Purification and kinetic characterization of the mitogen-activated protein kinase phosphatase rVH6.

The dual specificity protein-tyrosine phosphatase rVH6 belongs to a subfamily of enzymes that have in vivo and in vitro catalytic activity against mitogen-activated protein kinases. A method was developed for the expression and efficient purification of recombinant rVH6 in quantities sufficient for physical and kinetic characterization of the enzyme. Matrix-assisted laser desorption mass spectrometry verified the mass of purified rVH6 to be 43,500 +/- 150, and NH2-terminal sequence analysis confirmed the predicted amino acid sequence. Kinetic characterization of full-length rVH6 identified the critical ionizations involved in the kcat/Km parameter (apparent pKa values 5.1 and 6.6) and revealed a pH-independent kcat value of 0.014 s-1. In an attempt to define the essential catalytic core of this enzyme, amino acids 134-381 of rVH6 were expressed, purified, and characterized enzymatically. Kinetic analysis revealed that the truncated enzyme exhibited a turnover value similar to that of the full-length enzyme (kcat = 0.017 s-1), with p-nitrophenyl phosphate as substrate. Secondary structure prediction and molecular modeling of rVH6 based on the x-ray structure of the dual specificity protein tyrosine phosphatase, VHR, further supported the assignment of residues 134-381 to the core catalytic domain of rVH6. These results demonstrate that the NH2 terminus of rVH6 (residues 1-133) is not required for full enzyme activity and comprises a separate domain that may play a distinct physiological function.

A novel cytoplasmic dual specificity protein tyrosine phosphatase implicated in muscle and neuronal differentiation.

Dual specificity protein tyrosine phosphatases (dsPTPs) are a subfamily of protein tyrosine phosphatases implicated in the regulation of mitogen-activated protein kinase (MAPK). In addition to hydrolyzing phosphotyrosine, dsPTPs can hydrolyze phosphoserine/threonine-containing substrates and have been shown to dephosphorylate activated MAPK. We have identified a novel dsPTP, rVH6, from rat hippocampus. rVH6 contains the conserved dsPTP active site sequence, VXVHCX2GX2RSX5AY(L/I)M, and exhibits phosphatase activity against activated MAPK. In PC12 cells, rVH6 mRNA is induced during nerve growth factor-mediated differentiation but not during insulin or epidermal growth factor mitogenic stimulation. In MM14 muscle cells, rVH6 mRNA is highly expressed in proliferating cells and declines rapidly during differentiation. rVH6 expression correlates with the inability of fibroblast growth factor to stimulate MAPK activity in proliferating but not in differentiating MM14 cells. rVH6 protein localizes to the cytoplasm and is the first dsPTP to be localized outside the nucleus. This novel subcellular localization may expose rVH6 to potential substrates that differ from nuclear dsPTPs substrates.

Cultural competence--an essential hybrid for delivering high quality care in the 1990's and beyond.

Establishing guidelines for culturally competent medical care will help all physicians to fulfill their mandate to meet the health care needs of the individual patient as well as of patient populations, and to lower costs by encouraging a healing partnership with the patient, thus increasing patient responsibility for his or her own health. Being culturally sensitive is not enough. Nor is simply classifying patients according to race adequate. Many researchers in social science and health services increasingly agree that race in our heterogeneous U.S. population has limited biological meaning and more often than not is just a poor proxy for culture or socioeconomic status. Guidelines and quality indicators that seek to measure and improve cultural competence must take into account, in an integrated fashion, these three necessary components in the delivery of high-quality services to populations: 1) the health-related cultural factors; 2) the incidence and prevalence of diseases in the population; and 3) treatment outcomes peculiar to that population. To be culturally competent is to incorporate and integrate these critical factors into caring for diverse populations. If health care systems, individually and collectively, are to provide care of high quality that is cost-effective to all populations, researchers and funders of research must invest in an aggressive agenda that pursues two directions: the validation of existing quality indicators in minority populations, and the development of new quality indicators that assess the organization's ability to develop culturally competent care. Only in this way will those of us in the medical profession be able to fulfill our calling to relieve suffering without discriminating against some populations.

