Properties of enzymes from bacteria grown in the 70–100°C range

In chemostat cultures of Bacillus caldolyticus, adaptation in a single step from 70–100°C was followed under aerobic and oxygen-limited conditions and was found to proceed more smoothly under the latter circumstances. Variations of the medium (e.g. yeast extract or silicate concentrations) showed that growth at 100°C is in all respects similar to that of cultures at moderate temperatures.Enzyme preparations derived from cultures at 5°C intervals between 70 and 100°C were used to determine the temperature range. For all nine enzymes tested, the optimum temperature was found to be 67°C; the latter was independent of the growth temperature. Differences were found, however, with respect to the maximum temperature of individual enzymes, and three groups, with maxima between 70 and 80°C, 80 and 90°C and 90 and 100°C can be distinguished. Again, there was no correlation with the growth temperature.Stability experiments also revealed that enzymes from the same organism can have different thermal properties: Some were found to be quite thermolabile (e.g. the pyruvate kinase), while others (e.g. hexokinase and glutamate-pyruvate transaminase) exhibited a high thermostability. These properties were not related to the growth temperature within the 70–100°C range, too.Six of the enzymes tested could be stabilized by their respective substrates, but the degree of protection varied for individual enzymes. Three enzymes (acetate kinase, glutamate dehydrogenase and myokinase) could not be stabilized by their substrates.Comparative experiments with the hexokinase suggested, that the thermal integrity of the enzymes is better protected within the cell as compared to the stability of the enzyme preparations.Abbreviations used AK acetate kinase - Ala-DH alanine dehydrogenase - Ald aldolase - GIDH glutamate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - GTP glutamate-pyruvate transaminase - HK hexokinase - ICDH isocitrate dehydrogenase - MK myokinase - PK pyruvate kinase     

Effect of balance taping on trunk stabilizer muscles for back extensor muscle endurance: A randomized controlled study

Objective:The purpose of this study was to investigate the difference in back extensor muscle endurance before and after kinesiology tape application to all back stabilizer muscles and to the erector spinae alone.Methods:We assessed 32 adults (16 men and 16 women), randomly divided into two groups. In the erector spinae taping (EST) group, kinesiology tape was applied only to the erector spinae, and in the total muscle taping (TMT) group, kinesiology tape was applied to the erector spinae, latissimus dorsi, lower trapezius, internal oblique abdominis, and external oblique abdominis.Results:Both groups showed significant difference in terms of back extensor muscle endurance after kinesiology tape application (p<0.05). Between-group comparison revealed that the TMT group had more back extensor muscle endurance than the EST group (p<0.05) after kinesiology tape application.Conclusions:These findings indicate that, to improve back extensor muscle endurance, kinesiology tape should be applied to all back stabilizer muscles, rather than to the erector spinae muscles alone.     

Stabilized dried blood spot collection

During the collection phase of the dried blood spot method, practitioners need to ensure that there is no smearing of the blood sample on the filter paper or else readings from it will be invalid. This can be difficult to accomplish in the field if there is relative motion between the site of blood discharge on the finger and the filter paper. In this article, a gyroscope stabilization method is introduced and demonstrated to provide consistent and improved dried blood spot collection within a circular guide region notwithstanding the presence of rocking.     

ERK-mediated Cytoplasmic Retention of USP11 Contributes to Breast Cancer Cell Proliferation by Stabilizing Cytoplasmic p21

Breast cancer ranks as the most frequently diagnosed cancer among women worldwide. Elevated cytoplasmic p21 levels are often found in breast cancer tissues and related to a poor prognosis. However, the underlying mechanisms that lead to the stabilization of cytoplasmic p21 protein, which normally has a very short half-life, remain obscure. In this study, we found that there was a strong correlation between p21 and USP11 in the cytoplasm of breast cancer tissues and cells. Furthermore, we revealed that ERK1/2 phosphorylated USP11 at the Ser905 site, which promoted the cytoplasmic localization of USP11. In the cytoplasm, USP11 colocalized and interacted with p21. As a result, USP11 catalyzed the removal of polyubiquitin chains bound to cytoplasmic p21 and resulted in its stabilization. Functionally, USP11-mediated stabilization of cytoplasmic p21 induced breast cancer cell proliferation in vitro and in vivo. Our findings provide the first evidence that ubiquitinated p21 in the cytoplasm can be recycled through USP11-mediated deubiquitination, and we identified the USP11-p21 axis in the cytoplasm as a potential therapeutic target for breast cancer control.     

Investigations on unconventional hydrogen bonds in RNA binding proteins: the role of CH...O=C interactions

We have investigated the roles played by C-H...O=C interactions in RNA binding proteins. There was an average of 78 CH...O=C interactions per protein and also there was an average of one significant CH...O=C interactions for every 6 residues in the 59 RNA binding proteins studied. Main chain-Main chain (MM) CH...O=C interactions are the predominant type of interactions in RNA binding proteins. The donor atom contribution to CH...O=C interactions was mainly from aliphatic residues. The acceptor atom contribution for MM CH...O=C interactions was mainly from Val, Phe, Leu, Ile, Arg and Ala. The secondary structure preference analysis of CH...O=C interacting residues showed that, Arg, Gln, Glu and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Ile, Leu, Lys, Met, Phe, Trp and Val preferred to be in strand conformation. Most of the CH...O=C interacting polar amino acid residues were solvent exposed while, majority of the CH...O=C interacting non polar residues were excluded from the solvent. Long and medium-range CH...O=C interactions are the predominant type of interactions in RNA binding proteins. More than 50% of CH...O=C interacting residues had a higher conservation score. Significant percentage of CH...O=C interacting residues had one or more stabilization centers. Sixty-six percent of the theoretically predicted stabilizing residues were also involved in CH...O=C interactions and hence these residues may also contribute additional stability to RNA binding proteins.     

