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Background, Aim and Scope: In a gradualist approach to the introduction of crop biotechnology, the findings of experimentation at one scale are used to predict the outcome of moving to a higher scale of deployment. Movement through scales had occurred for certain genetically modified herbicide-tolerant (GMHT) crops in the UK as far as large-scale field trials. However, the land area occupied by these trials was still <1% of the area occupied by the respective non-GM crops. Some means is needed to predict the direction and size of the effect of increasing the area of GMHT cropping on ecological variables such as the diversity among species and trophic interactions. Species-accumulation curves are examined here as a method of indicating regional-scale impacts on botanical diversity from multiple field experiments., Materials and Methods: Data were used from experiments on the effect of (GMHT) crops and non-GM, or conventional, comparators in fields sown with four crop types (beet, maize, spring and winter oilseed rape) at a total of 250 sites in the UK between 2000 and 2003. Indices of biodiversity were measured in a split-field design comparing GMHT with the farmers' usual weed management. In the original analyses based on the means at site level, effects were detected on the mass of weeds in the three spring crops and the proportion of broadleaf and grass weeds in winter oilseed rape, but not on indices of plant species diversity. To explore the links between site means and total taxa, accumulation curves were constructed based on the number of plant species (a pool of around 250 species in total) and the number of plant functional types (24), inferred from the general life-history characteristics of a species., Results: Species accumulation differed between GMHT and conventional treatments in direction and size, depending on the type of crop and its conventional management. Differences were mostly in the asymptote of the curve, indicative of the maximum number of species found in a treatment, rather than the steepness of the curve. In winter oilseed rape, 8% more species were accumulated in the GMHT treatment, mainly as a result of the encouragement of grass species by the herbicide when applied in the autumn. (Overall, GMHT winter oilseed rape had strong negative effects on both the food web and the potential weed burden by increasing the biomass of grasses and decreasing that of broadleaf weeds.) In maize, 33% more species-a substantial increase-were accumulated in the GMHT than in the conventional, consistent with the latter's highly suppressive weed management using triazine herbicides. In the spring oilseed rape and beet, fewer species (around 10%) were accumulated in the GMHT than the conventional. The GMHT treatments did not remove or add any functional (life history) types, however. Differences in species accumulation between treatments appeared to be caused by loss or gain of rarer species. The generality of this effect was confirmed by simulations of species accumulation in which the species complement at each of 50 sites was drawn from a regional pool and subjected to reducing treatment at each site. Shifts in the species-accumulation parameters, comparable to those measured, occurred only when a treatment removed the rarer species at each site., Discussion: Species accumulation provided a set of simple curve-parameters that captured the net result of numerous local effects of treatments on plant species and, in some instances, the balance between grass and broadleaf types. The direction of effect was not the same in the four crops and depended on the severity of the conventional treatment and on complex interactions between season, herbicide and crop. The accumulation curves gave an indication of potential positive or negative consequences for regional species pools of replacing a conventional practice with GMHT weed management. In this and related studies, a range of indicators, through which diversity was assessed by both species and functional type, and at both site and regional scales, gave more insight into effects of GMHT treatment than provided by any one indicator., Conclusions: Species accumulation was shown to discriminate at the regional scale between agronomic treatments that had little effect on species number at the field scale. While a comprehensive assessment of GM cropping needs to include an examination of regional effects, as here, the costs of doing this in all instances would be prohibitive. Simulations of diversity-reducing treatments could provide a theoretical framework for predicting the likely regional effects from in-field plant dynamics., Recommendations and Perspectives: Accumulation curves potentially offer a means of linking within-site effects to regional impacts on biodiversity resulting from any change in agricultural practice. To guide empirical measurement, there is a scope to apply a methodology such as individual-based modelling at the field scale to explore the links between agronomic treatments and the relative abundance of plant types. The framework needs to be validated in practice, using species-based and functional taxonomies, the latter defined by measured rather than inferred traits.

Ypt/Rab GTPases act as key regulators of intracellular traffic through the conformational differences exhibited by their GTP or GDP-bound forms. In this paper, two Arabidopsis Ypt6 homologues, AtRAB-H1(b) and AtRAB-H1(c) were characterized and compared. Using a live cell imaging approach, it is shown that yellow fluorescent protein-fusions (YFP) of AtRAB-H1(b) and AtRAB-H1(c) locate to the Golgi and to the cytosol in both Nicotiana tabacum and in Arabidopsis thaliana. In addition, YFP-AtRAB-H1(b) targets an as yet unknown compartment not labelled by YFP-AtRAB-H1(c) or Golgi markers. It is also shown that the subcellular location of YFP-AtRAB-H1(b) and YFP-AtRAB-H1(c) is affected by the state of GTP-binding and that expression of a GTP-deficient mutant results in increased apoplastic fluorescence of a secretory form of YFP.

The interface between the endoplasmic reticulum (ER) and the Golgi apparatus is a critical junction in the secretory pathway mediating the transport of both soluble and membrane cargo between the two organelles. Such transport can be bidirectional and is mediated by coated membranes. In this review, we consider the organization and dynamics of this interface in plant cells, the putative structure of which has caused some controversy in the literature, and we speculate on the stages of Golgi biogenesis from the ER and the role of the Golgi and ER on each other's motility.

