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Complete Loss (complete + loss)
Selected AbstractsNecrobiotic xanthogranuloma with paraproteinemia; an atypical caseJOURNAL DER DEUTSCHEN DERMATOLOGISCHEN GESELLSCHAFT, Issue 1 2008Yoshiyuki Ito Summary Necrobiotic xanthogranuloma (NXG) is a rare marker for paraproteinemia. An 86-year-old woman had a one year history of large red-yellow to brown annular plaques involving all limbs. Biopsies showed a non-palisading granuloma with numerous multinucleated giant cells showing prominent elastophagocytosis and extensive areas of necrobiosis throughout the entire dermis. Complete loss of elastic fibers was observed in the central atrophic area of an annular plaque. Small vascular thromboses were also present. Laboratory findings revealed paraproteinemia of IgG-lambda type. Immunohistochemical staining detected the presence of roughly equal numbers of IgG-lambda-and IgG-kappa-staining plasma cells in the dermis. We diagnosed NXG with paraproteinemia with monoclonal gammopathy (IgG-lambda type) of unknown significance. [source] The emptiness of zero: representations of loss, absence, anxiety and desire in the late twentieth centuryCRITICAL QUARTERLY, Issue 1 2004Kathy Smith As the new millenium approached, the anxiety which this moment generated found resonance in various cultural representations, and the appearance of spectral imagery seemed to indicate an anxiety about subjectivity, and about its fragmentation or complete loss. Unable to comtemplate a state beyond this loss, there came into being a crisis in subjectivity itself, and the existence of the destabilized millenium subject. This subject approached the moment by both fixating on the threshold and by disavowing what lay beyond. These strategies, and the underlying anxiety which brought them into existence, were resonated through many of the cultural representations of the time. This paper argues that it is through nachträglichkeit - a 'making sense' in retrospect of earlier disparate experiences - that we can begin to examine, contextualise and account for the phenomenon of 'millenium frenzy' which came about at the end of 1999. It constructs a reading of this moment, and of two particular filmic representations which resonate the concerns of the time, examining in the process how - from a psychoanalytic perspective - culture might be understood through its representations, and how these representations can be understood through culture. [source] Long-Term Follow-Up After Autologous Fat Grafting: Analysis of Results from 116 Patients Followed at Least 12 Months After Receiving the Last of a Minimum of Two TreatmentsDERMATOLOGIC SURGERY, Issue 12 2000Sorin Eremia MD Background. The effectiveness of long-term results for correction of facial rhytides with single or multiple autologous fat transplants remains controversial. Objective. This study is a retrospective review of short- and long-term results for 116 patients who underwent multiple autologous fat grafting sessions into the nasolabial and melolabial (lateral oral commissure) fold, and in some cases additional sites such as lips and glabella. Methods. Criteria for inclusion into the study included at least two treatment sessions and at least a 12-month follow-up evaluation after the last treatment received. A 14-gauge needle cannula was used to aspirate the donor fat and to inject the fat grafts. Results. For the nasolabial and melolabial folds, short-term results at 3,4 months were uniformly excellent. Gradual correction loss was noted between 5 and 8 months, with 25% of patients still rated as excellent and 40% as good. Most patients continued to show correction loss between 9 and 14 months. Only 3,4% of the patients truly maintained long-term correction for more than 14 months. Multiple re-treatments did not significantly increase the percent of patients showing long-term results. For the glabella, the results were very disappointing, with most patients showing complete loss of correction after 3,4 months. For lip augmentation, correction loss was slower than in the glabella, but most patients showed complete loss of correction by 5,8 months. Complications were minimal. Conclusion. Autologous fat grafting is most effective for relatively short-term improvement of facial aging changes in the nasolabial and oral commissures areas. It is less effective for lip augmentation and completely ineffective for the glabella area. [source] The vesicular integral protein-like gene is essential for development of a mechanosensory system in zebrafishDEVELOPMENTAL NEUROBIOLOGY, Issue 12 2008Mabel Chong Abstract The zebrafish hi472 mutation is caused by a retroviral insertion into the vesicular integral protein-like gene, or zVIPL, a poorly studied lectin implicated in endoplasmic reticulum (ER)-Golgi trafficking. A mutation in the shorter isoform of zVIPL (zVIPL-s) results in a reduction of mechanosensitivity and consequent loss of escape behavior. Here we show that motoneurons and hindbrain reticulospinal neurons, which normally integrate mechanosensory inputs, failed to fire in response to tactile stimuli in hi472 larvae, suggesting a perturbation in sensory function. The hi472 mutant larvae in fact suffered from a severe loss of functional neuromasts of the lateral line mechanosensory system, a reduction of zVIPL labeling in support cells, and a reduction or even a complete loss of hair cells in neuromasts. The Delta-Notch signaling pathway is implicated in cellular differentiation of neuromasts, and we observed an increase in Notch expression in neuromasts of hi472 mutant larvae. Treatment of hi472 mutant larvae with DAPT, an inhibitor of Notch signaling, or overexpression of the Notch ligand deltaB in hi472 mutant blastocysts produced partial rescue of the morphological defects and of the startle response behavior. We conclude that zVIPL-s is a necessary component of Delta-Notch signaling during neuromast development in the lateral line mechanosensory system. