A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus.

The Golgi apparatus is a dynamic organelle whose structure is sensitive to vesicular traffic and to cell cycle control. We have examined the potential role for rab1a, a GTPase previously associated with ER to Golgi and intra-Golgi transport, in the formation and maintenance of Golgi structure. Bacterially expressed, recombinant rab1a protein was microinjected into rat embryonic fibroblasts, followed by analysis of Golgi morphology by fluorescence and electron microscopy. Three recombinant proteins were tested: wild-type rab, mutant rab1a(S25N), a constitutively GDP-bound form (Nuoffer, C., H. W. Davidson, J. Matteson, J. Meinkoth, and W. E. Balch, 1994. J. Cell Biol. 125: 225-237), and mutant rab1a(N124I) defective in guanine nucleotide binding. Microinjection of wild-type rab1a protein or a variety of negative controls (injection buffer alone or activated ras protein) did not affect the appearance of the Golgi, as visualized by immunofluorescence of alpha-mannosidase II (Man II), used as a Golgi marker. In contrast, microinjection of the mutant forms promoted the disassembly of the Golgi stacks into dispersed vesicular structures visualized by immunofluorescence. When S25N-injected cells were analyzed by EM after immunoperoxidase labeling, Man II was found in isolated ministacks and large vesicular elements that were often surrounded by numerous smaller unlabeled vesicles resembling carrier vesicles. Golgi disassembly caused by rab1a mutants differs from BFA-induced disruption, since beta-COP remains membrane associated, and Man II does not redistribute to the ER. BFA can still cause these residual Golgi elements to fuse and disperse, albeit at a slower rate. Moreover, BFA recovery is incomplete in the presence of rab1 mutants or GTP gamma S. We conclude that GTP exchange and hydrolysis by GTPases, specifically rab1a, are required to form and maintain normal Golgi stacks. The similarity of Golgi disassembly seen with rab1a mutants to that occurring during mitosis, may point to a molecular basis involving rab1a for fragmentation of the Golgi apparatus during cell division.

SecA homolog in protein transport within chloroplasts: evidence for endosymbiont-derived sorting.

The SecA protein is an essential, azide-sensitive component of the bacterial protein translocation machinery. A SecA protein homolog (CPSecA) now identified in pea chloroplasts was purified to homogeneity. CPSecA supported protein transport into thylakoids, the chloroplast internal membrane network, in an azide-sensitive fashion. Only one of three pathways for protein transport into thylakoids uses the CPSecA mechanism. The use of a bacteria-homologous mechanism in intrachloroplast protein transport provides evidence for conservative sorting of proteins within chloroplasts.

Disruption of endoplasmic reticulum to Golgi transport leads to the accumulation of large aggregates containing beta-COP in pancreatic acinar cells.

When transport between the rough endoplasmic reticulum (ER) and Golgi complex is blocked by Brefeldin A (BFA) treatment or ATP depletion, the Golgi apparatus and associated transport vesicles undergo a dramatic reorganization. Because recent studies suggest that coat proteins such as beta-COP play an important role in the maintenance of the Golgi complex, we have used immunocytochemistry to determine the distribution of beta-COP in pancreatic acinar cells (PAC) in which ER to Golgi transport was blocked by BFA treatment or ATP depletion. In controls, beta-COP was associated with Golgi cisternae and transport vesicles as expected. Upon BFA treatment, PAC Golgi cisternae are dismantled and replaced by clusters of remnant vesicles surrounded by typical ER transitional elements that are generally assumed to represent the exit site of vesicular carriers for ER to Golgi transport. In BFA-treated PAC, beta-COP was concentrated in large (0.5-1.0 micron) aggregates closely associated with remnant Golgi membranes. In addition to typical ER transitional elements, we detected a new type of transitional element that consists of specialized regions of rough ER (RER) with ribosome-free ends that touched or extended into the beta-COP containing aggregates. In ATP-depleted PAC, beta-COP was not detected on Golgi membranes but was concentrated in similar large aggregates found on the cis side of the Golgi stacks. The data indicate that upon arrest of ER to Golgi transport by either BFA treatment or energy depletion, beta-COP dissociates from PAC Golgi membranes and accumulates as large aggregates closely associated with specialized ER elements. The latter may correspond to either the site of entry or exit for vesicles recycling between the Golgi and the RER.

