A discrete stage of baculovirus GP64-mediated membrane fusion

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH-induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.     

Diversity of mycorrhizal fungi of terrestrial orchids: compatibility webs, brief encounters, lasting relationships and alien invasions

The diversity of mycorrhizal fungi associated with an introduced weed-like South African orchid (Disa bracteata) and a disturbance-intolerant, widespread, native West Australian orchid (Pyrorchis nigricans) were compared by molecular identification of the fungi isolated from single pelotons. Molecular identification revealed both orchids were associated with fungi from diverse groups in the Rhizoctonia complex with worldwide distribution. Symbiotic germination assays confirmed the majority of fungi isolated from pelotons were mycorrhizal and a factorial experiment uncovered complex webs of compatibility between six terrestrial orchids and 12 fungi from Australia and South Africa. Two weed-like (disturbance-tolerant rapidly spreading) orchids — D. bracteata and the indigenous Australian Microtis media, had the broadest webs of mycorrhizal fungi. In contrast, other native orchids had relatively small webs of fungi (Diuris magnifica and Thelymitra crinita), or germinated exclusively with their own fungus (Caladenia falcata and Pterostylis sanguinea). Orchids, such as D. bracteata and M. media, which form relationships with diverse webs of fungi, had apparent specificity that decreased with time, as some fungi had brief encounters with orchids that supported protocorm formation but not subsequent seedling growth. The interactions between orchid mycorrhizal fungi and their hosts are discussed.     

Seed Coat Dormancy in Two Species of Grevillea(Proteaceae)

The role played by the seed coat in seed dormancy of Grevillealinearifolia(Cav.) Druce and G. wilsonii(A. Cunn.) was testedby a series of manipulations in which the seed coat was dissectedand removed, dissected and returned to the decoated seed, ordissected, removed and given a heat shock, and returned to thedecoated seed. Germination of intact seeds of both species wasalso examined after exposure to heat shock, smoke, or heat shockand smoke combined. Water permeability of the seed coat wasinvestigated by examining imbibition. For intact seeds, virtuallyno germination occurred under any treatment (G. wilsonii), orgermination was increased by exposure to either heat or smoke(G. linearifolia). Removal of the seed coat led to germinationof all decoated seeds for G. linearifolia, or a proportion ofdecoated seeds for G. wilsonii. Inclusion of smoked water inthe incubation medium led to a higher proportion of decoatedseeds germinating for G. wilsonii. Returning the seed coat,either with or without heat shock to the seed coat, did notsignificantly affect germination in either species. Seed coatswere permeable to water in both species. For the two Grevilleaspecies, there were different dormancy mechanisms that werecontrolled by the seed coat (G. linearifolia) or by both theseed coat and embryo (G. wilsonii). Copyright 2000 Annals ofBotany Company Grevillea linearifolia, Grevillea wilsonii, dormancy, seed coat dormancy, seed coat permeability, smoke, heat shock, germination     

International principles and standards for native seeds in ecological restoration

The growing demand for native seeds in ecological restoration and rehabilitation, whether for mining, forest, or ecosystem restoration, has resulted in a major global industry in the sourcing, supply, and sale of native seeds. However, there are no international guidance documents for ensuring that native seeds have the same standards of quality assurance that are regular practice in the crop and horticultural industries. Using the International Principles and Standards for the Practice of Ecological Restoration as a foundation document, we provide for the first time a synthesis of general practices in the native seed supply chain to derive the Principles and Standards for Native Seeds in Ecological Restoration (“Standards”). These practices and the underpinning science provide the basis for developing quality measures and guidance statements that are adaptable at the local, biome, or national scale. Importantly, these Standards define what is considered native seed in ecological restoration and highlight the differences between native seeds versus seeds of improved genetics. Seed testing approaches are provided within a logical framework that outline the many different dormancy states in native seed that can confound restoration outcomes. A “pro‐forma” template for a production label is included as a practical tool that can be customized for local needs and to standardize reporting to end‐users on the level of seed quality and germinability to be expected in a native seed batch. These Standards are not intended to be mandatory; however, the guidance statements provide the foundation upon which regulatory approaches can be developed by constituencies and jurisdictions.     

Bromocriptine in management of large pituitary tumours.

Bromocriptine has an accepted place in the management of small pituitary tumours that secrete either prolactin or growth hormone. The treatment of large tumours with extrasellar extensions is more difficult, however: though surgery is the standard treatment, it is often unsuccessful in returning excessive hormone secretion to normal and may cause hypopituitarism. A prospective trial was undertaken to assess the frequency with which changes in pituitary function and size of large tumours occurs. Nineteen patients were studied before and during treatment with bromocriptine (7.5 to 60 ml/day) for three to 22 months, using contrast radiology and a detailed assessment of pituitary function. Eighteen patients had hyperprolactinaemia and two of these also had raised concentrations of growth hormones; one patient had an apparently non-functioning tumour. In 12 patients (63%) tumour size decreased with bromocriptine and no tumour enlarged. Nine patients had visual-field defects, which improved in seven, becoming normal in five. Pituitary function improved in nine patients (47%) becoming entirely normal in three. Bromocriptine should be the treatment of choice in patients with large pituitary tumours with extrasellar extensions, provided close supervision is maintained.     

