98 research outputs found
C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins
No full textThe human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency
N‐terminus of hMLH1 confers interaction of hMutLα and hMutLβ with hMutSα
No full textMismatch repair is a highly conserved system that ensures replication fidelity by repairing mispairs after DNA synthesis. In humans, the two protein heterodimers hMutSα (hMSH2‐hMSH6) and hMutLα (hMLH1‐hPMS2) constitute the centre of the repair reaction. After recognising a DNA replication error, hMutSα recruits hMutLα, which then is thought to transduce the repair signal to the excision machinery. We have expressed an ATPase mutant of hMutLα as well as its individual subunits hMLH1 and hPMS2 and fragments of hMLH1, followed by examination of their interaction properties with hMutSα using a novel interaction assay. We show that, although the interaction requires ATP, hMutLα does not need to hydrolyse this nucleotide to join hMutSα on DNA, suggesting that ATP hydrolysis by hMutLα happens downstream of complex formation. The analysis of the individual subunits of hMutLα demonstrated that the hMutSα–hMutLα interaction is predominantly conferred by hMLH1. Further experiments revealed that only the N‐terminus of hMLH1 confers this interaction. In contrast, only the C‐terminus stabilised and co‐immunoprecipitated hPMS2 when both proteins were co‐expressed in 293T cells, indicating that dimerisation and stabilisation are mediated by the C‐terminal part of hMLH1. We also examined another human homologue of bacterial MutL, hMutLβ (hMLH1–hPMS1). We show that hMutLβ interacts as efficiently with hMutSα as hMutLα, and that it predominantly binds to hMutSα via hMLH1 as well
Phosphorylation-dependent signaling controls degradation of DNA mismatch repair protein PMS2
No full textMutLα, a heterodimer consisting of MLH1 and PMS2, plays an important role in DNA mismatch repair and has been shown to be additionally involved in several other important cellular mechanisms. Previous work indicated that AKT could modulate PMS2 stability by phosphorylation. Still, the mechanisms of regulation of MutLα remain unclear. The stability of MutLα subunits was investigated by transiently overexpression of wild type and mutant forms of MLH1 and PMS2 using immunoblotting for measuring the protein levels after treatment. We found that treatment with the cell-permeable serine/threonine phosphatase inhibitor, Calyculin, leads to degradation of PMS2 when MLH1 or its C-terminal domain is missing or if amino acids of MLH1 essential for PMS2 interaction are mutated. In addition, we discovered that the C-terminal tail of PMS2 is relevant for this Calyculin-dependent degradation. A direct involvement of AKT, which was previously described to be responsible for PMS2 degradation, could not be detected. The multi-kinase inhibitor Sorafenib, in contrast, was able to avoid the degradation of PMS2 which postulates that cellular phosphorylation is involved in this process. Together, we show that pharmacologically induced phosphorylation by Calyculin can induce the selective proteasome-dependent degradation of PMS2 but not of MLH1 and that the PMS2 degradation could be blocked by Sorafenib treatment. Curiously, the C-terminal Lynch Syndrome-variants MLH1L749P and MLH1Y750X make PMS2 prone to Calyculin induced degradation. Therefore, we conclude that the specific degradation of PMS2 may represent a new mechanism to regulate MutLα.Fil: Hinrichsen, Inga. University Clinic Frankfurt; AlemaniaFil: Weßbecher, Isabel M.. University Clinic Frankfurt; AlemaniaFil: Huhn, Meik. University Clinic Frankfurt; AlemaniaFil: Passmann, Sandra. University Clinic Frankfurt; AlemaniaFil: Zeuzem, Stefan. University Clinic Frankfurt; AlemaniaFil: Plotz, Guido. University Clinic Frankfurt; AlemaniaFil: Biondi, Ricardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University Clinic Frankfurt; AlemaniaFil: Brieger, Angela. University Clinic Frankfurt; Alemani
DNA mismatch repair activity of MutLα is regulated by CK2-dependent phosphorylation of MLH1 (S477)
