Neocarzinostatin abstracts a hydrogen during formation of nucleotide 5'-aldehyde on DNA

The oxidative reaction of polydeoxyadenylic-deoxythymidylic acid [poly(dA-dT)] with neocarzinostatin that produces 5'-thymidine aldehyde esterified to the 5'-end of strand breaks proceeds with hydrogen abstraction. The abstracted hydrogen is covalently bound to the non-protein component of neocarzinostatin; only a small amount (5%) is washed out into solvent. These data rule out a peroxyl radical as the primary DNA damaging species involved in the production of the 5'-aldehyde group. In contrast to earlier reports, it is demonstrated that alpha-tocopherol is not an inhibitor of the reaction.     

Enzyme conformational alterations detected by partition column chromatography

In this paper, we demonstrate the ability of liquid-liquid partition chromatography (LLPC) to detect conformational alterations occurring in well-characterized enzymes. The conformational changes induced in dehydrogenases such as alcohol dehydrogenase (ADH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenases (LDH) and malate dehydrogenase (MDH) upon binding of ligand(s) were detectable by LLPC. The ligand-dependent equilibrium between two forms of citrate synthase (CS), glutamate-oxaloacetate transaminase (GOT), hexokinase (HK) and 3-phosphoglycerate kinase (PGK) could also be demonstrated. Furthermore, different conformational forms of some of the apoenzymes could also be detected and separated by LLPC. The results obtained here are discussed in relation to those obtained by other methods.     

Comparative Network Biology Discovers Protein Complexes That Underline Cellular Differentiation in Anabaena sp.

The filamentous cyanobacterium Anabaena sp. PCC 7120 can differentiate into heterocysts to fix atmospheric nitrogen. During cell differentiation, cellular morphology and gene expression undergo a series of significant changes. To uncover the mechanisms responsible for these alterations, we built protein–protein interaction (PPI) networks for these two cell types by cofractionation coupled with mass spectrometry. We predicted 280 and 215 protein complexes, with 6322 and 2791 high-confidence PPIs in vegetative cells and heterocysts, respectively. Most of the proteins in both types of cells presented similar elution profiles, whereas the elution peaks of 438 proteins showed significant changes. We observed that some well-known complexes recruited new members in heterocysts, such as ribosomes, diflavin flavoprotein, and cytochrome c oxidase. Photosynthetic complexes, including photosystem I, photosystem II, and phycobilisome, remained in both vegetative cells and heterocysts for electron transfer and energy generation. Besides that, PPI data also reveal new functions of proteins. For example, the hypothetical protein Alr4359 was found to interact with FraH and Alr4119 in heterocysts and was located on heterocyst poles, thereby influencing the diazotrophic growth of filaments. The overexpression of Alr4359 suspended heterocyst formation and altered the pigment composition and filament length. This work demonstrates the differences in protein assemblies and provides insight into physiological regulation during cell differentiation.     

Novel insights into ATP-Stimulated Cleavage of branched DNA and RNA Substrates through Structure-Guided Studies of the Holliday Junction Resolvase RuvX

Much of our understanding of the homologous recombination (HR) machinery hinges on studies using Escherichia coli as a model organism. Interestingly enough, studies on the HR machinery in different bacterial species casts doubt on the universality of the E. coli paradigm. The human pathogen Mycobacterium tuberculosis encodes two Holliday junction (HJ)‐resolvase paralogues, namely RuvC and RuvX; however, insights into their structural features and functional relevance is still limited. Here, we report on structure-guided functional studies of the M. tuberculosis RuvX HJ resolvase (MtRuvX). The crystalline MtRuvX is a dimer in the asymmetric unit, and each monomer has a RNAse H fold vis-à-vis RuvC-like nucleases. Interestingly, MtRuvX also contains some unique features, including the residues essential for ATP binding/coordination of Mg²⁺ ions. Indeed, MtRuvX exhibited an intrinsic, robust ATPase activity, which was further accentuated by DNA cofactors. Structure-guided substitutions of single residues at the ATP binding/Mg²⁺coordination sites while markedly attenuating the ATPase activity completely abrogated HJ cleavage, indicating an unanticipated relationship between ATP hydrolysis and DNA cleavage. However, the affinity of ATPase-deficient mutants for the HJ was not impaired. Contrary to RuvC, MtRuvX exhibits relaxed substrate specificity, cleaving a variety of branched DNA/RNA substrates. Notably, ATP hydrolysis plays a regulatory role, rendering MtRuvX from a canonical HJ resolvase to a DNA/RNA non-sequence specific endonuclease, indicating a link between HJ resolvase and nucleic acid metabolism. These findings provide novel insights into the structure and dual-functional activities of MtRuvX, and suggest that it may play an important role in DNA/RNA metabolism.     