BMP receptor signaling is required for postnatal maintenance of articular cartilage

Articular cartilage plays an essential role in health and mobility, but is frequently damaged or lost in millions of people that develop arthritis. The molecular mechanisms that create and maintain this thin layer of cartilage that covers the surface of bones in joint regions are poorly understood, in part because tools to manipulate gene expression specifically in this tissue have not been available. Here we use regulatory information from the mouse Gdf5 gene (a bone morphogenetic protein [BMP] family member) to develop new mouse lines that can be used to either activate or inactivate genes specifically in developing joints. Expression of Cre recombinase from Gdf5 bacterial artificial chromosome clones leads to specific activation or inactivation of floxed target genes in developing joints, including early joint interzones, adult articular cartilage, and the joint capsule. We have used this system to test the role of BMP receptor signaling in joint development. Mice with null mutations in Bmpr1a are known to die early in embryogenesis with multiple defects. However, combining a floxed Bmpr1a allele with the Gdf5-Cre driver bypasses this embryonic lethality, and leads to birth and postnatal development of mice missing the Bmpr1a gene in articular regions. Most joints in the body form normally in the absence of Bmpr1a receptor function. However, articular cartilage within the joints gradually wears away in receptor-deficient mice after birth in a process resembling human osteoarthritis. Gdf5-Cre mice provide a general system that can be used to test the role of genes in articular regions. BMP receptor signaling is required not only for early development and creation of multiple tissues, but also for ongoing maintenance of articular cartilage after birth. Genetic variation in the strength of BMP receptor signaling may be an important risk factor in human osteoarthritis, and treatments that mimic or augment BMP receptor signaling should be investigated as a possible therapeutic strategy for maintaining the health of joint linings.     

Increased incidence of pertussis and parapertussis in HIV-1-positive adolescents vaccinated previously with whole-cell pertussis vaccine

We investigated 296 adolescents (11–18 years), who had been immunized previously with the three doses of DPT vaccines. 48 were diagnosed positive for HIV-1. Nasopharyngeal swabs were obtained from 296 adolescents who presented with persistent cough and nasopharyngeal secretions. Nasopharyngeal swabs (calcium alginate) specimens were collected by passing the swabs through the nares into the posterior nasopharynx and rotating the swabs for a few seconds. The swabs were plated for culture of Bordetella organisms in charcoal cephalexin blood agar (CCBA). The CCBA plates were incubated for 2–6 days at 35 °C in a humid aerobic atmosphere. The suspected, shiny (mercury-like) colonies were tested by slide agglutination with antisera to B. pertussis and B. parapertussis, and urease, oxidase activities were performed. Results indicate that out of 48 HIV-1-positive adolescents, 18 had positive cultures for Bordetella organisms (14, Bordetella pertussis, and 4, Bordetella parapertussis). Of 248 HIV-1-negative subjects, 3 had Bordetella organisms (2, Bordetella pertussis, 1, Bordetella bronchiseptica). One of the subjects, a boy, aged 14 years, with Bordetella bronchiseptica had a dog as pet, which was found to be infected. The results indicate that adolescents with HIV-1 infection, despite being vaccinated against pertussis have a higher rate of infection when exposed to pertussis bacteria than HIV-1-negative adolescents. This revised version was published online in August 2006 with corrections to the Cover Date.     

An extensive 3' regulatory region controls expression of Bmp5 in specific anatomical structures of the mouse embryo.

Bone morphogenetic proteins (BMPs) are secreted signaling molecules that control important developmental events in many different organisms. Previous studies have shown that BMPs are expressed at the earliest stages of skeletal development, and are required for formation of specific skeletal features, strongly suggesting that they are endogenous signals used to control formation of skeletal tissue. Despite the importance of BMP signaling in normal development, very little is known about the mechanisms that control the synthesis and distribution of BMP signals in vertebrates. Here, we identify a large array of cis-acting control sequences that lay out expression of the mouse Bmp5 gene in specific skeletal structures and soft tissues. Some of these elements show striking specificity for particular anatomical features within the skeleton, rather than for cartilage and bone in general. These data suggest that the vertebrate skeleton is built from the sum of many independent domains of BMP expression, each of which may be controlled by separate regulatory elements driving expression at specific anatomical locations. Surprisingly, some of the regulatory sequences in the Bmp5 gene map over 270 kb from the Bmp5 promoter, making them among the most distant elements yet identified in studies of eukaryotic gene expression.     

The SER Standards: a globally relevant and inclusive tool for improving restoration practice—a reply to Higgs et al.

In response to a critique by Higgs et al., this article clarifies the content and intent of the Society for Ecological Restoration's (SER) International Standards for the Practice of Ecological Restoration. Higgs et al. expressed concern that the SER Standards are not sufficiently underpinned by principles and risk disenfranchising some practitioners by narrowing what qualifies as ecological restoration. To demonstrate that these concerns are unfounded, we discuss the policy context and principles on which the Standards are based, its organizational structure, the innovative and inclusive approach used for development, and highlight significant errata by Higgs et al.