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Aims Chronic stress is a well-known risk factor for the development of hypertension. However, the underlying mechanisms remain unclear. Corticotropin-releasing hormone (CRH) neurons in the central nucleus of the amygdala (CeA) are involved in the autonomic responses to chronic stress. Here, we determined the role of CeA-CRH neurons in chronic stress-induced hypertension. Methods and results Borderline hypertensive rats (BHRs) and Wistar-Kyoto (WKY) rats were subjected to chronic unpredictable stress (CUS). Firing activity and M-currents of CeA-CRH neurons were assessed, and a CRH-Cre-directed chemogenetic approach was used to suppress CeA-CRH neurons. CUS induced a sustained elevation of arterial blood pressure (ABP) and heart rate (HR) in BHRs, while in WKY rats, CUS-induced increases in ABP and HR quickly returned to baseline levels after CUS ended. CeA-CRH neurons displayed significantly higher firing activities in CUS-treated BHRs than unstressed BHRs. Selectively suppressing CeA-CRH neurons by chemogenetic approach attenuated CUS-induced hypertension and decreased elevated sympathetic outflow in CUS-treated BHRs. Also, CUS significantly decreased protein and mRNA levels of Kv7.2 and Kv7.3 channels in the CeA of BHRs. M-currents in CeA-CRH neurons were significantly decreased in CUS-treated BHRs compared with unstressed BHRs. Blocking Kv7 channel with its blocker XE-991 increased the excitability of CeA-CRH neurons in unstressed BHRs but not in CUS-treated BHRs. Microinjection of XE-991 into the CeA increased sympathetic outflow and ABP in unstressed BHRs but not in CUS-treated BHRs. Conclusions CeA-CRH neurons are required for chronic stress-induced sustained hypertension. The hyperactivity of CeA-CRH neurons may be due to impaired Kv7 channel activity, which represents a new mechanism involved in chronic stress-induced hypertension.

Aims Elevated sympathetic outflow is associated with primary hypertension. However, the mechanisms involved in heightened sympathetic outflow in hypertension are unclear. The central amygdala (CeA) regulates autonomic components of emotions through projections to the brainstem. The neuronal Kv7 channel is a non-inactivating voltage-dependent K+ channel encoded by KCNQ2/3 genes involved in stabilizing the neuronal membrane potential and regulating neuronal excitability. In this study, we investigated if altered Kv7 channel activity in the CeA contributes to heightened sympathetic outflow in hypertension. Methods and results The mRNA and protein expression levels of Kv7.2/Kv7.3 in the CeA were significantly reduced in spontaneously hypertensive rats (SHRs) compared with Wistar-Kyoto (WKY) rats. Lowering blood pressure with celiac ganglionectomy in SHRs did not alter Kv7.2 and Kv7.3 channel expression levels in the CeA. Fluospheres were injected into the rostral ventrolateral medulla (RVLM) to retrogradely label CeA neurons projecting to the RVLM (CeA-RVLM neurons). Kv7 channel currents recorded from CeA-RVLM neurons in brain slices were much smaller in SHRs than in WKY rats. Furthermore, the basal firing activity of CeA-RVLM neurons was significantly greater in SHRs than in WKY rats. Bath application of specific Kv7 channel blocker 10, 10-bis (4-pyridinylmethyl)-9(10H)-anthracnose (XE-991) increased the excitability of CeA-RVLM neurons in WKY rats, but not in SHRs. Microinjection of XE-991 into the CeA increased arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA), while microinjection of Kv7 channel opener QO-58 decreased ABP and RSNA, in anesthetized WKY rats but not SHRs. Conclusions Our findings suggest that diminished Kv7 channel activity in the CeA contributes to elevated sympathetic outflow in primary hypertension. This novel information provides new mechanistic insight into the pathogenesis of neurogenic hypertension. Translational perspective This study reveals a novel mechanistic insight into the pathogenesis of primary hypertension and provides a rationale to develop therapeutic strategies targeting Kv7 channels in the amygdala for treating hypertension. Viral vector-mediated overexpression of Kv7 channels in the amygdala is a potential approach to decrease blood pressure and inhibit sympathetic outflow in hypertension. However, this approach is not feasible to apply to hypertension patients at the current stage. Further studies to determine the molecular mechanism underlying downregulation of Kv7 channels may identify agents that increase expression levels of Kv7 channels in hypertension.