Immunization of melanoma patients with BEC2 anti-idiotypic monoclonal antibody that mimics GD3 ganglioside: enhanced immunogenicity when combined with adjuvant.

Previous attempts to immunize melanoma patients against GD3 ganglioside have been unsuccessful because of the poor immunogenicity of GD3. BEC2, an anti-idiotypic monoclonal antibody that mimics GD3, can induce anti-GD3 IgG in rabbits. Since clinical trials with BEC2 in melanoma patients demonstrated that BEC2 alone is not highly immunogenic, we have carried out sequential clinical trials exploring the use of two immunological adjuvants, BCG and QS21, administered with BEC2. Melanoma patients free of disease after surgical resection but at high risk for recurrence were immunized either with BEC2/BCG (14 patients) or BEC2/QS21 (6 patients). All patients developed high-titer IgG antibodies against BEC2, demonstrating that both adjuvants effectively enhanced the immunogenicity of BEC2. Anti-GD3 antibodies were induced in 3 of 14 patients immunized with BEC2/BCG; no patient immunized with BEC2/QS21 developed detectable anti-GD3 antibodies. After a median follow-up of 2.4 years, 71% of the patients immunized with BEC2/BCG remain alive and 64% are free of disease. In patients immunized with BEC2/BCG, no apparent association was observed between class II HLA type and either development of anti-GD3 antibodies or survival. We are encouraged by the results with BEC2/BCG, which suggest that further enhancement of the immune response to BEC2 will result in more frequent anti-GD3 antibody responses among immunized patients.

Sorting of cyst wall proteins to a regulated secretory pathway during differentiation of the primitive eukaryote, Giardia lamblia.

Giardia lamblia, which belongs to the earliest identified lineage to diverge from the eukaryotic line of descent, is one of many protists reported to lack a Golgi apparatus. Our recent finding of a developmentally regulated secretory pathway in G. lamblia makes it an ideal organism with which to test the hypothesis that the Golgi may be more readily demonstrated in actively secreting cells. These ultrastructural studies now show that a regulated pathway of transport and secretion of cyst wall antigens via a novel class of large, osmiophilic secretory vesicles, the encystation-specific vesicles (ESV), is assembled during encystation of G. lamblia. Early in encystation, cyst antigens are localized in simple Golgi membrane stacks and concentrated within enlarged Golgi cisternae which appear to be precursors of ESV. This would represent an unusual mechanism of secretory vesicle biogenesis. Later in differentiation, cyst antigens are localized within ESV, which transport them to the plasma membrane and release them by exocytosis to the nascent cell wall. ESV are not observed after completion of the cyst wall. In contrast to the regulated transport of cyst wall proteins, we demonstrate a distinct constitutive lysosomal pathway. During encystation, acid phosphatase activity is localized in endoplasmic reticulum, Golgi, and small constitutive peripheral vacuoles which function as lysosomes. However, acid phosphatase activity is not detectable in ESV. These studies show that G. lamblia, an early eukaryote, is capable of carrying out Golgi-mediated sorting of proteins to distinct regulated secretory and constitutive lysosomal pathways.

Isolation and partial characterization of the luminal plasmalemma of microvascular endothelium from rat lungs.

This paper describes a procedure for isolating in high yield and at a high degree of purity the endothelial luminal plasmalemma from the microvasculature of the rat lung. The procedure relies on the modification of the density of the luminal plasmalemma obtained by coating it by perfusion in situ first, with cationized colloidal silica and then with Na polyacrylate. These steps generate a strongly adhering coat to the luminal plasmalemma that resists tissue homogenization to yield, upon repeated centrifugation through Nycodenz density gradients, a nearly homogeneous fraction of coated luminal plasmalemmal fragments still carrying their associated plasmalemmal vesicles. The fraction is enriched in the luminal plasmalemmal antigen, angiotensin converting enzyme, contains gp60, an antigen expected to occur on both plasmalemmal domains, is not enriched in either alkaline phosphatase or 5'-nucleotidase activity and is free of the mitochondrial and endoplasmic reticulum antigens so far tested. This procedure, that can be extended--in principle--to any vascular bed, obviates the use of cultured cells for studying the biochemistry of the endothelium, at least as far as the luminal endothelial plasmalemma is concerned.

