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Objective: To describe the reliability of new assessment items and their clinical utility as judged by experienced nurse assessors, based on the results from the field test of Version 2.0 of the Resident Assessment Instrument (RAI)., Design: Independent dual assessment of residents of nursing facilities by staff nurses using a draft of Version 2.0 of the minimum data set (MDS)., Setting and Participants: A total of 187 randomly selected residents from 21 nursing homes in seven states volunteered to test Version 2.0 of the MDS., Measurement: The full array of MDS assessment items included measures in the following areas: Background information, cognitive patterns, communication/hearing, vision, mood and behavior, psychosocial well-being, physical functioning and structural problems, continence, disease diagnoses, health condition, oral/nutritional status, dental status, skin condition, activity pursuit patterns, medications, special treatments and procedures, and discharge potential and overall status., Results: Evaluative data address issues of MDS item utility and reliability. For new items, almost all achieved a reasonably high-weighted Kappa interrater reliability; revised items also surpassed earlier items, and with the updated training materials, even the non-changed items had higher average reliability levels. Based on the success of the field test and the positive response of the industry, Version 2.0 of the RAI has been adopted, and HCFA has initiated a more long-range process to update further the RAI when necessary., Conclusion: Findings support the reliability and clinical utility of the new and revised assessment items incorporated by HCFA in Version 2.0 of the MDS.

Objective: To evaluate the effect of the implementation of the National Resident Assessment Instrument (RAI) system on selected conditions representing outcomes for nursing home residents., Design: Quasi-experimental, pre-/post-design, with assessments at baseline and 6-month follow-up., Sample: Two thousand one hundred twenty-eight residents from 268 nursing homes in 10 states before RAI implementation, and 2,088 from 254 of the same nursing homes after implementation., Measures: From the full RAI Minimum Data Set, measures of dehydration, falls, decubitus, vision problems, stasis ulcer, pain, dental status (poor teeth), and malnutrition were examined at baseline and 6 months later. Poor nutrition was evaluated using a body mass index score below 20 and vision using a 4-level scale; other conditions were represented by their presence or absence. Decline and improvement were computed as the changes in level between baseline and follow-up, limiting the sample to those who could manifest each such change., Main Results: Of eight health conditions representing poorer health status, dehydration and stasis ulcer had significantly lower prevalence after the implementation of the RAI (1993) compared with 1990. At the same time, there was an increase in the prevalence of daily pain. Fewer residents declined over 6 months in nutrition and vision after implementation. Although for these two conditions there were also significantly reduced rates of improvement, the net was an overall reduction in the 6-month rate of decline for all residents. Pain also demonstrated a decline in the postimplementation rate of improvement. The combined eight conditions showed reductions in the rates of both decline and improvement., Conclusions: Several outcomes for nursing home residents improved after implementation of the RAI. Of the four conditions for which there are significant declines in prevalence or outcome changes, three are specifically addressed in the care planning guidelines incorporated the RAI system (all except stasis ulcer, although there is a RAP for decubitus ulcer). Pain, the only other condition with a significant result --an increase in baseline prevalence--also has no RAP. Although the changes might be ascribed otherwise, they support the premise that the RAI has directly contributed to improved outcomes for nursing home residents.

Objective: To characterize changes in key aspects of process quality received by nursing home residents before and after the implementation of the national nursing home Resident Assessment Instrument (RAI) and other aspects of the Omnibus Budget Reconciliation Act (OBRA) nursing home reforms., Design: A quasi-experimental study using a complex, multistage probability-based sample design, with data collected before (1990) and after (1993) implementation of the RAI and other OBRA provisions., Setting and Participants: Two independent cohorts (n > 2000) of residents in a random sample of 254 nursing facilities located in metropolitan statistical areas in 10 states., Intervention: OBRA-87 enhanced the regulation of nursing homes and included new requirements on quality of care, resident assessment, care planning, and the use of neuroleptic drugs and physical restraints. One of the key provisions, used to help implement the OBRA requirements in daily nursing home practice, was the mandatory use of a standardized, comprehensive system, known as the RAI, to assist in assessment and care planning. OBRA provisions went into effect in federal law on October 1, 1990, although delays issuing the regulations led to actual implementation of the RAI during the Spring of 1991. MEASUREMENTS AND ANALYSES: Research nurses spent an average of 4 days per facility in each data collection round, assessing a sample of residents, collecting data through interviews with and observations of residents, interviews with multiple shifts of direct staff caregivers for the sampled residents, and review of medical records, including physician's orders, treatment and care plans, nursing progress notes, and medication records. The RNs collected data on the characteristics of the sampled residents, on the care they received, and on facility practices. The effect of being a member of the 1990 pre-OBRA or the 1993 post-OBRA cohort was assessed on the accuracy of information in the residents' medical records, the comprehensiveness of care plans, and on other key aspects of process quality while controlling for any changes in resident case-mix. The data were analyzed using contingency tables and logistic regression and a special statistical software (SUDAAN) to assure proper variance estimation., Results: Overall, the process of care in nursing homes improved in several important areas. The accuracy of information in residents' medical records increased substantially, as did the comprehensiveness of care plans. In addition, several problematic care practices declined during this period, including use of physical restraints (37.4 to 28.1% (P < .001)) and indwelling urinary catheters (9.8 to 7% (P < .001)). There were also increases in good care practices, such as the presence of advanced directives, participation in activities, and use of toileting programs for residents with bowel incontinence. These results were sustained after controlling for differences in the resident characteristics between 1990 and 1993. Other practices, such as use of antipsychotic drugs, behavior management programs, preventive skin care, and provision of therapies were unaffected, or the differences were not statistically significant, after adjusting for changes in resident case-mix., Conclusion: The OBRA reforms and introduction of the RAI constituted an unprecedented implementation of comprehensive geriatric assessment in Medicare- and Medicaid-certified nursing homes. The evaluation of the effects of these interventions demonstrates significant improvements in the quality of care provided to residents. At the same time, these findings suggest that more needs to be done to improve process quality. The results suggest the RAI is one tool that facility staff, therapists, pharmacy consultants, and physicians can use to support their continuing efforts to provide high quality of care and life to the nation's 1.7 million nursing home residents.

