Transforming growth factor-beta inhibits the expression of clock genes

Disturbances of sleep-wake rhythms are an important problem in Alzheimer's disease (AD). Circadian rhythms are regulated by clock genes. Transforming growth factor-beta (TGF-β) is overexpressed in neurons in AD and is the only cytokine that is increased in cerebrospinal fluid (CSF). Our data show that TGF-β2 inhibits the expression of the clock genes Period (Per)1, Per2, and Rev-erbα, and of the clock-controlled genes D-site albumin promoter binding protein (Dbp) and thyrotroph embryonic factor (Tef). However, our results showed that TGF-β2 did not alter the expression of brain and muscle Arnt-like protein-1 (Bmal1). The concentrations of TGF-β2 in the CSF of 2 of 16 AD patients and of 1 of 7 patients with mild cognitive impairment were in the dose range required to suppress the expression of clock genes. TGF-β2-induced dysregulation of clock genes may alter neuronal pathways, which may be causally related to abnormal sleep-wake rhythms in AD patients.

Dephosphorylation of p-ERK1/2 in relation to tumor remission after HER-2 and Raf1 blocking therapy in a conditional mouse tumor model

Several studies have shown that HER-2/neu (erbB-2) blocking therapy strategies can cause tumor remission. However, the responsible molecular mechanisms are not yet known. Both ERK1/2 and Akt/PKB are critical for HER-2-mediated signal transduction. Therefore, we used a mouse tumor model that allows downregulation of HER-2 in tumor tissue by administration of anhydrotetracycline (ATc). Switching-off HER-2 caused a rapid tumor remission by more than 95% within 7 d of ATc administration compared to the volume before switching-off HER-2. Interestingly, HER-2 downregulation caused a dephosphorylation of p-ERK1/2 by more than 80% already before tumor remission occurred. Levels of total ERK protein were not influenced. In contrast, dephosphorylation of p-Akt occurred later, when the tumor was already in remission. These data suggest that in our HER-2 tumor model dephosphorylation of p-ERK1/2 may be more critical for tumor remission than dephosphorylation of p-Akt. To test this hypothesis we used a second mouse tumor model that allows ATc controlled expression of BXB-Raf1 because the latter constitutively signals to ERK1/2, but cannot activate Akt/PKB. As expected, downregulation of BXB-Raf1 in tumor tissue caused a strong dephosphorylation of p-ERK1/2, but did not decrease levels of p-Akt. Interestingly, tumor remission after switching-off BXB-Raf1 was similarly efficient as the effect of HER-2 downregulation, despite the lack of p-Akt dephosphorylation. In conclusion, two lines of evidence strongly suggest that dephosphorylation of p-ERK1/2 and not that of p-Akt is critical for the rapid tumor remission after downregulation of HER-2 or BXB-Raf1 in our tumor model: (i) dephosphorylation of p-ERK1/2 but not that of p-Akt precedes tumor remission after switching-off HER-2 and (ii) downregulation of BXB-Raf1 leads to a similarly efficient tumor remission as downregulation of HER-2, although no p-Akt dephosphorylation was observed after switching-off BXB-Raf1.

Analysis and mapping of CACNB4, CHRNA1, KCNJ3, SCN2A and SPG4, physiological candidate genes for porcine congenital progressive ataxia and spastic paresis

The cause of porcine congenital progressive ataxia and spastic paresis (CPA) is unknown. This severe neuropathy manifests shortly after birth and is lethal. The disease is inherited as a single autosomal recessive allele, designated cpa. In a previous study, we demonstrated close linkage of cpa to microsatellite SW902 on porcine chromosome 3 (SSC3), which corresponds syntenically to human chromosome 2. This latter chromosome contains ion channel genes (Ca(2+), K(+) and Na(+)), a cholinergic receptor gene and the spastin (SPG4) gene, which cause human epilepsy and ataxia when mutated. We mapped porcine CACNB4, KCNJ3, SCN2A and CHRNA1 to SSC15 and SPG4 to SSC3 with the INRA-Minnesota porcine radiation hybrid panel (IMpRH) and we sequenced the entire open reading frames of CACNB4 and SPG4 without finding any differences between healthy and affected piglets. An anti-epileptic drug treatment with ethosuximide did not change the severity of the disease, and pigs with CPA did not exhibit the corticospinal tract axonal degeneration found in humans suffering from hereditary spastic paraplegia, which is associated with mutations in SPG4. For all these reasons, the hypothesis that CACNB4, CHRNA1, KCNJ3, SCN2A or SPG4 are identical with the CPA gene was rejected.

Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania

The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.

