Synthesis of rhodamine 6G-based compounds for the ATRP synthesis of fluorescently labeled biocompatible polymers

Facile derivatization of rhodamine 6G in the 2' position by direct reaction with secondary amines is reported. If the secondary amine contains a hydroxy group, the hydroxyl-functional intermediate can be readily esterified to give either fluorescent initiators for atom transfer radical polymerization (ATRP) or a fluorescent methacrylic comonomer. In contrast to rhodamine dyes functionalized using primary amines, which are only fluorescent at low pH, these compounds are highly fluorescent at physiological pH. These new compounds were subsequently used to prepare a range of fluorescently labeled biocompatible polymers based on the biomimetic monomer, 2-(methacryloyloxy)ethyl phosphorylcholine (MPC), for biomedical studies.     

Identification of avidin and streptavidin binding motifs among peptides selected from a synthetic peptide library consisting solely of D-amino acids

Peptides consisting solely of D -amino acids (D -peptides) as opposed to their L -counterparts (L -peptides) are resistant towards proteolytic degradation in the organism and may therefore be useful in future efforts to develop new stable peptide-based drugs. Using the random synthetic peptide library technique several L - and D -peptides, capable of binding to both avidin and streptavidin, were found. The L -peptides contained the previously described HPQ/M motifis, and among the D -peptides three binding motifs could be identified, of which the most frequently found one contained an N-terminal aliphatic hydrophobic amino acid (V, L or I) and an aromatic amino acid (Y or F) on the second position. At the third position in this motif several different amino acid residues were found, although N was the most frequent. Peptides representing two of the D -motifs were synthesized as well as peptides containing the HPQ/M motifs, and their binding properties were examined. Although the D -peptides were originally selected using avidin they also inhibited binding between immobilized biotin and soluble streptavidin as well as avidin. The IC₅₀ of some of the peptides were approximately 10⁵ times higher than the IC₅₀ for biotin but some had a lower IC₅₀ than iminobiotin. The D -peptides, which were originally selected from the library using avidin, could also inhibit the binding between streptavidin and biotin. Likewise, L -peptides selected from a library screened with streptavidin, could inhibit the binding of both streptavidin and avidin to immobilized biotin. Furthermore, the D -peptide, VFSVQSGS, as well as biotin could inhibit binding of streptavidin to an immobilized L -peptide (RYHPQSGS). This indicates that the biotin-like structure mimicked by these two seemingly very different peptides may react with the same binding sites in the streptavidin molecule.     

Solutes in small amounts provide for lipid-bilayer softness: cholesterol,short-chain lipids,and bola lipids

The effect of the incorporation of small amounts (∼1 mole%) of amphiphilic solutes, such as cholesterol, a short-chain lipid (DC₁₀PC), and a bola lipid, into multilamellar DMPC bilayers is studied by small-angle neutron scattering and differential-scanning calorimetry. The anomalous swelling behavior observed in the transition region of pure DMPC bilayers is interpreted as an indication of bilayer softening and thermally reduced bending rigidity. Small amounts of the solutes are found to maintain or even enhance the bilayer softness. In the case of cholesterol, a systematic study shows that the well-known rigidification effect is observed only for cholesterol concentrations above 3–4 mole%. The results are discussed in relation to the physical properties of internal cell membranes. Received: 28 May 1996 / Accepted: 27 July 1996     

Probing the local lipid environment of the Rhodobacter sphaeroides cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc₁ complex (cytbc₁) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc₁ complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc₁ complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc₁ to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc₁ complexes retain their native dimeric structure and co-purify with 56 ± 6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b₆f complex (cytb₆f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc₁/cytb₆f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q₀ and Q_i sites.     

Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

Eukaryotic P-type plasma membrane H⁺-ATPases are primary active transport systems that are regulated at the post-translation level by cis-acting autoinhibitory domains, which can be relieved by protein kinase-mediated phosphorylation or binding of specific lipid species. Here we show that lysophospholipids specifically activate a plant plasma membrane H⁺-ATPase (Arabidopsis thaliana AHA2) by a mechanism that involves both cytoplasmic terminal domains of AHA2, whereas they have no effect on the fungal counterpart (Saccharomyces cerevisiae Pma1p). The activation was dependent on the glycerol backbone of the lysophospholipid and increased with acyl chain length, whereas the headgroup had little effect on activation. Activation of the plant pump by lysophospholipids did not involve the penultimate residue, Thr-947, which is known to be phosphorylated as part of a binding site for activating 14-3-3 protein, but was critically dependent on a single autoinhibitory residue (Leu-919) upstream of the C-terminal cytoplasmic domain in AHA2. A corresponding residue is absent in the fungal counterpart. These data indicate that plant plasma membrane H⁺-ATPases evolved as specific receptors for lysophospholipids and support the hypothesis that lysophospholipids are important plant signaling molecules.     

Metal Fluoride Inhibition of a P-type H+ Pump: STABILIZATION OF THE PHOSPHOENZYME INTERMEDIATE CONTRIBUTES TO POST-TRANSLATIONAL PUMP ACTIVATION*

The plasma membrane H⁺-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H⁺/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H⁺-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H⁺-ATPases is labile in the basal state, which may provide an explanation for the low H⁺/ATP coupling ratio of these pumps in the basal state.     

Health benefits of microalgae and their microbiomes

Microalgae comprise a phylogenetically very diverse group of photosynthetic unicellular pro- and eukaryotic organisms growing in marine and other aquatic environments. While they are well explored for the generation of biofuels, their potential as a source of antimicrobial and prebiotic substances have recently received increasing interest. Within this framework, microalgae may offer solutions to the societal challenge we face, concerning the lack of antibiotics treating the growing level of antimicrobial resistant bacteria and fungi in clinical settings. While the vast majority of microalgae and their associated microbiota remain unstudied, they may be a fascinating and rewarding source for novel and more sustainable antimicrobials and alternative molecules and compounds. In this review, we present an overview of the current knowledge on health benefits of microalgae and their associated microbiota. Finally, we describe remaining issues and limitation, and suggest several promising research potentials that should be given attention.