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.