Design, synthesis and biological activity of novel peptidyl benzyl ketone FVIIa inhibitors

Herein is described the synthesis of a novel class of peptidyl FVIIa inhibitors having a C-terminal benzyl ketone group. This class is designed to be potentially suitable as stabilization agents of liquid formulations of rFVIIa, which is a serine protease used for the treatment of hemophilia A and B inhibitor patients. A library of compounds was synthesized with different tripeptide sequences, N-terminals and d-amino acids in the P3 position. Cbz-d-Phe-Phe-Arg-bk (33) was found to be the best candidate with a potency of K_i = 8 μM and no substantial inhibition of related blood coagulation factors (thrombin and FXa). Computational studies revealed that 33 has a very stable binding conformation due to intramolecular hydrogen bonds, which cannot be formed with l-Phe in the P3 position. Nonpolar amino acids were found to be superior, probably due to a minimization of the cost of desolvation upon binding to FVIIa.     

Neuropeptide profiling of the bovine hypothalamus: thermal stabilization is an effective tool in inhibiting post-mortem degradation

The hypothalamus is the central regulatory region of the brain that links the nervous system to the endocrine system via the pituitary gland. It synthesizes and secretes neuropeptide hormones, which in turn act to stimulate or inhibit the secretion of pituitary hormones. We have undertaken a detailed MS investigation of the peptides present in the bovine hypothalamus by adapting a novel heat stabilization methodology, which improved peptide discovery to direct our studies into the molecular mechanisms involved in bovine reproduction. The untreated samples contained large numbers of protein degradation products that interfered with the analysis of the neuropeptides. In the thermally stabilized samples, we were able to identify many more neuropeptides that are known to be expressed in the bovine hypothalamus. Furthermore, we have characterized a range of post-translational modifications that indicate the presence of processed intact mature neuropeptides in the stabilized tissue samples, whereas we detected many trimmed or truncated peptides resulting from post-mortem degradation in the untreated tissue samples. Altogether, using an optimized workflow, we were able to identify 140 candidate neuropeptides. We also nominate six new candidate neuropeptides derived from proSAAS, secretogranin-2 and proTRH.     

Stabilization of native and double <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidases from <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> and <Emphasis Type="Italic">Trigonopsis variabilis</Emphasis> by immobilization on streptavidin-coated magnetic beads

Double d-amino acid oxidases (dRtDAO and dTvDAO) were previously genetically constructed by linking the C-terminus of one subunit of their corresponding native DAOs from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) to the N-terminus of the other identical subunit. We have now immobilized these double DAOs and their native counterparts onto streptavidin-coated magnetic beads through the interaction between biotin and streptavidin. The catalytic efficiencies (k_cat/K_M) of immobilized DAOs toward d-alanine and cepharosporin C remained similar to those of their soluble forms, except the catalytic efficiency of immobilized TvDAO toward d-alanine was decreased by 56%. After immobilization, the T_m value for RtDAO was shifted 15°C higher to 60°C, while those for dRtDAO, TvDAO and dTvDAO were increased by 5–8°C to 56, 60 and 60°C, respectively. In the presence of 10 mM H₂O₂, immobilized RtDAO, dRtDAO, TvDAO and dTvDAO exhibited half-lives of about 8, 10, 3 and 5 h, respectively, giving 16-, 10-, 6- and 7-fold greater stability than their soluble forms, respectively. Therefore, immobilization through biotin–streptavidin affinity binding enhances the thermal and oxidative stability of native and double DAOs studied, especially RtDAO. The additive stabilizing effect of subunit fusion and immobilization was more pronounced in the case of RtDAO than TvDAO.     

Adipogenic genes on induction and stabilization of commitment to adipose conversion

Most late events of adipose conversion are known, but those early events that lead to cell commitment, and important aspects of its mechanism remain unknown. We recently described that, in the absence of any other adipogenic factor, 4 h incubation with staurosporine promotes commitment of 3T3-F442A cells to adipogenesis. This commitment consists of two stages; a first stage of 4 h induction by staurosporine, and, in the absence of this drug, a second stage of stabilization which becomes completed after 40-48 h from staurosporine treatment. Here, we demonstrate that pparg2 gene is expressed early after induction stage but before commitment is stabilized, whereas cebpa is highly expressed during the last part of stabilization stage. A decrease of dlk1 expression, whose down-regulation is indispensable for adipogenesis, began to take place between 24 and 48 h of St-Dex incubation started, reaching the lowest levels well into the end of stabilization stage.     

Stabilization of a tetrameric enzyme (α-amino acid ester hydrolase from Acetobacter turbidans) enables a very improved performance of ampicillin synthesis

The stabilized derivative of the enzyme α-amino acid ester hydrolase from Acetobacter turbidans has been found to be very adequate as biocatalyst of the synthesis of the very relevant antibiotic ampicillin. This enzyme resulted much more adequate than the Penicillin G Acylase (PGA) from Escherichia coli (the most used enzyme). The stabilization of the enzyme was required because under optimal conditions (absence of phosphate and 40% of MeOH), no-stabilized derivatives or soluble enzyme from A. turbidans become very rapidly inactivated. Under these conditions, this new stabilized derivative exhibited a very high selectivity for the transferase activity compared to the esterase one, as well as a very low hydrolytic activity towards the antibiotic. Moreover, this new biocatalyst did not recognize -phenylglycine as substrate in the synthetic process. By using the racemic mixture of / phenylglycine methyl ester, 85% of the -ester could be transformed to ampicillin. In contrast, the enzyme from E. coli exhibited a high hydrolytic activity for the ampicillin yielding low synthetic yields. This enzyme also resulted much less enantioselective producing both isomers of the antibiotic.