Effect of amino acid analogs on the processing of the pancreatic polypeptide precursor in primary cell cultures

Amino acid analogs, which can be incorporated into nascent peptide chains were used in cultures of endocrine cells from canine pancreas to study the effect on processing of the metabolically labeled precursor for pancreatic polypeptide. Analogs for basic amino acids, canavanine, and aminoethylcysteine prevented the di-basic processing of the prohormone. The polar leucine analog, beta-hydroxyleucine, only partially perturbed the function and cleavage of the signal peptide but efficiently and unexpectedly blocked the dibasic cleavage of the prohormone. Other nonbasic amino acid analogs, beta-hydroxynorvaline and azetidine-2-carboxylic acid, which only could be incorporated into the prohormone at a distance from the processing site, also prevented dibasic cleavage of the prohormone. Although there are no phenylalanine residues in the prohormone, analogs for this amino acid, fluoro-phenylalanine and particularly phenylserine, could also block the processing of the prohormone at the dibasic site. This effect was prevented by addition of a small quantity of phenylalanine. It is concluded that amino acid analogs can interfere with precursor processing through altering both the primary and the secondary structure of the precursor but also through incorporation into cosynthesized protein(s) which are necessary for the precursor processing.     

Effect of temperature on the protein and nucleic acid content of thermotolerant yeasts

Summary The effect of growth at 30°, 35° and 40° on the biomass yield and on the nucleic acid and protein content of twelve isolates of yeast has been studied. Although yields of 41.6% and a true protein content of 34% were obtained, each of the strains exhibited a lower yield and protein content at 40° than at the lower temperatures. Nucleic acid levels were also reduced at 40°.     

Effect of thermotolerance on thermal radiosensitization in hepatoma cells

The interaction between hyperthermia and X irradiation was determined in cultured Reuber H35 hepatoma cells with different states of thermosensitivity. Incubation at 41 degrees C followed by 4-Gy X rays resulted after 2 hr in a stabilization of cell survival for heat or plus X rays, with a maximum synergism factor of 1.6. Thermotolerance did not develop during incubation at 41.7 or 42.5 degrees C. When heat treatment of cells was followed by irradiation, the synergism factor for thermal radiosensitization increased with both the amount of thermal cell killing and the amount of X-ray cell killing; the influence of thermal exposure on the synergism factor was greater than that of the X-ray dose. Cells were made thermotolerant either by incubation at 42.5 degrees C for 30 or 60 min followed by an interval at 37 degrees C, or by continuous incubation at 41 degrees C. In both cases thermotolerance was measured by incubation at 42.5 degrees C. No difference was observed between the maximum thermotolerance achieved with both methods. When cells were irradiated in addition to the second heat treatment, thermal radiosensitization was strongly reduced concomitant with the decreased sensitivity to killing by heat.     

Exploring the UDP pocket of LpxC through amino acid analogs

Lipopolysaccharide (LPS) biosynthesis is an attractive antibacterial target as it is both conserved and essential for the survival of key pathogenic bacteria. Lipid A is the hydrophobic anchor for LPS and a key structural component of the outer membrane of Gram-negative bacteria. Lipid A biosynthesis is performed in part by a unique zinc dependent metalloamidase, LpxC (UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase), which catalyzes the first non-reversible step in lipid A biosynthesis. The UDP portion of the LpxC substrate-binding pocket has been relatively unexplored. We have designed and evaluated a series of hydroxamate based inhibitors which explore the SAR of substitutions directed into the UDP pocket with a range of substituted α-amino acid based linkers. We also provide the first wild type structure of Pseudomonas aeruginosa LpxC which was utilized in the design of many of these analogs.     

Increased number of Arginine-based salt bridges contributes to the thermotolerance of thermotolerant acetic acid bacteria, Acetobacter tropicalis SKU1100

Constitution of oxidative defense systems and, correspondingly, oxidative stress prevention are highly dependent on amino acid supply. In vitro, experiments have demonstrated that amino acid availability participates to the homeostasis of reactive oxygen species. However the molecular mechanisms involved in the maintenance of redox homeostasis responsive to circulating amino acid levels remain unclear. As GCN2 is a protein kinase considered to be an important sensor for amino acids availability and a potential regulator of redox homeostasis, we hypothesized that this kinase can modulate redox homeostasis in vivo, in response to an amino acid-imbalanced diet.We investigated the response of GCN2+/+ and GCN2−/− mice to a long-term (24 weeks) leucine-imbalanced diet (EDΔLeu). In order to evaluate the oxidation level in each group of mice, we determined the degree of protein oxidation in the liver. Interestingly, GCN2−/− mice exhibited an increase in protein carbonylation, a marker of oxidative stress, in response to the EDΔLeu diet. These data correlate with a decrease in hepatic GPX1 expression, a major antioxidant enzyme, and a decrease in total GPX activity in the liver. Our results suggest that GCN2 and its downstream signaling pathway have an important role in the protection against oxidative injuries induced by an amino acid-imbalanced diet, and that it can play a critical role in the prevention of oxidative damage.     