Acute gastroenteritis in three community-based nursing homes.

BACKGROUND: Acute gastroenteritis is a cause of considerable morbidity and mortality in the elderly population. A prospective assessment of acute diarrhea in three community-based long-stay homes is described. METHODS: A cohort study of acute gastroenteritis was performed in three community-based nursing homes, involving 572 residents over an 8-month period. Diarrhea cases were enrolled on the basis of the acute onset of loose stools of > or = 24 hours, as well as one of the following: a rectal temperature of > or = 100 degrees F, dehydration, positive occult blood, > or = 48 hours duration, or as a part of any outbreak. Stool cultures for Clostridium difficile were performed on all NH 1 patients. RESULTS: Fifty-three gastroenteritis cases were ascertained, consistent with incidence rates of 14.6, 36.4, and 6.7 cases/100 patient years in NH 1, NH 2, and NH 3, respectively. Requiring a Foley catheter (OR = 2.57; 95% CI, 0.93, 7.09) increased diarrhea risk. Six Clostridium difficile enteritis cases and an episode attributable to Aeromonas/Pleisomonas species were diagnosed. One C. difficile diarrhea case was imported from hospital to NH 1. Ten of 12 fecal excretors resided in close geographic clusters in NH 1, where a majority of the latter were mobile and incontinent of stool. CONCLUSIONS: Acute gastroenteritis was a common disease in the study nursing homes, for which specific risk factors were identified. A predominant role for Clostridium difficile in the taxonomy of nursing home diarrhea was suggested.

Identification of a hormonally regulated protein tyrosine phosphatase associated with bone and testicular differentiation.

Absence of the tyrosine kinase activity of c-src and c-fms results in impairment of bone remodeling. Such dysfunction underscores the importance of tyrosine phosphorylation, yet the role of protein tyrosine phosphatases in bone metabolism remains unexamined. We have isolated the cDNA for a novel receptor-like tyrosine phosphatase expressed in bone and testis named osteotesticular protein tyrosine phosphatase (OST-PTP). The deduced 1711-residue protein possesses an extracellular domain with 10 fibronectin type III repeats and a cytoplasmic region with two catalytic domains. In primary rat osteoblasts, the 5.8-kilobase OST-PTP transcript is up-regulated in differentiating cultures and down-regulated in late stage mineralizing cultures. In addition, a presumed alternate transcript of 4.8-5.0 kilobases, which may lack PTP domains, is present in proliferating osteoblasts, but not detectable at other stages. Parathyroid hormone, a modulator of bone function, as well as cyclic AMP analogues, increase OST-PTP mRNA 5-8-fold in UMR 106 cells. In situ hybridization of adult rat testis revealed stage-specific expression of OST-PTP. OST-PTP may function in signaling pathways during bone remodeling, as well as serve a broader role in cell interactions associated with differentiation in bone and testis.

Protein tyrosine phosphatases: characterization of extracellular and intracellular domains.

Protein tyrosine phosphatases (PTPs) play an important role in the regulation of cell growth and differentiation. With over 30 PTPs identified, the specific functions of these enzymes are now being addressed. The identification of extracellular domain receptor-like PTP interactions and the characterization of intracellular PTP 'targeting' domains represent recent efforts in this pursuit.

Hospitals' motivations in establishing or closing geriatric evaluation management units: diffusion of a new patient-care technology in a changing health care environment.

