Identification of a rare subset of adipose tissue-resident progenitor cells, which express CD133 and TRPC3 as a VEGF-regulated Ca2+ entry channel

VEGF-induced Ca2+ signalling was investigated in CD133+/VEGFR-2+ progenitor cells isolated from human adipose stroma. Colonies derived from CD133+ immunoselected cells displayed inhomogenous Ca2+ signals, with variable magnitude of VEGF-induced Ca2+ entry, which positively correlated with expression of the Ca2+ channel protein TRPC3. High levels of VEGF-induced Ca2+ entry and TRPC3 expression were preferentially detected in rim areas of expanding colonies. Dominant negative suppression of TRPC3 inhibited VEGF-induced Ca2+ entry into CD133+ cells. Our results identify TRPC3 as a key Ca2+ entry channel in a subset of CD133+ stem cells. We suggest TRPC3 as an essential determinant of cell fate in CD133+ progenitor-derived colonies.     

Breeding system,pollination and demography in the rare granite endemic shrub Verticordia staminosa ssp. staminosa in south‐west Western Australia

Abstract Verticordia staminosa C. Gardner & A. S. George staminosa (Myrtaceae) is a rare granite endemic shrub that grows in discrete subpopulations on one isolated rock outcrop in a remnant of native vegetation in the Western Australian wheat belt. Key considerations in assessing the risk of extinction for rare plant species in fragmented landscapes are the reproductive dependence on a pollinator, breeding system, importance of seeds in demography, and regeneration niche. The present study determined the extent to which these factors constrain population growth in V. staminosa ssp. staminosa. Measurements across nine subpopulations on the breeding system, pollinator activity, rates of flowering, pollination and seed production, seedling demography, mature plant mortality and size‐class structure were undertaken over three consecutive years. The study species has a mixed mating system with similar rates of pollen tube development and fertilization observed in self‐, cross‐ and open‐pollinated flowers. Floral morphology, orientation and the concentration and volume of nectar produced suggest some degree of specialization associated with pollination by birds, which were occasionally seen visiting flowers. However, feral honey bees were the most commonly observed flower visitor and they seem to have replaced honeyeaters as the primary pollinator. Honey‐bee abundance increased with subpopulation size. However, rates of pollination and the subsequent proportion of flowers that produced viable seeds were independent of subpopulation size. Germination and seedling emergence occurred each winter but were greatest in the wettest winter. Recruitment was heavily biased towards individuals growing in or over cracks/fissures in the rock. Over the 3‐year study, recruitment exceeded mortality. A relatively unspecialized flower and mixed mating system have buffered the taxon against the effects of pollinator disruption. Seed production does not constrain population growth. The environmental variables of climate and suitable establishment crevices appear to be the major constraints to population growth.     

BioJava: an open-source framework for bioinformatics

SUMMARY: BioJava is a mature open-source project that provides a framework for processing of biological data. BioJava contains powerful analysis and statistical routines, tools for parsing common file formats and packages for manipulating sequences and 3D structures. It enables rapid bioinformatics application development in the Java programming language. AVAILABILITY: BioJava is an open-source project distributed under the Lesser GPL (LGPL). BioJava can be downloaded from the BioJava website (http://www.biojava.org). BioJava requires Java 1.5 or higher. All queries should be directed to the BioJava mailing lists. Details are available at http://biojava.org/wiki/BioJava:MailingLists.     

Heme oxygenase-1 prevents smoke induced B-cell infiltrates: a role for regulatory T cells?

Background

Smoking is the most important cause for the development of COPD. Since not all smokers develop COPD, it is obvious that other factors must be involved in disease development. We hypothesize that heme oxygenase-1 (HO-1), a protective enzyme against oxidative stress and inflammation, is insufficiently upregulated in COPD.The effects of HO-1 modulation on cigarette smoke induced inflammation and emphysema were tested in a smoking mouse model.

Methods

Mice were either exposed or sham exposed to cigarette smoke exposure for 20 weeks. Cobalt protoporphyrin or tin protoporphyrin was injected during this period to induce or inhibit HO-1 activity, respectively. Afterwards, emphysema development, levels of inflammatory cells and cytokines, and the presence of B-cell infiltrates in lung tissue were analyzed.

