Conformational analysis in solution of gastrin releasing peptide

Gastrin releasing peptide (GRP) is the first peptide isolated from porcine gastric and intestinal tissues and is homologous to the carboxyl terminus of bombesin (Bn) isolated from the skin of the frog Bombina bombina. It is a member of the Bn-like peptides, which are important in numerous biological and pathological processes. The Bn-like peptides show high sequence homology in their C-terminal regions, but they have different selectivity for their receptors. In particular, GRP selectively binds to the GRP receptor (GRPR). However, the molecular basis for this selectivity remains largely unknown. Here, we report the three-dimensional structure of GRP. Hopefully, it could be helpful in a better understanding of the binding selectivity between GRP and GRPR.     

ABCA1 mediates high-affinity uptake of 25-hydroxycholesterol by membrane vesicles and rapid efflux of oxysterol by intact cells

ATP Binding Cassette (ABC) transporter, ABCA1, plays a pivotal role in reverse cholesterol transport by mediating the cellular efflux of phospholipid and cholesterol. Studies using intact cells strongly suggest that ABCA1 acts as a phospholipid floppase, but there has been no direct demonstration that the protein is a primary active sterol transporter. Using membrane vesicles from insect Sf21 cells, we found that ABCA1 mediated ATP-dependent uptake of [³H]25-hydroxycholesterol with an apparent K_m of 0.7 µM. Consistent with this high apparent affinity, expression of ABCA1 in human embryonic kidney cells both increased rapid efflux of 25-hydroxcholesterol and prevented oxysterol-mediated repression of low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase mRNAs. Comparison of wild-type and ABCA1^–/– murine fibroblasts indicates that 25-hydroxycholesterol is effluxed 5-fold more rapidly by wild-type cells. In addition, the rate of efflux from the wild-type but not the ABCA1^–/– fibroblasts is increased a further twofold by inducers of ABCA1 expression. Thus under the experimental conditions employed, endogenous ABCA1 is a major contributor to 25-hydroxycholesterol efflux from wild-type fibroblasts. Evidence from in vitro studies indicates that oxysterols are potent inducers of genes involved in cellular cholesterol efflux and metabolism, including the ABCA1 gene, and repressors of genes involved in cholesterol synthesis or uptake. Our observations raise the possibility that efflux of oxysterols by ABCA1 could contribute to a homeostatic mechanism, which both attenuates oxysterol-induced expression of its cognate gene and alleviates repression of genes encoding proteins, such as HMG-CoA reductase and LDL receptor. active transport; cholesterol homeostasis     

Arthropod phylogeny: onychophoran brain organization suggests an archaic relationship with a chelicerate stem lineage

Neuroanatomical studies have demonstrated that the architecture and organization among neuropils are highly conserved within any order of arthropods. The shapes of nerve cells and their neuropilar arrangements provide robust characters for phylogenetic analyses. Such analyses so far have agreed with molecular phylogenies in demonstrating that entomostracans+malacostracans belong to a clade (Tetraconata) that includes the hexapods. However, relationships among what are considered to be paraphyletic groups or among the stem arthropods have not yet been satisfactorily resolved. The present parsimony analyses of independent neuroarchitectural characters from 27 arthropods and lobopods demonstrate relationships that are congruent with phylogenies derived from molecular studies, except for the status of the Onychophora. The present account describes the brain of the onychophoran Euperipatoides rowelli, demonstrating that the structure and arrangements of its neurons, cerebral neuropils and sensory centres are distinct from arrangements in the brains of mandibulates. Neuroanatomical evidence suggests that the organization of the onychophoran brain is similar to that of the brains of chelicerates.     

Contrasting levels of genetic diversity between the historically rare orchid Cypripedium japonicum and the historically common orchid Cypripedium macranthos in South Korea

Many terrestrial orchids are historically rare and occur in small, spatially isolated populations. Theory predicts that such species will harbour low levels of genetic variation within populations and will exhibit a high degree of population genetic divergence, primarily as a result of genetic drift. If the origin of the present‐day populations is relatively recent from the same genetically depauperate source population, a complete lack of genetic differentiation between conspecific populations is expected. If a terrestrial orchid was historically common with moderate or high levels of genetic diversity, but has experienced more recent anthropogenic disturbance as a result of over‐collection, it would still exhibit initial levels of genetic variation within populations and a low degree of genetic divergence between populations. To test these predictions, we examined the genetic diversity in six populations (N = 131) of the historically and currently rare Cypripedium japonicum and in four populations (N = 94) of the historically common but now rare C. macranthos from South Korea. Fourteen putative allozyme loci resolved from eight enzyme systems revealed no variation either within or among populations of C. japonicum, which supports the first prediction. In contrast, populations of C. macranthos harboured high levels of genetic variation (mean percentage of polymorphic loci %P = 46.7; mean expected heterozygosity H_e = 0.185) and exhibited a low degree of population genetic divergence (G_ST = 0.059), supporting the second prediction. The lack of genetic variation both within and among conspecific populations of C. japonicum may suggest that populations originated from the same genetically depauperate ancestral population. The high levels of genetic diversity maintained in populations of C. macranthos suggest that the collection‐mediated decrease in the number of individuals is still too recent for long‐term effects on genetic variation. Based on current demographic and genetic data, in situ and ex situ conservation strategies should be provided to preserve genetic variation and to ensure the long‐term survival of the two species in the Korean Peninsula. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 119–129.     