Objective: To evaluate the impact of the Resident Assessment Instrument (RAI) on changes in nursing home residents' functional status, cognitive status, and psychosocial well-being., Design: A quasi-experiment involving the collection of longitudinal data on two cohorts of nursing home residents. One cohort was assessed before the implementation of the RAI, and the other was assessed after the implementation of the new assessment process., Setting and Participants: Over 2000 nursing home residents in 267 nursing homes located in 10 geographic areas were assessed during the pre-RAI period. In the post-RAI period, 2000 new residents in 254 of the same facilities were assessed., Intervention: RAI implementation began in October 1990 and continued until October 1991. The RAI includes a structured, multidimensional resident assessment and problem identification system designed to form the basis for residents' care plans., Measurements: All residents were assessed at baseline and at 6 months using the Minimum Data Set for Nursing Home Resident Assessment and Care Screening (MDS) and its protocols. All data were collected by research nurses employed and trained by the research team., Results: Implementation of the RAI significantly reduced the rate of decline in seven of the nine outcomes under consideration. Reductions in improvement were also observed in all outcomes. In activities of daily living, social engagement, and cognitive function, the reduced decline far outweighed any reductions in improvement. In mood problems, problem behaviors, and understanding others, however, reductions in improvement were greater than any reductions in decline. Changes in the rates of decline and improvement were not uniform across all residents., Conclusion: The RAI may have improved the quality of care of nursing home residents by reducing overall rates of decline in important areas of resident function. However, this innovation may have generated trade-offs in that it may have reduced improvement rates in some areas of function. The system's implementation also seems to have focused staff's attention on the needs and strengths of specific subpopulations of residents. Revisions of the RAI must assist staff in generalizing their efforts to all residents and to increasing improvement rates, especially in areas related to mood and behavior.

Objective: To compare the rates of hospitalization among cohorts of nursing home residents assembled before and after the implementation of the federally mandated Resident Assessment Instrument (RAI)., Sample: Subjects were nursing home residents chosen from 268 facilities in major Metropolitan Statistics Areas in 10 states and representing more than 1500 facilities and 60,000 residents. Two resident cohorts (1990 and 1993) were sampled (8 to 16 residents per facility, depending upon facility size) as part of an evaluation of the impact of implementing the RAI., Methods: Research nurses reviewed records, interviewed staff, observed patients, and completed an RAI at baseline and 6 months later. All transitions during this interval (hospital admissions, nursing home transfers, returns home, death, etc.) were tracked. Using polytomous logistic regression, we tested the effect of cohort on the probability of being hospitalized in light of the competing risks of dying or remaining in the home, controlling for demographic and casemix variables, and having a DNR order in the chart., Results: A total of 4196 residents were studied, 2118 in 1990 (age 81.3, female 77.7%, LOS 6+ months 49.8%) and 2078 in 1993 (age 81.7, females 75.5%, LOS 6+ months 50.2%). The unadjusted probability of hospitalization dropped from .205 to .151. Multivariate analyses revealed a significant adjusted odds of hospitalization of .74 (95% CI .60-.91) and no cohort effect on home discharge or death. Among severely cognitively impaired residents, the adjusted odds of hospitalization in 1993 compared with the 1990 cohort was 0.74 (.53-1.03). Finally, among survivors in both cohorts who had a follow-up MDS performed, and whose ADL remained stable, 15.9% were hospitalized in 1990, whereas only 10.9% were hospitalized in 1993. On the other hand, ADL decliners were more likely to have been hospitalized in 1993 than in 1990 (40.6% vs 25.2%)., Conclusions: Although other changes in the industry, clinical practice, and health care policy may have influenced hospitalization of nursing home residents, the substantial reductions observed among the cognitively impaired and those with stable ADL suggest superior and uniform assessment information in the form of the RAI contributed significantly to this decline.

Objective: The Patient Self-Determination Act (PSDA) implemented in 1991 has focused national attention on the right of patients to be involved in decision-making and on the use of written advance directives. We report changes in advance care planning with the PSDA and other historical events in nursing homes in 10 states., Design: Pre- and Post-observational cohort study., Patients: Nursing home residents, residing in 270 long-term care facilities in 10 states, stratified to ensure representation of urban and rural facilities in each state. In 1990, 2175 patients were sampled, and 2088 different patients from the same facilities were sampled in 1993. Six-month follow-up was obtained at both time periods., Main Outcome Measures: Advance care planning was defined as the documentation in the medical record of a living will, a durable power of attorney, a "Do Not Resuscitate" (DNR) order, a "Do Not Hospitalize" (DNH) order, or an order to forgo artificial nutrition or hospitalization., Results: The rate of chart documentation of living wills increased from 4.2% in 1990 to 13.3% in 1993, and DNR orders increased dramatically from 31.1% to 51.5%. The rates of DNH and orders to forgo artificial hydration and nutrition remained less than 8% in both years. We found striking variations in advance care planing among the 10 states. In 1990, having a DNR order varied from 10.1% to 69.2% across the 10 states. With the exception of Oregon, where 69.2% of patients already had a DNR order, the states saw a 1.5 to 3.1 times increase in the rate of DNR orders in 1993 compared with 1990., Conclusion: With the implementation of the PSDA, there was modest increase in documentation of living wills, but DNH and orders to forgo artificial hydration and nutrition remained the same. There was a substantial increase in DNR orders that began before the PSDA implementation. This increase was associated both with the implementation of the PSDA and the increased debate about the appropriateness of CPR for nursing home residents. This increase varied considerably among geographic areas from the 10 states. Future research is needed to understand this geographic variation.