BACKGROUND: Although Geriatric Evaluation Management Units (GEMs) are beneficial to patients, they are still new and their adoption by hospitals is unknown. This study describes the adoption of GEMs in a large sample of hospitals, and explores the reasons underlying hospitals' decisions to open (and sometimes close) an inpatient GEM. METHODS: A nationwide mail survey was conducted of 3,655 hospitals. The survey asked whether the hospital had an operating GEM, had a GEM that closed, had considered opening a GEM (but had not done so), or had not considered opening a GEM. The survey also requested specific information about operating or closed GEMs. Descriptive statistics, chi-square, t-tests, one-way analysis of variance, and Tukey's standardized range test for multiple comparison of means were used to analyze the responses. RESULTS: Among the 1,639 responding hospitals, 159 had established GEMs, 200 were evaluating the possibility of opening a GEM, and 1,263 had neither opened nor considered opening a GEM. Adopters were more likely to be large, urban, teaching hospitals. Evaluators were more optimistic than adopters about GEM's potential to meet financial goals. GEMs that closed tended to be located in hospitals experiencing budget deficits. Among adopters, space and nonphysician staffing were the most critical barriers to establishing a GEM whereas, for evaluators, identifying reimbursement sources and physician staffing were the greatest barriers. VA GEMs are smaller and initiated for different reasons than non-VA hospital GEMs. CONCLUSIONS: Despite their demonstrated usefulness, the adoption of GEMs has been limited. The reasons underlying decisions to adopt this new technology or close a GEM are often related to financial, not clinical concerns.

Inositol 1,4,5-trisphosphate receptors: immunohistochemical localization to discrete areas of rat central nervous system.

The second messenger inositol 1,4,5-trisphosphate triggers the release of intracellular Ca2+ stores upon binding to the inositol 1,4,5-trisphosphate receptor protein, a calcium channel that has been purified and molecularly cloned. To clarify the roles of inositol 1,4,5-trisphosphate receptor in the central nervous system, we have examined in detail the distribution of inositol 1,4,5-trisphosphate receptors in the rat brain and spinal cord using immunohistochemical methods. Inositol 1,4,5-trisphosphate receptors are present in neuronal cells, fibers and terminals in a wide distribution of areas throughout the central nervous system. These include a number of areas not previously reported, such as the olfactory bulb, thalamic nuclei and dorsal horn of the spinal cord. In addition, we have noted a strikingly high density of inositol 1,4,5-trisphosphate receptors in circumventricular organs and neuroendocrine structures such as the area postrema, choroid plexus, subcommisural organ, pineal gland and pituitary. The distribution of inositol 1,4,5-trisphosphate receptors in discrete structures throughout the central nervous system, including interconnected neuronal systems and neuroendocrine and circumventricular organ structures, presumably reflects the importance of Ca2+ release mediated by the phosphoinositide second messenger system in control of diverse physiological processes.

Inositol 1,4,5-trisphosphate receptors: labeling the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands.

We have photolabeled the inositol 1,4,5-trisphosphate (IP3) receptor and probed the IP3 ligand binding site using two novel photoaffinity ligands, [125I] (azidosalicyl)aminopropyl-IP3 ([125I]ASA-IP3) and [3H] (benzoyldihydrocinnamyl)aminopropyl-IP3 ([3H]BZDC-IP3). Both ligands have high affinity for the IP3 receptor and, when photoactivated, label the IP3 receptor protein with appropriate inositol phosphate selectivity. The high specific activity of [125I]ASA-IP3 allowed identification of a single photolabeling site within the IP3R by two-dimensional peptide analysis. Substantially higher levels of incorporation into the receptor are achieved with [3H]BZDC-IP3 (50-60% efficiency) than with [125I]ASA-IP3 (3%), facilitating the use of [3H]BZDC-IP3 as a better ligand for the high-efficiency labeling and purification of IP3R-labeled peptides. Peptides were generated from photolabeled IP3 receptor by trypsin digestion and purified by high-pressure liquid chromatography (HPLC). A single purified [3H]BZDC-IP3-labeled peptide, corresponding to IP3R amino acids 476-501, was sequenced and shown to match specific sequences in the N-terminal 20% of the IP3 receptor, an area suggested on the basis of mutagenesis studies to contain the IP3 recognition site.

[3H]noscapine binding sites in brain: relationship to indoleamines and the phosphoinositide and adenylyl cyclase messenger systems.

