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Choroid plexus, located in brain ventricles, is the site of blood-cerebrospinal fluid barrier that contains endothelial cells and an epithelial monolayer separated by stroma. We established a two-cell-type model of the human choroid plexus consisting of immortalized endothelial cells (iHCPEnC) and epithelial papilloma (HIBCPP) cells grown on opposite sides of filter supports. In this protocol, we describe the preparation of this model, the measurement of transepithelial electrical resistance (TEER), and immunofluorescence imaging-based analysis to determine the barrier function.

MEIS1 is a three–amino acid loop extension class homeodomain-containing homeobox (HOX) cofactor that plays key roles in normal hematopoiesis and leukemogenesis. Expression of Meis1 is rate-limiting in MLL-associated leukemias and potently interacts with Hox and NUP98-HOX genes in leukemic transformation to promote self-renewal and proliferation of hematopoietic progenitors. The oncogenicity of MEIS1 has been linked to its transcriptional activation properties. To further reveal the pathways triggered by Meis1, we assessed the function of a novel engineered fusion form of Meis1, M33-MEIS1, designed to confer transcriptional repression to Meis1 target genes that are otherwise up-regulated in normal and malignant hematopoiesis. Retroviral overexpression of M33-Meis1 resulted in the rapid and complete eradication of M33-Meis1–transduced normal and leukemic cells in vivo. Cell-cycle analysis showed that M33-Meis1 impeded the progression of cells from G1-to-S phase, which correlated with significant reduction of cyclin D3 levels and the inhibition of retinoblastoma (pRb) hyperphosphorylation. We identified cyclin D3 as a direct downstream target of MEIS1 and M33-MEIS1 and showed that the G1-phase accumulation and growth suppression induced by M33-Meis1 was partially relieved by overexpression of cyclin D3. This study provides strong evidence linking the growth-promoting activities of Meis1 to the cyclin D-pRb cell-cycle control pathway.

MEIS1 is a three–amino acid loop extension class homeodomain-containing homeobox (HOX) cofactor that plays key roles in normal hematopoiesis and leukemogenesis. Expression of Meis1is rate-limiting in MLL-associated leukemias and potently interacts with Hoxand NUP98-HOXgenes in leukemic transformation to promote self-renewal and proliferation of hematopoietic progenitors. The oncogenicity of MEIS1 has been linked to its transcriptional activation properties. To further reveal the pathways triggered by Meis1, we assessed the function of a novel engineered fusion form of Meis1, M33-MEIS1, designed to confer transcriptional repression to Meis1target genes that are otherwise up-regulated in normal and malignant hematopoiesis. Retroviral overexpression of M33-Meis1resulted in the rapid and complete eradication of M33-Meis1–transduced normal and leukemic cells in vivo. Cell-cycle analysis showed that M33-Meis1impeded the progression of cells from G1-to-S phase, which correlated with significant reduction of cyclin D3levels and the inhibition of retinoblastoma (pRb) hyperphosphorylation. We identified cyclin D3as a direct downstream target of MEIS1 and M33-MEIS1 and showed that the G1-phase accumulation and growth suppression induced by M33-Meis1was partially relieved by overexpression of cyclin D3. This study provides strong evidence linking the growth-promoting activities of Meis1to the cyclin D-pRbcell-cycle control pathway.

Mycophenolic acid (MPA) is a potent inhibitor of the inosine monophosphate dehydrogenase and used as an immunosuppressive drug in transplantation. MPA inhibits proliferation of T- and B-lymphocytes by guanosine depletion. Since fibroblasts rely on the de novosynthesis of guanosine nucleotides, it is assumed that MPA interacts with fibroblasts causing an increased frequency of wound healing problems. We show a downregulation of the cytoskeletal proteins vinculin, actin and tubulin in fibroblasts exposed to pharmacological doses of MPA using microarray technology, real-time polymerase chain reaction (PCR) and Western blot. This reduction in RNA and protein content is accompanied by a substantial rearrangement of the cytoskeleton in MPA-treated fibroblasts as documented by immunofluorescence. The dysfunctional fibroblast growth was validated by scratch test documenting impaired migrational capacity. In contrast, cell adhesion was increased in MPA-treated fibroblasts. The results of the cultured human fibroblasts were applied to skin biopsies of renal transplant recipients. Skin biopsies of patients treated with MPA expressed less vinculin, actin and tubulin as compared to control biopsies that could explain potential wound healing problems posttransplantation. The perspective of MPA-induced cytoskeletal dysfunction may go beyond wound healing disturbances and may have beneficial effects on (renal) allografts with respect to scarring.