A heavy-metal-accumulating Citrobacter sp. has been used for the treatment of metal-laden industrial wastes. Metal uptake is mediated via a cell-bound phosphatase that liberates inorganic phosphate which precipitates with heavy metals as cell-bound metal phosphate. A phosphatase-efficient mutant accumulated little UO(2)2+, while a phosphatase-overproducing mutant accumulated correspondingly more metal, with a uranium loading equivalent to the bacterial dry weight achieved after 6 h exposure of resting cells to uranyl ion in the presence of phosphatase substrate (glycerol 2-phosphate). The phosphatase, visualized by immunogold labelling in the parent and overproducing strains, but not seen in the deficient mutant, was held within the periplasmic space with, in some cells, a higher concentration at the polar regions. Enzyme was also associated with the outer membrane and found extracellularly. Accumulated uranyl phosphate was visible as cell-surface- and polar-localized deposits, identified by energy-dispersive X-ray analysis (EDAX), proton-induced X-ray emission analysis (PIXE) and X-ray diffraction analysis (XRD) as polycrystalline HUO2PO4.4H2O. Nucleation sites for initiation of biocrystallization were identified at the cytoplasmic and outer membranes, prompting consideration of an in vitro biocatalytic system for metal waste remediation. Phosphatidylcholine-based liposomes with entrapped phosphatase released phosphate comparably to whole cells, as shown by 31P NMR spectroscopy in the presence of 'NMR-silent' 112Cd2+. Application of liposome-immobilized enzyme to the decontamination of uranyl solutions was, however, limited by rapid fouling of the biocatalyst by deposited uranyl phosphate. It is suggested that the architecture of the bacterial cell surface provides a means of access of uranyl ion to the inner and outer membranes and enzymically liberated phosphate in a way that minimizes fouling in whole cells.

Growth of Pseudomonas aeruginosa ATCC 15692 was promoted when the strain was cultured in an iron-depleted succinate medium, supplemented with transferrin at 30%, 60% and 100% and lactoferrin at 60% and 100% iron-saturation. No significant differences between cell growth and pyoverdin production were observed when transferrin iron saturation was increased from 30% to 100%; however, cell growth and pyoverdin production were strongly dependent on lactoferrin iron saturation. Lower lactoferrin iron saturation (< 30%) resulted in more pyoverdin production and reduced cell growth. Incubation of pyoverdin (1.0 microM) with 10.0 microM transferrin (30%, 60% and 100% iron-saturated) or lactoferrin (60% and 100% iron-saturated) led to quenching of pyoverdin fluorescence. Also, 24 h incubation of pyoverdin (20.0 microM) with these two proteins (20.0 microM, 30%, 60% and 100% iron-saturated transferrin and 60% and 100% iron-saturated lactoferrin) at 25 degrees C resulted in increased absorbance at 460 nm. Both the fluorescence quenching and absorbance increases were iron-saturation-dependent. Taken together, these results support the conclusion that at physiological pH, P. aeruginosa pyoverdin can acquire from partially iron-saturated transferrin or lactoferrin.

An unknown sequence that may encode a fragment of the Ser/Thr protein phosphatase (designated PP6Zm) related to PPT/rdgC phosphatases was identified using PCR on maize genomic DNA. A dbEST search using a partial amino acid sequence of PP6Zm revealed a putative homolog of PP6Zm expressed in Arabidopsis thaliana (EMBL AT6726). A search of the SwissProt database indicated that the partial amino acid sequence of AT6726 has the highest identity (54.3%) to the rdgC phosphatase from Drosophila melanogaster. The maize phosphatase PP1Zm6, described previously as a PP1 isoform (EMBO J., 1993, vol. 12, p. 3497), was found by us to be plant homolog of mammalian PPT. In addition, six fragments of new (pseudo) genes homologous to the phosphatase genes encoding PP1, PP2A, and PPX isoforms were detected in the maize genome. The existence in maize of a multigene PP2A family, reported only for dicotyledons, and of a PP1 multigene family, found earlier in both di- and monocotyledons, was shown.

Objectives: Using 1993 data, this study examines the prevalence of presumptively inappropriate prescriptions among residents, aged 65 and older, of board and care homes., Methods: Inappropriate drug prescriptions were identified through the use of established criteria developed for application to older nursing home residents and to community-dwelling elderly. This research used a sample of 2054 elderly residents from 410 facilities in 10 states. Weighted analyses were performed with SUDAAN, which accounted for the complex, multistage sampling design., Results: Depending on the criterion applied, between 20% and 25% of residents had at least one inappropriate prescription. Propoxyphene, long-acting benzodiazepines, dipyridamole, and amitriptyline were prescribed most frequently. Residents with inappropriate drug prescriptions had more complex drug regimens prescribed on a routine basis., Conclusions: The results are a conservative estimate of the extent of inappropriate drug prescribing and utilization in board and care facilities. Increased involvement by pharmacists and physicians in systematic drug utilization review is warranted.

rab1 cDNA coding for a small GTP binding protein Rab1 was isolated from cDNA library of tobacco (Nicotiana tabacum) leaves. The primary structure of this protein was deduced from the rab1 structure. Tobacco rab1 cDNA was expressed in Escherichia coli, and the product was purified and shown to exhibit GTPase activity. A set of Rab1 mutants with altered GTP binding and/or GTPase activities was obtained. Polyclonal antipeptide antibodies were raised against a sequence in the C-terminal region of the tobacco Rab1 capable of recognizing this protein.

Proteins are co-translationally transferred into the endoplasmic reticulum (ER) and then either retained or transported to different intracellular compartments or to the extracellular space. Various molecular signals necessary for retention in the ER or targeting to different compartments have been identified. In particular, the HDEL and KDEL signals used for retention of proteins in yeast and animal ER have also been described at the C-terminal end of soluble ER processing enzymes in plants. The fusion of a KDEL extension to vacuolar proteins is sufficient for their retention in the ER of transgenic plant cells. However, recent results obtained using the same strategy indicate that HDEL does not contain sufficient information for full retention of phaseolin expressed in tobacco. In the present study, an HDEL C-terminal extension was fused to the vacuolar or extracellular (delta pro) forms of sporamin. The resulting SpoHDEL or delta proHDEL, as well as Spo and delta pro, were expressed at high levels in transgenic tobacco cells (Nicotiana tabacum cv BY2). The intracellular location of these different forms of recombinant sporamin was studied by subcellular fractionation. The results clearly indicate that addition of an HDEL extension to either Spo or delta pro induces accumulation of these sporamin forms in a compartment that co-purifies with the ER markers NADH cytochrome C reductase, binding protein (BiP) and calnexin. In addition, a significant SpoHDEL or delta proHDEL fraction that escapes the ER retention machinery is transported to the vacuole. From these results, it may be proposed that, in addition to its function as an ER retention signal, HDEL could also act in quality control by targeting chaperones or chaperone-bound proteins that escape the ER to the plant lysosomal compartment for degradation.

