Long term cryostorage of UC blood units: ability of the integral segment to confirm both identity and hematopoietic potential.

BACKGROUND: With the maturation of UC blood banking, cord blood (CB) units stored for years prior to use in transplantation present a new set of issues in clinical transplantation, including interval improvements in immune typing and confirmation of product identity and viability. A preliminary analysis of the transplants supported by the St Louis Cord Blood Bank, looking for an impact of length of CB storage time and transplant outcome was performed. We evaluated the utility of an integral segment containing an aliquot of cryopreserved product that has been exposed to the same post-processing storage conditions as the unit as a quality control tool for CB banking. METHODS: Engraftment and survival following unrelated donor UC blood transplant were evaluated based on length of CB product storage at the St Louis Cord Blood Bank. A strategy of routine testing of the contiguous segment for high-resolution HLA typing (also confirming identity) and CFU analysis was tested in 283 consecutive CB searches. Comparison between CB unit and contiguous segment viability and hematopoietic potential was performed on 30 research CB units that had been stored up to 5 years. RESULTS: There was no statistical difference in engraftment or survival following unrelated donor cord blood transplant employing units banked < 1 year or > 3 years. Confirmatory HLA typing, CFU and viability testing was successfully performed from the same segment as part of a strategy for product release evaluation. When comparing the segment with its corresponding CB unit, the total colony-forming units (CFU) measured in the two was similar (P = 0.51, paired t-test). Three research units purposely sabotaged by an overnight thaw and refreeze had no CFU growth, but viability as measured by Trypan was still 68-98%. DISCUSSION: No deterioration of hematopoietic potential has been detected with storage up to 5 years. The contiguous segment CFU is representative of the product, and thus is a useful tool for quality control and confirmation of product viability. Viability, as measured by Trypan blue dye exclusion may be falsely reassuring.

A sodium channel signaling complex: modulation by associated receptor protein tyrosine phosphatase beta.

Voltage-gated sodium channels in brain neurons were found to associate with receptor protein tyrosine phosphatase beta (RPTPbeta) and its catalytically inactive, secreted isoform phosphacan, and this interaction was regulated during development. Both the extracellular domain and the intracellular catalytic domain of RPTPbeta interacted with sodium channels. Sodium channels were tyrosine phosphorylated and were modulated by the associated catalytic domains of RPTPbeta. Dephosphorylation slowed sodium channel inactivation, positively shifted its voltage dependence, and increased whole-cell sodium current. Our results define a sodium channel signaling complex containing RPTPbeta, which acts to regulate sodium channel modulation by tyrosine phosphorylation.

Integrating outcomes into computerized information systems. It's time to get guidelines off the shelf.

The tools used to measure outcomes in the 21st century probably will look nothing like the tools used to measure outcomes during the past century. These guideposts (guidelines, information systems, and vocabulary) are only three of the many new tools that will be available to us through technology. The three guideposts described in this column are necessary ingredients for success in managing outcomes and demonstrating accountability. They look like the lampposts to a bright future.

The extracellular domain of the beta1 subunit is both necessary and sufficient for beta1-like modulation of sodium channel gating.

The type IIA voltage-gated sodium Na(+) channel from rat brain is composed of a large, pore-forming alpha subunit and the auxiliary subunits beta1 and beta2. When expressed in Xenopus oocytes, the beta1 subunit modulates the gating properties of the type IIA alpha subunit, resulting in acceleration of both inactivation and recovery from inactivation and in a negative shift in the voltage dependence of fast inactivation. The beta1 subunit is composed of an extracellular domain with a single immunoglobulin-like fold, a single transmembrane segment, and a small intracellular domain. A series of chimeras with exchanges of domains between the Na(+) channel beta1 and beta2 subunits and between beta1 and the structurally related protein myelin P0 were constructed and analyzed by two-microelectrode voltage clamp in Xenopus oocytes. Only chimeras containing the beta1 extracellular domain were capable of beta1-like modulation of Na(+) channel gating. Neither the transmembrane segment nor the intracellular domain was required for modulation, although mutation of Glu(158) within the transmembrane domain altered the voltage dependence of steady-state inactivation. A truncated beta1 subunit was engineered in which the beta1 extracellular domain was fused to a recognition sequence for attachment of a glycosylphosphatidylinositol membrane anchor. The beta1(ec)-glycosylphosphatidylinositol protein fully reproduced modulation of Na(+) channel inactivation and recovery from inactivation by wild-type beta1. Our findings demonstrate that extracellular domain of the beta1 subunit is both necessary and sufficient for the modulation of Na(+) channel gating.