Release of N-Acetylaspartylglutamate from Slices of Rat Cerebellum, Striatum, and Spinal Cord, and the Effect of Climbing Fiber Deprivation

Abstract: The release of endogenous N -acetylaspartylglutamate (NAAG) from slices of rat cerebellum, striatum, and spinal cord upon depolarization with 50 m M K⁺ was investigated. NAAG in superfusates was prepurified using an ion exchanger, esterified, and then quantified by gas chromatography-mass spectrometry. Deuterated NAAG was used as internal standard. A depolarization-induced release of NAAG was found in all three regions. The release was Ca²⁺ dependent to over 85% in cerebellum and striatum, but only to approximately 70% in spinal cord. In addition, the effect of lesions of the olivocerebellar pathway on the K⁺-induced release of NAAG was studied: Treatment of the animals with 3-acetylpyridine reduced the release of NAAG from cerebellar hemispheres significantly, by about 40% compared with controls. These results suggest that part of the NAAG released from cerebellar slices on depolarization is related to climbing fibers. Implications of these findings concerning possible physiological roles of NAAG in the three CNS regions are discussed.     

Comparative study of the antioxidant activity of the essential oils of five plants against the H2O2 induced stress in Saccharomyces cerevisiae

The purpose of this work was to investigate the protective effect of five essential oils (EOs); Rosmarinus officinalis, Thymus vulgaris, Origanum compactum Benth., Eucalyptus globulus Labill. and Ocimum basilicum L.; against oxidative stress induced by hydrogen peroxide in Saccharomyces cerevisiae. The chemical composition of the EOs was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). The in vitro antioxidant activity was evaluated and the protective effect of EOs was investigated. Yeast cells were pretreated with different concentrations of EOs (6.25–25 µg/ml) for an hour then incubated with H₂O₂ (2 mM) for an additional hour. Cell viability, antioxidants (Catalase, Superoxide dismutase and Glutathione reductase) and metabolic (Succinate dehydrogenase) enzymes, as well as the level of lipid peroxidation (LPO) and protein carbonyl content (PCO) were evaluated. The chemical composition of EOs has shown the difference qualitatively and quantitatively. Indeed, O. compactum mainly contained Carvacrol, O. basilicum was mainly composed of Linalool, T. vulgaris was rich in thymol, R. officinalis had high α-Pinene amount and for E. globulus, eucalyptol was the major compound. The EOs of basil, oregano and thyme were found to possess the highest amount of total phenolic compounds. Moreover, they have shown the best protective effect on yeast cells against oxidative stress induced by H₂O₂. In addition, in a dose dependent manner of EOs in yeast medium, treated cells had lower levels of LPO, lower antioxidant and metabolic enzymes activity than cells exposed to H₂O₂ only. The cell viability was also improved. It seems that the studied EOs are efficient natural antioxidants, which can be exploited to protect against damages and serious diseases related to oxidative stress.     