The localisation of maize (Zea mays L.) auxin-binding protein (ABP1) has been studied using a variety of techniques. At the whole-tissue level, tissue printing indicated that ABP1 is expressed to similar levels in all cells of the maize coleoptile and in the enclosed leaf roll. Within cells, the signals from immunofluorescence and immunogold labelling of ultrathin sections both indicated that ABP1 is confined to the endoplasmic reticulum (ER), none being detected in either Golgi apparatus or cell wall. This distribution is consistent with targeting motifs in its sequence. These observations are discussed with reference to the various reports which place a population of ABP1 on the outer face of the plasma membrane, including those suggesting that it is necessary on the cell surface for rapid, auxin-mediated protoplast hyperpolarisation. We have tested the ER, namely that auxin binding induces a conformational change in ABP1 leading to concealment of the KDEL retention motif. Using double-label immunofluorescence the characteristic auxin-induced rise in Golgi-apparatus signal was found, yet no change in the distribution of the ABP1 signal was detected. Maize suspension cultures were used to assay for auxin-promoted secretions of ABP1 into the medium, but secretion was below the limit of detection. This can be ascribed at least partly to the very active acidification of the medium by these cells and the instability of ABP 1 in solution below pH 5.0. In the insect-baculovirus expression system, in which cell cultures maintain pH 6.2, a small amount of ABP1 secretion, less than 1% of the total, was detected under all conditions, Insect cells were shown to take up auxin and no inactivation of added auxin was detected, but auxin did not affect the level of ABP1 in the medium. Consequently, no evidence was found to support the model for auxin promotion of ABP1 secretion. Finally, quantitative glycan analysis was used to determine what proportion of ABP1 might reach the plasma membrane in maize coleoptile tissue. The results suggest that less than 15% of ABP1 ever escapes from the ER as far as the cis-Golgi and less than 2% passes further through the secretory pathway. Such leakage rates probably do not require a specialised mechanism allowing ABP1 past the KDEL retrieval pathway, but we are not able to rule out the possibility that some ABP1 is carried through associated with other proteins. The data are consistent with the presence of ABP1 both on the plasma membrane and in the ER. The relative sizes of the two pools explain the results obtained with immunofluorescence and immunogold labelling and illustrate the high efficiency of ER retention in plants.

Objectives: Various individual characteristics increase a nursing home resident's likelihood of being mechanically restrained. However, there is not good information on the role played by facility characteristics and geographic area in determining the likelihood of physical restraint use. This study investigates the importance of individual, facility, and area indicators in determining the likelihood of restraint use., Methods: This research uses data on 2,000 nursing home residents receiving care in more than 250 nursing homes in 10 different areas. Resident-level data were collected in 1990 by research nurses using the Minimum Data Set for Nursing Home Resident Assessment and Care Screening. Facility-level data were gathered by telephone interviews with facility staff. The relations among the variables were investigated using contingency tables and multivariate logistic regression models., Results: Controlling for residents' physical and cognitive function, facility characteristics and location had a significant impact on restraint practices. Those facilities with low nurse staffing were more likely to restrain residents. Furthermore, controlling for these factors, restraint practices varied significantly across the geographic areas in the study., Conclusions: Like hospitals, nursing homes exhibit significant interfacility and regional variation in their clinical practices. Future analyses of nursing home care might benefit from considering both factors in much finer detail.

MDL 105,519 [(E)-3-(2-phenyl-2-carboxyethenyl)-4, 6-dichloro-1H-indole-2-carboxylic acid] is a potent ligand at the N-methyl-D-aspartate (NMDA) receptor-associated glycine recognition site and is a noncompetitive inhibitor of NMDA receptor-mediated responses in vitro and in vivo. For purposes of characterizing its action at the glycine binding site, a halogenated analog was reduced with tritium to form radiolabeled MDL 105,519. [3H]MDL 105,519 bound to rat brain membranes with high affinity (Kd = 3.77 nM) and capacity (Bmax = 12.1 pmol/mg protein). Isolation of bound ligand by filtration gave identical levels of specific binding as did centrifugation techniques. The kinetics of the binding reaction were studied. Association was monophasic with Kon equal to 7.0 x 10(7) M-1.min-1. Dissociation was also monophasic with the Koff value calculated from association experiments (0.257 min-1) being similar to that measured directly in dissociation experiments (0.232 min-1). A kinetically derived value for the equilibrium dissociation constant was calculated with the two values for Koff and the association rate constant. The respective values (Kd = 3.67 and 3.31 nM, respectively) agreed well with that obtained from the saturation experiments. The pharmacology of the site labeled by [3H]MDL 105,519 matched that of the glycine recognition site labeled by [3H]glycine. A strong relationship existed between the pKi values of a series of glycine site agonists, partial agonists and antagonists obtained by use of these two radioligands (r = 0.90; P < .0005; slope = 0.997). No effect on specific binding of [3H]MDL 105,519 was observed with ligands (10 microM) interacting with other sites on the NMDA receptor complex or with non-NMDA glutamate recognition sites.

A cocktail of trophoblast-reactive monoclonal antibodies (MAbs) is required for efficient isolation of trophoblasts from maternal blood. A modified antibody screening procedure was used to identify a clone, in a COS cell placental cDNA expression library, that expressed the gene product recognized by MAb FDO202N. The antigen recognised by MAb FDO202N was identified as human placental lactogen (hPL) hormone. hPL hormone is secreted into the maternal blood by trophoblasts at high levels during pregnancy. Immunohistochemical localization of hPL hormone was consistent with expression in the syncytiotrophoblast and extravillous cytotrophoblast. A model system was used where known numbers of syncytiotrophoblast sprouts were seeded into saline or maternal blood, bound by trophoblast-specific MAbs, recovered magnetically, and then counted. MAb FDO202N was shown to facilitate the efficient recovery of trophoblast sprouts from saline and maternal blood.