No full textMutLα, a heterodimer consisting of MLH1 and PMS2, is a key player of DNA mismatch repair (MMR), yet little is known about its regulation. In this study, we used mass spectrometry to identify phosphorylated residues within MLH1 and PMS2. The most frequently detected phosphorylated amino acid was serine 477 of MLH1. Pharmacological treatment indicates that Casein kinase II (CK2) could be responsible for the phosphorylation of MLH1 at serine 477 in vivo. In vitro kinase assay verified MLH1 as a substrate of CK2. Most importantly, using in vitro MMR assay we could demonstrate that p-MLH1S477 lost MMR activity. Moreover, we found that levels of p-MLH1S477 varied during the cell cycle. In summary, we identified that phosphorylation of MLH1 by CK2 at amino acid position 477 can switch off MMR activity in vitro. Since CK2 is overexpressed in many tumors and is able to inactivate MMR, the new mechanism here described could have an important impact on tumors overactive in CK2.Fil: Weßbecher, Isabel M.. Goethe Universitat Frankfurt; AlemaniaFil: Hinrichsen, Inga. Goethe Universitat Frankfurt; AlemaniaFil: Funke, Sebastian. Klinikum Der Johannes-gutenberg-universität Und Fachbereich Medizin; AlemaniaFil: Oellerich, Thomas. Goethe Universitat Frankfurt; AlemaniaFil: Plotz, Guido. Goethe Universitat Frankfurt; AlemaniaFil: Zeuzem, Stefan. Goethe Universitat Frankfurt; AlemaniaFil: Grus, Franz H.. Klinikum Der Johannes-gutenberg-universität Und Fachbereich Medizin; AlemaniaFil: Biondi, Ricardo Miguel. Goethe Universitat Frankfurt; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Brieger, Angela. Goethe Universitat Frankfurt; Alemani
The Matilda Wilson College Proposal of December 1956: The First Curricular Source for Oakland University
No full textIn November 1956, Matilda Dodge Wilson and Alfred Wilson decided to donate their Meadow Brook Farms estate and $2 million to the state of Michigan for the purpose of founding an educational institution. One month later, an 11-page memo envisioning the nature of the future Michigan State University Oakland (MSUO) emerged from the office of the Michigan State University (MSU) Vice President for Academic Affairs, Thomas Hamilton. The document was the first conceptualization of an academic program for the new institution which became MSUO, although the author called it the Matilda Wilson College
CK2 and the Hallmarks of Cancer
No full textCancer is a leading cause of death worldwide. Casein kinase 2 (CK2) is commonly dysregulated in cancer, impacting diverse molecular pathways. CK2 is a highly conserved serine/threonine kinase, constitutively active and ubiquitously expressed in eukaryotes. With over 500 known substrates and being estimated to be responsible for up to 10% of the human phosphoproteome, it is of significant importance. A broad spectrum of diverse types of cancer cells has been already shown to rely on disturbed CK2 levels for their survival. The hallmarks of cancer provide a rationale for understanding cancer’s common traits. They constitute the maintenance of proliferative signaling, evasion of growth suppressors, resisting cell death, enabling of replicative immortality, induction of angiogenesis, the activation of invasion and metastasis, as well as avoidance of immune destruction and dysregulation of cellular energetics. In this work, we have compiled evidence from the literature suggesting that CK2 modulates all hallmarks of cancer, thereby promoting oncogenesis and operating as a cancer driver by creating a cellular environment favorable to neoplasia
The Books I Wanted to Write
No full textAuthor lists book titles he would have liked to have written and humorous descriptions of them
Considerações sôbre o emprego de variedades sintéticas no melhoramento do milho: I - sintéticos simples
No full text1) Inicialmente foi dado um breve resumo dos métodos básicos do melhoramento no milho os quais podem ser reunidos em dois grupos principais: o processo do milho híbrido, com as suas variantes, e os processos dos sintéticos. Estes últimos podem ainda ser subdivididos em duas categorias: os sintéticos simples e os sintéticos balançados. Na obtenção dos sintéticos simples toma-se inicialmente em consideração a capacidade combinatória das linhagens a serem misturadas, e se executa em cada geração de sintético uma seleção massal de conservação. Nos balançados devemos acrescentar uma forte seleção, na fase preparatória, contra todos os híbridos que dão segregações mendelianas fortes demais. 2) No curso de um breve resumo histórico ficou evidente que a idéia de se aproveitarem os sintéticos no melhoramento do milho, formulada pela primeira vez por Hayes e Garber (1919) deu resultados práticos apreciáveis. Assim Hayes, Rinke e Tsinang (1944) obtiveram produções de sintéticos que eram equivalentes de um híbrido duplo, Minhybrid 403. Lonnquist (1949) registrou produções de sintéticos idênticos ao híbrido duplo, US 13. Roberts, Wellhausen, Palácios e Guaves (1949) e Wellhausen (1950) relataram resultados bastante satisfatórios, obtidos no México. 3) Ficou demonstrado que as fórmulas de Sewall Wright (1932) e de Mangelsdorf (1939) não podem ser consideradas como explicações gerais do método, pois pela sua derivação pode-se mostrar facilmente que elas exigem certas premissas que nem sempre são justificáveis. 