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008 [source] Electroconductive Hydrogels: Electrical and Electrochemical Properties of Polypyrrole-Poly(HEMA) CompositesELECTROANALYSIS, Issue 7 2005Sean Brahim Abstract Composites of inherently conductive polypyrrole (PPy) within highly hydrophilic poly(2-hydroxyethyl methacrylate)-based hydrogels (p(HEMA)) have been fabricated and their electrochemical properties investigated. The electrochemical characteristics observed by cyclic voltammetry suggest less facile reduction of PPy within the composite hydrogel compared to electropolymerized PPy, as shown by the shift in the reduction peak potential from ,472,mV for electropolymerized polypyrrole to ,636,mV for the electroconductive composite gel. The network impedance magnitude for the electroconductive hydrogel remains quite low, ca. 100,,, even upon approach to DC, over all frequencies and at all offset potentials suggesting retained electronic (bipolaronic) conductivity within the composite. In contrast, sustained application of +0.7 V (vs. Ag/AgCl, 3,M Cl,) for typically 100,min. (conditioning) to reduce the background amperometric current to <1.0,,A, resulted in complete loss of electroactivity. Nyquist plots suggest that sustained application of such a modest potential to the composite hydrogel results in impedance characteristics that resembles p(HEMA) without evidence of the conducting polymer component. PPy composite gels supported a larger ferrocene monocarboxylate diffusivity (Dappt=7.97×10,5,cm2,s,1) compared to electropolymerized PPy (Dappt=5.56×10,5,cm2,s,1), however a marked reduction in diffusivity (Dappt=1.01×10,5,cm2,s,1) was observed with the conditioned hydrogel composite. Cyclic voltammograms in buffer containing H2O2 showed an absence of redox peaks for electrodes coated with PPy-containing membranes, suggesting possible chemical oxidation of polypyrrole by the oxidant [source] Transcription dynamics of the functional tfdA gene during MCPA herbicide degradation by Cupriavidus necator AEO106 (pRO101) in agricultural soilENVIRONMENTAL MICROBIOLOGY, Issue 3 2008Mette Haubjerg Nicolaisen Summary A modified protocol for simultaneous extraction of RNA and DNA, followed by real-time polymerase chain reaction quantification, was used to investigate tfdA gene expression during in situ degradation of the herbicide MCPA (4-chloro-2-methylphenoxy-acetic acid) in soil. tfdA encodes an ,-ketoglutarate-dependent dioxygenase catalysing the first step in the degradation pathway of MCPA and 2,4-D (2,4-dichlorophenoxy-acetic acid). A linear recovery of tfdA mRNA over three orders of magnitude was shown, and the tfdA mRNA level was normalized using the tfdA mRNA/DNA ratio. The density of active cells required for tfdA mRNA detection was 105 cells g,1 soil. Natural soil microcosms inoculated with Cupriavidus necator (formerly Ralstonia eutropha) AEO106 (pRO101) cells were amended with four different MCPA concentrations (2, 20, 50 and 150 mg kg,1). Mineralization rates were estimated by quantification of 14CO2 emission from degradation of 14C-MCPA. tfdA mRNA was detected 1 h after amendment at all four concentrations. In soils amended with 2 and 20 mg kg,1, the mRNA/DNA ratio for tfdA demonstrated a sharp transient maximum of tfdA expression from no to full expression within 3 and 6 h respectively, followed by a decline and complete loss of expression after 19 and 43 h. A more complex pattern of tfdA expression was observed for the higher 50 and 150 mg kg,1 amendments; this coincided with growth of C. necator AEO106 (pRO101) in the system. Repeated amendment with MCPA after 2 weeks in the 20 mg kg,1 scenario revealed a sharp increase of tfdA mRNA, and absence of a mineralization lag phase. For all amendments, tfdA mRNA was detectable only during active mineralization, and thus revealed a direct correlation between tfdA mRNA presence and microbial degrader activity. The present study demonstrates that direct analysis of functional gene expression dynamics by quantification of mRNA can indeed be made in natural soil. [source] Antiphospholipid syndrome and endocrine damage: why bilateral adrenal thrombosis?EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2003Kaspar Berneis Abstract: We describe a rare case of bilateral hemorrhagic infarction of the adrenal glands diagnosed in the context of positive antiphospholipid antibodies (aPL). The patient presented atypical clinical symptoms of adrenal insufficiency. Laboratory investigation showed complete adrenal failure and increased aPL, both manifestations persisted 1 yr after the initial event. MR imaging at baseline was compatible with bilateral hemorrhagic infarction and showed almost complete loss of viable adrenal tissue 1 yr later. Although no direct causal effect can be proved, the sequence of events and the exclusion of other common causes of bilateral adrenal hemorrhage (e.g. tuberculosis, severe coagulation disorder) support an association between aPL and adrenal hemorrhagic infarction. A unique link between particular anatomical characteristics of the adrenal fascicular zone and a novel, previously described, explanation model of aPL-thrombosis is hypothesized. It is based on the properties of late endosomes, which are important organelles participating in cholesterol trafficking and protein sorting within cells and express epitopes recognized by aPL. It would be interesting to investigate adrenal tissue for presence of late endosomes and their aPL relevant epitopes for proof of this tempting hypothesis. Focal accumulation of aPL and isolated, simultaneous, bilateral adrenal infarctions could thus be explained. [source] Generating functional CD8+ T cell memory response under transient CD4+ T cell deficiency: Implications for vaccination of immunocompromised individualsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2008Corey Smith Abstract Studies based on either MHC class II-knockout or CD4+ T cell-depleted murine models have demonstrated a critical role for CD4+ T cells in the generation of CD8+ T cell memory. However, it is difficult to extend these findings to immunocompromised humans where a complete loss of CD4+ T cells is rarely observed. Here, we have developed a model setting, which allows studies on the generation of CD8+ T cell memory responses in a transient CD4+ T cell-deficient setting similar to that seen in immunocompromised