Summary: In order to further understand the production and intracellular trafficking of pharmaceutical proteins in plants, the light and heavy chains (LC and HC) of the human immunodeficiency virus neutralizing monoclonal antibody 2G12 were fused to fluorescent proteins [Venus and monomeric red fluorescent protein (mRFP)] to enable the visualization of their passage through the plant cell. Co-expression of LC and HC with various markers of the endomembrane system demonstrated that LC fusions were found in mobile punctate structures, which are likely to be pre-vacuolar compartments (PVCs) as a proportion of the LC fusions were found to be located in the vacuole. In addition, apoplast labelling was also observed with a 2G12LC-RFP fusion. The HC fusion expressed alone was found only in the endoplasmic reticulum (ER). When the LC and HC fusions were expressed together, they were found to co-locate to larger punctate structures, which were morphologically distinct from any observed on expression of LC or HC alone. These structures appeared to be in close association with the ER and their labelling partially overlapped with PVC marker fluorescence, but no increase in apoplast labelling was observed. Co-immunoprecipitation data demonstrated that the presence of the fluorescent proteins did not affect the assembly of the antibody, and also showed the association of BiP with the antibody chains. The antigen-binding activity of the Venus-fused 2G12 antibody was confirmed by enzyme-linked immunosorbent assay.

Brefeldin A (BFA) is one of the most popular drugs used by researchers for studies on secretion and endocytosis because it interferes with specific vesicle coat proteins via action on a guanine nucleotide exchange factor. Due to its range of morphological effects on the Golgi apparatus in a variety of plant tissues, we believe that there is more to the BFA response than the primary molecular targets so far identified.

The levels of accumulation of recombinant vaccines in transgenic plants are protein specific and strongly influenced by the subcellular compartment of destination. The human immunodeficiency virus protein Nef (negative factor), a promising target for the development of an antiviral vaccine, is a cytosolic protein that accumulates to low levels in transgenic tobacco and is even more unstable when introduced into the secretory pathway, probably because of folding defects in the non-cytosolic environment. To improve Nef accumulation, a new strategy was developed to anchor the molecule to the cytosolic face of the endoplasmic reticulum (ER) membrane. For this purpose, the Nef sequence was fused to the C-terminal domain of mammalian ER cytochrome b5, a long-lived, tail-anchored (TA) protein. This consistently increased Nef accumulation by more than threefold in many independent transgenic tobacco plants. Real-time polymerase chain reaction of mRNA levels and protein pulse-chase analysis indicated that the increase was not caused by higher transcript levels but by enhanced protein stability. Subcellular fractionation and immunocytochemistry indicated that Nef-TA accumulated on the ER membrane. Over-expression of mammalian or plant ER cytochrome b5 caused the formation of stacked membrane structures, as observed previously in similar experiments performed in mammalian cells; however, Nef-TA did not alter membrane organization in tobacco cells. Finally, Nef could be removed in vitro by its tail-anchor, taking advantage of an engineered thrombin cleavage site. These results open up the way to use tail-anchors to improve foreign protein stability in the plant cytosol without perturbing cellular functions.

Personal care services are often provided to clients in community settings through highly discretionary processes. Such processes provide little guidance for caseworkers concerning how public resources should be allocated. The results of such processes almost guarantee that individuals with very similar needs will receive very different levels of care resources. Such disparities in treatment open the door to inequity and ineffectiveness. One way to address this problem is through case-mix classification systems that allocate hours of care according to client needs. This paper outlines the preliminary steps taken by one state in its movement toward such a system.

Although organelle movement in higher plants is predominantly actin-based, potential roles for the 17 predicted Arabidopsis myosins in motility are only just emerging. It is shown here that two Arabidopsis myosins from class XI, XIE, and XIK, are involved in Golgi, peroxisome, and mitochondrial movement. Expression of dominant negative forms of the myosin lacking the actin binding domain at the amino terminus perturb organelle motility, but do not completely inhibit movement. Latrunculin B, an actin destabilizing drug, inhibits organelle movement to a greater extent compared to the effects of AtXIE-T/XIK-T expression. Amino terminal YFP fusions to XIE-T and XIK-T are dispersed throughout the cytosol and do not completely decorate the organelles whose motility they affect. XIE-T and XIK-T do not affect the global actin architecture, but their movement and location is actin-dependent. The potential role of these truncated myosins as genetically encoded inhibitors of organelle movement is discussed.

We have cloned a member of the reticulon (RTN) family of Arabidopsis thaliana (RTNLB13). When fused to yellow fluorescent protein (YFP) and expressed in tobacco leaf epidermal cells, RTNLB13 is localized in the endoplasmic reticulum (ER). Coexpression of a soluble ER luminal marker reveals that YFP-tagged, myc-tagged or untagged RTNLB13 induces severe morphological changes to the lumen of the ER. We show, using fluorescence recovery after photobleaching (FRAP) analysis, that RTNLB13 overexpression greatly reduces diffusion of soluble proteins within the ER lumen, possibly by introducing constrictions into the membrane. In spite of this severe phenotype, Golgi shape, number and dynamics appear unperturbed and secretion of a reporter protein remains unaffected.

Brefeldin A (BFA) treatment stops secretion and leads to the resorption of much of the Golgi apparatus into the endoplasmic reticulum. This effect is reversible upon washing out the drug, providing a situation for studying Golgi biogenesis. In this investigation Golgi regeneration in synchronized tobacco BY-2 cells was followed by electron microscopy and by the immunofluorescence detection of ARF1, which localizes to the rims of Golgi cisternae and serves as an indicator of COPI vesiculation. Beginning as clusters of vesicles that are COPI positive, mini-Golgi stacks first become recognizable 60 min after BFA washout. They continue to increase in terms of numbers and length of cisternae for a further 90 min before overshooting the size of control Golgi stacks. As a result, increasing numbers of dividing Golgi stacks were observed 120 min after BFA washout. BFA-regeneration experiments performed on cells treated with BFA (10 microg mL(-1)) for only short periods (30-45 min) showed that the formation of ER-Golgi hybrid structures, once initiated by BFA treatment, is an irreversible process, the further incorporation of Golgi membranes into the ER continuing during a subsequent drug washout. Application of the protein kinase A inhibitor H-89, which effectively blocks the reassembly of the Golgi apparatus in mammalian cells, also prevented stack regeneration in BY-2 cells, but only at very high, almost toxic concentrations (>200 microm). Our data suggest that under normal conditions mitosis-related Golgi stack duplication may likely occur via cisternal growth followed by fission.

