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Selected AbstractsFull Length or Fragments in Hormone Studies?JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2006Geertje van der Horst No abstract is available for this article. [source] Axoneme-dependent tubulin modifications in singlet microtubules of the Drosophila sperm tailCYTOSKELETON, Issue 4 2008Henry D. Hoyle Abstract Drosophila melanogaster sperm tubulins are posttranslationally glutamylated and glycylated. We show here that axonemes are the substrate for these tubulin C-terminal modifications. Axoneme architecture is required, but full length, motile axonemes are not necessary. Tubulin glutamylation occurs during or shortly after assembly into the axoneme; only glutamylated tubulins are glycylated. Tubulins in other testis microtubules are not modified. Only a small subset of total Drosophila sperm axoneme tubulins have these modifications. Biochemical fractionation of Drosophila sperm showed that central pair and accessory microtubules have the majority of poly-modified tubulins, whereas doublet microtubules have only small amounts of mono- and oligo-modified tubulins. Glutamylation patterns for different ,-tubulins experimentally assembled into axonemes were consistent with utilization of modification sites corresponding to those identified in other organisms, but surrounding sequence context was also important. We compared tubulin modifications in the 9 + 9 + 2 insect sperm tail axonemes of Drosophila with the canonical 9 + 2 axonemes of sperm of the sea urchin Lytichinus pictus and the 9 + 0 motile sperm axonemes of the eel Anguilla japonica. In contrast to Drosophila sperm, L. pictus sperm have equivalent levels of modified tubulins in both doublet and central pair microtubule fractions, whereas the doublets of A. japonica sperm exhibit little glutamylation but extensive glycylation. Tubulin C-terminal modifications are a prevalent feature of motile axonemes, but there is no conserved pattern for placement or amount of these modifications. We conclude their functions are likely species-specific. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Light and scanning microscopic studies of integument differentiation in the grass snake Natrix natrix L. (Lepidosauria, Serpentes) during embryogenesisACTA ZOOLOGICA, Issue 1 2009Elwira Swad Abstract We analysed the differentiation of body cover in the grass snake (Natrix natrix L.) over the full length of the embryo's body at each developmental stage. Based on investigations using both light and scanning electron microscopes, we divided the embryonic development of the grass snake integument into four phases. The shape of the epidermal cells changes first on the caudal and ventral parts of the embryo, then gradually towards the rostral and dorsal areas. In stage V on the ventral side of the embryo the gastrosteges are formed from single primordia, but on the dorsal side the epidermis forms the scale primordia in stage VII. This indicates that scalation begins on the ventral body surface, and spreads dorsally. The appearance of melanocytes between the cells of the stratum germinativum in stage VII coincides with changes in embryo colouration. The first dermal melanocytes were detected in stage XI so in this stage the definitive skin pattern is formed. In the same stage the epidermis forms the first embryonic shedding complex and the periderm layer begins to detach in small, individual flakes. This process coincides with rapid growth of the embryos. [source] Comparison of the aggregation properties, secondary structure and apoptotic effects of wild-type, Flemish and Dutch N-terminally truncated amyloid , peptidesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2001N. Demeester Abstract The Dutch (E22Q) and Flemish (A21G) mutations in the ,APP region of the amyloid precursor protein (APP) are associated with familial forms of Alzheimer dementia. However, patients with these mutations express substantially different clinical phenotypes. Therefore, secondary structure and cytotoxic effects of the three A,(12,42) variants [wild-type (WT), Dutch and Flemish] were tested. At a concentration of 5 µm the aggregation of these peptides followed the order: A,(1,42) WT > A,(12,42) WT > A,(12,42) Flemish >,A,(12,42) Dutch. The stability of the secondary structure of these peptides upon decreasing the trifluoroethanol (TFE) concentration in the buffer was followed by circular dichroism measurements. WT peptides progressively lost their ,-helical structure; this change occurred faster for both the Flemish and Dutch peptides, and at higher percentages of TFE in the buffer, and was accompanied by an increase in ,-sheet and random coil content. Apoptosis was induced in neuronal