patients. Immunisation with an adenoviral vaccine under transient helpless or help-deficient conditions showed varying degrees of impact on the priming of CD8+ T cell responses. Antigen-specific T cells generated under normal CD4+ T cell help and transient help-deficient conditions showed similar effector phenotype and were capable of proliferation upon secondary antigen encounter. Most importantly, in spite of CD4+ T cell deficiency, the long-term CD8+ T cell memory response remained functionally stable and showed comparable cytotoxic effector function as seen in CD8+ T cells generated with normal CD4+ T cell numbers. These findings provide evidence that in spite of partially impaired activation of a primary CD8+ T cell response, a fully functional and stable memory CTL response can be induced under conditions of severe transient CD4+ T cell deficiency. [source] Capsaicin-sensitive sensory fibers in the islets of Langerhans contribute to defective insulin secretion in Zucker diabetic rat, an animal model for some aspects of human type 2 diabetesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2007Dorte X. Gram Abstract The system that regulates insulin secretion from ,-cells in the islet of Langerhans has a capsaicin-sensitive inhibitory component. As calcitonin gene-related peptide (CGRP)-expressing primary sensory fibers innervate the islets, and a major proportion of the CGRP-containing primary sensory neurons is sensitive to capsaicin, the islet-innervating sensory fibers may represent the capsaicin-sensitive inhibitory component. Here, we examined the expression of the capsaicin receptor, vanilloid type 1 transient receptor potential receptor (TRPV1) in CGRP-expressing fibers in the pancreatic islets, and the effect of selective elimination of capsaicin-sensitive primary afferents on the decline of glucose homeostasis and insulin secretion in Zucker diabetic fatty (ZDF) rats, which are used to study various aspects of human type 2 diabetes mellitus. We found that CGRP-expressing fibers in the pancreatic islets also express TRPV1. Furthermore, we also found that systemic capsaicin application before the development of hyperglycemia prevents the increase of fasting, non-fasting, and mean 24-h plasma glucose levels, and the deterioration of glucose tolerance assessed on the fifth week following the injection. These effects were accompanied by enhanced insulin secretion and a virtually complete loss of CGRP- and TRPV1-coexpressing islet-innervating fibers. These data indicate that CGRP-containing fibers in the islets are capsaicin sensitive, and that elimination of these fibers contributes to the prevention of the deterioration of glucose homeostasis through increased insulin secretion in ZDF rats. Based on these data we propose that the activity of islet-innervating capsaicin-sensitive fibers may have a role in the development of reduced insulin secretion in human type 2 diabetes mellitus. [source] Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and l - allo -Thr cleavageFEBS JOURNAL, Issue 18 2008Crystal structure, biochemical studies Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus (bsSHMT) is a pyridoxal 5,-phosphate-dependent enzyme that catalyses the conversion of l -serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids and transamination. In this article, we have examined the mechanism of the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional structure and biochemical properties of Y51F and Y61A bsSHMTs and their complexes with substrates, especially l - allo -Thr, show that the cleavage of 3-hydroxy amino acids could proceed via C, proton abstraction rather than hydroxyl proton removal. Both mutations result in a complete loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities. The mutation of Y51 to F strongly affects the binding of pyridoxal 5,-phosphate, possibly as a consequence of a change in the orientation of the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely reconstituted with pyridoxal 5,-phosphate. However, there was an alteration in the ,max value of the internal aldimine (396 nm), a decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with methoxyamine (MA). The mutation of Y61 to A results in the loss of interaction with C, and C, of the substrates. X-Ray structure and visible CD studies show that the mutant is capable of forming an external aldimine. However, the formation of the quinonoid intermediate is hindered. It is suggested that Y61 is involved in the abstraction of the C, proton from 3-hydroxy amino acids. A new mechanism for the cleavage of 3-hydroxy amino acids via C, proton abstraction by SHMT is proposed. [source] Homologous expression of a bacterial phytochromeFEBS JOURNAL, Issue 8 2007The cyanobacterium Fremyella diplosiphon incorporates biliverdin as a genuine, functional chromophore Bacteriophytochromes constitute a light-sensing subgroup of sensory kinases with a chromophore-binding motif in the N-terminal half and a C-terminally located histidine kinase activity. The cyanobacterium Fremyella diplosiphon (also designated Calothrix sp.) expresses two sequentially very similar bacteriophytochromes, cyanobacterial phytochrome A (CphA) and cyanobacterial phytochrome B (CphB). Cyanobacterial phytochrome A has the canonical cysteine residue, by which covalent chromophore attachment is accomplished in the same manner as in plant phytochromes; however, its paralog cyanobacterial phytochrome B carries a leucine residue at that position. On the basis of in vitro experiments that showed, for both cyanobacterial phytochrome A and cyanobacterial phytochrome B, light-induced autophosphorylation and phosphate transfer to their cognate response regulator proteins RcpA and RcpB [Hübschmann T, Jorissen HJMM, Börner T, Gärtner W & deMarsac NT (2001) Eur J Biochem268, 3383,3389], we aimed at the identification of a chromophore that is incorporated in vivo into cyanobacterial phytochrome B within the cyanobacterial cell. The approach was based on the introduction of a copy of cphB into the cyanobacterium via triparental conjugation. The His-tagged purified, recombinant protein (CphBcy) showed photoreversible absorption bands similar to those