Purpose: This study had two goals: (a) to assess the validity of the Online Survey Certification and Reporting (OSCAR) staffing data by comparing them to staffing measures from audited Medicaid Cost Reports and (b) to identify systematic differences between facilities that over-report or underreport staffing in the OSCAR., Design and Methods: We merged the 2002 Texas Nursing Facility Cost Report, the OSCAR for Texas facilities surveyed in 2002, and the 2003 Area Resource File. We eliminated outliers in the OSCAR using three decision rules, resulting in a final sample size of 941 of the total of 1,017 non-hospital-based facilities. We compared OSCAR and Medicaid Cost Report staffing measures for three staff types. We examined differences between facilities that over-reported or underreported staffing levels in the OSCAR by using logistic regression., Results: Average staffing levels were higher in the OSCAR than in the Medicaid Cost Report data. The two sets of measures exhibited correlations ranging between 0.5 and 0.6. For-profit and larger facilities consistently over-reported registered nurse staffing levels. Factors associated with increased odds of over-reporting licensed vocational nursing or certified nursing assistant staffing were lower Medicare or Medicaid censuses and less market competition. Facility characteristics associated with over-reporting were consistent across different levels of over-reporting. Underreporting was much less prevalent., Implications: Certain types of facilities consistently over-report staffing levels. These reporting errors will affect the validity of consumer information systems, regulatory activities, and health services research results, particularly research using OSCAR data to examine the relationship between staffing and quality. Results call for a more accurate reporting system.

Background: Nursing home performance measurement systems are practically ubiquitous. The vast majority of these systems aspire to rank order all nursing homes based on quantitative measures of quality. However, the ability of such systems to identify homes differing in quality is hampered by the multidimensional nature of nursing homes and their residents. As a result, the authors doubt the ability of many nursing home performance systems to truly help consumers differentiate among homes providing different levels of quality. We also argue that, for consumers, performance measurement models are better at identifying problem facilities than potentially good homes., Discussion: In response to these concerns we present a proposal for a less ambitious approach to nursing home performance measurement than previously used. We believe consumers can make better informed choice using a simpler system designed to pinpoint poor-quality nursing homes, rather than one designed to rank hundreds of facilities based on differences in quality-of-care indicators that are of questionable importance. The suggested performance model is based on five principles used in the development of the Consumers Union 2006 Nursing Home Quality Monitor., Summary: We can best serve policy-makers and consumers by eschewing nursing home reporting systems that present information about all the facilities in a city, a state, or the nation on a website or in a report. We argue for greater modesty in our efforts and a focus on identifying only the potentially poorest or best homes. In the end, however, it is important to remember that information from any performance measurement website or report is no substitute for multiple visits to a home at different times of the day to personally assess quality.

Purpose: The U.S. Department of Veterans Affairs has adopted two functional assessment systems that guide care planning: one for nursing home residents (the Resident Assessment Instrument [RAI]) and a compatible one for home care clients (RAI-HC). The purpose of this article is to describe the RAI-HC (often referred to as the Minimum Data Set-Home Care or MDS-HC) and its uses and offer lessons learned from implementation experiences in other settings., Design and Methods: We reviewed implementation challenges associated both with the RAI and the RAI-HC in the United States, Canada, and other adopter countries, and drew on these to suggest lessons for the Department of Veterans Affairs as well as other entities implementing the RAI-HC., Results: Beyond its clinical utility, there are a number of evidence-based uses for the assessment system. The resident-level data can be aggregated and analyzed, and scales identify clinical conditions and risk for various types of negative outcomes. In addition, the data can be used for other programmatic and research purposes, such as determining eligibility, setting payment rates for contract care, and evaluating clinical interventions. At the same time, there are a number of implementation challenges the Department of Veterans Affairs and other organizations may face., Implications: Policy makers and program managers in any setting, including state long-term-care programs, who wish to implement an assessment system must anticipate and address a variety of implementation problems with a clear and consistent message from key leadership, adequate training and clinical support for assessors, and appropriate planning and resources for data systems.

Over the past 40 years there have been marked shifts in arable farmland management that are widely believed to have had a considerable impact on flowering plants and invertebrates and the small mammals and birds that rely upon them. It is not yet possible to predict the dynamics of plants and invertebrates either with past or future changes in farmland management. This study investigates whether a basic invertebrate classification, formed of broad trophic groups, can be used to describe interactions between invertebrates and their resource plants and evaluate management impacts for genetically modified, herbicide-tolerant (GMHT) and conventional herbicide management in both spring- and winter-sown oilseed rape. It is argued that the analyses validate trophic-based approaches for describing the dynamics of invertebrates in farmland and that linear models might be used to describe the changes in invertebrate trophic group abundance in farmland when driven by primary producer abundance or biomass and interactions between invertebrates themselves. The analyses indicate that invertebrate dynamics under GMHT management are not unique, but similar to conventional management occurring over different resource ranges, and that dynamics differed considerably between spring- and winter-sown oilseed rape. Thus, herbicide management was of much lower impact on trophic relationships than sowing date. Results indicate that invertebrate dynamics in oilseed rape are regulated by a combination of top-down and bottom-up trophic processes.