High level expression of the major auxin-binding protein (ABP1) from maize (Zea mays L.) has been used to demonstrate that the machinery for retaining proteins in the endoplasmic reticulum (ER) of insect cells functions efficiently throughout the baculovirus infection cycle. Immunolocalization showed wild-type ABP1 (ABP1-KDEL) to be targeted to the lumen of the ER, in accordance with its signal peptide and carboxyterminal KDEL ER-retention signal. The protein accumulated in dilations of the ER, and none was detected at the cell surface. Immunoblotting of concentrated culture medium confirmed that ABP1-KDEL was not secreted at a detectable level. In contrast, when the carboxyterminus was mutated to KEQL, secretion of the baculovirus-expressed protein was readily detected. Immunolocalization and immunoblotting demonstrated that a high proportion of the ABP1-KEQL protein was secreted at the cell surface and into the culture medium. The data demonstrate that the ER of insect cells has a great capacity to retain proteins and that this property is largely unaffected by the cellular disruption caused by baculovirus replication.

We recently reported that 2-substitution of N 6 -benzyladenosine-5'-uronamides greatly enhances selectivity of agonists for rat A 3 adenosine receptors J. Med. Chem. 1994 , 37 , 3614-3621). Specifically, 2-Chloro- N 6 -(3-iodobenzyl)adenosine-5'- N -methyluronamide (2-CI-IB-MECA), which displayed a K 1 value of 0.33 nM, is the most selective for A 3 receptors yet reported with selectivity versus A 1 and A 2a receptors of 2500- and 1400-fold, respectively. In order to obtain pharmacological tools for the study of A 3 H]methylamine in methanol at 60 °C for 2 h. The specific activity of [ N 6 -(3-iodobenzyl)-2-chloro-2',3'-di- O -acetyl-5'-(methoxycarbonyl)adenosine (17) with [ 3 H]methylamine in methanol at 60 °C for 2 h. The specific activity of [ 3 H]2-CI-IB-MECA was 29 Ci/mmol with a radiochemical purity of 99%.

Background: Largely unsupervised administration of drugs and the potential for overuse of psychotropic agents in residential care facilities have emerged as major public policy concerns. In a large multistate study, we examined patterns of psychotropic prescription and use by facility licensure status and the extensiveness of state regulations., Methods: Descriptive analyses were based on a sample of 2,949 residents from 493 board-and-care facilities in 10 states, drawn via a complex, multistage sampling design. States were purposively selected based on the stringency of their board-and-care regulatory system, and samples of facilities were drawn, stratified by licensure status and home size. Residents were randomly selected within the sampled facilities. Weighted analyses were performed with Software for Survey Data Analysis (SUDAAN), accounting for the complex sampling design., Results: Approximately 43% of the residents were prescribed and 41% used at least one psychotropic agent, primarily on a routinely scheduled basis. Antipsychotics were prescribed to 22% and used by 21%; antidepressants were prescribed to 17% and taken by 16%; antimanic agents were prescribed to and used by 4%; and anxiolytics, sedatives, or hypnotics were prescribed to 17% and used by 14%. Among psychotropic users, over 50% had not had mental health services in the prior year; 25% had no psychiatric history. Among licensed facilities, prescription and use of psychotropics, particularly antipsychotics, was significantly higher among residents of homes located in states with limited regulatory systems., Conclusions: Results revealed high rates of psychotropic prescription and use, and not inconsequential levels of polypharmacy - within and across therapeutic classes - among board-and-care facility residents. Frequently, psychotropics were not used as an adjunct to alternative treatments, and were not associated with a psychiatric history. Extent of psychotropic use was related to the regulatory environment, suggesting that more extensive regulations and monitoring may reduce psychoactive drug use in board-and-care facilities, and more adequately ensure the appropriateness of such treatment.

Whilst the function and organization of the secretory machinery in eukaryotic cells exhibit basic similarities, the compartmentation of the endomembrane system can show significant differences between the fungal, plant and animal kingdoms. The use of the antibiotic brefeldin A (BFA) as an inhibitor of secretion in both animal and yeast cells has resulted in a remarkable advance in our understanding of the modes of action of vesicle shuttles between the endoplasmic reticulum and Golgi apparatus and within the Golgi apparatus itself. It is now apparent that application of the drug to filamentous fungi and plants will also help unravel the workings of the secretory system in these organisms. In this paper we review recent progress in our laboratories on elucidating the effects of BFA on the morphology of the Golgi apparatus and compare these with recently published data on fungal and plant cells. Variation in the response to BFA are reported, which may not all be attributed to differences in drug concentration and time of treatment. These may reflect differences in cellular sensitivity or multiple sites of action of the drug, and the existence of a specific molecular target for BFA is questioned.

Objective: To describe patterns of psychotropic use as well as clinically relevant and concurrent nonpsychotropic use among older residents of board and care facilities in 10 states., Methods: Weighted use rates were based on data from a sample of 2054 residents aged 65 and older from 410 facilities in 10 states, drawn via a complex, multistage sampling design. Data, including drug name, use frequency, and PRN use, were collected on all drugs taken by the resident during a 7-day period. During face-to-face interviews with residents and/or proxies, data were also collected on resident characteristics., Subjects: The majority of subjects were female, white, and widowed. Average age was 82 years. Approximately 25% had a current mental or emotional condition, and 8% had been hospitalized for a psychiatric condition during the year before admission. More than half needed help with at least one activity of daily living. Forty percent had moderate or severe cognitive impairment, and 18% had received mental health care during the previous year., Analyses: Weighted descriptive analyses were performed with Software for Survey Data Analysis (SUDAAN), which accounts for the complex, multistage sampling design., Results: Approximately 35% of the older residents used at least one psychoactive agent, with 30% of psychotropic users receiving two to four different psychotropic medications. Use rates decline with age, for psychotropics overall and for therapeutic classes. Results revealed potentially problematic polypharmacy in relation to drug duplication within therapeutic classes, use of multiple psychotropics across classes, and concurrent nonpsychotropic use., Conclusions: Compared with community-dwelling older adults, this study revealed high psychotropic use rates among older board and care facility residents. Overall, our results suggest that serious consideration be given to the apparent need for systematic drug utilization review, a potentially useful program that is mandated in other settings.

To achieve continuous expression of the major maize auxin-binding protein (ABP1) in insect cells, the ABP1 gene coding region was placed under control of a baculovirus immediate-early gene promoter and transfected into Spodoptera frugiperda Sf9 cells. The ABP1 gene was detected in twelve cell lines, one of which was selected for detailed analysis. Immunolocalisation demonstrated that ABP1 was targeted to and retained in the endoplasmic reticulum (ER), in accordance with its signal peptide and carboxy-terminal KDEL ER-retention signal. We discuss the advantages of stable-transformation over transient expression systems for characterising proteins targeted to the secretory system of insect cells.