of plant and bacterial phytochromes, but with remarkably red-shifted maxima [,max 700 and 748 nm, red-absorbing (Pr) and far red-absorbing (Pfr) forms of phytochrome, respectively]. A comparison of the absorption maxima with those of the heterologously generated apoprotein, assembled with phycocyanobilin (,max 686 and 734 nm) or with biliverdin IX, (,max 700 and 750 ± 2 nm), shows biliverdin IX, to be a genuine chromophore. The kinase activity of CphBcy and phosphotransfer to its cognate response regulator was found to be strictly Pr -dependent. As an N-terminally located cysteine was found as an alternative covalent binding site for several bacteriophytochrome photoreceptors that bind biliverdin and lack the canonical cysteine residue (e.g. Agrobacterium tumefaciens and Deinococcus radiodurans), this corresponding residue in heterologously expressed cyanobacterial phytochrome B was mutated into a serine (C24S); however, there was no change in its spectral properties. On the other hand, the mutation of His267, which is located directly after the canonical cysteine, into alanine (H267A), caused complete loss of the capability of cyanobacterial phytochrome B to form a chromoprotein. [source] Assignment of the [4Fe-4S] clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site-directed mutantsFEBS JOURNAL, Issue 18 2005Lucia Forzi Ech hydrogenase from Methanosarcina barkeri is a member of a distinct group of membrane-bound [NiFe] hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). The sequence of the enzyme predicts the binding of three [4Fe-4S] clusters, one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 105 Pa of H2 at pH 7 giving rise to two distinct S½ electron paramagnetic resonance (EPR) signals, designated as the g = 1.89 and the g = 1.92 signal. Redox titrations at different pH values demonstrated that these two clusters had a pH-dependent midpoint potential indicating a function in ion pumping. To assign these signals to the subunits of the enzyme a set of M. barkeri mutants was generated in which seven of eight conserved cysteine residues in EchF were individually replaced by serine. EPR spectra recorded from the isolated mutant enzymes revealed a strong reduction or complete loss of the g = 1.92 signal whereas the g = 1.89 signal was still detectable as the major EPR signal in five mutant enzymes. It is concluded that the cluster giving rise to the g = 1.89 signal is the proximal cluster located in EchC and that the g = 1.92 signal results from one of the clusters of subunit EchF. The pH-dependence of these two [4Fe-4S] clusters suggests that they simultaneously mediate electron and proton transfer and thus could be an essential part of the proton-translocating machinery. [source] Transcriptional activity of interferon regulatory factor (IRF)-3 depends on multiple protein,protein interactionsFEBS JOURNAL, Issue 24 2002Hongmei Yang Virus infection results in the activation of a set of cellular genes involved in host antiviral defense. IRF-3 has been identified as a critical transcription factor in this process. The activation mechanism of IRF-3 is not fully elucidated, yet it involves a conformational change triggered by the virus-dependent phosphorylation of its C-terminus. This conformational change leads to nuclear accumulation, DNA binding and transcriptional transactivation. Here we show that two distinct sets of Ser/Thr residues of IRF-3, on phosphorylation, synergize functionally to achieve maximal activation. Remarkably, we find that activated IRF-3 lacks transcriptional activity, but activates transcription entirely through the recruitment of the p300/CBP coactivators. Moreover, we show that two separate domains of IRF-3 interact with several distinct regions of p300/CBP. Interference with any of these interactions leads to a complete loss of transcriptional activity, suggesting that a bivalent interaction is essential for coactivator recruitment by IRF-3. [source] Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3bGENES TO CELLS, Issue 7 2006Akiko Tsumura DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1,/, or Dnmt3a,/,Dnmt3b,/, ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases. [source] Homozygous deletions within the 11q13 cervical cancer tumor-suppressor locus in radiation-induced, neoplastically transformed human hybrid cellsGENES, CHROMOSOMES AND CANCER, Issue 4 2004Marc S. Mendonca Studies on nontumorigenic and tumorigenic human cell hybrids derived from the fusion of HeLa (a cervical cancer cell line) with GM00077 (a normal skin fibroblast cell line) have demonstrated "functional" tumor-suppressor activity on chromosome 11. It has been shown that several of the neoplastically transformed radiation-induced hybrid cells called GIMs (gamma ray induced mutants), isolated from the nontumorigenic CGL1 cells, have lost one copy of the fibroblast chromosome 11. We hypothesized, therefore, that the remaining copy of the gene might be mutated in the cytogenetically intact copy of fibroblast chromosome 11. Because a cervical cancer tumor suppressor locus has been localized to chromosome band 11q13, we performed deletion-mapping analysis of eight different GIMs using a total of 32 different polymorphic and microsatellite markers on the long arm (q arm) of chromosome 11. Four irradiated, nontumorigenic hybrid cell lines, called CONs, were also analyzed. Allelic deletion was ascertained by the loss of a fibroblast allele in the hybrid cell lines. The analysis confirmed the loss of a fibroblast chromosome 11 in five of the GIMs. Further, homozygous deletion (complete loss) of chromosome band 11q13 band sequences, including that of D11S913, was observed in two of the GIMs. Detailed mapping with genomic sequences localized the homozygous deletion to a 5.7-kb interval between EST AW167735 and EST F05086. Southern blot hybridization using genomic DNA probes from the D11S913 locus confirmed the existence of homozygous deletion in the two GIM cell lines. Additionally, PCR analysis showed a reduction in signal intensity for a marker mapped 31 kb centromeric of D11S913 in four other GIMs. Finally, Northern blot hybridization with the genomic probes revealed the presence of