The US National Seed Strategy for Rehabilitation and Restoration: progress and prospects.

The National Seed Strategy for Rehabilitation and Restoration was developed by a partnership of 12 federal agencies and over 300 non-federal cooperators in the United States and launched in 2015. Implementation aims to ensure the availability of genetically appropriate native seed for ecological restoration across the country. Ecological restoration is required in response to a wide range of human impacts. The four main goals of the strategy are: identification of seed needs and availability of genetically appropriate seed; research to improve seed production and ecosystem restoration; development of decision support tools for ecological restoration; and communication and outreach. With the increasing occurrence and intensity of extreme weather events including drought and related wild fires, hurricanes and flooding, native seed is increasingly required in large quantities to build ecological resilience. Acceptance of this need will be crucial to implementation of the National Seed Strategy.

An Experimental and Numerical Investigation of CO2 Distribution in the Upper Airways During Nasal High Flow Therapy.

Nasal high flow (NHF) therapy is used to treat a variety of respiratory disorders to improve patient oxygenation. A CO2 washout mechanism is believed to be responsible for the observed increase in oxygenation. In this study, experimentally validated Computational Fluid Dynamics simulations of the CO2 concentration within the upper airway during unassisted and NHF assisted breathing were undertaken with the aim of exploring the existence of this washout mechanism. An anatomically accurate nasal cavity model was generated from a CT scan and breathing was reproduced using a Fourier decomposition of a physiologically measured breath waveform. Time dependent CO2 profiles were obtained at the entrance of the trachea in the experimental model, and were used as simulation boundary conditions. Flow recirculation features were observed in the anterior portion of the nasal cavity upon application of the therapy. This causes the CO2 rich gas to vent from the nostrils reducing the CO2 concentration in the dead space and lowering the inspired CO2 volume. Increasing therapy flow rate increases the penetration depth within the nasal cavity of the low CO2 concentration gas. A 65% decrease in inspired CO2 was observed for therapy flow rates ranging from 0 to 60 L min(-1) supporting the washout mechanism theory.

Morphine-induced internalization of the L83I mutant of the rat µ-opioid receptor.

BACKGROUND AND PURPOSE: Naturally occurring single-nucleotide polymorphisms (SNPs) within GPCRs can result in alterations in various pharmacological parameters. Understanding the regulation and function of endocytic trafficking of the µ-opioid receptor (MOP receptor) is of great importance given its implication in the development of opioid tolerance. This study has compared the agonist-dependent trafficking and signalling of L83I, the rat orthologue of a naturally occurring variant of the MOP receptor. EXPERIMENTAL APPROACH: Cell surface elisa, confocal microscopy and immunoprecipitation assays were used to characterize the trafficking properties of the MOP-L83I variant in comparison with the wild-type receptor in HEK 293 cells. Functional assays were used to compare the ability of the L83I variant to signal to several downstream pathways. KEY RESULTS: Morphine-induced internalization of the L83I MOP receptor was markedly increased in comparison with the wild-type receptor. The altered trafficking of this variant was found to be specific to morphine and was both G-protein receptor kinase- and dynamin-dependent. The enhanced internalization of L83I variant in response to morphine was not due to increased phosphorylation of serine 375, arrestin association or an increased ability to signal. CONCLUSIONS AND IMPLICATIONS: These results suggest that morphine promotes a specific conformation of the L83I variant that makes it more liable to internalize in response to morphine, unlike the wild-type receptor that undergoes significantly less morphine-stimulated internalization, providing an example of a ligand-selective biased receptor. The presence of this SNP within an individual may consequently affect the development of tolerance and analgesic responses. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Adverse experience during early life and adulthood interact to elevate tph2 mRNA expression in serotonergic neurons within the dorsal raphe nucleus.