cells by the A,(12,42) WT and Flemish peptides at concentrations as low as 1,5 µm, as evidenced by propidium iodide (PI) staining, DNA laddering and caspase-3 activity measurements. Even when longer incubation times and higher peptide concentrations were applied the N-truncated Dutch peptide did not induce apoptosis. Apoptosis induced by the full length A,(1,42) peptide was weaker than that induced by its N-truncated variant. These data suggest that N-truncation enhanced the cytotoxic effects of A, WT and Flemish peptides, which may play a role in the accelerated progression of dementia. [source] The first CH domain of affixin activates Cdc42 and Rac1 through ,PIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factorGENES TO CELLS, Issue 3 2004Wataru Mishima Rho GTPases, Cdc42 and Rac1, play pivotal roles in cell migration by efficiently integrating cell-substrate adhesion and actin polymerization. Although it has been suggested that integrins stimulate these Rho GTPases via some of integrin binding proteins such as focal adhesion kinase (FAK) and paxillin, the precise molecular mechanism is largely unknown. In this study, we showed that the over-expression of RP1 corresponding to the first CH domain (CH1) of affixin, an integrin-linked kinase (ILK)-binding protein, induced a significant actin reorganization in MDCK cells by activating Cdc42/Rac1. Affixin full length and RP1 co-immunoprecipitated with ,PIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor (GEF), and they co-localized at the tips of lamellipodia in motile cells. The involvement of ,PIX in the RP1-induced Cdc42 activation was demonstrated by the significant dominant negative effect of a point mutant of ,PIX, ,PIX (L383R, L384S), lacking GEF activity. Our data strongly support that ILK and affixin provide a novel signalling pathway that links integrin signalling to Cdc42/Rac1 activation. [source] Role of hepatitis C virus proteins (C, NS3, NS5A) in hepatic oncogenesisHEPATOLOGY RESEARCH, Issue 1 2008Aldona Kasprzak In recent years, the effects of hepatitis C virus (HCV) proteins on hepatocarcinogenesis have undergone intense investigations. The potentially oncogenic proteins include at least three HCV proteins: core (C) protein, NS3, and NS5A. Several authors indicated relationships between subcellular localization, concentration, a specific molecular form of the proteins (full length, truncated, phosphorylated), the presence of specific domains (the nuclear localization signal homologous to e.g. Bcl-2) and their effects on the mechanisms linked to oncogenesis. The involvement of all the proteins has been described as being in control of the cell cycle, through interactions with key proteins of the process (p53, p21, cyclins, proliferating cell nuclear antigen), transcription factors, proto-oncogenes, growth factors/cytokines and their receptors, and proteins linked to the apoptotic process. Untilnow, the involvement of the core protein of HCV in liver carcinogenesis is the most recognized. One of the most common proteins affected by HCV proteins is the p53 tumor-suppressor protein. The p21/WAF1 gene is a major target of p53, and the effect of HCV proteins on the gene is frequently considered in parallel. The results of studies on the effects of HCV proteins on the apoptotic process are controversial. This work summarizes the information collected thus far in the field of HCV molecular virology and principal intracellular signaling pathways in which HCV oncogenic proteins are involved. [source] Y-position cysteine substitution in type I collagen (,1(I) R888C/p.R1066C) is associated with osteogenesis imperfecta/Ehlers-Danlos syndrome phenotype,,HUMAN MUTATION, Issue 4 2007Wayne A. Cabral Abstract The most common mutations in type I collagen causing types II,IV osteogenesis imperfecta (OI) result in substitution for glycine in a Gly-Xaa-Yaa triplet by another amino acid. We delineated a Y-position substitution in a small pedigree with a combined OI/Ehlers-Danlos Syndrome (EDS) phenotype, characterized by moderately decreased DEXA z-score (,1.3 to ,2.6), long bone fractures, and large-joint hyperextensibility. Affected individuals have an ,1(I)R888C (p.R1066C) substitution in one COL1A1 allele. Polyacrylamide gel electrophoresis (PAGE) of [3H]-proline labeled steady-state collagen reveals slight overmodification of the ,1(I) monomer band, much less than expected for a substitution of a neighboring glycine residue, and a faint ,1(I) dimer. Dimers form in about 10% of proband type I collagen. Dimer formation is inefficient compared to a possible 25%, probably because the SH-side chains have less proximity in this Y-position than when substituting for a glycine. Theoretical stability calculations, differential scanning