Glutamate N-methyl-D-aspartate (NMDA) receptors (NMDARs) and Kv7/M channels are importantly involved in regulating neuronal activity involved in various physiological and pathological functions. Corticotropin-releasing hormone (CRH)-expressing neurons in the central nucleus of the amygdala (CeA) critically mediate autonomic response during stress. However, the interaction between NMDA receptors and Kv7/M channels in the CRH(CeA) neurons remains unclear. In this study, we identified rat CRH(CeA) neurons through the expression of an AAV viral vector-mediated enhanced green fluorescent protein (eGFP) driven by the rat CRH promoter. M-currents carried by Kv7/M channels were recorded using the whole-cell patch-clamp approach in eGFP-tagged CRH(CeA) neurons in brain slices. Acute exposure to NMDA significantly reduced M-currents recorded from the CRH(CeA) neurons. NMDA-induced suppression of M-currents was eliminated by chelating intracellular Ca(2+), supplying phosphatidylinositol 4,5-bisphosphate (PIP2) intracellularly, or blocking phosphoinositide3-kinase (PI3K). In contrast, inhibiting protein kinase C (PKC) or calmodulin did not alter NMDA-induced suppression of M-currents. Sustained exposure of NMDA decreased Kv7.3 membrane protein levels and suppressed M-currents, while the Kv7.2 expression levels remained unaltered. Pre-treatment of brain slices with PKC inhibitors alleviated the decreases in Kv7.3 and reduction of M-currents in CRH(CeA) neurons induced by NMDA. PKC inhibitors did not alter Kv7.2 and Kv7.3 membrane protein levels and M-currents in CRH(CeA) neurons. These data suggest that transient activation of NMDARs suppresses M-currents through the Ca(2+)-dependent PI3K-PIP2 signaling pathway. In contrast, sustained activation of NMDARs reduces Kv7.3 protein expression and suppresses M-currents through a PKC-dependent pathway.

Sentrin/small ubiquitin-like modifier (SUMO) is protein modification pathway that regulates multiple biological processes, including cell division, DNA replication/repair, signal transduction, and cellular metabolism. In this review, we will focus on recent advances in the mechanisms of disease pathogenesis, such as cancer, diabetes, seizure, and heart failure, which have been linked to the SUMO pathway. SUMO is conjugated to lysine residues in target proteins through an isopeptide linkage catalyzed by SUMO-specific activating (E1), conjugating (E2), and ligating (E3) enzymes. In steady state, the quantity of SUMO-modified substrates is usually a small fraction of unmodified substrates due to the deconjugation activity of the family Sentrin/SUMO-specific proteases (SENPs). In contrast to the complexity of the ubiquitination/deubiquitination machinery, the biochemistry of SUMOylation and de-SUMOylation is relatively modest. Specificity of the SUMO pathway is achieved through redox regulation, acetylation, phosphorylation, or other posttranslational protein modification of the SUMOylation and de-SUMOylation enzymes. There are three major SUMOs. SUMO-1 usually modifies a substrate as a monomer; however, SUMO-2/3 can form poly-SUMO chains. The monomeric SUMO-1 or poly-SUMO chains can interact with other proteins through SUMO-interactive motif (SIM). Thus SUMO modification provides a platform to enhance protein-protein interaction. The consequence of SUMOylation includes changes in cellular localization, protein activity, or protein stability. Furthermore, SUMO may join force with ubiquitin to degrade proteins through SUMO-targeted ubiquitin ligases (STUbL). After 20 yr of research, SUMO has been shown to play critical roles in most, if not all, biological pathways. Thus the SUMO enzymes could be targets for drug development to treat human diseases.

Background Continuous infusion of doxorubicin or dexrazoxane pre-treatment prior to bolus doxorubicin are proven strategies to protect against doxorubicin-induced cardiotoxicity. Recently, global longitudinal peak systolic strain (GLS) measured with speckle tracking echocardiography (STE) and high-sensitivity troponin T (hs-TnT) have been validated as sensitive indicators of doxorubicin-induced cardiotoxicity. Here, we asked whether changes in hs-TnT and/or GLS can be detected in patients who were treated with continuous infusion of doxorubicin or pre-treated with dexrazoxane followed by bolus doxorubicin. Methods Twenty-nine patients with newly diagnosed sarcoma were assigned to receive either 72-h doxorubicin infusion or dexrazoxane pre-treatment before bolus doxorubicin. Eight patients received dexrazoxane pre-treatment; eleven patients received continuous doxorubicin infusion; ten patients crossed over from continuous infusion to dexrazoxane. Bloods were collected for hs-TnT at baseline, 24 h or 72 h after initiation of doxorubicin treatment in each chemotherapy cycle. All blood samples were assayed in batch using hs-TnT kit from Roche diagnostics. 2D Echo and STE were performed before doxorubicin, after cycle 3, and at the end of chemotherapy. Results Seven patients in the cross-over group have at least one hs-TnT measurement between 5 ng/L to 10 ng/L during and after chemotherapy. Ten patients have at least one hs-TnT measurement above 10 ng/ml during and after chemotherapy (six in dexrazoxane group, three in continuous infusion group, one in cross-over group). The average hs-TnT level increases with each additional cycle of doxorubicin treatment. Eight patients had a more than 5% reduction in LVEF at the end of chemotherapy (four in dexrazoxane group, three in continuous infusion group, and one in cross-over group). Four out of these eight patients had a change of GLS by more than 15% (three in the dexrazoxane group). Conclusion Elevation in hs-TnT levels were observed in more than 59% of patients who had received either continuous doxorubicin infusion or dexrazoxane pre-treatment before bolus doxorubicin. However, changes in LVEF and GLS were less frequently observed. Thus, continuous doxorubicin infusion or dexrazoxane pre-treatment do not completely ameliorate subclinical doxorubicin-induced cardiotoxicity as detected by more sensitive techniques.