a novel >15-kb transcript in six of the GIMs. These transcripts were not observed in the nontumorigenic hybrid cell lines. Because the chromosome 11q13 band deletions in the tumorigenic hybrid cell lines overlapped with the minimal deletion in cervical cancer, the data suggest that the same gene may be involved in the development of cervical cancer and in radiation-induced carcinogenesis. We propose that a gene localized in proximity to the homozygous deletion is the candidate tumor-suppressor gene. © 2004 Wiley-Liss, Inc. [source] Heregulin and forskolin-induced cyclin D3 expression in Schwann cells: Role of a CCAAT promoter element and CCAAT enhancer binding proteinGLIA, Issue 3 2004Luis Fuentealba Abstract Heregulin, a polypeptide growth factor, and forskolin, an adenylyl cyclase activator, synergistically stimulate expression of cyclin D3 and cell division in Schwann cells. Heregulin induces expression in Schwann cells of a luciferase reporter gene linked to the cyclin D3 promoter. Forskolin markedly augments reporter expression in the presence of heregulin. Deletion analysis identified several promoter sites that contribute to high-level reporter expression in heregulin- and forskolin-treated Schwann cells. A promoter fragment that contains 103 bp of 5,-flanking sequence produced significant reporter expression in heregulin- and forskolin-stimulated cells. Deletion of a consensus CCAAT site within this promoter fragment caused a nearly complete loss of reporter expression. Similar results were obtained when CCAAT site mutations were introduced into the promoter. Heregulin and forskolin increased steady-state levels of CCAAT/enhancer binding protein-, (C/EBP,) in Schwann cells. Mobility shift assays identified proteins in Schwann cell nuclear extracts that formed stable complexes with the cyclin D3 CCAAT promoter element and were disrupted by anti-C/EBP, antibody. Transfection of Schwann cells with C/EBP, cDNA increased cyclin D3 reporter expression. In contrast to these results, mutation of a cAMP response element in the cyclin D3 promoter had only a modest effect on heregulin- and forskolin-stimulated reporter expression. These findings demonstrate that C/EBP, plays a key role in the heregulin and cAMP-dependent regulation of cyclin D3 expression in Schwann cells. © 2003 Wiley-Liss, Inc. [source] Effects of afforestation on water yield: a global synthesis with implications for policyGLOBAL CHANGE BIOLOGY, Issue 10 2005Kathleen A. Farley Abstract Carbon sequestration programs, including afforestation and reforestation, are gaining attention globally and will alter many ecosystem processes, including water yield. Some previous analyses have addressed deforestation and water yield, while the effects of afforestation on water yield have been considered for some regions. However, to our knowledge no systematic global analysis of the effects of afforestation on water yield has been undertaken. To assess and predict these effects globally, we analyzed 26 catchment data sets with 504 observations, including annual runoff and low flow. We examined changes in the context of several variables, including original vegetation type, plantation species, plantation age, and mean annual precipitation (MAP). All of these variables should be useful for understanding and modeling the effects of afforestation on water yield. We found that annual runoff was reduced on average by 44% (±3%) and 31% (±2%) when grasslands and shrublands were afforested, respectively. Eucalypts had a larger impact than other tree species in afforested grasslands (P=0.002), reducing runoff (90) by 75% (±10%), compared with a 40% (±3%) average decrease with pines. Runoff losses increased significantly with plantation age for at least 20 years after planting, whether expressed as absolute changes (mm) or as a proportion of predicted runoff (%) (P<0.001). For grasslands, absolute reductions in annual runoff were greatest at wetter sites, but proportional reductions were significantly larger in drier sites (P<0.01 and P<0.001, respectively). Afforestation effects on low flow were similar to those on total annual flow, but proportional reductions were even larger for low flow (P<0.001). These results clearly demonstrate that reductions in runoff can be expected following afforestation of grasslands and shrublands and may be most severe in drier regions. Our results suggest that, in a region where natural runoff is less than 10% of MAP, afforestation should result in a complete loss of runoff; where natural runoff is 30% of precipitation, it will likely be cut by half or more when trees are planted. The possibility that afforestation could cause or intensify water shortages in many locations is a tradeoff that should be explicitly addressed in carbon sequestration programs. [source] Specific Processes and Scrambling in the Dehydrogenation of Ethane and the Degenerate Hydrogen Exchange in the Gas-Phase Ion Chemistry of the Ni(C,H3,O)+/C2H6 CoupleHELVETICA CHIMICA ACTA, Issue 5 2007Maria Schlangen Abstract A mechanistically unprecedented situation characterizes the gas-phase ion chemistry of Ni(C,H3,O)+ when reacted under thermal, single-collision conditions with ethane. A dehydrogenation channel leading to Ni(C3,H7,O)+ is to 90% preceded by a complete loss of positional identity of all nine H-atoms of the encounter complex (,scrambling'), whereas ca. 10% of the reaction exhibit a selective CH bond activation of the alkane. In addition, a degenerate H exchange between ethane and the (C,H3,O) unit occurs as a side reaction, the mechanistic details of which remain unknown for the time being. [source] Variants of two major T cell epitopes within the hepatitis B surface antigen are not recognized by specific T helper cells of vaccinated individualsHEPATOLOGY, Issue 2 2002Tanja Bauer Several naturally occurring variants of immunogenic T cell epitopes were identified within the hepatitis B surface antigen (HBsAg). The effect of these variants on the cellular immune response was studied in individuals vaccinated against HBV. Class-II restricted T-cell responses of 30 vaccinees were analyzed after stimulation of peripheral blood mononuclear cells (PBMCs) with 4 