Anxiety disorders, depression and animal models of vulnerability to a depression-like syndrome have been associated with dysregulation of brain serotonergic systems. These effects could result from genetic influences, adverse early life experiences (ELE), or acute stressful life events, all of which can alter serotonergic neurotransmission and have been implicated in determining vulnerability to neuropsychiatric disorders. To evaluate the effects of ELE, adverse experiences during adulthood, and potential interactions between these factors on neuronal tryptophan hydroxylase 2 (tph2) mRNA expression, we investigated in rats the effects of maternal separation (MS)(separation from the dam for 180 min/day from postnatal day 2-14; MS180, a model of vulnerability to a depression-like syndrome), neonatal handling (separation from the dam for 15 min/day from postnatal day 2-14; MS15, a model of decreased stress sensitivity), or normal animal facility rearing (AFR) control conditions, with or without subsequent exposure to adult social defeat, on tph2 mRNA expression in the dorsal raphe nucleus (DR). Among rats exposed to social defeat, MS180 rats had increased tph2 mRNA expression in the DR, while MS15 rats had decreased tph2 mRNA expression compared to AFR rats. Social defeat increased tph2 mRNA expression, but only in MS180 rats and only in the "lateral wings" of the DR, a subdivision of the DR that is part of a sympathomotor command center. Overall, these data demonstrate that ELE and stressful experience during adulthood interact to determine tph2 mRNA expression. These changes in tph2 mRNA expression represent a potential mechanism through which adverse ELEs and stressful life experiences during adulthood may interact to increase vulnerability to stress-related psychiatric disease.

Involvement of PKC alpha and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of mu-opioid receptors in mature brain neurons.

BACKGROUND AND PURPOSE: The ability of an agonist to induce desensitization of the mu-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist). EXPERIMENTAL APPROACH: MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)alpha knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs). KEY RESULTS: Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCalpha, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCalpha knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM. CONCLUSIONS AND IMPLICATIONS: In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCalpha-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.

Lubrication regime of the contact between fat and bone in bovine tissue.

Fat pads are masses of encapsulated adipose tissue located throughout the human body. Whilst a number of studies describe these soft tissues anatomically little is known about their biomechanics, and surgeons may excise them arthroscopically if they hinder visual inspection of the joint or bursa. By measuring the coefficient of friction between, and performing Sommerfeld analysis of, the surfaces approximating the in vivo conjuncture, this contact has been shown to have a coefficient of friction of the order of 0.01. The system appears to be lubricated hydrodynamically, thus possibly promoting low levels of wear. It is suggested that one of the functions of fat pads associated with subtendinous bursae and synovial joints is to generate a hydrodynamic lubricating layer between the opposing surfaces.

The development of synovial joints.

During vertebrate evolution, successful adaptation of animal limbs to a variety of ecological niches depended largely on the formation and positioning of synovial joints. The function of a joint is to allow smooth articulation between opposing skeletal elements and to transmit biomechanical loads through the structure, and this is achieved through covering the ends of bones with articular cartilage, lubricating the joint with synovial fluid, using ligaments to bind the skeletal elements together, and encapsulating the joint in a protective fibrous layer of tissue. The diversity of limb generation has been proposed to occur through sequential branching and segmentation of precartilaginous skeletal elements along the proximodistal axis of the limb. The position of future joints is first delimited by areas of higher cell density called interzones initially through an as yet unidentified inductive signal, subsequently specification of these regions is controlled hierarchically by wnt14 and gdf5, respectively. Joint-forming cell fate although specified is not fixed, and joints will fuse if growth factor signaling is perturbed. Cavitation, the separation of the two opposing skeletal elements, and joint morphogenesis, the process whereby the joint cells organize and mature to establish a functional interlocking and reciprocally shaped joint, are slowly being unraveled through studying the plethora of molecules that make up the unique extracellular matrix of the forming structure. The joint lining tissue, articular cartilage, is avascular, and this limits its reparative capacity such that arthritis and associated joint pathologies are the single largest cause of disability in the adult population. Recent discoveries of adult stem cells and more specifically the isolation of chondroprogenitor cells from articular cartilage are extending available therapeutic options, though only with a more complete understanding of synovial joint development can such options have greater chances of success.