Functional roles of the extracellular segments of the sodium channel alpha subunit in voltage-dependent gating and modulation by beta1 subunits.

Voltage-gated sodium channels consist of a pore-forming alpha subunit associated with beta1 subunits and, for brain sodium channels, beta2 subunits. Although much is known about the structure and function of the alpha subunit, there is little information on the functional role of the 16 extracellular loops. To search for potential functional activities of these extracellular segments, chimeras were studied in which an individual extracellular loop of the rat heart (rH1) alpha subunit was substituted for the corresponding segment of the rat brain type IIA (rIIA) alpha subunit. In comparison with rH1, wild-type rIIA alpha subunits are characterized by more positive voltage-dependent activation and inactivation, a more prominent slow gating mode, and a more substantial shift to the fast gating mode upon coexpression of beta1 subunits in Xenopus oocytes. When alpha subunits were expressed alone, chimeras with substitutions from rH1 in five extracellular loops (IIS5-SS1, IISS2-S6, IIIS1-S2, IIISS2-S6, and IVS3-S4) had negatively shifted activation, and chimeras with substitutions in three of these (IISS2-S6, IIIS1-S2, and IVS3-S4) also had negatively shifted steady-state inactivation. rIIA alpha subunit chimeras with substitutions from rH1 in five extracellular loops (IS5-SS1, ISS2-S6, IISS2-S6, IIIS1-S2, and IVS3-S4) favored the fast gating mode. Like wild-type rIIA alpha subunits, all of the chimeric rIIA alpha subunits except chimera IVSS2-S6 were shifted almost entirely to the fast gating mode when coexpressed with beta1 subunits. In contrast, substitution of extracellular loop IVSS2-S6 substantially reduced the effectiveness of beta1 subunits in shifting rIIA alpha subunits to the fast gating mode. Our results show that multiple extracellular loops influence voltage-dependent activation and inactivation and gating mode of sodium channels, whereas segment IVSS2-S6 plays a dominant role in modulation of gating by beta1 subunits. Evidently, several extracellular loops are important determinants of sodium channel gating and modulation.

Molecular determinants of Na+ channel function in the extracellular domain of the beta1 subunit.

The rat brain voltage-gated Na+ channel is composed of three glycoprotein subunits: the pore-forming alpha subunit and two auxiliary subunits, beta1 and beta2, which contain immunoglobulin (Ig)-like folds in their extracellular domains. When expressed in Xenopus oocytes, beta1 modulates the gating properties of the channel-forming type IIA alpha subunit, resulting in an acceleration of inactivation. We have used a combination of deletion, alanine-scanning, site-directed, and chimeric mutagenesis strategies to examine the importance of different structural features of the beta1 subunit in the modulation of alphaIIA function, with an emphasis on the extracellular domain. Deletion analysis revealed that the extracellular domain is required for function, but the intracellular domain is not. The mutation of four putative sites of N-linked glycosylation showed that they are not required for beta1 function. Mutations of hydrophobic residues in the core beta sheets of the Ig fold disrupted beta1 function, whereas substitution of amino acid residues in connecting segments had no effect. Mutations of acidic residues in the A/A' strand of the Ig fold reduced the effectiveness of the beta1 subunit in modulating the rate of inactivation but did not significantly affect the association of the mutant beta1 subunit with the alphaIIA subunit or its effect on recovery from inactivation. Our data suggest that the Ig fold of the beta1 extracellular domain serves as a scaffold that presents the charged residues of the A/A' strands for interaction with the pore-forming alpha subunit.

Formation of the b subunit dimer is necessary for interaction with F1-ATPase.

In earlier work, we [McCormick, K. A., et al. (1993) J. Biol. Chem. 268, 24683-24691] observed that mutations at Ala-79 of the b subunit affect assembly of F1F0 ATP synthase. Polypeptides modeled on the soluble portion of the b subunit (bsol) with substitutions at the position corresponding to Ala-79 have been used to investigate secondary structure and dimerization of the b subunit. Circular dichroism spectra and chymotrypsin digestion experiments suggested that the recombinant polypeptides with Ala-79 substitutions assumed conformations similar to the bsol polypeptide. However, cross-linking studies of the Ala-79 substitution bsol polypeptides revealed defects in dimerization. The efficiency of dimer formation appeared to be related to the capacity of the altered bsol polypeptides for competing with F1-ATPase for binding to F1-depleted membrane vesicles. Ala-79 substitution polypeptides displaying limited dimerization, such as bsol Ala-79-->Leu, were shown to elute with F1-ATPase during size exclusion chromatography, suggesting a specific interaction. Sedimentation equilibrium studies indicated that 8% of the bsol Ala-79-->Leu polypeptide was in the form of a 30.6 kDa dimer and 92% a 15.3 kDa monomer. When the dimer concentration of bsol Ala-79-->Leu was normalized to the concentration of bsol, both had virtually identical capacities for competing with F1-depleted membrane vesicles for binding F1-ATPase. The result indicated that the amount of dimer formed is directly proportional to its ability to bind F1-ATPase. This suggests that formation of the b subunit dimer may be a necessary step preceding F1-ATPase binding in the assembly of the enzyme complex.

