Preparation and Properties of Recombinant Rat and Human Procholecystokinin57 –95

Recombinant human progastrin_6–80 binds two ferric ions with an apparent dissociation constant of 2.2 ± 0.1 μM [Baldwin (2004) Protein J 23:65–70]. The aims of the present study were to express fragments of recombinant procholecystokinin and to determine whether or not they bound ferric ions. Recombinant rat and human procholecystokinin_57–95 were expressed as glutathione S-transferase fusion proteins in E. coli. The fusion proteins were bound to glutathione-agarose, cleaved with thrombin, and purified by reverse phase HPLC. Recombinant procholecystokinin_57–95 did not bind to either the CCK1 or CCK2 receptor with high affinity. No change in absorption spectrum was observed on addition of ferric ions, and analysis of the quenching of tryptophan fluorescence observed in the presence of ferric ions indicated that binding to procholecystokinin_57–95 was at least 40–fold weaker than the binding of ferric ions to progastrin_6–80.     

VDAC activation by the 18 kDa translocator protein (TSPO), implications for apoptosis

The voltage dependent anion channel (VDAC), located in the outer mitochondrial membrane, functions as a major channel allowing passage of small molecules and ions between the mitochondrial inter-membrane space and cytoplasm. Together with the adenine nucleotide translocator (ANT), which is located in the inner mitochondrial membrane, the VDAC is considered to form the core of a mitochondrial multiprotein complex, named the mitochondrial permeability transition pore (MPTP). Both VDAC and ANT appear to take part in activation of the mitochondrial apoptosis pathway. Other proteins also appear to be associated with the MPTP, for example, the 18 kDa mitochondrial Translocator Protein (TSPO), Bcl-2, hexokinase, cyclophylin D, and others. Interactions between VDAC and TSPO are considered to play a role in apoptotic cell death. As a consequence, due to its apoptotic functions, the TSPO has become a target for drug development directed to find treatments for neurodegenerative diseases and cancer. In this context, TSPO appears to be involved in the generation of reactive oxygen species (ROS). This generation of ROS may provide a link between activation of TSPO and of VDAC, to induce activation of the mitochondrial apoptosis pathway. ROS are known to be able to release cytochrome c from cardiolipins located at the inner mitochondrial membrane. In addition, ROS appear to be able to activate VDAC and allow VDAC mediated release of cytochrome c into the cytosol. Release of cytochrome c from the mitochondria forms the initiating step for activation of the mitochondrial apoptosis pathway. These data provide an understanding regarding the mechanisms whereby VDAC and TSPO may serve as targets to modulate apoptotic rates. This has implications for drug design to treat diseases such as neurodegeneration and cancer.     

Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina

Two novel 21-residue antimicrobial peptides, arenicin-1 and arenicin-2, exhibiting activity against Gram-positive and Gram-negative bacteria and fungi, were purified from coelomocytes of marine polychaeta Arenicola marina (lugworm) by preparative gel electrophoresis and RP-HPLC. Molecular masses (2758.3 and 2772.3 Da) and complete amino acid sequences (RWCVYAYVRVRGVLVRYRRCW and RWCVYAYVRIRGVLVRYRRCW) were determined for each isoform. Each arenicin has one disulfide bond (Cys3-Cys20). The total RNA was isolated from the lugworm coelomocytes, RT-PCR and cloning were performed, and cDNA was sequenced. A 202-residue preproarenicin contains a putative signal peptide (25 amino acids) and a long prodomain. Arenicins have no structure similarity to any previously identified antimicrobial peptides.     

Genetic re-engineering of polyunsaturated phospholipid profile of Saccharomyces cerevisiae identifies a novel role for Cld1 in mitigating the effects of cardiolipin peroxidation

Cardiolipin (CL) is a unique phospholipid localized almost exclusively within the mitochondrial membranes where it is synthesized. Newly synthesized CL undergoes acyl remodeling to produce CL species enriched with unsaturated acyl groups. Cld1 is the only identified CL-specific phospholipase in yeast and is required to initiate the CL remodeling pathway. In higher eukaryotes, peroxidation of CL, yielding CL_OX, has been implicated in the cellular signaling events that initiate apoptosis. CL_OX can undergo enzymatic hydrolysis, resulting in the release of lipid mediators with signaling properties. Our previous findings suggested that CLD1 expression is upregulated in response to oxidative stress, and that one of the physiological roles of CL remodeling is to remove peroxidized CL. To exploit the powerful yeast model to study functions of CLD1 in CL peroxidation, we expressed the H. brasiliensis Δ¹²-desaturase gene in yeast, which then synthesized poly unsaturated fatty acids(PUFAs) that are incorporated into CL species. Using LC-MS based redox phospholipidomics, we identified and quantified the molecular species of CL and other phospholipids in cld1Δ vs. WT cells. Loss of CLD1 led to a dramatic decrease in chronological lifespan, mitochondrial membrane potential, and respiratory capacity; it also resulted in increased levels of mono-hydroperoxy-CLs, particularly among the highly unsaturated CL species, including tetralinoleoyl-CL. In addition, purified Cld1 exhibited a higher affinity for CL_OX, and treatment of cells with H₂O₂ increased CLD1 expression in the logarithmic growth phase. These data suggest that CLD1 expression is required to mitigate oxidative stress. The findings from this study contribute to our overall understanding of CL remodeling and its role in mitigating oxidative stress.     

