Interaction of PLD1b with actin in antigen-stimulated mast cells.

Phosphatidic acid, the product of phospholipase D catalysed phosphatidylcholine hydrolysis is an important signalling molecule that has been implicated in regulation of actin cytoskeleton remodelling and secretion from mast cells. We show that human PLD1b (hPLD1b) is an actin-binding protein and the N-terminus is predominantly involved in this interaction. Protein kinase C (PKC) is a major upstream regulator of PLD activity and PKC phosphorylation sites have been identified within the N-terminus of PLD1b at serine 2 and threonine 147. Over-expression of wild type hPLD1b in mast cells showed that antigen stimulation significantly enhanced co-localisation of PLD1b with actin structures. Mutation of serine 2 to alanine abolished antigen-induced co-localisation whereas mutation of threonine 147 had less dramatic effects on co-localisation. The absence of co-localisation of PLD1b (S2A) with actin coincides with a significant decrease in PLD activity in cells expressing the PLD1b (S2A) mutant. In resting RBL-2H3 cells, mutation of serine 2 to aspartate resulted in constitutive co-localisation of PLD with the actin cytoskeleton, coincident with restored PLD activity. These results reveal that serine 2 is an important regulatory site involved in controlling PLD enzyme activity and the interaction between PLD and actin.

Declining critical care research publications by authors from U.S. Institutions, 1990-1999.

PURPOSE: To determine whether the proportion of authors from U.S. institutions to those from non-U.S. institutions has changed for published critical care research in three critical care journals over the past ten years. METHOD: The authors of designated critical care clinical or laboratory investigations published from 1990 to 1999 in the three leading U.S. critical care journals, American Journal of Respiratory and Critical Medicine, Chest, and Critical Care Medicine, were evaluated according to the locations of their institutions (U.S. versus non-U.S.) through a review of these publications. RESULTS: The proportion of authorship by investigators from U.S. institutions has declined for critical care research publications from 61% of all authors in 1990 to 41% in 1999 (p < .00001). Statistically significant declines in the proportions of authors from U.S. institutions to those from non-U.S. institutions occurred in the American Journal of Respiratory and Critical Care Medicine (p < .05) and Critical Care Medicine (p < .00001), but not in Chest (p = .69). CONCLUSIONS: The reasons for the decline in authorship by investigators from U.S. institutions are speculative and likely multifactorial. They are, however, consistent with other published data showing limited non-clinical time allocated for education activities for critical care faculty. Anecdotal concerns expressed by many faculty that rising clinical commitments necessitated by current health care and reimbursement pressures preclude research and educational academic activities are supported by these data.

Credentialing for critical care in small hospitals.

OBJECTIVE: To assess credentialing practices for critical care admissions and procedures in smaller hospitals within the United States. METHODS: A questionnaire was sent to credentialing coordinators of 500 randomly selected American Hospital Association hospitals with fewer than 300 beds. MEASUREMENTS AND MAIN RESULTS: Most hospitals validate qualifications for intensive care unit (ICU) admitting and procedural privileges through recommendations only. Fewer (16%) require a specified prior number of procedures to have been performed, and 9% require prospective supervision before privileges are granted. Critical care subspecialists are present in 57% of these hospitals and tend to be in the larger facilities with more critical care beds. Criteria for ICU admission and procedure privileges appear to be inclusive, because family medicine, obstetrics-gynecology, orthopedic surgery, and neurology specialists are often credentialed. The presence of a critical care subspecialist is associated with fewer hospitals credentialing family medicine specialists for ICU admission and procedures but not obstetrician-gynecologists, orthopedic surgeons, or neurologists. CONCLUSIONS: This is a brief descriptive report of hospital policies that define which physicians are permitted to care for critically ill/injured patients in small U.S. hospitals. The presence of a critical care specialist appears to influence only slightly the ICU credentialing processes for other selected specialists.

Challenges in donor care.

This article is provided as a self-study feature on care of organ donors. Questions and possible responses are provided. Review all the responses for each question.

Using pressure-limited mechanical ventilation in caring for organ donors.

