Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Deletion mapping and heterogenote analysis of a mutation responsible for osmosis-sensitive growth, spectinomycin resistance, and alteration of cytoplasmic membrane in Escherichia coli

Lambda transducing phages carrying Escherichia coli deoxyribonucleic acid of various lengths from the aroE-rpsL region were lysogenized into the F'3 plasmid and were used for heterogenote analysis of YM101, a sucrose-dependent, spectinomycin-resistant mutant of E. coli. Three characteristics of the mutant strain, resistance to spectinomycin, sucrose dependence of growth, and lack of I-19 protein in the cytoplasmic membrane, were shown to be the result of a mutation in a region designated delta 53-spcl. This region extends over 3.6-kilobase pairs and is located within a cluster of ribosomal genes. The mutation is recessive to the wild-type allele.     

Isolation and some properties of copper-binding proteins found in a copper-resistant strain of yeast

Copper-binding proteins were extracted from a copper-resistantstrain of Saccharomyces cerevisiae which was obtained by repeatedsubculturing in a copper-containing medium. They were separatedinto three types through purification steps such as salt fractionation,gel filtration and preparative polyacrylamide gel electrophoresis.They resembled each other in amino acid composition. Acidicamino acids, lysine, serine, glycine and half-cystine constituteda large part of the protein, with a small amount of hydrophobicamino acids. Aromatic amino acids and methionine were almostabsent. The molecular weight of the components was estimatedto be about 10,000 by Sephadex gel filtration and electrophoresison polyacrylamide gel (slope method). Absorption spectra ofthe components exhibited a broad band at 275 nm, but none inthe visible region, thus resembling that of copper-thionein.Moreover, the absorption band at 275 nm changed markedly onaddition of Ag⁺, Hg2⁺, CN^– or H₂O₂, which are well knownas thiol reagents. These components were abo produced in theparent cells, if they could grow in a copper-containing medium.Based the results of experiments using various culture conditionsand some other yeast species, a possible role of the componentsis discussed. (Received July 13, 1976; )     

Integration of high-throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi-subunit vaccine antigen

Mammalian cell line generation typically includes stable pool generation, single cell cloning and several rounds of clone selection based on cell growth, productivity and product quality criteria. Individual clone expansion and phenotype-based ranking is performed initially for hundreds or thousands of mini-scale cultures, representing the major operational challenge during cell line development. Automated cell culture and analytics systems have been developed to enable high complexity clone selection workflows; while ensuring traceability, safety, and quality of cell lines intended for biopharmaceutical applications. Here we show that comprehensive and quantitative assessment of cell growth, productivity, and product quality attributes are feasible at the 200–1,200 cell colony stage, within 14 days of the single cell cloning in static 96-well plate culture. The early cell line characterization performed prior to the clone expansion in suspension culture can be used for a single-step, direct selection of high quality clones. Such clones were comparable, both in terms of productivity and critical quality attributes (CQAs), to the top-ranked clones identified using an established iterative clone screening approach. Using a complex, multi-subunit antigen as a model protein, we observed stable CQA profiles independently of the cell culture format during the clonal expansion as well as in the batch and fed-batch processes. In conclusion, we propose an accelerated clone selection approach that can be readily incorporated into various cell line development workstreams, leading to significant reduction of the project timelines and resource requirements.     

Regulation of polo-like kinase 1 by DNA damage and PP2A/B55α

In addition to governing mitotic progression, Plk1 also suppresses the activation of the G2 DNA damage checkpoint and promotes checkpoint recovery. Previous studies have shown that checkpoint activation after DNA damage requires inhibition of Plk1, but the underlying mechanism of Plk1 regulation was unknown. In this study we show that the specific phosphatase activity toward Plk1 Thr-210 in interphase Xenopus egg extracts is predominantly PP2A-dependent, and this phosphatase activity is upregulated by DNA damage. Consistently, PP2A associates with Plk1 and the association increases after DNA damage. We further revealed that B55α, a targeting subunit of PP2A and putative tumor suppressor, mediates PP2A/Plk1 association and Plk1 dephosphorylation. B55α and PP2A association is greatly strengthened after DNA damage in an ATM/ATR and checkpoint kinase-dependent manner. Collectively, we report a phosphatase-dependent mechanism that responds to DNA damage and regulates Plk1 and checkpoint recovery.     