Mycophenolic acid (MPA) is a potent inhibitor of the inosine monophosphate dehydrogenase and used as an immunosuppressive drug in transplantation. MPA inhibits proliferation of T- and B-lymphocytes by guanosine depletion. Since fibroblasts rely on the de novo synthesis of guanosine nucleotides, it is assumed that MPA interacts with fibroblasts causing an increased frequency of wound healing problems. We show a downregulation of the cytoskeletal proteins vinculin, actin and tubulin in fibroblasts exposed to pharmacological doses of MPA using microarray technology, real-time polymerase chain reaction (PCR) and Western blot. This reduction in RNA and protein content is accompanied by a substantial rearrangement of the cytoskeleton in MPA-treated fibroblasts as documented by immunofluorescence. The dysfunctional fibroblast growth was validated by scratch test documenting impaired migrational capacity. In contrast, cell adhesion was increased in MPA-treated fibroblasts. The results of the cultured human fibroblasts were applied to skin biopsies of renal transplant recipients. Skin biopsies of patients treated with MPA expressed less vinculin, actin and tubulin as compared to control biopsies that could explain potential wound healing problems posttransplantation. The perspective of MPA-induced cytoskeletal dysfunction may go beyond wound healing disturbances and may have beneficial effects on (renal) allografts with respect to scarring.

Across the 42 counties of Romania, the total suicide rate and the population percentage of ethnic Hungarians were strongly positively interrelated (79% attributable variance). Counties with the strongest Hungarian minority had suicide rates converging to (or exceeding) the suicide rate for Hungary, which rate is high. Of a set of about 20 vital statistics and socioeconomic indicators, only life expectancy predicted a significant increment of further variance in the suicide rates. However, this effect was small, adding merely 3% further variance explained to 79% already accounted for. Overall, the findings are supportive of the Finno-Ugrian Suicide Hypothesis, i.e., the notion that geographic patterns of suicide prevalence may be partially due to genetic differences between populations. Supplemental analyses of a questionnaire item which specifically queried this study's main finding indicated widespread disbelief of this fact of suicide prevalence across a variety of samples, including two samples from Romania.

Hantavirus serotype Puumala(PUUV) is the etiologic agent of Nephropathia epidemica(NE), a mild variant of Hemorrhagic Fever with Renal Syndrome (HFRS) in Europe, with lethality rates up to 1% among human patients. The serotype PUUV is composed of numerous geographically restricted strains forming a great number of phylogeographic lineages, sublineages, variants, and clusters. We describe a new, geographically and phylogenetically well-defined pathogenic PUUV sublineage in Southwest Germany (strain Heidelberg/hu) originated from an HFRS patient. The genetic analysis of PUUV strain Heidelberg/hu may contribute in future to better diagnostics, the development of vaccines, and the understanding of the factors that determine hantavirus pathogenesis.

AbstractVon diesem Kommentar zum Muwa??a'des Malik b. Anas, der in der Erforschung der malikitischen Rechtsliteratur bisher weitgehend unbenutzt geblieben ist, sind nach meinen heutigen Kenntnissen nur zwei Handschriften erhalten. Ein Exemplar davon liegt in der ?izana al-?ammain Rabat unter der Katalognummer 64 ? (davon Mikrofilm, Nr. 911) und ist bis auf einige fehlende Seiten am Schluss vollständig. Die andere Handschrift habe ich 1998 in der Moscheebibliothek von Qairawan (Raqqada) unter mehreren ungeordneten Blättern und Heftresten unterschiedlicher Provenienz identifiziert; sie trägt bisher keine endgültige Katalognummer. Der Fund, wie so oft unerwartet und zufällig, bestätigt allerdings die vagen und nur wenig informativen, vor allem aber nicht mehr aktuellen Angaben im alten Sigillder Bibliothek aus dem Jahr 693/1294, in dem der Muwa??a'-Kommentar von al-Qanazi?i angeführt wird.