Recognition of invading pathogens by Toll-like receptors (TLRs) activates innate immunity through signaling pathways that involved multiple protein kinases and phosphatases. We previously demonstrated that somatic nuclear autoantigenic sperm protein (sNASP) binds to TNF receptor–associated factor 6 (TRAF6) in the resting state. Upon TLR4 activation, a signaling complex consisting of TRAF6, sNASP, interleukin (IL)-1 receptor–associated kinase 4, and casein kinase 2 (CK2) is formed. CK2 then phosphorylates sNASP to release phospho-sNASP (p-sNASP) from TRAF6, initiating downstream signaling pathways. Here, we showed that protein phosphatase 4 (PP4) is the specific sNASP phosphatase that negatively regulates TLR4-induced TRAF6 activation and its downstream signaling pathway. Mechanistically, PP4 is directly recruited by phosphorylated sNASP to dephosphorylate p-sNASP to terminate TRAF6 activation. Ectopic expression of PP4 specifically inhibited sNASP-dependent proinflammatory cytokine production and downstream signaling following bacterial lipopolysaccharide (LPS) treatment, whereas silencing PP4 had the opposite effect. Primary macrophages and mice infected with recombinant adenovirus carrying a gene encoding PP4 (Ad-PP4) showed significant reduction in IL-6 and TNF-α production. Survival of Ad-PP4–infected mice was markedly increased due to a better ability to clear bacteria in a sepsis model. These results indicate that the serine/threonine phosphatase PP4 functions as a negative regulator of innate immunity by regulating the binding of sNASP to TRAF6.

Introduction: Doxorubicin-induced cardiotoxicity is a serious complication of cancer therapy recognized 40 years ago and remains to be a major clinical challenge today. Doxorubicin poisons topoisomerase 2a to kill cancer cells, whereas doxorubicin induces cardiotoxicity through topoisomerase 2b. When topoisomerase 2b was genetically deleted in the cardiomyocytes of adult mice, doxorubicin-induced cardiomyopathy can be prevented. Hypothesis: Pharmacological depletion of topoisomerase 2b in cardiac tissue could prevent doxorubicin-induced cardiomyopathy. Methods: Cell lines were treated with dexrazoxane (100 μM) for different time intervals with or without the proteasome inhibitor MG132 (10 μM). Cell lysates were immunoblotted with anti-topoisomerase 2a or 2b antibodies or anti-actin. Mice were injected with dexrazoxane (100 mg/kg IP) and cardiac tissues were analyzed for expression of topoisomerase 2b over time using western blot analysis. To study the effect of dexrazoxane pre-treatment on doxorubicin-induced chronic heart failure, C57BL/6 mice (n=7) were pre-treated with dexrazoxane (100 mg/kg IP) 8 hours before doxorubicin (5 mg/kg IP). Single injection of dexrazoxane (n=6), doxorubicin (n=8), or saline control (n=6) were also administered to three different cohorts of mice. This protocol was repeated weekly for 5 weeks. Pre-treatment and post-treatment cardiac MRI were obtained to evaluate the change in left ventricular ejection fraction. Results: Dexrazoxane induced a ubiquitin-dependent degradation of topoisomerase 2b, but not 2a in cell lines. In dexrazoxane-treated mice, topoisomerase 2b was not detectable 8 hours after dexrazoxane administration, but gradually returned to normal levels by 48 hours. Dexrazoxane pre-treatment 8 hours before doxorubicin prevented doxorubicin-induced reduction in ejection fraction as determined by cardiac MRI in a chronic heart failure model. Conclusions: Targeted degradation of topoisomerase 2b by dexrazoxane prevented doxorubicin-induced cardiotoxicity. Through selective degradation of topoisomerase 2b, but not 2a, doxorubicin's tumoricidal activity is preserved. This preventive strategy will be tested in a prospective clinical trial in breast cancer patients.

The opening session of Second International Colloquium on Cardio-Oncology addressed two areas of vital interest. The first reviewed new thoughts related to established agents. While anthracycline cardiotoxicity has been studied and reviewed extensively, ongoing research attempting to understand why it appears the mechanism(s) of toxicity differs from that of oncologic efficacy continue to evoke comment and intriguing speculation. Better understanding of the role of β-topoisomerase II in toxicity has advanced our understanding of the cascade of events that lead to heart failure. Additionally, the cardioprotective role of dexrazoxane fits well with our new understanding of how β-topoisomerase II works. Beyond the anthracyclines, new insight is providing us insight to better understand the impact on cardiac function seen with other agents including those targeting HER2 and several tyrosine-kinase inhibitors. Unlike the anthracyclines, these agents affect cardiac function in ways that are less direct, and therefore have different characteristics and should be thought of in alternate ways. This new knowledge regarding established agents furthers our understanding of the spectrum of cardiotoxicity and cardiac dysfunction in the cancer patient. The session also addressed cardiovascular toxicities of newer and established agents beyond myocardial dysfunction including effects on the vasculature. These agents cause changes that may be temporary or permanent, and that range from subclinical to life-threatening. The session ended with a discussion of the cardiac effects of immune checkpoint inhibitors. These agents can cause rare and sometimes fatal cardiac inflammation, for which long-term follow up may be required.