Comparison of two protocols of periosteal distraction osteogenesis in a rabbit calvaria model

The regenerative pathways during periosteal distraction osteogenesis may be influenced by the local environment composed by cells, growth factors, nutrition and mechanical load. The aim of the present study was to evaluate the influence of two protocols of periosteal distraction on bone formation. Custom made distraction devices were surgically fixed onto the calvariae of 60 rabbits. After an initial healing period of 7 days, two groups of animals were submitted to distraction rates of 0.25 and 0.5 mm/24 h for 10 days, respectively. Six animals per group were sacrificed 10 (mid-distraction), 17 (end-distraction), 24 (1-week consolidation), 31 (2-week consolidation) and 77 days (2-month consolidation) after surgery. Newly formed bone was assessed by means of micro-CT and histologically. Expression of transcripts encoding tissue-specific genes (BMP-2, RUNX2, ACP5, SPARC, collagen I α1, collagen II α1 and SOX9) was analyzed by quantitative PCR. Two patterns of bone formation were observed, originating from the old bone surface in Group I and from the periosteum in Group II. Bone volume (BV) and bone mineral density (BMD) significantly increased up to the 2-month consolidation period within the groups (p < 0.05). Significantly more bone was observed in Group II compared to Group I at the 2-month consolidation period (p < 0.001). Expression of transcripts encoding osteogenic genes in bone depended on the time-point of observation (p < 0.05). Low level of transcripts reveals an indirect role of periosteum in the osteogenic process. Two protocols of periosteal distraction in the present model resulted in moderate differences in terms of bone formation.

Conserved regulation of proximodistal limb axis development by Meis1/Hth.

Vertebrate limbs grow out from the flanks of embryos, with their main axis extending proximodistally from the trunk. Distinct limb domains, each with specific traits, are generated in a proximal-to-distal sequence during development. Diffusible factors expressed from signalling centres promote the outgrowth of limbs and specify their dorsoventral and anteroposterior axes. However, the molecular mechanism by which limb cells acquire their proximodistal (P-D) identity is unknown. Here we describe the role of the homeobox genes Meis1/2 and Pbx1 in the development of mouse, chicken and Drosophila limbs. We find that Meis1/2 expression is restricted to a proximal domain, coincident with the previously reported domain in which Pbx1 is localized to the nucleus, and resembling the distribution of the Drosophila homologues homothorax (hth) and extradenticle (exd); that Meis1 regulates Pbx1 activity by promoting nuclear import of the Pbx1 protein; and that ectopic expression of Meis1 in chicken and hth in Drosophila disrupts distal limb development and induces distal-to-proximal transformations. We suggest that restriction of Meis1/Hth to proximal regions of the vertebrate and insect limb is essential to specify cell fates and differentiation patterns along the P-D axis of the limb.

Complete genome of a Puumala virus strain from Central Europe

Puumala virus (PUUV) is one of the predominant hantavirus species in Europe causing mild to moderate cases of haemorrhagic fever with renal syndrome. Parts of Lower Saxony in north-western Germany are endemic for PUUV infections. In this study, the complete PUUV genome sequence of a bank vole-derived tissue sample from the 2007 outbreak was determined by a combined primer-walking and RNA ligation strategy. The S, M and L genome segments were 1,828, 3,680 and 6,550 nucleotides in length, respectively. Sliding-window analyses of the nucleotide sequences of all available complete PUUV genomes indicated a non-homogenous distribution of variability with hypervariable regions located at the 3′-ends of the S and M segments. The overall similarity of the coding genome regions to the other PUUV strains ranged between 80.1 and 84.7 % at the level of the nucleotide sequence and between 89.5 and 98.1 % for the deduced amino acid sequences. In comparison to the phylogenetic trees of the complete coding sequences, trees based on partial segments revealed a general drop in phylogenetic support and a lower resolution. The Astrup strain S and M segment sequences showed the highest similarity to sequences of strains from geographically close sites in the Osnabrück Hills region. In conclusion, a primer-walking-mediated strategy resulted in the determination of the first complete nucleotide sequence of a PUUV strain from Central Europe. Different levels of variability along the genome provide the opportunity to choose regions for analyses according to the particular research question, e.g., large-scale phylogenetics or within-host evolution.