Temperature and urea induced denaturation of the TRP‐cage mini protein TC5b: A simulation study consistent with experimental observations

The effects of temperature and urea denaturation (6M urea) on the dominant structures of the 20‐residue Trp‐cage mini‐protein TC5b are investigated by molecular dynamics simulations of the protein at different temperatures in aqueous and in 6M urea solution using explicit solvent degrees of freedom and the GROMOS force‐field parameter set 45A3. In aqueous solution at 278 K, TC5b is stable throughout the 20 ns of MD simulation and the trajectory structures largely agree with the NMR‐NOE atom–atom distance data available. Raising the temperature to 360 K and to 400 K, the protein denatures within 22 ns and 3 ns, showing that the denaturation temperature is well below 360 K using the GROMOS force field. This is 40–90 K lower than the denaturation temperatures observed in simulations using other much used protein force fields. As the experimental denaturation temperature is about 315 K, the GROMOS force field appears not to overstabilize TC5b, as other force fields and the use of continuum solvation models seem to do. This feature may directly stem from the GROMOS force‐field parameter calibration protocol, which primarily involves reproduction of condensed phase thermodynamic quantities such as energies, densities, and solvation free energies of small compounds representative for protein fragments. By adding 6M urea to the solution, the onset of denaturation is observed in the simulation, but is too slow to observe a particular side‐chain side‐chain contact (Trp6‐Ile4) that was experimentally observed to be characteristic for the denatured state. Interestingly, using temperature denaturation, the process is accelerated and the experimental data are reproduced.     

Sudden expansion of a single brown bear maternal lineage across northern continental Eurasia after the last ice age: a general demographic model for mammals?

The brown bear has proved a useful model for studying Late Quaternary mammalian phylogeography. However, information is lacking from northern continental Eurasia, which constitutes a large part of the species' current distribution. We analysed mitochondrial DNA sequences (totalling 1943 bp) from 205 bears from northeast Europe and Russia in order to characterize the maternal phylogeography of bears in this region. We also estimated the formation times of the sampled brown bear lineages and those of its extinct relative, the cave bear.
Four closely related haplogroups belonging to a single mitochondrial subclade were identified in northern continental Eurasia. Several haplotypes were found throughout the whole study area, while one haplogroup was restricted to Kamchatka. The haplotype network, estimated divergence times and various statistical tests indicated that bears in northern continental Eurasia recently underwent a sudden expansion, preceded by a severe bottleneck. This brown bear population was therefore most likely founded by a small number of bears that were restricted to a single refuge area during the last glacial maximum. This pattern has been described previously for other mammal species and as such may represent one general model for the phylogeography of Eurasian mammals. Bayesian divergence time estimates are presented for different brown and cave bear clades. Moreover, our results demonstrate the extent of substitution rate variation occurring throughout the phylogenetic tree, highlighting the need for appropriate calibration when estimating divergence times.     

Phylogeography of lions (Panthera leo ssp.) reveals three distinct taxa and a late Pleistocene reduction in genetic diversity

Lions were the most widespread carnivores in the late Pleistocene, ranging from southern Africa to the southern USA, but little is known about the evolutionary relationships among these Pleistocene populations or the dynamics that led to their extinction. Using ancient DNA techniques, we obtained mitochondrial sequences from 52 individuals sampled across the present and former range of lions. Phylogenetic analysis revealed three distinct clusters: (i) modern lions, Panthera leo ; (ii) extinct Pleistocene cave lions, which formed a homogeneous population extending from Europe across Beringia (Siberia, Alaska and western Canada); and (iii) extinct American lions, which formed a separate population south of the Pleistocene ice sheets. The American lion appears to have become genetically isolated around 340 000 years ago, despite the apparent lack of significant barriers to gene flow with Beringian populations through much of the late Pleistocene. We found potential evidence of a severe population bottleneck in the cave lion during the previous interstadial, sometime after 48 000 years, adding to evidence from bison, mammoths, horses and brown bears that megafaunal populations underwent major genetic alterations throughout the last interstadial, potentially presaging the processes involved in the subsequent end-Pleistocene mass extinctions.     

Expression of human interferon alpha-1 with enhanced stability via the tagging system of a stabilizing peptide

Human interferon α-1 (hIFNA1) is one of several interferon α subtypes that have been studied and commercialized to treat various viral diseases including hepatitis B and C as well as malignant melanoma. Protein aggregation has been problematic for every step in commercial production, from purification to the packaging and delivery of pharmaceutical proteins. In a previous study, we demonstrated that a stabilizing peptide from the C-terminal acidic tail of α-synuclein (ATS) could be used as an effective fusion tag to increase the stability of target proteins such as human growth hormone (hGH) and granulocyte colony-stimulating factor (G-CSF). In this study, we applied this ATS fusion system to hIFNA1 in order to protect against the aggregation of hIFNA1 by environmental stresses, since hIFNA1 aggregates elicit an undesirable immune response in humans. As expected, ATS-fused hIFNA1 (hIFNA1-ATS) protein showed enhanced stability against thermal stress, agitation stress, and repetitive freeze/thawing stress in comparison with native hIFNA1. More importantly, hIFNA1-ATS fusion protein appeared to be 1.6 times more active than hIFNA1 in a cell anti-proliferation assay. Furthermore, the solubility of hIFNA1-ATS appeared to be 1.7 times higher than that of native protein. Our results suggest that the ATS-tag system could be a useful means for protecting hIFNA1 protein from aggregation by various external stresses and could be used to increase the solubility of protein.     

Highly stable trypsin‐aggregate coatings on polymer nanofibers for repeated protein digestion

A stable and robust trypsin‐based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300‐fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC‐MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in “real‐world” proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.