Many Toll-like receptors (TLRs) signal through TNF receptor–associated factor 6 (TRAF6) to activate innate immune responses. Here, we show that somatic nuclear autoantigenic sperm protein (sNASP) binds to TRAF6 to prevent TRAF6 autoubiquitination in unstimulated macrophages. Following LPS stimulation, a complex consisting of sNASP, TRAF6, IRAK4, and casein kinase 2 (CK2) is formed. CK2 phosphorylates sNASP at serine 158, allowing sNASP to dissociate from TRAF6. Free TRAF6 is then autoubiquitinated, followed by activation of downstream signaling pathways. In sNasp S158A knockin (S158A-KI) mice, LPS-treated macrophages could not phosphorylate sNASP, which remained bound to TRAF6. S158A-KI mice were more susceptible to sepsis due to a marked reduction in IL-1β, TNF-α, and IFN-γ production accompanied by an inability to clear bacteria and recruit leukocytes. Furthermore, phosphorylation-regulated release of sNASP from TRAF6 is observed following activation of TLR-1, -2, -4, -5, and -6. Thus, sNASP is a negative regulator of TLR signaling to modulate the innate immune response.

In this second part of a 2-part review, we will review cancer or cancer therapy–associated systemic and pulmonary hypertension, QT prolongation, arrhythmias, pericardial disease, and radiation-induced cardiotoxicity. This review is based on a MEDLINE search of published data, published clinical guidelines, and best practices in major cancer centers. Newly developed targeted therapy can exert off-target effects causing hypertension, thromboembolism, QT prolongation, and atrial fibrillation. Radiation therapy often accelerates atherosclerosis. Furthermore, radiation can damage the heart valves, the conduction system, and pericardium, which may take years to manifest clinically. Management of pericardial disease in cancer patients also posed clinical challenges. This review highlights the unique opportunity of caring for cancer patients with heart problems caused by cancer or cancer therapy. It is an invitation to action for cardiologists to become familiar with this emerging subspecialty.

Session II of the Second International Colloquium on Cardio-Oncology, chaired by Dr Breccia (Rome, Italy) and Dr Jurczak (Krakow, Poland), focused on mechanisms and clinical course of cardiovascular toxicity of cancer treatment. Whereas the venerable anthracyclines keep challenging patients and clinicians with risk of left ventricular dysfunction and heart failure, other newer drugs cause substantially different clinical phenotypes of cardiovascular toxicity. In particular, Session II not only focused on arterial thrombosis and venous thromboembolism, but also hypertension or cardiomyopathy or atrial fibrillation induced by many otherwise life-saving drugs. Dr Breccia (Rome, Italy) reviewed incidence, mechanisms, risk factors, and principles for prevention of cardiovascular events induced by tyrosine kinase inhibitors of hematologic interest, such as those used to treat chronic myeloid leukemia. Dr Carver (Philadelphia) reviewed the incidence, predisposing factors, and principles for proactive management of cardiovascular events in patients treated by conventional chemotherapy or new drugs for treatment of multiple myeloma. Dr Szmit (Warsaw, Poland) discussed on how coagulation disorders should be classified according to patient- or drug-related factors and how they should be diagnosed and treated in patients with solid or hematologic tumors. Dr Minotti (Rome. Italy) illustrated some potential pitfalls of accelerated drug development and approval and their possible impact on clinical incidence of cardiovascular events induced by tyrosine kinase inhibitors of hematologic interest. Session II therefore offered a broad perspective of the risk-benefit ratio of new drugs that are plagued with concerns about cardiovascular events.

INTRODUCTION Approximately 50% of patients with sepsis-induced acute lung injury and acute respiratory distress syndrome require mechanical ventilation. Patients with extended mechanical ventilator use routinely develop reinfections, which increases hospital stay, mortality, and health care cost. Some studies have pointed out inflammatory factors concentrations can affect ventilator weaning, but do not indicate changed inflammatory factors related to ventilator weaning during using ventilators. OBJECTIVES This study aimed to investigate during period of septic patients using ventilators, the inflammatory cytokines concentrations related with weaning rate. METHODS Blood was collected from 35 septic patients before and during ventilator use on days 1, 7, 14, and 21 (or weaning). RESULTS 58.3% (N = 20) of septic patients with mechanical ventilators were weaned successfully within 21 days (ventilator weaned group, VW), 16.7% (N = 6) did not wean within 21 days (ventilator dependent group, VD), and 25% died (death group) in hospital. Before ventilator use, higher C-reactive protein (CRP), IL-6, and IL-8 levels were measured in the death group than in all other groups (P