Current strategies for articular cartilage repair.

Defects of articular cartilage that do not penetrate to the subchondral bone fail to heal spontaneously. Defects that penetrate to the subchondral bone elicit an intrinsic repair response that yields a fibrocartilaginous repair tissue which is a poor substitute for hyaline articular cartilage. Many arthroscopic repair strategies employed utilise this intrinsic repair response to induce the formation of a repair tissue within the defect. The goal, however, is to produce a repair tissue that has the same functional and mechanical properties of hyaline articular cartilage. To this end, autologous osteochondral transfer can provide symptomatic relief. This technique involves the excision of healthy cartilage plugs from 'non-load bearing' regions of the joint for implantation into the defect. Cell based transplantation methods currently involve the transplantation of expanded autologous chondrocytes to the defects to form a repair tissue. This technique again involves the excision of healthy cartilage from the joint for expansion. Current research is exploring the potential use of mesenchymal stem cells as a source for tissue engineering, as well as the combination of cells with biodegradable scaffolds. Although current repair strategies improve joint function, further research is required to prevent future degeneration of repair tissue.

Screening ABCG1, the human homologue of the Drosophila white gene, for polymorphisms and association with bipolar affective disorder.

ABCG1 encodes a transporter protein that may be involved in the cellular uptake of tryptophan. Tryptophan is the precursor for serotonin, which is implicated in the regulation of mood. The gene maps to chromosome 21q22.3, a region implicated in bipolar disorder I (BPI) in genetic linkage studies. ABCG1 is thus a suitable candidate gene for study in BP1. We screened all 15 exons and 700 bases of the 5' flanking region of ABCG1 for mutations, using Denaturing High Performance Liquid Chromatography (DHPLC). A total of 13 single nucleotide polymorphisms (SNPs) were identified. Ten of the SNPs were intronic, two lie within the 5' flanking region and one within the 3' UTR. We identified a GCC repeat within Exon 1 and two novel intronic VNTRs. Eight of the SNPs, the two VNTRs, the GCC repeat and two known microsatellite markers within the gene were tested for association with BP1 in a sample of 110 parent-offspring trios using the Extended Transmission Disequilibrium Test (ETDT). No alleles or haplotypes were significantly preferentially transmitted from parents to affected offspring. However, the trend for preferential transmission of markers in the 3'UTR is in the same direction as in a previous report for association with mood and panic disorders and therefore warrants attempts at replication. Marker-to-marker linkage disequilibrium (LD) showed that strong LD was present over relatively short distances of up to 20 kb and was present for SNPs as well as for polymorphic repeats. The polymorphisms identified in this study will be useful in examining the role of this gene in other neuropsychiatric disorders and behavioural traits.

Identification of a prostaglandin E2 receptor splice variant and its expression in rat tissues.

The intercellular signalling actions of the lipid mediators, the eicosanoids, are transduced by a family of seven transmembrane domain receptors. Members of this receptor family with high affinity for PGE2 are termed EP receptors. There are four known EP receptor genes that are transcribed to generate EP1, EP2, EP3 and EP4 receptors. Two of these receptor transcripts, EP1 and EP3, are further modified by RNA splicing to give multiple receptor isoforms. The EP3 receptor is known to have multiple splice variants in human (9 variants), cow (4 variants), mouse (3 variants) and rat (3 variants). In the rat the three EP3 splice variants differ in the sequence of the intracellular C-terminus. We have identified a fourth splice variant of the rat prostaglandin EP3 receptor that has a greatly truncated intracellular C-terminus when compared to the other EP3 receptor isoforms. Using nested RT-PCR we have shown that this novel splice variant is strongly expressed in rat brain and is also found in spinal cord, kidney and spleen.