synthetic peptides representing the 4 T-cell epitopes of HBsAg known as of yet. The 2 epitopes P1 (aa 16-33) and P4 (aa 213-226) could be identified as the dominant ones in our vaccinees by proliferation assays and enzyme-linked immunospot assays. Responses to these epitopes were compared with responses to their naturally occurring variants found in HBV isolates of chronic virus carriers. Three of 11 variants of epitope P4 led to a complete loss of T-cell reactivity in 4 of 10 donors, all of whom reacted well to the corresponding wild-type sequence. The remaining 6 donors recognized these variants as well as the vaccine epitope. Similarly, 3 P1-variants of the 12 found induced only a significantly reduced reactivity in 4 of 10 donors, whereas they led to a normal response in the other 6 individuals. Stimulation of T cells also induced the secretion of antibody to HBsAg (anti-HBs) by specific B cells; however, those peptides that failed to activate T cells were also unable to cause any significant anti-HBs production. In conclusion, our results suggest an immune escape of certain mutant strains of HBV in vaccinated individuals could exist at the T-cell level. [source] Analysis of genotypic and phenotypic clinical cut-off levels for ritonavir-boosted saquinavirHIV MEDICINE, Issue 2 2006A Hill There is a need for new, clinically relevant interpretation algorithms for genotypic and phenotypic resistance for ritonavir-boosted saquinavir (SQV/r) at the current approved dosage [1000/100 mg twice a day (bid)]. Clinical cut-off levels, which correlate baseline measures of resistance with HIV RNA responses in large cohorts or clinical trials, are the ideal reference for developing such algorithms. Cut-off levels previously developed for unboosted saquinavir may no longer apply, as the plasma drug levels with SQV/r are significantly higher and may be able partially to overcome protease inhibitor-resistant HIV. Clinical cut-off levels for SQV/r, assessed in several cohort studies and clinical trials, also suggest that multiple genotypic mutations are required for complete loss of virological response. For phenotypic analysis of resistance, saquinavir cut-off levels 10,11-fold higher than the wild-type IC50 have best distinguished responders from non-responders in cohort studies. Using Virtual Phenotype, a 12.3-fold upper cut-off level was determined from analysis of large cohort databases. These genotypic and phenotypic algorithms need to be validated in larger prospective studies. [source] Plectin deficiency leads to both muscular dystrophy and pyloric atresia in epidermolysis bullosa simplex,HUMAN MUTATION, Issue 10 2010Ken Natsuga Abstract Plectin is a cytoskeletal linker protein which has a long central rod and N- and C-terminal globular domains. Mutations in the gene encoding plectin (PLEC) cause two distinct autosomal recessive subtypes of epidermolysis bullosa: EB simplex (EBS) with muscular dystrophy (EBS-MD), and EBS with pyloric atresia (EBS-PA). Previous studies have demonstrated that loss of full-length plectin with residual expression of the rodless isoform leads to EBS-MD, whereas complete loss or marked attenuation of expression of full-length and rodless plectin underlies the more severe EBS-PA phenotype. However, muscular dystrophy has never been identified in EBS-PA, not even in the severe form of the disease. Here, we report the first case of EBS associated with both pyloric atresia and muscular dystrophy. Both of the premature termination codon-causing mutations of the proband are located within exon 32, the last exon of PLEC. Immunofluorescence and immunoblot analysis of skin samples and cultured fibroblasts from the proband revealed truncated plectin protein expression in low amounts. This study demonstrates that plectin deficiency can indeed lead to both muscular dystrophy and pyloric atresia in an individual EBS patient. © 2010 Wiley-Liss, Inc. [source] Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations,HUMAN MUTATION, Issue 9 2010James J. Cox Abstract SCN9Aencodes the voltage-gated sodium channel Nav1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Nav1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Nav1.7-,R1370-L1374). Both of these mutations map to the pore region of the Nav1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Nav1.7. © 2010 Wiley-Liss, Inc. [source] First missense mutation in the SOST gene causing sclerosteosis by loss of sclerostin function,HUMAN MUTATION, Issue 7 2010Elke Piters Abstract Sclerosteosis is a rare bone dysplasia characterized by greatly increased bone mass, especially of the long bones and the skull. Patients are tall, show facial asymmetry and often have syndactyly. Clinical complications are due to entrapment of cranial nerves. The disease is thought to be due to loss-of-function mutations in the SOST gene. The SOST gene product, sclerostin, is secreted by osteocytes and transported to the bone surface where it inhibits osteoblastic bone formation by antagonizing Wnt signaling. In a small Turkish family with sclerosteosis, we identified a missense mutation (c.499T>C; p.Cys167Arg) in exon 2 of the SOST gene. This type of mutation has not been previously reported and using different functional approaches, we show that it has a devastating effect on the biological function of sclerostin. The affected cysteine is the last cysteine residue of the cystine-knot motif and loss of this residue leads to retention of the mutant protein in the ER, possibly as a consequence of impaired folding. Together with a significant reduced ability to bind to LRP5 and inhibit Wnt signaling, the p.Cys167Arg mutation leads to a complete loss of function of sclerostin and thus to the characteristic sclerosteosis phenotype. © 2010 Wiley-Liss, Inc. [source] Broad tumor spectrum in a mouse model of multiple endocrine neoplasia type 1INTERNATIONAL JOURNAL OF CANCER, Issue 2 2007Kelly A. Loffler Abstract Multiple endocrine neoplasia type 1 (MEN1) is an inherited cancer predisposition syndrome typified by development of tumors in parathyroid, pituitary and endocrine pancreas, as well as less common sites including both endocrine and nonendocrine organs. Deletion or mutation