A 2-year-7-month-old female intact African hedgehog was presented with a subcutaneous mass around the right side of the neck as well as an intra-abdominal mass found during palpation. Surgical excision and exploratory laparotomy were performed. A uterine mass was identified during laparotomy and ovariohysterectomy was performed. The botryoid mass measured [Formula: see text][Formula: see text]cm and was located in the right uterine horn. It had a meat-like texture and was yellow-white and dark red in color. The removed subcutaneous mass measured [Formula: see text][Formula: see text]cm. The mass was well-encapsulated and had a yellow-white homogeneous texture on the cut surface. Histologically, the myometrium was invaded by neoplastic cells and the tissue boundaries were not obvious. Neoplastic cells were arranged in a whirling or intersecting pattern, with strong angiogenesis present. Vacuolated nuclei were round and oval to cigar shaped, with one to multiple nucleoli present. Immunohistochemistry revealed a positive reaction for CD10, but a negative reaction for smooth muscle actin (SMA) and desmin in the uterine neoplastic cells. Histologically, well-differentiated adipocytes with sheets of undifferentiated polygonal neoplastic cells, which were characterized by vacuolated nuclei with prominent multiple nucleoli, were found in the subcutaneous mass. Neoplastic cells of the subcutaneous mass were positively stained with antibodies of MDM2 and estrogen receptor (ER), but failed to give a positive result for vimentin because the cross-species interaction was insufficient. The definitive diagnosis was endometrial stromal sarcoma and liposarcoma in an African hedgehog.

Modern cancer therapy has successfully cured many cancers and converted a terminal illness into a chronic disease. Because cancer patients often have coexisting heart diseases, expert advice from cardiologists will improve clinical outcome. In addition, cancer therapy can also cause myocardial damage, induce endothelial dysfunction, and alter cardiac conduction. Thus, it is important for practicing cardiologists to be knowledgeable about the diagnosis, prevention, and management of the cardiovascular complications of cancer therapy. In this first part of a 2-part review, we will review cancer therapy-induced cardiomyopathy and ischemia. This review is based on a MEDLINE search of published data, published clinical guidelines, and best practices in major cancer centers. With the number of cancer survivors expanding quickly, the time has come for cardiologists to work closely with cancer specialists to prevent and treat cancer therapy-induced cardiovascular complications.

Importance Oncocardiology is a medical discipline that focuses on the identification, prevention, and treatment of cardiovascular complications related to cancer therapy. This discipline has gained interest from the cardiology community in recent years because of a remarkable increase in the number of cancer survivors and the proliferation of new cancer therapies causing cardiovascular complications, such as hypertension, heart failure, vascular complications, and cardiac arrhythmia. In this review, we provide historical perspectives, highlight new discoveries, and speculate on the opportunity created by merging the research interests and clinical practices of cardiology and oncology. Observations The old paradigm of anthracycline cardiotoxic effects is replaced by new insights that anthracycline targets topoisomerase II β to cause DNA double-strand breaks and a profound change in the transcriptome leading to the generation of reactive oxygen species and the development of mitochondriopathy. Prevention of anthracycline cardiotoxic effects should be based on inhibiting or degrading topoisomerase II β. New challenges were posed by the introduction of trastuzumab and tyrosine kinase inhibitors that revolutionized cancer therapy. The on-target cardiotoxic effects of trastuzumab were owing to a prosurvival benefit of Her2 that binds to neuregulin, whereas the off-target effect of multitargeted tyrosine kinase inhibitors may be mediated by disruption of the vascular endothelial growth factor signaling pathway or the stress-induced angiogenesis. Sensitive imaging techniques, such as global strain, and biomarkers have allowed for early detection of cardiotoxic effects. Early treatment with heart failure medications may be beneficial in preventing the development of late cardiotoxic effects. Conclusions and Relevance Close collaboration between cardiologists and oncologists is required to meet the demand of an increasing number of cancer survivors. New insights based on mechanistic studies or genetic discoveries will pave the way for better prevention, diagnosis, and treatment of cancer therapy-induced cardiovascular complications.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder that affects both sexes equally. The biochemical defect in PNH resides in the incomplete enzymatic assembly of glycosylphosphatidylinositol (GPI) anchors used for surface protein attachment. In all PNH patients tested to date, the biosynthetic defect occurs at the addition of N-acetyl-glucosamine to the phosphatidylinositol molecule (class A defect). A human cDNA, Piga, that repairs cell lines with the class A GPI-anchor biosynthetic defect has been recently cloned. Mapping of Piga to the X chromosome suggests that a single acquired mutation within Piga could alter GPI-anchor synthesis and result in PNH. However, this finding does not explain why all PNH patients have the class A defect. In the current study, the chromosomal assignment of Piga, as well as of Pigf and Pigh, two additional genes involved in GPI-anchor biosynthesis, has been established using a mouse interspecific backcross mapping technique. In contrast to Piga, both human and mouse Pigf and Pigh genes map to autosomes. The location of Pigf and Pigh suggests that mutations on both alleles of these autosomal genes would be necessary to produce PNH. This helps to explain the predominant class A defect in PNH.