Golgi proteins persist in the tubulovesicular remnants found in brefeldin A-treated pancreatic acinar cells.

Brefeldin A (BFA) blocks protein export from the endoplasmic reticulum (ER) and causes dismantling of the Golgi cisternae with relocation of resident Golgi proteins to the ER in many cultured cell lines. We examined the effects of BFA on Golgi organization and the distribution of Golgi markers in the rat exocrine pancreas. Immediately after BFA addition, Golgi stacks began to disorganize and Golgi cisternae to vesiculate, and by 15 min no intact Golgi cisternae remained. However, even after prolonged BFA incubation, clusters of small vesicles surrounded by transitional elements of the ER persisted both in the Golgi region and dispersed throughout the apical cytoplasm. These vesicles were morphologically heterogeneous in the density of their content and in the presence of cytoplasmic coats. Immunogold labeling demonstrated that some vesicles within the clusters contained gp58, a cis Golgi marker, and some contained alpha-mannosidase II, a middle/trans Golgi marker in this cell type. Neither marker was detected in the rough ER by immunogold or immunofluorescence labeling. When AlF4- was added during BFA treatment some of the vesicles in the clusters appeared coated. When microsomes were subfractionated into Golgi (light) and rough ER (heavy) fractions on sucrose density gradients, greater than 65% of alpha-mannosidase II and galactosyltransferase activities were found in light fractions (1.14-1.16 g/ml) in both control and BFA-treated lobules. In both cases equally low enzyme activity was recovered in heavier fractions (1.2-1.23 g/ml) containing RNA and alpha-glucosidase activity. However, 5 to 8% of the total recovered RNA consistently codistributed with the Golgi enzyme peak. These results indicate that BFA rapidly inhibits secretion and causes dismantling of the Golgi stacks in pancreatic acinar cells, but clusters of vesicles consisting of bona fide Golgi remnants persist even with prolonged exposure to BFA. Many of the vesicles contain Golgi markers by immunolabeling. By cell fractionation Golgi membrane enzyme activities are recovered in equal amounts in light (Golgi) fractions in both controls and BFA-treated specimens. These findings indicate that in the exocrine pancreas there is a dissociation of BFA's effects on the exocytic pathway: there is a block in transport and Golgi organization is disrupted, but remnant Golgi vesicles and tubules persist and retain Golgi membrane antigens and enzyme activities.

Evidence for a loop mechanism of protein transport by the thylakoid Delta pH pathway.

The thylakoid Delta pH pathway is a protein transport system with unprecedented characteristics. To investigate its mechanism, the topology of precursor insertion was determined. A fusion protein comprising a large polypeptide domain fused to the amino terminus of pOE17 (a Delta pH pathway precursor) was efficiently processed by thylakoid membranes. The amino terminus, including the targeting peptide, remained on the cis side of the membrane. Mature OE17 was transported to the lumen. These experiments demonstrate that Delta pH directed precursors enter the thylakoid membrane in a loop, implying that the Delta pH pathway has evolved from an export-type protein translocation system.

Nurses' knowledge about cancer pain: a survey of five countries.

Surveys of nurses' knowledge of cancer pain management were conducted in five countries: Australia, Canada, Japan, Spain, and the United States. The results reveal that, in all countries, serious knowledge deficits exist that could adversely affect the care of patients with cancer pain. It appears, however, that the longer a country has been engaged in efforts to educate health-care professionals and the public and to establish palliative care programs, the more likely are nurses from that country to possess correct information about cancer pain. Nevertheless, survey results in all countries strongly suggested the need to continue aggressive measures to educate nurses, who are the cornerstone of palliative care.

Nurses' knowledge of pain assessment and management: how much progress have we made?

