Structural Insights into Saccharomyces cerevisiae Msh4–Msh5 Complex Function Using Homology Modeling

The Msh4–Msh5 protein complex in eukaryotes is involved in stabilizing Holliday junctions and its progenitors to facilitate crossing over during Meiosis I. These functions of the Msh4–Msh5 complex are essential for proper chromosomal segregation during the first meiotic division. The Msh4/5 proteins are homologous to the bacterial mismatch repair protein MutS and other MutS homologs (Msh2, Msh3, Msh6). Saccharomyces cerevisiae msh4/5 point mutants were identified recently that show two fold reduction in crossing over, compared to wild-type without affecting chromosome segregation. Three distinct classes of msh4/5 point mutations could be sorted based on their meiotic phenotypes. These include msh4/5 mutations that have a) crossover and viability defects similar to msh4/5 null mutants; b) intermediate defects in crossing over and viability and c) defects only in crossing over. The absence of a crystal structure for the Msh4–Msh5 complex has hindered an understanding of the structural aspects of Msh4–Msh5 function as well as molecular explanation for the meiotic defects observed in msh4/5 mutations. To address this problem, we generated a structural model of the S. cerevisiae Msh4–Msh5 complex using homology modeling. Further, structural analysis tailored with evolutionary information is used to predict sites with potentially critical roles in Msh4–Msh5 complex formation, DNA binding and to explain asymmetry within the Msh4–Msh5 complex. We also provide a structural rationale for the meiotic defects observed in the msh4/5 point mutations. The mutations are likely to affect stability of the Msh4/5 proteins and/or interactions with DNA. The Msh4–Msh5 model will facilitate the design and interpretation of new mutational data as well as structural studies of this important complex involved in meiotic chromosome segregation.     

Reciprocal regulation of nuclear import of the yeast MutSα DNA mismatch repair proteins Msh2 and Msh6

DNA mismatch recognition is performed in eukaryotes by two heterodimers known as MutSα (Msh2/Msh6) and MutSβ (Msh2/Msh3) that must reside in the nucleus to function. Two putative Msh2 nuclear localization sequences (NLS) were characterized by fusion to green fluorescent protein (GFP) and site-directed mutagenesis in the context of Msh2. One NLS functioned in GFP targeting assays and both acted redundantly within Msh2. We examined nuclear localization of each of the MutS monomers in the presence and absence of their partners. Msh2 translocated to the nucleus in cells lacking Msh3 and Msh6; however, cells lacking Msh6 showed significantly decreased levels of nuclear Msh2. Furthermore, the overall protein levels of Msh2 were significantly diminished in the absence of Msh6, particularly if Msh2 lacked a functional NLS. Msh3 localized in the absence of Msh2, but Msh6 localization depended on Msh2 expressing functional NLSs. Overall, the nuclear levels of Msh2 and Msh6 decline when the other partner is absent. The data suggest a stabilization mechanism to prevent free monomer accumulation in the cytoplasm.     

The Concerted Action of Msh2 and UNG Stimulates Somatic Hypermutation at A · T Base Pairs