of the tumor suppressor gene MEN1 on chromosome 11 has been identified in many cases of MEN1 as well as in sporadic tumors. The molecular biology of menin, the protein encoded by MEN1, remains poorly understood. Here we describe a mouse model of MEN1 in which tumors were seen in pancreatic islets, pituitary, thyroid and parathyroid, adrenal glands, testes and ovaries. The observed tumor spectrum therefore includes types commonly seen in MEN1 patients and additional types. Pancreatic pathology was most common, evident in over 80% of animals, while other tumor types developed with lower frequency and generally later onset. Tumors of multiple endocrine organs were observed frequently, but progression to carcinoma and metastasis were not evident. Tumors in all sites showed loss of heterozygosity at the Men1 locus, though the frequency in testicular tumors was only 36%, indicating that a different molecular mechanism of tumorigenesis occurs in those Leydig tumors that do not show loss of the normal Men1 allele. Menin expression was below the level of detection in ovary, thyroid and testis, but loss of nuclear menin immunoreactivity was observed uniformly in all pancreatic islet adenomas and in some hyperplastic islet cells, suggesting that complete loss of Men1 is a critical point in islet tumor progression in this model. © 2006 Wiley-Liss, Inc. [source] Deamination of adenosine by extracts of Penicillium politans NRC-510JOURNAL OF BASIC MICROBIOLOGY, Issue 2 2005Ali M. Elshafei Prof. Cell-free extracts of nitrate-grown Penicillium politans NRC-510 could catalyze the hydrolytic deamination of adenosine to inosine maximally at pH 6.0 and 45 °C. However the same extracts could not catalyze the N-glycosidic bond cleavage of adenosine at pH 4.0, 6.0 and 8.0. Incubation of the extracts at 55 °C for 30 minutes caused about 31% loss in activity whereas incubation of the extracts at 60 °C for 15 minutes caused a complete loss of enzyme activity. Results indicated the absence of the involvement of sulfhydryl groups in the catalytic site of adenosine deaminase. The enzyme is inhibited by ethylene diamine tetraacetate indicating that adenosine deaminase is a metalloenzyme. MnCl2 and MgCl2 had a remarkable activating effect, whereas HgCl2, CaCl2 and ZnSO4 showed an inhibitory effect on enzyme activity. Dialyzing the extracts for 24 hours significantly increase deaminase activity by about 33%. The apparent Km value was calculated for adenosine and found to be 3.63 × 10,3M, which indicates high affinity of adenosine deaminase for its substrate adenosine. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Crystal structures of thymidylate synthase mutant R166Q: Structural basis for the nearly complete loss of catalytic activity,JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2006Rogerio R. Sotelo-Mundo Abstract Thymidylate synthase (TS) catalyzes the folate-dependent methylation of deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP). We have investigated the role of invariant arginine 166, one of four arginines that contact the dUMP phosphate, using site-directed mutagenesis, X-ray crystallography, and TS from Escherichia coli. The R166Q mutant was crystallized in the presence of dUMP and a structure determined to 2.9 Å resolution, but neither the ligand nor the sulfate from the crystallization buffer was found in the active site. A second structure determined with crystals prepared in the presence of dUMP and the antifolate 10-propargyl-5,8-dideazafolate revealed that the inhibitor was bound in an extended, nonproductive conformation, partially occupying the nucleotide-binding site. A sulfate ion, rather than dUMP, was found in the nucleotide phosphate-binding site. Previous studies have shown that the substitution at three of the four arginines of the dUMP phosphate-binding site is permissive; however; for Arg166, all the mutations lead to a near-inactive mutant. The present structures of TS R166Q reveal that the phosphate-binding site is largely intact, but with a substantially reduced affinity for phosphate, despite the presence of the three remaining arginines. The position of Cys146, which initiates catalysis, is shifted in the mutant and resides in a position that interferes with the binding of the dUMP pyrimidine moiety. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:88,92, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20122 [source] Range-wide genetic structure in a north-east Asian spruce (Picea jezoensis) determined using nuclear microsatellite markersJOURNAL OF BIOGEOGRAPHY, Issue 5 2009Mineaki Aizawa Abstract Aim, We used microsatellite markers to determine the range-wide genetic structure of Picea jezoensis and to test the hypothesis that the past population history of this widespread cold-temperate spruce has resulted in a low level of genetic variation and in imprints of inbreeding and bottlenecks in isolated marginal populations. Location, The natural range of the three infraspecific taxa of P. jezoensis throughout north-east Asia, including isolated marginal populations. Methods, We analysed a total of 990 individuals across 33 natural populations using four nuclear microsatellite loci. Population genetic structure was assessed by analysing genetic diversity indices for each population, examining clustering (model-based and distance-based) among populations, evaluating signals of recent bottlenecks, and testing for isolation by distance (IBD). Results, The 33 populations were clustered into five groups. The isolated marginal groups of populations (in Kamchatka, Kii in Japan and South Korea) exhibited low levels of allelic richness and gene diversity and a complete or almost complete loss of rare alleles. A recent bottleneck was detected in the populations in Hokkaido across to mid-Sakhalin. The IBD analysis revealed that genetic divergence between populations was higher for populations separated by straits. Main conclusions,Picea jezoensis showed a higher level of genetic differentiation among populations (FST = 0.101) than that observed in the genus Picea in general. This might be attributable to the fact that historically the straits around Japan acted as barriers to the movement