Undertreatment of pain and lack of knowledge about pain management have been evident for approximately two decades. Because nurses are often the cornerstone of pain management, nurses' knowledge in this area is especially important. This paper explores indications of progress in the level of nursing knowledge about basic aspects of pain management. The literature is reviewed and findings from recent (1995) surveys of nurses' knowledge are compared with results of similar surveys conducted beginning in 1988. Improvements in nurses' knowledge of pain assessment, opioid dosing, and likelihood of addiction seem to have occurred. However, knowledge deficits continue. Fewer than one-half of the nurses surveyed understand that the patient's self report of pain is the single most reliable indicator of pain and that the nurse should increase a previously safe but ineffective dose of opioid. Findings from surveys on addiction reveal that the longer the patient receives opioids the more concerned nurses become about causing addiction. Nevertheless, results of current knowledge surveys of nurses suggest that educational efforts probably have been beneficial and should continue. To maximize the impact of educational efforts, content in basic and continuing education courses should be prioritized and critically evaluated for relevance and accuracy, especially content related to addiction. Early in the education of nurses, responsibility for pain assessment and use of analgesics must be instilled.

The experience of chronic nonmalignant pain.

This article reports the results of a survey of 204 persons with chronic nonmalignant pain who were members of a national self-help organization. The survey evaluated the organization, explored the perceived effect of pain on quality of life, and assessed experiences with and perceptions of health-care providers. Response rate was 40%. Of survey respondents, 50% reported inadequate pain relief. Respondents identified depression as one of the worst problems caused by their chronic pain: 50% reported that they had considered suicide due to feelings of hopelessness associated with their pain, 51% reported taking only as much medication as prescribed, and 44% reported taking less medication than prescribed. Further investigation is needed to describe the personal impact of chronic nonmalignant pain.

Pain and addiction: an urgent need for change in nursing education.

Lack of education of health care professionals, including nurses, is frequently cited as a major reason for undertreatment of patients with pain. A reason for undertreatment of pain with opioid analgesics is the irrational fear of creating opioid addiction. To characterize the information nurses receive in their basic education that could contribute to misinformation about this issue, the authors reviewed 14 nursing textbooks, published since 1985, including 8 pharmacology texts and 6 medical surgical texts. An analysis of content revealed that only one textbook correctly stated the definition of opioid addiction and its likelihood following use of opioid analgesics for pain control. Almost all of the texts used confusing terminology, and some erroneously promoted the fear of addiction when opioids are used for pain relief. A simple solution to this problem is to encourage nursing educators to use the American Pain Society publication "Guidelines for Analgesic Use" until textbooks have the opportunity to incorporate correct information.

Analysis of pain content in nursing textbooks.

This paper reports on one goal of a project designed to improve end-of-life (EOL) care in nursing education. The goal was to improve the content regarding pain and EOL care included in major nursing textbooks. A descriptive study used content analysis of 50 textbooks selected from a potential of over 700 texts used in nursing education. The 50 texts included 45,683 pages. The text review was conducted using an analysis framework and included content analysis as well as quantification of the content present in the texts. While pain was the most common topic in the texts, the 248 pages of pain content represents only 0.5% of total text content. All nine areas of EOL content (902 pages) comprised only 2% of overall text. Particular weaknesses in the review of pain content were recognized in pharmacologic management of pain. In summary, nursing texts have limited content on pain. Increased attention to this area is essential to prepare nurses to care for patients at the end of life.

Modification of cytosolic free calcium concentrations in human keratinocytes after sulfur mustard exposure.

Exposure of confluent cultures of human skin keratinocytes to sulfur mustard (SM) induced an immediate and irreversible rise in internal free Ca(2+) levels that was independent of external Ca(2+) concentrations. The response was rapid, beginning within 1min after addition of SM to the cells, and sensitive, with significant effects observed at 100 mum. The rise in [Ca(2+)](INT) was unaffected by zero external Ca(2+) but was blocked by prior incubation with thapsigargin. The sensitivity to and irreversibility of the effects of SM on Ca(2+) levels was paralleled by cellular toxicity as assessed using three different cell viability assays. In addition, the time course of the onset of irreversible toxicity in our cultures coincides with the time course of effects on [Ca(2+)](INT). SM was also found to displace specifically bound ATP from purinergic (P(2)) receptors. These results suggest that therapies aimed at protecting internal stores of Ca(2+) from disruption by SM, perhaps at P(2) receptors, may provide substantial benefit in protecting human skin cells from the toxic effects of this vesicant.

Nurses' knowledge about equianalgesia and opioid dosing.

