Effects of an in-house coordinator and practitioner referral rather than proxy referral on tissue donation rates.

INTRODUCTION: Timely referral of patients following asystolic death to an organ procurement organization (OPO) may increase tissue donation rates. Lack of education of health care providers and nonphysicians (admitting department) about timely referral to the OPO following asystolic death may adversely affect tissue donation rates. We hypothesized that using an in-house donation coordinator for provider education and changing the responsibility for calling the OPO from the admitting department to the licensed independent practitioner (LIP) declaring death would increase timely referral and tissue donation rates. METHODS: An education program was developed in 2005 by a newly hired in-house coordinator to highlight the importance of tissue donation. In addition, to improve timely referrals to the OPO after death, the instructions accompanying the working copy of the death certificate were altered to require the patient's LIP to call the OPO within 1 hour of death (early 2007). Rates for both timely referrals and tissue donors were modeled by a Poisson regression model with a log link function. RESULTS: Timely referral rates rose from 48% before the interventions to 72% after the intervention (P < .0001). The number of tissue donors per number of referrals also increased significantly (P = .025) over that period. CONCLUSIONS: An in-house donation coordinator initiated education program and LIP referral rather than referral by other parties following asystolic death results in higher tissue donation rates.

Quality and outcomes in anaesthesia: lessons from litigation.

Healthcare litigation in the UK continues to grow at an alarming rate, with claims against anaesthetists and critical care physicians increasing each year. This has led to a huge financial burden for the taxpayer and a sharp increase in professional indemnity fees for individual doctors. Although such litigation should provide valuable information to educate practitioners and reduce future similar claims, there appear to be significant barriers preventing important lessons from being learned. Detailed learning opportunities are available only to the healthcare providers being sued or the expert witnesses employed to analyse the claims. Most practitioners have to rely on indemnifiers' case reports, closed-claim analyses, and ad hoc publications for information. In this review, we suggest ways in which important lessons from litigation could be brought to the attention of all clinicians. Currently, most clinicians are unable to determine whether key components of their practice such as consent, clinical decision-making, and documentation are of an acceptable standard for legal scrutiny. By reporting outcomes of Coroners' inquests, clinical and criminal negligence cases, and referrals to the General Medical Council, it would be hoped that more explicit standards of performance could be derived. Ultimately, this may not only improve patient safety, but protect practitioners from unjustifiable claims. Finally, given the critical importance of experts in the above process, we believe that a system for professional registration and regulation should be explored to ensure that they offer accurate, representative, and unbiased opinions and have the appropriate expertise in the subject matter to be analysed.

Leitmotifs in the biochemistry of LTP induction: amplification, integration and coordination.

Hippocampal long-term potentiation (LTP) is a robust and long-lasting form of synaptic plasticity that is the leading candidate for a cellular mechanism contributing to mammalian learning and memory. Investigations over the past decade have revealed that the biochemistry of LTP induction involves mechanisms of great subtlety and complexity. This review highlights themes that have emerged as a result of our increased knowledge of the signal transduction pathways involved in the induction of NMDA receptor-dependent LTP in area CA1 of the hippocampus. Among these themes are signal amplification, signal integration and signal coordination. Here we use these themes as an organizing context for reviewing the profusion of signaling mechanisms involved in the induction of LTP.

Correlation between molecular and conventional genealogies in Aicuña: a rural population from Northwestern Argentina.

Aicuña is a village in the northwest of Argentina, located about 300 km south of La Rioja city, in the province of La Rioja. The population of Aicuña derives from a founder couple established in the uninhabited Aicuña valley in the early years of the 17th century. Due to land ownership litigation, the descendants maintained a well-documented genealogy that extends for 12 generations, comprising more than 8,000 individuals. From the historical pedigree of Aicuña, we selected 14 males with direct patrilineal descent from the 2 most ancient male founders, and 23 donors (9 females and 14 males) with direct matrilineal descent from the most ancient female founder. All 3 founders lived in the 17th century. We collected DNA from buccal swabs and characterized the mitochondrial DNA (mtDNA) and Y haplotypes using 14 Y-specific markers, 11 mtDNA polymorphic markers and sequencing of the mt hypervariable regions 1 and 2. We found four different Y haplotypes: Y1 and Y2 haplotypes of European origin corresponding to the founder ancestors Francisco Páez de Espinoza and Apolinario Ormeño, which were shared by 6 and 3 donors, respectively. Three males selected as Ormeño patrilineal descendants showed a different Y haplotype (Y3), probably originated by erroneous paternity registration due to illegitimacy. The remaining case (haplotype Y4), also assumed to belong to the Ormeño lineage, was probably also due to an erroneously registered paternity. Twenty-two donors showed an association of mtDNA markers corresponding to the Amerindian haplotype A2. The founder of this matrilineage could be traced back for more than 14 generations. The haplotype B of one remaining female did not correspond with the historical pedigree and could be due to an error in the genealogy registration. Our results showed an 85% agreement between conventional and molecular genealogies, with mtDNA markers being Amerindian, and Y markers being European. The methodology used in this report is a tool which could potentially be employed as a precedent for land ownership by Aicuña villagers and Amerindian populations.