High affinity [3H]noscapine binding sites are brain specific, ion insensitive, and present in a variety of species and show strict structure-activity requirements. Among neurotransmitter-related structures, indoleamines and beta-carbolines display highest affinity for [3H]noscapine sites. Noscapine inhibits carbachol-stimulated phosphoinositide turnover in guinea pig and rat brain slices, with structural analogs possessing similar relative potencies for binding to [3H]noscapine binding sites and inhibiting phosphoinositide turnover. Noscapine and its derivatives also markedly enhance the ability of forskolin to augment cAMP levels in brain slices, with relative potencies paralleling affinities for noscapine binding sites.

Consensus among geriatric experts on the components of a complete nursing-home admission assessment.

Standards of practice for nursing-home admissions have not been established, and it is not known if geriatric clinicians agree on the components of an appropriate patient-admission evaluation. This study describes the consensus expressed by experts in clinical geriatrics regarding the most important components of nursing-home admission assessments. Directors of all geriatric-nurse-practitioner and geriatric-medicine fellowship programs (n = 79) were sent a two-round questionnaire asking them to describe and rate the components of a "complete admission assessment for every elderly patient entering a nursing home." Nurse practitioners and physicians ranked medication review, evaluation of urinary incontinence, mental status, vision and bowel status the highest, and gave them equal priority. Routine laboratory tests were not included among the highest-ranked items by either nurses or doctors. An enhanced assessment focusing on the functional consequences of diseases rather than on the traditional "head-to-toe" systems approach emerged as the most appropriate assessment for elderly nursing-home patients.

Photoaffinity labeling and characterization of isolated inositol 1,3,4,5-tetrakisphosphate- and inositol hexakisphosphate-binding proteins.

We have isolated high affinity inositol (1,3,4,5)-tetrakisphosphate (IP4)- and inositol hexakisphosphate (IP6)-binding proteins from detergent-solubilized rat brain membranes using a P1-tethered IP4 derivative linked to an Affi-Gel support. To determine the identity, binding characteristics, and distribution of the individual IP4 recognition sites, we have synthesized an IP4 photoaffinity label probe, 125I-(D,L)-1-O-[N-(4-azidosalicyloxy)-3-aminopropyl-1-phospho]- IP4 (125I-ASA-IP4). Two apparently distinct IP4-binding proteins (IP4BP), isolated with the IP4 affinity column, display high affinity and selectivity for IP4 over inositol trisphosphate (IP3), inositol pentakisphosphate (IP5), and IP6. The first IP4-binding protein (IP4BP1) which has a KD for IP4 of 4 nM, is comprised of a protein at 182 kDa which is specifically photolabeled with high affinity by 125I-ASA-IP4. The second, IP4BP2, has an affinity for IP4 of 1.5 nM and contains proteins at 84 and 174 kDa, both of which are specifically photoaffinity labeled. A putative IP6-binding protein (IP6BP), also isolated with the IP4 affinity column, binds IP6 with a KD of 14 nM and comprises three proteins of 115, 105, and 50 kDa. The 115- and 105-kDa subunits, but not the 50-kDa subunit, specifically incorporate the photolabel. The IP4BP (182, 174, and 84 kDa) and IP6BP (115 and 105 kDa) proteins are specifically photolabeled in the crude membrane, partially purified, and purified fractions. These receptor-binding proteins vary in inositol phosphate specificity and in the effects of pH, Ca2+, and heparin on IP4 photoaffinity labeling. In addition, IP4BP and IP6BP are enriched in the brain but differ in their regional localizations within the brain.

Ability of surrogates to represent satisfaction of nursing home residents with quality of care.

