NMR relaxation studies of intracellular Na in red blood cells

The state of intracellular Na⁺ in human and dog erythrocytes was characterized by ²³Na-NMR using dysprosium complexes as shift reagents. Intracellular Na⁺ concentrations were determined using integration of the inner Na⁺ NMR signals and measurements of the intracellular volume using ⁵⁹Co-NMR of extracellular Co(CN)³⁻₆. T₂ was found to be significantly shorter than T₁, indicating some binding to macromolecules. While the longitudinal magnetization decay follows a single exponential the transverse magnetization could be fitted with a double-exponential function. It was shown that neither the binding to the inner side of the membrane nor binding to hemoglobin contributes to the relaxation enhancement.     

A novel approach to evaluate ELISA antibody coverage of host cell proteins—combining ELISA-based immunocapture and mass spectrometry

Monitoring host cell proteins (HCPs) is one of the most important analytical requirements in production of recombinant biopharmaceuticals to ensure product purity and patient safety. Enzyme-linked immunosorbent assay (ELISA) is the standard method for monitoring HCP clearance. It is important to validate that the critical reagent of an ELISA, the HCP antibody, covers a broad spectrum of the HCPs potentially present in the purified drug substance. Current coverage methods for assessing HCP antibody coverage are based on 2D-Western blot or immunoaffinity-purification combined with 2D gel electrophoresis and have several limitations. In the present study, we present a novel coverage method combining ELISA-based immunocapture with protein identification by liquid chromatography–tandem mass spectrometry (LC–MS/MS): ELISA-MS. ELISA-MS is used to accurately determine HCP coverage of an early process sample by three commercially available anti-Escherichia coli HCP antibodies, evading the limitations of current methods for coverage analysis, and taking advantage of the benefits of MS analysis. The results obtained comprise a list of individual HCPs covered by each HCP antibody. The novel method shows high sensitivity, high reproducibility, and enables tight control of nonspecific binding through inclusion of a species-specific isotype control antibody. We propose that ELISA-MS will be a valuable supplement to existing coverage methods or even a replacement. ELISA-MS will increase the possibility of selecting the best HCP ELISA, thus improving HCP surveillance and resulting in a final HCP profile with the lowest achievable risk. Overall, this will be beneficial to both the pharmaceutical industry and patient safety.     

Synthesis and biological evaluation of the natural product komaroviquinone and related compounds aiming at a potential therapeutic lead compound for high-risk multiple myeloma

Alternatives of treatments for multiple myeloma (MM) have become increasingly available with the advent of new drugs such as proteasome inhibitors, thalidomide derivatives, histone deacetylase inhibitors, and antibody drugs. However, high-risk MM cases that are refractory to novel drugs remain, and further optimization of chemotherapeutics is urgently needed.We had achieved asymmetric total synthesis of komaroviquinone, which is a natural product from the plant Dracocephalum komarovi. Similar to several leading antitumor agents that have been developed from natural compounds, we describe the antitumor activity and cytotoxicity of komaroviquinone and related compounds in bone marrow cells. Our data suggested that komaroviquinone-related agents have potential as starting compounds for anticancer drug development.     

Induction of multinucleation by β-glucosyl Yariv reagent in regenerated cells from <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> protoplasts and involvement of arabinogalactan proteins in cell plate formation

Arabinogalactan proteins (AGPs) are abundant plant cell surface proteoglycans widely distributed in plant species. Since high concentrations of β-glucosyl Yariv reagent (βglcY), which binds selectively to AGPs, inhibited cell division of protoplast-regenerated cells of the liverwort Marchantia polymorpha L. (Shibaya and Sugawara in Physiol Plant 130:271–279, 2007), we investigated the mechanism underlying the inability of the cells to divide normally by staining nuclei, cell walls and β-1,3-glucan. Microscopic observation showed that the diameter of regenerated cells cultured with βglcY was about 2.8-fold larger than that of cells cultured without βglcY. The cells cultured with βglcY were remarkably multinucleated. These results indicated that βglcY did not inhibit mitosis but induced multinucleation. In the regenerated cells cultured with low concentrations of βglcY (5 and 1 μg ml⁻¹), the cell plate was stained strongly by βglcY, suggesting abundant AGPs in the forming cell plate. In these cell plates, β-1,3-glucan was barely detectable or not detected. In multinucleated cells, cell plate-like fragments, which could not reach the cell wall, were frequently observed and they were also stained strongly by βglcY. Our results indicated that AGPs might have an important role in cell plate formation, and perturbation of AGPs with βglcY might result in remarkable multinucleation in protoplast-regenerated cells of M. polymorpha. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The effect of thiol reagents on GABA transport in rat brain synaptosomes