The defect in the biosynthesis of the glycosyl-phosphatidyl inositol (GPI) anchor in paroxysmal nocturnal hemoglobinuria (PNH) appears to be in the initial steps. In biosynthetic studies using [3H]mannose, abnormal granulocytes of eight patients, and B lymphocytes transformed by Epstein-Barr virus of six different patients synthesized dolichyl phosphoryl mannose, but little or no later mannosylated intermediates. When fused with murine cell lines known to be deficient at different biosynthetic steps of the GPI anchor, the GPI-anchor-deficient granulocytes of 21/21 patients and lymphocytes from 6/6 patients complemented all murine cell lines except those of class A; cells of this class are not able to add N-acetylglucosamine to phosphatidylinositol. These studies indicate that the defect in GPI- anchor synthesis in PNH is early in the pathway, and is the same as that of class A mutants, but may be partial in some patients, resulting in the production of small amounts of mannosylated intermediates.

Previous attempts to express glycosylphosphatidylinositol-anchored proteins in Ltk- cells have not been successful because Ltk- cells cannot synthesize N-acetylglucosamine-phosphatidylinositol, the first intermediate in anchor biosynthesis. Using complementation cloning, we have identified a human cDNA that corrects the defect in anchor biosynthesis and allows the expression of glycosylphosphatidylinositol-anchored proteins in Ltk- cells. The nucleotide sequence predicts a novel cytosolic protein of 188 amino acids.

Salvinorin C (1), a minor component from a biologically active TLC fraction, was isolated from the leaves of the Mexican mint Salvia divinorum. Its structure was elucidated on the basis of extensive proton and C-13 NMR experiments, as well as by comparison of the NMR data with those of the mono- and diacetate derivatives 5-7 of the major NaBH(4)-reduction product of salvinorin A (2). [structure: see text]

Salvinorin C (1), a minor component from a biologically active TLC fraction, was isolated from the leaves of the Mexican mint Salvia divinorum. Its structure was elucidated on the basis of extensive proton and C-13 NMR experiments, as well as by comparison of the NMR data with those of the mono- and diacetate derivatives 5−7 of the major NaBH4-reduction product of salvinorin A (2).

Pain is a major presenting symptom in patients with rheumatic diseases. It is difficult, however, to define pain precisely because there are both objective and subjective components to the experience of pain. Thus, patients with the same degree of tissue inflammation may experience different levels of pain. The variation in individual pain experience often presents us with a diagnostic and therapeutic dilemma. Pain management, therefore, must be based on a solid understanding of the pathophysiology of pain and on a careful review of the risks, benefits, cost, and efficacy of available treatments.

To investigate the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor in the granulocytes of paroxysmal nocturnal hemoglobinuria (PNH), the glycolipids of granulocytes from PNH patients and normal volunteers were biosynthetically labeled with [3H]mannose in the presence of tunicamycin. Extracted glycolipids were examined by thin- layer chromatography and compared with known biosynthetic intermediates. Normal granulocytes consistently showed [3H]mannose incorporation into the complete GPI core, several GPI biosynthetic intermediates, and dolichol phosphate mannose (DPM). The granulocytes of 10 patients with PNH that had no expression of CD55 and CD59 on greater than 95% of the cells were able to incorporate [3H]mannose into DPM, but were not able to incorporate detectable amounts into the complete GPI core. THus, PNH granulocytes do not synthesize detectable amounts of the complete GPI core and this defect likely accounts for the absence of GPI-linked membrane proteins on hematopoietic cells in this syndrome.

The glycosylphosphatidylinositol (GPI) anchor, potentially capable of generating a number of second messengers, such as diacylglycerol, phosphatidic acid, and inositol phosphate glycan, has been postulated to be involved in signal transduction in various cell types, including T-cells. We have identified a panel of T-cell hybridoma mutants that are defective at various steps of GPI anchor biosynthesis. Since they were derived from a functional T-T hybridoma, we were able to determine the precise role of the GPI anchor in T-cell activation. Two mutants were chosen for this analysis. The first mutant is defective at the first step of GPI anchor biosynthesis, i.e. in the transfer of N-acetylglucosamine to a phosphatidylinositol acceptor. Thus, it cannot form any GPI precursors or GPI-like compounds. Interestingly, this mutant can be activated by antigen, superantigen, and concanavalin A in a manner comparable to the wild-type hybridoma. These data strongly suggest that the GPI anchor, its precursor, or its potential cleavage product, inositol phosphate glycan, is not required for the early phase of T-cell activation. The second mutant is able to synthesize the first two GPI precursors, but is not able to add mannose residues to them due to a deficiency in dolichol-phosphate-mannose (Dol-P-Man) biosynthesis. Unexpectedly, all of the Dol-P-Man mutants are defective in activation by antigen, suprantigen, and concanavalin A despite normal T-cell receptor expression. Here, we show that the activation defect was due to a pleiotropic glycosylation abnormality because Dol-P-Man is required for both GPI anchor and N-linked oligosaccharide biosynthesis. When the yeast Dol-P-Man synthase gene was stably transfected into the mutants, full expression of surface GPI-anchored proteins was restored. However, N-linked glycosylation was either partially or completely corrected in different transfectants. Reconstitution of activation defects correlates well with the status of N-linked glycosylation, but not with the expression of GPI-anchored proteins. These results thus reveal an unexpected role of N-linked glycosylation in T-cell activation.