Radiation therapy and concomitant paclitaxel/carboplatin chemotherapy for muscle invasive transitional cell carcinoma of the bladder: a well-tolerated combination.

This review evaluates tolerance and disease control for eight patients with muscle invasive bladder cancer treated with pelvic radiotherapy and concomitant paclitaxel/carboplatin chemotherapy. From October 1996 through February 1998, eight patients were treated with pelvic radiotherapy and concomitant paclitaxel/carboplatin chemotherapy. All received from 39.60-41.40 Gy to the pelvis followed by a boost to the initial site of disease. Final tumor doses ranged from 64.80-68.40 Gy. Most patients received paclitaxel at 150 mg/m2 and carboplatin at an area under the curve (AUC) of 7 at 3-week cycles during the radiation therapy. No patient required treatment interruption. With a median follow-up of 27 months, three patients remain free of local and distant disease at follow-up intervals of 24, 25, and 31 months. No surviving locally controlled patient demonstrated late urinary or gastrointestinal morbidity. All patients with a visibly complete transurethral resection of bladder tumor (TURBT) prior to radiotherapy achieved local disease control. For this group of patients, the absolute 2-year pelvic tumor control rate is 57%. The 2-year disease-specific survival is 43%. Paclitaxel/carboplatin chemotherapy can be delivered with continuous course pelvic radiation therapy without severe acute or apparent late toxicity. This combination also appears to be effective in achieving disease control in the urinary bladder, particularly in those patients who have undergone a thorough TURBT. The authors believe that it would be reasonable to investigate this combination in future bladder conservation protocols. The combination of paclitaxel and carboplatin with radiotherapy may be of particular value in elderly patients or those with renal impairment.

The A-type potassium channel Kv4.2 is a substrate for the mitogen-activated protein kinase ERK.

The mitogen-activated protein kinase ERK has recently become a focus of studies of synaptic plasticity and learning and memory. Due to the prominent role of potassium channels in regulating the electrical properties of membranes, modulation of these channels by ERK could play an important role in mediating learning-related synaptic plasticity in the CNS. Kv4.2 is a Shal-type potassium channel that passes an A-type current and is localized to dendrites and cell bodies in the hippocampus. The sequence of Kv4.2 contains several consensus sites for ERK phosphorylation. In the present studies, we tested the hypothesis that Kv4.2 is an ERK substrate. We determined that the Kv4.2 C-terminal cytoplasmic domain is an effective ERK2 substrate, and that it is phosphorylated at three sites: Thr(602), Thr(607), and Ser(616). We used this information to develop antibodies that recognize Kv4.2 phosphorylated by ERK2. One of our phospho-site-selective antibodies was generated using a triply phosphorylated peptide as the antigen. We determined that this antibody recognizes ERK-phosphorylated Kv4.2 in COS-7 cells transfected with Kv4.2 and native ERK-phosphorylated Kv4.2 in the rat hippocampus. These observations indicate that Kv4.2 is a substrate for ERK in vitro and in vivo, and suggest that ERK may regulate potassium-channel function by direct phosphorylation of the pore-forming alpha subunit.

MAPK regulation of gene expression in the central nervous system.

Long-term potentiation (LTP), a cellular model for long-term memory, is generally acknowledged to consist of both a short-term phase that is characterized by a dependence on autonomous protein kinase activity, and a long-term phase that is characterized by a dependence on changes in gene expression and new protein synthesis. Similarly, long-term memory exhibits a dependence on gene expression and altered protein synthesis. Recent evidence indicates that the mitogen-activated protein kinase (MAPK) cascade plays a role in both LTP and long-term memory. The MAPK cascade has heretofore largely been studied in the context of cell division and proliferation and as such, mechanisms for the regulation of gene expression by the MAPK cascade have received considerable attention. Given the possible role of altered gene expression in the late phase of LTP and in long-term memory, we evaluated the capacity of the MAPK ERK (extracellular signal-regulated kinase) to regulate phosphorylation of the transcription factor cAMP response element binding protein (CREB) in hippocampal area CA1. Our studies indicate a critical role for the MAPK cascade in the regulation of CREB phosphorylation in the hippocampus.