Pressure-limited (controlled) ventilation is commonly employed to provide mechanical ventilation in the intensive care unit when lung compliance is poor or when airway resistance is irreversibly high. Modification of the inspiratory-expiratory ratio to include inspiratory-expiratory ratio reversal and permissive hypercapnia can also be used when lung disease or injury is severe. Because other donor organs often can be saved for transplantation even when the lungs have been badly damaged, the organ procurement coordinator should adopt pressure-limited ventilation as well as inspiratory-expiratory ratio reversal and permissive hypercapnia as potentially helpful methods while providing mechanical ventilation to selected donors.

Teaching critical appraisal during critical care fellowship training: a foundation for evidence-based critical care medicine.

OBJECTIVE: To determine whether fellowship training in critical care medicine with critical appraisal exercises improves the ability and confidence of fellows to evaluate the medical literature. DESIGN: Prospective, interventional pilot study. SETTING: Multidisciplinary critical care medicine training program at a large university hospital. INTERVENTION: Fellows were given three didactic sessions covering study design, analysis, and critical appraisal techniques. During the course of the year, each fellow was required to review one article from the literature and present a critique of this article to the group and faculty (Journal Club). Fellows were guided in the preparation of this presentation by one of the critical care medicine faculty. Finally, a written analysis and critique of the article was performed by each fellow. MEASUREMENTS AND MAIN RESULTS: A test was given to each fellow at the beginning and end of the academic year. This test consisted of two pairs of articles on therapy for acute lung injury. For the pretest, each fellow was assigned, at random, one pair of articles. Fellows were given 1 hr to review both articles and to fill out a six-point test to assess their ability and confidence to appraise each article. At the end of the year, each fellow was tested on the opposite pair, the tests were graded in a blinded fashion and the results of each test were compared. Six fellows completed both pre- and posttests. These paired results were analyzed separately, whereas results for another six fellows were conducted as an unpaired analysis. Mean scores increased both for the paired analysis (4.1+/-0.7 vs. 5.1+/-0.5; p = .015) and for the unpaired analysis (4.3+/-0.6 vs. 5.0+/-0.5; p = .012). Self-reported confidence in critical appraisal also increased (2.5+/-0.5 vs. 3.9+/-0.7; p = .004 and 2.6+/-0.5 vs. 3.9+/-0.6; p < .001, respectively). CONCLUSION: Critical appraisal exercises used in the training of critical care medicine fellows appear to improve both ability and confidence to appraise relevant medical literature.

Abnormalities in fluids, electrolytes, and metabolism of organ donors.

Abnormal serum concentrations of electrolytes, hormones, and glucose are common throughout donor care. The organ procurement coordinator must properly interpret and plan treatment for these changes to prevent intracellular dysfunction in donor organs. This article describes abnormalities in magnesium, phosphorous, calcium, sodium, potassium, and glucose levels; polyuria; and thyroid and pituitary changes. Their potential consequences are discussed, and recommendations for treatment options are presented.

Maintaining acid-base balance in organ donors.

An abnormal blood pH may cause the loss of donor organs through harmful physiological consequences. The organ procurement coordinator must correctly analyze the acid-base abnormality and treat its cause while normalizing the blood pH. We recommend that treatment of acidemia or alkalemia be first directed toward changing parameters on the mechanical ventilator, using the Paco2 to modify blood pH. Thereafter, hydrochloric acid or sodium bicarbonate may be administered to correct the calculated metabolic acid-base deficit. The types of acidosis or alkalosis, dead space effect during mechanical ventilation, base excess, base deficit, and the appropriate evaluation of blood lactate are also discussed as related to the correction of the acid-base status throughout donor care.

Management of variations in blood pressure during care of organ donors.

The organ procurement coordinator commonly must correct and maintain the arterial blood pressure during donor care. This article reviews considerations in the accurate measurement of the blood pressure, causes of hypertension and hypotension, and desirable standards to use in order to provide adequate organ perfusion. Recommendations are presented for treatment of hypotension in a titrated response of intravenous fluids, inotropic support, and vasopressor infusion to maintain the mean arterial pressure above 65 mm Hg. Collaborative interaction between the coordinator and physician consultant remains important throughout management of blood pressure changes during donor care.

Recommendations for mechanical ventilation during donor care.

The organ procurement coordinator usually directs adjustments to the mechanical ventilator during donor care. It is often difficult to achieve optimal oxygen uptake and carbon dioxide removal while avoiding barotrauma or undesirable effects on the cardiac output. Interrelationships among a variety of ventilator parameters must be understood in order to achieve the desired goal of providing the best organs possible. These recommendations review the key ventilator parameters of tidal volume; positive end-expiratory pressure; auto-positive end-expiratory pressure; fraction of inspired oxygen; and flowrate and frequency and their interactions in controlling peak, plateau, and mean and end-expiratory airway pressures.