Purpose: The purpose of this article is to discuss and describe various measures of quality, quality indicators, and uses of information on quality with specific reference to the role or purpose of assisted living., Design and Methods: We reviewed a variety of major studies of assisted living quality. We elaborated models of assisted living based on differing goals or claims made for it by providers and policy makers. We then searched for available quality measures that might indicate whether assisted living was meeting those goals or living up to those claims., Results: Any meaningful concept of "quality" must embrace a variety of dimensions, including quality of care, quality of life, the physical environment, and resident rights. The ability to use a multidimensional concept of quality is complicated by the lack of consensus, confusion, and disagreement among consumers, providers, and regulators about the role of assisted living. This disagreement significantly confounds the task of comparing quality among assisted living settings and between assisted living and other types of long-term care., Implications: We propose ways that researchers may compare quality along dimensions claimed to be intrinsic to assisted living as part of an effort to inform consumer information systems, quality monitoring and assurance systems, and policy-relevant research. Such comparisons would vary, depending on the intended use of the indicators and role defined for assisted living. However, all uses contain structural, process, and outcome quality indicators, including direct feedback from interviews with residents that go beyond satisfaction measures.

Golgins are large coiled-coil proteins that play a role in tethering of vesicles to Golgi membranes and in maintaining the overall structure of the Golgi apparatus. Six Arabidopsis proteins with the structural characteristics of golgins were isolated and shown to locate to Golgi stacks when fused to GFP. Two of these golgin candidates (GC1 and GC2) possess C-terminal transmembrane (TM) domains with similarity to the TM domain of human golgin-84. The C-termini of two others (GC3/GDAP1 and GC4) contain conserved GRAB and GA1 domains that are also found in yeast Rud3p and human GMAP210. GC5 shares similarity with yeast Sgm1p and human TMF and GC6 with yeast Uso1p and human p115. When fused to GFP, the C-terminal domains of AtCASP and GC1 to GC6 localized to the Golgi, showing that they contain Golgi localization motifs. The N-termini, on the other hand, label the cytosol or nucleus. Immuno-gold labelling and co-expression with the cis Golgi Q-SNARE Memb11 resulted in a more detailed picture of the sub-Golgi location of some of these putative golgins. Using two independent assays it is further demonstrated that the interaction between GC5, the TMF homologue, and the Rab6 homologues is conserved in plants.

As compared with other eukaryotic cells, plants have developed an endoplasmic reticulum (ER)-Golgi interface with very specific structural characteristics. ER to Golgi and Golgi to ER transport appear not to be dependent on the cytoskeleton, and ER export sites have been found closely associated with Golgi bodies to constitute entire mobile units. However, the molecular machinery involved in membrane trafficking seems to be relatively conserved among eukaryotes. Therefore, a challenge for plant scientists is to determine how these molecular machineries work in a different structural and dynamic organization. This review will focus on some aspects of membrane dynamics that involve coat proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment receptor proteins), lipids, and lipid-interacting proteins.

Protein localization in living plant cells is commonly studied using fluorescent protein fusions. Stable transformation of plant cells requires the use of binary vectors, which are larger and not as amenable to genetic manipulation as animal cell transfection vectors. Binary vectors containing fluorescent protein fusion constructs are prepared using standard molecular biological techniques. Fusion genes as well as promoters and selection markers are stably incorporated into the plant cell genome via Agrobacterium-mediated transfer. Presented here are a series of protocols that detail binary vector construction, bacterial transformation, and a rapid transient assay technique that can be used to evaluate fusion protein fluorescence in leaves.

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), alpha-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and beta-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a trans-Golgi marker (ST52-mRFP), suggesting roles for protein-lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein-protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.

Purpose: We examined the effects of facility and market-level characteristics on staffing levels and turnover rates for direct care staff, and we examined the effect of staff turnover on staffing levels., Design and Methods: We analyzed cross-sectional data from 1,014 Texas nursing homes. Data were from the 2002 Texas Nursing Facility Medicaid Cost Report and the Area Resource File for 2003. After examining factors associated with staff turnover, we tested the significance and impact of staff turnover on staffing levels for registered nurses (RNs), licensed vocational nurses (LVNs) and certified nursing assistants (CNAs)., Results: All three staff types showed strong dependency on resources, such as reimbursement rates and facility payor mix. The ratio of contracted to employed nursing staff as well as RN turnover increased LVN turnover rates. CNA turnover was reduced by higher administrative expenditures and higher CNA wages. Turnover rates significantly reduced staffing levels for RNs and CNAs. LVN staffing levels were not affected by LVN turnover but were influenced by market factors such as availability of LVNs in the county and women in the labor force., Implications: Staffing levels are not always associated with staff turnover. We conclude that staff turnover is a predictor of RN and CNA staffing levels but that LVN staffing levels are associated with market factors rather than turnover. Therefore, it is important to focus on management initiatives that help reduce CNA and RN turnover and ultimately result in higher nurse staffing levels in nursing homes.

A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributions to be determined. Consequently, genuine organelle residents have been distinguished from contaminating proteins and proteins in transit through the secretory pathway.

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.

The Farm Scale Evaluations (FSEs) showed that genetically modified herbicide-tolerant (GMHT) cropping systems could influence farmland biodiversity because of their effects on weed biomass and seed production. Recently published results for winter oilseed rape showed that a switch to GMHT crops significantly affected weed seedbanks for at least 2 years after the crops were sown, potentially causing longer-term effects on other taxa. Here, we seek evidence for similar medium-term effects on weed seedbanks following spring-sown GMHT crops, using newly available data from the FSEs. Weed seedbanks following GMHT maize were significantly higher than following conventional varieties for both the first and second years, while by contrast, seedbanks following GMHT spring oilseed rape were significantly lower over this period. Seedbanks following GMHT beet were smaller than following conventional crops in the first year after the crops had been sown, but this difference was much reduced by the second year for reasons that are not clear. These new data provide important empirical evidence for longer-term effects of GMHT cropping on farmland biodiversity.