The nature of gamma-aminobutyric acid (GABA) transport has been investigated in preparations of rat brain synaptosomes using a number of thiol reagents with varying membrane permeabilities. N-Ethylmaleimide, p-chloromercuribenzoate and p-chloromercuriphenylsulfonate effectively inhibited GABA transport in both directions (i.e., uptake and release) whereas 5,5'-dithiobis-2-nitrobenzoate, mercaptopropionate and N- nitroethylenediamine were much less effective, or ineffective, even at millimolar concentrations. For each of the thiol reagents, the inhibition profile for GABA uptake was approximately the same as that for its release. The effectiveness of the reagents indicates that there is an external, reactable SH-group on the transporter, that the thiol reagent must be somewhat lipophilic for it to react with the SH-group(s), and that the same synaptosomal transport system is responsible for both uptake and release of GABA.     

Recent progress in dissecting ubiquitin signals with chemical biology tools

Protein ubiquitination regulates almost all eukaryotic cellular processes, and is of very high complexity due to the diversity of ubiquitin (Ub) modifications including mono-, multiply mono-, homotypic poly-, and even heterotypic poly-ubiquitination. To accurately elucidate the role of each specific Ub signal in different cells with spatiotemporal resolutions, a variety of chemical biology tools have been developed. In this review, we summarize some recently developed chemical biology tools for ubiquitination studies and their applications in molecular and cellular biology.     

Antimicrobial therapeutic enhancement of levofloxacin via conjugation to a cell‐penetrating peptide: An efficient sonochemical catalytic process

Herein, we make an effort to enhance the antimicrobial activity of levofloxacin (LVX) antibiotic via conjugation to a cell‐penetrating peptide (CPP) including Cys‐Gly‐Ala‐Phe‐Pro‐His‐Arg. For this purpose, cysteine is used as a linker between the LVX and CPP chain, and two heterogeneous nanoscale catalytic systems are employed as the substantial alternatives for traditional peptide coupling reagents like N,N,N′,N′‐tetramethyl‐O‐(benzotriazol‐1‐yl)uronium tetrafluoroborate (TBTU). Briefly, it has been found out that the antimicrobial potency of the synthesized CPP‐LVX conjugate (on the gram‐positive and gram‐negative bacteria) is noticeably enhanced (~20% more). It has been revealed via zone of inhibition (ZOI) and optical density (OD) evaluations. As a convenient method for making this type of the effective conjugations, ultrasound waves (with a specific frequency and power density) activate the catalytic sites of the heterogeneous nanoparticles. Through this synergistic effect, peptide/amide bond is formed during a short time (10 min), and high reaction yield (>90%) is obtained under mild conditions. Moreover, as a simple purification process, the catalyst nanoparticles are collected and separated through their high magnetic property.     

PIG-B: A homemade monophasic cocktail for the extraction of RNA

An inexpensive monophasic reagent has been developed for the extraction of total RNA from cells or tissues. The main ingredients of the reagent arePhenol,Isoamyl alcohol,Guanidinium isothiocyanate, andBeta-mercaptoethanol (PIG-B). The quality and yield of RNA obtained by this reagent is at par with that obtained by TRIzol, an expensive but widely used monophasic reagent available commercially. The complete composition and method of preparation of PIG-B is provided to aid preparation of the reagent in the laboratory.