The aim of the present work was the development of phosphorylcholine-based treatments for biofiltration membranes and the demonstration that such treatments prevent or inhibit protein fouling. Microfiltration membranes of cellulose triacetate, polyether sulphone and polyvinylidene fluoride were etched with oxygen in a plasma chamber to generate surface hydroxyl groups and were then treated with the monomer 2-methacryloyloxyethyl phosphorylcholine. These membranes were evaluated with water, buffer, bovine serum albumin (BSA), yeast fermentation broth, beer and orange juice. The treatment of cellulose triacetate membranes reduced both the initial flux and the extent of water fouling. In terms of the integrated flux, these factors tended to cancel each other out. For protein, the membranes gave similar or higher fluxes but worse fouling. The cellular feed (yeast) reacted more favourably to the coating than the BSA. The polyether sulphone was scarcely affected by the coating; fouling remaining high with most 'real' feeds. There was lower initial flux but less flux decline with water and beer. Washing with water and cleaning with Tergazyme did not restore the initial flux. Polyvinylidene fluoride membranes gave the most positive results. In most cases, the coating both increased initial flux and decreased the rate of fouling. The coating was particularly effective for BSA and for beer and orange juice, where fouling is probably caused by a polysaccharide rather than by a protein. Electron microscopy showed, nonetheless, that fouling by proteins was accompanied by protein adsorption primarily on the upper surface of the membrane and that coated membranes showed less deposition and in different places than did untreated membranes.

The MDS is a core set of items, definitions, and response categories used to assess all of the nation's 1.5 million nursing home residents who reside in facilities participating in the Medicare or Medicaid programs. Further, the Health Care Financing Administration (HCFA) has proposed a rule that would require facilities to computerize MDS data and submit it to state and federal agencies, paving the way for a national database. This article describes the process of testing the reliability of the MDS items in 13 nursing homes in five states. The results demonstrate that MDS data gathered in a research effort attain reliabilities that make such data useful. MDS items met a standard for excellent reliability (i.e., intraclass correlation of .7 or higher) in key areas of functional status, such as cognition, ADLs, continence, and diagnoses. Sixty-three percent of the items achieved reliability coefficients of .6 or higher. Eighty-nine percent of the items in the MDS achieved .4 or higher.

Polymorphic genetic variation shows that the genes for human 3 beta-hydroxysteroid dehydrogenases (3 beta-HSD) types I and II are closely linked. The type II mutations A82T, S100N and L173R are associated with male pseudohermaphroditism and A82T is associated, with variable penetrance, with female premature puberty. When expressed in vitro A82T showed less than 5% of normal activity and L173R showed a 30-50% reduction in activity. PCR experiments and direct genomic cloning show that there is a larger family of 3 beta-HSD sequences which require to be tested for expression. The phenomenon of epitopic heterogeneity of 3 beta-HSD is discussed and is now shown to apply to testicular Leydig cells as well as extrauterine trophoblast. RT-PCR analyses indicate that the phenomenon is most likely to be due to post-translational modification affecting the carboxytermini 3 beta-HSD types I and II. This phenomenon may reflect a further level at which enzyme activity is regulated.

This research tests the reliability and construct validity of social engagement, a new quality of life measure embedded in the federally mandated Resident Assessment Instrument (RAI) for the nursing home population. The sample consisted of 1,848 residents from 268 homes in 10 states with data collected by trained research nurses. Three resident groups were formed based on residents' cognitive and ADL functioning. Social engagement was significantly related to average time spent in activities across all three groups. We hypothesized a four-factor model with social engagement distinct from mood problems, conflicted relationships, and behavior problems. LISREL confirmatory factor analysis found the data to be consistent with this hypothesis (Fit Index > .98). Intercorrelations between factors showed that for high-functioning residents, engagement was negatively related to conflict but was positively related to conflict among the most impaired. The validity of the social engagement measure and its stability across types of residents suggest its potential utility as a marker of nursing home quality.

We have synthesised the α-subunit of the chick nicotinic acetylcholine receptor (nAChR) in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovius immediate early gene promoter. Transformed cells were obtained by co-transfection of the insect cells with pIEK1.nAChRα, encoding the α-subunit cDNA, and pIEK1.neo, encoding the neomycin resistance gene. G-418-resistant clones were selected and expanded into continuous cell lines synthesising the chick nAChR α-subunit. Using fluorescence microscopy and ligand binding studies we were able to demonstrate efficient membrane targeting of the receptor subunit in the insect cell plasma membrane. Stable insect cell lines may thus have significant advantages over transient baculovirus vectors for the synthesis and characterisation of heterologous receptor proteins.

The major auxin-binding protein (ABP1) from maize (Zea mays L.) has been expressed in insect cells using the baculovirus expression system. The recombinant protein can be readily detected in total insect cell lysates by Coomassie blue staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Our data suggest that ABP1 is processed similarly in both insect cells and maize. The signal peptide is cleaved at the same position as in maize and the mature protein undergoes tunicamycin-sensitive glycosylation, yielding a product with the same mobility on SDS-PAGE as authentic maize ABP1. On immunoblots the expressed protein is recognized by anti-KDEL monoclonal antibodies. Immunofluorescence localization demonstrates that it is targeted to and retained in the endoplasmic reticulum of insect cells in accordance with its signal peptide and KDEL retention sequence. The expressed ABP1 also appears to be active, since extracts of insect cells expressing ABP1 contain a saturable high-affinity 1-naphthylacetic acid-binding site, whereas no saturable auxin-binding activity is detected in extracts from control cells.