4) Para eliminar confusões na terminologia foi desenvolvido um esquema básico da constituição de sintéticos supondo que se parte de linhagens autofecundadas e que foram plantadas em conjunto para a reprodução de cruzamento livre. A geração que consiste das plantas autofecundadas, plantadas em mistura, é denominada SyO. A geração seguinte, a qual contém uma maior percentagem de híbridos simples e uma menor per-centagem de descendentes de cruzamentos dentro de mesma linhagem (descendentes consanguíneos) representa assim a geração Syl. A geração que segue depois de novo cruzamento livre, Sy2, será então composta de híbridos entre quatro linhagens (híbridos duplos"), entre três linhagens ("three way crosses"), entre duas linhagens ("híbridos simples") e descendentes de combinações consanguíneas, ("inbreds"). Porém se houver uma seleção em Sy1 que elimina todos os descendentes de combinações consanguíneas, sobrevivendo apenas híbridos simples, então a geração Sy2 será composta de híbridos entre plantas que não tem nenhuma das linhagens originais em comum, os que têm uma linhagem em comum e finalmente aqueles que têm duas linhagens em comum. 5) Empregando esta classificação das gerações, podemos verificar que a geração Sy1 de Lonnquist corresponde à geração Sy1 do esquema básico, a geração Sy1 deHayes et al corresponde à geração Sy2 do esquema básico é a geração Sy1 de Wellhausen et al corresponde aproximadamente à geração Sy3 do esquema básico. 6) Uma teoria mais correta dos sintéticos deve-se basear nas regras da genética em populações, as quais foram empregadas por Brieger para justificar o processo dos sintéticos balançados. Uma discussão mais detalhada desta teoria será assim dada numa outra publicação que se ocupara especialmente com ossintéticos balançados.1) The author gives a short resume of the principal breeding methods in maize. Since mass selection cannot be considered as a method for improving corn, two groups of methods remain: the hybrid corn method and the method of the synthetics. Reasons are given why it seems important that the latter should be applied, after a further improvement of the breeding technique and its theoreticsl basis. The method may still be subdivided into the method of simple synthetics and of balanced synthetics. In the preparation of the former, only the following two points have to be considered: selection for combining ability before the constitution of the synthetic, and mass selection aganst weak descendants of consanguineous matings after the establishment of the synthetic. In the case of the balanced synthetics, a third element is added: selection against or rather previous elination of all hybrids which give too strong mendelian segregation in a synthetic. 2) The first proposal to use synthetics has been made by Hayes and Gardner in 1919. Positive results were obtained howewer only much later, since before 1940 the importance of selection for combining ability was not recognized. Hayes, Rinke and Tsiang (1944) obtained a synthetic which equalled the double hybrid Minhybrid 403.Lonnquist (1949) obtained a synthetic which eaqulled the double hybrid US13. Roberts, Wellhausen, Palacios and Cuevas (1949), Roberts and Wellhausen (1948) and Wellhausen (1950) reported on satisfatory results from Mexico. Brieger (1944 and later) produced balanced synthetics of subtropical sweet corn. 3) The author demostrates that the formulae of Sewall Wright (1922) and of P. C. Magelsdorf (1939) cannot be used satisfactorily to explain the composition of synthetics. Both these formulae start from certain assumptions which are not allways satisfeid, and disregard the principles of genetics in populations under selection. 4) In oder to avoid confusion in discussions on the theory of synthetics, a basic scheme is proposed for the identficiation of subsequent generations. The first generation of plants, planted out in mixture and left to free pollinisation should be called So. and it should be composed of the offspring of individual selected plants which had suffered at least one selfing or more. The first generation after free pollinisation, or generation Sy1, consists thus mainly of simple hybrids except for some individuals resulting from consanguineous matings between sister plants. If there were no selection, the next generation Sy2 should be composed of individuals from matings between individuals which may have none,, one, two, three or even four lines in their ancestry in common. Howewer if artificial selection against weaker and less productive plants is carried out in the generations Syl and Sy2, than we may assume that only individuals remain from matings wich had from none up to two ancestral lines in common. 5) Using this classification we can say that the generation Syl in Lonnquist\u27s experiment corresponds to the generation Syl of the basic scheme, Syl of Hayes, Rinke and Tsiang correspond to Sy2 of the basic scheme and Syl of the Mexican authors corresponds to a generation of about the order Sy3. 6) A correct theory of synthetics should take fully into consideration the principals of population genetics, taking furthermore into consideration modern theories on the genetic basis of heterosis in maize