New tools--new models to integrate outcomes into quality measurement.

Nurse managers are faced with a changing health care system that values measurement of outcomes and performance. Today's nurse managers are confronted with the need for information to make decisions that translate information into knowledge about health care outcomes, effectiveness, efficiency, and quality. These same nurse managers are being held accountable for the efforts of nurses to enhance the health of the public. New models of evidence and outcomes integrated into the models of nursing process are required to measure quality in this new environment. Yet the measurement of outcomes and the measurement of quality should be united in this new paradigm. The author describes new tools available from the Agency for Health Care Policy and Research that could assist nurse managers in delivering and developing computerized patient record systems that capture the evidence, the clinical indicators, and the performance measures to assist in describing the outcomes of care.

The role of the Agency for Health Care Policy and Research (AHCPR) in improving outcomes of care.

This article describes the various outcomes programs supported by the Agency for Health Care Policy and Research (AHCPR). The mission of the agency is to generate and disseminate information that improves the delivery and quality of health care. The agency is charged with helping consumers, providers, purchasers, health plans, and policy makers meet the challenge of improving the quality of health care services while reducing spending. AHCPR has been recognized as funding the development of "gold standard" clinical practice guidelines and the source of unbiased, science-based information on what works and does not work in health care.

Improving nursing documentation to include outcomes of care in computerized information systems.

Often times when models of information systems are developed, outcomes are either left out or described as an end product of treatment alone. However, in research demonstrating outcomes, evaluating whether outcomes are achieved can best be accomplished when the outcomes are integrated into the entire care process. This paper describes a model for nursing to consider when integrating outcomes during several components of nursing care delivery, and several nursing domains for achieving outcome of care.

Including oncology outcomes of care in the computer-based patient record.

Changes in the health care system have caused a shift in research to outcomes of care, effectiveness, efficiencies, clinical practice guidelines, and costs. The greater use of computer systems, including decision support systems, quality assurance systems, effectiveness systems, cost containment systems, and networks, will be required to integrate administrative and patient care data for use in determining outcomes and resource management. This article describes developments to look forward to in the decade ahead, including the integration of outcomes data and clinical practice guidelines as content into computer-based patient records; the development of review criteria from clinical practice guidelines to be used in translating guidelines into critical paths; and feedback systems to monitor performance measures and benchmarks of care, and ultimately cost out cancer care.

Modulation of cardiac Na+ channel expression in Xenopus oocytes by beta 1 subunits.

Voltage-gated Na+ channels consist of a large alpha subunit of 260 kDa associated with beta 1 and/or beta 2 subunits of 36 and 33 kDa, respectively. alpha subunits of rat cardiac Na+ channels (rH1) are functional when expressed alone in Xenopus oocytes or mammalian cells. beta 1 subunits are present in the heart, and localization of beta 1 subunit mRNA by in situ hybridization shows expression in the perinuclear cytoplasm of cardiac myocytes. Coexpression of beta 1 subunits with rH1 alpha subunits in Xenopus oocytes increases Na+ currents up to 6-fold in a concentration-dependent manner. However, no effects of beta 1 subunit coexpression on the kinetics or voltage dependence of the rH1 Na+ current were detected. Increased expression of Na+ currents is not observed when an equivalent mRNA encoding a nonfunctional mutant beta 1 subunit is coexpressed. Our results show that beta 1 subunits are expressed in cardiac muscle cells and that they interact with alpha subunits to increase the expression of cardiac Na+ channels in Xenopus oocytes, suggesting that beta 1 subunits are important determinants of the level of excitability of cardiac myocytes in vivo.

Toward a uniform language for nursing in the US: work of the American Nurses Association Steering Committee on databases to support clinical practice.

This paper reports on the work of the American Nurses Association Steering Committee on Databases to Support Clinical Practice, in existence since 1989. Responding to its broad charges, the Steering Committee has laid down the foundations for its work in declaring the nursing process as the framework for nursing data in database systems, and in endorsing the Nursing Minimum Data Set as the set of minimum elements for any system designed to carry health-related data that reflects nursing care. In addition, the Steering Committee has begun initiatives to: 1) promote the inclusion of nursing-related data in large health-related databases, and 2) develop a Uniform Language for nursing through a phased approach. The Steering Committee also works directly with the International Council of Nurses to promote the inclusion of nursing data in internationally used classification systems and to develop an international language that describes nursing care.