calorimetry (DSC) thermograms, and thermal denaturation curves showed only weak local destabilization from the Y-position substitution in one or two chains of a collagen helix, but greater destabilization is seen in collagen containing dimers. Y-position collagen dimers cause kinking of the helix, resulting in a register shift that is propagated the full length of the helix and causes resistance to procollagen processing by N-proteinase. Collagen containing the Y-position substitution is incorporated into matrix deposited in culture, including immaturely and maturely cross-linked fractions. In vivo, proband dermal fibrils have decreased density and increased diameter compared to controls, with occasional aggregate formation. This report on Y-position substitutions in type I collagen extends the range of phenotypes caused by nonglycine substitutions and shows that, similar to X- and Y-position substitutions in types II and III collagen, the phenotypes resulting from nonglycine substitutions in type I collagen are distinct from those caused by glycine substitutions. Hum Mutat 28(4), 396,405, 2007. Published 2007 Wiley-Liss, Inc. [source] Molecular cloning of two prophenoloxidase genes from the mosquito Aedes aegyptiINSECT MOLECULAR BIOLOGY, Issue 1 2001A. S. Taft Abstract The biosynthesis of melanotic materials is an important process in the life of a mosquito. Melanin production is critical for many diverse processes such as egg chorion tanning, cuticular sclerotization, and melanotic encapsulation of metazoan parasites. Prophenoloxidase plays a critical role in this biochemical cascade. Two cDNAs, one full length and one partial clone, and two genomic clones encoding prophenoloxidase (pro-PO) were isolated from the yellow fever mosquito, Aedes aegypti. The full-length cDNA, pAaProPO1, is 2286 bp long with a 2055 bp open reading frame encoding a 685 amino acid protein that shares 89% identity with Armigeres subalbatus pro - PO. It contains two putative copper binding domains (amino acids 197,243 and 346,423) that are homologous to other insect pro-POs. AaProPO1 messenger RNA (mRNA) was detected by reverse transcription polymerase chain reaction (RT-PCR) only from third-stage larvae and not in adult mosquitoes after blood feeding, during the melanotic encapsulation of Dirofilaria immitis microfilariae or following exposure to bacteria. A 750 bp fragment of the second cDNA (pAaProPO2) was cloned using RT-PCR from mRNA obtained from 14-day postovipostional eggs. AaProPO2 mRNA was not found in any other life stages, and may be in low abundance or transiently expressed. AaProPO2 and AaProPO1 each contain three introns that are 60, 68 and 58 bp and 61, 69 and 59 bp long, respectively, and the intron sequences of these two genes are not similar. [source] Influence of endodontic sealer cement on fibreglass post bond strength to root dentineINTERNATIONAL ENDODONTIC JOURNAL, Issue 6 2008M. S. Menezes Abstract Aim, To test the hypothesis that the composition of endodontic sealer cements and the time elapsed between root filling and fibreglass post fixation interferes with adhesion to root canal dentine. Methodology, Sixty bovine incisor roots were divided into five groups (n = 12): CI, unfilled; SI, filled with a calcium hydroxide-based cement-Sealer 26, and immediate post fixation; S7, Sealer 26 and post fixation after 7 days; EI, filled with a zinc oxide and eugenol-based cement-Endofill and immediate fixation; and E7 Endofill and post fixation after 7 days. The posts were cemented with adhesive system and dual resin cement. Ten roots were cross-sectioned to obtain two 1-mm-thick discs for each cervical (TC), middle (TM) and apical (TA) third of the prepared root portion. The posts were submitted to a micropush-out test. The other two teeth were evaluated using scanning electron microscopy to analyse the bond interface. Data were analysed using anova, Tukey and Dunnett tests (P < 0.05). Results, Group EI was associated with a significant reduction in bond strength values irrespective of the root region; TC = 3.50 MPa (P = 0.0001); TM = 2.22 MPa (P = 0.0043) and TA = 1.45 MPa (P = 0.003). Region of canal had an influence on the values for the cement used in group E7, in which only the TA presented differences from the CI. Conclusions, Endofill interfered negatively with the bond to root dentine along its full length and in the TA when post fixation was delayed for 7 days. Bond strength decreased from crown to apex in all groups. [source] Antarctic climate change during the last 50 yearsINTERNATIONAL JOURNAL OF CLIMATOLOGY, Issue 3 2005John Turner Abstract An erratum has been published for this article in International Journal of Climatology 25 (8) 2005, 1147,1148. The Reference Antarctic Data