Degradation of misfolded or unassembled proteins that are co-translationally inserted into the endoplasmic reticulum involves the cytosolic proteasome system. Different principles may exist for the export of proteins into the cytosol for proteasomal degradation. Here we studied the degradation pathway of the viral glycoprotein gp48, a type I transmembrane protein, encoded by them06gene of murine cytomegalovirus. In cells stably transfected with the cytomegalovirus m06gene or infected with the virus itself, two populations of gp48 can be distinguished that have different fates. Complexes of gp48 and the major histocompatibility complex (MHC) class I molecule, are transported to the lysosome for degradation. Unassembled gp48 is degraded by the cytosolic proteasome. Proteasomal inhibitors stabilize the unassembled gp48 in its core-glycosylated and membrane-associated form in the endoplasmic reticulum (ER)-Golgi intermediate compartment. This implicates that both endoplasmic reticulum and ER-Golgi intermediate compartment export gp48 and that degradation is coupled to a functional proteasome. Analysis of gp48 mutants revealed that the cytosolic part of gp48 was not responsible for the proteasome-dependent substrate transport out of the ER-Golgi intermediate compartment. Thus an indirect interaction between the proteasome and its substrate has to be discussed.

ABSTRACTDuring synthesis and export of protein, the majority of the human immunodeficiency virus type 1 (HIV-1) Env glycoprotein gp160 is retained in the endoplasmic reticulum (ER) and subsequently ubiquitinated and degraded by proteasomes. Only a small fraction of gp160 appears to be correctly folded and processed and is transported to the cell surface, which makes it difficult to identify negative sequence elements regulating steady-state surface expression of Env at the post-ER level. Moreover, poorly localized mRNA retention sequences inhibiting the nucleocytoplasmic transport of viral transcripts interfere with the identification of these sequence elements. Using two heterologous systems with CD4 or immunoglobulin extracellular/transmembrane domains in combination with the gp160 cytoplasmic domain, we were able to identify two membrane-distal, neighboring motifs, is1(amino acids 750 to 763) andis2(amino acids 764 to 785), which inhibited surface expression and induced Golgi localization of the chimeric proteins. To prove that these two elements act similarly in the homologous context of the Env glycoprotein, we generated a synthetic gp160 gene with synonymous codons, the transcripts of which are not retained within the nucleus. In accordance with the results in heterologous systems, an internal deletion of both elements considerably increased surface expression of gp160.

ABSTRACTMany steps in the replication cycle of cytomegalovirus (CMV), like cell entry, capsid assembly, and egress of newly synthesized virions, have not been completely analyzed yet. In order to facilitate these studies, we decided to construct a recombinant CMV that incorporates the green fluorescent protein (GFP) into the nucleocapsid. A comparable herpes simplex virus type 1 (HSV-1) mutant has recently been generated by fusion of the GFP open reading frame (ORF) with the HSV-1 ORF encoding small capsid protein (SCP) VP26 (P. Desai and S. Person, J. Virol. 72:7563–7568, 1998). Recombinant CMV genomes expressing a fusion protein consisting of GFP and the SCP were constructed by the recently established bacterial artificial chromosome mutagenesis procedure. In transfected cells, the SCP-GFP fusion protein localized to distinct foci in the nucleus that may represent sites for capsid assembly (assemblons). However, no viable progeny was reconstituted from these mutant CMV genomes. CMV genomes with deletion of the SCP ORF also did not give rise to infectious virus. Rescue of the mutation by insertion of the SCP gene at an ectopic position in an SCP knockout genome indicates that, in contrast to the HSV-1 SCP, the CMV SCP is essential for viral growth. Expression of the SCP-GFP fusion protein together with the authentic SCP blocked the CMV infection cycle, suggesting that the SCP-GFP fusion protein exerts a dominant-negative effect on the assembly of new virions. The results of this study are discussed with regard to recently published data about the structure of the CMV virion and its differences from the HSV-1 virion.

