The physiological basis of seed dormancy in Avena fatua. VIII. Action of malonic acid

Adkins, S. W., Symons, S. J. and Simpson, G. M. 1988. The physiological basis of seed dormancy in Avena fatua . VIII. Action of malonic acid - Physiol. Plant, 72: 477–482.
A low concentration of malonic acid (50 m M ) induced germination in four genetically pure dormant lines of Avena fatua L. Sensitivity to this treatment was poor immediately after harvest but increased markedly during after-ripening, indicating that the mode of action of malonic acid (50 m M ) was similar to that of another organic acid, citric acid. Over the concentration range (10–50 m M ) where malonic acid promoted germination, oxygen uptake was also stimulated, and this was before the first visible signs of germination. At higher concentrations (100–300 m M ) where there was no promotion of germination, malonic acid strongly inhibited oxygen uptake. These results show that malonic acid has a dual effect on oxygen uptake and subsequent germination. Low concentrations (10–50 m M ) act by stimulating the Krebs cycle and germination through an acidification reaction like citric acid, and high concentrations (100–300 m M ) act by inhibiting germination through enzymatic restraint of the Krebs cycle.
The stimulation of both oxygen uptake and germination by three established germination promoters (sodium nitrate, citric acid and ethanol) was inhibited by a high concentration of malonic acid (200 m M ) but unaffected by a low concentration (50 m M ). These results show that oxygen uptake, and hence the activity of the Krebs cycle, are important processes involved in the dormancy breaking mechanism of these three promotors.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.     

Synthesis of an Asymmetrically Substituted AZA Crown Ether as Metal and Amino Acid Binding Site in DNA Conjugates

Abstract

Crown ether 4 as a receptor core for protonated primary amines such as amino acids has been synthesized and incorporated into oligodeoxynucleotides as dangling ends.     

Microbial production of propionic acid from propionibacteria: Current state,challenges and perspectives

Propionic acid (PA) is an important building block chemical and finds a variety of applications in organic synthesis, food, feeding stuffs, perfume, paint and pharmaceutical industries. Presently, PA is mainly produced by petrochemical route. With the continuous increase in oil prices, public concern about environmental pollution, and the consumers’ desire for bio-based natural and green ingredients in foods and pharmaceuticals, PA production from propionibacteria has attracted considerable attention, and substantial progresses have been made on microbial PA production. However, production of PA by propionibacteria is facing challenges such as severe inhibition of end-products during cell growth and the formation of by-products (acetic acid and succinic acid). The integration of reverse metabolic engineering and systematic metabolic engineering provides an opportunity to significantly improve the acid tolerance of propionibacteria and reduce the formation of by-products, and makes it feasible to strengthen the commercial competition of biotechnological PA production from propionibacteria to be comparable to the petrochemical route.     

Poly(glutamic Acid) for Biomedical Applications

ABSTRACT:?

Paclitaxel is a widely used anti-cancer agent. Conjugates of paclitaxel with poly(glutamic acid) have shown great promise in preclinical trials, and clinical trials are now underway. Preclinical data suggest that more paclitaxel is preferentially delivered to tumor sites vs. nonconjugated paclitaxel. When poly(glutamic acid) is conjugated to other families of cancer drugs, similar improvements in effectiveness and reduced toxicity are observed. Optimization of poly(glutamic acid) for use in drug delivery applications is a key step in making this technology viable.     

Retinoic Acid Improves a Hybridoma Culture in a Fructose-Based Medium by Up-Regulation of Fructose Incorporation via Retinoid Nuclear Receptors

Fructose was focused on as an alternative sugar source to glucose in a hybridoma culture medium because it decreases lactate production during cultivation, leading to cell and product stability. But, not all human hybridoma cell lines grew well in a fructose-based serum-free medium. We found that the addition of all-trans-retinoic acid to the fructose-based medium improved the growth and monoclonal antibody production of hybridoma cell lines by up-regulation of fructose incorporation that represented increased expression of the fructose transporter, GLUT5. Selective activation of retinoid nuclear receptor by synthetic ligands showed that both retinoic acid receptors and retinoid X receptors might be related to the improvement of the fructose-based hybridoma culture. This study might be applicable to cell cultures susceptible to lactate and pH changes as well as hybridoma cultures.     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Abstract

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.     

Role of methyl groups in dynamics and evolution of biomolecules

Recent studies have discovered strong differences between the dynamics of nucleic acids (RNA and DNA) and proteins, especially at low hydration and low temperatures. This difference is caused primarily by dynamics of methyl groups that are abundant in proteins, but are absent or very rare in RNA and DNA. In this paper, we present a hypothesis regarding the role of methyl groups as intrinsic plasticizers in proteins and their evolutionary selection to facilitate protein dynamics and activity. We demonstrate the profound effect methyl groups have on protein dynamics relative to nucleic acid dynamics, and note the apparent correlation of methyl group content in protein classes and their need for molecular flexibility. Moreover, we note the fastest methyl groups of some enzymes appear around dynamical centers such as hinges or active sites. Methyl groups are also of tremendous importance from a hydrophobicity/folding/entropy perspective. These significant roles, however, complement our hypothesis rather than preclude the recognition of methyl groups in the dynamics and evolution of biomolecules.