Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. In an effort to identify all genes regulated by Gcn4p during amino acid starvation, we performed cDNA microarray analysis. Data from 21 pairs of hybridization experiments using two different strains derived from S288c revealed that more than 1,000 genes were induced, and a similar number were repressed, by a factor of 2 or more in response to histidine starvation imposed by 3-aminotriazole (3AT). Profiling of a gcn4Δ strain and a constitutively induced mutant showed that Gcn4p is required for the full induction by 3AT of at least 539 genes, termed Gcn4p targets. Genes in every amino acid biosynthetic pathway except cysteine and genes encoding amino acid precursors, vitamin biosynthetic enzymes, peroxisomal components, mitochondrial carrier proteins, and autophagy proteins were all identified as Gcn4p targets. Unexpectedly, genes involved in amino acid biosynthesis represent only a quarter of the Gcn4p target genes. Gcn4p also activates genes involved in glycogen homeostasis, and mutant analysis showed that Gcn4p suppresses glycogen levels in amino acid-starved cells. Numerous genes encoding protein kinases and transcription factors were identified as targets, suggesting that Gcn4p is a master regulator of gene expression. Interestingly, expression profiles for 3AT and the alkylating agent methyl methanesulfonate (MMS) overlapped extensively, and MMS induced GCN4 translation. Thus, the broad transcriptional response evoked by Gcn4p is produced by diverse stress conditions. Finally, profiling of a gcn4Δ mutant uncovered an alternative induction pathway operating at many Gcn4p target genes in histidine-starved cells.

Transcriptional Profiling Shows that Gcn4p Is a Master Regulator of Gene Expression during Amino Acid Starvation in Yeast

The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid and (4) D-glutamic acid and dipeptide D-alanyl-D-alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.

Cytoplasmic steps of peptidoglycan biosynthesis.

The phenomenon of dispersion (transverse and longitudinal) in packed beds is summarized and reviewed for a great deal of information from the literature. Dispersion plays an important part, for example, in contaminant transport in ground water flows, in miscible displacement of oil and gas and in reactant and product transport in packed bed reactors. There are several variables that must be considered, in the analysis of dispersion in packed beds, like the length of the packed column, viscosity and density of the fluid, ratio of column diameter to particle diameter, ratio of column length to particle diameter, particle size distribution, particle shape, effect of fluid velocity and effect of temperature (or Schmidt number). Empirical correlations are presented for the prediction of the dispersion coefficients (D
 T and D
 L) over the entire range of practical values of Sc and Pem, and works on transverse and longitudinal dispersion of non-Newtonian fluids in packed beds are also considered.

https://paginas.fe.up.pt/~jdelgado/2006_HMT__JD.pdf

A critical review of dispersion in packed beds

The network structure and the metabolic fluxes in central carbon metabolism were characterized in aerobically grown cells of Saccharomyces cerevisiae. The cells were grown under both high and low glucose concentrations, i.e., either in a chemostat at steady state with a specific growth rate of 0.1 h−1 or in a batch culture with a specific growth rate of 0.37 h−1. Experiments were carried out using [1-13C]glucose as the limiting substrate, and the resulting summed fractional labelings of intracellular metabolites were measured by gas chromatography coupled to mass spectrometry. The data were used as inputs to a flux estimation routine that involved appropriate mathematical modelling of the central carbon metabolism of S. cerevisiae. The results showed that the analysis is very robust, and it was possible to quantify the fluxes in the central carbon metabolism under both growth conditions. In the batch culture, 16.2 of every 100 molecules of glucose consumed by the cells entered the pentose-phosphate pathway, whereas the same relative flux was 44.2 per 100 molecules in the chemostat. The tricarboxylic acid cycle does not operate as a cycle in batch-growing cells, in contrast to the chemostat condition. Quantitative evidence was also found for threonine aldolase and malic enzyme activities, in accordance with published data. Disruption of the MIG1 gene did not cause changes in the metabolic network structure or in the flux pattern.

Network Identification and Flux Quantification in the Central Metabolism of Saccharomyces cerevisiae under Different Conditions of Glucose Repression

Redox reactions are important steps in the metabolism and energy conversion of living cells. Besides the enzymes necessary, a number of coenzymes are involved in such reactions: ferrodoxins, lipoic acid, NAD(H), NADP(H), flavins and cytochromes. The coenzymes differ in their redox potential, in the binding constants and the mode of regeneration [1]. NADH/NADPH are the most frequently encountered coenzymes. In general they dissociate easily and need a second reaction with another metabolite for regeneration. These properties are one of the means by which nature directs the flow of intermediates in response to biosynthetic needs. Because of the spectral properties of the coenzyme moiety, dehydrogenases have been extensively studied in the past [2] and have found widespread applications in clinical and food analysis [3].

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1989.tb14983.x

Dehydrogenases for the synthesis of chiral compounds

c-Ski has been known to be phosphorylated at serine residue(s), which results in slower migration of c-Ski in SDS-polyacrylamide gel electrophoresis. The position(s) of phosphorylation, however, has not been determined. In the present study, we identified a phosphorylation site of c-Ski which affects its electrophoretic motility as serine 515 using MALDI-TOF mass spectrometry. A phosphorylation-resistant mutant, c-Ski S515A, did not exhibit a phosphatase-sensitive band shift. In addition, we confirmed that endogenous c-Ski is phosphorylated at serine 515, using a specific antibody. The phosphorylation status of c-Ski, however, does not appear to affect its stability or effects on TGF-β signalling. Identification of the phosphorylation site of c-Ski would allow us further examination of physiological significance of c-Ski phosphorylation.