OBJECTIVE: To measure the ability of surrogates to accurately represent nursing home residents' satisfaction with the nursing home care. DESIGN: Comparison by correlation analysis of questionnaire answers by nursing-home residents and their designated surrogates. SETTING: Four non-profit community nursing homes. PARTICIPANTS: One-hundred fifty-two resident-surrogate pairs were included, based on the following criteria: (1) the resident was able to respond to questions verbally and in English, had cognitive abilities sufficient to understand the questions, and had a responsible party who had a telephone number in the medical record; (2) both the resident and the surrogate agreed to be interviewed. OUTCOME MEASURES: A 26-item instrument (21 specific and 5 global items) was developed to measure surrogates' perceptions of residents' satisfaction with the quality of the physician services, nursing care, and the nursing home environment. The instrument was scored on a 4-point Likert scale in which higher scores indicated greater satisfaction and paralleled a similar instrument designed for nursing home residents. Correlation of residents' with surrogates' scores on the satisfaction instruments was examined. RESULTS: The mean score for most items was greater than 3.0, indicating overall satisfaction with the care. Correlations between surrogates and residents on specific items ranged from 0.1 to 0.55. Correlations were highest for global items and items addressing satisfaction with the environment. CONCLUSION: We conclude that nursing home residents' surrogates cannot accurately express the residents' satisfaction with all areas of nursing home care and that their evaluations should not be taken in lieu of the residents' opinions.

Amantadine-related adverse reactions among African-American elderly nursing home residents.

To identify factors associated with the development of adverse reactions to amantadine prophylaxis for influenza in a predominantly African-American nursing home population, we retrospectively reviewed the records of 100 residents who did and 45 who did not receive amantadine. During the 4 weeks of amantadine treatment, three independent observers rated all new symptoms as either related or unrelated to amantadine. Two types of comparison were made. Among patients receiving amantadine, those who did or did not develop new symptoms believed to be related to amantadine were compared by age, underlying diagnoses, type and number of medications, and renal function. Similar comparisons were made between patients who did and those who did not receive amantadine. The 100 residents who received amantadine prophylaxis had significantly more nonspecific symptoms than the 45 who did not receive amantadine. Otherwise, the two groups were comparable with regard to age, renal function, number of diagnoses, and medications. Of the 100 residents given amantadine, 16% were judged to experience amantadine-related adverse reactions. Those developing amantadine-related symptoms were significantly more likely to have nonspecific symptoms and to use psychotropic medications.

Purification and characterization of the inositol 1,4,5- trisphosphate receptor protein from rat vas deferens.

Among rat peripheral tissues examined, Ins(1,4,5)P(3) receptor binding is highest in the vas deferens, with levels about 25% of those of the cerebellum. We have purified the InsP(3) receptor binding protein from rat vas deferens membranes 600-fold. The purified protein displays a single 260 kDa band on SDS/PAGE, and the native protein has an apparent molecular mass of 1000 kDa, the same as in cerebellum. The inositol phosphate specificity, pH-dependence and influence of various reagents are the same for purified vas deferens and cerebellar receptors. Whereas particulate InsP(3) binding in cerebellum is potently inhibited by Ca(2+), particulate and purified vas deferens receptor binding of InsP(3) is not influenced by Ca(2+). Vas deferens appears to lack calmedin activity, but the InsP(3) receptor is sensitive to Ca(2+) inhibition conferred by brain calmedin. The vas deferens may prove to be a valuable tissue for characterizing functional aspects of InsP(3) receptors.

Financing long-term care. An insurance-based approach.

A joint public-private insurance program is the best approach to resolving the problem of financing long-term care. In this report, we describe one possible approach in detail. A modest expansion of the current (ie, after repeal of the Medicare Catastrophic Coverage Law of 1988) Medicare benefit for persons needing relatively short-term nursing home and home care services would be a first step. For those with extended long-term service needs, a non-means tested, publicly funded program with joint federal-state financing and administration would provide coverage after a substantial elimination period and with an income-related copayment. Private long-term care insurance purchased through employers before retirement or in the periretirement period, through use of income or equity accumulated in life insurance, pension funds, or home ownership, would be used to fund the exclusionary period or copayments of the public program by those who wish to have greater protection for income or assets. The role of Medicaid would be limited to paying for the deductible, copayments, and initial long-stay expenses of those with low incomes and limited assets.