Induction of apoptosis and enhancement of chemosensitivity in human prostate cancer LNCaP cells using bispecific antisense oligonucleotide targeting Bcl-2 and Bcl-xL genes

OBJECTIVE: To determine whether a specifically designed bispecific (Bcl-2/Bcl-xL) antisense oligonucleotide (ASO) induces apoptosis and enhances chemosensitivity in human prostate cancer LNCaP cells, as Bcl-2 and Bcl-xL are both anti-apoptotic genes associated with treatment resistance and tumour progression in many malignancies, including prostate cancer. MATERIALS AND METHODS: Inhibition of Bcl-2 and Bcl-xL expression by the bispecific ASO was evaluated using real-time reverse transcription-polymerase chain reaction and Western blotting, while growth inhibition and induction of apoptosis were analysed by a crystal violet assay, flow cytometry and Western blotting of apoptosis-relevant proteins. The effect of combined treatment with bispecific ASO and chemotherapy or small-interference RNA (siRNA) targeting the clusterin gene was also investigated. RESULTS: Bispecific ASO reduced Bcl-2 and Bcl-xL expression in LNCaP cells in a dose-dependent manner. There was cell growth inhibition, increases in the sub-G0-G1 fraction, and cleavage of caspase-3 and poly(ADP-Ribose) polymerase proteins in LNCaP cells after bispecific ASO treatment. Interestingly, Bcl-2/Bcl-xL bispecific ASO treatment also resulted in the down-regulation of Mcl-1 and up-regulation of Bax. The sensitivity of LNCaP cells to mitoxantrone, docetaxel or paclitaxel was significantly increased, reducing the 50% inhibitory concentration by 45%, 80% or 90%, respectively. Furthermore, the apoptotic induction by Bcl-2/Bcl-xL bispecific ASO was synergistically enhanced by siRNA-mediated inhibition of clusterin, a cytoprotective chaperone that interacts with and inhibits activated Bax. CONCLUSIONS: These findings support the concept of the targeted suppression of Bcl-2 anti-apoptotic family members using multitarget inhibition strategies for prostate cancer, through the effective induction of apoptosis.

BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel

OBJECTIVE: According to recent reports, the synovial membrane may contain mesenchymal stem cells with the potential to differentiate into chondrocytes under appropriate conditions. In order to assess the usefulness of synovium-derived progenitor cells for the purposes of cartilage tissue engineering, we explored their requirements for the expression of chondrocyte-specific genes after expansion in vitro. DESIGN: Mesenchymal progenitor cells were isolated from the synovial membranes of bovine shoulder joints and expanded in two-dimensions on plastic surfaces. They were then seeded either as micromass cultures or as single cells within alginate gels, which were cultured in serum-free medium. Under these three-dimensional conditions, chondrogenesis is known to be supported and maintained. Cell cultures were exposed either to bone morphogenetic protein-2 (BMP-2) or to isoforms of transforming growth factor-beta (TGF-beta). The levels of mRNA for Sox9, collagen types I and II and aggrecan were determined by RT-PCR. RESULTS: When transferred to alginate gel cultures, the fibroblast-like synovial cells assumed a rounded form. BMP-2, but not isoforms of TGF-beta, stimulated, in a dose-dependent manner, the production of messenger RNAs (mRNAs) for Sox9, type II collagen and aggrecan. Under optimal conditions, the expression levels of cartilage-specific genes were comparable to those within cultured articular cartilage chondrocytes. However, in contrast to cultured articular cartilage chondrocytes, synovial cells exposed to BMP-2 continued to express the mRNA for alpha1(I) collagen. CONCLUSIONS: This study demonstrates that bovine synovium-derived mesenchymal progenitor cells can be induced to express chondrocyte-specific genes. However, the differentiation process is not complete under the chosen conditions. The stimulation conditions required for full transformation must now be delineated.

NDRG1/2 expression is inhibited in primary acute myeloid leukemia

Expression of N-myc downregulated gene 1 (NDRG1) is associated with growth arrest and differentiation of tumor cells. In hematopoietic cells, NDRG1 was identified in a screen for differentiation-related genes in human myelomonocytic leukemic U937 cells. In the present study, we found significantly higher NDRG1 mRNA levels in granulocytes of healthy donors than in primary acute myeloid leukemia (AML) cells. Another NDRG family member, NDRG2, was significantly higher expressed in normal macrophages compared to primary AML cells. Moreover, NDRG1 mRNA levels increased in two acute promyelocytic leukemia (APL) patients as well as in NB4 and HT93 APL cells upon all-trans retinoic acid (ATRA) therapy. In line with these observations, silencing of NDRG1 diminished neutrophil differentiation of leukemic cell lines. In conclusion, we found an association of low NDRG1 levels with an immature cell phenotype and provide evidence that NDRG1 is functionally involved in neutrophil maturation.