Identification of a phosphorylation site in c-Ski as serine 515

Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties, and its peptidoglycans have the potential for applications in the fields of food science and medicine. We investigated the bifidobacterial alanine racemase, which is essential in the synthesis of d -alanine as an essential component of the peptidoglycans. Alanine racemase was purified to homogeneity from a crude extract of B. bifidum NBRC 14252. It consisted of two identical subunits with a molecular mass of 50 kDa. The enzyme required pyridoxal 5′-phosphate (PLP) as a coenzyme. The activity was lost in the presence of a thiol-modifying agent. The enzyme almost exclusively catalyzed the alanine racemization; other amino acids tested, except for serine, were inactive as substrates. The kinetic parameters of the enzyme suggested that the B. bifidum alanine racemase possesses comparatively low affinities for both the coenzyme (9.1 μM for PLP) and substrates (44.3 mM for l -alanine; 74.3 mM for d -alanine). The alr gene encoding the alanine racemase was cloned and sequenced. The alr gene complemented the d -alanine auxotrophy of Escherichia coli MB2795, and an abundant amount of the enzyme was produced in cells of the E. coli MB2795 clone. The enzymologic and kinetic properties of the purified recombinant enzyme were almost the same as those of the alanine racemase from B. bifidum NBRC 14252.

Molecular characterization of alanine racemase from Bifidobacterium bifidum

NADP(+)-dependent D-threonine dehydrogenase (EC 1.1.1.-), which catalyzes the oxidation of the 3-hydroxyl group of D-threonine, was purified to homogeneity from a crude extract of Pseudomonas cruciviae IFO 12047. The enzyme had a molecular mass of about 60,000 Da and consisted of two identical subunits. In addition to D-threonine, D-threo-3-phenylserine, D-threo-3-thienylserine, and D-threo-3-hydroxynorvaline were also substrates. However, the other isomers of threonine and 3-phenylserine were inert. The enzyme showed maximal activity at pH 10.5 for the oxidation of D-threonine. The enzyme required NADP+. NAD+ showed only slight activity. The enzyme was not inhibited by EDTA, o-phenanthroline, alpha,alpha'-dipyridyl, HgCl2, or p-chloromercuribenzoate but was inhibited by tartronate, malonate, pyruvate, and DL-2-hydroxybutyrate. The inhibition by these organic acids was competitive against D-threonine. Initial-velocity and product inhibition studies suggested that the oxidation proceeded through a sequential ordered Bi Bi mechanism. The Michaelis constants for D-threonine and NADP+ were 13 and 0.12 mM, respectively.

https://aem.asm.org/content/aem/59/9/2963.full.pdf

NADP(+)-dependent D-threonine dehydrogenase from Pseudomonas cruciviae IFO 12047.

The velocity distribution of liquid in a cylindrical mixing vessel with flat-plate-baffles like those commonly used was measured by the method similar to the one adopted for the agitator without baffles.1)Some of the experimental results are shown in Figs. 3, 4, 5, 6, 7, 8 and 9.Variations of the liquid velocity distributions caused by the baffle-plates inserted in the agitated vessel are shown in Fig. 10. Obviously, the insertion of baffle-plates reduces the circulating flow round the impeller axis (the circumferential component υt of liquid velocity) and promotes the circulation flow in the vertical direction (caused by the discharge flow from the tip of the impeller).The discharging performance of various impellers is represented by the ratio NPB/Nq1, which is a dimensionless factor corresponding to the relative power required for a unit quantity discharge. The ratios for various impellers are listed in Table 3 together with those in the non-baffled condition. It is to be noted that, in spite of a considerable increase in Nq1, the circulation efficiency of agitators is lowered by the insertion of baffle-plates.Furthermore, the power consumption in the neighbourhood of the impeller (NPimp) was calculated and compared with that consumed in the outer region of the vessel (ΔNP) as shown in Table 4.It may be concluded from above that the improvement in the circulating capacity can be accomplished to a certain extent by a proper design of baffle-plates.

https://www.jstage.jst.go.jp/article/kakoronbunshu1953/23/9/23_9_595/_pdf

Flow Patterns of Liquid in a Cylindrical Mixing Vessel without Baffles

An inducible NADP(+)-dependent D-phenylserine dehydrogenase [EC 1.1.1.-], which catalyzes the oxidation of the hydroxyl group of D-threo-beta-phenylserine, was purified to homogeneity from a crude extract of Pseudomonas syringae NK-15 isolated from soil. The enzyme consisted of two subunits identical in molecular weight (about 31,000). In addition to D-threo-beta-phenylserine, it utilized D-threo-beta-thienylserine, D-threo-beta-hydroxynorvaline, and D-threonine as substrates but was inert towards other isomers of beta-phenylserine and threonine. It showed maximal activity at pH 10.4 for the oxidation of D-threo-beta-phenylserine, and it required NADP+ as a natural coenzyme. NAD+ showed a slight coenzyme activity. The enzyme was inhibited by p-chloromercuribenzoate, HgCl2, and monoiodoacetate but not by the organic acids such as tartronate. The Michaelis constants for D-threo-beta-phenylserine and NADP+ were 0.44 mM and 29 microM, respectively. The N-terminal 27 amino acids sequence was determined. It suggested that the NADP(+)-binding site was located in the N-terminal region of the enzyme.

Shinji Nagata

Papers

Identification of a phosphorylation site in c-Ski as serine 515

Molecular characterization of alanine racemase from Bifidobacterium bifidum

NADP(+)-dependent D-threonine dehydrogenase from Pseudomonas cruciviae IFO 12047.

Flow Patterns of Liquid in a Cylindrical Mixing Vessel without Baffles

An Inducible NADP+-Dependent D-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15: Purification and Biochemical Characterization