Vitamin K‐dependent protein S, which is a cofactor for activated protein C and thus important for down‐regulation of the coagulation cascade, contains several Ca2+‐binding sites with unusually high affinity. The 89 amino acid fragment constituting the third and fourth epidermal growth factor‐like (EGF) modules of protein S is the smallest fragment that retains high‐affinity Ca2+binding and is therefore useful for investigating the structural basis of this property. Heteronuclear multidimensional nuclear magnetic resonance experiments were used to obtain extensive assignments of the 1H, 15N and 13C resonances of the module pair with one Ca2+bound in EGF 4. In addition, nearly complete assignments of the 1H resonances of the isolated Ca2+‐free EGF 3 module were obtained. The assignment process was complicated by broadening of several resonances, spectral heterogeneity caused by cis‐transisomerisation of the peptide bond preceding Pro‐168, and dimerisation. Analysis of weighted average secondary chemical shifts, 3JHNHαcoupling constants, and NOE connectivities suggest that both EGF modules in this fragment adhere to the classical secondary structure of EGF modules, consisting of one major and one minor anti‐parallel β‐sheet.

Calcium-binding epidermal growth factor (EGF)-like modules are found in numerous extracellular and membrane proteins involved in such diverse processes as blood coagulation, lipoprotein metabolism, determination of cell fate, and cell adhesion. Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme activated protein C, has four EGF-like modules in tandem with the three C-terminal modules each harbouring a Ca2+-binding consensus sequence. Recombinant fragments containing EGF modules 1–4 and 2–4 have two Ca2+-binding sites with dissociation constants ranging from 10−8to 10−5M. Module-module interactions that greatly influence the Ca2+affinity of individual modules have been identified. As a step towards an analysis of the structural basis of the high Ca2+affinity, we expressed the Ca2+-binding EGF pair 3–4 from human protein S. Correct folding was shown by 1H NMR spectroscopy. Calcium-binding properties of the C-terminal module were determined by titration with chromophoric chelators; binding to the low-affinity N-terminal site was monitored by 1H-15N NMR spectroscopy. At physiological pH and ionic strength, the dissociation constants for Ca2+binding were 1.0 × 10−6M and 4.8 × 10−3M for modules 4 and 3, respectively, i.e. the calcium affinity of the C-terminal site was about 5000-fold higher than that of the N-terminal site. Moreover, the Ca2+affinity of EGF 4, in the pair 3–4, was about 9000-fold higher than that of synthetic EGF 4. The EGF modules in protein S are known to mediate the interaction with factor Xa. We have now found modules 3–4 to be involved in this interaction. However, the individual modules 3 and 4 manifested no measurable activity.

Programme viewing gives an accurate picture of the interests and aspirations of the viewing household. With the advent of digital television, viewers' programme-watching patterns can be tracked and analysed and the opportunity will be in place to build new targeting systems which go a stage further than today's geodemographic, lifestyle or psychographic tools. This paper examines the significance of the development of digital television in the world of marketing and direct marketing, and will discuss how the datasets can be combined to provide a new super-powerful targeting system, building on today's foundations, and applicable throughout the marketing mix.

A novel Ca 2+binding protein, named caligulin, was extracted from the heat-treated 100 000 × gsupernatant of bovine brain and purified to electrophoretic homogeneity. The apparent Mrof caligulin determined on sodium dodecyl sulfate polyacrylamide gels was 24 000. Analysis by gel filtration chromatography indicated an apparent Mrof 33 000, suggesting a monomeric protein. Amino acid composition data demonstrated the presence of 25% acidic residues, 12% basic residues and 10% leucine. In the presence of 1 mM MgCl 2and 0.15 M KCl, caligulin bound 1 mol Ca 2+/mol protein with half-maximal binding at about 0.2 μM Ca 2+.

Autoantibodies against nucleolar components are a common serological feature of patients suffering from scleroderma, a collagen vascular autoimmune disease. An important target of these autoantibodies is a protein with an apparent molecular weight of 36 kDa and a pI value of 8.5, located in the dense fibrillar component of the nucleolus and therefore termed fibrillarin. Animal models in which abundant anti-nucleolar antibodies appear spontaneously have not yet been described; however, high levels of anti-fibrillarin antibodies can be induced by treating susceptible strains of mice with sub-toxic amounts of mercuric chloride. In this study, we have analysed the specificity of anti-fibrillarin autoantibodies of human and murine origin. Our results suggest that both species have similar, if not identical conformational epitopes that are the target of anti-fibrillarin autoantibodies; these epitopes require the presence of a 30-kDa fragment of the fibrillarin molecule. Post-translational modifications such as the dimethylation of arginines in the N terminus of the protein are not essential for antibody recognition.

