The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

Summary A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both -2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.     

Comparison of lodging safety factor of untreated and succinic Acid 2,2-dimethylhydrazide-treated shoots of mulberry tree

This study examined the lodging resistance of mulberry tree (Morus bombycis Koidz. cv Kenmochi) shoots treated or not treated with succinic acid 2,2-dimethylhydrazide (SADH). The lodging safety factor, an indicator of lodging resistance, was defined as the ratio of critical lodging load to the leaf fresh weight observed, provided that the distribution of the critical lodging load along the stem was similar to that of the leaf fresh weight observed. The critical lodging load was experimentally estimated by loading weights onto the stems. In the untreated trees, the lodging safety factor was maintained at about 3.2. In the SADH-treated trees, the stem elongation was inhibited to about 80% of that in the untreated trees, and the percentage of shoot dry matter partitioned into the leaves was always larger than that of the untreated trees. This dwarfing of the stem caused by SADH increased the critical lodging load supported by the unit stem dry weight, while this large investment of materials in leaves increased the leaf fresh weight supported by the unit stem dry weight. Since the increments canceled each other, the lodging safety factor of the SADH-treated shoots was similar to that of the untreated ones. These results suggest that the shoot formation of the mulberry tree is controlled to maintain the lodging safety factor at a constant level.     

A structured model of dual-limitation kinetics

A structured model of substrate-utilization kinetics that encompasses dual-limitation conditions, caused by simultaneously low concentrations of the electron donor and the electron acceptor, is developed by incorporating the internal cofactor responses into the kinetic variables. The structured model is based on an assumption that the maximum specific electron-donor-oxidation rate (q(md)) is not a constant, but is linearly controlled by the intracellular chemical potentials, log(NAD/NADH) and log(ATP/ADP . P(i)). Determination of the kinetic parameters for the dual-limitation model, using experimental data from the companion article, verifies that q(md) varies and demonstrates that the NAD/NADH ratio affects q(md) in a positive direction; thus, an increase of the ratio increases the rate of electron-donor utilization. Because the internal NAD/NADH ratio rises with an increase in S(ar) the specific electron-donor-utilization rate is accelerated by high S(a). Since the ratio also increases as the specific electron-donor-utilization rate falls, the specific rate is intrinsically accelerated by the cofactor response when it becomes low due to a depletion of electron donor. Because the cofactor responses upon changes of the external substrate concentrations are systematic, the dual-limitation model can be expressed as a function of only external concentrations of electron donor and electron acceptor, which results in a multiplicative (double-Monod) form. Thus, dual limitation by both substrates reduces the overall reaction rate below the rate expected from single limitation by only one, the most severely limiting, substrate. (c) 1996 John Wiley & Sons, Inc.     

Immunocytochemical localization of casein kinase II during interphase and mitosis

We have developed specific antibodies to synthetic peptide antigens that react with the individual subunits of casein kinase II (CKII). Using these antibodies, we studied the localization of CKII in asynchronous HeLa cells by immunofluorescence and immunoelectron microscopy. Further studies were done on HeLa cells arrested at the G1/S transition by hydroxyurea treatment. Our results indicate that the CKII alpha and beta subunits are localized in the cytoplasm during interphase and are distributed throughout the cell during mitosis. Further electron microscopic investigation revealed that CKII alpha subunit is associated with spindle fibers during metaphase and anaphase. In contrast, the CKII alpha' subunit is localized in the nucleus during G1 and in the cytoplasm during S. Taken together, our results suggest that CKII may play significant roles in cell division control by shifting its localization between the cytoplasm and nucleus.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast

The Schizosaccharomyces pombe pfh1⁺ gene (PIF1 homolog) encodes an essential enzyme that has both DNA helicase and ATPase activities and is implicated in lagging strand DNA processing. Mutations in the pfh1⁺ gene suppress a temperature-sensitive allele of cdc24⁺, which encodes a protein that functions with Schizosaccharomyces pombe Dna2 in Okazaki fragment processing. In this study, we describe the enzymatic properties of the Pfh1 helicase and the genetic interactions between pfh1 and cdc24, dna2, cdc27 or pol 3, all of which are involved in the Okazaki fragment metabolism. We show that a full-length Pfh1 fusion protein is active as a monomer. The helicase activity of Pfh1 displaced only short (<30 bp) duplex DNA regions efficiently in a highly distributive manner and was markedly stimulated by the presence of a replication-fork-like structure in the substrate. The temperature-sensitive phenotype of a dna2-C2 or a cdc24-M38 mutant was suppressed by pfh1-R20 (a cold-sensitive mutant allele of pfh1) and overexpression of wild-type pfh1⁺ abolished the ability of the pfh1 mutant alleles to suppress dna2-C2 and cdc24-M38. Purified Pfh1-R20 mutant protein displayed significantly reduced ATPase and helicase activities. These results indicate that the simultaneous loss-of-function mutations of pfh1⁺ and dna2⁺ (or cdc24⁺) are essential to restore the growth defect. Our genetic data indicate that the Pfh1 DNA helicase acts in concert with Cdc24 and Dna2 to process single-stranded DNA flaps generated in vivo by pol δ-mediated lagging strand displacement DNA synthesis.