for Environmental Research (READER) project data set of monthly mean Antarctic near-surface temperature, mean sea-level pressure (MSLP) and wind speed has been used to investigate trends in these quantities over the last 50 years for 19 stations with long records. Eleven of these had warming trends and seven had cooling trends in their annual data (one station had too little data to allow an annual trend to be computed), indicating the spatial complexity of change that has occurred across the Antarctic in recent decades. The Antarctic Peninsula has experienced a major warming over the last 50 years, with temperatures at Faraday/Vernadsky station having increased at a rate of 0.56 °C decade,1 over the year and 1.09 °C decade,1 during the winter; both figures are statistically significant at less than the 5% level. Overlapping 30 year trends of annual mean temperatures indicate that, at all but two of the 10 coastal stations for which trends could be computed back to 1961, the warming trend was greater (or the cooling trend less) during the 1961,90 period compared with 1971,2000. All the continental stations for which MSLP data were available show negative trends in the annual mean pressures over the full length of their records, which we attribute to the trend in recent decades towards the Southern Hemisphere annular mode (SAM) being in its high-index state. Except for Halley, where the trends are constant, the MSLP trends for all stations on the Antarctic continent for 1971,2000 were more negative than for 1961,90. All but two of the coastal stations have recorded increasing mean wind speeds over recent decades, which is also consistent with the change in the nature of the SAM. Copyright © 2005 Royal Meteorological Society [source] Regulation of Human Skeletal Stem Cells Differentiation by Dlk1/Pref-1JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2004Basem M Abdallah Abstract Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. Introduction: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. Materials and Methods: As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. Results: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPAR,2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor ,1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected. Conclusion: During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes. [source] Chromium (VI) inhibits heme oxygenase-1 expression in vivo and in arsenic-exposed human airway epithelial cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2006Kimberley A. O'Hara Inhaled hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms. One hypothesis for this lung pathogenesis is that Cr(VI) silences induction of cytoprotective genes, such as heme oxygenase-1 (HO-1), whose total lung mRNA levels were reduced 21 days after nasal instillation of potassium dichromate in C57BL/6 mice. To investigate the mechanisms for this inhibition, Cr(VI) effects on basal and arsenic (As(III))-induced HO-1 expression were examined in cultured human bronchial epithelial (BEAS-2B) cells. An effect of Cr(VI) on the low basal HO-1 mRNA and protein levels in BEAS-2B cells was not detectible. In contrast, Cr(VI) added to the cells before As(III), but not simultaneously with As(III), attenuated As(III)-induced HO-1 expression. Transient transfection with luciferase reporter gene constructs controlled by the full length ho-1 promoter or deletion mutants demonstrated that this inhibition occurred in the E1 enhancer region containing critical antioxidant response elements (ARE). Cr(VI) pretreatment inhibited As(III)-induced activity of a transiently expressed reporter construct regulated by three ARE tandem repeats. The mechanism for this Cr(VI)-attenuated transactivation appeared to be Cr(VI) reduction of the nuclear levels of the transcription factor Nrf2 and As(III)-stimulated Nrf2 transcriptional complex binding to the ARE cis element. Finally, exposing cells to Cr(VI) prior to co-exposure with As(III) synergized for apoptosis and loss of membrane integrity. These data suggest that Cr(VI) silences induction of ARE-driven genes required for protection from secondary insults. The data also have important implications for understanding the toxic mechanisms of low level, mixed metal exposures in the lung. J. Cell. Physiol. 