Mismatch repair plays an essential role in reducing the cellular mutation load. Paradoxically, proteins in this pathway produce A·T mutations during the somatic hypermutation of immunoglobulin genes. Although recent evidence implicates the translesional DNA polymerase η in producing these mutations, it is unknown how this or other translesional polymerases are recruited to immunoglobulin genes, since these enzymes are not normally utilized in conventional mismatch repair. In this report, we demonstrate that A·T mutations were closely associated with transversion mutations at a deoxycytidine. Furthermore, deficiency in uracil-N-glycolase (UNG) or mismatch repair reduced this association. These data reveal a previously unknown interaction between the base excision and mismatch repair pathways and indicate that an abasic site generated by UNG within the mismatch repair tract recruits an error-prone polymerase, which then introduces A·T mutations. Our analysis further indicates that repair tracts typically are ∼200 nucleotides long and that polymerase η makes ∼1 error per 300 T nucleotides. The concerted action of Msh2 and UNG in stimulating A·T mutations also may have implications for mutagenesis at sites of spontaneous cytidine deamination.The affinity maturation of the antibody response depends on the somatic hypermutation (SHM) process. The enzyme activation-induced cytidine deaminase (AID) initiates SHM in germinal center B cells by deaminating C within immunoglobulin (Ig) genes, yielding a G·U lesion that is resolved by several mechanisms (29). Replication across the U generates G·C to A·T transition mutations, while the removal of the U by uracil-N-glycolase (UNG) leads to transversion and transition mutations at the original G·C base pair (33). The AID-generated G·U lesion is also a substrate for the mismatch repair (MMR) proteins Msh2, Msh6, and Exo1. Unlike their normal role in DNA repair, the processing of this lesion by these MMR proteins during SHM paradoxically leads to the production of mutations at A·T base pairs (see below).MMR is a DNA repair process utilized by prokaryotes and eukaryotes (25). This pathway repairs DNA errors caused by the misincorporation of nucleotides during DNA synthesis. The initial mismatch is detected by MutSα, which consists of Msh2 and Msh6 in mammalian cells. The ability of MMR to discriminate between the mutated and unmutated strands of DNA is thought to be dictated by nicks or gaps on the newly synthesized lagging strand between Okazaki fragments or by strand ends on the leading strand at the replication fork (18). The MutLα endonuclease (Mlh1/Pms2) uses the DNA nick or end as a marker of the newly synthesized, and therefore mutated, strand to introduce a new nick on either side of the mismatch (15). This nicked strand is then excised by the 5′-to-3′ exonuclease Exo1, and the ensuing gap is repaired by the replicative polymerase δ. However, since AID acts primarily during G₁ of the cell cycle (11, 36), it is unclear whether Msh2/6 is capable of distinguishing between the AID-mutated and unmutated strands prior to strand excision.Consistently with their role in DNA repair, deficiency in Msh2, Msh6, or Exo1 generally leads to an increase in mutation frequencies in different tissues (40). However, in the case of SHM of Ig genes, the loss of these MMR proteins reduces the frequency of mutations at A·T base pairs (4, 5, 10, 16, 22, 30, 32, 37, 41, 42). One possible difference between conventional and mutagenic MMR is the involvement of the error-prone DNA polymerase η in the latter process. Indeed, both mice and humans lacking polymerase η resemble Msh2-deficient mice, in that mutations at A·T base pairs in the V region are less frequent (6, 7, 47). Moreover, the error spectrum of polymerase η on undamaged DNA matches the mutation spectrum of A·T mutations in the V region (35). While it is now well established that mutations at A·T base pairs are produced largely by proteins involved in the MMR pathway, it is not known how DNA polymerase η is recruited during SHM.One possible explanation for the use of error-prone polymerases is the occurrence of replication-blocking lesions, such as an abasic site or a modified nucleotide, in the V region of Ig genes. Evidence that a replication block leads to mutagenic MMR comes from recent studies showing the requirement of ubiquitinated PCNA for mutagenic MMR (1, 19, 34). Monoubiquitination at the K164 residue of PCNA in response to DNA damage leads to translesional synthesis (1), and SHM at A·T base pairs is reduced in PCNA^K164R/K164R mice to levels observed in MMR-deficient mice (19, 34). In addition, the finding that translesional DNA polymerases are involved in SHM (9, 31, 45-47) suggests that replication-blocking lesions are common at the Ig locus during SHM. Taken together, these observations suggest a model in which replication-blocking lesions recruit error-prone polymerases, which then generate mutations at nearby A·T base pairs. As reported here, we have tested this model by examining the correlated mutations in V region sequences from hypermutating Ramos cells and in murine centroblasts.     

Biocomputing — tools,aims, perspectives

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Microproteins—Discovery,structure, and function

Advances in proteogenomic technologies have revealed hundreds to thousands of translated small open reading frames (sORFs) that encode microproteins in genomes across evolutionary space. While many microproteins have now been shown to play critical roles in biology and human disease, a majority of recently identified microproteins have little or no experimental evidence regarding their functionality. Computational tools have some limitations for analysis of short, poorly conserved microprotein sequences, so additional approaches are needed to determine the role of each member of this recently discovered polypeptide class. A currently underexplored avenue in the study of microproteins is structure prediction and determination, which delivers a depth of functional information. In this review, we provide a brief overview of microprotein discovery methods, then examine examples of microprotein structures (and, conversely, intrinsic disorder) that have been experimentally determined using crystallography, cryo-electron microscopy, and NMR, which provide insight into their molecular functions and mechanisms. Additionally, we discuss examples of predicted microprotein structures that have provided insight or context regarding their function. Analysis of microprotein structure at the angstrom level, and confirmation of predicted structures, therefore, has potential to identify translated microproteins that are of biological importance and to provide molecular mechanism for their in vivo roles.     

Barley—Botany,production, harvesting,processing, utilization and economics

Barley, probably the oldest cultivated cereal, is widely grown in cooler areas of the world. The annual world production of nearly two and a half billion bushels exceeds that of rye but is less than that of rice, wheat, corn and oats, respectively. Most of the annual 300 million dollar crop of the U.S. is fed to livestock, but about one- third is manufactured into malt.     

A prourokinase-RGDS chimera——Construction, expression and characterization

A tetrapeptide, RGDS, was inserted into proUK kringle domain G118-L119 by the construction of a mutant proUK-RGDS gene. The gene was expressed in the baculovirus expression system. Immunoaffinity chromatography was used to purify the chimera and protein with purity over 90% was achieved. The chimera was tested for its platelet membrane binding function and showed a calcium-dependent platelet binding activity. Amidolytic activity of the chimera was tested. The result indicated that specific amidolytic activity of plasmin activated chimera was 62000 IU/mg, comparable to the previously reported 65 355 IU/mg of plasmin activated natural proUK. Activation of plasminogen by the chimera after plasmin treatment followed Micbieal-Menten kinetics, and the Km was 0.97 μmol/L, which was also comparable to 1.64 μmol/L of native urokinase. The chimera also showed intensive ability to inhibit platelet aggregation in vitro. These results indicate that this chimera might be useful as a bifunctional thrombolytic agent.     