<Emphasis Type="Italic">Proteinivorax hydrogeniformans</Emphasis> sp. nov., an anaerobic,haloalkaliphilic bacterium fermenting proteinaceous compounds with high hydrogen production

A search for the organisms responsible for the degradation of biomass of primary producers in Tanatar lakes resulted in the isolation of a novel anaerobic, haloalkaliphilic microorganism, strain Z-710^T. The strain grows on proteinaceous substrates (peptides) but not on proteins. A rather limited range of substances of other classes can be utilised together with tryptone but not individually. An interesting physiological feature of the novel strain is a high capacity for hydrogen production (up to 30% v/v) during proteolytic fermentation. Phylogenetic analysis based on the 16S rRNA gene sequence similarity revealed that the organism can be assigned to the previously described genus Proteinivorax. According to its physiological features and the low DNA–DNA hybridisation level of the strain with the type strain of the only previously described Proteinivorax species—Proteinivorax tanatarense Z-910^T—strain Z-710^T is described here as representing a novel species with the name Proteinivorax hydrogeniformans sp. nov. The type strain is Z-710^T (= DSM 102085^T = VKM B-3042^T).     

The evolutionary convergence of mid-Mesozoic lacewings and Cenozoic butterflies

Mid-Mesozoic kalligrammatid lacewings (Neuroptera) entered the fossil record 165 million years ago (Ma) and disappeared 45 Ma later. Extant papilionoid butterflies (Lepidoptera) probably originated 80–70 Ma, long after kalligrammatids became extinct. Although poor preservation of kalligrammatid fossils previously prevented their detailed morphological and ecological characterization, we examine new, well-preserved, kalligrammatid fossils from Middle Jurassic and Early Cretaceous sites in northeastern China to unravel a surprising array of similar morphological and ecological features in these two, unrelated clades. We used polarized light and epifluorescence photography, SEM imaging, energy dispersive spectrometry and time-of-flight secondary ion mass spectrometry to examine kalligrammatid fossils and their environment. We mapped the evolution of specific traits onto a kalligrammatid phylogeny and discovered that these extinct lacewings convergently evolved wing eyespots that possibly contained melanin, and wing scales, elongate tubular proboscides, similar feeding styles, and seed–plant associations, similar to butterflies. Long-proboscid kalligrammatid lacewings lived in ecosystems with gymnosperm–insect relationships and likely accessed bennettitalean pollination drops and pollen. This system later was replaced by mid-Cretaceous angiosperms and their insect pollinators.     

Membrane interactions of the hydrophobic segment of diacylglycerol kinase epsilon

Diacylglycerol kinase epsilon (DGKepsilon) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids as a putative membrane anchor. To model the conformation, and stoichiometry of this segment in membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of DGKepsilon of sequence KKKKLILWTLCSVLLPVFITFWKKKKK-NH(2). Flanking Lys residues mimic the natural setting of this peptide in DGKepsilon, while facilitating peptide synthesis and characterization. Circular dichroism and fluorescence spectroscopic analysis demonstrated that the peptide has increased helical content and significant blue shifts in the presence of anionic--but not zwitterionic--bilayer membranes. When labeled with fluorophores that can undergo fluorescence resonance energy transfer, the peptide was found to dimerize--a result also observed from migration rates on SDS-PAGE gels under both reducing and non-reducing disulfide bridge conditions. The peptide was shown to preferentially interact with cholesterol in lipid films comprised of homogeneous mixtures of cholesterol and phosphatidylcholine, yet the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. The peptide was also able to inhibit the activity of DGKepsilon protein in vitro. Our overall findings suggest that the peptide ultimately cannot leave the bulk water for attachment/insertion into the outer leaflet of an erythrocyte-like bilayer, yet its core sequence is sufficiently hydrophobic to insert into membrane core regions when membrane attachment is promoted by electrostatic attraction to anionic lipid head groups of the inner leaflet of an erythrocyte-like bilayer.