Expression and regulation of prostaglandin E receptor subtype mRNAs in rat sensory ganglia and spinal cord in response to peripheral inflammation.

Prostaglandins are known to act via seven transmembrane domain receptors to exert actions on both peripheral and central neurons resulting in changes in neuronal excitability. Prostaglandin E2, the prostaglandin most often associated with inflammation, itself acts on a family of closely related receptors, the EP receptors. Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), we have shown that rat primary afferent neurons express the mRNA for all EP receptor subtypes, and that some, but not all EP receptor subtype mRNAs are down-regulated in sensory neurons in response to an acute peripheral inflammation. We also show for the first time that all EP receptor subtype mRNAs are expressed in rat lumbar spinal cord. Spinal cord EP receptor subtype mRNAs are also regulated in acute inflammation in a pattern distinct from the changes seen in sensory ganglia in response to the same inflammatory stimulus.

Parent-child patterns of coping.

Coping of parents and children in a children's surgical area, where members had the same or different coping styles, was investigated using self-report and observation. Differences in effectiveness of coping strategies were found between groups of children, mainly in relation to emotion-focused coping. Parent groups were mainly distinguished from each other by the reported effectiveness of their spiritual and distracting strategies. Observational data showed group differences in patterns of parent-child interaction relating to coping behaviours prior to surgery. The findings indicate reciprocal parent-child influences of coping strategies and behaviours.

Aurally and visually guided visual search in a virtual environment.

We investigated the time participants took to perform a visual search task for targets outside the visual field of view using a helmet-mounted display. We also measured the effectiveness of visual and auditory cues to target location. The auditory stimuli used to cue location were noise bursts previously recorded from the ear canals of the participants and were either presented briefly at the beginning of a trial or continually updated to compensate for head movements. The visual cue was a dynamic arrow that indicated the direction and angular distance from the instantaneous head position to the target. Both visual and auditory spatial cues reduced search time dramatically, compared with unaided search. The updating audio cue was more effective than the transient audio cue and was as effective as the visual cue in reducing search time. These data show that both spatial auditory and visual cues can markedly improve visual search performance. Potential applications for this research include highly visual environments, such as aviation, where there is risk of overloading the visual modality with information.

Phase II study of dexverapamil plus anthracycline in patients with metastatic breast cancer who have progressed on the same anthracycline regimen.

The purpose of this study is to evaluate whether metastatic breast cancer that has progressed on an anthracycline-containing drug regimen will subsequently respond to that identical regimen if dexverapamil, a modulator of P-glycoprotein-mediated drug resistance, is given concomitantly. Eligible patients received 180 mg/m2 dexverapamil every 6 h for 15 doses with the anthracycline administered 30 min after the seventh dose. Blood for dexverapamil levels was drawn before and 30 min after this dose. When possible, biopsies were obtained to measure mdr-1 expression by reverse transcription-PCR and by image cytometry. Of the 21 patients entered onto the trial, 20 were evaluable for response. There were two partial responses (10%) that both lasted for 6 months, and two additional patients had stable disease. Seven patients had asymptomatic cardiotoxicity consisting of hypotension (24%), bradycardia (5%), or prolongation of the P-R interval (14%). Two patients developed acute congestive heart failure, one on dexverapamil and one 10 days after stopping it. Dexverapamil did not seem to increase anthracycline toxicity. The median trough dexverapamil plus norverapamil level on day 3 was 1110 ng/ml (range, 186-3385 ng/ml), and the median peak level was 2164 ng/ml (range, 964-8382 ng/ml). There was poor correlation between reverse transcription-PCR and image cytometry for the level of mdr-1 expression. Because dexverapamil has been shown to affect doxorubicin pharmacokinetics subsequent to the initiation of this trial, it cannot be concluded that the responses seen were necessarily due to P-glycoprotein inhibition. Additional studies are necessary to determine whether mdr-1 modulators can reverse clinical drug resistance in breast cancer patients. The intrinsic cardiotoxicity of dexverapamil makes it less suitable for such studies than several other available agents.