蛋白质纯化虚拟仿真课程的教学设计

目的 蛋白质纯化是医学与生物学实验教学课程中的一个重要内容，本文通过设计新的蛋白质纯化虚拟仿真教学内容，开发虚拟仿真教学系统，希望学生能够更有效率地掌握蛋白质纯化技术的要点，提升教学质量。方法 利用3D虚拟仿真技术，构建虚拟仿真实验室。结合线下教学经验，确定虚拟仿真教学中需要体现的教学内容、教学重点、核心仪器的特色、评分指标、考核方式、操作体验等。结果 本文基于3D虚拟仿真技术构建了蛋白质纯化虚拟仿真实验室，和传统教学相比，虚拟仿真教学可以提高教学效率，节省教学成本和场地占用；教学设计和虚拟仿真教学系统融合了教学、练习和考核模块，根据以往的教学经验优化了评分体系，保证学生更加有效、严谨地掌握技术细节；涵盖了全新的教学内容——荧光检测联用的分子排阻层析，添加了蛋白质层析的常用案例，保证了内容的新颖与实用；完全再现了现实实验室中仪器设备的搭建模式，实现虚拟学习与实际操作的无缝衔接。结论 蛋白质纯化虚拟仿真教学系统由虚及实，由点及面，把控细节，保证学生动手能力和探索能力的提高，学以致用，并且拥有传统教学模式不可比拟的优势。     

Preparation of 3-Methyl-3-(substituted phenyl)-pyruvic Acid

The relationships between dietary levels of the essential amino acids and hepatic polysome profiles of rats were investigated with special attention to the amino acid requirement pattern for the maximum rat growth as determined by other investigators. The basal diet contained a 7% essential amino acid mixture and a 3% non-essential amino acid mixture, with appropriate amounts of other nutrients. Rats were fed test diet for 5 hours and then the polysome profile was determined. The amounts of essential amino acids needed for maximum aggregation of polysome were low for methionine-cystine, leucine and tryptophan as compared with requirements for maximum growth. But in other essential amino acids, the amounts were in almost the same range as those reported for maximum growth by others. The differences between the amino acid requirement patterns for maximum aggregation of hepatic ribosomes and for maximum growth of rats might be due to a difference in amino acid requirements of the liver and whole body. Therefore, the hepatic polysome profile might be used to measure the effect of amino acid supplementation on dietary proteins. The requirement pattern of essential amino acids in other organs may be studied by polysome profile determination.     

A radioisotope method for assays of amylomaltase and D-enzyme

A method has been developed for the assay of amylomaltase based on the incorporation of a [14C]glucose moiety of uniformly 14C-labeled maltose into a maltodextrin fraction insoluble in aqueous ethanol. The presence of dextrin at a high concentration greatly enhances [14C]glucose incorporation and serves to minimize interference with the assay by contaminating enzymes that hydrolyze substrates and products in the assay mixture. Since a number of other enzymes are capable of forming glucose in the assay mixture, the 14C incorporation is a more specific method of enzyme assay than are previously reported assays based on glucose release.     

Purification and immunohistochemical localization of the auxin binding site i protein from maize coleoptiles

An auxin binding protein fraction prepared by means of affinity chromatography on 2-OH-3,5-diiodobenzoic acid-Sepharose and gel filtration was used as antigen. The obtained rabbit antisera contained antibodies against the auxin, binding protein (ABP) and several contaminating proteins (nonABP). The nonABP could be separated on an appropriate affinity matrix omitting the TIBA analogue. After their immobilization on Sepharose antibodies directed towards contaminating, the proteins were isolated and immobilized, too. This IgGanti nonABP-Sepharose retains almost all contaminating proteins present in the specific eluates of the auxin affinity matrix. In a final affinity chromatography step on IgG-Sepharose a highly purified ABP could be eluted. This ABP was immobilized on Sepharose for the separation of monospecific antibodies against ABP (IgGanti abp). Using these antibodies the ABP could be localized within the outer epidermal cells of the coleoptile by immunofluorescence microscopy. From the inhibition of auxin induced elongation of coleoptile tissue by IgGanti abp it is concluded that the ABP is localized at the plasmalemma of the epidermal cells and that the ABP is involved in auxin action as a true hormone receptor. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.