Purpose: Previous research in specific locales indicates that individuals admitted to rural nursing homes have lower care needs than individuals admitted to nursing homes in urban areas, and that rural nursing homes differ in their mix of short-stay and chronic-care residents. This research investigates whether differences in acuity are a function of differences in resident payer status and occur for both individuals admitted for short stays, with Medicare as payer, and those needing chronic care., Design and Methods: We used a representative 10% sample of national resident assessments (Minimum Data Set) for calendar year 2000 (N = 197,589). We conducted statistical analyses (means, percentages, and logistic regression) to investigate differences in Medicare and non-Medicare admissions to facilities in metropolitan and nonmetropolitan areas., Results: Non-Medicare residents admitted to rural nursing facilities have lower acuity scores than non-Medicare residents admitted to metropolitan nursing homes. However, individuals admitted under Medicare were similar in rural and urban areas., Implications: Differences in resident acuity at admission among facilities in different locales were largely a function of lower acuity levels for individuals admitted to rural nursing homes for long-term or chronic care, although differences in Medicare census also played some role in facility-level differences in acuity. Other factors must be explored to determine why this lower acuity occurs and whether higher use of rural nursing homes by less impaired older persons meets their needs and preferences and represents good public policy.

Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.

Individuals receiving end-of-life (EOL) care may have needs that are unrecognized or treated inappropriately. Yet, very little is known about differences in pain and special-care needs of EOL patients admitted to rural nursing facilities compared with urban nursing facilities, and whether the differing payer mix in urban and rural facilities affects the treatment ordered on admission. We examine a nationally representative sample of 6084 EOL patients upon admission to nursing homes to examine differences in diseases, pain assessments, and treatment orders. We found that rural EOL residents have higher rates of congestive heart failure, cancer, renal failure, and emphysema than urban EOL residents and are significantly more likely to report frequent pain, however, they are less likely to receive treatments such as IV medications, dialysis, and wound care.

Components of the plant cell secretory pathway, including the endoplasmic reticulum and Golgi apparatus, are in constant motion. The photoactivation of GFP has been used to determine that proteins within the membrane of the ER flow as the ER is remodelled. Measurement of the rate at which activated GFP moves away from the activation spot shows that this motion is much faster than would be expected if membrane components moved simply by diffusion. Treatment with latrunculin to depolymerize the actin cytoskeleton stops ER remodelling and reduces the rate of GFP movement to that expected from diffusion alone. This suggests that myosin binds directly or indirectly to ER membrane proteins and actively moves them around over the actin scaffold. Tracking of Golgi body movement was used to demonstrate that they move at the same rate and in the same direction as do photoactivated ER surface proteins. Golgi bodies, therefore, move with, and not over, the surface of the ER. These observations support the current theory of continuity between Golgi bodies and discrete ER exit sites in the ER membrane.

A combination of electron microscopy and fluorescence microscopy has provided us with a global picture of the structure of the plant Golgi apparatus. However, the components that shape this structure remain elusive. In other organisms, members of the golgin family of coiled-coil proteins are essential for Golgi structure and organisation. Putative Arabidopsis and rice homologues of some golgin family members can be identified using database searches. Likewise, the heterogeneous group of multi-subunit-tethering complexes is responsible for crucial transport steps that affect Golgi structure and cisternal organisation in animals and yeasts. The Arabidopsis genome harbours possible homologues for the majority of the subunits of these complexes, suggesting that they also operate in the plant kingdom.

GRIP domain proteins are a class of golgins that have been described in yeast and animals. They locate to the trans-Golgi network and are thought to play a role in endosome-to-Golgi trafficking. The Arabidopsis GRIP domain protein, AtGRIP, fused to the green fluorescent protein (GFP), locates to Golgi stacks but does not exactly co-locate with the Golgi marker sialyl transferase (ST)-mRFP, nor with the t-SNAREs Memb11, SYP31 and BS14a. We conclude that the location of AtGRIP is further to the trans side of the stack than STtmd-mRFP. The 185-aa C-terminus of AtGRIP containing the GRIP domain targeted GFP to the Golgi, although a proportion of the fusion protein was still found in the cytosol. Mutation of a conserved tyrosine (Y717) to alanine in the GRIP domain disrupted Golgi localization. ARL1 is a small GTPase required for Golgi targeting of GRIP domain proteins in other systems. An Arabidopsis ARL1 homologue was isolated and shown to target to Golgi stacks. The GDP-restricted mutant of ARL1, AtARL1-T31N, was observed to locate partially to the cytosol, whereas the GTP-restricted mutant AtARL1-Q71L labelled the Golgi and a population of small structures. Increasing the levels of AtARL1 in epidermal cells increased the proportion of GRIP-GFP fusion protein on Golgi stacks. We show, moreover, that AtARL1 interacted with the GRIP domain in a GTP-dependent manner in vitro in affinity chromatography and in the yeast two-hybrid system. This indicates that AtGRIP and AtARL1 interact directly. We conclude that the pathway involving ARL1 and GRIP domain golgins is conserved in plants.

Distinct sets of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are distributed to specific intracellular compartments and catalyze membrane fusion events. Although the central role of these proteins in membrane fusion is established in nonplant systems, little is known about their role in the early secretory pathway of plant cells. Analysis of the Arabidopsis (Arabidopsis thaliana) genome reveals 54 genes encoding SNARE proteins, some of which are expected to be key regulators of membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. To gain insights on the role of SNAREs of the early secretory pathway in plant cells, we have cloned the Arabidopsis v-SNAREs Sec22, Memb11, Bet11, and the t-SNARE Sed5, and analyzed their distribution in plant cells in vivo. By means of live cell imaging, we have determined that these SNAREs localize at the Golgi apparatus. In addition, Sec22 was also distributed at the ER. We have then focused on understanding the function of Sec22 and Memb11 in comparison to the other SNAREs. Overexpression of the v-SNAREs Sec22 and Memb11 but not of the other SNAREs induced collapse of Golgi membrane proteins into the ER, and the secretion of a soluble secretory marker was abrogated by all SNAREs. Our studies suggest that Sec22 and Memb11 are involved in anterograde protein trafficking at the ER-Golgi interface.