Meta-analyses qualify metzincins and related genes as acute rejection markers in renal transplant patients

Definition of acute renal allograft rejection (AR) markers remains clinically relevant. Features of T-cell-mediated AR are tubulointerstitial and vascular inflammation associated with excessive extracellular matrix (ECM) remodeling, regulated by metzincins, including matrix metalloproteases (MMP). Our study focused on expression of metzincins (METS), and metzincins and related genes (MARGS) in renal allograft biopsies using four independent microarray data sets. Our own cases included normal histology (N, n = 20), borderline changes (BL, n = 4), AR (n = 10) and AR + IF/TA (n = 7). MARGS enriched in all data sets were further examined on mRNA and/or protein level in additional patients. METS and MARGS differentiated AR from BL, AR + IF/TA and N in a principal component analysis. Their expression changes correlated to Banff t- and i-scores. Two AR classifiers, based on METS (including MMP7, TIMP1), or on MARGS were established in our own and validated in the three additional data sets. Thirteen MARGS were significantly enriched in AR patients of all data sets comprising MMP7, -9, TIMP1, -2, thrombospondin2 (THBS2) and fibrillin1. RT-PCR using microdissected glomeruli/tubuli confirmed MMP7, -9 and THBS2 microarray results; immunohistochemistry showed augmentation of MMP2, -9 and TIMP1 in AR. TIMP1 and THBS2 were enriched in AR patient serum. Therefore, differentially expressed METS and MARGS especially TIMP1, MMP7/-9 represent potential molecular AR markers.

Polyunsaturated fatty acid-enriched diets used for the treatment of canine chronic enteropathies decrease the abundance of selected genes of cholesterol homeostasis

Lipids are important for cell function and survival, but abnormal concentrations may lead to various diseases. Cholesterol homeostasis is greatly dependent on the active transport by membrane proteins, whose activities coordinate lipid status with cellular function. Intestinal Niemann-Pick C1-Like 1 protein (NPC1L1) and scavenger receptor B1 (SR-B1) participate in the uptake of extracellular cholesterol, whereas ATP binding cassette A1 (ABCA1) mediates the efflux of excessive intracellular cholesterol. Caveolin-1 binds cholesterol and fatty acids (FA) and participates in cholesterol trafficking. Sterol response element binding protein-2 (SREBP-2) is a sensor that regulates intracellular cholesterol synthesis. Given that cholesterol is a constituent of chylomicrons, whose synthesis is enhanced with an increased FA supply, we tested the hypothesis that feeding polyunsaturated FA (PUFA)-enriched diets in treatment of canine chronic enteropathies alters the mRNA expression of genes involved in cholesterol homeostasis. Using quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we compared the mRNA abundance of NPC1L1, SR-B1, ABCA1, caveolin-1, and SREBP-2 in duodenal mucosal biopsies of dogs with food-responsive diarrhea (FRD; n=14) and inflammatory bowel disease (IBD; n=7) before and after treatment with cholesterol-free PUFA-enriched diets and in healthy controls (n=14). The abundance of caveolin-1, ABCA1, and SREBP-2 were altered by PUFA-enriched diets (P<0.05), whereas that of NPC1L1 and SR-B1 mRNA remained unchanged. The gene expression of caveolin-1, ABCA1, and SREBP-2 was down-regulated (P<0.05) by PUFA-enriched diets in IBD dogs only. Our results suggest that feeding PUFA-enriched diets may alter cholesterol homeostasis in duodenal mucosal cells of dogs suffering from IBD.

CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression

Chronic myelogenous leukemia (CML) results from a chromosomal translocation in hematopoietic stem or early progenitor cells that gives rise to the oncogenic BCR/ABL fusion protein. Clinically, CML has a chronic phase that eventually evolves into an accelerated stage and blast crisis. A CML-specific immune response is thought to contribute to the control of disease. Whether the immune system can also promote disease progression is not known. In the present study, we investigated the possibility that the TNF receptor family member CD27 is present on leukemia stem cells (LSCs) and mediates effects of the immune system on CML. In a mouse model of CML, BCR/ABL+ LSCs and leukemia progenitor cells were found to express CD27. Binding of CD27 by its ligand, CD70, increased expression of Wnt target genes in LSCs by enhancing nuclear localization of active β-catenin and TRAF2- and NCK-interacting kinase (TNIK). This resulted in increased proliferation and differentiation of LSCs. Blocking CD27 signaling in LSCs delayed disease progression and prolonged survival. Furthermore, CD27 was expressed on CML stem/progenitor cells in the bone marrow of CML patients, and CD27 signaling promoted growth of BCR/ABL+ human leukemia cells by activating the Wnt pathway. Since expression of CD70 is limited to activated lymphocytes and dendritic cells, our results reveal a mechanism by which adaptive immunity contributes to leukemia progression. In addition, targeting CD27 on LSCs may represent an attractive therapeutic approach to blocking the Wnt/β-catenin pathway in CML.