Neurite Outgrowth of PC12 Cells by 4′-O-β-d-Glucopyranosyl-3′,4-Dimethoxychalcone from Brassica rapa L. ‘hidabeni’ was Enhanced by Pretreatment with p38MAPK Inhibitor

The cellular effects of eleven compounds including chalcone glycosides isolated from Brassica rapa L. ‘hidabeni’ and their synthetic derivatives were studied in rat pheochromocytoma PC12 cells. Of the compounds tested, 4′-O-β-d-glucopyranosyl-3′,4-dimethoxychalcone (A2) significantly increased the levels of the phosphorylated forms of extracellular signal-regulated kinases 1/2 (ERK 1/2), p38 mitogen-activated protein kinase (p38MAPK), and stress-activated protein kinases/Jun amino-terminal kinases (JNK/SAPK), but it did not affect Akt. Nerve growth factor (NGF), a well-known neurotrophic factor, increased the levels of phosphorylated ERK1/2, JNK/SAPK, and Akt but not p38MAPK, which may mediate marked neurite outgrowth. Signals evoked by A2 shared common characteristics with those induced by NGF; therefore, we evaluated the neuritogenic activity of A2 and found it induced only weak neurite outgrowth. However, this effect was enhanced by pre-treatment with a p38MAPK inhibitor, suggesting that the phosphorylation of p38MAPK down-regulated neurite outgrowth. From the results of this study, it was found that A2 in combination with a p38MAPK inhibitor can induce NGF-like effects. Hence, a combination of chalcone glycosides containing A2 and a p38MAPK inhibitor increases the likelihood that chalcone glycosides could be put to practical use in the form of drugs or alternative medicines to maintain neural health.     

Bacillus brevis,a Host Bacterium for Efficient Extracellular Production of Useful Proteins

Abstract

Since its discovery in 1998 RNA interference (RNAi), a potent and highly selective gene silencing mechanism, has revolutionized the field of biological science. The ability of RNAi to specifically down-regulate the expression of any cellular protein has had a profound impact on the study of gene function in vitro. This property of RNAi also holds great promise for in vivo functional genomics and interventions against a wide spectrum of diseases, especially those with “undruggable” therapeutic targets. Despite the enormous potential of RNAi for medicine, development of in vivo applications has met with significant problems, particularly in terms of delivery. For effective gene silencing to occur, silencing RNA must reach the cytoplasm of the target cell. Consequently, various strategies using chemically modified siRNA, liposomes, nanoparticles and viral vectors are being developed to deliver silencing RNA. These approaches, however, can be expensive and in many cases they lack target cell specificity or clinical compatibility. Recently, we have shown that RNAi can be activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture     

Rap1 potentiates endothelial cell junctions by spatially controlling myosin II activity and actin organization

Reorganization of the actin cytoskeleton is responsible for dynamic regulation of endothelial cell (EC) barrier function. Circumferential actin bundles (CAB) promote formation of linear adherens junctions (AJs) and tightening of EC junctions, whereas formation of radial stress fibers (RSF) connected to punctate AJs occurs during junction remodeling. The small GTPase Rap1 induces CAB formation to potentiate EC junctions; however, the mechanism underlying Rap1-induced CAB formation remains unknown. Here, we show that myotonic dystrophy kinase–related CDC42-binding kinase (MRCK)-mediated activation of non-muscle myosin II (NM-II) at cell–cell contacts is essential for Rap1-induced CAB formation. Our data suggest that Rap1 induces FGD5-dependent Cdc42 activation at cell–cell junctions to locally activate the NM-II through MRCK, thereby inducing CAB formation. We further reveal that Rap1 suppresses the NM-II activity stimulated by the Rho–ROCK pathway, leading to dissolution of RSF. These findings imply that Rap1 potentiates EC junctions by spatially controlling NM-II activity through activation of the Cdc42–MRCK pathway and suppression of the Rho–ROCK pathway.     

Synthesis and properties of thymidines with six-membered amide bridge

Artificial thymidine monomers possessing amide or N-methylamide bridges were designed, synthesized, and introduced into oligonucleotides. UV-melting experiments showed that these oligonucleotides preferred single-stranded RNA (ssRNA) to single-stranded DNA (ssDNA) in duplex formation. Both amide- and N-methylamide-modified oligonucleotides led to a significant increase in the binding affinity to ssRNA by up to +4.7 and +3.7 °C of the T_m value per modification, respectively, compared with natural oligonucleotide. In addition, their oligonucleotides showed high stability against 3′-exonuclease.