Controversy exists in the literature over the involvement of the endoplasmic reticulum (ER) in the delivery of membrane proteins to peroxisomes. In this study, the involvement of the ER in the trafficking of two Arabidopsis (Arabidopsis thaliana) peroxisomal membrane proteins was investigated using confocal laser scanning microscopy of living cells expressing fusions between enhanced yellow fluorescent protein (eYFP) and AtPEX2 and AtPEX10. The fusion proteins were always detected in peroxisomes and cytosol irrespective of the location of the eYFP tag or the level of expression. The cytosolic fluorescence was not due to cleavage of the eYFP reporter from the C-terminal fusion proteins. Blocking known ER transport routes using the fungal metabolite Brefeldin A or expressing dominant negative mutants of Sar1 or RabD2a had no effect on the trafficking of AtPEX2 and AtPEX10 to peroxisomes. We conclude that AtPEX2 and AtPEX10 are inserted into peroxisome membranes directly from the cytosol.

Responses of key invertebrates within Farm Scale Evaluations (FSEs) of maize reflected advantageous effects for weeds under genetically modified herbicide-tolerant (GMHT) management. Triazine herbicides constitute the main weed control in current conventional systems, but will be withdrawn under future EU guidelines. Here, we reappraise FSE data to predict effects of this withdrawal on invertebrate biodiversity under alternative management scenarios. Invertebrate indicators showed remarkably consistent and sensitive responses to weed abundance. Their numbers were consistently reduced by atrazine used prior to seedling emergence, but at reduced levels compared to similar observations for weeds. Large treatment effects were, therefore, maintained for invertebrates when comparing other conventional herbicide treatments with GMHT, despite reduced differences in weed abundance. In particular, benefits of GMHT remained under comparisons with best estimates of future conventional management without triazines. Pitfall trapped Collembola, seed-feeding carabids and a linyphiid spider followed closely trends for weeds and may, therefore, prove useful for modelling wider biodiversity effects of herbicides. Weaker responses to triazines applied later in the season, at times closer to the activity and capture of invertebrates, suggest an absence of substantial direct effects. Contrary responses for some suction-sampled Collembola and the carabid Loricera pilicornis were probably caused by a direct deleterious effect of triazines.

The plant Golgi apparatus is composed of many separate stacks of cisternae which are often associated with the endoplasmic reticulum and which in many cell types are motile. In this review, we discuss the latest data on the molecular regulation of Golgi function. The concept of the Golgi as a distinct organelle is challenged and the possibility of a continuum between the endoplasmic reticulum and Golgi is proposed.

The plant Golgi apparatus is composed of many separate stacks of cisternae which are often associated with the endoplasmic reticulum and which in many cell types are motile. In this review, we discuss the latest data on the molecular regulation of Golgi function. The concept of the Golgi as a distinct organelle is challenged and the possibility of a continuum between the endoplasmic reticulum and Golgi is proposed.

Objective: To provide preliminary data on Medicare expenditures for assisted living facility (ALF) residents and to investigate whether ALF characteristics were related to Medicare expenditures for ALF residents., Data Sources/study Setting: Data from the National Study of Assisted Living for the Frail Elderly conducted in 1998-1999. This analysis was restricted to the 40 percent of ALFs in that sample that adhered to the assisted living (AL) philosophy by offering more than minimal levels of services and privacy. This study involved the approximately 1,200 residents who remained in an ALF from baseline to follow-up data collection. Six months of postbaseline Medicare claims were acquired for 545 of these residents, who did not differ significantly from the larger sample., Data Collection: Baseline individual and facility data were collected in personal interviews with residents and a combination of personal and telephone interviews with facility staff. Medicare claims data were acquired from the Centers for Medicare and Medicaid Services., Study Design: Cross-sectional analyses using logistic and ordinary least squares regression techniques were used to determine the relationships among individual and facility characteristics and Medicare utilization and expenditures., Principal Findings: On an annualized basis, AL residents incurred Medicare costs of approximately US dollars 4,800. Just less than 15 percent of AL residents accounted for over 75 percent of total Medicare costs. Both the likelihood of utilizing Medicare-covered services and the intensity of service use were largely unaffected by the characteristics of the ALF in which residents lived. Utilization was largely a function of individual characteristics. The only exception to this general finding was that those individuals who utilized services and resided in smaller ALFs had significantly lower average expenditures than did individuals in larger ALFs., Conclusions: These preliminary data imply that both the level and distribution of Medicare expenditures among ALF residents were similar to those among the general community-dwelling Medicare beneficiary population. No significant relationships were observed between ALF characteristics and Medicare expenditures, except the effect of facility size. This result may imply that how the AL industry eventually defines itself in terms of services and amenities, other than size, may have little impact on Medicare expenditures for ALF residents. However, this is a single, initial study, so caution must be exercised when considering the implications of these results.