A temperature-sensitive Krp1 allows in vivo characterization of kexin activation.

Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway. The enzymes are themselves made as inactive precursors and we have investigated the activation of Krp1, a kexin from the fission yeast Schizosaccharomyces pombe. As Krp1 is essential for cell growth, we have used a krp1ts strain to investigate the role of the prosequence in the activation process. Mutations that reduce either the efficiency with which the prosequence is released or the rate at which the released prosegment is subsequently cleaved at an internal site are less active when assayed in vivo. We also show that prosegments lacking an internal dibasic motif can act as autoinhibitors and prevent activation of the catalytic fragment. Krp1 constructs containing prosequences based on these inhibitors do not become active in vitro. Surprisingly, the same constructs do become active in the intact cell and appear to suggest that alternative activation processes can be used by these enzymes.

Compensation for teaching in critical care.

OBJECTIVES: To determine the financial or nonclinical time critical care program directors or teaching faculty members receive as compensation for their educational activities. To compare compensation types and amounts among critical care specialties and between university vs. nonuniversity sponsoring institutions. DATA SOURCES AND EXTRACTION: Survey returns (46%) from critical care fellowship directors listed in the American Medical Association Graduate Medical Education Directory. Information was stratified according to fellowship specialty and type of sponsoring hospital and compared by chi-square analysis and the Kruskal-Wallis test. CONCLUSIONS: Most program directors (77%) and faculty (82%) receive no specified compensation for education-related activities. Multidisciplinary programs are more likely to compensate faculty members than other specialty-specific programs (p = .006). Most programs sponsored by university or military/federal hospitals do not provide specified compensation (79% and 100%, respectively). Overall, community hospital-based programs provide a greater percentage of compensation to directors and faculty than university programs (for directors, p = .02; odds ratio, 3.85; for faculty, p = .001; odds ratio, 8.4). When compensation is specified, it is most often financial and it averages 18% of the salary (range, 5% to 100%) for directors and 19% of the salary for faculty (range, 5% to 50%). When reduced clinical time is provided (5% of program directors, 2% of faculty), it averages 13% (range, 8% to 18%) for directors and 18% (range, 10% to 25%) for faculty. Alternative methods for assigning educational compensation are discussed.

Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-biphosphate-specific PH domain.

The signalling pathway leading, for example, to actin cytoskeletal reorganisation, secretion or superoxide generation involves phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, which appears to mediate the messenger functions of this pathway. Two PLD genes (PLD1 and PLD2) with similar domain structures have been doned and progress has been made in identifying the protein regulators of PLD1 activation, for example Arf and Rho family members. The activities of both PLD isoforms are dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and our sequence analysis suggested the presence of a pleckstrin homology (PH) domain in PLD1, although its absence has also been daimed. Investigation of the inositide dependence showed that a bis-phosphorylated lipid with a vicinal pair of phosphates was required for PLD1 activity. Furthermore, PLD1 bound specifically and with high affinity to lipid surfaces containing PI(4,5)P2 independently of the substrate phosphatidylcholine, suggesting a key role for the PH domain in PLD function. Importantly, a glutathione-S-transferase (GST) fusion protein comprising GST and the PH domain of PLD1 (GST-PLD1-PH) also bound specifically to supported lipid monolayers containing PI(4,5)P2. Point mutations within the PLD1 PH domain inhibited enzyme activity, whereas deletion of the domain both inhibited enzyme activity and disrupted normal PLD1 localisation. Thus, the functional PH domain regulates PLD by mediating its interaction with polyphosphoinositide-containing membranes; this might also induce a conformational change, thereby regulating catalytic activity.

Regulation of coagulation abnormalities and temperature in organ donors.

The 3 most common reasons for abnormal coagulation of blood in organ donors result from prior medications, consumption or dilution of coagulation factors and platelets during massive transfusion, and disseminated intravascular coagulation. Evaluation and treatment of these conditions are reviewed, and recommendations are provided for ordering appropriate laboratory tests and blood bank products.

Current considerations in the issue of brain death.