Results

Smoke exposure induced emphysema and increased the numbers of inflammatory cells and numbers of B-cell infiltrates, as well as the levels of inflammatory cytokines in lung tissue. HO-1 modulation had no effects on smoke induced emphysema development, or the increases in neutrophils and macrophages and inflammatory cytokines. Interestingly, HO-1 induction prevented the development of smoke induced B-cell infiltrates and increased the levels of CD4⁺CD25⁺ T cells and Foxp3 positive cells in the lungs. Additionally, the CD4⁺CD25⁺ T cells correlated positively with the number of Foxp3 positive cells in lung tissue, indicating that these cells were regulatory T cells.

Conclusion

These results support the concept that HO-1 expression influences regulatory T cells and indicates that this mechanism is involved in the suppression of smoke induced B-cell infiltrates. The translation of this interaction to human COPD should now be pursued.     

A hybrid method for peptide identification using integer linear optimization, local database search, and quadrupole time-of-flight or OrbiTrap tandem mass spectrometry

A novel hybrid methodology for the automated identification of peptides via de novo integer linear optimization, local database search, and tandem mass spectrometry is presented in this article. A modified version of the de novo identification algorithm PILOT, is utilized to construct accurate de novo peptide sequences. A modified version of the local database search tool FASTA is used to query these de novo predictions against the nonredundant protein database to resolve any low-confidence amino acids in the candidate sequences. The computational burden associated with performing several alignments is alleviated with the use of distributive computing. Extensive computational studies are presented for this new hybrid methodology, as well as comparisons with MASCOT for a set of 38 quadrupole time-of-flight (QTOF) and 380 OrbiTrap tandem mass spectra. The results for our proposed hybrid method for the OrbiTrap spectra are also compared with a modified version of PepNovo, which was trained for use on high-precision tandem mass spectra, and the tag-based method InsPecT. The de novo sequences of PILOT and PepNovo are also searched against the nonredundant protein database using CIDentify to compare with the alignments achieved by our modifications of FASTA. The comparative studies demonstrate the excellent peptide identification accuracy gained from combining the strengths of our de novo method, which is based on integer linear optimization, and database driven search methods.     

Neurosteroids in the fetus and neonate: potential protective role in compromised pregnancies

Complications during pregnancy and birth asphyxia lead to brain injury, with devastating consequences for the neonate. In this paper we present evidence that the steroid environment during pregnancy and at birth aids in protecting the fetus and neonate from asphyxia-induced injury. Earlier studies show that the placental progesterone production has a role in the synthesis and release of neuroactive steroids or their precursors into the fetal circulation. Placental precursor support leads to remarkably high concentrations of allopregnanolone in the fetal brain and to a dramatic decline with the loss of the placenta at birth. These elevated concentrations influence the distinct behavioral states displayed by the late gestation fetus and exert a suppressive effect that maintains sleep-like behavioral states that are present for much of fetal life. This suppression reduces CNS excitability and suppresses excitotoxicity. With the availability of adequate precursors, mechanisms within the fetal brain ultimately control neurosteroid levels. These mechanisms respond to episodes of acute hypoxia by increasing expression of 5alpha-reductase and P450scc enzymes and allopregnanolone synthesis in the brain. This allopregnanolone response, and potentially that of other neurosteroids including 5alpha-tetrahydrodeoxycorticosterone (TH-DOC), reduces hippocampal cell death following acute asphyxia and suggests that stimulation of neurosteroid production may protect the fetal brain. Importantly, inhibition of neurosteroid synthesis in the fetal brain increases the basal cell death suggesting a role in controlling developmental processes late in gestation. Synthesis of neurosteroid precursors in the fetal adrenal such as deoxycorticosterone (DOC), and their conversion to active neurosteroids in the fetal brain may also have a role in neuroprotection. This suggests that the adrenal glands provide precursor DOC for neurosteroid synthesis after birth and this may lead to a switch from allopregnanolone alone to neuroprotection mediated by allopregnanolone and TH-DOC.     