We evaluated the effects of the herbicide management associated with genetically modified herbicide-tolerant (GMHT) winter oilseed rape (WOSR) on weed and invertebrate abundance and diversity by testing the null hypotheses that there is no difference between the effects of herbicide management of GMHT WOSR and that of comparable conventional varieties. For total weeds, there were few treatment differences between GMHT and conventional cropping, but large and opposite treatment effects were observed for dicots and monocots. In the GMHT treatment, there were fewer dicots and monocots than in conventional crops. At harvest, dicot biomass and seed rain in the GMHT treatment were one-third of that in the conventional, while monocot biomass was threefold greater and monocot seed rain almost fivefold greater in the GMHT treatment than in the conventional. These differential effects persisted into the following two years of the rotation. Bees and Butterflies that forage and select for dicot weeds were less abundant in GMHT WORS management in July. Year totals for Collembola were greater under GMHT management. There were few other treatment effects on invertebrates, despite the marked effects of herbicide management on the weeds.

Context: Expanding the availability of long-term care (LTC) services and making them more responsive to consumer preferences is an important goal, particularly for elderly people living in rural areas who tend to be older and have greater functional limitations but less access to the range of LTC options available in metropolitan areas. One option that has been growing in popularity is assisted-living facilities (ALFs)., Purpose and Methods: This paper describes rural ALFs and compares them with metropolitan ALFs. Data were collected using a multistage sample design that yielded a nationally representative sample of ALFs. Telephone interviews were completed with administrators of 1,251 ALFs in 1998., Findings: Nationwide, assisted living was largely administered by private payment, and there was an undersupply in rural areas. Compared with metropolitan ALFs, rural ALFs were smaller and less likely to offer the types of services and accommodations associated with the philosophy of assisted living. They were more likely to offer accommodations with little privacy, and while similar in the services they offered, rural ALFs were less likely to have nurses on staff, particularly licensed practical nurses. Moreover, they were less likely to offer a combination of high services and high privacy. Finally, rural ALFs charged lower prices than urban ALFs; however, the average price was still unaffordable for most elderly rural residents., Conclusions: These findings suggest that assisted living, as currently structured, will make only a marginal contribution to meeting the needs of frail elders in rural areas.

The higher plant Golgi apparatus, comprising many individual stacks of membrane bounded cisternae, is one of the most enigmatic of the cytoplasmic organelles. Not only can the stacks receive material from the endoplasmic reticulum, process it and target it to the correct cellular destination, but they can also synthesise and export complex carbohydrates and lipids and most likely act as one end point of the endocytic pathway. In many cells such processing and sorting can take place while the stacks are moving within the cytoplasm and, remarkably, the organelle manages to retain its structural integrity. This review considers some of the latest data and views on transport both to and from the Golgi and the mechanisms by which such activity is regulated.

Rab GTPases are universal key regulators of intracellular secretory trafficking events. In particular, Rab 5 homologues have been implicated in endocytic events and in the vacuolar pathway. In this study, we investigate the location and function of a member of this family, AtRabF2b (Ara7) in tobacco (Nicotiana tabacum) leaf epidermal cells using a live cell imaging approach. Fluorescent-tagged AtRabF2b[wt] localized to the prevacuolar compartment and Golgi apparatus, as determined by coexpression studies with fluorescent markers for these compartments. Mutations that impair AtRabF2b function also alter the subcellular location of the GTPase. In addition, coexpression studies of the protein with the vacuole-targeted aleurain-green fluorescent protein (GFP) and rescue experiments with wild-type AtRabF2b indicate that the dominant-negative mutant of AtRabF2b causes the vacuolar marker to be secreted to the apoplast. Our results indicate a clear role of AtRabF2b in the vacuolar trafficking pathway.

Objectives: We examined differences in quality of care among nursing homes in locales of varying degrees of rurality., Methods: We classified locales into 4 classes according to rurality. We analyzed a 10% sample of nursing home admissions in the United States in 2000 (n=198613) to estimate survival models for 9 quality indicators., Results: For postacute admissions, we observed significant differences in rates of decline for residents in facilities in large towns compared with urban areas, but differences in quality were both negative and positive. Among admissions for long-term or chronic care, rates of decline in 2 of 9 quality areas were lower for residents in isolated areas., Conclusions: We observed significant differences in a number of quality indicators among different classes of nursing home locations, but differences varied dramatically according to type of admission. These differences did not exhibit the monotonicity that we would have expected had they derived solely from rurality. Also, quality indicators exhibited more similarities than differences across the 4 classes of locales. The results underscore the importance, in some instances, of emphasizing the effects of specific settings rather than some continuum of rurality and of moving beyond the assumption that nursing home residents constitute a homogeneous population.

We have studied the transport of proricin and pro2S albumin to the protein storage vacuoles of developing castor bean (Ricinus communis L.) endosperm. Immunoelectron microscopy and cell fractionation reveal that both proteins travel through the Golgi apparatus and co-localize throughout their route to the storage vacuole. En route to the PSV, the proteins co-localize in large (>200 nm) vesicles, which are likely to represent developing storage vacuoles. We further show that the sequence-specific vacuolar sorting signals of both proricin and pro2SA bind in vitro to proteins that have high sequence similarity to members of the VSR/AtELP/BP-80 vacuolar sorting receptor family, generally associated with clathrin-mediated traffic to the lytic vacuole. The implications of these findings in relation to the current model for protein sorting to storage vacuoles are discussed.