of seeds and pollen. The low levels of genetic diversity in the isolated marginal population groups may reflect genetic drift that has occurred after isolation. Evidence of a significant bottleneck between the Hokkaido and mid-Sakhalin populations implies that the cold, dry climate in the late Pleistocene resulted in the decline and contraction of populations, and that there was a subsequent expansion followed by a founder effect when conditions improved. The high polymorphism observed in P. jezoensis nuclear microsatellites revealed cryptic genetic structure that organellar DNA markers failed to identify in a previous study. [source] To go or not to go: Migration of human mesenchymal progenitor cells stimulated by isoforms of PDGFJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2004Jörg Fiedler Abstract The recruitment of mesenchymal progenitor cells (MPCs) and their subsequent differentiation to osteoblasts is mandatory for bone development, remodeling, and repair. To study the possible involvement of platelet-derived growth factor (PDGF) isoforms, primary human MPCs and osteogenic differentiated progenitor cells (dOB) were examined for chemotaxic response to homodimeric human platelet-derived growth factor AA, -BB, and heterodimeric PDGF-AB. The role of PDGF receptors was addressed by preincubation with PDGF receptor alpha and beta chain specific antibodies. Migration of MPCs, dOB, and primary osteoblasts (OB) was stimulated by the addition of rhPDGF-AA, rhPDGF-BB, and rhPDGF-AB. The effect was highest in MPCs and for rhPDGF-BB, and declining with osteogenic differentiation. Preincubation with the receptor alpha specific antibody decreased the CI to borderline values while pretreatment with the receptor beta specific antibody led to a complete loss of chemotactic response to PDGF isoforms. In control experiments, basal migration values and rhBMP-2 as well as rxBMP-4 induced chemotaxis of MPC were not influenced by the addition of receptor alpha or beta antibodies. Interestingly, without preincubation the parallel exposure of MPC to rhTGF-,1 instantaneously leads to a selective loss of migratory stimulation by rhPDGF-AA. The chemotactic effect of PDGF isoforms for primary human MPCs and the influence of osteogenic differentiation suggest a functional role for recruitment of MPCs during bone development and remodeling. Moreover, these observations may be useful for novel approaches towards guided tissue regeneration or tissue engineering of bone. © 2004 Wiley-Liss, Inc. [source] Molecular dynamics simulation of clustered DNA damage sites containing 8-oxoguanine and abasic siteJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 8 2005Hirofumi Fujimoto Abstract Clustered DNA damage sites induced by ionizing radiation have been suggested to have serious consequences to organisms, such as cancer, due to their reduced probability to be repaired by the enzymatic repair machinery of the cell. Although experimental results have revealed that clustered DNA damage sites effectively retard the efficient function of repair enzymes, it remains unclear as to what particular factors influence this retardation. In this study, approaches based on molecular dynamics (MD) simulation have been applied to examine conformational changes and energetic properties of DNA molecules containing clustered damage sites consisting of two lesioned sites, namely 7,8-dihydro-8-oxoguanine (8-oxoG) and apurinic/apyrimidinic (AP) site, located within a few base pairs of each other. After 1 ns of MD simulation, one of the six DNA molecules containing a clustered damage site develops specific characteristic features: sharp bending at the lesioned site and weakening or complete loss of electrostatic interaction energy between 8-oxoG and bases located on the complementary strand. From these results it is suggested that these changes would make it difficult for the repair enzyme to bind to the lesions within the clustered damage site and thereby result in a reduction of its repair capacity. © 2005 Wiley Periodicals, Inc. J Comput Chem 26: 788,798, 2005 [source] Chromogenic in situ hybridization analysis of melastatin mRNA expression in melanomas from American Joint Committee on Cancer stage I and II patients with recurrent melanomaJOURNAL OF CUTANEOUS PATHOLOGY, Issue 9 2006L. Hammock Objective:, To determine whether loss of melastatin (MLSN) is a universal phenomenon in American Joint Committee on Cancer (AJCC) stage I and II melanoma patients who experienced recurrence. Material and methods:, Paraffin blocks of primary melanomas (PMs) were retrieved from 30 patients who had a negative sentinel lymph node biopsy and developed recurrent melanoma (AJCC stage I and II). Chromogenic in situ hybridization (CISH) methods were utilized to evaluate the expression of MLSN mRNA. These results were correlated with clinicopathologic data. Results:, Variable, heterogeneous expression of MLSN mRNA was identified in normal, in situ and invasive melanocytes within and between cases. For the invasive PM component, 24 (80%) had focal, regional or complete loss of MLSN mRNA. The remaining 20% had either regional or total partial downregulation of MLSN mRNA. Intact MLSN mRNA expression was present regionally in 14/30 (47%), with mean relative tumor area of 38%, range 5,85%. Increasing loss of MLSN mRNA significantly correlated with increasing tumor depth and microsatellites (r = 0.1/0.4, p = 0.04). However, thin, AJCC T stage 1a PM had higher relative mean loss than intermediate AJCC T stage 2a/2b/3a thickness PM (65% vs. 34%/48%/25%). Increasing loss of MLSN mRNA significantly impacted on disease free survival (DFS) by multivariate analysis (58 vs. 0% 2 years DFS, , 75 vs. >75% mRNA loss, p = 0.02). Decreased overall survival significantly correlated with increasing age and vascular invasion on multivariate analysis. Conclusion:, Extensive loss of MLSN in PM correlated with aggressive metastatic melanoma. Ancillary testing for MLSN mRNA expression by CISH could offer a means to more accurately identify AJCC stage I and II patients at risk for metastatic disease, who could benefit from adjuvant therapy. [source] | ||||||||||||||||||||||||||||||||||||||||