An Exclusive Increase in the Concentration of ATP as a Result of Osmotic Stress in Escherichia coli B

The ATP level was exclusively increased among the nucleotides in Escherichia coli under osmotic stress, concomitant with a decrease of guanosine phosphates levels. The profile of ATP formation was different from that resulting from chemicals. E. coli contains a specific system to enhance the ATP level and it might be possible that the source of the increased ATP is guanosine phosphates.     

Effective cancer therapy based on selective drug delivery into cells across their membrane using receptor-mediated endocytosis

Cancer is one of the major causes of death globally. The current treatment options are insufficient, leading to unmet medical needs in cancer treatment. Off-target side effects, multidrug resistance, selective distribution to cancerous tissues, and cell membrane permeation of anti-cancer agents are critical problems to overcome. There is a method to solve these problems by using receptor-mediated endocytosis (RME). It is well known that proteins such as integrin, HER2, EGFR, or other cancer biomarkers are specifically overexpressed on the surface of target cancer cells. By taking advantage of such specific receptors, payloads can be transported into cells through endocytosis using a conjugate composed of the corresponding ligands connected to the payloads by an appropriate linker. After RME, the payloads released by endosomal escape into the cytoplasm can exhibit the cytotoxic activity against cancer cells. Cell-penetrating peptides (CPPs), tumor-homing peptides (THPs), and monoclonal antibodies (mAbs) are utilized as ligands in this system. Antibody drug conjugates (ADCs) based on RME have already been used to cure cancer. In addition to the canonical conjugate method, nanocarriers for spontaneous accumulation in cancer tissue due to enhanced permeability and retention (EPR) effect are extensively used. In this review, I introduce the possibilities and advantages of drug design and development based on RME for the treatment of cancer.     

The role of glycine in determining the rate of glutathione synthesis in poplar. Possible implications for glutathione production during stress

The terminal step of glutathione (GSH) synthesis is the condensation of γ-glutamyl-cysteine (γ-EC) with glycine. Relatively little information exists concerning the importance of photorespiratory glycine in determining the rate of conversion of γ-EC to GSH. Consequently, the effect of exogenous glycine and of illumination on foliar contents of γ-EC and GSH was studied in excised leaves and leaf discs from untransformed poplar ( Populus tremula × P. alba ) and poplar overexpressing γ-glutamylcysteine synthetase (γ-ECS; EC 6.3.2.2). Poplars strongly overexpressing γ-ECS (ggs28) had enhanced levels of γ-EC and GSH compared to untransformed poplars. The relationship between γ-EC and GSH contents in ggs28 was light dependent. In illuminated leaves, GSH contents were up to 50-fold higher than γ-EC. On darkening, γ-EC accumulated markedly and GSH declined, so that the GSH:γ-EC ratio was close to 1. These dark-induced changes were prevented by supplying glycine through the petiole or by incubation of leaf discs on glycine. Dark accumulation of γ-EC in leaf discs from untransformed poplar was also prevented by supplying glycine. Supplying cysteine in the dark to discs from untransformed poplar and ggs28 increased γ-EC levels markedly but GSH levels only slightly. Subsequent illumination caused γ-EC to decrease and GSH to increase. Supplying glycine in concert with cysteine had similar effects to illumination. The data suggest that photorespiratory glycine is essential for GSH synthesis, especially under stress conditions, where increased amounts of GSH are required.     

Somatic embryo cycling: Evaluation of a novel transformation and assay system for seed-specific gene expression in soybean

Somatic embryo cycling, a modification of soybean somatic embryogenic suspension culture, was developed as an efficient and rapid method of producing tissue suitable for stable transformation of soybean germplasm by biolistic particle bombardment. Instead of using immature seed explants, cotyledon-staged somatic embryo hypocotyls were placed on auxin-containing medium, where they initiated new somatic embryos primarily from single epidermal cells. By bombarding hypocotyls prior to initiation of subsequent embryo formation, we have effectively transformed soybean somatic embryos with the reporter genes neomycin phosphotransferase,gb-glucuronidase, and a mammalian stearyl CoA delta-9 desaturase, controlled by a seed-specific promoter. These embryos contain significantly reduced levels of saturated palmitic and stearic fatty acids, and significant amounts of monounsaturated palmitoleic acid, which is not normally abundant in soybean seeds. This study demonstrates the effectiveness of somatic embryo cycling for soybean transformation, and for testing expression of genes for seed-specific proteins. Abnormal flower development in recovered plants is a limitation for application of the technique to produce transgenic seed at present.     