In contrast with animals, plant cells contain multiple mobile Golgi stacks distributed over the entire cytoplasm. However, the distribution and dynamics of protein export sites on the plant endoplasmic reticulum (ER) surface have yet to be characterized. A widely accepted model for ER-to-Golgi transport is based on the sequential action of COPII and COPI coat complexes. The COPII complex assembles by the ordered recruitment of cytosolic components on the ER membrane. Here, we have visualized two early components of the COPII machinery, the small GTPase Sar1p and its GTP exchanging factor Sec12p in live tobacco (Nicotiana tabacum) leaf epidermal cells. By in vivo confocal laser scanning microscopy and fluorescence recovery after photobleaching experiments, we show that Sar1p cycles on mobile punctate structures that track with the Golgi bodies in close proximity but contain regions that are physically separated from the Golgi bodies. By contrast, Sec12p is uniformly distributed along the ER network and does not accumulate in these structures, consistent with the fact that Sec12p does not become part of a COPII vesicle. We propose that punctate accumulation of Sar1p represents ER export sites (ERES). The sites may represent a combination of Sar1p-coated ER membranes, nascent COPII membranes, and COPII vectors in transit, which have yet to lose their coats. ERES can be induced by overproducing Golgi membrane proteins but not soluble bulk-flow cargos. Few punctate Sar1p loci were observed that are independent of Golgi bodies, and these may be nascent ERES. The vast majority of ERES form secretory units that move along the surface of the ER together with the Golgi bodies, but movement does not influence the rate of cargo transport between these two organelles. Moreover, we could demonstrate using the drug brefeldin A that formation of ERES is strictly dependent on a functional retrograde transport route from the Golgi apparatus.

The UK Farm-Scale Evaluations (FSE) compared the effects on biodiversity of management of genetically modified herbicide-tolerant (GMHT) spring-sown crops with conventional crop management. The FSE reported larger weed abundance under GMHT management for fodder maize, one of three crops studied. Increased seed production may be important for the long-term persistence of these arable weeds and may benefit invertebrates, small mammals and seed-eating birds. In three-quarters of FSE maize fields, growers used atrazine on the conventionally managed half, reflecting contemporary commercial practice. Withdrawal of the triazine herbicides atrazine, simazine and cyanazine from approved lists of EU chemicals could therefore reduce or even reverse the reported benefits of GMHT maize. Here we analyse effects of applications of triazine herbicides in conventional maize regimes on key indicators, using FSE data. Weed abundances were decreased greatly relative to all other regimes whenever atrazine was applied before weeds emerged. Here, we forecast weed abundances in post-triazine herbicide regimes. We predict weed abundances under future conventional herbicide management to be considerably larger than that for atrazine used before weeds emerged, but still smaller than for the four FSE sites analysed that used only non-triazine herbicides. Our overall conclusion is that the comparative benefits for arable biodiversity of GMHT maize cropping would be reduced, but not eliminated, by the withdrawal of triazines from conventional maize cropping.

We describe the use of a secreted form of Aequoria victoria green fluorescent protein (secGFP) in a non-invasive live cell assay of membrane traffic in Arabidopsis thaliana. We show that in comparison to GFP-HDEL, which accumulates in the endoplasmic reticulum (ER), secGFP generates a weak fluorescence signal when transported to the apoplast. The fluorescence of secGFP in the apoplast can be increased by growth of seedlings on culture medium buffered at pH 8.1, suggesting that apoplastic pH is responsible, at least in part, for the low fluorescence intensity of seedlings expressing secGFP. Inhibition of secGFP transport between the ER and plasma membrane (PM), either by Brefeldin A (BFA) treatment or by genetic intervention results in increased intracellular secGFP accumulation accompanied by an increase in the secGFP fluorescence intensity. secGFP thus provides a valuable tool for forward and reverse genetic analysis of membrane traffic and endomembrane organisation in Arabidopsis. Using this assay for quantitative sublethal perturbation of secGFP transport, we identify a role for root hair defective 3 (RHD3) in transport of secreted and Golgi markers between the ER and the Golgi apparatus.

Peroxisomes participate in many important functions in plants, including seed reserve mobilization, photorespiration, defense against oxidative stress, and auxin and jasmonate signaling. In mammals, defects in peroxisome biogenesis result in multiple system abnormalities, severe developmental delay, and death, whereas in unicellular yeasts, peroxisomes are dispensable unless required for growth of specific substrates. PEX10 encodes an integral membrane protein required for peroxisome biogenesis in mammals and yeast. To investigate the importance of PEX10 in plants, we characterized a Ds insertion mutant in the PEX10 gene of Arabidopsis (AtPEX10). Heterozygous AtPEX10::dissociation element mutants show normal vegetative phenotypes under optimal growth conditions, but produce about 20% abnormal seeds. The embryos in the abnormal seeds are predominantly homozygous for the disruption allele. They show retarded development and some morphological abnormalities. No viable homozygous mutant plants were obtained. AtPEX10 fused to yellow fluorescent protein colocalized with green fluorescent protein-serine-lysine-leucine, a well-documented peroxisomal marker, suggesting that AtPEX10 encodes a peroxisomal protein that is essential for normal embryo development and viability.

Purpose: Throughout the 1990s, assisted living was the most rapidly growing form of senior housing. The purpose of this paper is to describe the existing supply of assisted living facilities (ALFs) and examine the extent to which they matched the philosophy of assisted living., Design and Methods: The study involved a multistage sample design to produce nationally representative estimates for the ALF industry. Administrators of nearly 1,500 eligible ALFs were interviewed by telephone., Results: As of 1998, there were an estimated 11,459 ALFs nationwide, with 611,300 beds and 521,500 residents. Nearly 60% offered a combination of low services and low or minimal privacy, whereas only 11% offered relatively high services and high privacy. Seventy-three percent of the resident rooms or apartments were private. Aging-in-place was limited by discharge policies in most ALFs for residents who needed help with transfers, had moderate to severe cognitive impairment, had any behavioral symptoms, or needed nursing care. The industry is largely private pay and unaffordable for low- or moderate-income persons aged >/=75 unless they use assets as well as income to pay., Implications: ALFs differed widely in ownership, size, policies, and the degree to which they manifested the philosophy of assisted living. This diversity represents a challenge for consumers in terms of selecting an appropriate facility and for policy makers in terms of deciding what role they want assisted living to play in long-term care.