Nurses are recognized as the cornerstone of palliative care. Yet, surveys of nurses' knowledge of cancer pain management reveal serious knowledge deficits that could adversely affect the care of patients with cancer pain. Previous research has explored basic pain management issues such as pain assessment and myths and misconceptions surrounding pain, and principles of analgesic use. Advances in recent years have increased the demand for continuing education that will extend scientific advances in pain to clinical practice. The purpose of this article is to share results from a study which evaluated nurses knowledge regarding three methods of analgesic delivery that have become common in clinical practice: intravenous morphine, extended release morphine, and transdermal fentanyl. Several resources are provided to assist clinicians in the appropriate use of these analgesic methods.

Clinical decision making and pain.

Pain management is intimately linked to decision making. Nurses play a key role in making decisions regarding pain and its management. This paper presents data from a preliminary study (N = 53) extending the investigators' pain research into the areas of clinical decision making and ethical dilemmas related to pain management. Nurses completed surveys focused on actual experiences of caring for patients in pain. The results identify common clinical decisions related to pain, barriers to providing optimum pain relief, and ethical/professional conflicts in pain management. Decisions related to the amount of pain intensity, when to give medications, and choice of analgesics. Nurses identified verbal and nonverbal cues central to their decisions regarding pain assessment. Barriers to effective pain management included physician knowledge and cooperation, patient/family knowledge and cooperation, as well as nursing knowledge and time. Respondents identified ethical dilemmas about overmedication or undermedication, conflicts with physicians or patients, and concern over opioid side effects. A tentative model of decision making related to pain is presented.

Nurses' knowledge of opioid analgesic drugs and psychological dependence.

Inadequate knowledge of opioid analgesic drugs and the incidence of psychological dependence are major barriers to nursing management of patients in pain. This study analyzed data obtained from 27 workshops on pain across 14 states (2,459 nurses) to determine current nursing knowledge of pharmacological management of pain. Results indicate that nurses lack knowledge in classification of opioids ranging from 23 to 98% correct response across seven analgesic drugs. Less than 25% of nurses correctly identified the frequency of psychological dependence. Further analysis revealed significant differences in basic versus advanced learners and geographical differences in knowledge. Implications are made for nursing education and practice.

Nursing approaches to nonpharmacological pain control.

A combination of pharmacological and nonpharmacological methods of pain control probably yield the most effective pain relief for the patient. The nurse may make a significant contribution to pain control by being able to offer a variety of nonpharmacological methods of pain relief that the patient may use in combination with the more traditional methods of analgesia or local anesthesia. Recent research supports some of the older methods of nonpharmacological pain control such as distraction, especially humor; relaxation using the patient's own memory of peaceful events; and cutaneous stimulation, especially use of cold. Cutaneous stimulation may even be effectively used at sites other than the site of pain. Specific examples of these techniques are presented.

Assessment and relief of pain in chemically dependent patients.

The growing number of patients admitted to acute care settings with the dual problem of pain and chemical dependency is bringing this major dilemma to the forefront of clinical nursing practice. Orthopaedics is one of the clinical areas likely to encounter an increased proportion of such patients. The care of chemically dependent patients with pain is not only often enormously challenging but also potentially frustrating. Planning care for these patients is best accomplished with a team approach that includes the expertise of several specialties, particularly pain and addiction. Although considerably more research is needed to identify the most effective approaches to the care of chemically dependent patients with pain, sufficient information now exists to support suggestions for guidelines. This article addresses definitions of pain and chemical dependency, methods of identifying the chemically dependent patient, the prevalence of pain and addiction, clinical conflicts and dilemmas related to relieving pain in addicted patients, a framework for planning care, and specific guidelines for individualizing the plan of care. Orthopaedic nurses are challenged to accept a leadership role in improving the care of chemically dependent patients with pain by evaluating these guidelines and developing additional approaches.

Multimodal balanced analgesia in the critically ill.

Pain is common but is often undertreated in critically ill patients. A multimodal balanced analgesic approach is recommended for the management of pain in these patients. Balanced analgesia uses combined analgesic regimens, thereby reducing the likelihood of significant effects from a single agent or method. It may include several different drugs given to prevent or aggressively treat continuous and breakthrough pain as well as pain from procedures.