Purification and Partial Characterization of Arginine Decarboxylase from Brassica campestris

Arginine decarboxylase (EC 4.1.1.19) has been purified and characterized from Brassica campestris cv B-9. The enzyme was purified 1120 fold and the recovery was 9%. The mol wt of the enzyme determined by gel filtration was 240 kD with identical subunits of 60 kD. The pH and temperature optima for the enzyme were 8.0 and 30°C respectively. The Km was 0.31mM. Polyamines inhibited the enzyme activity significantly. Immunodiffusion with ADC-specific antibodies showed cross reactivity against purified ADC from Brassica.     

Fast and simple anion-exchange chromatography for large-scale purification of self-complementary oligonucleotides.

A fast and simple anion-exchange chromatography method is described for large-scale purification of synthetic oligonucleotides. Using a single matrix and aqueous solvent system, the two-step chromatographic procedure can handle complex separation problems of self-complementary or G-rich sequences without the use of urea or formaldehyde. The work also demonstrates the complication encountered, possibly due to hairpin formation, in one of the oligomers.     

Microbial electron uptake in microbial electrosynthesis: a mini-review

Journal of Industrial Microbiology & Biotechnology - Microbial electron uptake (EU) is the biological capacity of microbes to accept electrons from electroconductive solid materials. EU has...     

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.     

A transient reversal of miRNA‐mediated repression controls macrophage activation

In mammalian macrophages, the expression of a number of cytokines is regulated by miRNAs. Upon macrophage activation, proinflammatory cytokine mRNAs are translated, although the expression of miRNAs targeting these mRNAs remains largely unaltered. We show that there is a transient reversal of miRNA‐mediated repression during the early phase of the inflammatory response in macrophages, which leads to the protection of cytokine mRNAs from miRNA‐mediated repression. This derepression occurs through Ago2 phosphorylation, which results in its impaired binding to miRNAs and to the corresponding target mRNAs. Macrophages expressing a mutant, non‐phosphorylatable AGO2—which remains bound to miRNAs during macrophage activation—have a weakened inflammatory response and fail to prevent parasite invasion. These findings highlight the relevance of the transient relief of miRNA repression for macrophage function.     

Surface components of HeLa cells that inhibit cytadherence of Chlamydia trachomatis

Abstract Isolated HeLa plasma membrane (PM) preparations and extracts containing either cell-surface proteins or lipids were examined for inhibition of adherence of radiolabeled Chlamydia trachomatis serovar E elementary bodies to glutaraldehydefixed HeLa monolayers. A dose-dependent adherence-inhibitory activity could be demonstrated with the PM. A urea extract as well as lipids from HeLa cells also inhibited chlamydial cytadherence. The inhibitory activity of the PM was trypsin-sensitive. It was absent when the urea extract was prepared from trypsin-treated HeLa cells. The urea extract was subjected to electrophoresis and protein blotting using a native gel system. Probing with radiolabeled chlamydial cytadhesin showed a single protein present in the urea extract that could represent a HeLa cell protein receptor for the chlamydiae.