Glycosylphosphatidylinositol (GPI) serves as a membrane anchor for a large number of eukaryotic proteins. A genetic approach was used to investigate the biosynthesis of GPI anchor precursors in mammalian cells. T cell hybridoma mutants that cannot synthesize dolichol-phosphate-mannose (Dol-P-Man) also do not express on their surface GPI-anchored proteins such as Thy-1 and Ly-6A. These mutants cannot form mannose-containing GPI precursors. Transfection with the yeast Dol-P-Man synthase gene rescues the synthesis of both Dol-P-Man and mannose-containing GPI precursors, as well as the surface expression of Thy-1 and Ly-6A, suggesting that Dol-P-Man is the donor of at least one mannose residue in the GPI core.

Bleomycin (BLM) is an anticancer drug that generates reactive oxygen species (ROS) after interacting with iron and oxygen. We hypothesized that BLM could cause a different status of oxidative stress in normal versus tumor cells due to possible altered redox status and gene expression in cells following transformation. In this study, the extent of cytotoxicity, levels of ROS, and activities of antioxidant enzymes were compared between normal WI38 cells and SV40-transformed WI38 (VA13) cells following BLM treatment. Basal activities of MnSOD and catalase were lower in VA13 cells and basal ROS levels were higher in VA13 cells. Although BLM caused greater growth inhibition and apoptosis in VA13 cells, it increased ROS levels at an earlier time point in WI38 cells. Moreover, BLM treatment (100 μg/ml) had no effect on the activities of MnSOD, CuZnSOD, and catalase, but increased the activities of glutathione peroxidase (GPX) in WI38 cells after a 48-h treatment and in VA13 cells after a 24- and 48-h treatment. Northern blot analysis indicated that the increase in GPX activities was due to increased transcript levels of GPX1 but not GPX4 in both cells. Our results indicate selective induction of the GPX1 gene by BLM and different redox responses to BLM between WI38 and VA13 cells.

Pain is the most common reason patients seek medical care. While pain may be a symptom of an underlying disease or injury, pain can also become a persistent symptom of an autonomous neurophysiological process constituting a unique neurobiological disorder. It has been demonstrated that all forms of pain, including acute, chronic, and that associated with terminal illness, are often poorly managed with consequent needless suffering, functional impairment and disability. The American Academy of Pain Medicine, in its position …

Cancer is the second leading cause of death in the United States. About 1.2 million new cancer cases were diagnosed in 1998. One out of every four deaths, approximately half a million people, will be the result of cancer in 1999. Pain is often inadequately treated in patients with cancer. Sixty-seven percent of outpatients with metastatic cancer have pain, and 36% of these patients have pain severe enough to impair their ability to function. 6 Sixty percent of terminally ill cancer patients suffer from pain. 11 Eighty-six percent of practicing physicians surveyed believed that the majority of cancer patients with pain were undermedicated. 32 On a global scale, the World Health Organization Cancer Pain Relief Program reported that approximately 5 million people worldwide experience cancer pain on a daily basis, with 25% of them dying without relief from severe pain. Cancer pain can be controlled through relatively simple means in most patients. Unrelieved pain can lessen the patients' activity, function, appetite, and sleep. It can also induce fear and increase suffering in patients with cancer. Chronic unrelieved pain depresses patients and increases the risk of suicide. In addition to playing a major role in the quality of care for cancer patients at the end of their life, pain management is also vital to cancer survivors and patients with stable disease status. Although they have survived the disease, patients may have chronic pain from disease or therapy. Adequate pain control is important to ensure that patients are able to function productively, maintain social relationships, and improve their quality of life. The most recent report on cancer incidence and mortality showed the first decline after nearly 20 years of a steady increase 35 ; thus, pain control in cancer survivors and patients with stable disease is an important part of the total care of cancer patients. The majority of cancer pain can be controlled with oral pharmacologic management. Both physicians and patients should understand that the long-term use of opioids for pain relief is different from substance abuse. Studies have shown that the number of individuals who become addicted to drugs by introducing them through medical use is extremely small. 24 Clinicians should assess patients and, if pain is present, provide them with optimal relief throughout the course of illness. The treatment plan should include patient and family education about pain and its management and encourage patients to be active participants in pain management. 7 Comprehensive management of cancer patients with pain is best accomplished through interdisciplinary coordination. Whenever possible, the primary care physician should coordinate the services of medical, surgical, and radiation oncologists; pain specialists; psychologists; psychiatrists; physical therapists; social workers; nurses; and hospice workers.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder that affects both sexes equally. The biochemical defect in PNH resides in the incomplete enzymatic assembly of glycosylphosphatidylinositol (GPI) anchors used for surface protein attachment. In all PNH patients tested to date, the biosynthetic defect occurs at the addition of N-acetyl-glucosamine to the phosphatidylinositol molecule (class A defect). A human cDNA, Piga, that repairs cell lines with the class A GPI-anchor biosynthetic defect has been recently cloned. Mapping of Piga to the X chromosome suggests that a single acquired mutation within Piga could alter GPI-anchor synthesis and result in PNH. However, this finding does not explain why all PNH patients have the class A defect. In the current study, the chromosomal assignment of Piga, as well as of Pigf and Pigh, two additional genes involved in GPI-anchor biosynthesis, has been established using a mouse interspecific backcross mapping technique. In contrast to Piga, both human and mouse Pigf and Pigh genes map to autosomes. The location of Pigf and Pigh suggests that mutations on both alleles of these autosomal genes would be necessary to produce PNH. This helps to explain the predominant class A defect in PNH.