Background: Chronic cognitive impairment is a major problem in U.S. nursing homes, yet traditional assessment systems in most facilities included only limited information on cognitive status. Following the Congressional mandate in the Omnibus Reconciliation Act of 1987 (OBRA '87), U.S. nursing homes now complete the Minimum Data Set (MDS), a standardized, comprehensive assessment of each resident's functional, medical, psychosocial, and cognitive status. We designed a Cognitive Performance Scale (CPS) that uses MDS data to assign residents into easily understood cognitive performance categories., Methods: Information was drawn from three data sets, including two multistate data sets constructed for the Health Care Financing Administration. The prevalence and reliability of the MDS cognitive performance variables were established when assessed by trained nursing personnel. Five selected MDS items were combined to create the single, functionally meaningful seven-category hierarchical Cognitive Performance Scale., Results: The CPS scale corresponded closely with scores generated by the Mini-Mental State Examination and the Test for Severe Impairment, nursing judgments of disorientation, and neurological diagnoses of Alzheimer's disease and other dementias., Conclusions: The new CPS provides a functional view of cognitive performance, using readily available MDS data. It should prove useful to clinicians and investigators using the MDS to determine a resident's cognitive assets.

A new monoclonal antibody (FDO26G) is described which was raised against purified human 3 beta-hydroxysteroid dehydrogenase type I (3 beta-HSD type I). FDO26G reacted strongly with villous syncytiotrophoblast, weakly with some trophoblast cells in chorion laeve, and not at all with extravillous trophoblast in cytotrophoblast cell islands and decidual trophoblast. All these types of trophoblast reacted strongly with monoclonal antibody FDO161G, which has previously been shown to react with 3 beta-HSD type I and, like FDO26G, reacts strongly with adrenal cells. Mapping experiments using a combination of lacZ fusion polypeptides and synthetic peptides located the FDO26G epitope to residues 354-366 at the C-terminal end of the molecule, a sequence that is identical in the type I and type II forms of the enzyme. The epitope contains a consensus for a casein kinase-II site with serine 359 as the candidate phosphorylation site. This suggested that the lack of reactivity of FDO26G to 3 beta-HSD in extravillous trophoblast might be due to phosphorylation at serine 359. Peptide 354-366 was synthesized with phosphoserine at residue 359 and its binding to FDO26G was compared with that of the unphosphorylated peptide. FDO26G bound the phosphopeptide at least as strongly as the unphosphorylated peptide. It is concluded that the lack of staining of extravillous trophoblast by FDO26G is due to the presence of a different sequence at residues 354-366 and that a hitherto unidentified third isoform of human 3 beta-HSD is expressed in these cells.

Objective: Our purpose was to evaluate the morphologic characteristics of cells separated from peripheral maternal blood with immunomagnetic beads coated with trophoblast-reactive monoclonal antibodies of restricted specificity., Study Design: Blood samples were collected from 14 pregnant women at 9 to 12 or 16 to 18 weeks' gestation. Immunomagnetic beads coated with trophoblast-reactive monoclonal antibodies were used to isolate cells from the blood. The isolated cells were then fixed, embedded, and sectioned to enable morphologic identification., Results: Multinucleate cells identical to syncytiotrophoblast sprouts were identified in 12 of 14 samples., Conclusion: Multinucleate syncytiotrophoblast can be isolated from the peripheral blood of normal pregnant women in both the first and second trimesters of pregnancy. The isolated trophoblast may allow prenatal genetic diagnosis by a noninvasive technique.

The aim of this study was to develop a computer expert system that could reproduce a pathologist's diagnosis of iron deficiency from the data obtained from blood tests. 275 cases were collected for construction and testing of the expert system. The expert system used a combination of fuzzy set logic and cut-off points from 14 parameters to arrive at one of 5 diagnostic categories graded from "iron deficient" to "no evidence of iron deficiency". The agreement between pathologist and expert system was 0.91 (Spearman rank correlation coefficient) in the learning population; this dropped to 0.79 in the test population. Absolute agreement on diagnostic category was reached in 71% of cases. In no case was there disagreement by more than 3 grades.

Objectives: Reducing the widespread use of physical restraints in nursing homes is a primary goal of the federal nursing home reforms enacted as part of the Omnibus Budget Reconciliation Act of 1987. However, some nursing home operators assert that reducing restraint use could be prohibitively expensive, costing payors perhaps as much as $1 billion annually. We investigated whether nursing home residents free from physical restraint require more care and resources than similar residents who are restrained., Methods: We examined the major component of nursing home costs--staff time--and its allocation among residents who were physically restrained and those who were not restrained. The multivariate analysis used staff-time-study data on the care of 11,932 nursing home residents in 276 facilities in seven states., Results: The analyses indicate that, when differences in impairment and care needs are controlled for, residents who are physically restrained require more nursing care than other residents. Higher levels of nursing-assistant time were consistently provided to restrained residents., Conclusions: Residents free of restraints are less costly to care for than restrained residents. The 1987 federal requirements concerning restraint use, which are aimed at improving quality of care and quality of life, can be implemented without engendering a major increase in care costs.

Objective: To explore the relationship between an elderly subject's cognitive status and the reliability of multidimensional assessment data., Design: Survey, with cognitive status as the independent variable and interrater reliability as dependent variable., Setting: Medicare/Medicaid-certified nursing homes., Participants: 147 residents age 65 or older., Measurements: Dual assessments of elderly nursing home residents were performed by nurse assessors using the Health Care Financing Administration's new Minimum Data Set for Nursing Home Resident Assessment and Care Screening (MDS). Assessments were classified on the basis of residents' cognitive status, and levels of disagreement between assessors were analyzed., Main Results: Overall assessment reliability, agreement concerning a resident's activities of daily living status, and the reliability of estimates of his or her communication skills and sensory abilities were significantly affected by a resident's cognitive status. The presence of cognitive impairment made these measurements less reliable--especially those related to communication skills, vision, and hearing., Conclusions: Assessments of residents suffering from cognitive impairment were significantly less reliable than assessments of cognitively intact residents. However, these differences in reliability were not uniform across all assessment domains. When treating the cognitively impaired elderly, clinicians must exercise caution in their reliance on standardized measurements that may be less reliable for this population.

Levels of acute phase and other plasma proteins were measured in 21 men with major depression, 28 men with alcohol dependence, and 12 men who acted as controls. The depressed men had significantly elevated levels of the acute phase proteins, haptoglobin and alpha-1-antichymotrypsin, and of immunoglobulin G. The elevations in haptoglobin and alpha-1-antichymotrypsin were highly correlated with each other, and were correlated with the severity of depression and negatively correlated with the thyroid stimulating hormone response to thyrotropin. The alcoholic men had elevated haptoglobin levels, but significantly decreased levels of immunoglobulin G. These findings provide further evidence for an inflammatory response during depression.