Input-specific immunolocalization of differentially phosphorylated Kv4.2 in the mouse brain.

Voltage-gated A-type potassium channels such as Kv4.2 regulate generation of action potentials and are localized abundantly in the hippocampus and striatum. Phosphorylation consensus sites for various kinases exist within the sequence of the potassium channel subunit Kv4.2, including consensus sites for extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK), protein kinase A (PKA), protein kinase C (PKC), and calcium/calmodulin-dependent kinase II (CaMKII), and kinase assays have shown that particular amino acids of the consensus sites are bonafide phosphorylation sites in vitro. We have developed antibodies recognizing Kv4.2 triply phosphorylated at the three ERK sites as well as two antibodies recognizing singly phosphorylated Kv4.2 channels at the PKA sites (one amino-terminal and one carboxy-terminal). In the present study, we report the development of reliable immunohistochemistry protocols to study the localization of these phosphorylated versions of Kv4.2, as well as total Kv4.2 in the mouse brain. A general description of the areas highlighted by these antibodies includes the hippocampus, amygdala, cortex, and cerebellum. Such areas display robust synaptic plasticity and have been implicated in spatial, associative, and motor learning. Interestingly, in the hippocampus, the antibodies to differentially phosphorylated Kv4.2 channels localize to specific afferent pathways, indicating that the Kv4.2 phosphorylation state may be input specific. For example, the stratum lacunosum moleculare, which receives inputs from the entorhinal cortex via the perforant pathway, displays relatively little ERK-phosphorylated Kv4.2 or PKA carboxy-terminal-phosphorylated Kv4.2. However, this same layer is highlighted by antibodies that recognize Kv4.2 that has been phosphorylated by PKA at the amino terminus. Similarly, of the three antibodies tested, the soma of CA3 neurons are primarily recognized by the ERK triply phosphorylated Kv4.2 antibody, and the mossy fiber inputs to CA3 are primarily recognized by the carboxy-terminal PKA-phosphorylated Kv4.2. This differential phosphorylation is particularly interesting in two contexts. First, phosphorylation may be serving as a mechanism for targeting. For example, the amino-terminal PKA phosphorylation may be acting as a tag for a discrete pool of Kv4.2 to enter stratum lacunosum moleculare. Second, as phosphorylation may regulate channel biophysical properties, differential phosphorylation of Kv4.2 in the dendrites of pyramidal neurons may confer unique biophysical properties upon particular dendritic input layers.

Kv4.2 phosphorylation by cyclic AMP-dependent protein kinase.

Recent evidence suggests that K(+) channels composed of Kv4.2 alpha-subunits underlie a transient current in hippocampal CA1 neurons and ventricular myocytes, and activation of the cAMP second messenger cascade has been shown to modulate this transient current. We determined if Kv4.2 alpha-subunits were directly phosphorylated by cAMP-dependent protein kinase (PKA). The intracellular domains of the amino and carboxyl termini of Kv4.2 were expressed as glutathione S-transferase fusion protein constructs; we observed that both of these fusion proteins were substrates for PKA in vitro. By using phosphopeptide mapping and amino acid sequencing, we identified PKA phosphorylation sites on the amino- and carboxyl-terminal fusion proteins corresponding to Thr(38) and Ser(552), respectively, within the Kv4.2 sequence. Kinetic characterization of the PKA sites demonstrated phosphorylation kinetics comparable to Kemptide. To evaluate PKA site phosphorylation in situ, phospho-selective antisera for each of the sites were generated. By using COS-7 cells expressing an EGFP-Kv4.2 fusion protein, we observed that stimulation of the endogenous PKA cascade resulted in an increase in phosphorylation of Thr(38) and Ser(552) within Kv4.2 in the intact cell. We also observed modulation of PKA phosphorylation at these sites within Kv4.2 in hippocampal area CA1. These results provide insight into likely sites of regulation of Kv4.2 by PKA.

The mitogen-activated protein kinase cascade couples PKA and PKC to cAMP response element binding protein phosphorylation in area CA1 of hippocampus.

Activation of the mitogen-activated protein kinase (MAPK) cascade recently was discovered to play an important role in synaptic plasticity in area CA1 of rat hippocampus. However, the upstream mechanisms regulating MAPK activity and the downstream effectors of MAPK in the hippocampus are uncharacterized. In the present studies we observed that hippocampal MAPK activation is regulated by both the PKA and PKC systems; moreover, we found that a wide variety of neuromodulatory neurotransmitter receptors (metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, and beta-adrenergic receptors) couple to MAPK activation via these two cascades. In additional studies we observed that PKC is a powerful regulator of CREB phosphorylation in area CA1. MAPK plays a critical role in transcriptional regulation by PKC, because MAPK activation is a necessary component for increased CREB phosphorylation in response to the activation of this kinase. Surprisingly, we also observed that MAPK activation is necessary for PKA coupling to CREB phosphorylation in area CA1. Overall, these studies indicate an unexpected richness of diversity in the regulation of MAPK in the hippocampus and suggest the possibility of a broad role for the MAPK cascade in regulating gene expression in long-term forms of hippocampal synaptic plasticity.