The role of contact chemoreception in egg-laying behaviour of locusts

Following selection of an appropriate egg-laying site desert locusts lay their eggs at depths in soil by digging their abdomen into the substrate using rhythmic movements of their abdomen and hard, sclerotised ovipositor valves. We have analysed the role of contact chemoreception on egg-laying behaviour and on the rhythmic digging movements of the valves. All chemicals tested acted aversively and reduced both the duration spent egg-laying and the number of eggs laid, with the concentration at which they became aversive being dependent on whether the chemical was normally present in the diet. Chemicals such as sucrose and a lysine glutamate salt prevented egg-laying only at much higher concentrations than known anti-feedants such as nicotine hydrogen tartrate and hydroquinine. Similarly for animals in which fictive digging movements were induced all chemicals stopped the digging rhythm, with sucrose and sodium chloride inhibiting the rhythm at relatively high concentrations compared to NHT and hydroquinone. We conclude that for both egg-laying behaviour and rhythmic digging that the aversiveness of a chemical rather than its identity per se plays a major role in regulating behaviour.     

A catalytic loop within Pseudomonas aeruginosa exotoxin A modulates its transferase activity.

Mutagenesis techniques were used to replace two loop regions within the catalytic domain of Pseudomonas aeruginosa exotoxin A (ETA) with functionally silent polyglycine loops. The loop mutant proteins, designated polyglycine Loops N and C, were both less active than the wild-type enzyme. However, the polyglycine Loop C mutant protein, replaced with the Gly(483)-Gly(490) loop, showed a much greater loss of enzymatic activity than the polyglycine Loop N protein. The former mutant enzyme exhibited an 18,000-fold decrease in catalytic turnover number (k(cat)), with only a marginal effect on the K(m) value for NAD(+) and the eukaryotic elongation factor-2 binding constant. Furthermore, alanine-scanning mutagenesis of this active-site loop region revealed the specific pattern of a critical region for enzymatic activity. Binding and kinetic data suggest that this loop modulates the transferase activity between ETA and eukaryotic elongation factor-2 and may be responsible for stabilization of the transition state for the reaction. Sequence alignment and molecular modeling also identified a similar loop within diphtheria toxin, a functionally and structurally related class A-B toxin. Based on these results and the similarities between ETA and diphtheria toxin, we propose that this catalytic subregion represents the first report of a diphthamide-specific ribosyltransferase structural motif. We expect these findings to further the development of pharmaceuticals designed to prevent ETA toxicity by disrupting the stabilization of the transition state during the ADP-ribose transfer event.     

Involvement of the amino-terminal beta-hairpin of the Aspergillus ribotoxins on the interaction with membranes and nonspecific ribonuclease activity

Ribotoxins are a family of potent cytotoxic proteins from Aspergillus whose members display a high sequence identity (85% for about 150 amino acid residues). The three-dimensional structures of two of these proteins, alpha-sarcin and restrictocin, are known. They interact with phospholipid bilayers, according to their ability to enter cells, and cleave a specific phosphodiester bond in the large subunit of ribosome thus inhibiting protein biosynthesis. Two nonconservative sequence changes between these proteins are located at the amino-terminal beta-hairpin of alpha-sarcin, a characteristic structure that is absent in other nontoxic structurally related microbial RNases. These two residues of alpha-sarcin, Lys 11 and Thr 20, have been substituted with the equivalent amino acids in restrictocin. The single mutants (K11L and T20D) and the corresponding K11L/T20D double mutant have been produced in Escherichia coli and purified to homogeneity. The spectroscopic characterization of the purified proteins reveals that the overall native structure is preserved. The ribonuclease and lipid-perturbing activities of the three mutants and restrictocin have been evaluated and compared with those of alpha-sarcin. These proteins exhibit the same ability to specifically inactivate ribosomes, although they show different activity against nonspecific substrate analogs such as poly(A). The mutant variant K11L and restrictocin display a lower phospholipid-interacting ability correlated with a decreased cytotoxicity. The results obtained are interpreted in terms of the involvement of the amino-terminal beta-hairpin in the interaction with both membranes and polyadenylic acid.