Antimicrobial peptides with stability toward tryptic degradation

The inherent instability of peptides toward metabolic degradation is an obstacle on the way toward bringing potential peptide drugs onto the market. Truncation can be one way to increase the proteolytic stability of peptides, and in the present study the susceptibility against trypsin, which is one of the major proteolytic enzymes in the gastrointestinal tract, was investigated for several short and diverse libraries of promising cationic antimicrobial tripeptides. Quite surprisingly, trypsin was able to cleave very small cationic antimicrobial peptides at a substantial rate. Isothermal titration calorimetry studies revealed stoichiometric interactions between selected peptides and trypsin, with dissociation constants ranging from 1 to 20 microM. Introduction of hydrophobic C-terminal amide modifications and likewise bulky synthetic side chains on the central amino acid offered an effective way to increased half-life in our assays. Analysis of the degradation products revealed that the location of cleavage changed when different end-capping strategies were employed to increase the stability and the antimicrobial potency. This suggests that trypsin prefers a bulky hydrophobic element in S1' in addition to a positively charged side chain in S1 and that this binding dictates the mode of cleavage for these substrates. Molecular modeling studies supported this hypothesis, and it is shown that small alterations of the tripeptide result in two very different modes of trypsin binding and degradation. The data presented allows for the design of stable cationic antibacterial peptides and/or peptidomimetics based on several novel design principles.     

Organic acids reduce aluminum toxicity in maize root membranes

Application of 10–50 μ M Al ions to a plasma membrane-enriched microsomal fraction, isolated from roots of maize ( Zea mays L.), resulted in decreased Mg²-ATPase activity. This was probably caused by changes in membrane structure as detected by the use of spin probes. Both enzymatic activity and membrane structure could in part be protected from Al injury when organic acids, similar to those found in maize root tissue, were administered prior to the metal. When stressed by application of Al ions, the Al-tolerant maize hybrid, W64, maintained higher concentrations of organic acids, especially malic and trans -aconitic, than the Al-sensitive maize hybrid, A632. We hypothesize that citric and malic acid, because of their high stability constants with Al and/or the acid's concentration reduce Al toxicity in maize root tissue, especially in the Al-tolerant line.     

Intracellular pH of Thermoplasma acidophila.

Thermoplasma acidophila, a mycoplasma-like organism, was grown at 56 degrees C and pH 2. The intracellular pH of this organism is close to neutral as measured by the distribution of a radioactive weak organic acid, 5,5-dimethyl-2,4-oxazolidinedione, across the plasma membrane. The cell can maintain the pH gradient when subjected to heat or to metabolic inhibitors. Our experiments seem to indicate that a major portion of the pH gradient is not maintained by active processes, but rather by a Donnan potential across the cell membrane.     

Why the term “larva” is ambiguous,or what makes a larva?

The term “larva” is used for many different metazoans. Although this implies a uniform meaning, the term has in fact been used to address immatures with very different characteristics. For providing more precise reference how the term larva is applied in a specific context, I outline here different criteria that have been used to identify an immature as a larva. These include larvae that (a) differ morphologically from their adult (morpho-larva s. l.); (b) differ morphologically from their adult and additionally possess structures that become reduced during ontogeny (morpho-larva s. str.); (c) have a different ecological niche than their adult (eco-larva s. l.); (d) have a different ecological niche than their adult and additionally fulfil a dispersal function (eco-larva s. str.); (e) transform by a metamorphosis to the non-larval immature or adult (metamorph-larva); (f) differ from the adult by having evolved new structures in the early stages (apo-larva); (g) differ from the adult as the adult has evolved new structures (plesio-larva). The differentiation of these criteria will provide a more precise reference reducing possible misunderstanding and allowing a more precise communication.     

Growth inhibition of leukemia cell line CEM-C1 by farnesol: effects of phosphatidylcholine and diacylglycerol.

Acute leukemia cells of the established line CEM-C1 were treated during growth in serum-free medium with various concentrations of trans-trans farnesol. At concentrations ranging from 9.0 to 31.5 microM, farnesol inhibited growth of these cells without causing cell lysis. This effect was preceded by very rapid inhibition of choline incorporation in cellular lipid fraction. The growth inhibitory effect was prevented to a large extent by incubation with phosphatidylcholine or diacylglycerol.     

An amphipathic cyclic tetrapeptide scaffold containing halogenated β2,2‐amino acids with activity against multiresistant bacteria

The present study describes the synthesis and biological studies of a small series of head‐to‐tail cyclic tetrapeptides of the general structure c(Lys‐β^2,2‐Xaa‐Lys) containing one lipophilic β^2,2‐amino acid and Lys, Gly, Ala, or Phe as the Xaa residue in the sequence. The peptides were investigated for antimicrobial activity against gram‐positive and gram‐negative reference strains and 30 multiresistant clinical isolates including strains with extended spectrum β‐lactamase—carbapenemase (ESBL‐CARBA) production. Toxicity was determined against human red blood cells. The most potent peptides showed high activity against the gram‐positive clinical isolates with minimum inhibitory concentrations of 4–8 μg/mL and low haemolytic activity. The combination of high antimicrobial activity and low toxicity shows that these cyclic tetrapeptides containing lipophilic β^2,2‐amino acids form a valuable scaffold for designing novel antimicrobial agents.     

Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membrane after treatment of intact tissues and cell free extracts with triacontanol

Ca²⁺- and Mg²⁺-dependent ATPase activity (EC 3.6.1.3) in a plasma membrane-enriched fraction increased rapidly after in vivo application of physiologically active concentrations of triacontanol (TRIA) to the roots of barley ( Hordeum vulgare L. cv. Conquest) seedlings. Ca²⁺- and Mg²⁺-dependent ATPase activity was 64 and 85% higher, respectively, in the roots of seedlings germinated in the presence of growth-promoting concentrations of TRIA compared to controls. The increase in vivo was concentration dependent, with the greatest increase obtained at 2.3 n M TRIA. Maximal stimulation of ATPase activity of excised tissue treated with TRIA coincided with the temperature at which the barley was grown. At this temperature the plasma membrane is primarily in a mixed gel/liquid crystalline state. Pretreatment of barley roots with cyclohexamide did not alter ATPase stimulation by TRIA. Two to three times more [¹⁴C]-TRIA (mg membrane protein)⁻¹ was found associated with plasma membrane-enriched vesicles treated with TRIA than with vesicles enriched for mitochondrial membranes or for vesicles enriched for tonoplast, Golgi and rough endoplasmic reticulum. Both Ca²⁺- and Mg²⁺-dependent ATPase activity increased by 40–60% within 30 min of the addition of 2.3 n M TRIA to cell-free extracts of barley roots. The addition of octacosanol, the C₂₈ analogue of TRIA, to cell-free extracts did not affect metal-dependent ATPase activity. Consistent with many studies in the green-house, simultaneous additions of equimolar amounts of TRIA and octacosanol to cell-free extracts resulted in inhibition of ATPase stimulation by TRIA. TRIA may directly affect plasma membrane function in barley roots.