49th Discussion forum on LCA—sustainable consumption patterns—September 18, 2012, Zurich, Switzerland

There are different ways and strategies to reduce the environmental impacts caused today. One starting point for reducing the environmental impacts of today is the private consumption. Finally, all goods and services provided in a country contribute to fulfil the needs and demands of consumers. Several national and international initiatives therefore aim for a considerable reduction of the environmental impacts of consumption patterns. The 49th LCA Discussion Forum analysed the present consumption patterns of households and their consequent environmental impacts. Based on this, potentials for a reduction of the environmental impacts were identified and discussed. In this context, the possible applications, advantages and drawbacks of the life cycle assessment (LCA) methodology were analysed. National and international speakers provided qualified insights on the topic. The 49th Discussion Forum concentrated on different aspects of sustainable consumption patterns. The focus lay on private households and the environmental impacts caused by their consumption patterns. In the first session, the idea of the “2000-Watt society” was introduced as an example of a concept of a “sustainable lifestyle”. Another way of analysing the total impacts is the consumption perspective introduced in a second presentation. Based on an analysis of environmental impacts due to final demand of Swiss households, different measures for a reduction of environmental impacts were proposed and analysed. The second session examined similar activities in Germany. The short presentations covered the communication of LCA results. The third session focused on web-based eco-calculators. In the last session, two scientific inputs were given on the modelling of household consumption patterns and on the impact of rebound effects on the environmental impact of private consumption. The most important consumption domains are nutrition, mobility and energy use in households. Apart from different modelling approaches and boundary conditions, the majority of the presentations showed that today’s consumption patterns in Switzerland and Germany are far away from a sustainable level. Considerable reduction measures are needed in order to reach this goal. Eco-calculators and similar tools provide an effective way to raise customer awareness. In general, it is very important to communicate LCA results in a simple, clear and transparent way.     

牛磺酸,羟脯氨酸,γ—氨基丁酸,鸟氨酸和色氨酸的测定

在日立８３５－５０型氨基酸分析仪上,用７２ｍｉｎ程序进行分析,通常一次只能分析１７种氨基酸。采用本文所述方法则一次能分析２２种氨基酸。在不延长分析时间,不增加成本的情况下,一次多分析了五种氨基酸,提高了仪器工作效率。     

Nanotechnology Nexus—Intersection of Research,Science, Technology,and Regulation

Not a day passes where nanotechnology does not make headlines in the popular press, scientific journals, as well as in the regulatory arena. Environmental and public health activists are voicing a growing concern and focus on the risks potentially posed by nanotechnology and the ability of the government to regulate these new and exciting technologies. Whereas such concerns state the need for stringent, precautionary, and almost exclusionary approaches to the regulation of nanotechnology, many entities believe that a voluntary approach to these often novel materials and technologies is the appropriate and sensible path. In this editorial, we discuss the importance of nanotechnology, who cares, and the available options for approaching the regulation of this often novel technology. We focus on the U.S. Environmental Protection Agency (USEPA) and its voluntary regulatory data submission program as the preferred alternative. ^{2 2}Comments are those of the authors and do not necessarily represent those of their employers. View all notes     

D—氨基酸在人体的来龙去脉：人体中D—氨基酸,D—氨基酸氧化酶,D…

本文分析近年,特别是近两年有关人体、食物中Ｄ－氨基酸、Ｄ－氨基酸氧化酶、Ｄ－天冬氨酸氧化酶研究进展,强调：１．必须研究食品中Ｄ－氨基酸水平;２．Ｄ－氨基酸氧化酶、Ｄ．天冬氨酸氧化酶作为药物治疗因Ｄ－氨基酸过量所致疾病。     

乳腺癌HSV—1,HSV—2与c—erbB—2的免疫组化研究

采用免疫组织化学Ｓ－Ｐ法检测５２例手术切除乳腺癌组织ｃ－ｅｒｂＢ－２蛋白和ＨＳＶ－１、ＨＳＶ－２表达情况。结果发现癌组织中ｃ－ｅｒｂＢ－２阳性３４例（６５．４％）;ＨＳＶ－１阳性３８例（７３．１％）;ＨＳＶ－２阳性１５例（２８．８％）。癌旁组织３２例,阳性分别为３例（９．４％）;１２例（３７．５％）;２例（６．３％）。乳腺癌中ｃ－ｅｒｂＢ－２阳性率明显高于癌旁组织。乳腺癌及癌旁的ＨＳＶ－１阳性率明显高于ＨＳＶ－２,乳腺癌ｃ－ｅｒｂＢ－２阳性组中ＨＳＶ－１和ＨＳＶ－２的表达有显著差异,而在阴性组二者无差异,提示乳腺癌的发生可能和ＨＳＶ－１感染密切相关,ｃ－ｅｒｂＢ－２表达也可能和ＨＳＶ－１感染有关。     

β—1,—3葡聚糖的结构与功能

β－１,３－葡聚糖是一类具有抗癌及免疫调节等生物活性的生物大分子其高级结构为单股和３股螺旋体,它的生物活性,理化特性与其分子结构有一定对应关系。