Brain death is an anatomically and physiologically complex process. The societal and psychological implications of brain death and organ donation are equally complex, and they have profound ramifications. Because the vast majority of organ donors die as a consequence of catastrophic intracranial processes, neurosurgeons are in a unique position to positively influence the supply of transplantable organs. Enhanced knowledge of the physiology of evolving brain death will improve the care of potential organ donors and increase the probability of successful transplantation. Likewise, better information about patient and family directives, beliefs, grieving, concurrent exposure to other health care workers, and experiences in the hospital environment will assist the neurosurgeon in providing the family with the opportunity for donation. Neurosurgeons can also play a leading role in the multidisciplinary approach required to support the families of potential organ donors during the transition from neurointerventional therapy to somatic support. New federal regulations on organ donation and a review of the literature about the "art of asking" are presented.

Phospholipase D1 localises to secretory granules and lysosomes and is plasma-membrane translocated on cellular stimulation.

Phospholipase D (PLD) activity has been implicated in the regulation of membrane trafficking [1,2], superoxide generation and cytoskeletal remodelling [3,4]. Several PLD genes have now been identified and it is probable that different isoforms regulate distinct functions. Defining the subcellular localisation of each isoform would facilitate understanding of their roles. Previous PLD localisation studies have been based largely on enzyme activity measurements, which cannot distinguish between isoforms [2,5]. We have cloned the cDNAs encoding human PLD1a and PLD1b from an HL60 cell cDNA library and expressed them as catalytically active fusion proteins with green fluorescent protein (GFP) in COS-1 cells and RBL-2H3 cells, a mast cell model which degranulates upon cross-linking of the high-affinity immunoglobulin E (IgE) receptor. In unstimulated cells, GFP-PLD1b colocalised with secretory granule and lysosomal markers; it was not found at the plasma membrane or nucleus and did not colocalise with markers for the Golgi. Stimulation or RBL-2H3 cells through IgE receptor cross-linking caused plasma membrane recruitment of GFP-PLD1b. Inhibition of IgE-receptor-stimulated, PLD-catalysed phosphatidate formation suppressed secretion of granule and lysosomal contents, but did not affect translocation of GFP-PLD1b. These experiments suggest that PLD1 plays a role in regulated exocytosis rather than endoplasmic reticulum (ER) to Golgi membrane transport.

The processing of yeast pheromones.

Yeast provides an attractive system in which to study proprotein processing. Many of their processing events are remarkably similar to those in higher eukaryotes and their amenability to experimental manipulation permits approaches that are not always feasible in multicellular organisms. Analysing the biosynthesis of the yeast mating pheromones has been particularly rewarding and has provided insights into both the proteolytic aspects of processing and other post-translational events such as prenylation and carboxymethylation.

Activation of the kexin from Schizosaccharomyces pombe requires internal cleavage of its initially cleaved prosequence.

Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway. The enzymes themselves are made as inactive precursors, and we investigated the activation process by studying the maturation of Krp1, a kexin from the fission yeast Schizosaccharomyces pombe. Using a cell-free translation-translocation system prepared from Xenopus eggs, we found that Krp1 is made as a preproprotein that loses the presequence during translocation into the endoplasmic reticulum. The prosequence is also rapidly cleaved in a reaction that is autocatalytic and probably intramolecular and is inhibited by disruption of the P domain. Prosequence cleavage normally occurs at Arg-Tyr-Lys-Arg102/ (primary cleavage site) but can occur at Lys-Arg82 (internal cleavage site) and/or Trp-Arg99 when the basic residues are removed from the primary site. Cleavage of the prosequence is necessary but not sufficient for activation, and Krp1 is initially unable to process substrates presented in trans. Full activation is achieved after further incubation in the extract and is coincident with the addition of O-linked sugars. O glycosylation is not, however, essential for activity, and the crucial event appears to be cleavage of the initially cleaved prosequence at the internal site. Our results are consistent with a model in which the cleaved prosequence remains noncovalently associated with the catalytic domain and acts as an autoinhibitor of the enzyme. Inhibition is then relieved by a second (internal) cleavage of the inhibitory prosequence. Further support for this model is provided by our finding that overexpression of a Krp1 prosequence lacking a cleavable internal site dramatically reduced the growth rate of otherwise wild-type S. pombe cells, an effect that was not seen after overexpression of the normal, internally cleavable, prosequence or prosequences that lack the Lys-Arg102 residues.