Shoot inversion-induced ethylene in pharbitis nil induces the release of apical dominance by restricting shoot elongation

Shoot inversion induces outgrowth of the highest lateral bud (HLB) adjacent to the bend in the stem in Pharbitis nil. In order to determine whether or not ethylene produced by shoot inversion plays a direct role in promoting or inhibiting bud outgrowth, comparisons were made of endogenous levels of ethylene in the HLB and HLB node of plants with and without inverted shoots. That no changes were found suggests that the control of apical dominance does not involve the direct action of ethylene. This conclusion is further supported by evidence that the direct application of ethylene inhibitors or ethrel to inactive or induced lateral buds has no significant effect on bud outgrowth. The hypothesis that ethylene evolved during shoot inversion indirectly promotes the outgrowth of the highest lateral bud (HLB) by restricting terminal bud (TB) growth is found to be supported by the following observations: (1) the restriction of TB growth appears to occur before the beginning of HLB outgrowth; (2) the treatment of the inverted portion of the shoot with AgNO₃, an inhibitor of ethylene action, dramatically eliminates both the restriction of TB growth and the promotion of HLB outgrowth which usually accompany shoot inversion; and (3) the treatment of the upper shoot of an upright plant with ethrel mimics shoot inversion by retarding upper shoot growth and inducing outgrowth of the lateral bud basipetal to the treated region.     

Generation of one-carbon units in methionine-supplemented Euglena cells

The possible effect of L-methionine supplements on the folate metabolism of division-synchronized Euglena gracilis (strain Z) cells has been examined. Cells receiving 1 mM L-methionine for four cell cycles were examined for folate derivatives, prior to and during cell division. Before cell division, methionine-supplemented cells contained less formylfolate but more methylfolate than unsupplemented cells. During division, both types of folates were present in lower concentrations in the supplemented cells. Growth in methionine for 10 and 34 hr also increased the levels of free aspartate, threonine, serine, cysteine and methionine relative to the controls. Methionine-supplemented cells contained ca 50% of the 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) activity per cell of unsupplemented control cultures and specific enzyme activity was reduced ca 90%. Supplemented cells contained almost twice as much serine hydroxymethyltransferase (EC 2.1.2.1) activity per cell but comparable levels of glycollate dehydrogenase. Growth in methionine also reduced the incorporation of formate-¹⁴C] into serine, RNA, DNA, adenine and protein methionine. In contrast, incorporation of glycine-[2-¹⁴C] and serine-[3-¹⁴C] into folate-related products was not greatly altered by this treatment. Levels of radioactivity in these products suggested that formate was a more important C₁ unit source than glycine or serine when growth occurred in unsupplemented medium. It is concluded that methionine reduces formylfolate production by an effect on the cellular levels of formyltetrahydrofolate synthetase.     

Glycine origin of the methyl substituent on C7'-N of octodiose for the biosynthesis of apramycin

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicating that glycine and/or serine might be involved in the biosynthesis of apramycin. The 13C-NMR analysis of [2-13C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH3 substituent of apramycin on the C7' of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes as previously reported for the case of rapamycin. The 15N NMR spectra of [2-13C,15N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the -NH2 substituents of apramycin.     

Kinetic investigation of the effect of the amino acid side chains in the selective acidolysis of N-acyl-N,alpha,alpha-trialkyl glycine amides.

Accurate kinetic measurements of the rate constants for the acidolysis of five N-acetyl-N-(4-methoxybenzyl)-alpha,alpha-trialkyl glycine cyclohexyl amides in TFA were performed at 25 degrees C and the reactions monitored by HPLC. In all cases the results were consistent with a first order behaviour with respect to the substrate. No direct correlation was obtained with these data between the rate constant values and structure, but a good correlation coefficient was obtained when a multiple regression analysis was applied by taking advantage of a Taft equation using appropriate polar and steric substituent parameters. In a plot of the values observed for log k against those calculated by this equation all five points fell very close to the line of perfect correlation. The calculated sensitivity coefficients to polar and steric contributions were used to discuss the experimental results and showed that the acidolysis were comparatively less affected by steric effects than expected.     

On the activity of ribulosediphosphate carboxylase with CO2 and O2 from leaf extracts of Zea mays

Ribulosediphosphate carboxylase, partially purified from corn leaves, demonstrates a low K_m(CO₂) of 19 μM if stabilized with ribose-5-phosphate during extraction. It also exhibits a ribulosediphosphate dependent uptake of oxygen, similar to that observed with spinach carboxylase. The low K_m(CO₂) is similar to the apparent K_m(CO₂) for photosynthesis by intact corn tissue and requires reconsideration of the hypothesis that CO₂ is concentrated in the bundle sheath cell by the C₄ pathway during photosynthesis.     

Cytochromes bd-I and bo3 are essential for the bactericidal effect of microcin J25 on Escherichia coli cells

Microcin J25 has two targets in sensitive bacteria, the RNA polymerase, and the respiratory chain through inhibition of cellular respiration. In this work, the effect of microcin J25 in E. coli mutants that lack the terminal oxidases cytochrome bd-I and cytochrome bo₃ was analyzed. The mutant strains lacking cytochrome bo₃ or cytochrome bd-I were less sensitive to the peptide. In membranes obtained from the strain that only expresses cytochrome bd-I a great ROS overproduction was observed in the presence of microcin J25. Nevertheless, the oxygen consumption was less inhibited in this strain, probably because the oxygen is partially reduced to superoxide. There was no overproduction of ROS in membranes isolated from the mutant strain that only express cytochrome bo₃ and the inhibition of the cellular respiration was similar to the wild type. It is concluded that both cytochromes bd-I and bo₃ are affected by the peptide. The results establish for the first time a relationship between the terminal oxygen reductases and the mechanism of action of microcin J25.