The role of nonerythroid spectrin αII in cancer
No full textNonerythroid spectrin αII (SPTAN1) is an important cytoskeletal protein that ensures vital cellular properties including polarity and cell stabilization. In addition, it is involved in cell adhesion, cell-cell contact, and apoptosis. The detection of altered expression of SPTAN1 in tumors indicates that SPTAN1 might be involved in the development and progression of cancer. SPTAN1 has been described in cancer and therapy response and proposed as a potential marker protein for neoplasia, tumor aggressiveness, and therapeutic efficiency. On one hand, the existing data suggest that overexpression of SPTAN1 in tumor cells reflects neoplastic and tumor promoting activity. On the other hand, nuclear SPTAN1 can have tumor suppressing effects by enabling DNA repair through interaction with DNA repair proteins. Moreover, SPTAN1 cleavage products occur during apoptosis and could serve as markers for the efficacy of cancer therapy. Due to SPTAN1’s multifaceted functions and its role in adhesion and migration, SPTAN1 can influence tumor growth and progression in both positive and negative directions depending on its specific regulation. This review summarizes the current knowledge on SPTAN1 in cancer and depicts several mechanisms by which SPTAN1 could impact tumor development and aggressiveness
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Social Networks, Poverty and Development: An Analysis of Capacity Building in Arizona and New Mexico Colonias
No full textSince the early 1960s, scholars and policymakers have struggled to understand the appropriate role of government in effectively using resources to alleviate poverty. While early U.S. anti-poverty efforts emphasized place-based strategies, such as government-directed infrastructure investments, approaches have gradually shifted to favor civil sector efforts that build community capacity. Efforts to strengthen community capacity have emphasized enhancement of community participation, improvement of governance and strengthening of accountability. Yet, despite the growing emphasis on capacity building, rural regions such as the US-Mexico border region, Appalachia, the Mississippi Delta and Native American country have remained poor.This dissertation argues that government institutions have failed to improve conditions in poor, rural regions because current efforts ignore the broader context and fail to understand the needs of both formal organizations and informal participants. This research argues that without an adequate conceptual framework for assessing these three factors - the structural environment, community-based organizations, and local society -- federal investments cannot change local conditions. A conceptual model integrating these factors is applied to the case of poor, unincorporated colonias located in Arizona and New Mexico counties bordering Mexico. The empirical application of the conceptual model relies on methods integrating both regional and local analysis. The regional analysis is used to develop a socioeconomic index of deprivation. The index accomplishes two objectives. First, it reveals patterns of deprivation, uncovering the relationship between the impact of location (proximity to metropolitan and border areas) on the level of socioeconomic deprivation. Second, it is applied to select five cases for further analysis. The local analysis integrates qualitative research and formal social network methods. Unlike other studies of community capacity, which mostly rely on qualitative case studies, formal social network analysis is used to identify structural differences regarding how community organizations and individuals build autonomy and linkage with local and non-local organizations to improve the quality of life.This research improves understanding - both from conceptual and methodological perspectives -- of how to analyze rural poverty so as to better design federal government programs that will better serve poor communities, especially those in unincorporated areas
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