin in concert with the glycogen synthase kinase 3beta (GSK-3beta) and Axin proteins. Loss of APC function, inactivation of Axin or activating beta-catenin mutations result in the cellular accumulation of beta-catenin. Upon translocation to the nucleus beta-catenin serves as an activator of T-cell factor (Tcf)-dependent transcription leading to an increased expression of several specific target genes including c-Myc, cyclin D1, MMP-7, and ITF-2. While APC mutations are almost exclusively found in colorectal cancers, deregulation of Wnt/beta-catenin/Tcf signaling is also common in other gastrointestinal and extra-gastrointestinal human cancers. In a fraction of hepatocellular carcinomas the Writ pathway is deregulated by inactivation of Axin or stabilizing mutations of beta-catenin. The majority of hepatoblastomas and a group of gastric cancers also carry beta-catenin mutations. Clearly, this pathway harbors great potential for future applications in cancer diagnostics, staging, and therapy. Copyright (C) 2002 S. Karger AG, Basel
Author response: Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9
No full textStereoselective preparation of 2-silylated 1,3-diols and the regioselectivity of their Peterson olefination
No full textThe reduction of the carbonyl group of ct-silylated aldols with complex hydrides was shown to proceed with high stereoselectivity. The center of chirality in the ¢t-position to the ketone, at the C-atom where the silicon group is attached, usually dominated the stereochemical control of the reaction. The presence of the ~-hydroxy functionality, however, also seems to be necessary for a high degree of selectivity. Peterson olefination of 2-silylated 1,3-diols afforded stereoselectively (E)-configured allylic alcohols as the major products. With KH as the base, the reaction proceeds predominantly in a syn-fashion, preferring to eliminate a syn- rather than an ant/-configured 13-hydroxysilane unit. Under 'silico-nucleophilic' conditions (OH- or F-), an anti-configured ~-hydroxysilane moiety can also be eliminated in an anti-fashion. This reaction is strongly preferred over the corresponding syn-elimination, but is still less prominent than a competitive syn-elimination of a syn-configured 13-hydroxysilane uni
Evaluation of the hCMEC/D3 cell line, a new "in vitro" model of the human blood-brain barrier for transport and gene regulation studies
No full textBrain endothelial capillary cells form the blood-brain barrier (BBB), a highly selective
membrane between the peripheral blood and the central nervous system. The main
functions of the BBB are to protect the brain tissue by preventing the entry of toxic
compounds and to supply it with nutrients in order to assure proper function. Tight
junctions are the key elements for the establishment of a tight barrier and seal the
intercellular gaps against passive diffusion of hydrophilic compounds. A second important
characteristic of the brain capillary endothelial cells are transport proteins that prevent
brain penetration of their substrates by pumping them back in the blood. These
compounds include a series of clinically used drugs. Important drug efflux transporters
located at the BBB are P-glycoprotein (P-gp), the breast cancer resistance protein
(BCRP) and the family of multidrug resistance proteins (MRP).
During drug development, the question of whether a drug candidate reaches the brain
tissue is of great importance. Therefore, models are needed to predict the BBB
permeability of new compounds. In the past, in vitro models have been developed to
address this question. These models include isolated brain capillaries, isolated primary
brain capillary endothelial cells and BBB cell lines of various origins. A major problem
encountered with these cell lines was an insufficient paracellular resistance.
Recently, the hCMEC/D3 cell line was generated by immortalizing primary human brain
endothelial cells. In culture this cell line shows a morphology that closely resembles to
primary cells, forms tight monolayers and expresses BBB markers such as chemokine
receptors, tight junctional molecules and ATP binding cassette (ABC)-transporters.
The aim of this thesis was to evaluate the hCMEC/D3 cell line as an in vitro model of the
human BBB to study 1) permeability properties including para- and transcellular diffusion
as well as active transport 2) the influence of endo- and exogenous factors on the
paracellular permeability and 3) the regulation of breast cancer resistance protein and Pglycoprotein
by pro-inflammatory cytokines.
The first study describes the characterization of the hCMEC/D3 cells as an in vitro model
of the human BBB for permeability studies (section Error! Reference source not
found.). The ability of the cells to allow discrimination between para- and transcellular
diffusion was investigated by measuring the transport of a series of compounds with
different physicochemical properties. A ratio of 2.8 was observed when comparing the
permeabilities of the compounds with the highest and the lowest diffusion rate. The
passive permeability of sucrose could be reduced significantly by replacing fetal calf
serum with human serum. Furthermore, quantitative mRNA expression of the ABCtransporters
P-gp, BCRP, MRP1, MRP2, MRP3, MRP4, MRP5 as well as the human
transferrin receptor (hTfR) was shown. Protein expression of P-gp, BCRP and the hTfR
was detected and functional activity of P-gp, BCRP and the MRPs was investigated in
efflux experiments. Furthermore, bidirectional P-gp transport activity was observed.
In a second project the impact of endo- and exogenous factors on the paracellular
permeability of hCMEC/D3 monolayers was assessed, since it is know that the molecular
assembly of tight junctions depends on the surrounding milieu (section Error! Reference
source not found.). Based on reports in the literature, the cells were incubated with a
variety of compounds that included anti-inflammatory drugs, growth factors and
antioxidants. The effects on the monolayer tightness of hCMEC/D3 were investigated by
measuring the transport of sucrose, a paracellular permeability marker. N-acetylcystein
(NAC), atorvastatin and sodium nitroprusside (SNP) reduced the sucrose permeability
significantly, and slightly increased zonula occludens protein (ZO-1) expression.
Additionally, NAC and SNP reduced the generation of reactive oxygen species (ROS),
which have been reported to disrupt the assembly of tight junctions.
The effect of the pro-inflammatory cytokines IL-1[beta], IL-6 and TNF-[alpha] on the expression and
activity of the ABC-transporters BCRP and P-gp was investigated in the hCMEC/D3 cell
line (section Error! Reference source not found.). IL-1[beta], IL-6 and TNF-[alpha], which are
know to be elevated during various diseases, suppressed significantly BCRP mRNA
expression. In addition, BCRP activity was reduced under the influence of all tested
cytokines, as shown by efflux experiments. P-gp mRNA levels were slightly reduced by
IL-6 but significantly increased after TNF-[alpha] treatment. TNF-[alpha] also increased the protein
expression of P-gp. This in vitro study indicates that expression levels of BCRP and P-gp
at the BBB might be altered during acute or chronic inflammation, resulting in a changed
brain penetration of their substrates.
In an isolated project, the pharmacokinetics and pharmacodynamics of increasing oral
doses of the satiety peptides GLP-1 and PYY3-36 were assessed in healthy male
volunteers. Oral administration of either peptide induced a rapid and dose-dependent
increase in plasma drug concentrations. Oral administration of GLP-1 induced a potent
effect on insulin release and both peptides suppressed ghrelin secretion. In conclusion,
this study showed, for the first time, that satiety peptides such as GLP-1 and PYY3-36
can be orally delivered safely and effectively in humans
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