209: 113,121, 2006. © 2006 Wiley-Liss, Inc. [source] Alternative splicing of cyclooxygenase-1 gene: altered expression in leucocytes from patients with bronchial asthma and association with aspirin-induced 15-HETE releaseALLERGY, Issue 6 2007M. L. Kowalski Background:, Cyclooxygenase-1 (COX-1) is a key enzyme involved in generation of prostanoids, important mediators and modulators of asthmatic inflammation. In a subpopulation of aspirin-sensitive asthmatics (ASA) inhibition of COX-1 by nonsteroidal anti-inflammatory drugs results in activation of inflammatory cells and development of symptoms. Alternatively spliced variants of COX-1 lacking 111 bp from exon 9 were described previously but have never been identified in human leucocytes peripheral blood leucocytes (PBL) or upper airway epithelial cells. We aimed to assess the expression of spliced variants of COX-1 mRNA in PBLs from patients with asthma and in healthy subjects (HS) referring the expression to patients characteristics (including ASA-sensitivity) and to aspirin-triggered 15-hydroxyeicosatetraenoic acid (15-HETE) generation. Methods:, The study included 30 patients with ASA, 30 patients with aspirin-tolerant asthma (ATA) and 30 HS serving as controls. Nasal polyps for epithelial cell cultures were obtained from 10 patients with chronic rhinosinusitis. Expression of full length and spliced variants of COX-1 enzyme was detected by RT-PCR and presented as the ratio of full-length COX-1 to alternatively spliced COX-1 mRNA [COX-1 alternative splicing index (COX-1 AS index)]. Release of eicosanoids (PGE2 and 15-HETE) by PBLs was measured with enzyme immunoassay. Results:, In both PBLs and airway epithelial cells the expression of full-length product prevailed over spliced variants of COX-1 enzyme. Cyclooxygenase-1 AS index was significantly lower in asthmatics as compared to HS (1.96 ± 0.71 vs 2.41 ± 0.99, P < 0.05) indicating the relatively higher expression of the alternative transcript in asthmatic patients. Cyclooxygenase-1 AS index was not different between ASA and ATA groups (mean 1.90 ± 0.66 vs 2.02 ± 0.76, respectively, P = 0.39). There was no significant association between COX-1 AS index and mean daily dose of inhaled glucocorticosteroids or pulmonary function tests (FEV1, FVC) but in ASA group a weak correlation with daily dose of oral glucocorticosteroids was found (r = 0.39; P = 0.03). In ASA patients there was a significant positive correlation between the COX-1 AS index and the percentage of aspirin-triggered increase in 15-HETE generation (r = 0.51; P < 0.03). Conclusions:, Alternatively spliced variants of COX-1 mRNA are differently expressed in patients with bronchial asthma and may be associated with aspirin-triggered 15-HETE generation. [source] Photoluminescence microscopy of as-grown individual single-walled carbon nanotubes on Si/SiO2 substratesPHYSICA STATUS SOLIDI (B) BASIC SOLID STATE PHYSICS, Issue 13 2006Oliver Kiowski Abstract We present far-field photoluminescence (PL) imaging at room temperature of tens of micrometer long individual single-walled carbon nanotubes (SWNTs) grown and measured directly on a Si/SiO2 surface. The SWNTs are grown by Chemical Vapour Deposition (CVD) with ethanol as carbon source and contact the surface with their full length. We detect the PL and its variations along SWNTs in a home-built laser microscope with a spatial resolution of ,400 nm. We are able to reliably assign (n, m)-structure of SWNTs by measuring PL spectra in the range of 800,1600 nm as function of the excitation wavelength varying between 710 and 860 nm. This allows us to check structural integrity (changes of helicity) along the tube. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Changes in the invasive and metastatic capacities of HT-29/M3 cells induced by the expression of fucosyltransferase 1CANCER SCIENCE, Issue 7 2007Raquel Mejías-Luque Lewis antigens are terminal fucosylated oligosaccharides synthesized by the sequential action of several glycosyltransferases. The fucosyltransferases are the enzymes responsible for the addition of terminal fucose to precursor oligosaccharides attached to proteins or lipids. These oligosaccharides, defined as cell surface markers, have been implicated in different types of intercellular interactions and in adhesion and invasion processes. Transfection of HT-29/M3 colon cancer cells with the full length of human fucosyltransferase (FUT1), induces the synthesis of H type 2 and Lewis y antigens, associated with a decrease of sialyl-Lewis x. The capacity to develop primary tumors when cells were injected intrasplenically was similar in parental and FUT1-transfected cells, but the capacity to colonize the liver after spleen removal was significantly reduced in M3/FUT1 transfected cells. These results indicate that the expression of FUT1 induces changes in the metastatic capacity of HT-29/M3 colon cancer cells, as a consequence of the altered expression pattern of type 2 Lewis antigens. Also, an association between MUC5AC expression and the degree of gland differentiation in both primary splenic tumors and hepatic metastases was detected. (Cancer Sci 2007; 98: 1000,1005) [source] | |||||||||||||