Effect of dietary amino acid on the amino acid pool of Aedes aegypti

Ion-exchange chromatography analysis of whole body extracts of Aedes aegypti mosquitoes which had received amino acids in their diet revealed that generally there were changes in the titre of two or more amino acids. Cysteine produced the greatest number of changes and was toxic to the insect. Of the ten amino acids provided, none resulted in the significant change in the concentration of tyrosine following a blood meal as was observed in previous studies. Evidence is presented for the conversion of arginine to ornithine and for the synthesis of arginine from glutamic acid. The data presented tend to support the hypothesis of lysine synthesis from α-ketoglutarate and for the use of proline as an energy reserve in the insect.     

Effect of amino acid starvation on UV sensitivity ofLactobacillus acidophilus cells

InLactobacillus acidophilus cultures UV irradiated in the exponential phase of growth, the dosesurvival curve was of the simple exponential type, without any shoulder. If the bacteria were subjected to amino acid starvation prior to irradiation, an shoulder corresponding to a quasi-treshold dose (D_q) of about 780 ergs/mm² appeared in the curve. The administration of protein or RNA-synthesia inhibitors prior to irradiation had the same effect. The effect of pre-irradiation amino acid starvation was abolished by simultaneous thymidine starvation. It was likewise abolished if amino acid starvation was followed by incubation in the presence of amino acids (without thymidine) and then by irradiation of the cells. Post-irradiation amino acid starvation did not lead to the formation of an shoulder but if combined with thymidine starvation it did. It can be concluded from the results that post-irradiation repair processes are facilitated or promoted if, during the post-irradiation interval DNA synthesis is delayed. This delay represents a compensation of the pre-irradiation increase of cellular DNA-content, taking place during inhibition of proteosynthesis. The postirradiation administration of caffeine did not abolish the formation of the shoulder induced by pre-irradiation amino acid starvation; on the contrary, it induced its formation even in exponentially growing, irradiated control bacteria.     

Blood-brain barrier transport of the alpha-keto acid analogs of amino acids

A number of alpha-keto acid analogs of amino acids have been found to penetrate the blood-brain barrier (BBB). Pyruvate, alpha-ketobutyrate, alpha-ketoisocaproate, and alpha-keto-gamma-methiolbutyrate all cross the BBB by a carrier-mediated process and by simple diffusion. Under normal physiological conditions, diffusion accounts for roughly 15% or less of total transport. Aromatic alpha-keto acids, phenylpyruvate, and p-hydroxyphenylpyruvate do not penetrate the BBB, nor do they inhibit the transport of other alpha-keto acids. Evidence based primarily on inhibition studies indicates that the carrier-mediated transport of alpha-keto acids occurs via the same carrier demonstrated previously for propionate, acetoacetate, and beta-hydroxybutyrate transport, commonly referred to as the monocarboxylate carrier. As a group, the alpha-keto acid analogs of the amino acids have the highest affinity for the carrier, followed by propionate and beta-hydroxybutyrate. Starvation for 4 days induces transport of alpha-keto acids, but transport is suppressed in rats fed commercial laboratory rations and subjected to portacaval shunts. The mitochondrial pyruvate translocator inhibitor alpha-cyanocinnamate has no effect on the BBB transport of alpha-keto acids.     

Comparison of the inhibitory effects of diverse amino acids and amino acid analogs on 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity in isolated epidermal cells