Use of monoclonal antibodies to study the structure and function of eukaryotic protein synthesis initiation factor eIF-2B.

The eukaryotic protein synthesis initiation factor, eIF-2B, is a multimeric protein of five different subunits termed alpha, beta, gamma, delta and epsilon, which facilitates recycling of a further factor, eIF-2, and is an important control point in the initiation process. In order to investigate the structure and function of eIF-2B, monoclonal antibodies have been prepared to the beta, delta and epsilon subunits of the factor from rabbit reticulocytes. All three antibodies are active in Western blotting, ELISA and immunoprecipitation. The anti-epsilon antibody inhibits both the guanine nucleotide exchange activity of eIF-2B and protein synthesis in the rabbit reticulocyte lysate at the level of initiation. The other two antibodies do not inhibit either guanine nucleotide exchange or protein synthesis. The monoclonal antibodies and a polyclonal anti-(rabbit reticulocyte eIF-2B) serum were used to investigate the subunit size and the antigenic structure of eIF-2B from a variety of rabbit tissues and from a variety of mammalian species. eIF-2B from all rabbit tissues tested was indistinguishable from that prepared from rabbit reticulocytes. Quantitative studies showed substantial variation in the relative concentrations of eIF-2 and eIF-2B between different rabbit tissues. Marked variation in both the sizes of the subunits and their reaction with the antibodies was observed between eIF-2B from rabbit, rat, guinea pig and man.

The role of the beta-subunit of initiation factor eIF-2 in initiation complex formation.

The functional properties of preparations of protein synthesis initiation factor eIF-2 which lack the beta-subunit (as confirmed immunologically) were compared with those of the heterotrimeric factor. The former can bind guanine nucleotides but not initiator tRNA, and also exhibits a substantially reduced rate of initiation factor eIF-2B-mediated GDP/GTP-exchange.

Phosphorylation of elongation factor-2 from the lepidopteran insect, Spodoptera frugiperda.

In mammalian cells, protein synthesis can be regulated at the level of elongation by the phosphorylation of elongation factor 2 (eEF-2) by a highly specific Ca2+/calmodulin-dependent kinase. In this report, we show that eEF-2 from a cell line derived from the insect, Spodoptera frugiperda, is a substrate for mammalian eEF-2 kinase and that phosphorylation is Ca(2+)-dependent. Furthermore, two-dimensional peptide mapping shows that the kinase phosphorylates the same sites in Spodoptera eEF-2 as those phosphorylated in the rabbit protein. However, we were unable to detect an eEF-2 kinase in Spodoptera cells.

Purification and characterisation of an initiation-factor-2 kinase from uninduced mouse erythroleukaemia cells.

Mouse erythroleukaemia (MEL) cells, which have not been induced into erythroid development, contain a protein kinase (MKu) which phosphorylates the alpha subunit of protein-synthesis-initiation factor 2 (eIF-2 alpha). In this paper, we show that this kinase phosphorylates both eIF-2 alpha and a synthetic peptide based on the phosphorylation site in eIF-2 alpha at Ser51, the target residue for other eIF-2 alpha kinases. Consistent with this, prior treatment of eIF-2 with MKu impaired the exchange of bound GDP for GTP which is catalysed by the exchange factor eIF-2B. Using a modified cell-free translation system, we have shown that MKu inhibits translation, consistent with the above observations concerning the site of phosphorylation and the effect of phosphorylation on eIF-2B-mediated guanine-nucleotide exchange. MKu has been purified and its properties have been compared with those of the haem-controlled repressor eIF-2 alpha kinase (HCR) from rabbit reticulocytes. Its behaviour on gel filtration is similar to that of HCR, while its behaviour on anion exchange resembles that of certain phosphorylated species of HCR. Highly purified preparations of MKu contain a protein with an apparent molecular mass of 98 kDa which comigrates with HCR on SDS/PAGE. This protein undergoes phosphorylation when incubated in the presence of Mg(2+)-ATP, and both this apparent autophosphorylation and the activity of the kinase against eIF-2 alpha are inhibited by the same, low, (10 microM) concentrations of haemin. Phosphorylation of the 98-kDa components present in the MEL-cell kinase preparation and in purified rabbit reticulocyte HCR occurs on serine and threonine residues. Analysis of these phosphoproteins by peptide mapping reveals significant differences in their structures, indicating that they may be closely related, but are certainly not identical.

