A Native Chemical Ligation Handle that Enables the Synthesis of Advanced Activity‐Based Probes: Diubiquitin as a Case Study

We present the development of a native chemical ligation handle that also functions as a masked electrophile that can be liberated during synthesis when required. This handle can thus be used for the synthesis of complex activity‐based probes. We describe the use of this handle in the generation of linkage‐specific activity‐based deubiquitylating enzyme probes that contain substrate context and closely mimic the native ubiquitin isopeptide linkage. We have generated activity‐based probes based on all seven isopeptide‐linked diubiquitin topoisomers and demonstrated their structural integrity and ability to label DUBs in a linkage‐specific manner.     

Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach

Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.     

Protection and Isolation of Bioorthogonal Metal Catalysts by Using Monolayer-Coated Nanozymes

We demonstrate here the protection of biorthogonal transition metal catalysts (TMCs) in biological environments by using self-assembled monolayers on gold nanoparticles (AuNPs). Encapsulation of TMCs in this hydrophobic environment preserves catalytic activity in presence of pH conditions and complex biological media that would deactivate free catalyst. Significantly, the protection affords by these nanozymes extends to isolation of the catalyst active site, as demonstrated by the independence of rate over a wide pH range, in strong contrast to the behavior of the free catalyst.     

多肽液相分段合成及其进展

本文综述了多肽的液相分段合成方法,这些合成方法是近年来多肽和蛋白质合成领域中的一种发展趋势.详细介绍了天然化学连接、化学区域选择连接、施陶丁格连接等方法,并对这些方法进行了比较,指出其优点及不足并提出改进办法.最后对多肽合成技术的发展做了进一步展望.     

New methods for the generation of carbohydrate arrays on glass slides and their evaluation

Glycosides, having spacers functionalized with an aldehyde or a carboxylic group, were immobilized through reductive amination or amidation, respectively, onto amino-functionalized glass slides. Hybridization experiments with lectins exhibited very little nonspecific protein binding, hence precluding the necessity for the blocking of unreacted functional groups on the glass slide. The covalency and the concentration dependency of the sugar ligation to the glass slide were demonstrated; the reversibility and the selectivity of lectin-carbohydrate interactions were shown.     

Erratum to Stricker.

Reports an error in the article "The The Renin-Angiotensin System and Thirst: A Reevaluation. II. Drinking Elicited in Rats by Caval Ligation or Isoproterenol" by Edward M. Stricker (Journal of Comparative and Physiological Psychology, 1977, Vol. 91, No. 6, pp. 1220-1231), one line was printed incorrectly. On page 1228, the third line in the left-hand column reads "protin after 2 U/100 g hog renin"; the entire first sentence should read as follows: Three other nephrectomized rats were anesthetized 50 min after .33 mg/kg isoproterenol, 5 min after 2 U/100 g hog renin had been given. (The following abstract originally appeared in record 2005-09077-001) Ligation of the inferior vena cava and administration of isoproterenol have been shown to stimulate renin secretion and to augment water intake in rats. However, the present experiments suggested that the plasma renin activities produced by these treatments do not account for more than 20% of the observed drinking behavior. Direct measurements of arterial blood pressure further indicated that nephrectomized rats go into hypotensive shock after caval ligation or isoproterenol treatment. Drinking was elicited in these hypotensive animals by systemic injection of hypertonic NaCl solution, renin, or Pitressin, or by intracranial injection of angiotensin, but in each case a rapid increase in blood pressure also was observed. Thus, it appears that nephrectomy reduces water intake in these animals by undermining their general capacity to behave rather than by removing a specific dipsogenic stimulus. These and other results suggested that drinking elicited in rats by caval ligation or isoproterenol is not mediated by the renin-angiotensin system. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Vapor‐Based Multicomponent Coatings for Antifouling and Biofunctional Synergic Modifications

A general concept is introduced featuring an ideal multifunctional surface that can avoid fouling problems while allowing the installed groups to perform with the high efficacy and accuracy necessary for delivering cascading and spontaneous biological activities. The idea is realized by using a direct synthesis of a multicomponent coating containing the two functionalities of 4‐methyl‐propiolate and 4‐N‐maleimidomethyl that is achieved via chemical vapor deposition copolymerization on various substrates. The novel coating can simultaneously perform specific bio‐orthogonal reactions, including the azide‐alkyne click reaction and a thiol‐maleimide coupling reaction. In the study, azide‐terminated polyethylene glycols are first immobilized on the methyl propiolate groups to impart an antifouling property, while bioactivity is enabled by tethering biotinylated thiols or Cys‐Arg‐Glu‐Asp‐Val (CREDV) peptides on the maleimide groups. The induced antifouling properties and bioactivities are confirmed by quartz crystal microbalance and cell culture studies. Finally, precisely manipulated endothelial cells, namely, human umbilical vein endothelial cells and bovine arterial endothelial cells, are observed on a complex stent substrate and on confined areas of the poly(methyl methacrylate) substrates.     

Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier

The highly efficient yeast lithium acetate transformation protocol of Schiestl and Gietz (1989) was tested for its applicability to some of the most important needs of current yeast molecular biology. The method allows efficient cloning of genes by direct transformation of gene libraries into yeast. When a random gene pool ligation reaction was transformed into yeast, the LEU2, HIS3, URA3, TRP1 and ARG4 genes were found among the primary transformants at a frequency of approximately 0.1%. The RAD4 gene, which is toxic to Escherichia coli, was also identified among the primary transformants of a ligation library at a frequency of 0.18%. Non-selective transformation using this transformation protocol was shown to increase the frequency of gene disruption three-fold. Co-transformation showed that 30-40% of the transformation-competent cells take up more than one DNA molecule which can be used to enrich for integration and deletion events 30- to 60-fold. Co-transformation was used in the construction of simultaneous double gene disruptions as well as disrupting both copies of one gene in a diploid which occurred at 2-5% the frequency of the single event.