At a concentration of 1.25 mM, 14 amino acids were capable of inhibiting the induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in isolated epidermal cells. The greatest percentages of inhibition of TPA-induced epidermal ornithine decarboxylase activity were as follows: cysteine, 98%; tryptophan, 74%; methionine, 64%; phenylalanine, 51%; glycine, 44%; asparagine, 43%; glutamic acid, 42%; leucine, 40%; and arginine, 39%. These amino acid treatments did not alter the time- and concentration-response curves for induction of ornithine decarboxylase activity by TPA. Moreover, there was no difference between the rates at which [3H]arginine, [3H]leucine, [3H]phenylalanine, [3H]methionine, [3H]tryptophan and [14C]cysteine were taken up by freshly isolated epidermal cells or incorporated into epidermal proteins. Arginine, phenylalanine and methionine inhibited the induction of ornithine decarboxylase activity by the tumor promoter to degrees comparable to those elicited by their analogs canavanine and homoarginine, beta-2-thienyl-DL-alanine, and ethionine, respectively. These amino acids and amino acid analogs did not alter the overall rate of protein synthesis. In contrast, both the amino acids and their analogs increased the rates of proteolysis in isolated epidermal cells, an effect which correlated well with the abilities of these different compounds to inhibit TPA-induced ornithine decarboxylase activity. Moreover, both methionine and phenylalanine decreased the half-life and increased the rate of heat denaturation of the TPA-induced enzyme, a result identical to that obtained after treatment with the analogs ethionine and beta-2-thienyl-DL-alanine, respectively. Taken together, these results suggest that millimolar concentrations of exogenous amino acids might induce the synthesis of abnormal proteins and nonfunctional enzymes. Therefore, it is speculated that the uptake of unbalanced amounts of amino acids into the epidermal target cells might alter the stability and the ultrastructure of the TPA-stimulated enzyme just as the amino acid analogs do.     

Effect of pH and cell cycle progression on development and decay of thermotolerance

Synchronous Chinese hamster ovary cells were heated in G1 and incubated at 37 degrees C at pH 6.75 or pH 7.4 before they were heated a second time. The magnitude and rate of development and decay of thermotolerance were greatly reduced at pH 6.75. This was also observed for asynchronous cells. Furthermore, the heat-induced delay in cell cycle progression was greatly enhanced at low pH and correlated with the reduced rate for development and decay of thermotolerance. However, studies with [3H]TdR to kill cells entering S phase showed that the decay of thermotolerance is relatively independent of the cell cycle. Therefore, low pH apparently slows many cell processes, including those associated with heat-induced cell cycle delay and the rate of development and decay of thermotolerance.     

Enhanced glycosyltransferase activity during thermotolerance development in mammalian cells

The cellular heat shock response leads to the enhanced synthesis of a family of heat shock proteins and the development of thermotolerance. In CHO cells, however, heat shock also leads to enhanced synthesis of a 50 kD glycoprotein and elevated activity of N-acetylgalactosaminyltransferase (GalNAcT). In this study we showed increased GalNAcT activity during thermotolerance expression in all of five mammalian cell lines included in the study. However, there was no simple correlation between cellular heat sensitivity of unheated control cells and basal levels of GalNAcT activity, measured toward the same exogenous acceptor apomucin. Although GalNAcT was elevated in thermotolerant cells, GalNAcT activity itself did not exhibit thermotolerance in terms of reduced sensitivity to heat inactivation. The increase in GalNAcT activity after heating was similar in exponentially growing and plateau-phase cultures and was inhibited neither by cycloheximide nor actinomycin D. However, the inhibitors by themselves also increased GalNAcT activity in unheated control cells. Chemical inducers of thermotolerance (arsenite and diamide) increased GalNAcT activity, but the increase was modest when compared to that following hyperthermia. In addition to GalNAcT, two other glycosyltransferases with specificity for O-glycans, alpha 1,2-fucosyltransferase and alpha 2,6-sialyltransferase, also showed increased activity after hyperthermia and during thermotolerance development. Together with previously published data, these results support the hypothesis that heat-induced activation of O-glycan-specific glycosyltransferases plays a physiological role in the cellular heat shock response and in thermotolerance development.     

Effect of pH on quercetin-induced suppression of heat shock gene expression and thermotolerance development in HT-29 cells.

When cells were heated for 15 min at 45 degrees C, they became thermotolerant to a second heat exposure at 45 degrees C. Thermotolerance developed rapidly, reached its maximum 6 hr after heat shock, and then gradually decayed. The development of thermotolerance was partially suppressed by treatment with various concentrations of quercetin (0.05-0.2 mM) at pH 7.4 after the initial heat treatment. In contrast, the drug markedly inhibited thermotolerance development at pH 6.5. Furthermore, a combination of low pH and quercetin treatment distinctively altered the expression of HSP70 gene compared with that of HSP28 or HSP90 gene. These results demonstrate a good correlation between the amount of HSP70 gene expression and development of thermotolerance.     

Global analysis of the genes involved in the thermotolerance mechanism of thermotolerant Acetobacter tropicalis SKU1100

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.     

Variable denaturation of ovalbumin by incorporation of amino acid analogs

A novel firefly luciferin- enhanced luminescent procedure for the quantitation of horseradish peroxidase labels has been directly incorporated into established enzyme immunoassays. The procedure is rapid and sensitive and uses readily available reagents. Light emission from the enhanced reaction is high and relatively constant and thus easily measured. The luminescence procedure has been successfully incorporated into immunometric assays for rubella antibody and human IgE and into a competitive immunoassay for digoxin.