The epitopes of two monoclonal antibodies raised to a putative auxin receptor have been mapped. Carboxy-peptidase A digestion of the antigen, auxin-binding protein (ABP) purified from maize, completely abolished binding of antibody MAC 256 and impaired binding of MAC 259, suggesting that they both recognise C-terminal epitopes. Published sequences of ABP showed that the C terminus was KDEL, a tetrapeptide used for targeting proteins to the ER in animal cells. We have used this short homology to confirm that the two monoclonals recognise C-terminal KDEL, showing that animal KDEL proteins and synthetic KDEL peptides are recognised and that animal cell ER is stained strongly and specifically. Sucrose density gradient fractionation of maize microsomal membranes showed that plant KDEL proteins, including ABP, fractionated with markers for the endoplasmic reticulum. However, few proteins are stained by anti-KDEL monoclonals in plants. For comparison, a monoclonal antibody raised to a synthetic HDEL peptide was also used and found to stain a set of proteins in all plant species tested. The anti-HDEL and anti-KDEL monoclonals were sequence specific, staining different proteins. On density gradient fractionation HDEL proteins also banded with ER marker activities. However, the intracellular distribution of HDEL and KDEL proteins determined by immunofluorescence was different. Whereas HDEL proteins showed a distribution characteristic of plant ER, and this localisation was confirmed by immunogold labelling of ultrathin sections and electron microscopy, KDEL proteins showed strong fluorescence in discrete parts of the cell cortex. These observations are discussed in terms of the potential these monoclonal antibodies have as markers for ER and of the role ABP plays in plant cell signalling.

Case-mix reimbursement systems for nursing home care are now in use or under consideration in over 20 states. Though many in the long-term care field still debate the relative merits of the various systems, those systems based on the Resource Utilization Groups-II (RUG-II) system used in New York seem to be increasingly popular. However, one of the more serious questions raised concerning RUG-like systems is whether they fully acknowledge the role played by cognitive deficits in determining nursing home residents' care needs. The results of this research indicate: 1) that resource provision and cognitive status are correlated, 2) that residents with higher levels of cognitive impairment appear most frequently in RUG-II categories that have higher case-mix weights, 3) that simply adding some measure of cognitive function does not significantly increase the power of these models, and 4) that the RUG-II model predicts the level of care resources provided to the cognitively intact much better than it predicts those provided to residents suffering from relatively severe disorientation. These results imply that such classification systems may create disincentives for the admission of the most cognitively impaired residents and that the care of these individuals, more than others, may be dependent on facility-to-facility variation.

The monoclonal antibody FDO161G reacts with a 43-kDa protein found in human extravillous trophoblast, syncytiotrophoblast, adrenal cortex, interstitial cells of the testis and ovarian follicle cumulus cells. cDNAs for this protein have been isolated from the lambda gt11 library, sequenced, and expressed in COS-7 cells. The protein was identified as 3 beta-hydroxy-5-ene steroid dehydrogenase (HSD). The sequence of the HSD protein raises questions about its association with cell membrane systems. The lack of reactivity of FDO161G with other tissues suggests that HSD has a limited tissue distribution and that other enzymes may exist in peripheral tissues, which can convert delta 5 3-hydroxysteroids to delta 4 3-ketosteroids.

Fetal trophoblast cells were isolated from maternal peripheral blood by means of murine monoclonal antibodies of high specificity and affinity for human syncytiotrophoblast and nonvillous cytotrophoblast cells. The cells were isolated in sufficient numbers to allow polymerase chain reaction (PCR) amplification of the Y-chromosome-specific DNA sequence from the peripheral blood of thirteen pregnant women. The fetal sex predicted by PCR analysis of the isolated trophoblast cells accorded with that ascertained by karyotyping of chorionic villus samples in eleven of twelve women studied in early pregnancy and with the sex of the baby on delivery in one woman studied at 34 weeks' gestation. Isolation of these fetal cells could allow noninvasive diagnosis of a wide range of inherited disorders.

In response to the Omnibus Reconciliation Act of 1987 mandate for the development of a national resident assessment system for nursing facilities, a consortium of professionals developed the first major component of this system, the Minimum Data Set (MDS) for Resident Assessment and Care Screening. A two-state field trial tested the reliability of individual assessment items, the overall performance of the instrument, and the time involved in its application. The trial demonstrated reasonable reliability for 55% of the items and pinpointed redundancy of items and initial design of scales. On the basis of these analyses and clinical input, 40% of the original items were kept, 20% dropped, and 40% altered. The MDS provides a structure and language in which to understand long-term care, design care plans, evaluate quality, and describe the nursing facility population for planning and policy efforts.

The detection of the lymphokine monocyte chemotactic factor by determination of the leading front of monocyte migration is described. Stimulated and control supernatants were placed on the attractant side of cellulose ester filters with normal human monocytes as responder cells. Lymphokine activity was assessed by comparing the leading front of monocyte migration in response to control and stimulated culture supernatants. More monocytes migrated to all levels within the filters towards PHA stimulated culture supernatant than to control supernatant, indicating that the lymphokine acted on the entire migrating cell population and not only the cells at the leading front. Analysis of cell migration in a range of concentration gradients demonstrated that the lymphokine induced by PHA was chemotactic for monocytes. There was a significant association (p less than 0.01) between the production of monocyte chemotactic factor in vitro and skin test reactivity in vivo to three soluble antigens, SKSD, Candida albicans extract and tetanus toxoid. It is concluded that the detection of monocyte chemotactic factor production in vitro provides good evidence of sensitisation, however due to a number of false negative reactions, failure to detect the lymphokine does not necessarily indicate lack of reactivity in vivo.

The in vitro random migration and chemotactic activity of peripheral blood monocytes and neutrophils was compared with the delayed hypersensitivity skin test to streptokinase-streptodornase in fourteen normal subjects. A significant positive correlation (P less than 0.001) was observed between the random migration of monocytes and the size of the skin test reaction. No significant correlation was found with random neutrophil migration or monocyte and neutrophil chemotactic responses. These results indicate that the in vitro random mobility of monocytes is related to the in vivo expression of a delayed-type hypersensitivity skin reaction.