Adult mice of strains C3Hf and XVII/G received 2, 4, 5-T by continuous oral administration (80 ppm in the diet). The 2, 4, 5-T preparation contained less than 0-05 ppm of dioxins. In 2, 4, 5-T -treated C3Hf mice a significant increase in the incidence of neoplastic lesions was found. No significant difference was found in the XVII/G strain between the treated and control mice. Rare forms of tumours, which were not observed in the controls, were present in the 2, 4, 5-t -treated C3Hf mice.

The human foamy or spumaretrovirus (HSRV) is a complex retrovirus that encodes the three retroviral genes gag, pol, and env and, in addition, at least three bel genes. The HSRV Bel-1 protein was identified as a transcriptional trans-activator. HSRV transcription starts in the 5' long terminal repeat at a defined guanine residue. We report here that a second efficiently utilized start site of transcription is contained within a HSRV env DNA sequence upstream of the bel genes. Bel-specific transcripts that initiate at the internal transcriptional start site at nt 9196 were identified in HSRV-infected cells by primer extension and S1 nuclease analysis, and the intragenic promoter was shown to be constitutively activated by Bel-1 in the HSRV provirus. In transient expression assays with indicator gene constructs, expression by the HSRV intragenic promoter/enhancer is Bel-1 dependent. The data provide evidence for an intragenic start site of transcription in the genome of a complex, exogenous human retrovirus and are discussed in terms of a model for regulating spumaretroviral gene expression.

Mapping of transcripts of the human foamy virus genome was carried out in permissive human embryonic fibroblast cells by Northern blot hybridization and S1 nuclease analysis. Since several splice sites that are localized within a relatively narrow genomic region were detected, the polymerase chain reaction (PCR) was employed, and cloning and sequencing of the splice site junctions of the corresponding viral cDNAs were subsequently performed. All spumavirus transcripts have a common but relatively short leader RNA. Genomic, singly spliced env mRNAs and several singly and multiply spliced subgenomic transcripts were identified. The multiply spliced viral mRNAs consist of various exons located in the central or 3' part of the viral genome. At least four novel gene products, termed Bet, Bes, Beo, and Bel3, are predicted to exist. The poly(A) addition site that defines the boundary of the R and U5 region in the 3' long terminal repeat was determined. The pattern of spumavirus splicing is more complex than that of oncoviruses and more similar to that of lentiviruses. One of the characteristic features of spumavirus transcription is the existence of singly spliced bel1 and bel2 mRNAs that alternatively are multiply spliced, thereby generating a complexity comparable to, but different from, that of lentiviruses and from that of other known retroviruses. The complex spumavirus transcriptional pattern of human spumavirus and the coding potential of the 10 exons identified are discussed.

We performed fetectomies, according to the technique of Sobis and Vandeputte for the surgical induction of teratomas, in female mice mated with syngeneic, congeneic, or allogeneic males. Benign teratomas including multiple tissue formations derived from the three germinal layers were observed only in syngeneic pregnancies. In addition, the frequency of induced teratomas was lower in some strains (A/Sn, A.By, and A.SW) than in others (A.CA, B10. A, C57BL/6, and C3H/He). This variable frequency may be explained by postulating the existence of fetal, strain-specific antigens. In hybrid pregnancies we observed no complete teratomas, but only cartilaginous formations considered as vestiges of early teratoma differentiation. The maternal immune reaction against either major (H-2) or minor (non-H-2) paternal histocompatibility antigens was sufficient to suppress the development of teratomas from visceral yolk sac of hybrid fetuses. This reaction, including both a serological and a cell-mediated activity, was measured in vitro. These results suggest that a maternal immune reaction is not efficiently altered by the normal first half of pregnancy and that differentiated teratoma tissues express the paternal antigens sufficiently to induce their immune rejection.