Since 1997, the Institute of Medicine of the National Academy of Sciences has released two reports regarding the need to provide more educational programs that stress the importance of end-of-life care and that train clinicians to provide such care [1,2]. The Summary and Recommendations for Improving Palliative Care for Cancer, released by the Institute of Medicine in 2001, listed “inadequate training of health care personnel in symptom management and other palliative care skills” as a barrier to ensure excellent palliative and end-of-life care [2]. Pain is a common symptom of patients with cancer, and pain control is an important clinical goal for this patient group as well as for patients with nonmalignant diseases. All patients expect reliable skillful care from their physicians when experiencing pain and approaching death. The Joint Commission on Accreditation of Healthcare Organizations ( JCAHO) first established pain management standards for all hospitals in 2001. These standards require all physicians to assess and manage pain for all patients. However, pain medicine and palliative care are not a part of the required curriculum at most of the United State's medical schools. Billings et al. [3] reviewed articles, and Barzansky et al. [4] reviewed the Liaison Committee on Medical Education (LCME) and Association of American Medical Colleges (AAMC) Annual Medical School Questionnaires regarding medical education … Reprint requests to: Hui-Ming Chang, MD, MPH, Department of Palliative Care and Rehabilitation, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard Box 8, Houston, TX 77030. Tel: (713) 500-3559; Fax: (713) 500-0336; E-mail: Hui-Ming.Chang{at}uth.tmc.edu.

A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized. The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level. We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level. The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway. Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants. These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences. Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.

Fas/APO-1 and TNF receptor 1 share a common signaling motif in their cytoplasmic tail called the "death domain." Using the death domain as bait in the yeast two-hybrid system, several death domain-containing proteins that participate in cell death signaling have been identified. Here we report the isolation of a novel protein, sentrin, which interacts with Fas/APO-1 and TNF receptor 1 but not with FADD/MORT1 or CD40. Two-hybrid interaction assays reveal that sentrin associates only with the signal-competent forms of Fas/APO-1 or TNF receptor 1 death domains. Sentrin is a novel protein of 101 amino acids with homology to ubiquitin, Nedd8, and a Saccharomyces cerevisiae protein, Smt3. When overexpressed, sentrin provides protection against both anti-Fas/APO-1 and TNF-induced cell death.

A number of eukaryotic proteins are anchored to the membrane by glycosylphosphatidylinositol (GPI), of which the core structure is conserved from protozoan to mammalian cells. Here, we used a panel of thymoma mutants, which synthesize Thy-1 but cannot express it on the cell surface, to study the GPI biosynthetic pathway in mammalian cells. These mutants have been assigned into six complementation classes (A, B, C, E, F, H) by the technique of somatic cell hybridization. Using a combination of metabolic labeling and chemical/enzymatic tests, the biosynthetic defects were mapped to four different steps. Class A, C, and H mutants cannot transfer N-acetylglucosamine (GlcNAc) to a phosphatidylinositol acceptor, suggesting that the first step of GPI synthesis is regulated by at least three genes. The Class E mutant does not synthesize dolichol-phosphate-mannose, the donor for the first mannose residue transferred to the GPI core, and thus cannot form any mannose-containing GPI precursors. Class B and F mutants are defective in the addition of the third mannose residue or ethanolamine phosphate, respectively, to the elongating GPI core. Our findings have implications for the biosynthesis and attachment of the mammalian GPI anchor.

A large number of mammalian proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Biosynthetic intermediates of the GPI anchor have been identified in mammalian cells. The early GPI precursors are sensitive to phosphatidylinositol (PI)-specific phospholipase C (PLC). However, all of the later GPI precursors, which contain 1 or more mannose residues, are PI-PLC-resistant, suggesting that there is another unidentified precursor. Here, we report the identification of this missing link. This GPI precursor can only be labeled with glucosamine and inositol, and is resistant to PI-PLC but sensitive to GPI-phospholipase D. It accumulates in large quantity only in mutants which are defective in the addition of the first mannose residue to the elongating GPI core. Thus, fatty acylation of glucosaminylphosphatidylinositol, to render it PI-PLC-resistant, is an obligatory step in the biosynthesis of mammalian GPI anchor precursors.