Toward standard classification schemes for nursing language: recommendations of the American Nurses Association Steering Committee on Databases to Support Clinical Nursing Practice.

The American Nurses Association (ANA) Cabinet on Nursing Practice mandated the formation of the Steering Committee on Databases to Support Clinical Nursing Practice. The Committee has established the process and the criteria by which to review and recommend nursing classification schemes based on the ANA Nursing Process Standards and elements contained in the Nursing Minimum Data Set (NMDS) for inclusion of nursing data elements in national databases. Four classification schemes have been recognized by the Committee for use in national databases. These classification schemes have been forwarded to the National Library of Medicine (NLM) for inclusion in the Unified Medical Language System (UMLS) and to the International Council of Nurses for the development of a proposed International Classification of Nursing Practice.

Urinary incontinence: a program that works.

Using a simple and inexpensive intervention, improvement in dryness was apparent by the first week of the study. More importantly, results were achieved by changing staff behavior, rather than residents' behavior.

The effects of changing prompted voiding schedules in the treatment of incontinence in nursing home residents.

OBJECTIVE: To determine the effects of different prompted voiding schedules on urinary incontinence on a continence unit (CU) and the maintenance of benefits on normal nursing units. DESIGN: Multiphase study with both intra- and inter-subject comparisons. PARTICIPANTS: Subjects were 41 consenting incontinent nursing home residents. Based on clinical criteria, subjects were assigned to one of four treatment groups that varied as to the schedule of prompted voiding received. MEASUREMENT: The study used chart review, Katz ADL, and MMSE. Urologic status, self-initiated toileting, urine volumes voided, and incontinence assessed by pad/pants checks were measured by research nurses. Baseline pad check data were collected on residents' normal nursing units. Residents were transferred to the CU where baseline measurement was repeated, and the effects of different prompted voiding schedules were then assessed. Indigenous staff were trained to use prompted voiding, and nurse supervisors were instructed in special procedures for enhancing maintenance of the intervention. Residents were returned to their normal units and the maintenance of improvements in continence status was assessed at 2 weeks and 3 months post-CU discharge. RESULTS: One of the four groups showed significant improvement on the CU in response to the 2-hour schedule; two groups improved on the less intensive 3-hour schedule (P < 0.05). Two groups maintained this improvement on their normal nursing units (P < 0.05); one group showed a non-significant trend toward improvement. Self-initiated toileting decreased (P < 0.05) and volume voids in an appropriate receptacle increased (P < 0.05) during training. CONCLUSIONS: Prompted voiding is an effective treatment for urinary incontinence, and a less intensive 3-hour schedule may be superior to the standard 2-hour schedule for some residents. These improvements in dryness can be maintained by normal nursing home staff if formal staff management procedures are utilized by nurse supervisors.

Characterization of mutations in the b subunit of F1F0 ATP synthase in Escherichia coli.

Site-directed mutagenesis was used to investigate the restrictions on Ala-79 of the b subunit in F1F0 adenosine triphosphate synthase. This amino acid had been previously identified as particularly sensitive to mutation (McCormick, K. A., and Cain, B. D. (1991) J. Bacteriol. 173, 7240-7248). Mutant uncF (b) genes were placed under control of the lac promoter and monitored for F1F0 ATP synthase function in an uncF(b) deletion strain. Three deleterious bAla-79 mutations were moved to the unc operon in the chromosome by homologous recombination. Decreases in enzymatic activity in the uncF (b) mutant strains resulted from reduced amounts of enzyme. With the exception of the bAla-79-->Pro mutation, high expression of mutant uncF (b) genes resulted in increases in F1F0 ATP synthase activity which were sufficient to overcome the defects. In addition to the decrease in the amount of enzyme, the bAla-79-->Lys mutation affected ATP synthesis to a much greater extent than ATP-driven proton translocation. The evidence supports our earlier hypothesis, in which bAla-79 was proposed to play an important, but not essential, structural role in F1F0 ATP synthase assembly or stability.

Behavioral management strategies for urinary incontinence.

Behavioral interventions are recommended by Agency for Health Care Policy and Research Clinical Guideline as the first line of treatment for most types of urinary incontinence. This article defines and briefly describes the six behavioral treatment techniques and discusses the research base for the efficacy of each type of intervention. Caveats and needed research are also presented.