Permanency planning by older parents who care for adult children with mental retardation.

In a study of 57 older parent caregivers of adult children with mental retardation, more than half had not planned for their child's future care. Income, race, child's gender and level of adaptive behavior, and degree of parental interaction with relatives and friends were significantly related to future care-planning activity.

The effect of calcium ions on the activated partial thromboplastin time of heparinized plasma.

The effect of calcium chloride (CaCl2) on the activated partial thromboplastin time (aPTT) of heparinized plasma was studied. The aPTT ratio (heparinized plasma:control plasma) increased as the CaCl2 concentration to recalcify the plasma was increased from 15 to 35 mmol/L CaCl2. Platelet-poor plasma from patients receiving intravenous heparin, and in vitro heparinized plasmas from either coumarinized patients or plasma depleted of the vitamin K-dependent factors, displayed the calcium-dependent increase in the aPTT ratio. The magnitude of the calcium-dependent change in the aPTT ratio was similar for the three partial thromboplastins studied. Heparinized blood collected in 3.2% and 3.8% citrate demonstrated the calcium-dependent increase in the aPTT ratio. The authors have also studied the effect of the divalent cations (Ca+2, Mg+2, Zn+2, and Sr+2) on the anti-Factor Xa activity of heparin to determine whether the calcium-dependent increase in the aPTT was due to an increase in the anti-Factor Xa activity. The anti-Factor Xa activity of heparin was measured using chromogenic substrate S-2251, purified Factor Xa, and excess antithrombin III. The anti-Factor Xa activity of heparinized plasma increased 2.4-2.8-fold as the divalent cation concentration was increased from 0-5 mmol/L. Similar results were obtained using purified bovine Factor Xa, antithrombin III, and heparin in the absence of plasma. These results suggest that divalent cations play an important role in modulating heparin's anticoagulant activity in vitro. In addition, the CaCl2 concentration used to recalcify plasma is an important variable that modifies heparin sensitivity of the aPTT. Furthermore, divalent cations play an important role in regulating the anti-Factor Xa activity of heparin in vitro.

A laser diffraction method for measuring muscle sarcomere length in vivo for application to tendon transfers.

A technique that uses laser light diffraction to measure muscle sarcomere length allows direct determination of optimal muscle length during tendon transfers. Forearm muscle sarcomere length with the hand in the position of function is 2.4, and muscle length corresponds directly to sarcomere length. We have used these observations to restore optimal muscle length during tendon transfers. Standard high radial nerve tendon transfers in six fresh cadaver forearms demonstrated the efficacy of the laser diffraction method in accurately measuring sarcomere length. In two clinical trials with the laser, standard high radial nerve palsy tendon transfers were performed. In each case the clinical tendency was to overpull the muscle during the transfer. With the laser it was possible to identify excessive muscle stretch and restore optimal muscle length.

Malignant hyperthermia: a possible new variant.

A young healthy male, who had three consecutive episodes of postoperative hyperthermia was anaesthetized with special precautions to prevent malignant hyperthermia. Despite neuroleptic anaesthesia and dantrolene pretreatment, the patient experienced post-anaesthetic hyperthermia. The patient's clinical picture was almost identical to the symptoms experienced by two of his maternal relatives. All three experienced nausea, vomiting, muscle cramps and high fever which occurred between five to seven hours after general anaesthesia. The serum potassium (K) and creatinine phosphokinase (CPK) levels determined during the hyperthermic episode and on the next day were not elevated. On the basis of the patient's family history, his clinical picture, and his laboratory data, we speculate that this patient might have a form of malignant hyperthermia or a possible new variant.

Potential for Growth of Nonproteolytic Types of Clostridium botulinum in Pasteurized Restructured Meat Products: A Review 1.

Type E and nonproteolytic type B strains of Clostridium botulinum can grow and produce toxin at temperatures below 5°C. Recent publications describing the greater heat resistance of nonproteolytic type B C. botulinum spores than type E spores are discussed in relation to suitable proess lethalities required for a safe pasteurized product. The incidences of botulism in Europe caused by nonproteolytic type B spores were compared to the lack of such incidences in the U.S. and to published procedures for isolating the causative agent for botulism. The incidence of C. botulinum spores in meat products in the U.S. also is reviewed.