Purification, phosphorylation and control of the guanine-nucleotide-exchange factor from rabbit reticulocyte lysates.

A simple, improved procedure for the isolation of guanine-nucleotide-exchange factor (GEF) and for eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocyte lysates has been developed using ion-exchange chromatography on S-Sepharose, Q-Sepharose, Mono Q and Mono S. The majority of the eIF-2 is separated from GEF at an early stage in the procedure and the remaining small amount of eIF-2.GEF complex is separated from the bulk of the GEF by FPLC on Mono S. The procedure yields approximately 2 mg each of eIF-2 and GEF, of 90% and greater than 80% purity, respectively, from the blood of ten rabbits. All fractions of purified GEF contain four subunits of molecular masses 84, 66, 54 and 39 kDa, with various amounts of a fifth, 30-kDa subunit. The modulation of GEF activity was investigated using the highly purified factor in a guanine-nucleotide-exchange assay. The activity of GEF was stimulated by physiological concentrations of the polyamines, spermine and spermidine, but was unaffected by another polycationic compound, polylysine. Activity was also found to be inhibited by 1 mM NADP+ or NAD+, and this inhibition was overcome by the presence of 1 mM NADPH. Stoichiometric amounts of GEF were unable to release GDP from eIF-2.GDP complexes in the absence of free guanine nucleotides, suggesting that GEF operates by a ternary-complex mechanism. Casein kinase 1 or casein kinase 2 can each phosphorylate the largest subunit (84 kDa) of GEF. These enzymes both phosphorylate serine residues in GEF but they phosphorylate distinct sites, as demonstrated by phosphopeptide mapping following proteolytic or cyanogen bromide digestion. Neither of these kinases phosphorylated any of the other subunits of GEF to any significant extent and several other kinases were inactive against GEF. No effect of phosphorylation on activity could be demonstrated.

Expression of the outer capsid protein VP5 of two bluetongue viruses, and synthesis of chimeric double-shelled virus-like particles using combinations of recombinant baculoviruses.

We have previously reported the assembly of virus-like particles (VLPs), consisting of the four major structural proteins of bluetongue virus (BTV), in Spodoptera frugiperda cells coinfected with recombinant baculoviruses (French et al. (1990). J. Virol. 64, 5695-5700). In this paper we report further studies using this system to assemble heterologous VLPs containing the outer capsid proteins (VP2 and VP5) of a range of different BTV serotypes. S. frugiperda cells were coinfected with three recombinant baculoviruses; a dual recombinant expressing VP3 and VP7 (of BTV-17 and -10, respectively) in combination with a single recombinant expressing VP2 of BTV-1, -2, -10, -11, 13, or -17 and an additional single recombinant expressing VP5 of BTV-2, BTV-10, or BTV-13. The resultant VLPs were purified and analyzed by electronmicroscopy, Western immunoblotting, and hemagglutination assays to determine whether double-shelled VLPs had been assembled. In the course of these experiments the VP2 proteins of all six available serotypes were successfully incorporated into VLPs. Particles from two different combinations of chimeric VLPs (having VP2 derived from BTV-1 or that of BTV-17) were used to raise antisera in guinea pigs. Both of these sera showed high neutralizing antibody titers against live BTV, indicating that heterologous VLPs may have potential for use in anti-BTV vaccines.